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Manuela SIMONI

Professore Ordinario
Dipartimento di Scienze Biomediche, Metaboliche e Neuroscienze sede ex-Medicina, Endocrinologia, Metabolismo e Geriatria


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Pubblicazioni

2022 - Accurate and time-saving, two-step intracavernosal injection procedure to diagnose psychological erectile dysfunction [Articolo su rivista]
Santi, Daniele; Spaggiari, Giorgia; Simoni, Manuela; Granata, Antonio R M
abstract

Background: The recognition of the erectile dysfunction pathogenesis is essential to identify the appropriate erectile dysfunction management. As vascular erectile dysfunction could be a manifestation of a systemic arterial damage, the watershed in the erectile dysfunction diagnostic framework is the discrimination between psychological erectile dysfunction and vascular erectile dysfunction. However, reliable tools to directly diagnose psychological erectile dysfunction are currently lacking. Objective: To identify which parameters could predict psychological erectile dysfunction. Moreover, we suggest a new intracavernosal injection procedure to optimize the erectile dysfunction diagnostic workup. Materials and methods: A retrospective, real-world analysis was carried out including all men who underwent intracavernosal injection procedure at the Modena Andrology Unit from 2018 to 2021. A first intracavernosal injection procedure with 5 µg of prostaglandin E-1 (PGE-1) was performed. In the absence of a full drug-induced erection (immediate or delayed), an echo-color Doppler penile evaluation after administration of PGE-1 10 µg was conducted, measuring intracavernosal blood flows, to document a possible vascular etiology. Hormonal evaluations were performed. Results: Out of 179 enrolled patients, 70.4% showed psychological erectile dysfunction, 21.7% vascular erectile dysfunction, and 7.8% hormonal genesis. Multinomial logistic regression analysis identified absence of cardiovascular disease (p = 0.017), presence of spontaneous morning erections (p = 0.018), and normal penile erections with masturbation (p = 0.035) as predictors of psychological erectile dysfunction. Clinically, normal intracavernosal injection test response was detected in 86 patients and abnormal response in 93 subjects. Among the latter, 54 patients experienced a delayed response. The combination of intracavernosal injection test with late penile erections evaluation was able to diagnose psychological erectile dysfunction (sensitivity 97%, specificity 100%), avoiding unnecessary retesting. Discussion: We propose a two-step intracavernosal injection procedure that allows to recognize psychological erectile dysfunction with a high sensitivity/specificity, saving costs and time, and limiting adverse events. Moreover, the presence of spontaneous morning erections and valid penile erections after masturbation could guide the diagnostic workup, indirectly identifying those patients deserving of a deeper evaluation of vascular health.


2022 - COVID-19 lockdown negatively impacted on adherence to denosumab therapy: incidence of non-traumatic fractures and role of telemedicine [Articolo su rivista]
De Vincentis, S; Domenici, D; Ansaloni, A; Boselli, G; D'Angelo, G; Russo, A; Taliani, E; Rochira, V; Simoni, M; Madeo, B
abstract

Purpose Coronavirus disease (COVID-19) lockdowns have impacted on management of osteoporosis and the use of telemedicine is increasingly widespread albeit supported by little evidence so far. The aim of the study is to assess adherence to denosumab and incidence of non-traumatic fractures during the lockdown compared to the pre-COVID-19 year and to explore the effectiveness of telemedicine in the management of osteoporotic patients. Methods Retrospective, longitudinal, single-center study on patients receiving subcutaneous denosumab therapy every 6 months. Each patient was scheduled to undergo 2 visits: one during the pre-COVID-19 period (March 2019-March 2020) and another visit during the lockdown period (March 2020-March 2021). Data on new fractures, adherence, risk factors for osteoporosis and the modality of visit (telemedicine or face-to-face) were collected. Results The prevalence of non-adherent patients was higher during the lockdown (35 of 269 patients, 13.0%) than the pre-COVID-19 period (9 of 276 patients, 3.3%) (p < 0.0001). During the lockdown, the number of new non-traumatic fractures was higher than the pre-COVID-19 year (p < 0.0001): 10 patients out of 269 (3.7%) experienced a fragility fracture and 2 patients (0.7%) a probable rebound fracture during the lockdown period, whereas no patient had fragility/rebound fractures during the pre-COVID-19 period. No difference was found in the prevalence of non-adherence and new non-traumatic fractures comparing patients evaluated with tele-medicine to those evaluated with face-to-face visit. Conclusion Non-adherent patients and new non-traumatic fractures (including rebound fractures) were more prevalent during the lockdown in comparison to the pre-COVID-19 period, regardless of the modality of medical evaluation.


2022 - Changes in quality of life after thyroidectomy in subjects with thyroid cancer in relation to the dose of levothyroxine [Articolo su rivista]
Monzani, M. L.; Piccinini, F.; Boselli, G.; Corleto, R.; Margiotta, G.; Peeters, R. P.; Simoni, M.; Brigante, G.
abstract

Previous studies demonstrated decreased quality of life (QoL) in differentiated thyroid cancer (DTC) survivors and suggested QoL variability related to time from thyroidectomy and levothyroxine dosage. The aims of this study were to evaluate QoL in thyroidectomized subjects in different levothyroxine states and to evaluate the association between TSH and thyroid hormones and QoL.


2022 - Diabetic Neuropathic Cachexia: A Clinical Case and Review of Literature [Articolo su rivista]
Bellelli, A.; Santi, D.; Simoni, M.; Greco, C.
abstract

A 46-year-old man was admitted to the surgical department because of abdominal pain and anemia, with the radiological finding of a perforated duodenal ulcer, and underwent laparoscopic surgical treatment. Type 2 diabetes mellitus (T2DM) had been diagnosed 5 years earlier and treated with diet. At clinical investigation, the patient was depressed and anorexic; moreover, he complained of lower extremity weakness and bilateral feet pain, burning in nature and accompanied by allodynia. This painful sensation had been preceded by an 8-month history of fatigue and anorexia with profound weight loss of 35 kg. After clinical evaluation and a nerve conduction study, diagnosis of diabetic cachectic neuropathy was made based on the rapid onset of severe neuropathic pain in the context of diabetic neuropathy, marked weight loss, and depressed mood. The therapy with pregabalin and duloxetine had scarce effect and was gradually discontinued. The patient, however, obtained progressive relief and amelioration of neuropathic lower-limb pain concomitant with weight gain. This clinical trend also confirmed the diagnosis of this rare form of diabetic neuropathy. A few cases of diabetic neuropathic cachexia have been reported in the literature and are briefly reviewed here.


2022 - Editorial: Follicle-stimulating hormone: Fertility and beyond-volume II [Articolo su rivista]
Simoni, M.; Huhtaniemi, I.; Casarini, L.; Santi, D.
abstract


2022 - Effect of Genetic Variants of Gonadotropins and Their Receptors on Ovarian Stimulation Outcomes: A Delphi Consensus [Articolo su rivista]
Conforti, A.; Tuttelmann, F.; Alviggi, C.; Behre, H. M.; Fischer, R.; Hu, L.; Polyzos, N. P.; Chuderland, D.; Rama Raju, G. A.; D'Hooghe, T.; Simoni, M.; Sunkara, S. K.; Longobardi, S.
abstract

Background: A Delphi consensus was conducted to evaluate the influence of single nucleotide polymorphisms (SNPs) in genes encoding gonadotropin and gonadotropin receptors on clinical ovarian stimulation outcomes following assisted reproductive technology (ART) treatment. Methods: Nine experts plus two Scientific Coordinators discussed and amended statements plus supporting references proposed by the Scientific Coordinators. The statements were distributed via an online survey to 36 experts, who voted on their level of agreement or disagreement with each statement. Consensus was reached if the proportion of participants agreeing or disagreeing with a statement was >66%. Results: Eleven statements were developed, of which two statements were merged. Overall, eight statements achieved consensus and two statements did not achieve consensus. The statements reaching consensus are summarized here. (1) SNP in the follicle stimulating hormone receptor (FSHR), rs6166 (c.2039A>G, p.Asn680Ser) (N=5 statements): Ser/Ser carriers have higher basal FSH levels than Asn/Asn carriers. Ser/Ser carriers require higher amounts of gonadotropin during ovarian stimulation than Asn/Asn carriers. Ser/Ser carriers produce fewer oocytes during ovarian stimulation than Asn/Asn or Asn/Ser carriers. There is mixed evidence supporting an association between this variant and ovarian hyperstimulation syndrome. (2) SNP of FSHR, rs6165 (c.919G>A, p.Thr307Ala) (N=1 statement): Few studies suggest Thr/Thr carriers require a shorter duration of gonadotropin stimulation than Thr/Ala or Ala/Ala carriers. (3) SNP of FSHR, rs1394205 (−29G>A) (N=1 statement): Limited data in specific ethnic groups suggest that A/A allele carriers may require higher amounts of gonadotropin during ovarian stimulation and produce fewer oocytes than G/G carriers. (4) SNP of FSH β-chain (FSHB), rs10835638 (−211G>T) (N=1 statement): There is contradictory evidence supporting an association between this variant and basal FSH levels or oocyte number. (5) SNPs of luteinizing hormone β-chain (LHB) and LH/choriogonadotropin receptor (LHCGR) genes (N=1 statement): these may influence ovarian stimulation outcomes and could represent potential future targets for pharmacogenomic research in ART, although data are still very limited. Conclusions: This Delphi consensus provides clinical perspectives from a diverse international group of experts. The consensus supports a link between some variants in gonadotropin/gonadotropin receptor genes and ovarian stimulation outcomes; however, further research is needed to clarify these findings.


2022 - Effect of the Glucagon-Like Peptide-1 Receptor Agonists on Autonomic Function in Subjects with Diabetes: A Systematic Review and Meta-Analysis [Articolo su rivista]
Greco, Carla; Santi, Daniele; Brigante, Giulia; Pacchioni, Chiara; Simoni, Manuela
abstract

Background: In addition to the metabolic effects in diabetes, glucagon-like peptide 1 receptor (GLP-1R) agonists lead to a small but substantial increase in heart rate (HR). However, the GLP-1R actions on the autonomic nervous system (ANS) in diabetes remain debated. Therefore, this meta-analysis evaluates the effect of GLP-1R agonist on measures of ANS function in diabetes. Methods: According to the Cochrane Collaboration and Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) statement, we conducted a meta-analysis considering clinical trials in which the autonomic function was evaluated in diabetic subjects chronically treated with GLP-1R agonists. The outcomes were the change of ANS function measured by heart rate variability (HRV) and cardiac autonomic reflex tests (CARTs). Results: In the studies enrolled, HR significantly increased after treatment (P<0.001), whereas low frequency/high frequency ratio did not differ (P=0.410); no changes in other measures of HRV were detected. Considering CARTs, only the 30:15 value derived from lying-to-standing test was significantly lower after treatment (P=0.002), but only two studies reported this measurement. No differences in other CARTs outcome were observed. Conclusion: The meta-analysis confirms the HR increase but seems to exclude an alteration of the sympatho-vagal balance due to chronic treatment with GLP-1R agonists in diabetes, considering the available measures of ANS function.


2022 - Follicle stimulating hormone effectiveness in male idiopathic infertility: what happens in daily practice? [Articolo su rivista]
Romeo, Marilina; Spaggiari, Giorgia; Nuzzo, Federico; Granata, Antonio R M; Simoni, Manuela; Santi, Daniele
abstract

To assess the effectiveness of follicle-stimulating hormone (FSH) administration in male idiopathic infertility in a clinical setting.


2022 - Genetic signature of differentiated thyroid carcinoma susceptibility: a machine learning approach [Articolo su rivista]
Brigante, Giulia; Lazzaretti, Clara; Paradiso, Elia; Nuzzo, Federico; Sitti, Martina; Tüttelmann, Frank; Moretti, Gabriele; Silvestri, Roberto; Gemignani, Federica; Försti, Asta; Hemminki, Kari; Elisei, Rossella; Romei, Cristina; Zizzi, Eric Adriano; Deriu, Marco Agostino; Simoni, Manuela; Landi, Stefano; Casarini, Livio
abstract

To identify a peculiar genetic combination predisposing to differentiated thyroid carcinoma (DTC), we selected a set of single nucleotide polymorphisms (SNPs) associated with DTC risk, considering polygenic risk score (PRS), Bayesian statistics and a machine learning (ML) classifier to describe cases and controls in three different datasets. Dataset 1 (649 DTC, 431 controls) has been previously genotyped in a genome-wide association study (GWAS) on Italian DTC. Dataset 2 (234 DTC, 101 controls) and dataset 3 (404 DTC, 392 controls) were genotyped. Associations of 171 SNPs reported to predispose to DTC in candidate studies were extracted from the GWAS of dataset 1, followed by replication of SNPs associated with DTC risk (P < 0.05) in dataset 2. The reliability of the identified SNPs was confirmed by PRS and Bayesian statistics after merging the three datasets. SNPs were used to describe the case/control state of individuals by ML classifier. Starting from 171 SNPs associated with DTC, 15 were positive in both datasets 1 and 2. Using these markers, PRS revealed that individuals in the fifth quintile had a seven-fold increased risk of DTC than those in the first. Bayesian inference confirmed that the selected 15 SNPs differentiate cases from controls. Results were corroborated by ML, finding a maximum AUC of about 0.7. A restricted selection of only 15 DTC-associated SNPs is able to describe the inner genetic structure of Italian individuals, and ML allows a fair prediction of case or control status based solely on the individual genetic background.


2022 - Human fertility and sleep disturbances: A narrative review [Articolo su rivista]
Spaggiari, Giorgia; Romeo, Marilina; Casarini, Livio; Granata, Antonio R M; Simoni, Manuela; Santi, Daniele
abstract

Introduction: Many factors may be hidden behind the global fertility decline observed in Western countries. Alongside the progressively increased age of infertile couples, environmental and behavioural factors, including non-optimal lifestyle habits, should be considered. Among these, sleep disorders have been suggested to be linked to human fertility. Methods: This is a narrative review, describing first sleep physiology, its disturbances, and the tools able to quantify sleep dysfunction. Then, we consider all available studies aimed at investigating the connection between sleep disorders and human fertility, providing a comprehensive view on this topic. Results: Forty-two studies investigating the relationship between sleep habits and human reproduction were included. All the published evidence was grouped according to the aspect of human fertility considered, i.e. i) female reproductive functions, ii) male reproductive functions, iii) natural conception and iv) assisted reproduction. For each of the sub-groups considered, the connection between sleep dysregulation and human fertility was classified according to specific sleep characteristics, such as sleep duration, quality, and habits. In addition, possible physio-pathological mechanisms proposed to support the link between sleep and fertility were summarized. Conclusion: This review summarizes the most relevant findings about the intricate and still largely unknown network of molecular pathways involved in the regulation of circadian homeostasis, to which sleep contributes, essential for reproductive physiology. Thus, many mechanisms seem correlate sleep disorders to reproductive health, such as adrenal activation, circadian dysregulation, and genetic influences. This review highlights the need to properly designed trials on the topic.


2022 - Ketogenic state improves testosterone serum levels-results from a systematic review and meta-analysis [Articolo su rivista]
Furini, Chiara; Spaggiari, Giorgia; Simoni, Manuela; Greco, Carla; Santi, Daniele
abstract

It is widely demonstrated that obesity and hypogonadism are bi-directionally correlated, since the hypogonadism prevalence is higher in obese population, while weight loss increases testosterone serum levels. Several approaches are available to contrast weight excess, from simple dietary regimens to more complex surgical procedures. Ketogenic diets (KD) fit in this context and their application is growing year after year, aiming to improve the metabolic and weight patterns in obese patients. However, KD influence on testosterone levels is still poorly investigated.


2022 - Management of male factor infertility: position statement from the Italian Society of Andrology and Sexual Medicine (SIAMS): Endorsing Organization: Italian Society of Embryology, Reproduction, and Research (SIERR) [Articolo su rivista]
Ferlin, A.; Calogero, A. E.; Krausz, C.; Lombardo, F.; Paoli, D.; Rago, R.; Scarica, C.; Simoni, M.; Foresta, C.; Rochira, V.; Sbardella, E.; Francavilla, S.; Corona, G.
abstract

Purpose: Infertility affects 15–20% of couples and male factors are present in about half of the cases. For many aspects related to the diagnostic and therapeutic approach of male factor infertility, there is no general consensus, and the clinical approach is not uniform. Methods: In the present document by the Italian Society of Andrology and Sexual Medicine (SIAMS), endorsed by the Italian Society of Embryology, Reproduction, and Research (SIERR), we propose evidence-based recommendations for the diagnosis, treatment, and management of male factor infertility to improve patient and couple care. Results: Components of the initial evaluation should include at minimum medical history, physical examination, and semen analysis. Semen microbiological examination, endocrine assessment, and imaging are suggested in most men and recommended when specific risk factors for infertility exist or first-step analyses showed abnormalities. Full examination including genetic tests, testicular cytology/histology, or additional tests on sperm is clinically oriented and based on the results of previous investigations. For treatment purposes, the identification of the specific cause and the pathogenetic mechanism is advisable. At least, distinguishing pre-testicular, testicular, and post-testicular forms is essential. Treatment should be couple-oriented, including lifestyle modifications, etiologic therapies, empirical treatments, and ART on the basis of best evidence and with a gradual approach. Conclusion: These Guidelines are based on two principal aspects: they are couple-oriented and place high value in assessing, preventing, and treating risk factors for infertility. These Guidelines also highlighted that male infertility and in particular testicular function might be a mirror of general health of a man.


2022 - Neither rationale nor scientific evidence exist to support that double stimulation is potentially unsafe [Articolo su rivista]
Casarini, Livio; Vaiarelli, Alberto; Cimadomo, Danilo; Santi, Daniele; Simoni, Manuela; Garcìa-Velasco, Juan Antonio; Alviggi, Carlo; La Marca, Antonio; Rienzi, Laura; Ubaldi, Filippo Maria
abstract


2022 - Phosphodiesterase (PDE) 5 inhibitors sildenafil, tadalafil and vardenafil impact cAMP-specific PDE8 isoforms-linked second messengers and steroid production in a mouse Leydig tumor cell line [Articolo su rivista]
Limoncella, S.; Lazzaretti, C.; Paradiso, E.; D'Alessandro, S.; Barbagallo, F.; Pacifico, S.; Guerrini, R.; Tagliavini, S.; Trenti, T.; Santi, D.; Simoni, M.; Sola, M.; Di Rocco, G.; Casarini, L.
abstract

Type 5 phosphodiesterase (PDE5) blockade by inhibitors (PDE5i) results in intracellular cyclic guanosine monophosphate (cGMP) increase and smooth muscle relaxation and are used for the treatment of men erectile dysfunction. Although they have high specificity for PDE5, these inhibitors are suspected to cross-interact also with cyclic adenosine monophosphate (cAMP)-specific PDEs, inducing the intracellular accumulation of this cyclic nucleotide and related testosterone increase, positively impacting male reproductive parameters. However, the link between the use of PDE5i and the activation of cAMP-mediated steroidogenesis is still unclear. We have investigated whether three PDE5i, sildenafil, tadalafil and vardenafil, cross-interacts with the high affinity cAMP-specific enzymes type 8A and 8B PDEs (PDE8A and PDE8B), in live, transfected mouse Leydig tumor (mLTC1) and human embryonic kidney (HEK293) cell lines in vitro. The PDE5i-induced production of cAMP-dependent testosterone and its precursor progesterone was evaluated as well. We have developed PDE8A/B biosensors and modified cyclic nucleotides confirming enzyme binding to cAMP, but not to cGMP, in our cell models. cAMP binding to PDE8A/B was displaced upon cell treatment with PDE5i, revealing that sildenafil, tadalafil and vardenafil have similar effectiveness in live cells, in vitro. The cross-interaction between PDE5i and PDE8A/B supports the gonadotropin-enhanced intracellular cAMP increase, occurring together with cGMP increase, as well as steroid synthesis. Indeed, we found that Leydig cell treatment by PDE5i increases progesterone and testosterone production triggered by gonadotropins. We demonstrated that PDE5i may interact with the cAMP-specific PDE8A and PDE8B, possibly inducing intracellular cAMP and sex steroid hormone increase. These findings support clinical data suggesting that PDE5i might increase testosterone levels in men.


2022 - Qualitative and quantitative analysis of doctor-patient interactions during andrological consultations [Articolo su rivista]
Santi, Daniele; Spaggiari, Giorgia; Romeo, Marilina; Ebert, Riccardo; Corradini, Federico; Baraldi, Claudio; Granata, Antonio R M; Rochira, Vincenzo; Simoni, Manuela; Gavioli, Laura; Niemants, Natacha S A
abstract

Although a trustworthy connection between doctor and patient is crucial in clinical practice, it could be hindered by different cultural and linguistic backgrounds. Moreover, an effective doctor-patient interaction could be even more challenging in andrological fields, in which psychological and social components are predominant.


2022 - Real-world evidence analysis of the follicle-stimulating hormone use in male idiopathic infertility [Articolo su rivista]
Santi, D.; Spaggiari, G.; Granata, A. R. M.; Simoni, M.
abstract

Male idiopathic infertility remains a therapeutic challenge in the couple infertility management. In this setting, an empirical treatment with follicle-stimulating hormone (FSH) is allowed, although not recommended. Twenty-one clinical trials and four meta-analyses highlighted an overall increased pregnancy rate in case of FSH administration, but the indiscriminate FSH prescription is still unsupported by clinical evidence in idiopathic infertility. This context could represent an example in which real-world data (RWD) could add useful information. From a nationwide clinical practice survey performed in Italy, emerged the clinicians’ attitude to prescribe FSH in the case of impaired semen with a significant improvement of semen parameters, identifying FSH treatment as a therapeutic card in the real-life management. Although more robust data are still needed to optimize FSH treatment in male idiopathic infertility, RWD should be included in the body of evidence considered in healthcare decision-making.


2022 - Regulation of antral follicular growth by an interplay between gonadotropins and their receptors [Articolo su rivista]
Casarini, L.; Paradiso, E.; Lazzaretti, C.; D'Alessandro, S.; Roy, N.; Mascolo, E.; Zareba, K.; Garcia-Gasca, A.; Simoni, M.
abstract

Knowledge of the growth and maturation of human antral follicles is based mainly on concepts and deductions from clinical observations and animal models. To date, new experimental approaches and in vitro data contributed to a deep comprehension of gonadotropin receptors’ functioning and may provide new insights into the mechanisms regulating still unclear physiological events. Among these, the production of androgen in the absence of proper LH levels, the programming of follicular atresia and dominance are some of the most intriguing. Starting from evolutionary issues at the basis of the gonadotropin receptor signal specificity, we draw a new hypothesis explaining the molecular mechanisms of the antral follicular growth, based on the modulation of endocrine signals by receptor-receptor interactions. The “heteromer hypothesis” explains how opposite death and life signals are delivered by gonadotropin receptors and other membrane partners, mediating steroidogenesis, apoptotic events, and the maturation of the dominant follicle.


2022 - Testosterone Serum Levels Are Related to Sperm DNA Fragmentation Index Reduction after FSH Administration in Males with Idiopathic Infertility [Articolo su rivista]
Lispi, Monica; Drakopoulos, Panagiotis; Spaggiari, Giorgia; Caprio, Francesca; Colacurci, Nicola; Simoni, Manuela; Santi, Daniele
abstract

Purpose: Although a robust physiological rationale supports follicle stimulating hormone (FSH) use in male idiopathic infertility, useful biomarkers to evaluate its efficacy are not available. Thus, the primary aim of the study was to evaluate if testosterone serum levels are related to sperm DNA fragmentation (sDF) index change after FSH administration. The secondary aim was to confirm sDF index validity as a biomarker of FSH administration effectiveness in male idiopathic infertility. Methods: A retrospective, post-hoc re-analysis was performed on prospectively collected raw data of clinical trials in which idiopathic infertile men were treated with FSH and both testosterone serum levels and sDF were reported. Results: Three trials were included, accounting for 251 patients. The comprehensive analysis confirmed FSH's beneficial effect on spermatogenesis detected in each trial. Indeed, an overall significant sDF decrease (p < 0.001) of 20.2% of baseline value was detected. Although sDF resulted to be unrelated to testosterone serum levels at baseline, a significant correlation was highlighted after three months of FSH treatment (p = 0.002). Moreover, testosterone serum levels and patients' age significantly correlated with sDF (p = 0.006). Dividing the cohort into responders/not responders to FSH treatment according to sDF change, the FSH effectiveness in terms of sDF improvement was related to testosterone and male age (p = 0.003). Conclusion: Exogenous FSH administration in male idiopathic infertility is efficient in reducing sDF basal levels by about 20%. In terms of sDF reduction, 59.2% of the patients treated were FSH-responders. After three months of FSH administration, a significant inverse correlation between sDF and testosterone was detected, suggesting an association between the FSH-administration-related sDF improvement and testosterone serum levels increase. These observations lead to the hypothesis that FSH may promote communications or interactions between Sertoli cells and Leydig cells.


2022 - The (decision) tree of fertility: an innovative decision-making algorithm in assisted reproduction technique [Articolo su rivista]
Villani, M. T.; Morini, D.; Spaggiari, G.; Furini, C.; Melli, B.; Nicoli, A.; Iannotti, F.; La Sala, G. B.; Simoni, M.; Aguzzoli, L.; Santi, D.
abstract

Purpose: Several mathematical models have been developed to estimate individualized chances of assisted reproduction techniques (ART) success, although with limited clinical application. Our study aimed to develop a decisional algorithm able to predict pregnancy and live birth rates after controlled ovarian stimulation (COS) phase, helping the physician to decide whether to perform oocytes pick-up continuing the ongoing ART path. Methods: A single-center retrospective analysis of real-world data was carried out including all fresh ART cycles performed in 1998–2020. Baseline characteristics, ART parameters and biochemical/clinical pregnancies and live birth rates were collected. A seven-steps systematic approach for model development, combining linear regression analyses and decision trees (DT), was applied for biochemical, clinical pregnancy, and live birth rates. Results: Of fresh ART cycles, 12,275 were included. Linear regression analyses highlighted a relationship between number of ovarian follicles > 17 mm detected at ultrasound before pick-up (OF17), embryos number and fertilization rate, and biochemical and clinical pregnancy rates (p < 0.001), but not live birth rate. DT were created for biochemical pregnancy (statistical power–SP:80.8%), clinical pregnancy (SP:85.4%), and live birth (SP:87.2%). Thresholds for OF17 entered in all DT, while sperm motility entered the biochemical pregnancy’s model, and female age entered the clinical pregnancy and live birth DT. In case of OF17 < 3, the chance of conceiving was < 6% for all DT. Conclusion: A systematic approach allows to identify OF17, female age, and sperm motility as pre-retrieval predictors of ART outcome, possibly reducing the socio-economic burden of ART failure, allowing the clinician to perform or not the oocytes pick-up.


2022 - Which sperm parameter limits could really guide the clinical decision in assisted reproduction? [Articolo su rivista]
Santi, Daniele; Spaggiari, Giorgia; Morini, Daria; Melli, Beatrice; Dalla Valentina, Leonardo; Aguzzoli, Lorenzo; Simoni, Manuela; Villani, Maria Teresa
abstract

Background The predictive role of sperm motility and morphology was recently detected in a large sample of more than 20000 assisted reproductive technology (ART) fresh cycles. However, the complete ART procedure consisted of both fresh and frozen-embryos transfers and only a comprehensive evaluation of the entire process could really confirm if these parameters really predict the ART success. The aim of the study was to evaluate which sperm parameter could predict the success of ART. Methods A retrospective, real-world data analysis was performed, enrolling all couples attending ART from 2008 to 2021, including both fresh and frozen cycles and both in vitro fertilization (IVF) and intra-cytoplasmic sperm injection (ICSI) procedures. Results Fresh cycles success (considering live birth rate) was predicted by female age (1.04 [1.02-1.06]), injected oocytes (0.96 [0.93-0.99]), embryo number (0.79 [0.75-0.83]) and progressive sperm motility (0.98 [0.97-0.99]). On the contrary, frozen cycle outcomes were predicted only by sperm motility (0.97 [0.95-0.99]). This prediction was confirmed in IVF but not in ICSI cycles. Conclusion Both female and male parameters predicted the ART success considering the entire path. However, frozen cycle success was predicted only by progressive sperm motility in IVF cycles, suggesting that the potential amelioration of this male parameter is relevant to improve ART success. Those couples expected to obtain the highest embryos after fertilization (low female age and better semen parameters) will have more attempts with frozen cycles and thus would benefit from a potential treatment focused to improve sperm parameters.


2021 - A prospective, observational clinical trial on the impact of COVID-19-related national lockdown on thyroid hormone in young males [Articolo su rivista]
Brigante, Giulia; Spaggiari, Giorgia; Rossi, Barbara; Granata, Antonio; Simoni, Manuela; Santi, Daniele
abstract

Trying to manage the dramatic coronavirus disease 2019 (COVID-19) infection spread, many countries imposed national lockdown, radically changing the routinely life of humans worldwide. We hypothesized that both the pandemic per se and the consequent socio-psychological sequelae could constitute stressors for Italian population, potentially affecting the endocrine system. This study was designed to describe the effect of lockdown-related stress on the hypothalamic-pituitary-thyroid (HPT) axis in a cohort of young men. A prospective, observational clinical trial was carried out, including patients attending the male infertility outpatient clinic before and after the national lockdown for COVID-19 pandemic. The study provided a baseline visit performed before and a follow-up visit after the lockdown in 2020. During the follow-up visit, hormonal measurements, lifestyle habits and work management were recorded. Thirty-one male subjects were enrolled (mean age: 31.6±6.0years). TSH significantly decreased after lockdown (p=0.015), whereas no significant changes were observed in the testosterone, luteinising hormone, follicle-stimulating hormone, estradiol and prolactin serum levels. No patient showed TSH serum levels above or below reference ranges, neither before nor after lockdown. Interestingly, TSH variation after lockdown was dependent on the working habit change during lockdown (p=0.042). We described for the first time a TSH reduction after a stressful event in a prospective way, evaluating the HPT axis in the same population, before and after the national lockdown. This result reinforces the possible interconnection between psychological consequences of a stressful event and the endocrine regulation.


2021 - Andrology Awards 2019 and 2020 [Articolo su rivista]
Hofmann, M. -C.; Simoni, M.
abstract


2021 - Apolipoprotein M and Sphingosine-1-Phosphate Receptor 1 Promote the Transendothelial Transport of High-Density Lipoprotein [Articolo su rivista]
Velagapudi, S.; Rohrer, L.; Poti, F.; Feuerborn, R.; Perisa, D.; Wang, D.; Panteloglou, G.; Potapenko, A.; Yalcinkaya, M.; Hulsmeier, A. J.; Hesse, B.; Lukasz, A.; Liu, M.; Parks, J. S.; Christoffersen, C.; Stoffel, M.; Simoni, M.; Nofer, J. -R.; Von Eckardstein, A.
abstract

Objective: ApoM enriches S1P (sphingosine-1-phosphate) within HDL (high-density lipoproteins) and facilitates the activation of the S1P1 (S1P receptor type 1) by S1P, thereby preserving endothelial barrier function. Many protective functions exerted by HDL in extravascular tissues raise the question of how S1P regulates transendothelial HDL transport. Approach and Results: HDL were isolated from plasma of wild-type mice, Apom knockout mice, human apoM transgenic mice or humans and radioiodinated to trace its binding, association, and transport by bovine or human aortic endothelial cells. We also compared the transport of fluorescently-labeled HDL or Evans Blue, which labels albumin, from the tail vein into the peritoneal cavity of apoE-haploinsufficient mice with (apoE-haploinsufficient mice with endothelium-specific knockin of S1P1) or without (control mice, ie, apoE-haploinsufficient mice without endothelium-specific knockin of S1P1) endothelium-specific knockin of S1P1. The binding, association, and transport of HDL from Apom knockout mice and human apoM-depleted HDL by bovine aortic endothelial cells was significantly lower than that of HDL from wild-type mice and human apoM-containing HDL, respectively. The binding, uptake, and transport of 125I-HDL by human aortic endothelial cells was increased by an S1P1 agonist but decreased by an S1P1 inhibitor. Silencing of SR-BI (scavenger receptor BI) abrogated the stimulation of 125I-HDL transport by the S1P1 agonist. Compared with control mice, that is, apoE-haploinsufficient mice without endothelium-specific knockin of S1P1, apoE-haploinsufficient mice with endothelium-specific knockin of S1P1 showed decreased transport of Evans Blue but increased transport of HDL from blood into the peritoneal cavity and SR-BI expression in the aortal endothelium. Conclusions: ApoM and S1P1 promote transendothelial HDL transport. Their opposite effect on transendothelial transport of albumin and HDL indicates that HDL passes endothelial barriers by specific mechanisms rather than passive filtration.


2021 - Are sperm parameters able to predict the success of assisted reproductive technologies? A retrospective analysis of over 22000 ART cycles [Articolo su rivista]
Villani, M T; Morini, D; Spaggiari, G; Falbo, A I; Melli, B; La Sala, G B; Romeo, M; Simoni, M; Aguzzoli, L; Santi, D
abstract

An explosive increase in couples attending assisted reproductive technologies (ART) has been recently observed, despite an overall success rate about 20-30%. Considering the ART-related economic and psycho-social costs, the improvement of these percentages is extremely relevant. However, in the identification of predictive markers of ART success, male parameters are largely underestimated so far.


2021 - Association of nonalcoholic fatty liver disease (Nafld) with peripheral diabetic polyneuropathy: A systematic review and meta-analysis [Articolo su rivista]
Greco, C.; Nascimbeni, F.; Carubbi, F.; Andreone, P.; Simoni, M.; Santi, D.
abstract

Aims. The relationship between nonalcoholic fatty liver disease (NAFLD) and diabetic polyneuropathy (DPN) has been demonstrated in many studies, although results were conflicting. This meta-analysis aims to summarize available data and to estimate the DPN risk among NAFLD patients. Materials and methods. We performed a comprehensive literature review until 4 June 2021. Clinical trials analyzing the association between NAFLD and DPN were included. Results. Thirteen studies (9614 participants) were included. DPN prevalence was significantly higher in patients with NALFD, compared to patients without NAFLD (OR (95%CI) 2.48 (1.42–4.34), p = 0.001; I2 96%). This finding was confirmed in type 2 diabetes (OR (95%CI) 2.51 (1.33–4.74), p = 0.005; I2 97%), but not in type 1 diabetes (OR (95%CI) 2.44 (0.85–6.99), p = 0.100; I2 77%). Also, body mass index and diabetes duration were higher in NAFLD subjects compared to those without NAFLD (p < 0.001), considering both type 2 and type 1 diabetes. Conclusion. Despite a high heterogeneity among studies, a significantly increased DPN prevalence among type 2 diabetes subjects with NAFLD was observed. This result was not found in type 1 diabetes, probably due to the longer duration of disease. Physicians should pay more attention to the early detection of DPN, especially in patients with NAFLD.


2021 - Endocrine Disruption of the Follicle-Stimulating Hormone Receptor Signaling During the Human Antral Follicle Growth [Articolo su rivista]
Roy, N.; Mascolo, E.; Lazzaretti, C.; Paradiso, E.; D'Alessandro, S.; Zareba, K.; Simoni, M.; Casarini, L.
abstract

An increasing number of pollutants with endocrine disrupting potential are accumulating in the environment, increasing the exposure risk for humans. Several of them are known or suspected to interfere with endocrine signals, impairing reproductive functions. Follicle-stimulating hormone (FSH) is a glycoprotein playing an essential role in supporting antral follicle maturation and may be a target of disrupting chemicals (EDs) likely impacting female fertility. EDs may interfere with FSH-mediated signals at different levels, since they may modulate the mRNA or protein levels of both the hormone and its receptor (FSHR), perturb the functioning of partner membrane molecules, modify intracellular signal transduction pathways and gene expression. In vitro studies and animal models provided results helpful to understand ED modes of action and suggest that they could effectively play a role as molecules interfering with the female reproductive system. However, most of these data are potentially subjected to experimental limitations and need to be confirmed by long-term observations in human.


2021 - From subjective to objective: A pilot study on testicular radiomics analysis as a measure of gonadal function [Articolo su rivista]
De Santi, Bruno; Spaggiari, Giorgia; Granata, Antonio Rm; Romeo, Marilina; Molinari, Filippo; Simoni, Manuela; Santi, Daniele
abstract

Background: The connection between testicular ultrasound (US) parameters and testicular function, including both spermato- and steroidogenesis has been largely suggested, but their predictive properties are not routinely applied. Radiomics, a new engineering approach to radiological imaging, could overcome the visual limit of the sonographer. Objectives: This study is aimed at extracting objective testicular US features, correlating with testicular function, including both spermato- and steroidogenesis, using engineering approach, in order to overcome the operator-dependent subjectivity. Materials and methods: Prospective observational pilot study from December 2019 to December 2020 on normozoospermic subjects and patients with semen variables alterations, excluding azoospermia. All patients underwent conventional semen analysis, pituitary-gonadal hormones assessment and testicular US, performed by the same operator. US images were analysed by Biolab (Turin) throughout image segmentation, image pre-processing and texture features extraction. Results: 170 US testicular images were collected from 85 patients (age 38.6±9.1 years). A total of 44 first-order and advanced features were extracted. US inhomogeneity defined by radiomics significantly correlate with the andrologist definition, showing for the first time a mathematical quantification of a subjective US evaluation. 13 US texture features correlated with semen parameters, predicting sperm concentration, total sperm number, progressive motility, total motility and morphology, and with gonadotropins serum levels, but not with total testosterone serum levels. Classification analyses confirmed that US textural features predicted patients' classification according to semen parameters alterations. Conclusions: Radiomics texture features qualitatively describe the testicular parenchyma with objective and reliable quantitative parameters, reflecting both the testicular spermatogenic capability and the action of pituitary gonadotropins. This is an innovative model in which US texture features represent a mirror of the pituitary-gonadal homeostasis in terms of reproductive function. This article is protected by copyright. All rights reserved.


2021 - Identification of key receptor residues discriminating human chorionic gonadotropin (Hcg)-and luteinizing hormone (lh)-specific signaling [Articolo su rivista]
Lazzaretti, C.; Secco, V.; Paradiso, E.; Sperduti, S.; Rutz, C.; Kreuchwig, A.; Krause, G.; Simoni, M.; Casarini, L.
abstract

(1) The human luteinizing hormone (LH)/chorionic gonadotropin (hCG) receptor (LHCGR) discriminates its two hormone ligands and differs from the murine receptor (Lhr) in amino acid residues potentially involved in qualitative discerning of LH and hCG. The latter gon-adotropin is absent in rodents. The aim of the study is to identify LHCGR residues involved in hCG/LH discrimination. (2) Eight LHCGR cDNAs were developed, carrying “murinizing” mutations on aminoacidic residues assumed to interact specifically with LH, hCG, or both. HEK293 cells expressing a mutant or the wild type receptor were treated with LH or hCG and the kinetics of cyclic adenosine monophosphate (cAMP) and phosphorylated extracellular signal-regulated ki-nases 1/2 (pERK1/2) activation was analyzed by bioluminescence resonance energy transfer (BRET). (3) Mutations falling within the receptor leucine reach repeat 9 and 10 (LRR9 and LRR10; K225S +T226I and R247T), of the large extracellular binding domain, are linked to loss of hormone-specific induced cAMP increase, as well as hCG-specific pERK1/2 activation, leading to a Lhr-like modulation of the LHCGR-mediated intracellular signaling pattern. These results support the hypothesis that LHCGR LRR domain is the interaction site of the hormone β-L2 loop, which differs between LH and hCG, and might be fundamental for inducing gonadotropin-specific signals. (4) Taken to-gether, these data identify LHCGR key residues likely evolved in the human to discriminate LH/hCG specific binding.


2021 - Improvement of sperm morphology after surgical varicocele repair [Articolo su rivista]
Morini, D.; Spaggiari, G.; Daolio, J.; Melli, B.; Nicoli, A.; De Feo, G.; Valli, B.; Viola, D.; Garganigo, S.; Magnani, E.; Pilia, A.; Polese, A.; Colla, R.; Simoni, M.; Aguzzoli, L.; Villani, M. T.; Santi, D.
abstract

Background: A causative relationship between varicocele and impairment of semen quality has been largely investigated in the context of male infertility, although its clinical benefit remains controversial. Objective: To investigate the effect of varicocele correction on detailed morphologic microscopic semen parameters in a large homogeneous cohort of patients and to evaluate which factors could predict semen improvement after the surgical treatment. Materials and methods: An observational, retrospective cohort study was carried out including all patients undergoing surgical treatment for varicocele from September 2011 to March 2020 in the same clinical centre. Enrolled males performed at least one semen analysis before and one after surgical varicocele correction. Primary outcome was the detailed morphologic microscopic sperm evaluation. Secondary outcomes were conventional semen analyses. Results: A total of 121 males (mean age 24.6 ± 6.1 years) were enrolled. Using detailed morphologic microscopic sperm evaluation, a significant morphological improvement was recorded, with a reduction in head and tail abnormalities. Moreover, a significant increase in sperm concentration (p = 0.015) and percentage of progressive and total motility (p = 0.022 and p = 0.039) were observed after surgery. The multivariate logistic analysis identified the ultrasonography varicocele degree before surgery as a main predictor of the sperm concentration improvement (p = 0.016), with the highest improvement for varicocele of I and II degree. Discussion: For the first time, the detailed morphologic microscopic sperm evaluation highlights a relevant reduction in sperm abnormalities after varicocele surgery, showing its potential application in clinical practice.


2021 - LH/hCG and the Receptor: A Single Receptor for Two Ligands [Capitolo/Saggio]
Casarini, L.; Santi, D.; Brigante, G.; Simoni, M.
abstract

Gonadotropins are glycoprotein hormones that regulate development and reproduction. They are a family of structurally similar hormones likely evolved from a common ancestral encoding gene and achieved relatively high complexity in humans. Two of these molecules are the luteinizing hormone (LH) and the choriogonadotropin (hCG). They are encoded by a common gene cluster and act on the same receptor (LHCGR), however, are produced in primates and specialized for regulating unique physiological functions, such as LH for gametogenesis and hCG for pregnancy. The action of the two hormones is differentiated by LHCGR, which mediates ligand-specific intracellular signals optimized for supporting LH and hCG physiological functions. Since the two hormones are used as drugs for the treatment of infertility, the knowledge of their mode of action should be helpful for interpreting gonadotropin-related pathological conditions and optimizing their clinical treatment.


2021 - L’ormone luteinizzante e la gonadotropina corionica umana: attività molecolari e cliniche mediate da un unico recettore [Articolo su rivista]
Sperduti, Samantha; Paradiso, Elia; Lazzaretti, Clara; Rochira, Vincenzo; Brigante, Giulia; Santi, Daniele; Simoni, Manuela; Casarini, Livio
abstract


2021 - Nuclear expression of VDR and AHR is mutually exclusive in glandular cells in endometriosis [Articolo su rivista]
De Pascali, F.; Casarini, L.; Kuhn, C.; Simoni, M.; Mahner, S.; Jeschke, U.; von Schonfeldt, V.
abstract

The vitamin D receptor (VDR) and aryl hydrocarbon receptor (AHR) are two nuclear receptors that exert their effects by binding with ligands and forming a molecular complex. These complexes translocate to the nucleus and activate the expression of a series of genes which have a response element to VDR or AHR. Both receptors have been identified in the pathogenesis of endometriosis, a common disease characterized by the formation of endometrium-like tissue in ectopic zones. Despite numerous therapies, there is no definitive cure for endometriosis at the pharmacological level. Our study aims to describe the location and the expression of VDR and AHR at the protein level. For this purpose, an evaluation was performed using tissue from the three normal phases of the endometrium (proliferative, early, and late secretory) and in endometriosis by immunohistochemistry, using anti-VDR and anti-AHR antibodies. We demonstrate that in the nuclei of glandular cells in endometriosis, the expression of VDR and AHR is mutually exclusive—when the expression of one receptor is high, the other one is low—suggesting a possible target in the treatment of endometriosis. We also identify a significant change in the expression of glandular cytoplasmic AHR between the proliferative and late secretory endometrium.


2021 - Pharmacological characterization of low molecular weight biased agonists at the follicle stimulating hormone receptor [Articolo su rivista]
De Pascali, F.; Ayoub, M. A.; Benevelli, R.; Sposini, S.; Lehoux, J.; Gallay, N.; Raynaud, P.; Landomiel, F.; Jean-Alphonse, F.; Gauthier, C.; Pellissier, L. P.; Crepieux, P.; Poupon, A.; Inoue, A.; Joubert, N.; Viaud-Massuard, M. -C.; Casarini, L.; Simoni, M.; Hanyaloglu, A. C.; Nataraja, S. G.; Yu, H. N.; Palmer, S. S.; Yvinec, R.; Reiter, E.
abstract

Follicle-stimulating hormone receptor (FSHR) plays a key role in reproduction through the activation of multiple signaling pathways. Low molecular weight (LMW) ligands composed of biased agonist properties are highly valuable tools to decipher complex signaling mechanisms as they allow selective activation of discrete signaling cascades. However, available LMW FSHR ligands have not been fully characterized yet. In this context, we explored the pharmacological diversity of three benzamide and two thiazolidinone derivatives compared to FSH. Concentration/activity curves were generated for Gαs, Gαq, Gαi, β-arrestin 2 recruitment, and cAMP production, using BRET assays in living cells. ERK phosphorylation was analyzed by Western blotting, and CRE-dependent transcription was assessed using a luciferase reporter assay. All assays were done in either wild-type, Gαs or β-arrestin 1/2 CRISPR knockout HEK293 cells. Bias factors were calculated for each pair of read-outs by using the operational model. Our results show that each ligand presented a discrete pharmacological efficacy compared to FSH, ranging from super-agonist for β-arrestin 2 recruitment to pure Gαs bias. Interestingly, LMW ligands generated kinetic profiles distinct from FSH (i.e., faster, slower or transient, depending on the ligand) and correlated with CRE-dependent transcription. In addition, clear system biases were observed in cells depleted of either Gαs or β-arrestin genes. Such LMW properties are useful pharmacological tools to better dissect the multiple signaling pathways activated by FSHR and assess their relative contributions at the cellular and physio-pathological levels.


2021 - Quantification of hormone membrane receptor FSHR, GPER and LHCGR transcripts in human primary granulosa lutein cells by real-time quantitative PCR and digital droplet PCR [Articolo su rivista]
Sperduti, S.; Lazzaretti, C.; Paradiso, E.; Anzivino, C.; Villani, M. T.; De Feo, G.; Simoni, M.; Casarini, L.
abstract

Background: Quantitative real time polymerase chain reaction (qPCR) and droplet digital PCR (ddPCR) are methods used for gene expression analysis in several contexts, including reproductive endocrinology. Objectives: Herein, we compared qPCR and ddPCR technologies for gene expression analysis of hormone membrane receptor-encoding genes (FSHR, GPER and LHCGR), as well as the commonly used RPS7 housekeeping gene, in order to identify the most reliable method to be applied for gene expression analysis in the context of human reproduction. Methods: Total RNA was extracted from human primary granulosa lutein cells of donor patients undergoing assisted reproduction and used for gene expression analysis by qPCR and ddPCR, after finding the optimal annealing temperature. Immunostaining for protein localization in cell membranes was also performed. Results: Both techniques provided results reflecting the low number of FSHR and GPER transcripts, although ddPCR quantified the low-expressed genes with major accuracy, thanks to its higher reaction efficiency. The absolute FSHR and GPER transcript number was also determined by ddPCR, resulting in 40- to 260-fold lower amount than LHCGR transcripts. qPCR and ddPCR data are convergent with immunofluorescence analysis of membrane receptor expression in human primary granulosa lutein cells. Conclusion: These results suggest that ddPCR is the candidate technology for analysis of genes with relatively low expression levels and provides useful insights for characterizing hormone receptor expression levels in the context of reproductive endocrinology.


2021 - Real-life use of BRAF-V600E mutation analysis in thyroid nodule fine needle aspiration: consequences on clinical decision-making [Articolo su rivista]
Brigante, G.; Craparo, A.; Pignatti, E.; Marino, M.; Monzani, M. L.; De Vincentis, S.; Casarini, L.; Sperduti, S.; Boselli, G.; Margiotta, G.; Ippolito, M.; Rochira, V.; Simoni, M.
abstract

Purpose: This study aimed to evaluate the real-life use of BRAF-V600E mutation analysis in washout liquid from thyroid nodule fine needle aspiration (FNA), and the consequences of genetic result on clinical decision-making. Methods: We retrospectively considered subjects tested for BRAF-V600E among those attending the Endocrinology Unit of Modena for FNA between 2014 and 2018. Washing fluid was collected together with cytological sample and stored at −20 °C. If the clinician deemed it necessary, the sample was thawed, DNA extracted, and genetic test performed by high-resolution melting technique. We collected data on cytology according to the Italian Consensus for the cytological classification of thyroid nodules, type of surgery (when performed), histology, and adverse events. Results: Out of 7112 subjects submitted to FNA, BRAF analysis was requested for 683 (9.6%). Overall, 896 nodules were analyzed: 74% were indeterminate at cytology, mainly TIR3A (low risk). Twenty-two nodules were mutant (BRAF+). Only 2% of indeterminate, mainly TIR3B, were BRAF+. Based on final histological diagnosis, BRAF test had high specificity (100%) but poor sensitivity (21%), also in indeterminate nodules. Mutant subjects underwent more extensive surgery compared to wild type (p = 0.000), with frequent prophylactic central lymph node dissection. One third had local metastases. Higher prevalence of hypoparathyroidism was found in BRAF+ compared to wild type (p = 0.018). Conclusions: The analysis of BRAF-V600E outside of gene panels has low sensitivity, especially in indeterminate nodules, and a positive result could lead to more extensive surgery with greater risk of hypoparathyroidism and questionable clinical utility.


2021 - Recent advances in understanding gonadotropin signaling [Articolo su rivista]
Casarini, Livio; Simoni, Manuela
abstract

Gonadotropins are glycoprotein sex hormones regulating development and reproduction and bind to specific G protein–coupled receptors expressed in the gonads. Their effects on multiple signaling cascades and intracellular events have recently been characterized using novel technological and scientific tools. The impact of allosteric modulators on gonadotropin signaling, the role of sugars linked to the hormone backbone, the detection of endosomal compartments supporting signaling modules, and the dissection of different effects mediated by these molecules are areas that have advanced significantly in the last decade. The classic view providing the exclusive activation of the cAMP/protein kinase A (PKA) and the steroidogenic pathway by these hormones has been expanded with the addition of novel signaling cascades as determined by high-resolution imaging techniques. These new findings provided new potential therapeutic applications. Despite these improvements, unanswered issues of gonadotropin physiology, such as the intrinsic pro-apoptotic potential to these hormones, the existence of receptors assembled as heteromers, and their expression in extragonadal tissues, remain to be studied. Elucidating these issues is a challenge for future research.


2021 - Reduced FSH and LH action: implications for medically assisted reproduction [Articolo su rivista]
Bosch, E.; Alviggi, C.; Lispi, M.; Conforti, A.; Hanyaloglu, A. C.; Chuderland, D.; Simoni, M.; Raine-Fenning, N.; Crepieux, P.; Kol, S.; Rochira, V.; D'Hooghe, T.; Humaidan, P.
abstract

Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) play complementary roles in follicle development and ovulation via a complex interaction in the hypothalamus, anterior pituitary gland, reproductive organs, and oocytes. Impairment of the production or action of gonadotropins causes relative or absolute LH and FSH deficiency that compromises gametogenesis and gonadal steroid production, thereby reducing fertility. In women, LH and FSH deficiency is a spectrum of conditions with different functional or organic causes that are characterized by low or normal gonadotropin levels and low oestradiol levels. While the causes and effects of reduced LH and FSH production are very well known, the notion of reduced action has received less attention by researchers. Recent evidence shows that molecular characteristics, signalling as well as ageing, and some polymorphisms negatively affect gonadotropin action. These findings have important clinical implications, in particular for medically assisted reproduction in which diminished action determined by the afore-mentioned factors, combined with reduced endogenous gonadotropin production caused by GnRH analogue protocols, may lead to resistance to gonadotropins and, thus, to an unexpected hypo-response to ovarian stimulation. Indeed, the importance of LH and FSH action has been highlighted by the International Committee for Monitoring Assisted Reproduction Technologies (ICMART) in their definition of hypogonadotropic hypogonadism as gonadal failure associated with reduced gametogenesis and gonadal steroid production due to reduced gonadotropin production or action. The aim of this review is to provide an overview of determinants of reduced FSH and LH action that are associated with a reduced response to ovarian stimulation.


2021 - Ruolo diagnostico del dosaggio della calcitonina su liquido di lavaggio da agoaspirato tiroideo nel carcinoma midollare della tiroide [Articolo su rivista]
Brigante, Giulia; Madeo, Bruno; Diazzi, Chiara; Simoni, Manuela; Rochira, Vincenzo
abstract


2021 - Sphingosine-1 phosphate induces cAMP/PKA-independent phosphorylation of the cAMP response element-binding protein (CREB) in granulosa cells [Articolo su rivista]
Paradiso, E.; Lazzaretti, C.; Sperduti, S.; Antoniani, F.; Fornari, G.; Brigante, G.; Di Rocco, G.; Tagliavini, S.; Trenti, T.; Morini, D.; Falbo, A. I.; Villani, M. T.; Nofer, J. -R.; Simoni, M.; Poti, F.; Casarini, L.
abstract

Background and aims: Sphingosine-1 phosphate (S1P) is a lysosphingolipid present in the ovarian follicular fluid. The role of the lysosphingolipid in gonads of the female is widely unclear. At nanomolar concentrations, S1P binds and activates five specific G protein-coupled receptors (GPCRs), known as S1P1-5, modulating different signaling pathways. S1P1 and S1P3 are highly expressed in human primary granulosa lutein cells (hGLC), as well as in the immortalized human primary granulosa cell line hGL5. In this study, we evaluated the signaling cascade activated by S1P and its synthetic analogues in hGLC and hGL5 cells, exploring the biological relevance of S1PR-stimulation in this context. METHODS AND RESULTS. hGLC and hGL5 cells were treated with a fixed dose (0.1 μM) of S1P, or by S1P1- and S1P3-specific agonists SEW2871 and CYM5541. In granulosa cells, S1P and, at a lesser extent, SEW2871 and CYM5541, potently induced CREB phosphorylation. No cAMP production was detected and pCREB activation occurred even in the presence of the PKA inhibitor H-89. Moreover, S1P-dependent CREB phosphorylation was dampened by the mitogen-activate protein kinase (MEK) inhibitor U0126 and by the L-type Ca2+ channel blocker verapamil. The complete inhibition of CREB phosphorylation occurred by blocking either S1P2 or S1P3 with the specific receptor antagonists JTE-013 and TY52156, or under PLC/PI3K depletion. S1P-dependent CREB phosphorylation induced FOXO1 and the EGF-like epiregulin-encoding gene (EREG), confirming the exclusive role of gonadotropins and interleukins in this process, but did not affect steroidogenesis. However, S1P or agonists did not modulate granulosa cell viability and proliferation in our conditions. Conclusions: This study demonstrates for the first time that S1P may induce a cAMP-independent activation of pCREB in granulosa cells, although this is not sufficient to induce intracellular steroidogenic signals and progesterone synthesis. S1P-induced FOXO1 and EREG gene expression suggests that the activation of S1P–S1PR axis may cooperate with gonadotropins in modulating follicle development.


2021 - Spontaneous pregnancies among infertile couples during assisted reproduction lockdown for COVID-19 pandemic [Articolo su rivista]
Villani, Maria Teresa; Morini, Daria; Spaggiari, Giorgia; Simoni, Manuela; Aguzzoli, Lorenzo; Santi, Daniele
abstract

The worldwide spread of the SARS-CoV-2 infection has profoundly affected all aspects of human life, with tangible consequences in several contexts, including reproduction. However, evidences on the inter-relation between psychological distress and reproductive medicine are still conflicting.


2021 - Striving for the best: Complying with the quality criteria required by ANDROLOGY is not optional [Articolo su rivista]
Simoni, M.; Hofman, M. -C.
abstract


2021 - The “hitchhiker’s guide to the galaxy” of endothelial dysfunction markers in human fertility [Articolo su rivista]
Santi, D.; Spaggiari, G.; Greco, C.; Lazzaretti, C.; Paradiso, E.; Casarini, L.; Poti, F.; Brigante, G.; Simoni, M.
abstract

Endothelial dysfunction is an early event in the pathogenesis of atherosclerosis and represents the first step in the pathogenesis of cardiovascular diseases. The evaluation of endothelial health is fundamental in clinical practice and several direct and indirect markers have been suggested so far to identify any alterations in endothelial homeostasis. Alongside the known endothelial role on vascular health, several pieces of evidence have demonstrated that proper endothelial functioning plays a key role in human fertility and reproduction. Therefore, this stateof‐the‐art review updates the endothelial health markers discriminating between those available for clinical practice or for research purposes and their application in human fertility. Moreover, new molecules potentially helpful to clarify the link between endothelial and reproductive health are evaluated herein.


2020 - An Update on Clinical and Surgical Interventions to Reduce Sperm DNA Fragmentation in Infertile Men [Articolo su rivista]
Esteves, Sandro C; Santi, Daniele; Simoni, Manuela
abstract

Sperm chromatin integrity is essential for normal embryo development and pregnancy outcome. Sperm DNA fragmentation (SDF) testing constitutes a diagnostic tool to measure the proportion of sperm with damaged chromatin in the ejaculate. SDF is associated with potentially treatable conditions, including varicocele, male accessory gland infections, inadequate lifestyle, and gonadotoxin exposure, thus prompting their treatment as a means of improving sperm DNA quality and the reproductive outcomes.


2020 - Announcing the Sixth Andrology Award [Capitolo/Saggio]
Carrell, D. T.; Simoni, M.
abstract


2020 - Clinical practice survey on BRAF V600E role in the therapeutic deci- sion in indeterminate thyroid cytology [Abstract in Atti di Convegno]
Brigante, G.; Craparo, A.; Pignatti, E.; Marino, M.; Casarini, L.; Sperduti, S.; Boselli, G.; Margiotta, G.; Rochira, V.; Simoni, M.
abstract

Introduction The use of multigene panels in thyroid nodule diagnosis is still limited, due to high costs and need for ad hoc sampling. Since BRAF-V600E is the com- monest genetic alteration in differentiated thyroid cancer, this is the mostly tested genetic parameter in clinical practice. Aim To evaluate the use of BRAF mutation analysis in wash-out liquid from fine needle aspiration (FNA) in clinical practice, characterizing the cases in which it is requested, and the consequences of genetic test result on thera- peutic decisions. Methods We considered all the subjects tested for BRAF-V600E among those attend- ing the Endocrinology Unit of Modena for FNA between January 2014 and November 2018. After written informed consent, washing fluid was collected together with cytological sample and stored at –20°C. If the clinician deemed it necessary, the sample was thawed, DNA was extracted and genetic test was performed by the high-resolution melting protocol previously described1. We collected cytology of nodules according to the 2010 SIAPEC-IAP Italian Consensus, and when surgical treatment was performed, histology. Results Out of a total of 7112 subjects submitted to FNA, BRAF analysis was re- quested for 681 (9.6%), for a total of 898 nodules: 97% of nodules were indeterminate at cytology, mainly TIR3A (low risk); 2% suspicious or di- agnostic for cancer, and genetic test was requested to estimate prognosis; 1% were suspect nodules at ultrasonography with unsuspicious cytology. Only 22 nodules were mutant (BRAF+).Most of them were already high risk or suspicious lesions at cytology (64%). One third were TIR3A. Con- sidering the prevalence of BRAF mutation among cytological classes of the whole group, only 1% of TIR3A were BRAF+. Twenty BRAF+ patients were addressed to surgery (one lost at follow-up, one refused): 5% underwent hemithyroidectomy, 25% total thyroidectomy and 70% total thyroidectomy plus central lymph nodes dissection. They all had papillary thyroid cancer. Since 64% of BRAF+ were TIR3B-4-5 at cytology, they had surgical indica- tion even before the genetic test. Among the 14 subjects treated with central neck dissection, only 2 had suspect metastasis before surgery; among those who would have had no indication, one third had metastases (only 1 among TIR3A and 2 among TIR3B). Conclusions Despite the development of panels, single gene tests are still requested, mainly for nodules with indeterminate low risk cytology. BRAF mutation in TIR3A is rare and leads clinicians to more invasive surgery, with question- able clinical utility.


2020 - De novo Lesions Frequently Develop in Adult Normal Thyroid Over Almost Six Years [Articolo su rivista]
Brigante, Giulia; Monzani, Maria Laura; Locaso, Michela; Gnarini, Valentina Luisa; Graziadei, Luigi; Kaleci, Shaniko; De Santis, Maria Cristina; Tagliavini, Simonetta; Simoni, Manuela; Rochira, Vincenzo; Madeo, Bruno
abstract

Purpose: In order to understand how thyroid abnormalities emerge over time in adults, we evaluated incidence of thyroid diseases in healthy subjects, after almost 6 years from a previous negative ultrasound. Methods: Anamnestic and physical data were collected. Ultrasound neck evaluation was performed by an experienced endocrinologist, recording detailed thyroid and nodules characteristics. Nodules were classified according to American Thyroid Association classification for prediction of cancer risk. Serum samples were collected for subsequent evaluations (TSH, free thyroid hormones, calcitonin, anti-thyroid antibodies). Anamnestic, clinical, sonographic, and serological characteristics were analyzed with logistic regression analysis for subjects with nodules vs. those without. Results: One hundred and eleven subjects were enrolled (43M, 68F). Half of them developed nodules, mainly smaller than 1 cm and without suspicious characteristics. Ninety-seven percent were euthyroid. Only 4% had serological diagnosis of thyroiditis. Incidence of thyroid diseases was higher in women, especially nulliparous. Comparing clinical characteristics of subjects with and without nodules, the only statistically significant difference concerned thyroid volume adjusted for body weight or surface (p < 0.05), but not residual volume excluding nodules. Multivariate logistic regression analysis showed that female gender, higher BMI-adjusted thyroid volume and residual thyroid volume excluding nodules, nulliparity, age, and fT3 increase the risk of developing nodules. Conclusions: These results demonstrate that adult thyroid tissue undergoes changes that are already detectable by US after almost 6 years. Half of the enrolled subjects developed de novo nodules or colloid cysts of poor clinical relevance.


2020 - Effects of acute hCG stimulation on serum INSL3 and 25-OH vitamin D in Klinefelter syndrome. [Articolo su rivista]
Santi, D.; Ivell, R.; Anand-Ivell, R.; De Toni, L.; Fanelli, F.; Mezzullo, M.; Pelusi, C.; Pagotto, U.; Belli, S.; M: Granata, A. R.; Roli, L.; Rochira, V.; Trenti, T.; Ferlin, A.; Simoni, M.
abstract

Background: It has recently been suggested that the hypergonadotropic hypogonadism characterizing Klinefelter syndrome (KS) might not be due to a steroidogenic dysfunction per se, but mainly to an altered testosterone (T) secretion into the bloodstream. However, the Leydig cell functionality remains incompletely studied in KS, and new markers should be considered. Previous data indicated that chronic hCG stimulation influence the production of both Insulin-like peptide 3 (INSL3) and 25-hydroxy-vitamin D (25-VD) in eugonadal men. Aim of the study: To evaluate INSL3 and 25-VD serum levels, as markers of Leydig cell functionality, in association with sex steroids, after an acute hCG test in a group of KS patients and healthy volunteers. Methods: A retrospective analysis of a prospective, case-control, clinical trial was carried out. KS patients (n=11) and age-matched healthy controls(n=11) provided a basal blood sample (V0) immediately followed by a single intramuscular injection of hCG 5000 IU. Blood samples were taken in the following five days(V1-V5). Results: At baseline, INSL3 was lower in KS patients compared to controls (p=0.007). When adjusted for INSL3 levels, the production of steroids was similar between KS patients and controls. 25-VD was in the insufficient range both in KS patients and controls and was not different (p=0.064). Acute hCG stimulation increased neither INSL3 nor 25-VD in both KS patients and controls. In controls, an inverse correlation was detected between INSL3 levels and body mass index (p=0.020) and waist circumference (p=0.020). Conclusions: INSL3 secretion is independent from steroidogenesis and its production is mostly not influenced by acute hCG stimulation both in KS men and controls. INSL3 serum levels should be considered as a marker of Leydig cell differentiation and numbers rather than steroidogenesis. 25-VD serum levels are also not increased by a single acute hCG administration, which was not able to restore the normal concentrations of 25-VD.


2020 - Elevated levels of nitrous dioxide are associated with lower AMH levels: A real-world analysis [Articolo su rivista]
La Marca, A.; Spaggiari, G.; Domenici, D.; Grassi, R.; Casonati, A.; Baraldi, E.; Trenti, T.; Simoni, M.; Santi, D.
abstract

STUDY QUESTION: Are there any associations between environmental pollutants and ovarian reserve, expressed by anti-Mullerian hormone (AMH) serum levels? SUMMARY ANSWER: In this first real-world approach to demonstrate the relationship between air pollutants and serum AMH levels, adverse associations were observed for nitrogen dioxide (NO2) but not with particulate matter. WHAT IS KNOWN ALREADY: In recent years, air pollution has emerged as a potential disrupter to the homeostasis of physiological hormones, possibly affecting human reproduction. Although the influence of age and smoking on AMH levels is largely accepted, the relationship between AMH and the environment has not currently been established. STUDY DESIGN, SIZE, DURATION: A longitudinal, observational, retrospective, real-world study was carried out, including all AMH measurements performed in a single laboratory from January 2007 to October 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS: Serum AMH data were connected to patients' age and residential address, to include air pollution data after geo-localisation. The air pollution considered daily particulate matter (PM) and NO2 values. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 1463 AMH measurements were collected (mean 1.94 ng/ml, median 0.90 ng/ml). AMH was inversely related to patients' age in women older than 25 years (adjusted R-squared 0.120, P < 0.001), but not in those younger than 25 years (adjusted R-squared 0.068, P = 0.055). AMH levels were inversely related to environmental pollutants, such as PM10 (Rho =-0.088, P = 0.001), PM2.5 (Rho =-0.062, P = 0.021) and NO2 (Rho =-0.111, P < 0.001). After subdividing the dataset into quartiles for PM10 and PM2.5, the influence of age on AMH serum levels was found to be a stronger influence than that exerted by PM (P = 0.833 and P = 0.370, respectively). On the contrary, considering NO2 quartiles, higher AMH levels were observed in third quartile compared to fourth quartile, even after adjustment for age (P = 0.028), indicating a stronger influence of NO2 exposure on AMH serum levels. Considering an AMH cut-off of 0.3 ng/ml, a significant higher frequency of women with severe ovarian reserve reduction in the fourth quartile was shown only for NO2 (P = 0.010). LIMITATIONS, REASONS FOR CAUTION: Several limitations should be underlined, such as the lack of information about work and life habits of each patient and the retrospective nature of the analysis performed on real-world data. WIDER IMPLICATIONS OF THE FINDINGS: Although the genetic component is highly predictive for defining the ovarian reserve at birth, potentially modifiable environmental factors could influence the rate of decline in AMH and ovarian reserve during adulthood. STUDY FUNDINGCOMPETING INTEREST(S): Authors have neither funding nor competing interests to declare. TRIAL REGISTRATION NUMBER: N/A.


2020 - Enhanced expression of the sphingosine-1-phosphate-receptor-3 causes acute myelogenous leukemia in mice [Articolo su rivista]
Vorbach, S.; Grunder, A.; Zhou, F.; Koellerer, C.; Jutzi, J. S.; Simoni, M.; Riccetti, L.; Valk, P. J.; Sanders, M. A.; Muller-Tidow, C.; Nofer, J. -R.; Pahl, H. L.; Poti, F.
abstract

Acute myeloid leukemia (AML) carries a 10–100 fold lower mutational burden than other neoplastic entities. Mechanistic explanations for why a low number of mutations suffice to induce leukemogenesis are therefore required. Here we demonstrate that transgenic overexpression of the wild type sphingosine-1-phosphate receptor 3 (S1P3) in murine hematopoietic stem cells is sufficient to induce a transplantable myeloid leukemia. In contrast, S1P3 expression in more mature compartments does not cause malignant transformation. Treatment with the sphingosine phosphate receptor modulator Fingolimod, which prevents receptor signaling, normalized peripheral blood cell counts and reduced spleen sizes in S1P3 expressing mice. Gene expression analyses in AML patients revealed elevated S1P3 expression specifically in two molecular subclasses. Our data suggest a previously unrecognized contribution of wild type S1P3 signaling to leukemogenesis that warrants the exploration of S1P3 antagonists in preclinical AML models.


2020 - Evolutionary, structural, and physiological differences between hCG and LH [Capitolo/Saggio]
Casarini, Livio; Lazzaretti, Clara; Paradiso, Elia; Santi, Daniele; Brigante, Giulia; Simoni, Manuela
abstract


2020 - FSH Treatment of male idiopathic infertility: Time for a paradigm change [Articolo su rivista]
Simoni, Manuela; Santi, Daniele
abstract

Follicle-stimulating hormone (FSH) has been used in inconclusive clinical trials for male idiopathic infertility in the past. FSH is sometimes prescribed empirically for male idiopathic infertility, showing some improvement in sperm parameters in about half of the patients. In this opinion article we briefly analyze the pathophysiological evidences in favor of a more aggressive approach in planning future studies on pharmacological FSH use in male infertility, in analogy with the FSH use for multiple follicular growth in women undergoing ovarian stimulation for assisted reproduction. There is sufficient evidence that spermatogenesis does not run at its top in the primate and that some extra FSH can stimulate spermatogenesis over its baseline. Existing data suggest that the pharmacological regimens applied so far were insufficient, both in dosage and in duration, to elicit this response in about half of the patients. A paradigm change is needed now: we should move away from the classical, endocrinological approach, which simply applied the substitutive, therapeutic regimen used in hypogonadotropic hypogonadism, towards testing a "testicular hyperstimulation" scheme for a time sufficient to cover more than only one spermatogenic cycle, a concept to be verified in an appropriately controlled, prospective, randomized clinical trial.


2020 - FSH for the Treatment of Male Infertility [Articolo su rivista]
Casarini, L.; Crépieux, P.; Reiter, E.; Lazzaretti, C.; Paradiso, E.; Rochira, V.; Brigante, G.; Santi, D.; Simoni, M.
abstract

Follicle-stimulating hormone (FSH) supports spermatogenesis acting via its receptor (FSHR), which activates trophic effects in gonadal Sertoli cells. These pathways are targeted by hormonal drugs used for clinical treatment of infertile men, mainly belonging to sub-groups defined as hypogonadotropic hypogonadism or idiopathic infertility. While, in the first case, fertility may be efficiently restored by specific treatments, such as pulsatile gonadotropin releasing hormone (GnRH) or choriogonadotropin (hCG) alone or in combination with FSH, less is known about the efficacy of FSH in supporting the treatment of male idiopathic infertility. This review focuses on the role of FSH in the clinical approach to male reproduction, addressing the state-of-the-art from the little data available and discussing the pharmacological evidence. New compounds, such as allosteric ligands, dually active, chimeric gonadotropins and immunoglobulins, may represent interesting avenues for future personalized, pharmacological approaches to male infertility.


2020 - Follicle-stimulating Hormone (FSH) Action on Spermatogenesis: A Focus on Physiological and Therapeutic Roles [Articolo su rivista]
Santi, Daniele; Crépieux, Pascale; Reiter, Eric; Spaggiari, Giorgia; Brigante, Giulia; Casarini, Livio; Rochira, Vincenzo; Simoni, Manuela
abstract

Background: Human reproduction is regulated by the combined action of the follicle-stimulating hormone (FSH) and the luteinizing hormone (LH) on the gonads. Although FSH is largely used in female reproduction, in particular in women attending assisted reproductive techniques to stimulate multi-follicular growth, its efficacy in men with idiopathic infertility is not clearly demonstrated. Indeed, whether FSH administration improves fertility in patients with hypogonadotropic hypogonadism, the therapeutic benefit in men presenting alterations in sperm production despite normal FSH serum levels is still unclear. In the present review, we evaluate the potential pharmacological benefits of FSH administration in clinical practice. Methods: This is a narrative review, describing the FSH physiological role in spermatogenesis and its potential therapeutic action in men. Results: The FSH role on male fertility is reviewed starting from the physiological control of spermatogenesis, throughout its mechanism of action in Sertoli cells, the genetic regulation of its action on spermatogenesis, until the therapeutic options available to improve sperm production. Conclusion: FSH administration in infertile men has potential benefits, although its action should be considered by evaluating its synergic action with testosterone, and well-controlled, powerful trials are required. Prospective studies and new compounds could be developed in the near future.


2020 - How Much Vitamin D is Too Much? A Case Report and Review of the Literature [Articolo su rivista]
De Vincentis, S.; Russo, A.; Milazzo, M.; Lonardo, A.; De Santis, M. C.; Rochira, V.; Simoni, M.; Madeo, B.
abstract

Background: The beneficial effects of vitamin D, together with the high prevalence of vitamin D deficiency, have led to an expanding use of vitamin D analogues. While inappropriate consumption is a recognized cause of harm, definition of doses at which vitamin D becomes toxic remain elusive. Case presentation: A 56-year woman was admitted to our Hospital following a 3-week history of nausea, vomiting and muscle weakness. The patient had been assuming very high dose of cholecalciferol since 20 months (cumulative 78,000,000UI, mean daily 130,000UI), as indicated by a non-conventional protocol for multiple sclerosis. Before starting vitamin D integration, serum calcium and phosphorus levels were normal, while 25OH-vitamin D levels were very low (12.25 nmol/L). On admission, hypercalcemia (3.23 mmol/L) and acute kidney injury (eGFR 20 mL/min) were detected, associated with high concentrations of 25OH-vitamin D (920 nmol/L), confirming the suspicion of vitamin D intoxication. Vitamin D integration was stopped and, in a week, hypercalcemia normalized. It took about 6 months for renal function and 18 months for vitamin D values to go back to normal. Conclusions: This case confirms that vitamin D intoxication is possible albeit with a really high dose. The doses used in clinical practice are far lower than these and, therefore, intoxication rarely occurs even in those individuals whose baseline vitamin D serum levels have never been assessed. Repeated measurements of vitamin D are not necessary in patients under standard integrative therapy. However, patients and clinicians should be aware of the potential dangers of vitamin D overdose.


2020 - Membrane Estrogen Receptor (GPER) and Follicle-Stimulating Hormone Receptor (FSHR) Heteromeric Complexes Promote Human Ovarian Follicle Survival [Articolo su rivista]
Casarini, L.; Lazzaretti, C.; Paradiso, E.; Limoncella, S.; Riccetti, L.; Sperduti, S.; Melli, B.; Marcozzi, S.; Anzivino, C.; Sayers, N. S.; Czapinski, J.; Brigante, G.; Poti, F.; La Marca, A.; De Pascali, F.; Reiter, E.; Falbo, A.; Daolio, J.; Villani, M. T.; Lispi, M.; Orlando, G.; Klinger, F. G.; Fanelli, F.; Rivero-Muller, A.; Hanyaloglu, A. C.; Simoni, M.
abstract

Molecular Biology; Female Reproductive Endocrinology; Endocrine Regulation


2020 - Multilevel approach to male fertility by machine learning highlights a hidden link between haematological and spermatogenetic cells [Articolo su rivista]
Santi, Daniele; Spaggiari, Giorgia; Casonati, Andrea; Casarini, Livio; Grassi, Roberto; Vecchi, Barbara; Roli, Laura; De Santis, Maria Cristina; Orlando, Giovanna; Gravotta, Enrica; Baraldi, Enrica; Setti, Monica; Trenti, Tommaso; Simoni, Manuela
abstract

Male infertility represents a complex clinical condition requiring an accurate multilevel assessment, in which machine learning (ML) technology, combining large data series in nonlinear and highly interactive ways, could be innovatively applied.


2020 - Polygenic Susceptibility to Papillary Thyroid Cancer in Italian Subjects. [Abstract in Atti di Convegno]
Brigante, G.; Lazzaretti, C.; Paradiso, E.; Foersti, A.; Hemminki, K.; Elisei, R.; Romei, C.; Rochira, V.; Simoni, M.; Landi, S.; Casarini, L.
abstract

olygenic Susceptibility to Papillary Thyroid Cancer in Italian Subjects INTRODUCTION AND AIM. Thyroid cancer is the most common endocrine neoplasia, with an estimated age- standardized incidence rate of 6.7 per 100000 worldwide in 2018 [1]. This rate is rapidly increasing and papillary thy- roid carcinoma (PTC) is the main histotype. PTC suscepti- bility is the result of genetic predisposition, environmental factors and lifestyle. We studied the genetic combination that characterizes PTC affected subjects, differentiating them from healthy controls. METHODS AND RESULTS. We considered the genetic variants (SNPs) significantly associated with PTC on the PubMed database. 184 informative SNPs were selected, considering linkage disequilibrium. Then, SNPs data were extracted from the online 1000 Genomes database,comprising genome of 2504 unselected individuals col- lected worldwide. The combination of 184 SNPs associ- ated with PTC was used to group individuals in different risk-clusters according to their genetic structure, calcu- lated by Bayesian statistics, as previously performed for polycystic ovary syndrome [2]. Individuals were distrib- uted among 7 groups worldwide, indicating different de- gree of genetic predisposition to PTC. We then considered genetic data from about 1200 individuals (697 PTC versus 497 healthy controls) of Central/South Italian origin reg- istered in a GWAS, specific for PTC [3]. This first analysis was refined using the 33 SNPs reasonably most causa- tive of genetic clustering (26 with p<0.05 at trend test in GWAS and 7 with p<0.05 in the model of recessive inher- itance). At multivariate logistic regression analysis, PTC and healthy controls resulted genetically different (ODDS RATIO 188.6, 95%CI 64.35-552.8), revealing diverse pre- disposition to develop cancer. Afterwards, these results have been confirmed in an independent cohort of Italian subjects (234 PTC and 100 controls). Then, the genetic structure of each subject was indicated as a percentage of affinity to each risk-cluster and re-analyzed together with other risk factors: sex, body-mass index, area of origin and familiarity (quantified in a growing score as the degree of kinship increases). These data were analyzed together by principal component analysis and clustering of the two groups was even more pronounced. The most contributive factors to the diversity between PTC and healthy controls were genetics and familiarity. CONCLUSION. We demonstrated that PTC affected subjects are genetically different from healthy controls, and that the difference is identifiable in a peculiar combi- nation of genetic variants.


2020 - Prospects for FSH Treatment of Male Infertility [Articolo su rivista]
Simoni, Manuela; Brigante, Giulia; Rochira, Vincenzo; Santi, Daniele; Casarini, Livio
abstract

Context: Despite the new opportunities provided by assisted reproductive technology (ART), male infertility treatment is far from being optimized. One possibility, based on pathophysiological evidence, is to stimulate spermatogenesis with gonadotropins. Evidence Acquisition: We conducted a comprehensive systematic PubMed literature review, up to January 2020, of studies evaluating the genetic basis of follicle-stimulating hormone (FSH) action, the role of FSH in spermatogenesis, and the effects of its administration in male infertility. Manuscripts evaluating the role of genetic polymorphisms and FSH administration in women undergoing ART were considered whenever relevant. Evidence Synthesis: FSH treatment has been successfully used in hypogonadotropic hypogonadism, but with questionable results in idiopathic male infertility. A limitation of this approach is that treatment plans for male infertility have been borrowed from hypogonadism, without daring to overstimulate, as is done in women undergoing ART. FSH effectiveness depends not only on its serum levels, but also on individual genetic variants able to determine hormonal levels, activity, and receptor response. Single-nucleotide polymorphisms in the follicle- stimulating hormone subunit beta (FSHB) and follicle-stimulating hormone receptor (FSHR) genes have been described, with some of them affecting testicular volume and sperm output. The FSHR p.N680S and the FSHB –211G>T variants could be genetic markers to predict FSH response. Conclusions: FSH may be helpful to increase sperm production in infertile men, even if the evidence to recommend the use of FSH in this setting is weak. Placebo-controlled clinical trials, considering the FSHB-FSHR haplotype, are needed to define the most effective dosage, the best treatment length, and the criteria to select candidate responder patients.


2020 - Seasonal Changes of Serum Gonadotropins and Testosterone in Men Revealed by a Large Data Set of Real-World Observations Over Nine Years [Articolo su rivista]
Santi, D.; Spaggiari, G.; Granata, A. R. M.; Setti, M.; Tagliavini, S.; Trenti, T.; Simoni, M.
abstract

Environmental rhythmicity is able to affect the hypothalamic-pituitary-gonadal axis in several animals to achieve reproductive advantages. However, conflicting results were obtained when assessing the environmental-dependent rhythmicity on reproductive hormone secretion in humans. This study was designed to evaluate seasonal fluctuations of the main hormones involved in the hypothalamic-pituitary-gonadal axis in men, using a big data approach. An observational, retrospective, big data trial was carried out, including all testosterone, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) measurements performed in a single laboratory between January 2010 and January 2019 using Chemiluminescent Microparticle Immunoassay. Subjects presenting any factor interfering with the hypothalamic-pituitary-gonadal axis were excluded. The trend and seasonal distributions were analyzed using autoregressive integrated moving average (ARIMA) models. A total of 12,033 data, accounting for 7,491 men (mean age 47.46 ± 13.51 years, range 18–91 years) were included. Testosterone serum levels (mean 5.34 ± 2.06 ng/dL, range 1.70–15.80 ng/dL) showed a seasonal distribution with higher levels in summer and a direct correlation to environmental temperatures and daylight duration. LH levels (mean 4.64 ± 2.54 IU/L, range 1.00–15.00 IU/L) presented 2 peaks of secretion in autumn and spring, independently from environmental parameters. FSH levels (mean 5.51 ± 3.24 IU/L) did not show any seasonal distribution. A clear seasonal fluctuation of both LH and testosterone was demonstrated in a large cohort of adult men, although a circannual seasonality of hypothalamic-pituitary-gonadal hormones in humans could be not strictly evolutionarily required. Testosterone seasonality seems independent from LH fluctuations, which could be regulated by cyclic central genes expression, and more sensible to environmental temperatures and daylight duration.


2020 - Terapia sostitutiva tiroidea e risposta tissutale: quando il TSH non basta [Articolo su rivista]
Monzani, Maria Laura; Piccinini, Francesca; Simoni, Manuela; Brigante, Giulia
abstract


2020 - Thanks Doug! Welcome Marie-Claude! [Articolo su rivista]
Simoni, M.
abstract


2020 - The COVID-19 pandemics: Shall we expect andrological consequences? A call for contributions to ANDROLOGY [Capitolo/Saggio]
Simoni, M.; Hofmann, M. -C.
abstract


2020 - The added value of operator’s judgement in thyroid nodule ultrasound classification arising from histologically based comparison of different risk stratification systems. [Articolo su rivista]
Madeo, B.; Brigante, G.; Ansaloni, A.; Taliani, E.; Kaleci, S.; Monzani, M. L.; Simoni, M.; Rochira, V.
abstract

Objective: Several ultrasound classifications for thyroid nodules were proposed but their accuracy is still debated, since mainly estimated on cytology and not on histology. The aim of this study was to test the diagnostic accuracy and the inter-classification agreement of AACE/ACE-AME, American Thyroid Association (ATA), British Thyroid Association (BTA), and Modena Ultrasound Thyroid Classification (MUT) that stratifies malignancy risk considering also the clinician subjective impression. Methods: A prospective study collecting thyroid nodule features at ultrasound and histological diagnosis was conducted. Ultrasound features were collected following a preformed checklist in candidates for surgery because of indeterminate, suspicious, or malignant cytology. All the nodules, besides the cytologically suspicious one, were blinded analyzed. MUT score was applied prospectively, and the others retrospectively. Sensitivity, specificity, diagnostic cut-off value, and accuracy of each classification were calculated. The overall agreement between classifications was tested by Bland-Altman, and agreement between single nodule analysis by different classifications by Weighted Cohen's Kappa. Results: In classifying a total of 457 nodules, MUT has the highest accuracy (AUC 0.808) and specificity (89%), followed by ATA and BTA, and finally by AACE/ACE-AME. ATA, BTA, and MUT are highly interchangeable. Considering agreement between single nodule analyses, ATA and BTA had the best (κ = 0.723); AACE/ACE-AME showed slight agreement with BTA (κ = 0.177) and MUT (κ = 0.183), and fair agreement with ATA (κ = 0.282); MUT had fair agreement with both ATA (κ = 0.291) and BTA (κ = 0.271). Conclusion: Classifications have an acceptable overall diagnostic accuracy, improved using a less rigid system that takes into consideration operator subjective impression.


2020 - The calcium-to-phosphorous (Ca/P) ratio in the diagnosis of primary hyperparathyroidism and hypoparathyroidism: a multicentric study. [Articolo su rivista]
Madeo, B.; De Vincentis, S.; Repaci, A.; Altieri, P.; Vicennati, V.; Kara, E.; Vescini, F.; Amadori, P.; Balestrieri, A.; Pagotto, U.; Simoni, M.; Rochira, V.
abstract

Purpose The diagnosis of primary hyperparathyroidism (PHPT) and chronic hypoparathyroidism (HypoPT) is still challenging, especially in patients asymptomatic or with non-classical phenotypes and for physicians not skilled in calcium-phosphorous (Ca–P) disorders. The serum calcium/phosphorous (Ca/P) ratio has been proposed as accurate index to identify PHPT, while it has never been tested in HypoPT. The aim of this study is to investigate the diagnostic power of the serum Ca/P ratio in the diagnosis of primary parathyroid dysfunctions (both PHPT and HypoPT) in a large series of data. Methods A multicentric, retrospective, cross-sectional study (ClinicalTrials.gov: NCT03747029) was carried out including 432 PHPT patients and 217 HypoPT patients compared with 389 controls. Serum Ca, P, creatinine, parathyroid hormone and 25OH-vitamin D were collected. Serum Ca and P were expressed in mmol/L. Ca/P diagnostic performance was evaluated by receiver operating characteristic (ROC) curve, sensitivity, specificity and accuracy. Results The Ca/P ratio was significantly higher in PHPT and lower in HypoPT patients than controls (p < 0.0001). At ROC curve analysis, the Ca/P ratio above 2.55 was defined to identify PHPT patients (sensitivity 85.7%, specificity 85.3%) and below 1.78 to identify HypoPT patients (sensitivity 88.2%, specificity 87.9%). Conclusions The Ca/P ratio is a highly accurate index to identify PHPT when Ca/P is above 2.55 and HypoPT when it is below 1.78. These results demonstrate the reliability of this index to rule in/out primary parathyroid dysfunctions and remark the importance of measuring serum P in clinical practice.


2020 - The physician's gender influences the results of the diagnostic workup for erectile dysfunction [Articolo su rivista]
Rastrelli, Giulia; Cipriani, Sarah; Craparo, Andrea; De Vincentis, Sara; Granata, Antonio Rm; Spaggiari, Giorgia; Simoni, Manuela; Maggi, Mario; Santi, Daniele
abstract

Despite the well-known influence of psychological and situational factors on erectile dysfunction (ED), the influence of the physician's gender on the andrological work-up has never been investigated so far.


2020 - Two human menopausal gonadotrophin (hMG) preparations display different early signaling in vitro [Articolo su rivista]
Casarini, L.; Riccetti, L.; Paradiso, E.; Benevelli, R.; Lazzaretti, C.; Sperduti, S.; Melli, B.; Tagliavini, S.; Varani, M.; Trenti, T.; Morini, D.; Falbo, A.; Villani, M. T.; Jonas, K. C.; Simoni, M.
abstract

Commercial hMG drugs are marketed for the treatment of infertility and consist of highly purified hormones acting on receptors expressed in target gonadal cells. Menopur® and Meriofert® are combined preparation of FSH and hCG and are compared in vitro herein. To this purpose, the molecular composition of the two drugs was analyzed by immunoassay. The formation of FSH receptor and LH/hCG receptor (FSHR; LHCGR) heteromer, intracellular Ca2+ and cAMP activation, β-arrestin 2 recruitment and the synthesis of progesterone and estradiol were evaluated in transfected HEK293 and human primary granulosa lutein cells treated by drugs administered within the pg-mg/ml concentration range. Molecular characterization revealed that Meriofert® has a higher FSH:hCG ratio than Menopur® which, in turn, displays the presence of LH molecules. While both drugs induced similar FSHR-LHCGR heteromeric formations and intracellular Ca2+ increase, Meriofert® had a higher potency than Menopur® in inducing a cAMP increase. Moreover, Meriofert® revealed a higher potency than Menopur® in recruiting β-arrestin 2, likely due to different FSH content modulating the tridimensional structure of FSHR-LHCGR-β-arrestin 2 complexes, as evidenced by a decrease in bioluminescence resonance energy transfer signal. This drug-specific activation of intracellular signaling pathways is consistent with the molecular composition of these preparations and impacts downstream progesterone and estradiol production, with Menopur® more potent than Meriofert® in inducing the synthesis of both the steroids. These findings are suggestive of distinct in-vivo activities of these preparations, but require cautious interpretation and further validation from clinical studies.


2020 - Use of follicle-stimulating hormone (FSH) for the male partner of idiopathic infertile couples in Italy: Results from a multicentre, observational, clinical practice survey [Articolo su rivista]
Santi, Daniele; De Vincentis, Sara; Alfano, Patrizia; Balercia, Giancarlo; Calogero, Aldo E; Cargnelutti, Francesco; Coccia, Maria Elisabetta; Condorelli, Rosita A; Dal Lago, Alessandro; De Angelis, Cristina; Gallo, Mariagrazia; Iannantuoni, Nicola; Lombardo, Francesco; Marino, Angelo; Mazzella, Marco; Pallotti, Francesco; Paoli, Donatella; Pivonello, Rosario; Rago, Rocco; Rampini, Mariarita; Salvio, Gianmaria; Simoni, Manuela
abstract

The management of male idiopathic infertility is heterogeneous. Although meta-analyses reported the effectiveness on pregnancy rate, the real clinical impact of follicle-stimulating hormone (FSH) was not evaluated so far. In Italy, FSH is approved by the national Medicines Agency (AIFA) for idiopathic infertile patients with FSH < 8 IU/L, independently of semen parameters.


2020 - β-arrestin 2 Is a Prognostic Factor for Survival of Ovarian Cancer Patients Upregulating Cell Proliferation [Articolo su rivista]
Czogalla, B.; Partenheimer, A.; Jeschke, U.; von Schonfeldt, V.; Mayr, D.; Mahner, S.; Burges, A.; Simoni, M.; Melli, B.; Benevelli, R.; Bertini, S.; Casarini, L.; Trillsch, F.
abstract

Establishing reliable prognostic factors as well as specific targets for new therapeutic approaches is an urgent requirement in advanced ovarian cancer. For several tumor entities, the ubiquitously spread scaffold protein β-arrestin 2, a multifunctional scaffold protein regulating signal transduction and internalization of activated G protein-coupled receptors (GPCRs), has been considered with rising interest for carcinogenesis. Therefore, we aimed to elucidate the prognostic impact of β-arrestin 2 and its functional role in ovarian cancer. β-arrestin 2 expression was analyzed in a subset of 156 samples of ovarian cancer patients by immunohistochemistry. Cytoplasmic expression levels were correlated with clinical as well as pathological characteristics and with prognosis. The biologic impact of β-arrestin 2 on cell proliferation and survival was evaluated, in vitro. Following transient transfection by increasing concentrations of plasmid encoding β-arrestin 2, different cell lines were evaluated in cell viability and death. β-arrestin 2 was detected in all histological ovarian cancer subtypes with highest intensity in clear cell histology. High β-arrestin 2 expression levels correlated with high-grade serous histology and the expression of the gonadotropin receptors FSHR and LHCGR, as well as the membrane estrogen receptor GPER and hCGβ. Higher cytoplasmic β-arrestin 2 expression was associated with a significantly impaired prognosis (median 29.88 vs. 50.64 months; P = 0.025). Clinical data were confirmed in transfected HEK293 cells, human immortalized granulosa cell line (hGL5) and the ovarian cancer cell line A2780 in vitro, where the induction of β-arrestin 2 cDNA expression enhanced cell viability, while the depletion of the molecule by siRNA resulted in cell death. Reflecting the role of β-arrestin 2 in modulating GPCR-induced proliferative and anti-apoptotic signals, we propose β-arrestin 2 as an important prognostic factor and also as a promising target for new therapeutic approaches in advanced ovarian cancer.


2019 - Advancing the cause of improved male reproductive health [Articolo su rivista]
Carrell, D. T.; Simoni, M.; Krausz, C.; Gerton, G. L.
abstract

N/A


2019 - Altered methylation pattern of the SRD5A2 gene in the cerebrospinal fluid of post-finasteride patients: A pilot study [Articolo su rivista]
Melcangi, R. C.; Casarini, L.; Marino, M.; Santi, D.; Sperduti, Samantha; Giatti, S.; Diviccaro, S.; Grimoldi, M.; Caruso, D.; Cavaletti, G.; Simoni, M.
abstract

Context: Post-finasteride syndrome (PFS) occurs in patients with androgenic alopecia after suspension of the finasteride treatment, leading to a large variety of persistent side effects. Despite the severity of the clinical picture, the mechanism underlying the PFS symptoms onset and persistence is still unclear. Objective: To study whether epigenetic modifications occur in PFS patients. Methods: Retrospective analysis of a multicentric, prospective, longitudinal, case–control clinical trial, enrolling 16 PFS patients, compared to 20 age-matched healthy men. Main outcomes were methylation pattern of SRD5A1 and SRD5A2 promoters and concentration of 11 neuroactive steroids, measured by liquid chromatography-tandem mass spectrometry, in blood and cerebrospinal fluid (CSF) samples. Results: SRD5A1 and SRD5A2 methylation analysis was performed in all blood samples (n = 16 PFS patients and n = 20 controls), in 16 CSF samples from PFS patients and in 13 CSF samples from controls. The SRD5A2 promoter was more frequently methylated in CSF of PFS patients compared to controls (56.3 vs 7.7%). No promoter methylation was detected in blood samples in both groups. No methylation occurred in the SRD5A1 promoter of both groups. Unmethylated controls compared to unmethylated SRD5A2 patients showed higher pregnenolone, dihydrotestosterone and dihydroprogesterone, together with lower testosterone CSF levels. Andrological and neurological assessments did not differ between methylated and unmethylated subjects. Conclusions: For the first time, we demonstrate a tissue-specific methylation pattern of SRD5A2 promoter in PFS patients. Although we cannot conclude whether this pattern is prenatally established or induced by finasteride treatment, it could represent an important mechanism of neuroactive steroid levels and behavioural disturbances previously described in PFS.


2019 - Classification of thyroid nodules by ultrasound in clinical practice: the added value of the judgement of the skilled endocrinologist [Abstract in Atti di Convegno]
Madeo, B.; Brigante, G.; Ansaloni, A.; Taliani, E.; Kaleci, S.; Monzani, M. L.; Simoni, M.; Rochira, V.
abstract


2019 - Classification of thyroid nodules by ultrasound in clinical practice: the added value of the judgment of the skilled endocrinologist [Abstract in Rivista]
Madeo, B.; Brigante, G.; Ansaloni, A.; Taliani, E.; Kaleci, S.; Monzani, M. L.; Simoni, M.; Rochira, V.
abstract


2019 - Concomitant medullary thyroid carcinoma with paraganglioma-like pattern and papillary thyroid carcinoma [Articolo su rivista]
Greco, C.; Brigante, G.; Taliani, E.; Corrado, S.; Simoni, M.; Madeo, B.
abstract

A 74-year-old man was referred to the Endocrinology Unit because of multinodular goiter. The dominant nodule (1.7 × 1.9 × 2.4 cm), at the medium-superior third of the left lobe, was inhomogeneously hypoechoic, with irregular margins, macrocalcifications and intranodular vascularization. Fine-needle aspiration biopsy (FNAB) was performed. The cytological diagnosis was TIR 2, benign, according to the 2013 Italian thyroid cytology classification system. Moderately high serum calcitonin (s-Ct) (61.5 pg/mL, n.r. 0–7.5) and normal CEA were detected. The Ct level in FNAB wash-out fluid (Ct-FNAB) was 1450 pg/mL. Based on s-Ct and Ct-FNAB levels, patient underwent total thyroidectomy. Macroscopically, a dominant circumscribed nodule of 2 ecm was described; the histological and immunohistochemical features identified medullary thyroid carcinoma (MTC) with paraganglioma (PG)-like pattern positive for Ct, CEA and chromogranin and negative for S-100 sustentacular cells (SC). Moreover, papillary carcinoma of 3 mm in the right lobe was also associated. No areas of hyperaccumulation of the tracer were documented at Ga68 PET/CT. No RET-proto-oncogene mutations were found. Post-surgery s-Ct levels were within normal range (4 pg/mL). Two years after thyroidectomy, the patient is still disease-free. We reported a case of sporadic and rare variant of MTC: this is the ninth described case of PG-like MTC. In this case, cytologically benign, the clinical suspicion arose from high Ct values at FNAB wash-out fluid. Even if clinical behavior of this variant seems indolent, additional studies are necessary to understand prognoses and predictive factors.


2019 - Diagnostic Accuracy of Different Thyroid Ultrasound Classification Systems and the Added Value of Operator Subjective Impression in Stratifying Nodule Risk [Abstract in Atti di Convegno]
Madeo, Bruno; Brigante, G.; Ansaloni, A.; Taliani, E.; Kaleci, S.; Monzani, M. L.; Simoni, M.; V. Rochira.,
abstract


2019 - Editorial: Follicle-Stimulating Hormone: Fertility and Beyond [Articolo su rivista]
Simoni, Manuela; Huhtaniemi, Ilpo; Santi, Daniele; Casarini, Livio
abstract

Editorial on the Research Topic


2019 - Expression and clinicopathological role of miR146a in thyroid follicular carcinoma. [Articolo su rivista]
Pignatti, E.; Vighi, E.; Magnani, E.; Kara, E.; Roncati, L.; Maiorana, A.; Santi, D.; Madeo, B.; Cioni, K.; Carani, C.; Rochira, V.; Simoni, M.; Brigante, Giulia
abstract

PURPOSE: Dysregulation of microRNA expression has been involved in the development and progression of follicular thyroid carcinoma (FTC). The aim of this work was to study the expression of miRNA146a in FTC and the association with clinicopathological features of the disease. METHODS: Thirty-eight patients affected by FTC were included in the study. Twenty patients carrying follicular thyroid adenoma (FA) were also enroled as the benign counterpart of FTC. Total RNA including miRNA146a was extracted from formalin-fixed paraffin-embedded (FFPE) pairs of affected/unaffected tissue and its expression was assessed by real-time PCR. Two selected target genes, TRAF6 (tumour necrosis factor receptor-associated factor 6) and IRAK1 (Il-1 receptor-associated kinase 1/2), were also analysed. RESULTS: miR146a expression in FTC tissue was overall not downregulated in malignant versus unaffected tissue, but its expression was inversely correlated with clinicopathological features of FTCs at diagnosis. A decreased expression of miR146a became apparent in FTC thyroid tissue of widely compared to minimally invasive tumours. However, miR146a expression differences between contralateral unaffected tissue (extra-FTC) and FTC were not observed regardless of clinicopathological features. IRAK1, a known target for miR146a, was upregulated in FTC and the increase was mainly appreciable in Hurtle FTC variant. Unexpectedly, miR146a did not correlate with TRAF6 showing an inverse trend compared to IRAK1 although both genes regulate the activity of nuclear factor- kB (NF-kB). CONCLUSION: The results of this study indicate that downregulation of miR146a, inversely correlated with clinicopathological features of FTCs at diagnosis and suggest a possible involvement of miR146a in FTC development. IRAK1 over-expression in FTC may be related to tumour development/progression. In vitro experiments are needed to support this hypothesis.


2019 - Glycosylation Pattern and in vitro Bioactivity of Reference Follitropin alfa and Biosimilars [Articolo su rivista]
Riccetti, Laura; Sperduti, Samantha; Lazzaretti, Clara; Klett, Danièle; De Pascali, Francesco; Paradiso, Elia; Limoncella, Silvia; Potì, Francesco; Tagliavini, Simonetta; Trenti, Tommaso; Galano, Eugenio; Palmese, Angelo; Satwekar, Abhijeet; Daolio, Jessica; Nicoli, Alessia; Villani, Maria Teresa; Aguzzoli, Lorenzo; Reiter, Eric; Simoni, Manuela; Casarini, Livio
abstract

Recombinant follicle-stimulating hormone (FSH) (follitropin alfa) and biosimilar preparations are available for clinical use. They have specific FSH activity and a unique glycosylation profile dependent on source cells. The aim of the study is to compare the originator (reference) follitropin alfa (Gonal-f (R))- with biosimilar preparations (Bemfola (R) and Ovaleap (R))-induced cellular responses in vitro. Gonadotropin N-glycosylation profiles were analyzed by ELISA lectin assay, revealing preparation specific-patterns of glycan species (Kruskal-Wallis test; p < 0.05, n = 6) and by glycotope mapping. Increasing concentrations of Gonal-f (R) or biosimilar (1 x 10(-3) -1 x 10(3) ng/ml) were used for treating human primary granulosa lutein cells (hGLC) and FSH receptor (FSHR)-transfected HEK293 cells in vitro. Intracellular cAMP production, Ca2+ increase and beta-arrestin 2 recruitment were evaluated by BRET, CREB, and ERK1/2 phosphorylation by Western blotting. 12-h gene expression, and 8- and 24-h progesterone and estradiol synthesis were measured by real-time PCR and immunoassay, respectively. We found preparation-specific glycosylation patterns by lectin assay (Kruskal-Wallis test; p < 0.001; n = 6), and similar cAMP production and beta-arrestin 2 recruitment in FSHR-transfected HEK293 cells (cAMP EC50 range = 12 +/- 0.9-24 +/- 1.7 ng/ml; beta-arrestin 2 EC50 range = 140 +/- 14.1-313 +/- 18.7 ng/ml; Kruskal-Wallis test; p >= 0.05; n = 4). Kinetics analysis revealed that intracellular Ca2+ increased upon cell treatment by 4 mu g/ml Gonal-f (R), while equal concentrations of biosimilars failed to induced a response (Kruskal-Wallis test; p < 0.05; n = 3). All preparations induced both 8 and 24 h-progesterone and estradiol synthesis in hGLC, while no different EC(50)s were demonstrated (Kruskal -Wallis test; p > 0.05; n = 5). Apart from preparation-specific intracellular Ca2+ increases achieved at supra-physiological hormone doses, all compounds induced similar intracellular responses and steroidogenesis, reflecting similar bioactivity, and overall structural homogeneity.


2019 - Gnrh antagonists produce differential modulation of the signaling pathways mediated by gnrh receptors [Articolo su rivista]
Sperduti, S.; Limoncella, S.; Lazzaretti, C.; Paradiso, E.; Riccetti, L.; Turchi, S.; Ferrigno, I.; Bertacchini, J.; Palumbo, C.; Poti, F.; Longobardi, S.; Millar, R. P.; Simoni, M.; Newton, C. L.; Casarini, L.
abstract

Commercial gonadotropin-releasing hormone (GnRH) antagonists differ by 1–2 amino acids and are used to inhibit gonadotropin production during assisted reproduction technologies (ART). In this study, potencies of three GnRH antagonists, Cetrorelix, Ganirelix and Teverelix, in inhibiting GnRH-mediated intracellular signaling, were compared in vitro. GnRH receptor (GnRHR)-transfected HEK293 and neuroblastoma-derived SH-SY5Y cell lines, as well as mouse pituitary LβT2 cells endogenously expressing the murine GnRHR, were treated with GnRH in the presence or absence of the antagonist. We evaluated intracellular calcium (Ca2+) and cAMP increases, cAMP-responsive element binding-protein (CREB) and extracellular-regulated kinase 1 and 2 (ERK1/2) phosphorylation, β-catenin activation and mouse luteinizing-hormone β-encoding gene (Lhb) transcription by bioluminescence resonance energy transfer (BRET), Western blotting, immunostaining and real-time PCR as appropriate. The kinetics of GnRH-induced Ca2+ rapid increase revealed dose-response accumulation with potency (EC50) of 23 nM in transfected HEK293 cells, transfected SH-SY5Y and LβT2 cells. Cetrorelix inhibited the 3 × EC50 GnRH-activated calcium signaling at concentrations of 1 nM–1 µM, demonstrating higher potency than Ganirelix and Teverelix,.


2019 - Identificazione ecografica di un nodulo sottocutaneo rilevato alla palpazione del collo e risultato un microcarcinoma papillare tiroideo persistente in sede sottocutanea atipica 25 anni dopo terapia chirurgica locale, tiroidectomia e linfadenectomia [Articolo su rivista]
Spaggiari, Giorgia; Piccinini, Francesca; Ansaloni, Anna; Madeo, Bruno; Zirilli, Lucia; Simoni, Manuela; Santi, Daniele
abstract

Descrizione di un caso clinico di particolare interesse


2019 - Multilevel approach to male infertility using machine learning [Abstract in Atti di Convegno]
Santi, D; Spaggiari, G; Michelangeli, M; Casarini, L; Grassi, R; Vecchi, B; Roli, L; De Santis, Mc; Baraldi, E; Setti, M; Trenti, T; Simoni., M
abstract


2019 - Probing the Effect of Sildenafil on Progesterone and Testosterone Production by an Intracellular FRET/BRET Combined Approach [Articolo su rivista]
Casarini, L.; Riccetti, L.; Limoncella, Silvia; Lazzaretti, C.; Barbagallo, F.; Pacifico, S.; Guerrini, R.; Tagliavini, S.; Trenti, T.; Simoni, M.; Sola, M.; Di Rocco, G.
abstract

Forster resonance energy transfer (FRET)-based biosensors have been recently applied to the study of biological pathways. In this study, a new biosensor was validated for the first time in live HEK293 and steroidogenic MLTC-1 cell lines for studying the effect of the PDE5 inhibitor on the hCG/LH-induced steroidogenic pathway. The sensor improves FRET between a donor (D), the fluorescein-like diarsenical probe that can covalently bind a tetracysteine motif fused to the PDE5 catalytic domain, and an acceptor (A), the rhodamine probe conjugated to the pseudosubstrate cGMPS. Affinity constant (Kd) values of 5.6 ± 3.2 and 13.7 ± 0.8 μM were obtained with HEK293 and MLTC-1 cells, respectively. The detection was based on the competitive displacement of the cGMPS-rhodamine conjugate by sildenafil; the Ki values were 3.6 ± 0.3 nM (IC50 = 2.3 nM) in HEK293 cells and 10 ± 1.0 nM (IC50 = 3.9 nM) in MLTC-1 cells. The monitoring of both cAMP and cGMP by bioluminescence resonance energy transfer allowed the exploitation of the effects of PDE5i on steroidogenesis, indicating that sildenafil enhanced the gonadotropin-induced progesterone-to-testosterone conversion in a cAMP-independent manner.


2019 - Semi-annual seasonal pattern of serum thyrotropin in adults [Articolo su rivista]
Santi, D.; Spaggiari, G.; Brigante, G.; Setti, M.; Tagliavini, S.; Trenti, T.; Simoni, M.
abstract

Circannual rhythmicity in thyroid-stimulating hormone (TSH) secretion is proposed, whereas evidences on seasonal peripheral thyroid hormones' fluctuation are contradictory. This study was designed to evaluate hypothalamic-pituitary-thyroid (HPT) seasonal secretion pattern using a big data approach. An observational, retrospective, big data trial was carried out, including all TSH measurements performed in a single laboratory between January 2010 and December 2017. A large dataset was created matching TSH data with patients' age, gender, environmental temperature exposure, and free triiodothyronine (fT3) and free thyroxine (fT4) when available. The trend and seasonal distributions were analysed using autoregressive integrated moving average models. A total of 1,506,495 data were included in the final database with patients mean age of 59.00 +/- 18.44 years. The mean TSH serum levels were 2.08 +/- 1.57 microIU/mL, showing a seasonal distribution with higher levels in summer and winter seasons, independently from age, gender and environmental temperatures. Neither fT3 nor fT4 showed a seasonal trend. TSH seasonal changes occurred independently from peripheral thyroid hormone variations, gender, age and environmental temperatures. Although seasonal TSH fluctuation could represent a residual ancestral mechanism to maintain HPT homeostasis, the underlying physiological mechanism remains unclear and specific studies are needed to clarify its impacting role in humans.


2019 - Thyroid function in Klinefelter syndrome: a multicentre study from KING group [Articolo su rivista]
Balercia, G.; Bonomi, M.; Giagulli, V. A.; Lanfranco, F.; Rochira, V.; Giambersio, A.; Accardo, G.; Esposito, D.; Allasia, S.; Cangiano, B.; De Vincentis, S.; Condorelli, R. A.; Calogero, A.; Pasquali, D.; Aversa, A.; Balercia, G.; Calogero, A.; Corona, G.; Giorgino, F.; Fabbri, A.; Ferlin, A.; Ferrante, E.; Francavilla, F.; Giagulli, V.; Jannini, E.; Lanfranco, F.; Maggi, M.; Pasquali, D.; Pivonello, R.; Pizzocaro, A.; Radicioni, A.; Rochira, V.; Vignozzi, L.; Barchi, M.; Condorelli, R. A.; Cordeschi, G.; D'Andrea, S.; Mambro, A. D.; Foresta, C.; Francavilla, S.; Garolla, A.; Giovannini, L.; Granata, A. R. M.; La Vignera, S.; Motta, G.; Negri, L.; Pelliccione, F.; Persani, L.; Salzano, C.; Santi, D.; Selice, R.; Simoni, M.; Tatone, C.; Tirabassi, G.; Tresoldi, A. S.; Vicari, E.
abstract

PURPOSE:The prevalence and the etiopathogenesis of thyroid dysfunctions in Klinefelter syndrome (KS) are still unclear. The primary aim of this study was to evaluate the pathogenetic role of hypogonadism in the thyroid disorders described in KS, with the scope to distinguish between patients with KS and hypogonadism due to other causes (Kallmann syndrome, idiopathic hypogonadotropic hypogonadism, iatrogenic hypogonadism and acquired hypogonadotropic hypogonadism after surgical removal of pituitary adenomas) called non-KS. Therefore, we evaluated thyroid function in KS and in non-KS hypogonadal patients. METHODS: This is a case-control multicentre study from KING group: Endocrinology clinics in university-affiliated medical centres. One hundred and seventy four KS, and sixty-two non-KS hypogonadal men were enrolled. The primary outcome was the prevalence of thyroid diseases in KS and in non-KS. Changes in hormonal parameters were evaluated. Exclusion criterion was secondary hypothyroidism. Analyses were performed using Student's t test. Mann-Whitney test and Chi-square test. RESULTS: FT4 was significantly lower in KS vs non-KS. KS and non-KS presented similar TSH and testosterone levels. Hashimoto's thyroiditis (HT) was diagnosed in 7% of KS. Five KS developed hypothyroidism. The ratio FT3/FT4 was similar in both groups. TSH index was 1.9 in KS and 2.3 in non-KS. Adjustment for differences in age, sample size and concomitant disease in multivariate models did not alter the results. CONCLUSIONS: We demonstrated in KS no etiopathogenic link to hypogonadism or change in the set point of thyrotrophic control in the altered FT4 production. The prevalence of HT in KS was similar to normal male population, showing absence of increased risk of HT associated with the XXY karyotype.


2019 - Ultrasound Changes of Healthy Thyroides over Six Years in Adults [Abstract in Atti di Convegno]
Brigante, G.; Monzani, M. L.; Locaso, M.; Gnarini, V. L.; Graziadei, L.; Kaleci, S.; De Santis, M. C.; Tagliavini, S.; Simoni, M.; Rochira, V.; Madeo, B.
abstract


2019 - Ultrasound changes of healthy thyroides over six years in adults [Abstract in Atti di Convegno]
Brigante, G.; Monzani, M. L.; Locaso, M.; Gnarini, V. L.; Graziadei, L.; Kaleci, S.; De Santis, M. C.; Tagliavini, S.; Simoni, M.; Rochira, V.; Madeo, B.
abstract


2019 - Welcome to the Chinese version of Andrology! [Articolo su rivista]
Simoni, M.; Carrell, D. T.
abstract

N/A


2018 - 'Spare' Luteinizing Hormone Receptors: Facts and Fiction. [Articolo su rivista]
Casarini, Livio; Santi, Daniele; Simoni, Manuela; Potì, Francesco.
abstract

It is common opinion that maximal activation of luteinizing hormone (LH)-dependent steroidogenic signal occurs at <1% of human LH/choriogonadotropin (hCG) receptor (LHCGR) occupancy. This effect would be a consequence of an excess of receptors expressed on the surface of theca cells, resulting in a pool of LHCGRs remaining unbound (spare). This concept was borrowed from historical pharmacological studies, when discrepancies between ligand-receptor binding and dose-response curves of cAMP were evaluated by treating mouse or rat Leydig cells with hCG in vitro. Recent findings demonstrated the specificity of LH- and hCG-dependent effects, receptor heterodimerization, and differing behaviors of rodent versus human gonadotropin-responsive cells, which may help to revise the 'spare' LHCGRs concept applied to human ovarian physiology and assisted reproduction.


2018 - Andrology Award 2017 [Articolo su rivista]
Simoni, M.; Carrell, D. T.
abstract

N/A


2018 - Approccio a multilivelli all’infertilità maschile utilizzando il machine learning. [Abstract in Atti di Convegno]
Santi, D; Spaggiari, G; Michelangeli, M; Casarini, L; Grassi, R; Vecchi, B; Roli, L; De Santis, Mc; Baraldi, E; Setti, M; Trenti, T; Simoni, M
abstract


2018 - Characteristics of a nationwide cohort of patients presenting with isolated hypogonadotropic hypogonadism (IHH) [Articolo su rivista]
Bonomi, Marco; Vezzoli, Valeria; Krausz, Csilla; Guizzardi, Fabiana; Vezzani, Silvia; Simoni, Manuela; Bassi, Ivan; Duminuco, Paolo; Di Iorgi, Natascia; Giavoli, Claudia; Pizzocaro, Alessandro; Russo, Gianni; Moro, Mirella; Fatti, Letizia; Ferlin, Alberto; Mazzanti, Laura; Zatelli, Maria Chiara; Cannavò, Salvo; Isidori, Andrea M.; Pincelli, Angela Ida; Prodam, Flavia; Mancini, Antonio; Limone, Paolo; Tanda, Maria Laura; Gaudino, Rossella; Salerno, Mariacarolina; Francesca, Pregnolato; Maghnie, Mohamad; Maggi, Mario; Persani, Luca; Aimaretti, G.; Altobell, M.; Ambrosio, M. R.; Andrioli, M.; Angelett, G.; Arecco, F.; Arnald, G.; Arosio, M.; Balsamo, A.; Baldassarr, M.; Bartalena, L.; Bazzon, N.; Beccari, L.; Beck-Peccoz, P.; Bellastella, G.; Bellizz, M.; Benedicent, F.; Bernasconi, S.; Bizzarri, C.; Bona, G.; Bonadonna, S.; Borrett, G.; Boschetti, M.; Brunani, A.; Brunelli, V.; Buz, F.; Cacciatore, C.; Cangiano, B.; Cappa, M.; Casalone, R.; Cassio, A.; Cavarzere, P.; Cherubini, V.; Ciampani, T.; Cicognan, D.; Cignarell, A.; Cisternin, M.; Colombo, P.; Corbetta, S.; Corciul, N.; Corona, G.; Cozzi, R.; Crivellaro, C.; Dalle Mule, I.; Danesi, L.; Eli, A. V. D.; Degli Uberti, E.; De Leo, S.; Della Valle, E.; De Marchi, M.; Di Iorgi, N.; Di Mambr, A.; Fabbri, A.; Foresta, C.; Forti, G.; Franceschi, A. R.; Garolla, A.; Ghezzi, M.; Giacomozzi, C.; Giusti, M.; Grosso, E.; Guabello, G.; Guarneri, M. P.; Grugni, G.; Isidori, A. M.; Lanfranco, F.; Lania, A.; Lanzi, R.; Larizza, L.; Lenzi, A.; Loche, S.; Loli, P.; Lombardi, V.; Maggi, M. C.; Mandrile, G.; Manieri, C.; Mantovani, G.; Marelli, S.; Marzullo, M.; Mencarelli, M. A.; Migone, N.; Motta, G.; Neri, G.; Padov, G.; Parenti, G.; Pasquino, B.; Pia, A.; Piantanida, E.; Pignatti, E.; Pilotta, A.; Pivett, B.; Pollazzon, M.; Pontecorvi, A.; Porcelli, P.; Pozza, G. B.; Pozzobon, G.; Radetti, G.; Razzore, P.; Rocchett, L.; Roncoron, R.; Rossi, G.; Sala, E.; Salvatoni, A.; Salvini, F.; Secc, A.; Segni, M.; Selice, R.; Sgaramella, P.; Sileo, F.; Sinisi, A. A.; Sirchia, F.; Spada, A.; Tresoldi, A.; Vigneri, R.; Weber, G.; Zucchini, S.
abstract

Objective: Isolated hypogonadotropic hypogonadism (IHH) is a rare disorder with pubertal delay, normal (normoosmic-IHH, nIHH) or defective sense of smell (Kallmann syndrome, KS). Other reproductive and nonreproductive anomalies might be present although information on their frequency are scanty, particularly according to the age of presentation. Design: Observational cohort study carried out between January 2008 and June 2016 within a national network of academic or general hospitals. Methods: We performed a detailed phenotyping of 503 IHH patients with: (1) manifestations of hypogonadism with low sex steroid hormone and low/normal gonadotropins; (2) absence of expansive hypothalamic/pituitary lesions or multiple pituitary hormone defects. Cohort was divided on IHH onset (PPO, pre-pubertal onset or AO, adult onset) and olfactory function: PPO-nIHH (n = 275), KS (n = 184), AO-nIHH (n = 36) and AO-doIHH (AO-IHH with defective olfaction, n = 8). Results: 90% of patients were classifed as PPO and 10% as AO. Typical midline and olfactory defects, bimanual synkinesis and familiarity for pubertal delay were also found among the AO-IHH. Mean age at diagnosis was signifcantly earlier and more frequently associated with congenital hypogonadism stigmata in patients with Kallmann's syndrome (KS). Synkinesis, renal and male genital tract anomalies were enriched in KS. Overweight/obesity are signifcantly associated with AO-IHH rather than PPO-IHH. Conclusions: Patients with KS are more prone to develop a severe and complex phenotype than nIHH. The presence of typical extra-gonadal defects and familiarity for PPO-IHH among the AO-IHH patients indicates a common predisposition with variable clinical expression. Overall, these fndings improve the understanding of IHH and may have a positive impact on the management of patients and their families.


2018 - Clinical relevance of genetic variants of gonadotrophins and their receptors in controlled ovarian stimulation: a systematic review and meta-analysis [Articolo su rivista]
Alviggi, Carlo; Conforti, Alessandro; Santi, Daniele; Esteves, Sandro C; Andersen, Claus Yding; Humaidan, Peter; Chiodini, Paolo; De Placido, Giuseppe; Simoni, Manuela
abstract

Genotype has been implicated in the outcome of ovarian stimulation. The analysis of patient-specific genotypes might lead to an individualized pharmacogenomic approach to controlled ovarian stimulation (COS). However, the validity of such an approach remains to be established.


2018 - Could chronic Vardenafil administration influence the cardiovascular risk in men with type 2 diabetes mellitus? [Articolo su rivista]
Santi, Daniele; Locaso, Michela; Granata, Antonio R; Trenti, Tommaso; Roli, Laura; Pacchioni, Chiara; Rochira, Vincenzo; Carani, Cesare; Simoni, Manuela
abstract

Introduction Appropriate algorithms for the prediction of cardiovascular risk are strongly suggested in clinical practice, although still controversial. In type 2 diabetes mellitus (T2DM), the benefi- cial effect of phosphodiesterase (PDE)-5 inhibitors is demonstrated on endothelial function but not on the estimation of cardiovascular risk. Aim To study whether the chronic Vardenafil administration to men with T2DM influences vari- ables correlated with the predicted long-term cardiovascular risk calculated by different vali- dated algorithms. Methods Per-protocol analysis of a longitudinal, prospective, randomized, placebo-controlled, dou- ble-blind, investigator-started, clinical trial. 54 male patients affected by T2DM were assigned to study (26patients) and control-group (28patients), respectively. The study included a treatment phase (24weeks) (Vardenafil/placebo 10mg twice-daily) and a follow- up phase (24weeks). Three time points were considered: baseline(V0), end of treatment (V1) and end of the study(V2). Parameters evaluated: endothelial health-related parameters and cardiovascular risk, assessed by calculating the Framingham (coronary hart disease [CHD], myocardial infarction [MI], stroke and cardiovascular disease [CVD]), ASSIGN and CUORE equations. Results Predicted cardiovascular risk at ten years resulted different using the three algorithms cho- sen, without differences between study and control groups and among visits. IL-6 was directly related to CHD, CVD and CUORE scores at V1 and with MI and STROKE at V2. Similarly, hs-CRP was directly related to CHD, MI, STROKE and CUORE only at V1 in the study group. Testosterone serum levels were inversely related to CHD and MI at V1 in study group. Discussion The predicted cardiovascular risk is different depending on the algorithm chosen. Despite no predictive risk reduction after six months of treatment, a possible effect of Vardenafil could be hypothesized through its action on inflammation markers reduction and through restoration of normal testosterone levels.


2018 - Editorial - Sex goes molecular! [Articolo su rivista]
Jannini, Emmanuele A.; Simoni, Manuela
abstract

N/A


2018 - Elevating Endogenous Sphingosine-1-Phosphate (S1P) Levels Improves Endothelial Function and Ameliorates Atherosclerosis in Low Density Lipoprotein Receptor-Deficient (LDL-R-/-) Mice [Articolo su rivista]
Feuerborn, Renata; Besser, Manuela; Potì, Francesco; Burkhardt, Ralph; Weißen-Plenz, Gabriele; Ceglarek, Uta; Simoni, Manuela; Proia, Richard L.; Freise, Hendrik; Nofer, Jerzy-Roch
abstract

Background Sphingosine-1-phosphate (S1P) is a bioactive lysosphingolipid and a constituent of high-density lipoprotein (HDL) exerting several atheroprotective effects in vitro. However, the few studies addressing anti-atherogenic effects of S1P in vivo have led to disparate results. We here examined atherosclerosis development in low-density lipoprotein receptor (LDL-R)-deficient (LDL-R-/-) mice with elevated endogenous S1P levels. Methods and Results Sub-lethally irradiated LDL-R-/-mice were transplanted with bone marrow deficient in sphingosine kinase 2 (SphK2), which led to the elevation of S1P concentrations in erythrocytes, plasma and HDL by approximately 1.5- to 2.0-fold in SphK2-/-/LDL-R-/-mice. Afterwards, mice were fed a Western diet for 14 weeks. Elevation of endogenous S1P significantly reduced atherosclerotic lesion formation by approximately half without affecting the plasma lipid profile. Furthermore, the macrophage content of atherosclerotic lesions and lipopolysaccharide-induced monocyte recruitment to the peritoneal cavity were reduced in SphK2-/-/LDL-R-/-mice. Studies using intra-vital microscopy revealed that endogenous S1P lowered leukocyte adhesion to capillary wall and decreased endothelial permeability to fluorescently labelled LDL. Moreover, SphK2-/-/LDL-R-/-mice displayed decreased levels of vascular cell adhesion molecule 1 in atherosclerotic lesions and in plasma. Studies in vitro demonstrated reduced monocyte adhesion and transport across an endothelial layer exposed to increasing S1P concentrations, murine plasma enriched in S1P or plasma obtained from SphK2-deficient animals. In addition, decreased permeability to fluorescence-labelled dextran beads or LDL was observed in S1P-treated endothelial cells. Conclusion We conclude that raising endogenous S1P levels exerts anti-atherogenic effects in LDL-R-/-mice that are mediated by favourable modulation of endothelial function.


2018 - FSH (Follicle-Stimulating Hormone) [Capitolo/Saggio]
Santi, Daniele; Casarini, Livio; R Marshall, Gary; Simoni, Manuela
abstract

Follicle-stimulating hormone (FSH) is a glycoprotein regulating development and reproduction. In both adult fertile males and females, FSH mediates spermatogenesis and folliculogenesis, acting through its G-protein coupled receptor (FSHR). Mutations and single nucleotide polymorphisms occurring within the genes encoding the hormone beta subunit and FSHR may modulate or even impair the physiological function of FSH in both males and females. Synthesis and secretion of FSH are described in the chapter, with a specific overview on the pathways activated upon FSH-FSHR interaction and the physiology of the hormone.


2018 - Fresh spring air for Andrology [Articolo su rivista]
Simoni, M.; Carrell, D. T.
abstract

N/A


2018 - LH (Luteinizing Hormone) [Capitolo/Saggio]
Casarini, Livio; Santi, Daniele; R Marshall, Gary; Simoni, Manuela
abstract

Luteinizing hormone (LH) is secreted by the pituitary gland as a heterodimeric glycoprotein acting on the gonads, regulating development and reproduction. In human of fertile age, it plays a central role in follicle development and spermatogenesis stimulating the production of steroid hormones and mediating proliferative signals. LH acts on a G-protein coupled receptor (LHCGR), shared together with the pregnancy hormone choriogonadotropin (hCG), which features specific intracellular signaling and physiological function. In this chapter, the role exerted by LH during fetal life and fertile age of humans is described.


2018 - La risposta degli steroidi allo stimolo con gonadotropina corionica (hCG) nei soggetti con sindrome di Klinefelter non cambia utilizzando gli immunodosaggi o la spettrometria di massa [Articolo su rivista]
Roli, L.; Santi, D.; Tagliavini, S.; Cavalieri, S.; De Santis, M. C.; Baraldi, E.; Fanelli, F.; Mezzullo, M.; Granata, A. R.; Pagotto, U.; Pasquali, R.; Rochira, V.; Carani, C.; Simoni, M.; Trenti, T.
abstract

Obiettivi. La cromatografia liquida associata alla spettrometria di massa tandem (LC-MS/MS) è stata sviluppata contemporaneamente agli immunodosaggi (IA) ed oggigiorno viene proposta come gold standard per il dosaggio degli steroidi. Le cellule del Leydig dei soggetti con sindrome di Klinefelter (KS) sono in grado di rispondere allo stimolo con gonadotropina corionica (hCG), anche se la produzione di testosterone (T) è difettosa. L'obiettivo dello studio è di valutare come i risultati ottenuti con gli IA e la LC-MS/MS possono differentemente impattare sui risultati di una ricerca clinica sulla steroidogenesi gonadica dopo stimolo con hCG. Metodi. E' stato condotto uno studio clinico longitudinale, prospettico, caso-controllo (clinicaltrial.gov NCT02788136), arruolando maschi con KS e controlli sani appaiati per età, sottoposti a stimolo con somministrazione di hCG. Gli steroidi sierici sono stati valutati in condizioni basali e per 5 giorni consecutivi ad una iniezione intramuscolare di 5000 IU di hCG utilizzando sia gli IA che la LC-MS/MS. Risultati. Sono stati arruolati 13 pazienti KS (36 ± 9 anni) non in terapia sostitutiva con T e 14 controlli sani (32 ± 8 anni). T, progesterone (P), cortisolo (C), 14-idrossi-progesterone (17OHP) e androstenedione (A) erano si- gnificativamente più elevati utilizzando di IA rispetto alla LC-MS/MS. I due metodi hanno dimostrato una relazione diretta sebbene con una costante sovrastima da parte degli IA. Entrambi i metodi hanno rilevato lo stesso profilo d'incremento di 17OHP e di T, con aree sotto la curva (AUC) equivalenti. Conclusioni. Sebbene sia stata dimostrata una relazione lineare tra IA e LC-MS/MS, quest'ultima è più sensibile ed accurata, mentre gli IA mostrano una costante sovrastima dei livelli di steroidi sierici. Questo risultato suggerisce la necessità di stabilire intervalli di riferimento metodo-specifici. La fondamentale differenza fra i due metodi apre una profonda riconsiderazione su cosa sia necessario per migliorare l'accuratezza dei dosaggi per gli steroidi.


2018 - Molecular basis of androgen action on human sexual desire [Articolo su rivista]
Santi, Daniele; Spaggiari, Giorgia; Gilioli, Lisa; Potì, Francesco; Simoni, Manuela; Casarini, Livio
abstract

Reproduction is a fundamental process for the species maintenance and the propagation of genetic information. The energy expenditure for mating is overtaken by motivational stimuli, such as orgasm, finely regulated by steroid hormones, gonadotropins, neurotransmitters and molecules acting in the brain and peripheral organs. These functions are often investigated using animal models and translated to humans, where the androgens action is mediated by nuclear and membrane receptors converging in the regulation of both long-term genomic and rapid non-genomic signals. In both sexes, testosterone is a central player of this game and is involved in the regulation of sexual desire and arousal, and, finally, in reproduction through cognitive and peripheral physiological mechanisms which may decline with aging and circadian disruption. Finally, genetic variations impact on reproductive behaviours, resulting in sex-specific effect and different reproductive strategies. In this review, androgen actions on sexual desire are evaluated, focusing on the molecular levels of interaction.


2018 - Pharmacogenetics of G-protein-coupled receptors variants: FSH receptor and infertility treatment [Articolo su rivista]
Santi, Daniele; Potì, Francesco; Simoni, Manuela; Casarini, Livio
abstract

Infertility treatment may represent a paradigmatic example of precision medicine. Follicle-stimulating hormone (FSH) has been proposed as a valuable therapeutic option both in males and in females, even if a standardized approach is far to be established. To date, several genetic mutations as well as polymorphisms have been demonstrated to significantly affect the pathophysiology of FSH-FSH receptor (FSHR) interaction, although the underlying molecular mechanisms remain unclear. This review aims to highlight possible aspects of FSH therapy that could benefit from a pharmacogenetic approach, providing an up-to-date overview of the variability of the response to FSH treatment in both sexes. Specific sections are dedicated to the clinical use of FSH in infertility and how FSHR polymorphisms may affect the therapeutic endpoints.


2018 - Response: Commentary: Efficacy of Follicle-Stimulating Hormone (FSH) Alone, FSH + luteinizing hormone, human menopausal gonadotropin or FSH + human chorionic gonadotropin on assisted reproductive technology outcomes in the "Personalized" Medicine Era: A meta-analysis [Articolo su rivista]
Santi, Daniele; Casarini, Livio; Alviggi, Carlo; Simoni, Manuela
abstract

N/A


2018 - Seasonal variation of semen parameters correlates with environmental temperature and air pollution: A big data analysis over 6 years [Articolo su rivista]
Santi, Daniele; Magnani, Elisa; Michelangeli, Marco; Grassi, Roberto; Vecchi, Barbara; Pedroni, Gioia; Roli, Laura; De Santis, Maria Cristina; Baraldi, Enrica; Setti, Monica; Trenti, Tommaso; Simoni, Manuela
abstract

Male fertility is progressively declining in many developed countries, but the relationship between male infertility and environmental factors is still unclear.


2018 - Serum Calcium to Phosphorous (Ca/P) Ratio is a simple, inexpensive, and accurate tool in the diagnosis of primary hyperparathyroidism [Articolo su rivista]
Madeo, B.; Kara, E.; Cioni, K.; Vezzani, S.; Trenti, T.; Santi, D.; Simoni, M.; Rochira, V.
abstract

Primary hyperparathyroidism (PHPT) diagnosis is challenging and is based on serum calcium (Ca) and parathyroid hormone (PTH). Because serum Ca and phosphorous (P) are inversely related in PHPT, we investigated the diagnostic value of the serum Ca/P ratio in the diagnosis of PHPT. We report a single-center, case-controlled, retrospective study including 97 patients with documented PHPT and compared them with those of 96 controls (C). The main outcome measures were: serum PTH, 25-OH vitamin D, Ca, P, albumin, and creatinine. Sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the serum Ca/P ratio were calculated. The results were verified using an independent, anonymous set of data extracted from a laboratory database containing over 900 million entries. A total of 35 (36.1%) PHPT patients had normocalcemic PHPT (NCHPT). Ca and PTH were significantly higher in PHPT than in C (p < 0.0001). P was significantly lower in PHPT than in C (p < 0.0001). The Ca/P ratio was significantly higher in PHPT than in C (p < 0.0001). Receiver-operating characteristic (ROC) curves analyses identified a cutoff of 2.71 (3.5 if Ca and P are expressed in mg/dL) for Ca/P ratio with a sensitivity and specificity of 86% and 87%, respectively (p < 0.0001), confirmed by the independent, big data approach. In conclusion, Ca/P is a valuable tool for the diagnosis of PHPT and is of superior value compared to serum Ca alone, especially in NCPHT. Because Ca/P is simple, inexpensive, and easily accessible worldwide, this ratio is useful for PHPT diagnosis, especially in laboratory/medical settings relying on limited resources, such as low-income countries. © 2017 The Authors. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.


2018 - Sperm DNA fragmentation index as a promising predictive tool for male infertility diagnosis and treatment management [Abstract in Rivista]
Santi, Daniele; Spaggiari, Giorgia; Simoni, Manuela
abstract

10.1530/endoabs.56.GP214


2018 - Sperm DNA fragmentation index as a promising predictive tool for male infertility diagnosis and treatment management – meta-analyses [Articolo su rivista]
Santi, Daniele; Spaggiari, Giorgia; Simoni, Manuela
abstract

Conventional semen analyses have limitations in male infertility diagnosis and prognosis. Assessment of sperm DNA fragmentation (sDF) has been proposed to discriminate fertile from infertile men and predict FSH treatment response in infertile men, although a comprehensive evaluation of this is not available. The aims of these meta-analyses were to assess the power of sDF in male infertility diagnosis and its role in predicting FSH therapy response in infertile men. Two literature searches were conducted in MEDLINE (PubMed), Embase, the Cochrane Library, Scopus and UpToDate. First, interventional/observational clinical trials comparing fertile to infertile/subfertile men were included. Second, interventional/observational clinical trials evaluating FSH-treated infertile men were assessed. sDF levels were significantly higher in infertile men considering 28 studies (P &lt; 0.001), independently of the sDF method applied. Receiver operator characteristics curves identified an sDF threshold of 20%, with sensitivity of 79% and specificity of 86%. Six studies showed significant sDF improvement of 4.24% (95% confidence interval: 0.24–8.25%) after 3 months of FSH treatment. These meta-analyses demonstrate the sDF relevance in male infertility, suggesting a higher accuracy in detecting sperm function than conventional semen parameters. Although larger prospective trials are needed, sDF represents a promising tool for clinical and research practice.


2018 - Testicular ultrasound inhomogeneity is more informative than testicular volume in fertility evaluation [Abstract in Atti di Convegno]
Spaggiari, G; Santi, D; Granata, Arm; Simoni, M.
abstract


2018 - The cAMP/PKA pathway: steroidogenesis of the antral follicular stage. [Articolo su rivista]
Riccetti, Laura; Sperduti, Samantha; Lazzaretti, Clara; Casarini, Livio; Simoni, Manuela
abstract

Pituitary gonadotropins, follicle-stimulating (FSH) and luteinizing hormone (LH) promote follicular recruitment and support antral follicle growth, maturation and selection, resulting in ovulation of the dominant follicle. FSH and LH biological functions are mediated by G protein-coupled receptors, FSHR and LHCGR, resulting in the activation of a number of signaling cascades, such as the cyclic AMP/protein kinase A (cAMP/PKA) pathway. Some in-vitro data are consistent with the dual, proliferative and pro-apoptotic role of cAMP, leaving unanswered questions on how cAMP/PKA signaling is linked to the follicle fate. Progression of the antral stage is characterized by the presence of dynamic serum gonadotropin and estrogen levels, accompanying proliferation and steroidogenesis of growing as well as apoptosis of atretic follicles. These events are parallel to changes of FSHR and LHCGR density at the cell surface occurring throughout the antral stage, reasonably modulating the cAMP/PKA activation pattern, cell metabolism and functions. Understanding whether gonadotropins and receptor expression levels impact on the steroidogenic pathway and play a role in determining the follicular fate, may put new light on molecular mechanisms regulating human reproduction. The aim of the present review is to update the role of major players modulating the cAMP/PKA pathway and regulating the balance between proliferative, differentiating and pro-apoptotic signals.


2018 - Thyroid ultrasound alterations occurrence in patients with previous negative examination: A 6-years observational follow-up trial [Abstract in Rivista]
Monzani, Maria Laura; Brigante, Giulia; Locaso, Michela; Santi, Daniele; Graziadei, Luigi; Gnarini, Valentina Luisa; Simoni, Manuela; Madeo, Bruno
abstract

10.1530/endoabs.56.P1051


2018 - Two hormones for one receptor: evolution, biochemistry, actions and pathophysiology of LH and hCG [Articolo su rivista]
Casarini, Livio; Santi, Daniele; Brigante, Giulia; Simoni, Manuela
abstract

Luteinizing hormone (LH) and chorionic gonadotropin (CG) are glycoproteins fundamental for sexual development and reproduction. Since they act on the same receptor (LHCGR), there is a general consensus that LH and hCG are equivalent. However, separate evolution of LHβ and hCGβ subunits occurred in primates, resulting in two molecules sharing ∼85% identity and regulating different physiological events. Pituitary, pulsatile LH production results in a ∼90 min half-life molecule targeting the gonads, to regulate gametogenesis and androgen synthesis. Trophoblast hCG, the "pregnancy hormone", exists in several isoforms and glycosylation variants with long half-lives (hours), angiogenic potential, and acts on luteinized ovarian cells as a progestational. The different molecular features of LH and hCG lead to hormone-specific LHCGR binding and intracellular signaling cascades. In ovarian cells, LH action is preferentially exerted through kinases, pERK1/2 and pAKT, resulting in irreplaceable proliferative/anti-apoptotic signals and partial agonism on progesterone production in vitro. In contrast, hCG displays notable cAMP/PKA-mediated steroidogenic and pro-apoptotic potential, which is masked by estrogen action in vivo. In vitro data are confirmed by large dataset from assisted reproduction, since the steroidogenic potential of hCG positively impacts on the number of retrieved oocytes, while LH impacts pregnancy rate (per oocyte number). Interestingly, Leydig cell in vitro exposure to hCG results in qualitatively similar cAMP/PKA and pERK1/2 activation as compared to LH, as well as testosterone. The supposed equivalence of LH and hCG is debunked by such data highlighting their sex-specific functions, thus deeming it an oversight caused by incomplete understanding of clinical data.


2018 - ‘Easier ways to get a publication’: the problem of low quality scientific publications [Articolo su rivista]
Carrell, D. T.; Simoni, M.
abstract

N/A


2017 - Andrology Award 2016 [Articolo su rivista]
Carrell, D. T.; Simoni, M.
abstract

N/A


2017 - Big clinical problem, big advancement, big questions to still address: the status of microTESE [Capitolo/Saggio]
Carrell, D. T.; Simoni, M.
abstract


2017 - Calcium to phosphorous ratio (Ca/P) as helpful index to recognize primary hyperparathyroidism, but not primary hypoparathyroidism: a big-data approach. [Abstract in Rivista]
De Vincentis, Sara; Santi, Daniele; Rochira, Vincenzo; Setti, M.; Tagliavini, S.; Varani, M.; Trenti, T.; Simoni, Manuela; Madeo, Bruno
abstract

Background Primary hyperparathyroidism (HyperPT) and primary hypoparathyroidism (HypoPT) are often underdiagnosed. Several strategies have been investigated in the past in order to identify diagnostic parameters, although the diagnosis of both HyperPT and HypoPT remains challenging so far, especially in asymptomatic patients. Calcium (Ca) and phosphorus (P) are inversely related together, thus the Ca/P ratio could be an useful tool to define these conditions. Recently, we proposed for the first time a cut-off of 3.5 for Ca/P ratio for the diagnosis of HyperPT. Aim To evaluate the diagnostic value of the Ca/P ratio for HyperPT and HypoPT through a big-data approach. Methodology A retrospective, observational, case-control study on big-data was carried out. All examinations of parathyroid hormone (PTH), Ca and P performed at the laboratory of Modena Hospital from 2010 to 2016 were consecutively included. We considered only patients between 18 and 90 years of age. Laboratory ranges of normality for both PTH and Ca were used to divide records in HyperPT, HypoPT and controls. Statistical analysis The diagnostic accuracy of Ca/P ratio was investigated using receiver operator characteristics (ROC) curves in order to define cut-off points, which show higher sensitivity and specificity for the identification of affected patients. Results 46 597 records were considered. 576 HyperPT (1.2%), 323 HypoPT (0.7%) and 45 698 controls (98.1%) were found. Ca/P ratio was significantly different among groups (P!0.001). In particular, Ca/P ratio was significantly higher in HyperPT than controls (P!0.001). For the diagnosis of HyperPT, the threshold of 3.17 for Ca/P ratio was obtained by means of the ROC curve analysis, with 85% of both sensitivity and specificity. HypoPT showed lower Ca/P ratio compared to controls (P!0.001), although no useful threshold for the diagnosis was found at ROC curve because of the low sensitivity. Conclusions We confirm the high sensitivity and specificity of Ca/P ratio for the diagnosis of HyperPT using the largest cohort of patients available so far in the literature. On the contrary, Ca/P ratio does not contribute to identify patients with HypoPT and further researches are needed to better describe this condition. In conclusion, Ca/P ratio is a simple and inexpensive diagnostic tool to recognize HyperPT.


2017 - Calcium to phosphorous ratio (Ca/P) as helpful index to recognize primary hyperparathyroidism, but not primary hypoparathyroidism: a big-data approach. [Abstract in Atti di Convegno]
Santi, Daniele; De Vincentis, Sara; Rochira, Vincenzo; Setti, M.; Tagliavini, S.; Varani, M.; Trenti, T.; Simoni, Manuela; Madeo, Bruno
abstract

BACKGROUND: Primary hyperparathyroidism (HyperPT) and primary hypoparathyroidism (HypoPT) are often underdiagnosed. Several strategies have been investigated in the past in order to identify diagnostic parameters, although the diagnosis of both HyperPT and HypoPT remains challenging so far, especially in asymptomatic patients. Calcium (Ca) and phosphorus (P) are inversely related together, thus the Ca/P ratio could be an useful tool to define these conditions. Recently, we proposed for the first time a cut-off of 3.5 for Ca/P ratio for the diagnosis of HyperPT. AIM: to evaluate the diagnostic value of the Ca/P ratio for HyperPT and HypoPT through a big-data approach. METHODOLOGY: a retrospective, observational, case-control study on big-data was carried out. All examinations of parathyroid hormone (PTH), Ca and P performed at the laboratory of Modena Hospital from 2010 to 2016 were consecutively included. We considered only patients between 18 and 90 years of age. Laboratory ranges of normality for both PTH and Ca were used to divide records in HyperPT, HypoPT and controls. Statistical analysis: The diagnostic accuracy of Ca/P ratio was investigated using receiver operator characteristics (ROC) curves in order to define cut-off points, which show higher sensitivity and specificity for the identification of affected patients. RESULTS: 46597 records were considered. 576 HyperPT (1.2%), 323 HypoPT (0.7%) and 45698 controls (98.1%) were found. Ca/P ratio was significantly different among groups (p<0.001). In particular, Ca/P ratio was significantly higher in HyperPT than controls (p<0.001). For the diagnosis of HyperPT, the threshold of 3.17 for Ca/P ratio was obtained by means of the ROC curve analysis, with 85% of both sensitivity and specificity. HypoPT showed lower Ca/P ratio compared to controls (p<0.001), although no useful threshold for the diagnosis was found at ROC curve because of the low sensitivity. CONCLUSIONS: We confirm the high sensitivity and specificity of Ca/P ratio for the diagnosis of HyperPT using the largest cohort of patients available so far in the literature. On the contrary, Ca/P ratio does not contribute to identify patients with HypoPT and further researches are needed to better describe this condition. In conclusion, Ca/P ratio is a simple and inexpensive diagnostic tool to recognize HyperPT.


2017 - Central hypogonadism due to a giant, “silent” FSH-secreting, atypical pituitary adenoma: effects of adenoma dissection and short-term Leydig cell stimulation by luteinizing hormone (LH) and human chorionic gonadotropin (hCG) [Articolo su rivista]
Santi, Daniele; Spaggiari, Giorgia; Casarini, Livio; Fanelli, Flaminia; Mezzullo, Marco; Pagotto, Uberto; Granata, Antonio R. M; Carani, Cesare; Simoni, Manuela
abstract

We present a case report of an atypical giant pituitary adenoma secreting follicle-stimulating hormone (FSH). A 55-year-old patient presented for erectile dysfunction, loss of libido and fatigue. The biochemical evaluation showed very high FSH serum levels in the presence of central hypogonadism. Neither testicular enlargement nor increased sperm count was observed, thus a secretion of FSH with reduced biological activity was supposed. The histological examination after neuro-surgery showed an atypical pituitary adenoma with FSH-positive cells. Hypogonadism persisted and semen analyses impaired until azoospermia in conjunction with the reduction in FSH levels suggesting that, at least in part, this gonadotropin should be biologically active. Thus, we hypothesized a concomitant primary testicular insufficiency. The patient underwent short-term treatment trials with low doses of either recombinant luteinizing hormone (LH) or human chorionic gonadotropin (hCG) in three consecutive treatment schemes, showing an equal efficacy in stimulating testosterone (T) increase. This is the first case of atypical, giant FSH-secreting pituitary adenoma with high FSH serum levels without signs of testicular hyperstimulation, in presence of hypogonadism with plausible combined primary and secondary etiology. Hypophysectomized patients may represent a good model to assess both pharmacodynamics and effective dose of LH and hCG in the male.


2017 - Diagnosi precoce nella Sindrome di Klinefelter [Abstract in Rivista]
Fisher, A. D.; Corona, G.; Rochira, V.; Maggi, M.; Simoni, M.; Santi, D.
abstract

The diagnosis of Klinefelter Syndrome is needed to guarantee adequate management, treatment and follow-up of the disease and to prevent the occurrence of associated comorbidities. The early diagnosis has the advantage to plan a continuous monitoring of the patients through periodic visits of follow-up. This allows to identify on time changes of the clinical pattern and to tailor treatment and counselling.


2017 - Effects of chronic administration of the phosphodiesterase inhibitor vardenafil on serum levels of adrenal and testicular steroids in men with type 2 diabetes mellitus [Articolo su rivista]
Santi, Daniele; Granata, A. R.; Pignatti, Elisa; Trenti, T.; Roli, L.; Bozic, R.; Zaza, S.; Pacchioni, C.; Rochira, Vincenzo; Carani, Cesare; Simoni, Manuela
abstract

To investigate whether long-term, chronic treatment with the phosphodiesterase-5 inhibitor vardenafil affects adrenal and testicular steroidogenesis in diabetic men, using liquid chromatography-tandem mass spectrometry. A longitudinal, prospective, investigator-started, randomized, placebo-controlled, double-blind, clinical-trial was carried out, enrolling 54 male patients affected by type 2 diabetes mellitus diagnosed within the last 5 years. In total, 26 and 28 patients were followed for 1 year and assigned to the study and placebo group, respectively. Progesterone, 17-hydroxyprogesterone, androstenedione, testosterone, dehydroepiandrosterone, dehydroepiandrosterone sulfate, corticosterone, 11-deoxycortisol and cortisol, were evaluated using liquid chromatography-tandem mass spectrometry. No differences were seen in sex testicular steroids between study and control group. As for the adrenal gland, steroids were considered according to the zona in which they are produced. No significant differences were seen in steroid produced in zona fasciculata. For the zona reticularis, dehydroepiandrosterone significantly decreased during treatment only in the study group (p = 0.007), with higher levels at visit 2 and 8 than other visits. The dehydroepiandrosterone sulfate/dehydroepiandrosterone ratio significantly increased during treatment only in the verum group. Considering the adrenal zona glomerulosa, corticosterone significantly changed among visits both in both groups (p < 0.001), with higher levels at visit 2 (p = 0.028), 8 (p = 0.003), and 10 (p = 0.044), i.e., in coincidence with the complete clinical and instrumental examination performed only at these visits according to the study protocol. Chronically administered vardenafil reduces dehydroepiandrosterone levels and increases dehydroepiandrosterone sulfate/dehydroepiandrosterone ratio as possible consequences of modulation of steroidogenic enzymes by tissue changes in cyclic adenosine monophosphate and cyclic guanosine monophosphate availability. A possibly stress-related increase in corticosterone is suggested for the first time.


2017 - Effects of probiotics assumption on serum thyroid hormone and TSH levels in hypothyroid patients on levothyroxine treatment. [Abstract in Atti di Convegno]
Spaggiari, Giorgia; Brigante, Giulia; De Vincentis, Sara; Cattini, Umberto; Roli, L.; De Santis, M. C.; Baraldi, Enrica; Tagliavini, S.; Varani, M.; Trenti, T.; Rochira, Vincenzo; Simoni, Manuela; Santi, Daniele
abstract

Background. Probiotics are live microorganisms able to confer a health benefit to the host, when administered in adequate amounts. Despite the widespread use of probiotics, their pharmacological interference remains unclear. The relationship between probiotics and levothyroxine (LT4) requirement has not yet been investigated. Objective. To assess whether a mixture of highly charged Lactobacilli and Bifidobacteria (VSL#3®) is able to influence LT4 metabolism acting on the gut microbiota. Methods. A prospective, randomized, single-blind, controlled, investigator-started clinical trial was carried out. Patients with primary hypothyroidism were randomly assigned to the study (VSL#3®+ LT4) and the control group (LT4). A two months treatment phase was followed by two months of follow-up. Clinical examination, blood tests for thyroid function and for peripheral tissue markers of thyroid hormones effect were performed monthly for 4 months. LT4 dose adjustments were performed during the study when necessary. Results. Thirty-nine patients were enrolled in the study group and 41 in the control group. No difference in thyroid function (thyroid-stimulating hormone (TSH), free triiodothyronine (fT3) and free thyroxine (fT4)) and peripheral tissue markers was found between groups and among visits. FT3/fT4 ratio was directly correlated to TSH at each visit in the control and in the study group, with the exception of the first evaluation of subjects treated with probiotics. LT4 daily dose adjustments occurred 10 times in 8 patients, more frequently in the control than in the study group, despite no differences in the mean LT4 daily dose. Conclusions. VSL#3® does not directly alter thyroid functional compensation. A probiotics- mediated influence on thyroid hormones homeostasis is suggested since probiotics supplementation could be able to prevent serum hormonal fluctuations.


2017 - Efficacy of FSH alone, FSH + LH, hMG or FSH + hCG on ART outcomes in the 'personalized' medicine era: a meta-analysis [Abstract in Rivista]
Santi, Daniele; Casarini, Livio; Alviggi, Carlo; Simoni, Manuela
abstract

Background: Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) act on the same receptor, activating different signal transduction pathways. The role of LH or hCG addition to follicle stimulating hormone (FSH) as well as menopausal gonadotropins (hMG) in controlled ovarian stimulation (COS) is debated. Aim: To compare FSH+LH, or FSH+hCG or hMG vs FSH alone on COS outcomes. Design: A meta-analysis according to PRISMA statement and Cochrane Collaboration was performed, including prospective, controlled clinical trials published until July 2016, enrolling women treated with FSH combined with other gonadotropins. Trials enrolling women with polycystic ovarian syndrome were excluded. Results: Considering 70 studies, the administration of FSH alone resulted in higher number of oocytes retrieved than FSH+LH or hMG. The MII oocytes number did not change when FSH alone was compared to FSH+LH, FSH+hCG or hMG. Embryo number and implantation rate were higher when hMG was used instead of FSH alone. Pregnancy rate was significantly higher in FSH+LH-treated group versus others. Only twelve studies reported live birth rate, not providing protocol-dependent differences. Patients’ stratification by age (median=32.5 years) and/or by GnRH agonist/antagonist identified patient subgroups benefiting from specific drug combinations. Conclusion: In COS, FSH alone results in higher oocyte number. However, hMG improves the collection of mature oocytes and embryos and increases implantation rate, although the final increased pregnancy rate is evident only in GnRH agonist protocol. On the other hand, LH addition leads to higher pregnancy rate. This study supports the concept of a different clinical action of gonadotropins in COS, reflecting previous in vitro data.


2017 - Efficacy of follicle-stimulating hormone (FSH) alone, FSH + luteinizing hormone, human menopausal gonadotropin or FSH + human chorionic gonadotropin on assisted reproductive technology outcomes in the "personalized" medicine era: A meta-analysis [Articolo su rivista]
Santi, Daniele; Casarini, Livio; Alviggi, Carlo; Simoni, Manuela
abstract

Setting: Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) act on the same receptor, activating different signal transduction pathways. The role of LH or hCG addition to follicle-stimulating hormone (FSH) as well as menopausal gonadotropins (human menopausal gonadotropin; hMG) in controlled ovarian stimulation (COS) is debated. Objective: To compare FSH + LH, or FSH + hCG or hMG vs. FSH alone on COS outcomes. Design: A meta-analysis according to PRISMA statement and Cochrane Collaboration was performed, including prospective, controlled clinical trials published until July 2016, enrolling women treated with FSH alone or combined with other gonadotropins. Trials enrolling women with polycystic ovarian syndrome were excluded (PROSPERO registration no. CRD42016048404). Results: Considering 70 studies, the administration of FSH alone resulted in higher number of oocytes retrieved than FSH + LH or hMG. The MII oocytes number did not change when FSH alone was compared to FSH + LH, FSH + hCG, or hMG. Embryo number and implantation rate were higher when hMG was used instead of FSH alone. Pregnancy rate was significantly higher in FSH + LH-treated group vs. others. Only 12 studies reported live birth rate, not providing protocol-dependent differences. Patients' stratification by GnRH agonist/antagonist identified patient subgroups benefiting from specific drug combinations. Conclusion: In COS, FSH alone results in higher oocyte number. HMG improves the collection of mature oocytes, embryos, and increases implantation rate. On the other hand, LH addition leads to higher pregnancy rate. This study supports the concept of a different clinical action of gonadotropins in COS, reflecting previous in vitro data.


2017 - Environmental temperature and particulate matter are correlated with semen parameters: a big-data approach [Abstract in Rivista]
Santi, Daniele; Magnani, E; Granata, Ar; Roli, L; De Santis, Mc; Baraldi, E; Trenti, T; Setti, M; Simoni, M
abstract

Environmental temperature and particulate matter are correlated with semen parameters: a big-data approach


2017 - Estrogen Modulates Specific Life and Death Signals Induced by LH and hCG in Human Primary Granulosa Cells In Vitro [Articolo su rivista]
Casarini, Livio; Riccetti, Laura; DE PASCALI, Francesco; Gilioli, Lisa; Marino, Marco; Vecchi, Eugenia; Morini, Daria; Nicoli, Alessia; LA SALA, Giovanni Battista; Simoni, Manuela
abstract

Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) are glycoprotein hormones used for assisted reproduction acting on the same receptor (LHCGR) and mediating different intracellular signaling. We evaluated the pro- and anti-apoptotic effect of 100 pM LH or hCG, in the presence or in the absence of 200 pg/mL 17β-estradiol, in long-term, serum-starved human primary granulosa cells (hGLC) and a transfected granulosa cell line overexpressing LHCGR (hGL5/LHCGR). To this purpose, phospho-extracellular-regulated kinase 1/2 (pERK1/2), protein kinase B (pAKT), cAMP-responsive element binding protein (pCREB) activation and procaspase 3 cleavage were evaluated over three days by Western blotting, along with the expression of target genes by real-time PCR and cell viability by colorimetric assay. We found that LH induced predominant pERK1/2 and pAKT activation STARD1, CCND2 and anti-apoptotic XIAP gene expression, while hCG mediated more potent CREB phosphorylation, expression of CYP19A1 and procaspase 3 cleavage than LH. Cell treatment by LH is accompanied by increased (serum-starved) cell viability, while hCG decreased the number of viable cells. The hCG-specific, pro-apoptotic effect was blocked by a physiological dose of 17β-estradiol, resulting in pAKT activation, lack of procaspase 3 cleavage and increased cell viability. These results confirm that relatively high levels of steroidogenic pathway activation are linked to pro-apoptotic signals in vitro, which may be counteracted by other factors, i.e., estrogens.


2017 - Genetics of gonadotropins and their receptors as markers of ovarian reserve and response in controlled ovarian stimulation [Articolo su rivista]
Riccetti, Laura; Pascali, Francesco De; Gilioli, Lisa; Santi, Daniele; Brigante, Giulia; Simoni, Manuela; Casarini, Livio
abstract

Several controlled ovarian stimulation (COS) protocols have been developed to increase the yield of mature oocytes retrieved in assisted reproductive techniques (ARTs). The ovarian reserve (OR) influences the COS response, and it represents the main parameter that helps clinicians in refining clinical treatments in the perspective of a "personalized" ART. This approach is even more needed in particular conditions such as poor OR or polycystic ovary syndrome. Follicle-stimulating hormone, luteinizing hormone, and human chorionic gonadotropin are currently used in COS at different combinations and with different efficacies, even if the best approach definition is controversial. Differences in individual-specific ovarian response to gonadotropin stimulation can be due to alterations of genes encoding for hormones or their receptors. In particular, FSHB c.-211G>T, FSHR p.Asn680Ser, and c.-29G>A SNP allelic combinations may be used as OR and COS response markers. The purpose of this review is to highlight the evidence-based relevance of mutations and polymorphisms in gonadotropins and their receptor genes as predictive markers of OR and COS response to achieve fine-tuned therapeutic regimens.


2017 - Gonadotropins beyond ART [Articolo su rivista]
De Vincentis, Sara; Casarini, Livio; Simoni, Manuela; Brigante, Giulia
abstract

Gonadotropins (LH, FSH and hCG) play a central role in controlling steroidogenesis and gametogenesis. For this reason, they are largely used in the treatment of infertility, especially in the setting of assisted reproductive technique. Beyond their important action in the regulation of reproduction, gona dotropins are also involved in other hormonal processes, closely interacting with other endocrine axes. Among them, the interaction between gonadal and thyroid axes is widely studied in the literature. There is evidence of an undeniable structural similarity of both hormones and receptors, maybe due to a common ancient origin. Indeed, altered levels of thyroid hormones could lead to different disorders of gonadal development and function throughout entire life, especially before and during pregnancy. Moreover, a complex interplay between insulin-like growth factors and gonadotropins has been described both at central and peripheral level. Finally, several tumors are able to produce gonadotropins or are regulated by them in their own growth. The role of gonadotropins in the regulation of cellular growth and apoptosis is evident by now, but still not fully understood.


2017 - Heterogeneous hCG and hMG commercial preparations result in different intracellular signalling but induce a similar long-term progesterone response in vitro [Articolo su rivista]
Riccetti, Laura; Klett, Danièle; Ayoub, Mohammed Akli; Boulo, Thomas; Pignatti, Elisa; Tagliavini, Simonetta; Varani, Manuela; Trenti, Tommaso; Nicoli, Alessia; Capodanno, Francesco; La Sala, Giovanni Battista; Reiter, Eric; Simoni, Manuela; Casarini, Livio
abstract

STUDY QUESTION: Are four urinary hCG/menotropin (hMG) and one recombinant preparation characterized by different molecular features and do they mediate specific intracellular signaling and steroidogenesis?SUMMARY ANSWER: hCG and hMG preparations have heterogeneous compositions and mediate preparation-specific cell signaling and early steroidogenesis, although similar progesterone plateau levels are achieved in 24 h-treated human primary granulosa cells in vitro.WHAT IS KNOWN ALREADY: hCG is the pregnancy hormone marketed as a drug for ARTs to induce final oocyte maturation and ovulation, and to support FSH action. Several hCG formulations are commercially available, differing in source, purification methods and biochemical composition.STUDY DESIGN, SIZE, DURATION: Commercial hCG preparations for ART or research purposes were compared in vitro.PARTICIPANTS/MATERIALS, SETTING, METHODS: The different preparations were quantified by immunoassay with calibration against the hCG standard (Fifth IS; NIBSC 07/364). Immunoreactivity patterns, isoelectric points and oligosaccharide contents of hCGs were evaluated using reducing and non-reducing Western blotting, capillary isoelectric-focusing immunoassay and lectin-ELISA, respectively. Functional studies were performed in order to evaluate intracellular and total cAMP, progesterone production and beta-arrestin 2 recruitment by ELISA and BRET, in both human primary granulosa lutein cells (hGLC) and luteinizing hormone (LH)/hCG receptor (LHCGR)-transfected HEK293 cells, stimulated by increasing hormone concentrations. Statistical analysis was performed using two-way ANOVA and Bonferroni post-test or Mann-Whitney's U-test as appropriate.MAIN RESULTS AND THE ROLE OF CHANCE: Heterogeneous profiles were found among preparations, revealing specific molecular weight patterns (20-75 KDa range), isoelectric points (4.0-9.0 pI range) and lectin binding (P &lt; 0.05; n = 7-10). These drug-specific compositions were linked to different potencies on cAMP production (EC50 1.0-400.0 ng/ml range) and beta-arrestin 2 recruitment (EC50 0.03-2.0 mu g/ml) in hGLC and transfected HEK293 cells (P &lt; 0.05; n = 3-5). In hGLC, these differences were reflected by preparation-specific 8-h progesterone production although similar plateau levels of progesterone were acheived by 24-h treatment (P &gt;= 0.05; n = 3).LARGE SCALE DATA: N/A.LIMITATIONS, REASONS FOR CAUTION: The biological activity of commercial hCG/hMG preparations is provided in International Units (IU) by in-vivo bioassay and calibration against an International Standard, although it is an unsuitable unit of measure for in-vitro studies. The re-calibration against recombinant hCG, quantified in grams, is based on the assumption that all of the isoforms and glycosylation variants have similar immunoreactivity.WIDER IMPLICATIONS OF THE FINDINGS: hCG/hMG preparation-specific cell responses in vitro may be proposed to ART patients affected by peculiar ovarian response, such as that caused by polycystic ovary syndrome. Otherwise, all the preparations available for ART may provide a similar clinical outcome in healthy women.


2017 - Human LH and hCG stimulate differently the early signalling pathways but result in equal testosterone synthesis in mouse Leydig cells in vitro [Articolo su rivista]
Riccetti, Laura; De Pascali, Francesco; Gilioli, Lisa; Potì, Francesco; Giva, LAVINIA BEATRICE; Marino, Marco; Tagliavini, Simonetta; Trenti, Tommaso; Fanelli, Flaminia; Mezzullo, Marco; Pagotto, Uberto; Simoni, Manuela; Casarini, Livio
abstract

BACKGROUND: Human luteinizing hormone (LH) and chorionic gonadotropin (hCG) are glycoprotein hormones regulating development and reproductive functions by acting on the same receptor (LHCGR). We compared the LH and hCG activity in gonadal cells from male mouse in vitro, i.e. primary Leydig cells, which is a common tool used for gonadotropin bioassay. Murine Leydig cells are naturally expressing the murine LH receptor (mLhr), which binds human LH/hCG. METHODS: Cultured Leydig cells were treated by increasing doses of recombinant LH and hCG, and cell signaling, gene expression and steroid synthesis were evaluated. RESULTS: We found that hCG is about 10-fold more potent than LH in cAMP recruitment, and slightly but significantly more potent on cAMP-dependent Erk1/2 phosphorylation. However, no significant differences occur between LH and hCG treatments, measured as activation of downstream signals, such as Creb phosphorylation, Stard1 gene expression and testosterone synthesis. CONCLUSIONS: These data demonstrate that the responses to human LH/hCG are only quantitatively and not qualitatively different in murine cells, at least in terms of cAMP and Erk1/2 activation, and equal in activating downstream steroidogenic events. This is at odds with what we previously described in human primary granulosa cells, where LHCGR mediates a different pattern of signaling cascades, depending on the natural ligand. This finding is relevant for gonadotropin quantification used in the official pharmacopoeia, which are based on murine, in vivo bioassay and rely on the evaluation of long-term, testosterone-dependent effects mediated by rodent receptor.


2017 - Human Luteinizing Hormone and Chorionic Gonadotropin Display Biased Agonism at the LH and LH/CG Receptors. [Articolo su rivista]
Riccetti, Laura; Romain, Yvinec; Danièle, Klett; Nathalie, Gallay; Yves, Combarnous; Eric, Reiter; Simoni, Manuela; Casarini, Livio; Mohammed, Akli Ayoub
abstract

Human luteinizing hormone (LH) and chorionic gonadotropin (hCG) have been considered biologically equivalent because of their structural similarities and their binding to the same receptor; the LH/CGR. However, accumulating evidence suggest that LH/CGR differentially responds to the two hormones triggering differential intracellular signaling and steroidogenesis. The mechanistic basis of such differential responses remains mostly unknown. Here, we compared the abilities of recombinant rhLH and rhCG to elicit cAMP, β-arrestin 2 activation, and steroidogenesis in HEK293 cells and mouse Leydig tumor cells (mLTC-1). For this, BRET and FRET technologies were used allowing quantitative analyses of hormone activities in real-time and in living cells. Our data indicate that rhLH and rhCG differentially promote cell responses mediated by LH/CGR revealing interesting divergences in their potencies, efficacies and kinetics: rhCG was more potent than rhLH in both HEK293 and mLTC-1 cells. Interestingly, partial effects of rhLH were found on β-arrestin recruitment and on progesterone production compared to rhCG. Such a link was further supported by knockdown experiments. These pharmacological differences demonstrate that rhLH and rhCG act as natural biased agonists. The discovery of novel mechanisms associated with gonadotropin-specific action may ultimately help improve and personalize assisted reproduction technologies.


2017 - Klinefelter syndrome (KS): genetics, clinical phenotype and hypogonadism [Articolo su rivista]
Bonomi, M.; Rochira, V.; Pasquali, D.; Balercia, G.; Jannini, E. A.; Ferlin, A.; Calogero, A.; Corona, G.; Fabbri, A.; Francavilla, F.; Giagulli, V.; Lanfranco, F.; Maggi, M.; Pivonello, R.; Pizzocaro, A.; Radicioni, A.; Accardo, L.; Cangiano, B.; Condorelli, R. A.; Cordeschi, G.; D'Andrea, S.; Mambro, A. D.; Esposito, D.; Foresta, C.; Francavilla, S.; Galdiero, M.; Garolla, A.; Giovannini, L.; Balercia, A. R. M.; La Vignera, S.; Motta, G.; Luciano, L.; Pelliccione, F.; Persani, L.; Santi, D.; Selice, R.; Simoni, M.; Tatone, C.; Tirabassi, G.; Tresoldi, A. S.; Vicari, E.
abstract

Klinefelter Syndrome (KS) is characterized by an extreme heterogeneity in its clinical and genetic presentation. The relationship between clinical phenotype and genetic background has been partially disclosed; nevertheless, physicians are aware that several aspects concerning this issue are far to be fully understood. By improving our knowledge on the role of some genetic aspects as well as on the KS, patients' interindividual differences in terms of health status will result in a better management of this chromosomal disease. The aim of this review is to provide an update on both genetic and clinical phenotype and their interrelationships.


2017 - Klinefelter syndrome: cardiovascular abnormalities and metabolic disorders [Articolo su rivista]
Calogero, A. E.; Giagulli, V. A.; Mongioi, L. M.; Triggiani, V.; Radicioni, A. F.; Jannini, E. A.; Pasquali, D.; Balercia, G.; Bonomi, M.; Corona, G.; Fabbri, A.; Ferlin, A.; Francavilla, F.; Giagulli, V.; Lanfranco, F.; Maggi, M.; Pivonello, R.; Pizzocaro, A.; Radicioni, A.; Rochira, V.; Vignozzi, L.; Accardo, G.; Cangiano, B.; Condorelli, R. A.; Cordeschi, G.; D'Andrea, S.; Di Mambro, A.; Esposito, D.; Foresta, C.; Francavilla, S.; Galdiero, M.; Garolla, A.; Giovannini, L.; Granata, A. R. M.; La Vignera, S.; Motta, G.; Negri, L.; Pelliccione, F.; Persani, L.; Salzano, C.; Santi, D.; Selice, R.; Simoni, M.; Tatone, C.; Tirabassi, G.; Tresoldi, A. S.; Vicari, E.
abstract

Klinefelter syndrome (KS) is one of the most common genetic causes of male infertility. This condition is associated with much comorbidity and with a lower life expectancy. The aim of this review is to explore more in depth cardiovascular and metabolic disorders associated to KS. KS patients have an increased risk of cerebrovascular disease (standardized mortality ratio, SMR, 2.2; 95% confidence interval, CI, 1.6–3.0), but it is not clear whether the cause of the death is of thrombotic or hemorrhagic nature. Cardiovascular congenital anomalies (SMR, 7.3; 95% CI, 2.4–17.1) and the development of thrombosis or leg ulcers (SMR, 7.9; 95% CI, 2.9–17.2) are also more frequent in these subjects. Moreover, cardiovascular abnormalities may be at least partially reversed by testosterone replacement therapy (TRT). KS patients have also an increased probability of endocrine and/or metabolic disease, especially obesity, metabolic syndrome and type 2 diabetes mellitus. The effects of TRT on these abnormalities are not entirely clear.


2017 - NEUROACTIVE STEROID LEVELS AND PSYCHIATRIC AND ANDROLOGICAL FEATURES IN POST-FINASTERIDE PATIENTS [Articolo su rivista]
Melcangi, Roberto Cosimo; Santi, Daniele; Spezzano, Roberto; Grimoldi, Maria; Tabacchi, Tommaso; Fusco, Maria Letizia; Diviccaro, Silvia; Giatti, Silvia; Carrà, Giuseppe; Caruso, Donatella; Simoni, Manuela; Cavaletti, Guido
abstract

Recent reports show that, in patients treated with finasteride for male pattern hair loss, persistent side effects including sexual side effects, depression, anxiety and cognitive complaints may occur. We here explored the psychiatric and andrological features of patients affected by post-finasteride syndrome (PFS) and verified whether the cerebrospinal fluid (CSF) and plasma levels of neuroactive steroids (i.e., important regulators of nervous function) are modified. We found that eight out of sixteen PFS male patients considered suffered from a DSM-IV major depressive disorder (MDD). In addition, all PFS patients showed erectile dysfunction (ED); in particular, ten patients showed a severe and six a mild-moderate ED. We also reported abnormal somatosensory evoked potentials of the pudendal nerve in PFS patients with severe ED, the first objective evidence of a neuropathy involving peripheral neurogenic control of erection. Testicular volume by ultrasonography was normal in PFS patients. Data obtained on neuroactive steroid levels also indicate interesting features. Indeed, decreased levels of pregnenolone, progesterone and its metabolite (i.e., dihydroprogesterone), dihydrotestosterone and 17beta-estradiol and increased levels of dehydroepiandrosterone, testosterone and 5alpha-androstane-3alpha,17beta-diol were observed in CSF of PFS patients. Neuroactive steroid levels were also altered in plasma of PFS patients, however these changes did not reflect exactly what occurs in CSF. Finally, finasteride did not only affect, as expected, the levels of 5alpha-reduced metabolites of progesterone and testosterone, but also the further metabolites and precursors suggesting that this drug has broad consequence on neuroactive steroid levels of PFS patients.


2017 - Primary Leydig cells naturally expressing mouse LHR do not discriminate between LH- and hCGmediated signaling in vitro [Articolo su rivista]
Riccetti, Laura; Gilioli, Lisa; Brigante, Giulia; Simoni, Manuela; Casarini, Livio
abstract

Human luteinizing hormone (LH) and chorionic gonadotropin (hCG) are glycoprotein hormones fundamental for development and reproduction. These hormones were considered biologically equivalent for decades due to structural similarities and binding to the same receptor (LHCGR), although they mediate different physiological roles. Previous reports demonstrated LH- and hCGspecific intracellular signaling mediated by LHCGR in human primary granulosa cells, but few studies using rodent receptor (Lhr) are available. We investigated the Lhr-mediated activation of the cAMP/PKA-pathway, ERK1/2 and CREB phosphorylation, gene expression and steroidogenesis, in murine Leydig cells treated with LH and hCG. We found that hCG is more potent than LH in inducing cAMP production, as well as downstream the pERK1/2 activation. However, similar levels of CREB phosphorylation, Stard1 gene expression and testosterone production occurred upon LH and hCG treatment in vitro. These findings revealed that rodent Lhr mediates quantitatively, but not qualitatively, different LH- and hCG-dependent signaling, which results in similar testosterone synthesis. These data suggest that in vivo bioassay using a model expressing rodent receptor, which rely on the evaluation of testosterone-dependent endpoints, may be not suitable to quantify gonadotropins activity for clinical purpose.


2017 - Probiotics ingestion does not directly affect thyroid hormonal parameters in hypothyroid patients on levothyroxine treatment [Articolo su rivista]
Spaggiari, Giorgia; Brigante, Giulia; Vincentis, Sara De; Cattini, Umberto; Roli, Laura; De Santis, Maria Cristina; Baraldi, Enrica; Tagliavini, Simonetta; Varani, Manuela; Trenti, Tommaso; Rochira, Vincenzo; Simoni, Manuela; Santi, Daniele
abstract

Purpose: The relationship between probiotics and levothyroxine (LT4) requirement has not yet been investigated. The aim of this study was to assess whether a mixture of highly charged Lactobacilli and Bifidobacteria (VSL#3®) is able to influence LT4metabolism acting on the gut microbiota. Methods: A prospective, randomized, single-blind, controlled, investigator-started clinical trial was carried out. Patients with primary hypothyroidism were randomly assigned to the study (VSL#3® + LT4) and the control group (LT4). A 2-month treatment phase was followed by 2 months of follow-up. Clinical examination, blood tests for thyroid function and for peripheral tissue markers of thyroid hormones (PTM) were performed monthly. LT4dose adjustments were performed when necessary. Results: Thirty-nine patients were enrolled in the study group and 41 in the control group. No difference in thyroid function [thyroid-stimulating hormone (TSH), free triiodothyronine (fT3), and free thyroxine (fT4)] and PTM was found between groups and among visits. FT3/fT4ratio was directly correlated to TSH at each visit in both groups, with the exception of the first evaluation of probiotics-treated subjects (rho = 0.287, p = 0.076). LT4daily dose adjustments occurred more frequently in the control than in the study group (p = 0.007), despite no differences in the mean LT4daily dose. In particular, LT4doses were increased six times in the control group and decreased four times in the study group. Conclusion: VSL#3® does not directly alter thyroid functional compensation. A probiotics-mediated influence on thyroid hormones homeostasis is suggested since probiotics supplementation could be able to prevent serum hormonal fluctuations.


2017 - Serum calcium to phosphorous ratio (Ca/P) as a simple, inexpensive screening tool in the diagnosis of primary hyperparathyroidism (PHPT). [Abstract in Atti di Convegno]
Madeo, Bruno; Kara, Elda; Cioni, K.; Vezzani, Silvia; Simoni, Manuela; Rochira, Vincenzo
abstract

Background The diagnosis of primary hyperparathyroidism (PHPT) is challenging and is based on serum calcium (Ca) and parathyroid hormone (PTH). As serum Ca and phosphorous (P) are inversely related in PHPT, the Ca/P ratio might be considered a good candidate tool in the diagnosis of PHPT. The aim of this study is to investigate the diagnostic value of the Ca/P ratio in the diagnosis of PHPT. Methods For this single-centre, case-controlled, retrospective study we collected data from review charts of 97 patients with documented PHPT (69 females; 28 males) and compared them with those of 96 controls (C) (44 females; 52 males). The retrospective nature of the study allows obtaining for certain the diagnosis of PHPT. Main outcome measures were: serum PTH, 25-OH Vitamin D, Ca, P, albumin, and creatinine. Results Among PHPT patients, 35 (36.1%) had normocalcemic PHPT (NCHPT). Ca was significantly higher in PHPT (median: 11; min-max: 9.4-15.5) than C (9.4; 8.3-10.2) (p<0.0001). P was significantly lower in PHPT (2.4; 1.4-3.9) than in C (3.5; 2.1-4.5) (p<0.0001). PTH was significantly higher in PHPT (135.2; 57.6-1748) than in C (32.1; 14-80.7) (p<0.0001). Ca/P ratio was significantly higher in PHPT (4.6; 2.7-8.8) than in C (2.7; 2.0-4.6) (p<0.0001). ROC curves analyses identified a of 3.5 for Ca/P ratio with a sensitivity and specificity of 86% and 87%, respectively (p<0.0001). Conclusions Ca/P ratio is a valuable highly sensitive, highly specific tool for the diagnosis of PHPT. Besides, Ca/P has the best diagnostic value in identifying normocalcemic PHPT (NCPHPT). Considering that Ca/P is simple to obtain, easily accessible in every clinical and laboratory setting worldwide, and inexpensive even when used in large sample size of patients, this diagnostic tool could be useful for screening PHPT, especially in patients accessing emergency rooms or in the general practitioner setting. This biochemical index has the advantage to be universally used in all clinical settings for both the diagnosis and the screening of PHPT thanks to its low costs and worldwide availability in any laboratory setting (even in developing countries).


2017 - Short term Leydig cell stimulation by LH and hCG in man with central hypogonadism [Abstract in Atti di Convegno]
Santi, Daniele; Spaggiari, G; Casarini, L; Fanelli, F; Mezzullo, M; Pagotto, U; Granata, Arm; Carani, C; Simoni, M
abstract

Short term Leydig cell stimulation by LH and hCG in man with central hypogonadism


2017 - Sperm recovery and ICSI outcomes in Klinefelter syndrome: A systematic review and meta-analysis [Articolo su rivista]
Corona, G.; Pizzocaro, A.; Lanfranco, F.; Garolla, A.; Pelliccione, F.; Vignozzi, L.; Ferlin, A.; Foresta, C.; Jannini, E. A.; Maggi, M.; Lenzi, A.; Pasquali, D.; Francavilla, S.; Balercia, G.; Bonomi, M.; Calogero, A.; Fabbri, A.; Francavilla, F.; Giagulli, V.; Pivonello, R.; Radicioni, A.; Rochira, V.; Accardo, G.; Cangiano, B.; Condorelli, R. A.; Cordeschi, G.; D'Andrea, S.; Di Mambro, A.; Esposito, D.; Galdiero, M.; Giovannini, L.; Granata, A. R. M.; La Vignera, S.; Motta, G.; Negri, L.; Persani, L.; Salzano, C.; Santi, D.; Selice, R.; Simoni, M.; Tatone, C.; Tirabassi, G.; Tresoldi, A. S.; Vicari, E.
abstract

BACKGROUND: Specific factors underlying successful surgical sperm retrieval rates (SRR) or pregnancy rates (PR) after testicular sperm extraction (TESE) in adult patients with Klinefelter syndrome (KS) have not been completely clarified. OBJECTIVE AND RATIONALE: The aim of this review was to meta-analyse the currently available data from subjects with KS regarding SRRs as the primary outcome. In addition, when available, PRs and live birth rates (LBRs) after the ICSI technique were also investigated as secondary outcomes. SEARCH METHODS: An extensive Medline, Embase and Cochrane search was performed. All trials reporting SRR for conventional- TESE (cTESE) or micro-TESE (mTESE) and its specific determinants without any arbitrary restriction were included OUTCOMES: Out of 139 studies, 37 trials were included in the study, enrolling a total of 1248 patients with a mean age of 30.9 ± 5.6 years. The majority of the studies (n = 18) applied mTESE, 13 applied cTESE and in one case testicular sperm aspiration (TESA) was used. Additionally, four studies used a mixed approach and in one study, the method applied for sperm retrieval was not specified. Overall, a SRR per TESE cycle of 44[39;48]% was detected. Similar results were observed when mTESE was compared to cTESE (SRR 43[35;50]% vs 45[38;52]% for cTESE vs micro-TESE, respectively; Q = 0.20, P = 0.65). Meta-regression analysis showed that none of the parameters tested, including age, testis volume and FSH, LH and testosterone (T) levels at enrollment, affected the final SRR. Similarly, no difference was observed when a bilateral procedure was compared to a unilateral approach. No sufficient data were available to evaluate the effect of previous T treatment on SRR. Information on fertility outcome after ICSI was available for 29 studies. Overall a total of 218 biochemical pregnancies after 410 ICSI cycles were observed (PR = 43[36;50]%). Similar results were observed when LBR was analyzed (LBR = 43[34;53]%). Similar to what was observed for SRR, no influence of KS age, mean testis volume, LH, FSH or total T levels on either PR and LBR was observed. No sufficient data were available to test the effect of the women's age or other female fertility problems on PR and LBR. Finally, no difference in PR or LBR was observed when the use of fresh sperm was compared to the utilization of cryopreserved sperm. WIDER IMPLICATIONS: The present data suggest that performing TESE/micro-TESE in subjects with KS results in SRRs of close to 50%, and then PRs and LBRs of close to 50%, with the results being independent of any clinical or biochemical parameters tested.


2017 - The steroid response to human chorionic gonadotropin (hCG) stimulation in men with Klinefelter syndrome does not change using immunoassay or mass spectrometry [Articolo su rivista]
Roli, L.; Santi, D.; Belli, S.; Tagliavini, S.; Cavalieri, S.; De Santis, M. C.; Baraldi, E.; Fanelli, F.; Mezzullo, M.; Granata, A. R.; Pagotto, U.; Pasquali, R.; Rochira, V.; Carani, C.; Simoni, M.; Trenti, T.
abstract

Purpose: Liquid-chromatography tandem mass-spectrometry (LC-MS/MS) was developed in parallel to Immunoassays (IAs) and today is proposed as the “gold standard” for steroid assays. Leydig cells of men with Klinefelter syndrome (KS) are able to respond to human chorionic gonadotropin (hCG) stimulation, even if testosterone (T) production was impaired. The aim was to evaluate how results obtained by IAs and LC-MS/MS can differently impact on the outcome of a clinical research on gonadal steroidogenesis after hCG stimulation. Methods: A longitudinal, prospective, case-control clinical trial. (clinicaltrial.gov NCT02788136) was carried out, enrolling KS men and healthy age-matched controls, stimulated by hCG administration. Serum steroids were evaluated at baseline and for 5 days after intramuscular injection of 5000 IU hCG using both IAs and LC-MS/MS. Results: 13 KS patients (36 ± 9 years) not receiving T replacement therapy and 14 controls (32 ± 8 years) were enrolled. T, progesterone, cortisol, 17-hydroxy-progesterone (17OHP) and androstenedione, were significantly higher using IAs than LC-MS/MS. IAs and LC-MS/MS showed direct correlation for all five steroids, although the constant overestimation detected by IAs. Either methodology found the same 17OHP and T increasing profile after hCG stimulation, with equal areas under the curves (AUCs). Conclusions: Although a linearity between IA and LC-MS/MS is demonstrated, LC-MS/MS is more sensitive and accurate, whereas IA shows a constant overestimation of sex steroid levels. This result suggests the need of reference intervals built on the specific assay. This fundamental difference between these two methodologies opens a deep reconsideration of what is needed to improve the accuracy of steroid hormone assays.


2017 - Thyroid nodules ultrasound classification and the importance of the endocrinologist clinical feeling. [Abstract in Rivista]
Madeo, Bruno; Brigante, Giulia; Ansaloni, Anna; Taliani, Erica; Simoni, Manuela; Rochira, Vincenzo
abstract

Background and aim of the study Several ultrasound (US) classifications for estimating thyroid nodules risk have been proposed. Since most of them are hardly applicable in clinical practice, we created a local tool, named Modena classification (MC), considering US characteristics and clinician subjective impression. The aim is to verify the diagnostic accuracy of MC and to compare it to US classifications of American Thyroid Association(ATA) (1) and British Thyroid Association(BTA) (2). Methods We prospectively enrolled 111 patients (33M, 78F; age 19–75; total 457 nodules) with an indeterminate, suspicious for malignancy or malignant cytology. All the patients underwent neck US before surgery and a score risk was assigned, according to MC: low (not certainly nodular or not suspect); intermediate (indeterminate); high (suspect or very suspect). Then, we retrospectively classified nodules according to ATA and BTA. The US pattern was related to hystology. Results All the classifications had low sensitivity and positive predictive value (PPV), and high specificity and negative predictive value (NPV) for low risk categories. For the intermediate risk category, BTA had the highest accuracy (68%). For higher risk categories, MC had good sensitivity (62%), high specificity (89%) and accuracy (81%); ATA had high sensitivity (83%), low specificity (48%), accuracy 58%; BTA had high sensitivity (88%), low specificity (44%), accuracy 57%. Conclusions A classification that considers the subjective impression of the clinician, in addition to the known US characteristics, has highest accuracy and specificity compared to guidelines classifications, particularly if the nodule has suspect US features. References (1) Haugen et al. Thyroid. 2016, 26: 1–133. (2) Perros et al. Clin Endocrinol (Oxf). 2014; 81 (Suppl 1):1–122. DOI: 10.1530/endoabs.49.EP1383


2016 - Clinical Applications of Gonadotropins in the Female: Assisted Reproduction and Beyond. [Articolo su rivista]
Casarini, Livio; Simoni, Manuela; Brigante, Giulia; Santi, Daniele
abstract

Gonadotropins (LH, FSH, and hCG) act in concert in the regulation of female reproductive system. Exploiting this influence, they are part of the assisted reproductive technique protocols. In this review we analyze the effectiveness of the different available gonadotropin formulations and the consequent adverse events. Moreover, different protocols for poor-responders and polycystic ovary syndrome affected women are explored. All these clinical different approaches have specific molecular bases, covered in this review starting from evolution and population genetics, getting to in vitro studies of gonadotropins action. Beyond their application in assisted reproductive technique, gonadotropins have also been largely studied for their intertwined network of interactions with other hormones, which all together contribute to the functioning of the reproductive system and other hormonal axes. In particular, there is both clinical and molecular evidence of interaction between thyroid hormones and insulin growth factors with gonadotropins. Finally, gonadotropins are widely studied for their role in the maintenance of the proper balance between cell proliferation and differentiation, and therefore in cancer.


2016 - Effects of chronic administration of the phosphodiesterase inhibitor vardenafil on serum levels of adrenal and testicular steroids in men with type 2 diabetes mellitus [Abstract in Rivista]
Santi, Daniele; Granata, A. R. M.; Pignatti, Elisa; Trenti, T.; Roli, L.; Bozic, R.; Zaza, S.; Rochira, Vincenzo; Carani, Cesare; Magnani, Elisa; Simoni, Manuela
abstract

Background. Steroidogenesis is a complex enzymatic pro- cess in which cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) play an important role. Phosphodiesterase-5 inhibitors(PDE5i) increase cGMP, improving NO availability. Objective. To investigate whether long-term, chronic treat- ment with the PDE5i Vardenafil affects adrenal and testicular steroidogenesis in diabetic men, using liquid chromatography- mass spectrometry (LC-MS/MS). Design. A longitudinal, prospective, investigator-started, randomized, placebo-controlled, double-blind, clinical-trial was carried out. Setting and participants. 54 male patients affected by T2DM diagnosed within the last 5 years were enrolled. 26 and 28 patients were assigned to the verum and placebo-group, re- spectively. Interventions. The study consisted of an enrolment phase, a treatment phase (24 weeks) (Vardenafil/placebo 10 mg twice- daily), and a follow-up phase (24 weeks). Outcome measurements. Progesterone (P), 17-hydroxy- progesterone (17OHP), androstenedione (A), testosterone (T), dehydroepiandrosterone (DHEA), DHEA sulphate (DHEAS), corticosterone, 11-deoxycortisol and cortisol (C), were evalu- ated using LC-MS/MS. Results. No differences were seen in sex testicular steroids between study and control group. For the adrenal gland, steroids were considered according to the zona in which they are pro- duced. Considering steroids produced in the zona fasciculata, no significant differences were seen in 11-deoxycortisol and C among visits, both in the study and in the control group. For the zona reticularis, DHEA significantly decreased during treatment only in the study group (p=0.007). At post-hoc test DHEA showed higher levels at visit 2 and 8 than in other visits. The DHEAS/DHEAS ratio significantly increased during treatment only in the verum group. Considering the adrenal zona glomeru- losa, corticosterone significantly changed among visits both in the study and in the control group (p&lt;0.001). At post-hoc test, in ПРОБЛЕМЫ ЭНДОКРИНОЛОГИИ, 5, 2016 both groups, corticosterone was significantly higher at visit 2 (p=0.028), 8 (p=0.003) and 10 (p=0.044), i.e. in coincidence with the complete clinical and instrumental examination per- formed only at these visits according to the study protocol. Conclusions. This is the first double-blind, placebo-con- trolled clinical-trial in which steroidogenesis is extensively in- vestigated by LC-MS/MS in T2DM men chronically treated with Vardenafil for 6 months, and followed-up for 6 months after therapy-withdrawal. Chronically administered Vardenafil reduces DHEA levels and increases DHEAS/DHEA ratio as possible consequences of modulation of steroidogenic enzymes by tissue changes in cGMP and/or cAMP availability. A possi- bly stress-related increase in corticosterone is suggested for the first time.


2016 - Follicle-stimulating hormone potentiates the steroidogenic activity of chorionic gonadotropin and the anti-apoptotic activity of luteinizing hormone in human granulosa-lutein cells in vitro [Articolo su rivista]
Casarini, Livio; Riccetti, Laura; DE PASCALI, Francesco; Nicoli, Alessia; Tagliavini, Simonetta; Trenti, Tommaso; LA SALA, Giovanni Battista; Simoni, Manuela
abstract

Luteinizing hormone (LH) and choriogonadotropin (hCG) are glycoprotein hormones regulating ovarian function and pregnancy, respectively. Since these molecules act on the same receptor (LHCGR), they were traditionally assumed as equivalent in assisted reproduction techniques (ART), although differences between LH and hCG were demonstrated at molecular and physiological level. In this study, we demonstrated for the first time that co-treatment with a follicle-stimulating hormone (FSH) dose in the ART therapeutic range potentiates different LH- and hCG-dependent responses in vitro, measured in terms of cAMP, phospho-CREB, -ERK1/2 and -AKT activation, gene expression, progesterone and estradiol production in human granulosa-lutein cells (hGLC). We show that in the presence of FSH, hCG biopotency is about 5-fold increased, in the presence of FSH, in terms of cAMP activation. Accordingly, CREB phosphorylation and steroid production is increased under hCG and FSH co-treatment. LH effects, evaluated as steroidogenic cAMP/PKA pathway activation, do not change in the presence of FSH, which, however, increases LH-dependent ERK1/2 and AKT, but not CREB phosphorylation, resulting in antiapoptotic effects. The different modulatory activity of FSH on LH and hCG action in vitro corresponds to their different physiological functions, reflecting proliferative effects exerted by LH during the follicular phase and before trophoblast development, and the high steroidogenic potential of hCG requested to sustain pregnancy from the luteal phase onwards.


2016 - Gonadotrophin Receptors [Capitolo/Saggio]
Casarini, Livio; Huhtaniemi, Ilpo; Simoni, Manuela; Rivero Müller, Adolfo
abstract

The two gonadotrophin receptors (GnRs), luteinizing hormone receptor (LHCGR) and follicle-stimulating receptor (FSHR), belong to the glycoprotein hormone receptor subgroup of type A G protein-coupled receptors (GPCRs). LHCGR binds specifically the two structurally similar gonadotrophins, luteinizing hormone (LH) and human chorionic gonadotrophin (hCG), and FSHR binds follicle-stimulating hormone (FSH). The receptors reside on plasma membrane and transmit the gonadotrophin signal to target cells using the classical Gs/adenylyl cyclase/cyclic AMP/protein kinase A signaling cascade. Other signaling pathways (e.g., inositol phosphate, calcium) are activated at pharmacological hormone concentrations or at high receptor density. LHCGR is expressed in testicular Leydig cells and in ovarian theca, luteinizing granulosa and luteal cells. FSHR is expressed in testicular Sertoli cells and ovarian granulosa cells. LHCGR activation stimulated Leydig cell steroidogenesis, in particular testosterone production, while FSHR maintains Sertoli cell metabolism, thereby indirectly stimulating spermatogenesis. Recent basic research, using GnR, expressing cells in vitro and genetically modified mice in vivo, has elucidated novel aspects of the molecular mechanisms of gonadotrophin receptor function. The crystal structure of GnRs has also been partly resolved. Numerous inactivating and activating GnR mutations that have been discovered in patients have unraveled the molecular basis of hypogonadism and other aberrations of reproductive endocrine functions. The purpose of this chapter is to review the recent trends of GnR research and how it has elucidated the molecular mechanisms of GnR function and the role of GnR in human reproductive physiology and pathophysiology.


2016 - Human chorionic gonadotropin stimulation gives evidence of differences in testicular steroidogenesis in Klinefelter syndrome, as assessed by liquid chromatography-tandem mass spectrometry [Articolo su rivista]
Belli, Serena; Santi, Daniele; Leoni, E; Dall'Olio, Enrico; Fanelli, F; Mezzullo, M; Pelusi, C; Roli, L; Tagliavini, Silvia; Trenti, T; Granata, A. R; Pagotto, U; Pasquali, R; Rochira, Vincenzo; Carani, Cesare; Simoni, Manuela
abstract

BACKGROUND: Men with Klinefelter syndrome (KS) show hypergonadotropic hypogonadism, but the pathogenesis of hypotestosteronemia remains unclear. Testicular steroidogenesis in KS men was evaluated over three decades ago after human chorionic gonadotropin (hCG) stimulation, but inconclusive results were obtained. Intriguingly, some recent studies show increased intratesticular testosterone concentrations in men with KS. OBJECTIVE: To analyze serum steroid profile, as a proxy of testicular steroidogenesis, after hCG stimulation in KS compared with control men. DESIGN: A prospective, longitudinal, case-control, clinical trial. METHODS: Thirteen KS patients (36±9 years) not receiving testosterone (TS) replacement therapy and 12 eugonadic controls (32±8 years) were enrolled. Serum steroids were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) at baseline and for five consecutive days after intramuscular injection of 5000IU hCG. RESULTS: Progesterone (P), 17-hydroxyprogesterone (17OHP), TS, and estradiol (E2) showed a significant increase (P<0.001) after hCG stimulation in both groups. On the contrary, androstenedione (AS) and dehydroepiandrosterone did not increase after hCG stimulation. The 17OHP/P ratio increased in both groups (P<0.001), the TS/AS ratio (17β-hydroxysteroid dehydrogenase type 3 (17βHSD3) activity) did not increase after hCG in any group, and the E2/TS ratio (aromatase activity) increased significantly in both groups (P=0.009 in KS and P<0.001 in controls). Luteinizing hormone decreased after hCG in both groups (P=0.014 in KS and P<0.001 in controls), whereas follicle-stimulating hormone decreased only in control men (P<0.001). CONCLUSION: This study demonstrates for the first time using LC-MS/MS that Leydig cells of KS men are able to respond to hCG stimulation and that the first steps of steroidogenesis are fully functional. However, the TS production in KS men is impaired, possibly related to reduced hydroxysteroid deydrogenase activity due to an unfavorable intratesticular metabolic state.


2016 - Is polycystic ovary syndrome a sexual conflict? A review [Articolo su rivista]
Casarini, Livio; Simoni, Manuela; Brigante, Giulia
abstract

Several studies have attempted to explain the high overall prevalence of polycystic ovary syndrome among women worldwide (about 4-10%) despite its link to subfertile phenotypes. For this reason, it is considered an evolutionary paradox. In this review, we show that several genetic loci associated with the disease differently modulate the reproductive parameters of men and women. This observation suggests that such genetic variants lead to opposite effects in the two sexes in reproductive success. Intralocus sexual conflict as a cause of the persistence polycystic ovary syndrome genotypes among humans is supported.


2016 - Is serum estradiol (E2) really increased in patients with Klinefelter Syndrome (KS)? Results from a meta-analysis study. [Abstract in Atti di Convegno]
Santi, Daniele; Scaltriti, Sara; Simoni, Manuela; Rochira, Vincenzo
abstract

BACKGROUND: KS has been classically described as characterized by hyperestrogenism and elevated serum E2 together with increased gonadotropins and low-to-normal serum testosterone (T). In literature, data on increased serum E2 are not solid. The aim of this study is to meta- analyse data from studies evaluating serum E2 in both KS and healthy subjects (HS) in order to verify if E2 is increased in KS. METHODS: An extensive MEDLINE was performed using ‘PubMed’ with the following key words: ‘KS’ and ‘E2’ or ‘T’ or ‘sex steroids’ from 1946 to January 2015 (Current Contents-ISI was used for searching oldest studies). All studies (case-control, case-series, case-reports) reporting E2 measurement were considered. Controlled-studies were used for meta-analysis. Only serum E2 at baseline (no ongoing treatments) was included. Meta-analysis was conducted according to the PRISMA statement using RevMan. RESULTS: Out of 956 articles, 26 case-control studies, 15 case-series and 21 case-reports had data on serum E2. A total of 878 KS and 1000 HS were included in the meta-analysis. Serum E2 was significantly higher in HS than in KS, with a mean difference of 7,93 pg/mL (CI: 2,24,13,61;p=0,006), with a chi-squared=688,32 (I-square=97%) (Figure 1). Serum T was significantly lower in KS than in HS, with a mean difference of -2,79 ng/mL (CI:-3,46,-2,11;p<0,001), with a chi-squared=198,29 (I-square=89%). Data from case-series and case-reports confirmed that E2 is not above the normal range in KS.CONCLUSIONS: Serum E2 is not increased in KS and is significantly lower than in HS in this meta-analysis. The limits of this study are the heterogeneity of methods for steroids measurement and the lack of studies having the comparison of serum E2 between KS and HS as primary endpoint. The traditional belief that KS is associated to elevated E2 should be reconsidered together with some pathophysiological and clinical issues.


2016 - Is serum estradiol (E2) really increased in patients with Klinefelter Syndrome (KS)? Results from a meta-analysisi study. [Abstract in Atti di Convegno]
Santi, Daniele; Scaltriti, Sara; Simoni, Manuela; Rochira, Vincenzo
abstract

Results from a meta-analysis on E2 in men with Klinefelter Syndrome


2016 - Serum Calcium to Phosphorous Ratio (Ca/P) As a Simple, Inexpensive Screening Tool in the Diagnosis of Primary Hyperparathyroidism (PHPT) [Abstract in Rivista]
Madeo, Bruno; Kara, Elda; Cion, K.; Vezzani, Silvia; Simoni, Manuela; Rochira, Vincenzo
abstract

Background: PHPT is the third most common endocrine disease, but it remains often overlooked and underdiagnosed. Several strategies, including biochemical markers used alone or combined in complex algorithms, have been investigated in the past with the aim to identify tools useful to easily diagnose or screen PHPT. At present, however, the diagnosis of PHPT remains challenging, especially in asymptomatic patients. As serum calcium (Ca) and phosphorous (P) are inversely related in PHPT, the Ca/P ratio might be considered a good candidate tool in the diagnosis of PHPT. Surprisingly, no data on Ca/P ratio are available in literature, despite they are very simple biochemical measurements largely available in any clinical laboratory setting. The aim of this study is to investigate the diagnostic value of the Ca/P ratio in the diagnosis of PHPT. Material and Methods: Data retrospectively obtained from review charts of 97 patients with documented PHPT (69 females; 28 males) were compared with those of 96 controls (C) (44 females; 52 males). Exclusion criteria: age <18yrs, severe chronic diseases, cancer, bone metabolic diseases, use of medications affecting serum Ca. Biochemical measurements included PTH, Vitamin D (LIASON, XL, Diasorin device), serum Ca, P, albumin, and creatinine (AU 680 Beckman device). Normal ranges were 15-88 pg/mL, 8.5-11, and 2.5-5.1 mg/dl for PTH, Ca, and P, respectively. SPSS 19.0 and SigmaPlot 11.0 were used for statistical analyses for group comparisons, ROC curves and cutoffs performance. Results: Among PHPT patients, 16 (17%) had severe hypercalcemia (>12 mg/dL), 44 (45%) mild hypercalcemia, and 36 (38%) normocalcemic PHPT (NCHPT). Ca was significantly higher in PHPT (median: 11; min-max: 9.4-15.5) than C (9.4;8.3-10.2) (p<0.0001). P was significantly lower in PHPT (2.4;1.4-3.9) than in C (3.5;2.1-4.5) (p<0.0001). PTH was significantly higher in PHPT (135.2;57.6-1748) than in C (32.1;14-106.1) (p<0.0001). Ca/P ratio was significantly higher in PHPT than in C. ROC curves analyses identified a cutoff of 3.5 for both Ca/P ratio and Ca/P ratio obtained by using Ca corrected by albumin. The sensitivity and specificity were 86% and 87%, respectively for Ca/P ratio and 89% and 93%, respectively for corrected Ca/P ratio (p<0.0001).The diagnostic value of Ca/P ratio was significantly better if compared with PTH and Ca used alone or in combination. Conclusions: Ca/P ratio is a valuable highly sensitive, highly specific tool for the diagnosis of PHPT. Considering that Ca/P is simple to obtain, easily accessible in every clinical and laboratory setting worldwide, and inexpensive even when used in large sample size of patients, this diagnostic tool could be useful for screening PHPT, especially in patients accessing emergency rooms or in the general practitioner setting. - See more at: http://press.endocrine.org/doi/abs/10.1210/endo-meetings.2016.BCHVD.9.PP26-4#sthash.Onic06Oc.dpuf


2016 - Serum calcium to phosphorous ratio (Ca/P) as a simple, inexpensive screening tool in the diagnosis of primary hyperparathyroidism (PHPT) [Abstract in Rivista]
Madeo, Bruno; Kara, Elda; Cioni, Katia; Vezzani, Silvia; Simoni, Manuela; Rochira, Vincenzo
abstract

Background PHPT is often overlooked/underdiagnosed. Several strategies (biochemical markers alone or combined in complex algorithms) have been investigated to easily diagnose/screen PHPT, but PHPT diagnosis remains challenging at present, especially in asymptomatic patients. As serum calcium (Ca) and phosphorous (P) are inversely related in PHPT, the Ca/P ratio could be a good candidate tool for PHPT diagnosis. Surprisingly, no literature data on Ca/P ratio are available, despite they are very simple biochemical measurements largely available in any clinical lab setting. Aim To investigate the Ca/P ratio diagnostic value in the diagnosis of PHPT. Methods Data retrospectively obtained from review charts of 97 patients with documented PHPT (69 females; 28 males) [16 (17%) with severe hypercalcemia (O12 mg/dl); 44 (45%) mild hypercalcemia, 36 (38%) normocalcemic PHPT (NCHPT)] were compared with those of 96 controls (C) (44 females; 52 males). Exclusion criteria: age !18 years, severe chronic diseases, cancer, bone metabolic diseases, use of medications affecting serum Ca. Biochemical measurements: PTH, Vitamin D, serum Ca, P, albumin, and creatinine. Normal ranges: PTH (15–88 pg/ml), Ca (8.5–11 mg/dl), P (2.5–5.1 mg/dl). SPSS 19.0 and SigmaPlot 11.0 were used for statistics (group comparisons, ROC curves, cutoffs performance). Results Ca and PTH were significantly higher in PHPT [(Ca median:11; min-max:9.4– 15.5); (PTH 135.2; 57.6–1748)] than C [(Ca 9.4; 8.3–10.2); (PTH 32.1; 14–106.1) (P!0.0001). P was significantly lower in PHPT (2.4; 1.4–3.9) than in C (3.5; 2.1– 4.5) (P!0.0001). Ca/P ratio was significantly higher in PHPT than in C. ROC curves analyses identified a cutoff of 3.5 for both Ca/P ratio and Ca/P ratio obtained by using albumin corrected-Ca. The sensitivity and specificity were 86 and 87%, respectively for Ca/P ratio and 89 and 93%, respectively for corrected Ca/P ratio (P!0.0001). The diagnostic value of Ca/P ratio performed better than PTH and Ca used alone or in combination. Conclusions Ca/P ratio is a valuable highly sensitive, highly specific tool for the diagnosis of PHPT. Since Ca/P is simple to obtain, easily accessible in every clinical and lab setting worldwide, and inexpensive even when used in large sample size of patients, this diagnostic tool could be useful for screening PHPT, especially in patients accessing emergency rooms or in the general practitioner setting.


2016 - Six months of daily treatment with Vardenafil improves parameters of endothelial inflammation and of hypogonadism in male patients with type 2 diabetes and erectile dysfunction: a randomized, double-blind, prospective trial. [Articolo su rivista]
Santi, Daniele; Granata, Ar; Guidi, Alessandro; Pignatti, Elisa; Trenti, T; Roli, L; Bozic, R; Zaza, S; Pacchioni, C; Romano, S; Nofer, Jr; Rochira, Vincenzo; Carani, Cesare; Simoni, Manuela
abstract

Abstract OBJECTIVE: Type 2 diabetes mellitus (T2DM) is associated with endothelial dysfunction, characterized by a reduction of nitric oxide (NO)-mediated relaxation. Phosphodiesterase-5 inhibitors (PDE5i) improve NO levels. The aim of the study is to investigate whether long-term, chronic treatment with the PDE5i Vardenafil improves systemic endothelial function in diabetic men. DESIGN: Prospective, investigator-initiated, randomized, placebo-controlled, double-blind, clinical-trial. METHODS: 54 male patients affected by T2DM, diagnosed within the last 5 years, and erectile dysfunction were enrolled, regardless of testosterone (T) levels. 26 and 28 patients were assigned to verum and placebo-group, respectively. The study consisted of an enrolment phase, a treatment phase (24weeks) (Vardenafil/placebo 10mg twice-daily), and a follow-up phase (24weeks). Parameters evaluated: International Index of Erectile Function (IIEF)-15, flow mediated dilation (FMD), serum interleukin (IL)-6, endothelin (ET)-1, gonadotropins and T (measured by liquid-chromatography/tandem mass-spectrometry). RESULTS: IIEF-15 erectile function improved during treatment (p&lt;0.001). At the end of the treatment both FMD (p=0.040) and IL-6 (p=0.019) significantly improved. FMD correlated with serum T levels (R2=0.299, p&lt;0.001). T increased significantly under Vardenafil treatment and returned in the eugonadal range only in hypogonadal men (n=13), without changes in gonadotropins. Chronic Vardenafil treatment did not result in relevant side effects. CONCLUSIONS: This is the first double-blind, placebo-controlled clinical-trial designed to evaluate the effects of chronic treatment of Vardenafil on endothelial health-related parameters and sexual hormones in patients affected by a chronic disease. Chronically administered Vardenafil is effective and improves endothelial parameters in T2DM patient. Moreover, chronic Vardenafil therapy improves hypogonadism in diabetic, hypogonadal men.


2016 - Sperm quality and environment: A retrospective, cohort study in a Northern province of Italy [Articolo su rivista]
Santi, Daniele; Vezzani, Silvia; Granata, Antonio; Roli, Laura; De Santis, Maria Cristina; Ongaro, Chiara; Donati, Federica; Baraldi, Enrica; Trenti, Tommaso; Setti, Monica; Simoni, Manuela
abstract

Background: Several studies proposed a relationship between environmental factors and semen quality, as well as the negative effect of air pollution on spermatogenesis and gonadal function. No specific studies evaluated the environmental influence on semen quality in a specific geographical area. Aim: to evaluate the environmental influence on male sperm parameters in a Northern Italian population referred for semen analysis in the National Health System. The objective of the study is the assessment of the relationship of both air pollution and environmental parameters with quality-related sperm variables, during the coldest months of the year when air is usually most polluted, due to low ventilation and poor rainfall. Study design: A retrospective, observational, cohort study was carried out in the province of Modena, located in the Emilia-Romagna region of Northern Italy. Methods: Semen analyses (n=406), environmental temperature, air humidity and air particulate matter (PM) measurements from the 1st of November, 2014 to the 19th of February, 2015 were acquired to the first database. Since spermatogenesis lasts over two months, a second, wider database was arranged, evaluating environmental exposure in the 3 months before semen collection (from August 1st 2014). All data included in the database were registered by geo-coding the residential address of the patients and the site of registration of environmental factors. The geo-codification of parameters was performed using Fusion Tables of Google available at https://www.google.com/fusiontables/data?dsrcid=implicit, considering the exact time of measurement. Results: Average air temperature was inversely related to sperm concentration and to total sperm number (p&lt;0.001). Semen volume was inversely related only to the minimum (p&lt;0.001) and not to maximum recorded temperature (p=0.110). Air humidity was not related to sperm quantity and quality. PM2.5 was directly related to total sperm number (p&lt;0.001). PM10 was directly related to both semen volume (0&lt;0.001), and typical forms (p&lt;0.001), inversely related to atypical forms (p&lt;0.001), but related neither to sperm concentration (p=0.430) nor to sperm motility. The extended analyses considering environmental parameters in the 3 months before semen collection, confirmed the relationship between air temperature and sperm quantity, whereas no influence was found between PM and sperm quality. Conclusion: An influence of environmental temperature on semen quantity is suggested, without a clear effect of air pollution, as assessed through PM10 levels, on sperm parameter variations.


2016 - The influence of environment on the sperm quality: a comprehensive, retrospective, cohort study. [Abstract in Rivista]
Santi, Daniele; Vezzani, S; Granata A., Roli L; De Santis, Mc; Baraldi, E; Trenti, T; Setti, M; Simoni, M.
abstract

influence of environment on the sperm quality: a comprehensive, retrospective, cohort study


2016 - Treatment with human, recombinant FSH improves sperm DNA fragmentation in idiopathic infertile men depending on the FSH receptor polymorphism p.N680S: A pharmacogenetic study [Articolo su rivista]
Simoni, Manuela; Santi, Daniele; Negri, Luciano; Hoffmann, Ivan; Muratori, Monica; Baldi, Elisabetta; Cambi, Marta; Marcou, Marios; Greither, Thomas; Baraldi, Enrica; Tagliavini, Simonetta; Carra, Daniela; Lombardo, Francesco; Gandini, Loredana; Pallotti, Francesco; Krausz, Csilla; Rastrelli, Giulia; Ferlin, Alberto; Menegazzo, Massimo; Pignatti, Elisa; Linari, Francesca; Marino, Marco; Benaglia, Renzo; Levi Setti, Paolo E.; Behre, Hermann M.
abstract

Study question: Does the spermDNAfragmentation index (DFI) improve depending on the FSH receptor (FSHR) genotype as assessed by the nonsynonymous polymorphisms rs6166 (p.N680S) after 3 months of recombinant FSH treatment in men with idiopathic infertility? summary answer: FSH treatment significantly improves sperm DFI only in idiopathic infertile men with the p.N680S homozygous N FSHR. what is known already: FSH, fundamental for spermatogenesis, is empirically used to treat male idiopathic infertility and several studies suggest that DFI could be a candidate predictor of response to FSH treatment, in terms of probability to conceive. Furthermore, it is known that the FSHR single nucleotide polymorphism (SNP) rs6166 (p.N680S) influences ovarian response in women and testicular volume in men. study design, size and duration: Amulticenter, longitudinal, prospective, open-label, two-arm clinical trial was performed. Subjects enrolled were idiopathic infertile men who received 150 IU recombinant human FSH s.c. every other day for 12 weeks and were followed-up for a further 12 weeks after FSH withdrawal. Patients were evaluated at baseline, at the end of treatment and at the end of follow-up. participants/materials, setting, methods: Eighty-nine men with idiopathic infertility carrier of the FSHR p.N680S homozygousNor S genotype, FSH ≤ 8 IU/l and DFI &gt;15%,were enrolled. A total of 66 patients had DFI analysis completed on at least two visits. DFI was evaluated in one laboratory by TUNEL/PI (propidium iodide) assay coupled to flow cytometry, resolving two different fractions of sperm, namely the 'brighter' and 'dimmer' sperm DFI fractions. main results and the roleof chance: Thirty-eightmen(57.6%)were carriers of the p.N680S homozygousNand 28 (42.4%) of the homozygous S FSHR. Sperm concentration/number was highly heterogeneous and both groups included men ranging from severe oligozoospermia to normozoospermia. Total DFI was significantly lower at the end of the study in homozygous carriers of the p.N680SNversus p.N680S S allele (P = 0.008). Total DFI decreased significantly from baseline to the end of the study (P = 0.021) only in carriers of the p.N680S homozygous N polymorphism, and this decrease involved the sperm population containing vital sperm (i.e. brighter sperm) (P = 0.008). The dimmer sperm DFI fraction, including only nonvital sperm, was significantly larger in p.N680S S homozygous patients than in homozygous N men (P = 0.018). Total DFIwas inversely related to total sperm number (P = 0.020) and progressive sperm motility (P = 0.014).Whenpatients were further stratified according to sperm concentration (normoozospermic versus oligozoospermic) or -211G&gt;T polymorphism in the FSHB gene (rs10835638) (homozygous Gversus others), the significant improvement of sperm DFI in FSHR p.N680S homozygousNmen was independent of sperm concentration and associated with the homozygous FSHB -211G&gt;T homozygous G genotype. limitations, reasons for caution: The statistical power of the study is 86.9% with alpha error 0.05. This is the first pharmacogenetic study suggesting that FSH treatment induces a significant improvement of total DFI in men carriers of the p.N680S homozygousNFSHR; however, the results need to be confirmed in larger studies using a personalized FSH dosage and treatment duration. wider implications of the findings: The evaluation of sperm DFI as a surrogate marker of sperm quality, and of the FSHR SNP rs6166 (p.N680S), might be useful to predict the response to FSH treatment in men with idiopathic infertility. study funding/competing interest(s): The study was supported by an unrestricted grant to M.S. and H.M.B. from Merck Serono that provided the drug used in the study. MS received additional grants from Merck Serono and IBSA as well as honoraria from Merck Serono. The remaining authors declare that no conflicts of interest are present. trial registration number: EudraCT number 2010-020240-35.


2016 - Will steroid measurements affect the outcomes of clinical trials? Comparison between immunoassayand mass spectrometry in men with Kinefelter Syndrome undergoing human corionic gonadotropin stimulation test. [Abstract in Atti di Convegno]
Santi, Daniele; Roli, L.; Belli, Serena; Tagliavini, Silvia; Cavalieri, Silvia; De Santis, M. C.; Baraldi, Enrica; Fanelli, F.; Mezzullo, M.; Granata, A. R.; Pagotto, U.; Pasquali, R.; Rochira, Vincenzo; Carani, Cesare; Trenti, T.; Simoni, Manuela
abstract

The abstract deal with the comparison between immunoassayand mass spectrometry in measuring sex steroids in men with Kinefelter Syndrome


2016 - β-arrestins regulate gonadotropin receptor-mediated cell proliferation and apoptosis by controlling different FSHR or LHCGR intracellular signaling in the hGL5 cell line. [Articolo su rivista]
Casarini, Livio; Reiter, Eric; Simoni, Manuela
abstract

Gonadotropin signaling classically involves proliferative, steroidogenic and apoptotic stimuli. In this study, we used the human granulosa cell line hGL5 to demonstrate how follicle-stimulating hormone (FSH) and luteinizing hormone (LH) differently control proliferative or apoptotic signals, revealing novel intrinsic properties of their receptors (FSHR, LHCGR). We found that, in this tumor-like cell line, the expression of endogenous FSHR and LHCGR is serum-dependent, but both receptors were unable to activate the canonical cAMP/PKA pathway upon gonadotropin stimulation, failing to produce cAMP, progesterone and G protein-coupled receptor (GPCR)-mediated apoptosis in vitro. Conversely, ligand treatment resulted in FSHR- and LHCGR-mediated ERK1/2 phosphorylation and cell proliferation due to receptor coupling to β-arrestins. The inactive cAMP/PKA pathway was unlocked by siRNA-mediated knock-down of β-arrestin 1 and 2, leading to progesterone synthesis and apoptosis. Surprisingly, FSH, but not LH treatment accelerated the cAMP/PKA-mediated apoptosis after β-arrestin silencing, an effect which could be reproduced by overexpressing the FSHR, but not the LHCGR. This work demonstrates that the expression of FSHR and LHCGR can be induced in hGL5 cells but that the FSHR-dependent cAMP/PKA pathway is constitutively silenced, possibly to protect cells from FSHR-cAMP-PKA-induced apoptosis. Also, we revealed previously unrecognized features intrinsic to the two structurally similar gonadotropin receptors, oppositely resulting in the regulation of life and death signals in vitro.


2015 - Chronic, long-term administration of Vardenafil improves endothelial function and corrects hypogonadism in patients with type 2 diabetes mellitus. A longitudinal, prospective, randomized, placebo-controlled, double blind, clinical trial [Abstract in Rivista]
Santi, Daniele; Guidi, Alessandro; Granata, Antonio; Pignatti, Elisa; Bozic, Roberto; Zaza, Stefano; Roli, Laura; Trenti, Tommaso; Carani, Cesare; Simoni, Manuela
abstract

10.1530/endoabs.37.OC4.3


2015 - Efficacy of follicle-stimulating hormone treatment in male idiopathic infertility: a meta-analysis [Abstract in Atti di Convegno]
Santi, Daniele; Granata, Antonio; Simoni, Manuela
abstract

Study question: To comprehensively evaluate whether follicle stimulating hor- mone (FSH) administration to the male partner of idiopathic infertile couples improves pregnancy rate, both spontaneous and after assisted reproductive tech- nique (ART), using a meta-analytic approach and including controlled clinical trial. Summary answer: The administration of FSH to infertile men is reported in the literature since 1991, improving fertilisation and pregnancy rate. A significant increase in pregnancy rate after ART and male treatment with FSH was already shown in several studies, since FSH improves the sperm quality. FSH treatment could improve sperm quality and pregnancy rate in idiopathic infertile men. What is known already: The Cochrane Collaboration recently estimated the overall effect of FSH treatment of the man in couples attending ART, enrolled in randomised, controlled, clinical-trials. That meta-analysis demonstrated that FSH treatment significantly improves spontaneous pregnancy rate, whereas no improvement of pregnancy rate was observed after ART, using fixed and strict inclusion criteria. They excluded all trials in which the randomization was not provided leading to potential loss of useful information that could help clini- cians in their routinely practice. Study design, size, duration: We conducted a comprehensive literature search for controlled clinical trials in which FSH was administered for male idiopathic infertility, compared with placebo or no treatment. The randomization was not considered as inclusion criterion. We considered studies in which men with idiopathic infertility or subfertility were enrolled, chronicallty treated with any type of FSH, compared with placebo or no treatment. Participants/materials, setting, methods: We found 15 controlled clinical stud- ies. Concerning the type of FSH, eight studies included in the meta-analysis used recombinant FSH, whereas seven studies used purified FSH. Pregnancy rate, when evaluated, was considered spontaneous or after ART. Selected trials gave details about 1275 infertile-men, 614 treated with FSH and 661 not-treated. Main results and the role of chance: Among the 15 studies included, nine studies evaluated the spontaneous pregnancy rate, resulting in an overall im- provement of about 4.5 (CI 2.17–9.33 and I2 = 0%) (p < 0.001). Eight stud- ies evaluated pregnancy rate after ART, showing a significant improvement of about 1.60 (CI 1.08–2.37 and I2 = 43%) (p = 0.002). Sub-dividing studies ac- cording to the FSH preparations (purified or recombinant), the pregnancy rate improvement remained significant (p = 0.007 and p = 0.002, respectively). Elev- en studies considered sperm quality after FSH treatment, finding a significant improvement of sperm concentration (mean improvement of 2.66x106 millions/ mL, with CI 0.47–4.84, p = 0.02), but not of sperm motility (mean improvement of 1.22x106 millions/mL, with CI -0.07–2.52, p = 0.06). Finally, three trials evaluated testicular volume, showing a non-significant increase in men treated (mean increase of 1.35 mL, with CI -0.44–3.14, p = 0,14). Limitations, reason for caution: The heterogeneity of studies, together with the high risk of biases in this field of research could limit the strength of these results. Wider implications of the findings: The results of controlled clinical trials available in literature indicate an improvement of pregnancy rate after FSH ad- ministration to the male partner of infertile couples, both spontaneous and after ART. Study funding/competing interest(s): Funding by University(ies) – University of Modena and Reggio Emilia. Trial registration number: NA. Keywords: FSH, idiopathic male infertility, reproduction


2015 - FSH treatment improves sperm DNA damage in men with idiopathic infertility carriers of the FSH receptor p.N680S homozygous N genotype: an interim analysis [Abstract in Atti di Convegno]
Simoni, M; Santi, D; Linari, F; Baldi, E; Cambi, M; Ferlin, A; Gandini, L; Garolla, A; Krausz, C; Levi Setti, Pe; Lombardo, F; Marino, M; Muratori, M; Negri, L; Pignatti, E; Rastrelli, G; and Behre, Hm
abstract

Study question: To assess whether in men with idiopathic infertility, the sperm DNA fragmentation (sDF) improves depending on the FSH receptor (FSHR) genotype as assessed by the non-synonymous polymorphisms (SNP) rs6166 (wild type or p.N680S). Summary answer: FSH treatment improves sDF in a subgroup of idiopathic infertile men, although 40% of these men do not show any significant improve- ment. The response of sDF, a surrogate marker of sperm quality, together with the evaluation of FSHR SNP p.N680S might be useful to predict the response to FSH treatment. What is known already: FSH is fundamental for spermatogenesis and is em- pirically used to treat male idiopathic infertility. Several studies suggest that sDF could be a candidate predictor of response to FSH treatment, in terms of probability to conceive. Furthermore, it is widely accepted that the FSHR SNP p.N680S influences ovarian response in women and testicular volume in men. Study design, size, duration: Multicenter, longitudinal, prospective, open-la- bel, two-arms clinical trial. Subjects enrolled were idiopathic infertile men and received 150 IU of recombinant FSH (Gonal f®) every other day for 12 weeks and were then followed-up for further 12 weeks after FSH-withdrawal. Patients were evaluated at baseline and at the end of the two phases. Participants/materials, setting, methods: Eighty-eight men with idiopathic male infertility carrier of the homozygous FSHR p.N680S N or S genotype, FSH < 8 IU/L and sDF > 15%, were enrolled. 66 patients completed the sDF analysis. sDF was centrally evaluated by TUNEL/PI assay coupled to flow cy- tometry, resolving two different sperm populations, namely: PIbrighter and PIdimmer. Main results and the role of chance: Thirty-seven men (56%) were carriers of the p.N680S homozygous-N and 29 (44%) of the homozygous-S genotype, respectively. Total sDF (PIbrighter + PIdimmer) was significantly lower at the end of the study in patients carriers of the p.N680S-N allele than patients carri- ers of p.N680S-S allele (p = 0.008). Only in patients carriers of the p.N680S-N allele, total sDF decreased significantly from baseline to the end of the study (p = 0.021) and this decrease was entirely sustained by the sperm population containing vital sperms (i.e., PIbrighter fraction) (p = 0.008). PIdimmer frac- tion, including only non-vital sperms, was significantly higher in patients car- riers of the p.N680S-S allele than in carriers of N allele (p = 0.018). Total sDF was inversely related to total sperm number (p = 0.020) and progressive sperm motility (p = 0.014). Limitations, reason for caution: The statistical power of the results obtained so far is 86.9%, with alpha-error 0.05. This is an interim-analysis. Wider implications of the findings: The study suggests that FSH treatment induces a significant improvement of total sDF in men carriers of the p.N680S homozygous N allele. This sDF decrease awaits confirmation, since the study will be completed by June 2015. Study funding/competing interest(s): Funding by commercial/corporate company(ies) – The study was supported by unrestricted grant by Merck Serono. Trial registration number: EudraCT number 2010-020240-35. Keywords: FSH treatment, male infertility, Sperm-DNA fragmentation


2015 - FSH treatment of male idiopathic infertility improves pregnancy rate: a meta-analysis [Articolo su rivista]
Santi, Daniele; Granata, Ar; Simoni, Manuela
abstract

INTRODUCTION: The aim of this study is to comprehensively evaluate whether FSH administration to the male partner of infertile couples improves pregnancy rate, spontaneously and/or after assisted reproductive techniques (ART). METHODS: Meta-analysis of controlled clinical trials in which FSH was administered for male idiopathic infertility, compared with placebo or no treatment. Randomization was not considered as an inclusion criterion. RESULTS: We found 15 controlled clinical studies (614 men treated with FSH and 661 treated with placebo or untreated). Concerning the type of FSH, eight studies used recombinant FSH, whereas seven studies used purified FSH. Nine studies evaluated spontaneous pregnancy rate, resulting in an overall odds ratio (OR) of about 4.5 (CI: 2.17-9.33). Eight studies evaluated pregnancy rate after ART, showing a significant OR of 1.60 (CI: 1.08-2.37). Sub-dividing studies according to the FSH preparations (purified/recombinant), pregnancy rate improvement remained significant for each preparation. Eleven studies considered sperm quality after FSH treatment, finding a significant improvement of sperm concentration (2.66×10(6)/ml, CI: 0.47-4.84), but not of concentration of sperm with progressive motility (1.22×10(6)/ml, CI: -0.07 to 2.52). Three trials evaluated testicular volume, showing a non-significant increase in men treated (1.35 ml, CI: -0.44 to 3.14). CONCLUSION: The results of controlled clinical trials available in the literature indicate an improvement of pregnancy rate after FSH administration to the male partner of infertile couples, both spontaneously and after ART. However, the heterogeneity of studies, the high risk of bias and the lack of precise criteria to guide FSH administration limit the strength of these results. Future studies should be designed to identify the markers of FSH response which are helpful in the decision-making process. Meanwhile, the use of FSH in the treatment of male infertility should be cautious.


2015 - Genetic markers of ovarian response. [Capitolo/Saggio]
Brigante, Giulia; Simoni, Manuela
abstract


2015 - High-Resolution Melting is a sensitive, cost-effective, time-saving technique for BRAF V600E detection in thyroid FNAB washing fluid: a prospective cohort study [Articolo su rivista]
Marino, Marco; Monzani, Maria Laura; Brigante, Giulia; Cioni, Katia; Madeo, Bruno; Santi, Daniele; Maiorana, Antonino; Bettelli, Stefania Raffaella; Moriondo, Valeria; Pignatti, Elisa; Bonacini, Lara; Carani, Cesare; Rochira, Vincenzo; Simoni, Manuela
abstract

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2015 - Primary hypoparathyroidism is common in adult patients with b-thalassemia and protect patients from osteoporosis. [Abstract in Rivista]
Ansaloni, Anna; Ferrara, F.; Diazzi, Chiara; Pietrangelo, Antonello; Simoni, Manuela; Rochira, Vincenzo
abstract

Introduction b-thalassemia (bT) is associated to several endocrine abnormalities mainly due to iron overload. With the increase in bT-patients life expectancy, due to progresses in iron chelation therapy, more patients enter into adulthood than in the past and the prevalence of endocrine diseases is being reconsidered. The aim of the study is to investigate the prevalence of primary hypoparathyroidism (pHPT) in adult bT-patients and to characterize the relative clinical phenotype with particular regard to bone health. Methods We enrolled 26 adult patients with major or intermedia bT (12M and 14F; mean ageGS.D. of 38.1G7.5 years). Serum PTH, 25-hydroxyvitamin D (25OHD), calcium, phosphorous, albumin, bone turnover markers, and bone mineral density (BMD) by dual-energy X-ray absorptiometry (Hologic) at lumbar and femoral site were measured. Results pHPT (PTH !15 pg/ml) was found in seven of the 26 patients (27%). Of them, four patients (57%) had hypocalcemia and two were on chronic calcium therapy. Lumbar BMD was significantly higher in patients with pHPT (0.884G 0.189 g/cm2) than in patients without pHPT (0.731G0.124 g/cm2) (PZ0.023). No significant difference was found in femoral BMD, even though a trend for higher BMD was present in pHPT (0.704G0.117 vs 0.670G0.143 g/cm2 in pHPT and no-pHPT respectively) (PZ0.578). The prevalence of osteoporosis was higher in patients without pHPT (68%) than in patients with pHPT (29%). Two patients had a history of bone osteoporotic fractures and both of them did not present pHPT. Bone turnover markers were no different in the two groups. Conclusions The prevalence of pHPT in adult bT-patients is higher if compared to that observed in pediatric bT-patients, the latter ranging from 8 to 11%. Moreover we found an higher prevalence of pHPT compared to that reported in literature on adult bT patients. As expected, pHPT seems to exert a protective role on the development of osteoporosis in these patients.


2015 - Proliferative versus apoptotic signals in granulosa cells: β-arrestins as switch between life and death signals in vitro [Abstract in Atti di Convegno]
Casarini, Livio; Simoni, Manuela
abstract

Background: The immortalized human granulosa cell line hGL5 is not responsive to FSH and LH/hCG, which fail to activate the steroidogenic cAMP/PKA pathway, CREB phosphorylation and progesterone production. Conversely, the activation of adenylyl cyclase by forskolin results in intracellular cAMP increase and steroid production, cell rounding and apoptosis, suggesting a possible FSHR and LHCGR downregulation. Curiously, in hGL5 cells the expression of some receptors (e.g. the oxytocin receptor) is absent in serum starvation conditions and increases in a serum concentration-dependent manner. Aim of the study: To investigate the mechanism of FSHR expression regulation in hGL5 cells and to evaluate whether it is associated with life/death signals in vitro. Methods. We evaluated the FSHR expression in hGL5 cells maintained under different serum concentrations (between 0 and 15%) by real time PCR and Western blotting. The response to 50 nM FSH or 100 pM LH was evaluated by measuring cAMP and progesterone production by ELISA, as well as ERK1/2 and CREB phosphorylation by Western blotting. Cell viability was assessed by proliferation assay and confocal imaging. These endpoints were evaluated in the presence and in the absence of selective inhibitors or agonists (i.e. the PKA inhibitor H-89, the phorbol ester PMA as a PKC-ERK1/2 activator, and siRNA against β-arrestin1/2). Results. The expression of FSHR and LHCGR was serum-dependent at both mRNA and protein level, being absent under starvation and increasing progressively with serum concentrations (linear regression of expression-fold values plotted against serum concentration; p<0.05; n=3). However, FSH/LH stimulation was ineffective both on cAMP and progesterone production and CREB phosphorylation (FSH/LH-stimulated cells vs controls; Mann-Whitney’s U-test; p≥0.05; n=3), suggesting uncoupling of the receptors to the Gs alpha protein. ERK1/2 phosphorylation was FSH/LH dose-dependent in the presence of serum (linear regression; p<0.05; n=3), resulting in a significant increase of cell proliferation over 4 days (FSH/LH-stimulated cells vs controls; Mann-Whitney’s U-test; p<0.05; n=3). A similar increase of cell proliferation and ERK1/2 phosphorylation was provoked by PMA (Mann-Whitney’s U-test; p<0.05; n=3). β-arrestin1/2 siRNA transfection unlocked the cAMP/PKA pathway, leading to cAMP and progesterone accumulation and CREB phosphorylation, at high basal levels (Mann-Whitney’s U-test; p<0.05; n=3). Moreover, siRNA-treated cells underwent cell rounding, pro-caspase 3 cleavage and apoptosis. The pro-apoptotic effects of cAMP/PKA pathway activation were augmented by FSH- but not LH treatment, and inhibited by selective PKA blockade by H-89. Accordingly, transfected hGL5 cells permanently overexpressing the FSHR (but not LHCGR) for 4-8 weeks showed high basal cAMP levels, cell rounding and apoptosis (Mann-Whitney’s U-test; p<0.05; n=3), revealing the dual role of the FSHR in the activation of proliferative and apoptotic signals. Conclusions. Our results suggest that β-arrestins determine the FSHR-mediated (but not LH-mediated) signaling in vitro towards proliferative- or cell death-related pathways. Our results corroborate the relationship between cAMP/PKA pathway activation and cell death in granulosa cells.


2015 - SKI-II--a sphingosine kinase 1 inhibitor--exacerbates atherosclerosis in low-density lipoprotein receptor-deficient (LDL-R-/-) mice on high cholesterol diet [Articolo su rivista]
Potì, F; Ceglarek, U; Burkhardt, R; Simoni, Manuela; Nofer, J. r.
abstract

Background: Sphingosine 1-phosphate (S1P) is a lysosphingolipid associated with high-density lipoproteins (HDL) that contributes to their anti-atherogenic potential. We investigated whether a reduction in S1P plasma levels affects atherosclerosis in low-density lipoprotein receptor deficient (LDL-R-/-) mice. Methods and Results: LDL-R-/- mice on Western diet containing low (0.25% w/w) or high (1.25% w/w) cholesterol were treated for 16 weeks with SKI-II, a sphingosine kinase 1 inhibitor that significantly reduced plasma S1P levels. SKI-II treatment increased atherosclerotic lesions in the thoracic aorta in mice on high but not low cholesterol diet. This compound did not affect body weight, blood cell counts and plasma total and HDL cholesterol, but decreased triglycerides. In addition, mice on high cholesterol diet receiving SKI-II showed elevated levels of tumor necrosis factor-α and endothelial adhesion molecules (sICAM-1, sVCAM-1). Conclusion: Prolonged lowering of plasma S1P produces pro-atherogenic effects in LDL-R-/- mice that are evident under condition of pronounced hypercholesterolemia.


2015 - Serum gonadotropins secretion is not reduced with advancing age in HIV-infected females: results of a case–control study in menopausal women. [Abstract in Rivista]
Diazzi, Chiara; Brigante, Giulia; Guaraldi, Giovanni; Simoni, Manuela; Rochira, Vincenzo
abstract

Introduction HIV infection treated with highly active antiretroviral therapy (HAART) seems to be associated with hypogonadism in men. Less is known in HIV-infected women gonadal status. The aim of this study is to investigate gonadal function, in menopausal HIV-infected women compared sex- and age-matched healthy subjects (HS). Methods We retrospectively compared 188 HIV-infected women with 192 HS selected reviewing record charts and laboratory database respectively. We considered only women older than 50 years and we grouped them according to their age (50–54; 55–59; and O60 years). Basal serum LH, FSH, estradiol, and testosterone were measured. The FSH cut-off of 40 UI/l for establishing menopausal status. Results The percentage of subjects with FSH levels O40 UI/l was higher in HIV-infected women (67.5%) than in healthy controls (59.4%). This difference was found also in the younger subgroup (38% vs 27%). FSH serum levels in HIV-infected women (54.08G31.47 mUI/ml) did not differ (PZ0.27) from HS (50.87G 31 mUI/ml). Accordingly, no significant differences were found in LH, estradiol, and testosterone levels. Conclusions Menopause seems to occur at a younger age than HS in HIV-infected women. Moreover, differently from what was documented in HIV-infected male counterpart, HIV-infected women seem to not develop hypogonadotropic hypogonadism, but have a tendency to higher serum FSH at a younger age (!54 years) suggesting premature hypergonadotropic hypogonadism. With this in view menopause may be considered an element of the process of premature aging associated with HIV infection and its comorbidities.


2015 - Telomerase in differentiated thyroid cancer: promoter mutations, expression and localization [Articolo su rivista]
Muzza, Marina; Colombo, Carla; Rossi, Stefania; Tosi, Delfina; Cirello, Valentina; Perrino, Michela; De Leo, Simone; Magnani, Elisa; Pignatti, Elisa; Vigo, Beatrice; Simoni, Manuela; Bulfamante, Gaetano; Vicentini, Leonardo; Fugazzola, Laura
abstract

Telomerase-reverse-transcriptase (TERT) promoter mutations have been recently described in tumors. In the present large series, TERT mutations were found in 12% of papillary thyroid cancers (PTCs) and in 14% of follicular thyroid cancers (FTCs), and were found to significantly correlate with older age at diagnosis and poorer outcome. Interestingly, the prognostic value of TERT mutations resulted to be significantly stronger than that of BRAF(V600E). Moreover, the outcome was not different among tumors with isolated TERT mutation and those with coexistent mutations (TERT/BRAF in PTCs or TERT/RAS in FTCs). TERT rs2853669 polymorphism was found in 44.4% of tumors. At WB, TERT was significantly more expressed in tumors than in normal samples, being the highest levels of expression recorded in TERT mutated cases. At IHC, in tumors and in metastatic lymph-nodes TERT staining was significantly higher in the cytoplasm than in the nucleus, whereas in normal tissue the degree of staining did not differ in the two cellular compartments. In conclusion, TERT mutations were shown to strongly correlate with a poorer outcome in differentiated thyroid tumors, and neither BRAF nor RAS mutation were found to confer an additional effect in the disease persistence. TERT protein was found to be more expressed in neoplastic than in normal tissues, and to display a different cellular localization, suggesting that it could contribute to thyroid cancer progression by mechanisms taking place in the cytoplasm.


2015 - Therapy of endocrine disease. Effects of chronic use of phosphodiesterase inhibitors on endothelial markers in type 2 diabetes mellitus: a meta-analysis. [Articolo su rivista]
Santi, Daniele; Giannetta, Elisa; Isidori, Andrea M; Vitale, Cristiana; Aversa, Antonio; Simoni, Manuela
abstract

OBJECTIVE: Diabetes mellitus (DM) is associated with endothelial dysfunction, reducing nitric oxide-dependent vasodilation, and increasing production of pro-inflammatory factors, leading to an increased risk of long-term cardiovascular disease. As the effects of phosphodiesterase 5 inhibitors (PDE5i) on endothelial function have not been systematically investigated, we conducted a meta-analysis of available randomized clinical trials (RCTs). DESIGN: A thorough search of the literature was carried out. Relevant studies were considered according to RCT study design, enrollment of men with type 2 DM, chronic administration of PDE5i, and evaluation of endothelial function through both hemodynamic and endothelial inflammation-related parameters. RESULTS: Fifteen studies fulfilled the eligibility criteria but only six RCTs met the inclusion criteria and were analyzed for 476 diabetic men, 239 randomized to Sildenafil, and 237 to placebo respectively. Four RCTs evaluated flow-mediated dilation (FMD), demonstrating a weighted mean increase of 2.19% (95% CI 0.48 to 3.90). This result showed a high heterogeneity (I(2): 98%). Thus, a further sub-group meta-analysis was performed and this analysis confirmed a significant, Sildenafil-related FMD improvement. Sildenafil improved endothelin 1 and high sensitivity C-reactive protein by ∼-0.94 pg/ml and -0.36 mg/l, respectively, not reaching statistical significance (P=0.69 and P=0.22 respectively). Finally, Sildenafil administration significantly reduced serum levels of interleukin 6 (IL6, -0.82 pg/ml; 95% CI -1.58 to -0.07). CONCLUSION: This meta-analysis suggests a beneficial effect of chronic PDE5i administration on endothelial function. Chronic Sildenafil administration seems to improve hemodynamic (FMD) and serum pro-inflammatory makers (IL6) in diabetic men. Larger studies are needed to confirm the effects of chronic PDE5i on endothelial function.


2015 - Unraveling the fertility knot in World Health Organization type 2 anovulatory women: another step toward a pharmacogenetic treatment choice [Articolo su rivista]
Simoni, Manuela
abstract

Follicle-stimulating hormone receptor polymorphism affects the outcome of ovulation induction in normogonadotropic (World Health Organization class 2) anovulatory subfertility.


2014 - Atheroprotective role of high-density lipoprotein (HDL)-associated sphingosine-1-phosphate (S1P) [Articolo su rivista]
Potì, Francesco; Simoni, Manuela; Nofer, Jerzy Roch
abstract

Numerous epidemiological studies documented an inverse relationship between plasma high-density lipoprotein (HDL) cholesterol levels and the extent of atherosclerotic disease. However, clinical interventions targeting HDL cholesterol failed to show clinical benefits with respect to cardiovascular risk reduction, suggesting that HDL components distinct from cholesterol may account for anti-atherogenic effects attributed to this lipoprotein. Sphingosine-1-phosphate (S1P)-a lysosphingolipid exerting its biological activity via binding to specific G protein-coupled receptors and regulating a wide array of biological responses in a variety of different organs and tissues including the cardiovascular system-has been identified as an integral constituent of HDL particles. In the present review, we discuss current evidence from epidemiological studies, experimental approaches in vitro, and animal models of atherosclerosis, suggesting that S1P contributes to atheroprotective effects exerted by HDL particles.


2014 - BRAF V600E mutation in washing liquid of thyroid fine-needle aspiration: a surprising tool in cytological benign nodules. [Abstract in Rivista]
Monzani, Maria Laura; Brigante, Giulia; Marino, Marco; L., Bonacini; Pignatti, Elisa; K., Cioni; Madeo, Bruno; Rochira, Vincenzo; Santi, Daniele; Maiorana, Antonino; Carani, Cesare; Simoni, Manuela
abstract

The good diagnostic value of a new method for BRAF V600E detection within the washing fluid of thyroid fine-needle aspiration biopsy has been demonstrated together with time and cost saving characteristics of this procedure.


2014 - Biosimilar recombinant follicle stimulating hormones in infertility treatment [Articolo su rivista]
Santi, Daniele; Simoni, Manuela
abstract

Follicle stimulating hormone (FSH) is a glycoprotein hormone essential for reproduction both in females and males and it is physiologically produced by the anterior pituitary gland in several isoforms. This heterogeneity is typical also of FSH-containing compounds, both urinary-derived and recombinant products. These compounds are widely used in assisted reproductive technologies (ART), to induce multifollicular development. Recently, the increased cost pressure on healthcare systems and the patent expiration date of widely used biotechnology-derived, recombinant FSH, prompted the pharmaceutical interest in FSH biosimilars.


2014 - Central hypogonadotropic hypogonadism: genetic complexity of a complex disease [Articolo su rivista]
Marino, Marco; Moriondo, Valeria; Vighi, Eleonora; Pignatti, Elisa; Simoni, Manuela
abstract

Central hypogonadotropic hypogonadism (CHH) is an emerging pathological condition frequently associated with overweight, metabolic syndrome, diabetes, and midline defects. The genetic mechanisms involve mutations in at least twenty-four genes regulating GnRH neuronal migration, secretion, and activity. So far, the mechanisms underlying CHH, both in prepubertal and in adulthood onset forms, remain unknown in most of the cases. Indeed, all detected gene variants may explain a small proportion of the affected patients (43%), indicating that other genes or epigenetic mechanisms are involved in the onset of CHH. The aim of this review is to summarize the current knowledge on genetic background of CHH, organizing the large amount of data present in the literature in a clear and concise manner, to produce a useful guide available for researchers and clinicians.


2014 - Delta-4 pathway steroid profiling by isotopic dilution, liquid chromatography-mass spectrometry after human chorionic gonadotropin stimulation in serum of men with Klinefelter Syndrome [Abstract in Atti di Convegno]
Belli, Serena; Antonio R. M., Granata; Rochira, Vincenzo; Flaminia, Fanelli; Carlotta, Pelusi; Marco, Mezzullo; Eleonora, Leoni; S., Tagliavini; Laura, Roli; Renato, Pasquali; Uberto, Pagotto; Simoni, Manuela
abstract

ABSTRACT Background: Klinefelter Syndrome (KS) is associated with primary hypogonadism. Recent studies demonstrate greater intratesticular testosterone (ITT) concentrations in KS than in controls. However, the steroidogenic reserve of the testis in KS has never been evaluated in detail. Only few and contradictory studies - mainly from the 80s of the last century - are available in the literature about stimulated serum steroid profile. Aim: To investigate whether an enzymatic block of steroidogenesis might explain hypogonadism in KS. Methods: 13 KS patients (34±8years) not receiving testosterone replacement therapy and 12 eugonadic controls (EC) (32±8years) were enrolled. Serum steroids were measured by Isotopic Dilution, Liquid Chromatography- Tandem Mass Spectrometry (ID-LC-MS/MS) at baseline and for 5 days after i.m. injection of Human Chorionic Gonadotropin (HCG), 5000 IU. Results: HCG raised significantly in serum of both KS and EC (ANOVA, p<0.001), without significant difference between the 2 groups (t-test, p=0.715). All Delta-4 pathway steroids showed a significant increase after HCG stimulation (ANOVA): progesterone (P) (p<0.001 in both groups), 17OH-progesterone (17OHP) (p<0.001 in both groups), androstenedione (A) (p=0.049 in SK and p<0.001 in EU), testosterone (T) (p<0.001 in both groups). The 17OHP/P ratio (p<0.001 in both groups) and T/A ratio (p=0.003 in KS and p<0.001 in EC) raised significantly after HCG. Comparison of response to HCG between patients and controls (t-test) resulted in significant differences for T (p=0.001), 17OHP/P (p=0.005) and T/A ratios (p=0.002). Conclusion: Leydig cells of KS patients are able to respond to HCG by increasing T production less than in EC. The different trend in the two groups of 17OHP/P and T/A ratios suggests a less efficient enzymatic activity of P450c17/POR and 17ßHSD3 in KS than in EC. This study supports a disturbed steroidogenesis in Leydig cells of KS. The simultaneous evaluation by ID-LC-MS/MS of both serum T and ITT levels after HCG in an experimental animal model of KS could help in clarifying the pathophysiology of hypogonadism in KS.


2014 - EAA/EMQN best practice guidelines for molecular diagnosis of Y-chromosomal microdeletions: state-of-the-art 2013. [Articolo su rivista]
Krausz, C; Hoefsloot, L; Simoni, Manuela; Tüttelmann, F; European, Academy of Andrology; European, Molecular Genetics Quality Network
abstract

The molecular diagnosis of Y-chromosomal microdeletions is a common routine genetic test which is part of the diagnostic workup of azoospermic and severe oligozoospermic men. Since 1999, the European Academy of Andrology (EAA) and the European Molecular Genetics Quality Network (EMQN) have been actively involved in supporting the improvement of the quality of the diagnostic assays by publication of the laboratory guidelines for molecular diagnosis of Y-chromosomal microdeletions and by offering external quality assessment trials. The present revision of the 2004 laboratory guidelines summarizes all the clinical novelties related to the Y chromosome (classic, partial and gene-specific deletions, genotype-phenotype correlations, methodological issues) and provides an update on the results of the quality control programme. These aspects also reflect the consensus of a large group of specialists present at a round table session during the recent Florence-Utah-Symposium on 'Genetics of male infertility' (Florence, 19-21 September, 2013). During the last 10 years the gr/gr deletion has been demonstrated as a significant risk factor for impaired sperm production. However, the screening for this deletion type in the routine diagnostic setting is still a debated issue among experts. The original basic protocol based on two multiplex polymerase chain reactions remains fully valid and appropriate for accurate diagnosis of complete AZF deletions and it requires only a minor modification in populations with a specific Y chromosome background. However, in light of novel data on genotype-phenotype correlations, the extension analysis for the AZFa and AZFb deletions is now routinely recommended. Novel methods and kits with excessively high number of markers do not improve the sensitivity of the test, may even complicate the interpretation of the results and are not recommended. Annual participation in an external quality control programme is strongly encouraged. The 12-year experience with the EMQN/EAA scheme has shown a steep decline in diagnostic (genotyping) error rate and a simultaneous improvement on reporting practice.


2014 - Espressione di miRNA-146a nel carcinoma follicolare della tiroide e correlazione con istotipo e stadiazione clinica [Abstract in Atti di Convegno]
Pignatti, Elisa; Vighi, Eleonora; Magnani, Elisa; Kara, Elda; Maiorana, Antonino; Rochira, Vincenzo; Carani, Cesare; Simoni, Manuela
abstract

This study investigates the relationship among miRNA-146a and clinical and histological correlates in follicular thyroid cancer


2014 - FSH treatment improves sperm DNA damage in men with idiopathic infertility carriers of the FSH receptor p.N680S homozygous N genotype: an interim analysis [Abstract in Atti di Convegno]
Santi, Daniele; Linari, Francesca; Baldi, E; Behre, Hm; Cambi, M; Ferlin, A; Gandini, L; Garolla, A; Krausz, C; Levi Setti, Pe; Lombardo, F; Marino, M; Muratori, M; Negri, L; Pignatti, E; Rastrelli, G; Simoni, Manuela
abstract

FSH treatment improves sperm DNA damage in men with idiopathic infertility carriers of the FSH receptor p.N680S homozygous N genotype: an interim analysis


2014 - FSHR polymorphism p.N680S mediates different responses to FSH in vitro [Articolo su rivista]
Casarini, Livio; Moriondo, Valeria; Marino, Marco; Adversi, Francesca; Capodanno, Francesco; Grisolia, Chiarina; LA MARCA, Antonio; LA SALA, Giovanni Battista; Simoni, Manuela
abstract

The single nucleotide polymorphism p.N680S of the follicle-stimulating hormone (FSH) receptor (FSHR) is a discrete marker of ovarian response but previous in vitro studies failed to demonstrate differences in the response to FSH between N and S carrier cells. Here we demonstrate that p.N680S mediates different kinetics of the response to FSH in vitro. Intracellular cAMP production is faster in p.N680S N than in S homozygous human granulosa cells (45 versus 90 min to achieve the plateau, respectively; Mann-Whitney's U-test; p < 0.005; n = 4). Reflecting the cAMP kinetics, phospho-ERK1/2 and -CREB activation, AREG and STARD1 gene expressions and progesterone production were qualitatively and quantitatively different in N versus S homozygous cells. Finally, the blockade of ERK pathway by U0126 abolishes the genotype-mediated different effects on gene expression and progesterone production (Mann-Whitney's U-test; p ≥ 0.005; n = 3).


2014 - Funzione gonadica e sessuale in uomini giovani/adulti con infezione da Human Immunodeficiency Virus (HIV) [Abstract in Atti di Convegno]
Santi, Daniele; Brigante, Giulia; C., Diazzi; S., De Vincentis; Zona, Stefano; Guaraldi, Giovanni; Simoni, Manuela; Rochira, Vincenzo
abstract

This study investigates male sexual function in men with HIV infection according with their gonadal status and circulating androgens


2014 - GH deficiency in HIV-infected patients compared to hypoopituitary patients [Abstract in Rivista]
Diazzi, Chiara; Brigante, Giulia; G., Ferrannini; Guaraldi, Giovanni; Ansaloni, Anna; Simoni, Manuela; Rochira, Vincenzo
abstract

The difference between HIV-infected patients with growth hormone deficiency (GHD) and GHD patients with hypopituitarism is in higher values go GH peak after GHRH+Arginine and IGF-1 in men with HIV.


2014 - GH deficiency in HIV-infected patients compared to hypopituitary patients [Abstract in Rivista]
Brigante, Giulia; Diazzi, Chiara; G., Ferrannini; S., De Vincentis; Guaraldi, Giovanni; Ansaloni, Anna; Simoni, Manuela; Rochira, Vincenzo
abstract

Gh peak after GHR+Arginine and IGF-1 are lower in HIV-infected patients with biochemical growth hormone deficiency than in patients with hypopituitarism and GHD.


2014 - Gene polymorphisms in female reproduction [Capitolo/Saggio]
Casarini, Livio; Simoni, Manuela
abstract

This chapter presents an overview of the gene polymorphisms underlying the functions of ovarian receptors and their clinical implications in the female fecundity. A selection of genetic studies revealing significant associations between receptor polymorphisms, gene mutations, and some pathological conditions (i.e., female infertility, premature ovarian failure, polycystic ovary syndrome, endometriosis) are reviewed.


2014 - Gonadal and sexual function in young/middle aged human immunodeficiency virus (HIV)-infected men [Abstract in Rivista]
Santi, Daniele; Brigante, Giulia; Diazzi, Chiara; S., De Vincentis; Zona, Stefano; Guaraldi, Giovanni; Simoni, Manuela; Rochira, Vincenzo
abstract

The study investigated male sexual function in men with HIV infection according with their gonadal status.


2014 - I pazienti con infezione da HIV hanno un reale deficit di GH (GHD) ? Confronto tra pazienti HIV positivi con documentato GHD biochimico e pazienti ipopituitarici HIV negativi con GHD [Abstract in Atti di Convegno]
Brigante, Giulia; Diazzi, Chiara; G., Ferrannini; Guaraldi, Giovanni; Ansaloni, Anna; Simoni, Manuela; Rochira, Vincenzo
abstract

The response to GHRH+Arginine is higher in HIV-infected patients with GHD when compared to patients with GHD and hypopituitarism


2014 - Identificazione di BRAF V600E nel liquido di lavaggio dell’agoaspirato: un nuovo strumento nella diagnostica del nodulo tiroideo [Abstract in Atti di Convegno]
Monzani, Maria Laura; Brigante, Giulia; Marino, Marco; L., Bonacini; Pignatti, Elisa; K., Cioni; Madeo, Bruno; Rochira, Vincenzo; Santi, Daniele; Maiorana, Antonino; Carani, Cesare; Simoni, Manuela
abstract

This study investigates the diagnostic value of a new methodology for the detection of the BRAF mutation in samples of washing fluid of thyroid fine needle aspiration biopsies. The study demonstrates that this method is valid, and time and cost saving.


2014 - Il volume tiroideo è ridotto in pazienti adulti affetti da beta-talassemia rispetto ai controlli [Abstract in Atti di Convegno]
Ansaloni, Anna; Diazzi, Chiara; Santi, Daniele; Brigante, Giulia; Ferrara, Francesca; Pietrangelo, Antonello; Simoni, Manuela; Rochira, Vincenzo
abstract

This study demonstrates that thyroid volume in adult patients with beta-thalassemia is reduced when compared with that of matched control subjects


2014 - Intratesticular testosterone is increased in men with Klinefelter syndrome and may not be released into the bloodstream owing to altered testicular vascularization– a preliminary report [Articolo su rivista]
Tuttelmann, F; Damm, O. S; Luetjens, C. M; Baldi, M; Zitzmann, M; Kliesch, S; Nieschlag, E; Gromoll, J; Wistuba, J; Simoni, Manuela
abstract

Klinefelter syndrome (KS, 47,XXY) is associated with low serum testosterone (T), long thought to arise from disturbed steroidogenesis in Leydig cells. However, intratesticular testosterone (ITT) concentrations were recently found to be normal in a KS mouse model(41,XXY*). So far, nothing was known about ITT concentrations in human patients with KS. Therefore, ITT, sex hormone-binding globulin (SHBG) and histological parameters were measured in human testicular biopsies of 11 KS patients, 30 azoospermic patients with Sertoli-cell-only syndrome and nine men with normal spermatogenesis as controls. ITT concentrations showed an overall pronounced excess over intratesticular SHBG in molar terms and were significantly increased in men with KS despite of reduced serum T levels. While the ratio of ITT/serum T was markedly increased in KS, the ITT/LH-ratio was comparable between all groups. After finding significantly increased ITT levels in men with KS, a finding even more striking than in the 41,XXY* KS mouse model, we set out to find a possible 'vascular' explanation for the lack of T release into the testicular blood stream. In testis biopsies from patients,reliable analysis of the vessels is, however, not possible because of the bias resulting from the dissection technique requiring avoidance of larger blood vessels to prevent bleeding. Consequently, the blood vessel constitution was evaluated in whole testis sections from adult male 41,XXY* and 40,XY*mice (n=5, each). Indeed, the blood vessel/testes surface ratio correcting for the smaller testes of XXY*mice was significantly lower in these mice compared with XY*controls. In conclusion, testicular T production does not seem to be impaired in men with KS. On the contrary, ITT concentrations are increased, but not because of increased SHBG activity. The data from the mouse model let us speculate that a reduced vascular bed might be involved in lower release of T into the blood stream.


2014 - Kallmann's syndrome and normosmic isolated hypogonadotropic hypogonadism: Two largely overlapping manifestations of one rare disorder [Articolo su rivista]
Bonomi, Marco; Cappa, Marco; Cariboni, Anna; Di Schiavi, Elia; Fabbri, Andrea; Ferlin, Alberto; Foresta, Carlo; Ghizzoni, Lucia; Jannini, Emmanuele; Krausz, Csilla; Loche, Sandro; Lombardo, Francesco; Maggi, Mario; Maggi, Roberto; Maghnie, Mohamad; Mancini, Antonio; Merlo, Giorgio; Panzica, Giancarlo; Radetti, Giorgio; Russo, Gianni; Simoni, Manuela; Sinisi, Antonio Agostino; Persani, Luca
abstract

Abstract


2014 - Lessons Learned in Andrology: Back to the future: making children in bed (at the right time) [Articolo su rivista]
Simoni, Manuela
abstract

Comment on personal view of current status of male infertility diagnosis and treatment


2014 - Mechanisms in endocrinology: Genetics of FSH action: a 2014-and-beyond view [Articolo su rivista]
Simoni, Manuela; Casarini, Livio
abstract

OBJECTIVE: To assess the pharmacogenetic potential of FSH for infertility treatment. DESIGN: Review of the literature and genomic databases. METHODS: Single-nucleotide polymorphism (SNP) assessed: rs6166 (c.2039A>G, p.N680S), rs6165 (c.919A>G, p.T307A), rs1394205 (c.-29G>A) in FSHR, and rs10835638 (c.-211G>T) in FSHB. Literature search via PubMed. Blast analysis of genomic information available in the NCBI nucleotide database. Comparison of allele frequency and haplotype distribution using the http://spsmart.cesga.estool. RESULTS: All these SNPs appear first in Homo, result in reduced FSH action, and are present with variable frequencies and combinations worldwide. Stringent clinical studies demonstrate that the FSHR genotype influences serum FSH levels and gonadal response in both sexes. Serum FSH levels depend on the -211G>T SNP, influencing transcriptional activity of the FSHB promoter. Genotypes reducing FSH action are overrepresented in infertile subjects. CONCLUSIONS: Although the clinical relevance of the FSHR polymorphisms alone is limited, the combination of FSHR and FSHB genotypes has a much stronger impact than either one alone in both sexes. About 20% of people are carriers of the alleles associated with lower serum FSH levels/reduced FSHR expression or activity, possibly less favorable for reproduction. Prospective studies need to investigate whether stratification of infertile patients according to their FSHR-FSHB genotypes improves clinical efficacy of FSH treatment compared with the current, naïve approach. A relative enrichment of less favorable FSHR-FSHB genotypes may be related to changes in human reproductive strategies and be a marker of some health-related advantage at the cost of reduced fertility.


2014 - Pre-miR146a expression in follicular carcinomas of the thyroid [Articolo su rivista]
Roncati, Luca; Pignatti, Elisa; Vighi, Eleonora; Magnani, Elisa; Kara, Elda; Rochira, Vincenzo; Carani, Cesare; Simoni, Manuela; Maiorana, Antonino
abstract

INTRODUCTION: Micro-RNA, a new class of small, non-coding RNAs, have been shown to be deregulated in several human carcinomas. In particular, SNP rs2910164 in pre-miR146a appears to be correlated with papillary thyroid carcinoma and may be involved in its genetic predisposition. Since data on follicular thyroid carcinomas (FTC) are lacking, we evaluated the involvement of SNP rs2910164 in FTC. METHODS: Thirty-nine cases of FTC and 20 follicular adenomas, defined according to WHO criteria, were selected. DNA and RNA were extracted from formalin-fixed paraffin-embedded blocks of both neoplastic and non-neoplastic areas. The DNA region of pre-miR146a, containing SNP rs2910164, was sequenced. Total RNA including miRNAs was used for stem-loop RT reactions, and applying a standard TaqMan PCR kit protocol for real-time PCR. Wilcoxon signed-rank test and Friedman test were used for statistical analyses. RESULTS: In 31% of FTC, the G allele was observed in neoplastic tissues, compared with the non-neoplastic areas (p < 0.05), whereas the CC phenotype was completely absent in tumours. Moreover, the expression of pre-miR146a was found to be significantly down-regulated in neoplastic tissues from FTC cases (p = 0.043), although no significant differences were seen in follicular thyroid adenomas. DISCUSSION: The expression profile of pre-miR146a can be correlated with FTC tumourigenesis. The G allele in SNP rs2910164 appears to be correlated with the transition from normal to neoplastic tissue. The GG and GC alleles appear to be associated with an increased risk for FTC, while the CC allele seems to play a protective role.


2014 - Pre-mir146a e FSHR sono marker di mosaicismo tiroideo nel carcinoma follicolare della tiroide [Abstract in Atti di Convegno]
Vighi, Eleonora; Pignatti, Elisa; Magnani, Elisa; Kara, Elda; Artuso, Lucia; Bernardis, Isabella; V., Cirello; Tagliafico, Enrico; Maiorana, Antonino; L., Fugazzola; Rochira, Vincenzo; Carani, Cesare; Simoni, Manuela
abstract

This study investigates the pre-mir146a within follicular thyroid cancer tissue and normal thyroid


2014 - Reassembly of somatic cells and testicular organogenesis in vitro [Articolo su rivista]
Reuter, Karin; Ehmcke, Jens; Stukenborg, Jan Bernd; Simoni, Manuela; Damm, Oliver S; Redmann, Klaus; Schlatt, Stefan; Wistuba, Joachim
abstract

Testicular organogenesis in vitro requires an environment allowing a reassembly of testicular cell types. Previous in vitro studies using male murine germ cells cultured in a defined three-dimensional environment demonstrated tubulogenesis and differentiation into spermatozoa. Combining scaffolds as artificial culture substrates with testicular cell culture, we analysed the colonization of collagen sponges by rat testicular cells focusing on cell survival and reassembly of tubule-like-structures in vitro. Isolated testicular cells obtained from juvenile Sprague Dawley and eGFP transgenic rats were cultured on collagen sponges (DMEM high glucose+Glutamax, 35°C, 5% CO2 with or without gonadotropins). Live cell imaging revealed the colonization of cells across the entire scaffold for up to 35 days. After two days, histology showed cell clusters attached to the collagen fibres and displaying signs of tubulogenesis. Clusters consisted mainly of Sertoli and peritubular cells which surrounded some undifferentiated spermatogonia. Flow cytometry confirmed lack of differentiation as no haploid cells were detected. Leydig cell activity was detected by a rise of testosterone after gonadotropin stimulation. Our approach provides a novel method which is in particular suitable to follow the somatic testicular cells in vitro an issue of growing importance for the analysis of germ line independent failure of spermatogenesis.


2014 - Relationship between testosterone and HIV-related comorbidities: secondary hypogonadism is associated with a poor health status in HIV-infected men [Abstract in Atti di Convegno]
Rochira, Vincenzo; Diazzi, Chiara; Brigante, Giulia; Santi, Daniele; Maria Chiara De, Caroli; Sara De, Vincentis; Simoni, Manuela; Carani, Cesare; Guaraldi, Giovanni
abstract

ABSTRACT INTRODUCTION: Testosterone (T) deficiency is very common in men with Human Immunodeficiency Virus (HIV), and it is more often associated with inappropriately low/normal luteinizing hormone (LH). However, the underlying causes remain poorly understood. Moreover, the role of HIV and/or HIV infection treatments, as well as the role of the general health status on the gonadal axis have been rarely investigated. AIM: The aim of this study is to evaluate the association between gonadal function and health status in men with HIV infection. METHODS: We performed a cross-sectional, observational study on 1359 consecutive HIV male outpatients. Morning serum Total T (TT), LH, estradiol, HIV parameters were measured. Frailty Index and number of comorbidities were extracted from the Clinical Database in which all patients data are recorded. TT<300 ng/dL was used as the threshold for biochemical T deficiency. RESULTS: T deficiency was found in 212 subjects (15.6%), and most of them (n=183; 13.4%) had secondary hypogonadism. TT resulted inversely related to Frailty Index in all patients (r=0.302, r2=0.091), this correlation being strengthened in HIV- infected men with secondary hypogonadism (r=0.403, r2=0.162). The percentage of HIVinfected men with TT <300 ng/dL increased progressively in accordance with the increase in the number of comorbidities (0.5%, 2.8%, 8.5%, 22.7%, 25.5%, 40% in men with 0, 1, 2, 3, 4, and >5 comorbidities, respectively). CONCLUSION: Poor health status in HIV-infected men might be involved in the pathogenesis of hypogonadism. This mechanism could reflect an adaptive response to illness in unhealthy patients similarly to what happens in other clinical conditions such as anorexia nervosa. Thus, low TT could be considered a biomarker of frailty and might confer an advantage for both the sick patients (in terms of sparing energy) and the species (preventing fatherhood). Furthermore, frailty related hypogonadism could be part of the process of premature aging already demonstrated in HIVinfected patients.


2014 - The FSHR polymorphism p.N680S mediates different response kinetics to FSH in vitro [Abstract in Atti di Convegno]
Casarini, Livio; Moriondo, Valeria; Marino, Marco; Adversi, Francesca; Grisolia, Chiarina; LA MARCA, Antonio; LA SALA, Giovanni Battista; Simoni, Manuela
abstract

Introduction: FSH acts on its receptor (FSHR) resulting in signal transduction activation, gene expression and steroidogenesis. The FSHR common SNP p.N680S is a marker of gonadal response in vivo. However, in vitro dose–response experiments failed to demonstrate the molecular basis thereof so far. In this study, we systematically investigated whether p.N680S mediates different kinetics of FSH response in vitro. Design: We evaluated the activation kinetics of cAMP, phERK1/2, phCREB by ELISA and western blotting in FSHR homozygous, primary, human granulosa lutein cells (hGLC-680N, -680S) stimulated by 50 nM r-FSH for up to 2 h (short-term stimulation). Following short-term stimulation the expression of target genes was evaluated by real-time PCR after 12 h, and progesterone production kinetics over 24 h. Specific inhibitors/agonists (U0126, PMA) were used in the presence and in the absence of FSH. Results: Intracellular cAMP increased within 5–10 min in hGLC-680N, reaching the plateau in about 45 min. cAMP increase was delayed in hGLC-680S, reaching the plateau in 120 min, revealing different activation kinetics (Mann–Whitney U test; P<0.05; n=4). r-FSH-dependent cAMP stimulation kinetics resulted in different ERK1/2 and CREB phosphorylation, reaching maximal levels in 5–30 min in hGLC-680N, whereas, in hGLC-680S, these were weaker and steady over 2 h (Mann–Whitney U test; P<0.05; n=3). hGLC-680N stimulation resulted in higher expression levels of AREG and StAR (Mann–Whitney U test; P<0.05; n=4) and in subsequently different progesterone production kinetics, achieving overall higher levels in hGLC-680N vs -680S (Mann–Whitney U test; P<0.05; n=3). Interestingly, the different kinetics of progesterone production between hGLC-680N and -680S were interchanged by selective phospho-ERK1/2 blockade/activation through specific inhibitor/agonist, revealing a short-term cross-talk mediated by ERK1/2. Conclusions: This study demonstrates for the first time in vitro, how FSHR p.N680S mediates different response to FSH, resulting in different kinetics of cAMP, phERK1/2 and phCREB activation, and progesterone production.


2014 - The PCOS evolutionary paradox: a GWAS-based, in silico, evolutionary explanation [Abstract in Atti di Convegno]
Casarini, Livio; Simoni, Manuela
abstract

Introduction: PCOS is a common endocrine disorder in women exhibiting characteristics ranging from hyperandrogenic to metabolic phenotypes, more prevalent in people of African/Caucasian and Asian ancestry, respectively. Since PCOS impairs fertility without diminishing in prevalence, it was discussed as an evolutionary paradox. GWAS identified 17 SNPs with different allele frequencies, depending on ethnicity, in various susceptibility loci (FSHR, LHCGR, DENND1A, THADA, C9ORF3, YAP1, HMGA2, RAB5B/SUOX, INSR, TOX3, and SUMO1P1). The aim of this study was to analyze in silico the PCOS phenotype–genotype relationship using these SNPs for analysis of genetic clustering and distance, two measures of the degree of similarity of genetic data. Methods: HapMap and HGDP databases (hapmap.ncbi.nlm.nih.gov; www.hagsc.org/hgdp/files.html) were used as source of allele frequencies of the 17 SNPs, using data from 622 male and female individuals of various populations, grouped in Africans, Americans, European-Caucasians, Mediterranean-Middle Easterns, Central Asians, Oceanians and East Asians. Genetic clustering was calculated from SNPs data by Bayesian analysis using the STRUCTURE software (burn-in=5000/50000 MCMC reps; iterations=20; 2<K<10). The inferred ancestry of individuals was matched with PCOS phenotype data of each group, extracted from a previous meta-analysis. The measure of genetic distance was plotted against the geographic distance between the populations. Results: The 622 male and female individuals were assigned to five genetic clusters, matching with different world regions (Kruskal–Wallis/Dunn’s post-test; P<0.0001), and converging in only two main PCOS phenotypes (Anova/Bonferroni post-test; P<0.0001). The overall genetic distance, calculated using PCOS markers, increased along with the geographic distance among the populations (linear regression; r2=0.2106; P<0.0001), in a phenotype-unrelated manner. Conclusions: Phenotype–genotype correlations were demonstrated for PCOS, suggesting that its genetic gradient results from genetic drift together with intralocus sexual conflict rather than natural selection of phenotypic traits in females. Recognizing the genetic background may be important for the correct pharmacological approach to PCOS treatment.


2014 - The TRHR Gene Is Associated with Hypothalamo-Pituitary Sensitivity to Levothyroxine [Articolo su rivista]
Brigante, Giulia; Spaggiari, Giorgia; Santi, Daniele; Cioni, Katia; Gnarini, Valentina; Diazzi, Chiara; Pignatti, Elisa; Casarini, Livio; Marino, Marco; Tüttelmann, Frank; Carani, Cesare; Simoni, Manuela
abstract

Thyroidectomized patients need variable doses of levothyroxine (LT4) to obtain target thyroid-stimulating hormone (TSH) levels. Individual feedback set-points have been hypothesized and the influence of several genes in the regulation of the pituitary-thyroid axis has been demonstrated.


2014 - Thyroid volume in adult beta-thalassemic patients is smaller than in controls [Abstract in Rivista]
Ansaloni, Anna; Diazzi, Chiara; Santi, Daniele; Brigante, Giulia; Ferrara, Francesca; Carani, Cesare; Pietrangelo, Antonello; Simoni, Manuela; Rochira, Vincenzo
abstract

Thyroid volume is significantly reduced in adult patients with beta-thalassemia than in matched controls


2013 - Are pre-miR-146a and PTTG1 associated with papillary thyroid cancer? [Articolo su rivista]
Marino, Marco; Valentina, Cirello; Gnarini, Valentina; Carla, Colombo; Pignatti, Elisa; Casarini, Livio; Diazzi, Chiara; Rochira, Vincenzo; Katia, Cioni; Madeo, Bruno; Carani, Cesare; Simoni, Manuela; Laura, Fugazzola
abstract

Papillary thyroid carcinoma (PTC) is the most common endocrine malignancy, with a steadily increasing incidence in the last few decades worldwide. The predisposition to developing this carcinoma by the heterozygous state of rs2910164 within the precursor of the miR-146a has been reported, but recently not confirmed. Interestingly, on the same chromosome, almost 50 kb separate the pre-miR-146a from the pituitary tumor-transforming gene 1 (PTTG1), a proto-oncogene involved in several tumors, including thyroid cancers. In this study, we analyzed, using a case–control design, the genetic association between PTC and the genomic region encompassing pre-miR-146a rs2910164 and PTTG1 rs1862391 and rs2910202. We enrolled 307 affected patients and 206 healthy controls. The possible presence of thyroid nodules in controls was excluded by ultrasonography. All the cases were submitted to single- nucleotide polymorphism (SNP) genotyping of pre-miR-146a and PTTG1, and risk association analyses were carried out. The genotypic and allelic frequencies of pre-miR-146a rs2910164 were not statistically different in the patients and controls, and this SNP was not in linkage disequilibrium with the investigated PTTG1 SNPs. Consistently, meta-analyses, the first including all the affected cases published to date, did not confirm the previously reported association of the heterozygous CG genotype with PTC. The PTTG1 SNPs exhibited the same allelic frequency in the patients and controls and were not associated with the disease. In conclusion, in a well-selected Italian population, neither pre-miR-146a rs2910164 nor PTTG1 rs1862391 and rs2910202 were found to be associated with the risk of developing PTC.


2013 - Association of pre-miR-146a rs2910164 GG genotype with papillary thyroid cancer: a new case control study on two adjacent genes on chromosome 5, pre-miR-146a and PTTG1 [Abstract in Rivista]
Marino, Marco; Valentina, Cirello; Gnarini, Valentina; Pignatti, Elisa; Casarini, Livio; Diazzi, Chiara; Rochira, Vincenzo; Katia, Cioni; Madeo, Bruno; Simoni, Manuela; Laura, Fugazzola
abstract

Role of the pre-miR-146a and PTTG1 on papilary thyroid cancer


2013 - Complete aromatase deficiency in four adult men: detection of a novel mutation and two known mutations in the CYP19A1 gene [Abstract in Rivista]
Pignatti, Elisa; K. M., Ajlouni; N., Khawaja; K., Unluhizarci; E., Kartal; Carani, Cesare; Simoni, Manuela; Marino, Marco; Vighi, Eleonora; Rochira, Vincenzo
abstract

The abstracts descibes four new cases of patients with aromatase deficiency. Both the clinical features and the results of the molecular studies are reported.


2013 - Complete aromatase deficiency in four adult men: detection of a novel mutation and two known mutations in the CYP19A1 gene [Abstract in Rivista]
Pignatti, Elisa; K., Ajlouni; N., Khawaja; K., Unluhizarci; E., Kartal; Carani, Cesare; Marino, Marco; Simoni, Manuela; Vighi, Eleonora; Rochira, Vincenzo
abstract

The study describes 4 new cases of the rare disease aromatase deficiency


2013 - Complete aromatase deficiency in four adult men: detection of a novel mutation and two known mutations in the CYP19A1 gene [Abstract in Rivista]
Pignatti, Elisa; Kursad, Unluhizarci; Ermine, Kartal; Kamel, Ajlouni; Nahla, Khawaja; Carani, Cesare; Marino, Marco; Simoni, Manuela; Vighi, Eleonora; Rochira, Vincenzo
abstract

The abstract deals with the clinical and genetic description of 4 new cases of aromatase deficiency.


2013 - Effects of treatment for acromegaly on Bone Mineral Density (BMD): is Pegvisomant protective on lumbar BMD? [Abstract in Rivista]
Brigante, Giulia; Santi, Daniele; Diazzi, Chiara; S., De Vincentis; G., Ferrannini; Madeo, Bruno; Simoni, Manuela; Carani, Cesare; Rochira, Vincenzo
abstract

The abstract deals with the different effects of treatments for acromegaly on bone


2013 - Effects of treatment for acromegaly on bone mineral density (BMD): is pegvisomant protective on lumbar BMD? [Abstract in Rivista]
Brigante, Giulia; Santi, Daniele; Diazzi, Chiara; S., De Vincetis; G., Ferrannini; Madeo, Bruno; Simoni, Manuela; Rochira, Vincenzo; Carani, Cesare
abstract

The type of medical therapy for acromegaly may have different effects on bone mineral density


2013 - Effects of treatment for acromegaly on bone mineral density: is pegvisomant protective on lumbar BMD? [Abstract in Rivista]
Brigante, Giulia; Santi, Daniele; Diazzi, Chiara; Sara De, Vincentis; Giulia, Ferrannini; Madeo, Bruno; Carani, Cesare; Simoni, Manuela; Rochira, Vincenzo
abstract

The type of pharmacological treatment used for the control of acromegaly might have different effect in the prevention of bone loss.


2013 - Experimental endocrine manipulation by contraceptive regimen in the male marmoset (Callithrix jacchus) [Articolo su rivista]
Wistuba, Joachim; Luetjens, C. Marc; Ehmcke, Jens; Redmann, Klaus; Damm, Oliver S; Steinhoff, Antje; Sandhowe Klaverkamp, Reinhild; Nieschlag, Eberhard; Simoni, Manuela; Schlatt, Stefan
abstract

Marmosets are used as preclinical model in reproductive research. In contrast to other primates, they display short gestation times rendering this species valid for exploration of effects on fertility. However, their peculiar endocrine regulation differs from a those of macaques and humans. We subjected male marmosets to previously clinically tested hormonal regimens that are known to effectively suppress spermatogenesis. Beside a control group, seven groups (each n=6) were investigated for different periods of up to 42 months: regimen I, (four groups) received testosterone undecanoate (TU) and norethisterone enanthate (NETE); regimen II, (two groups) received TU and NETE followed by NETE only; and regimen III, (one group) received NETE only. Testicular volume, cell ploidy and histology, endocrine changes and fertility were monitored weekly. TU and NETE and initial TU and NETE treatment followed by NETE failed to suppress spermatogenesis and fertility. Testicular volumes dropped, although spermatogenesis was only mildly affected; however, testicular cellular composition remained stable. Serum testosterone dropped when NETE was given alone but the animals remained fertile. Compared with controls, no significant changes were observed in sperm motility and fertility. Administration of TU and NETE affected testicular function only mildly, indicating that the regulatory role of chorionic gonadotrophin and testosterone on spermatogenesis is obviously limited and testicular function is maintained, although the endocrine axis is affected by the treatment. In conclusion, marmosets showed a different response to regimens of male contraception from macaques or men and have to be considered as a problematic model for preclinical trials of male hormonal contraception.


2013 - KRP-203, Sphingosine 1-Phosphate Receptor Type 1 Agonist, Ameliorates Atherosclerosis in LDL-R-/- Mice. [Articolo su rivista]
Poti', Francesco; Gualtieri, Fabio; Sacchi, Sandro; Weißen Plenz, G; Varga, G; Brodde, M; Weber, C; Simoni, Manuela; Nofer, Jr
abstract

Objective—Sphingosine 1-phosphate (S1P) partly accounts for antiatherogenic properties of high-density lipoproteins. We previously demonstrated that FTY720, a synthetic S1P analog targeting all S1P receptors but S1P receptor type 2, inhibits murine atherosclerosis. Here, we addressed the identity of S1P receptor mediating atheroprotective effects of S1P. Approach and Results—Low-density lipoprotein receptor–deficient mice on cholesterol-rich diet were given selective S1P receptor type 1 agonist KRP-203 (3.0 mg/kg per day; 6 and 16 weeks). KRP-203 substantially reduced atherosclerotic lesion formation without affecting plasma lipid concentrations. However, KRP-203 induced lymphopenia, reduced total (CD4+, CD8+) and activated (CD69+/CD8+, CD69+/CD4+) T cells in peripheral lymphoid organs, and interfered with lymphocyte function, as evidenced by decreased T-cell proliferation and interleukin-2 and interferon-γ production in activated splenocytes. Cyto- and chemokine (tumor necrosis factor-α, regulated and normal T cell expressed and secreted) levels in plasma and aortas were reduced by KRP-203 administration. Moreover, macrophages from KRP-203–treated mice showed reduced expression of activation marker MCH-II and poly(I:C)-elicited production of tumor necrosis factor-α, monocyte chemoattractant protein-1, and interleukin-6. In vitro studies demonstrated that KRP-203 reduced tumor necrosis factor-α, interleukin-6, and interferon-γ–induced protein-10 production; IκB and signal transducer and activator of transcription-1 phosphorylation; and nuclear factor κB and signal transducer and activator of transcription-1 activation in poly(I:C)-, lipopolysaccharide-, or interferon-γ–stimulated bone marrow macrophages, respectively. Conclusions—Present results demonstrate that activation of S1P signaling pathways inhibit atherosclerosis by modulating lymphocyte and macrophage function and suggest that S1P receptor type 1 at least partially mediates antiatherogenic effects of S1P.


2013 - Leydig cell tumor in an anabolic steroid abuser [Articolo su rivista]
Belli, Serena; Guidi, Alessandro; Simoni, Manuela; Carani, Cesare; Granata, A. R.
abstract

Case report


2013 - Mutations and somatic changes in the genotype of rs2910164 in pre-miR146a are frequent in follicular thyroid carcinoma [Abstract in Rivista]
Vighi, Eleonora; Pignatti, Elisa; Roncati, Luca; Rochira, Vincenzo; Kara, Elda; Madeo, Bruno; E., Magnani; Maiorana, Antonino; Carani, Cesare; Simoni, Manuela
abstract

Mutations and somatic changes in the genotype of rs2910164 in pre-miR146a are frequent in follicular thyroid carcinoma and may be associated with its biological behavior


2013 - New insights on follicular thyroid carcinoma: the role of pre-mir-146a [Abstract in Rivista]
Pignatti, Elisa; Vighi, Eleonora; Roncati, Luca; E., Magnani; Kara, Elda; Madeo, Bruno; Rochira, Vincenzo; Maiorana, Antonino; Carani, Cesare; Simoni, Manuela
abstract

Role of pre-mir-146a on follicular thyroid cancer


2013 - Papillary thyroid cancer: a new case-control study involving pre-mir-146a and PTTG1 genes [Abstract in Rivista]
Marino, Marco; V., Cirello; Gnarini, Valentina; Pignatti, Elisa; Casarini, Livio; Diazzi, Chiara; Rochira, Vincenzo; K., Cioni; Madeo, Bruno; Simoni, Manuela; L., Fugazzola
abstract

Studio genetico dei carcinomi papillari della tiroide


2013 - Polymorphisms in gonadotropin and gonadotropin receptor genes as markers of ovarian reserve and response in in vitro fertilization [Articolo su rivista]
LA MARCA, Antonio; Sighinolfi, Giovanna; Argento, Cindy; Grisendi, Valentina; Casarini, Livio; Volpe, Annibale; Simoni, Manuela
abstract

Since gonadotropins are the fundamental hormones that control ovarian activity, genetic polymorphisms may alter gonadal responsiveness to glycoproteins; hence they are important regulators of hormone activity at the target level. The establishment of the pool of primordial follicles takes place during fetal life and is mainly under genetic control. Consequently, single nucleotide polymorphisms (SNPs) in gonadotropins and their receptors do not seem to be associated with any significant modification in the endowment of nongrowing follicles in the ovary. Indeed, the age at menopause, a biological characteristic strongly related to ovarian reserve, as well as markers of functional ovarian reserve such as anti-Müllerian hormone and antral follicle count, are not different in women with different genetic variants. Conversely, some polymorphisms in FSH receptor (FSHR) seem to be associated with modifications in ovarian activity. In particular, studies suggest that the Ser680 genotype for FSHR is a factor of relative resistance to FSH stimulation resulting in slightly higher FSH serum levels, thus leading to a prolonged duration of the menstrual cycle. Moreover, some FSHR gene polymorphisms show a positive association with ovarian response to exogenous gonadotropin administration, hence exhibiting some potential for a pharmacogenetic estimation of the FSH dosage in controlled ovarian stimulation. The study of SNPs of the FSHR gene is an interesting field of research that could provide us with new information about the way each woman responds to exogenous gonadotropin administration during ovulation induction.


2013 - Pre-miR146a expression profiling of follicular thyroid carcinoma [Abstract in Rivista]
Pignatti, Elisa; Vighi, Eleonora; Roncati, Luca; Kara, Elda; Eleonora, Porcheddu; Elisa, Magnani; Rochira, Vincenzo; Maiorana, Antonino; Carani, Cesare; Simoni, Manuela
abstract

Pre-miR146a may be a marker for predicitng the phenotype of thyroid follicular carcinomas


2013 - Prevalence of olfactory and other developmental anomalies in patients with central hypogonadotropic hypogonadism [Articolo su rivista]
DELLA VALLE, Elisa; Vezzani, Silvia; Rochira, Vincenzo; Antonio R. M., Granata; Madeo, Bruno; Genovese, Elisabetta; Pignatti, Elisa; Marino, Marco; Carani, Cesare; Simoni, Manuela
abstract

Introduction: Hypogonadotropic hypogonadism (HH) is a heterogeneous disease caused by mutations in several genes. Based on the presence of hyposmia/anosmia it is distin- guished into Kallmann syndrome (KS) and isolated HH. The prevalence of other develop- mental anomalies is not well established. Methods: We studied 36 patients with HH (31 males, 5 females, mean age 41.5), 9 with familial and 27 with sporadic HH (33 congenital, 3 adult-onset), by physical examination, smell test (BSIT Sensonics), audiometry, renal ultrasound, and magnetic resonance imag- ing of the olfactory structures. Results: Based on the smell test, patients were classified as normosmic (n = 21, 58.3%) and hypo/anosmic (n = 15, 41.6%). Hypoplasia/agenesis of olfactory bulbs was found in 40% of patients (10/25; 75% hypo/anosmic, 7.6% normosmic, p<0.01, Fisher’s test). Remarkably, olfactory structures were normal in two anosmic patients, while one nor- mosmic patient presented a unilateral hypoplastic bulb. Fourteen of 33 patients (42.4%) presented neurosensorial hearing loss of various degrees (28.5% hypo/anosmic, 52.6% normosmic, p=NS). Renal ultrasound revealed 27.7% of cases with renal anomalies (26.6% hypo/anosmic, 28.5% normosmic, p = NS). At least one midline defect was found in 50% of the patients (53.3% hypo/anosmic, 47.6% normosmic, p = NS), including abnor- mal palate, dental anomalies, pectus excavatum, bimanual synkinesis, iris coloboma, and absent nasal cartilage. Anamnestically 4/31 patients reported cryptorchidism (25% hypo/anosmic, 5.2% normosmic, p = NS). Conclusion: Hypo/anosmia is significantly related to anatomical anomalies of the olfac- tory bulbs/tracts but the prevalence of other developmental anomalies, especially midline defects and neurosensorial hearing loss, is high both in HH and KS and independent of the presence of anosmia/hyposmia. From the clinical standpoint KS and normosmic HH should be considered as the same complex, developmental disease.


2013 - SNP RS2910164 in pre-mir146a undergoes somatic changes in follicular thyroid carcinoma [Abstract in Rivista]
Vighi, Eleonora; Pignatti, Elisa; Roncati, Luca; Rochira, Vincenzo; Kara, Elda; Madeo, Bruno; E., Magnani; Maiorana, Antonino; Carani, Cesare; Simoni, Manuela
abstract

Role of SNP in follicular thyroid cancer


2013 - The combination of genetic variants of the FSHB and FSHR genes affects serum FSH in women of reproductive age [Articolo su rivista]
LA MARCA, Antonio; Papaleo, E; Alviggi, C; Ruvolo, G; De Placido, G; Candiani, M; Cittadini, E; De Michele, F; Moriondo, Valeria; Catellani, V; Volpe, Annibale; Simoni, Manuela
abstract

What is the effect of FSHB-211G>T together with the FSHR 2039 A>G on serum FSH in women?


2013 - The diagnostic value of calcitonin measurement in wash-out fluid from fine-needle aspiration of thyroid nodules in the diagnosis of medullary thyroid cancer [Articolo su rivista]
Diazzi, Chiara; Madeo, Bruno; Taliani, Erica; Zirilli, Lucia; Romano, Stefania; Antonio R. M., Granata; Maria Cristina De, Santis; Simoni, Manuela; Katia, Cioni; Carani, Cesare; Rochira, Vincenzo
abstract

Objectives: The diagnostic value of calcitonin measurement in fine-needle aspiration biopsy (FNAB) wash-out fluid (Ct-FNAB) for medullary thyroid cancer (MTC) remains to be determined. This prospective study aims to assess the diagnostic value of Ct-FNAB in thyroid nodules in comparison with basal serum calcitonin (Ct), Pentagastrin-stimulated Ct (Pg-sCt), and cytology. Methods: Among patients with goiter addressed to US-FNAB having initial clinical suggestion for thyroidectomy, 27 patients with thyroid nodule/s (n=60), normal, borderline or increased Ct, fulfilled criteria for thyroidectomy. All 27 patients (enrolled according to exclusion/inclusion criteria) underwent ultrasonography (US), Ct, Pg-sCt, US-assisted FNAB of each patient’s nodule for both cytology, and Ct-FNAB before thyroidectomy. Results: Ct-FNAB resulted always >1000 pg/mL in MTC nodules at histology. For values between 36 and 1000 pg/mL, MTCs and nodular or micronodular C-cell Hyperplasia (CCH) results overlapped. Most of the nodules without MTC and/or CCH had Ct-FNAB<17 pg/mL. Ct-FNAB diagnostic power was superior to and similar to other diagnostic procedures (Ct, Pg-sCt, and cytology) in identifying both MTC and CCH, and MTC alone, respectively. Conclusion: The diagnostic power of Ct-FNAB is valuable compared with other routine procedures. Ct-FNAB is highly reliable for the early detection and accurate localization of MTC in thyroid nodules, but it does not differentiate between MTC and CCH. Ct-FNAB is an extremely valuable diagnostic tool, especially considering that other diagnostic procedures do not provide a definitive diagnosis, and it can be included in the clinical work up of thyroid nodules when MTC is suspected.


2013 - The prognostic value of miRNA146a in follicular thyroid carcinoma [Articolo su rivista]
Roncati, Luca; Simoni, Manuela; Maiorana, Antonino
abstract

The presence of miRNA 146a was studied in a series of follicular carcinomas of the thyroid. Its prognostic value is discussed.


2013 - Very high prevalence of ultrasound thyroid scan abnormalities in healthy volunteers in Modena, Italy [Articolo su rivista]
Gnarini, Valentina; Brigante, Giulia; DELLA VALLE, Elisa; Diazzi, Chiara; Madeo, Bruno; Carani, Cesare; Rochira, Vincenzo; Simoni, Manuela
abstract

Background: Italy is characterized by high preva- lence of goiter. To date, only limited data about the preva- lence of goiter in the Italian adult population are available. Aim: To investigate the prevalence of thyroid ultrasound ab- normalities in adults unaware of any thyroid disease and eval- uate the rate of differentiated thyroid cancer (DTC) obtained by this intervention. Methods: Ultrasound (US) thyroid scan was performed in adult volunteers recruited by advertisement in Modena, Italy. One hundred and thirty-five women and 66 men (no.= 201), unaware of any thyroid disease (mean age of 46±10.7 yr) underwent their first thyroid US scan. Results: US thyroid abnormalities were found in 101 subjects (50.3%): 91 nodular goiters (45.2%) and 13 US-thyroiditis (6.5%) associ- ated with positive auto-antibodies in 11 of them. Seventeen subjects (18%) with nodules underwent US-fine needle aspiration biopsy with the following cytological class (C) outcome: 14 patients C2 (82%), 1 patient C3 (6%), 2 patients had C4 (12%), the latter received histological confirmation. Conclu- sions: The prevalence of thyroid abnormalities is very high in subjects unaware of any thyroid disease. DTC was found in 1% of subjects and in 2% of those affected by nodular goi- ter. Compared to the detection rate of the well-established screening programs for breast (0.45%) and colorectal (0.27%) cancer, the prevalence of DTC seems to be much higher. Thy- roid US screening could allow the detection of DTC in asymp- tomatic subjects and this diagnosis often includes DTC at an advanced stage. Thus, US screening not necessarily results in the over-diagnosis of clinically not relevant thyroid diseases.


2012 - Aromatase expression in human peripheral blood leukocytes (PBLs) and in various tissues in primates: studies in elderly humans and cynomolgus monkeys [Articolo su rivista]
Pignatti, Elisa; Casarini, Livio; S., Scaltriti; J., Wistuba; S., Schlatt; A., Rossi; A., Lachhab; E., Taliani; Carani, Cesare; Simoni, Manuela
abstract

Background Previous analysis of aromatase gene and protein expression in PBLs, studied in children and adults, were extended to elderly subjects. In addition we assessed whether aromatase expression in PBLs could be used as a parameter of aromatase expression in other tissues, using the cynomolgus monkey as model. Methods Real-time analysis of aromatase gene expression and protein evaluation by Western blot were performed in PBLs of human elderly subjects and in various tissues from cynomolgus monkeys. Results No gender-related difference in CYP19A1 mRNA and protein expression in PBLs from human elderly women and men was found. In elderly male cynomolgus monkeys CYP19A1 mRNA and protein were expressed in all cells and tissues analysed, with the lowest levels in PBLs but no clear-cut correlation with other tissues. Conclusions Aromatase expression in PBLs in elderly human subjects is not gender-related and cannot be a surrogate of aromatase expression for other tissues.


2012 - Effect of sphingosine 1-phosphate (S1P) receptor agonists FTY720 and CYM5442 on atherosclerosis development in LDL receptor deficient (LDL-R(-/-) mice [Articolo su rivista]
F., Potì; S., Costa; V., Bergonzini; M., Galletti; Pignatti, Elisa; C., Weber; Simoni, Manuela; J. R., Nofer
abstract

Objectives: Sphingosine 1-phosphate (S1P) – a lysosphingolipid present in HDL – exerts atheroprotective ef- fects in vitro, while FTY720, a non-selective S1P mimetic inhibits atherosclerosis in LDL receptor-deficient (LDL-R−/−) mice under conditions of severe hypercholesterolemia. We here examined the effect of FTY720 and a selective S1P receptor type 1 agonist CYM5442 on atherosclerosis in moderately hypercholesterolemic LDL-R−/− mice.Methods and results: LDL-R−/− mice fed Western diet (0.25% cholesterol) were given FTY720 (0.4 mg/kg/day) or CYM5442 (2.0 mg/kg/day) for 18 weeks. FTY720 but not CYM5422 persistently lowered blood lympho- cytes, depleted CD4+ and CD8+ T cells in spleen and lymph nodes, and reduced splenocyte IL-2 secretion. However, both compounds reduced the activity of splenic and peritoneal macrophages as inferred from the down-regulated CD68 and MHC-II expression in CD11b+ cells and the reduced IL-6 secretion in response to LPS, respectively. CYM5442 and FTY720 reduced weight gain, white adipose tissue depots and fasting glu- cose suggesting improvement of metabolic control, but failed to influence atherosclerosis in LDL-R−/− mice. Conclusion: Despite down-regulating macrophage function and – in case of FTY720 – altering lymphocyte dis- tribution CYM5442 and FTY720 fail to affect atherosclerosis in moderately hypercholesterolemic LDL-R−/− mice. We hypothesize that S1P mimetics exert atheroprotective effects only under conditions of increased cholesterol burden exacerbating vascular inflammation.


2012 - LH and hCG Action on the Same Receptor Results in Quantitatively and Qualitatively Different Intracellular Signalling [Articolo su rivista]
Casarini, Livio; Lispi, M.; Longobardi, S.; Milosa, F.; LA MARCA, Antonio; Tagliasacchi, Daniela; Pignatti, Elisa; Simoni, Manuela
abstract

Human luteinizing hormone (hLH) and chorionic gonadotropin (hCG) act on the same receptor (LHCGR) but it is not known whether they elicit the same cellular and molecular response. This study compares for the first time the activation of cell-signalling pathways and gene expression in response to hLH and hCG. Using recombinant hLH and recombinant hCG we evaluated the kinetics of cAMP production in COS-7 and hGL5 cells permanently expressing LHCGR (COS-7/LHCGR, hGL5/LHCGR), as well as cAMP, ERK1/2, AKT activation and progesterone production in primary human granulosa cells (hGLC). The expression of selected target genes was measured in the presence or absence of ERK- or AKT-pathways inhibitors. In COS-7/LHCGR cells, hCG is 5-fold more potent than hLH (cAMP ED50: 107.1±14.3 pM and 530.0±51.2 pM, respectively). hLH maximal effect was significantly faster (10 minutes by hLH; 1 hour by hCG). In hGLC continuous exposure to equipotent doses of gonadotropins up to 36 hours revealed that intracellular cAMP production is oscillating and significantly higher by hCG versus hLH. Conversely, phospho-ERK1/2 and -AKT activation was more potent and sustained by hLH versus hCG. ERK1/2 and AKT inhibition removed the inhibitory effect on NRG1 (neuregulin) expression by hLH but not by hCG; ERK1/2 inhibition significantly increased hLH- but not hCG-stimulated CYP19A1 (aromatase) expression. We conclude that: i) hCG is more potent on cAMP production, while hLH is more potent on ERK and AKT activation; ii) hGLC respond to equipotent, constant hLH or hCG stimulation with a fluctuating cAMP production and progressive progesterone secretion; and iii) the expression of hLH and hCG target genes partly involves the activation of different pathways depending on the ligand. Therefore, the LHCGR is able to differentiate the activity of hLH and hCG.


2012 - Methodology for measuring testosterone, dihydrotestosterone and sex hormonebinding globulin in a clinical setting [Capitolo/Saggio]
Simoni, M.; Fanelli, F.; Roli, L.; Pagotto, U.
abstract

Testosterone is a hormone difficult to measure accurately. Yet, its accurate determination is the prerequisite for the correct diagnosis and clinical management of hypogonadism in males and hyperandrogenism in females. In the last decade a number of studies increased awareness of the poor performance of most of the current assays and identified some strategies to improve the accuracy of testosterone testing (Rosner et al. 2007; 2010). In the previous edition of this book a chapter was dedicated to the description of the principles, analytical performance and limitations of the existing methodologies for measuring testosterone, DHT and SHBG (Simoni 2004). Here, we will provide an update on the state of the art to help the reader choose the testosterone detection system most suitable for his/her needs in view of the current recommendations. Testosterone and DHT circulate in serum largely bound to transport proteins: that is albumin, which displays low affinity but very high binding capacity, and SHBG, with high affinity and low capacity. A systematic analysis of serum transport of steroid hormones and their interaction with binding proteins revealed an association constant of SHBG of 1.6 × 109 M-1 for testosterone and of 5.5 × 109 M-1 for DHT at 37 C (Dunn et al. 1981). By comparison the association constant of albumin for testosterone is five orders of magnitude lower (6 × 104 M-1) (Anderson 1974). The relative amounts of protein binding of circulating testosterone in men and women are shown in Table 4.1.


2012 - New understandings of the genetic basis of isolated idiopathic central hypogonadism [Articolo su rivista]
M., Bonomi; D. V., Libri; F., Guizzardi; E., Guarducci; E., Maiolo; Pignatti, Elisa; R., Asci; L., Persani; G., Aimaretti; M., Altobelli; G., Arnaldi; M., Baldi; L., Bartalena; L., Beccaria; P., Beck Peccoz; G., Bellastella; G., Borretta; F., Buzi; S., Cannavò; M., Cappa; A., Cariboni; T., Ciampani; A., Cicognani; M., Cisternino; S., Corbetta; N., Corciulo; R., Cozzi; A. V., D'Elia; E. D., Uberti; M., De Marchi; G., Forti; N., di Iorgi; A., Fabbri; A., Ferlin; R., Gaudino; E., Grosso; C., Krausz; F., Lanfranco; D., Larizza; P., Limone; M., Maggi; R., Maggi; M., Maghnie; A., Mancini; G., Mandrile; Marino, Marco; M. A., Mencarelli; N., Migone; G., Neri; L., Perroni; E., Pignatti; A., Pilotta; A. I., Pincelli; A., Pizzocaro; A., Pontecorvi; G., Radetti; P., Razzore; G., Russo; F., Salvini; A., Secco; M., Segni; Simoni, Manuela; A., Sinisi; R., Vigneri; G., Weber
abstract

Idiopathic hypogonadotropic hypogonadism is a rare disease that is characterized by delayed/absent puberty and/or infertility due to an insufficient stimulation of an otherwise normal pituitary-gonadal axis by gonadotrophin-releasing hormone (GnRH) action. Because reduced or normal luteinizing hormone (LH)/follicle-stimulating hormone (FSH) levels may be observed in the affected patients, the term idiopathic central hypogonadism (ICH) appears to be more appropriate. This disease should be distinguished from central hypogonadism that is combined with other pituitary deficiencies. Isolated ICH has a complex pathogenesis and is fivefold more prevalent in males. ICH frequently appears in a sporadic form, but several familial cases have also been reported. This finding, in conjunction with the description of numerous pathogenetic gene variants and the generation of several knockout models, supports the existence of a strong genetic component. ICH may be associated with several morphogenetic abnormalities, which include osmic defects that, with ICH, constitute the cardinal manifestations of Kallmann syndrome (KS). KS accounts for approximately 40% of the total ICH cases and has been generally considered to be a distinct subgroup. However, the description of several pedigrees, which include relatives who are affected either with isolated osmic defects, KS, or normo-osmic ICH (nICH), justifies the emerging idea that ICH is a complex genetic disease that is characterized by variable expressivity and penetrance. In this context, either multiple gene variants or environmental factors and epigenetic modifications may contribute to the variable disease manifestations. We review the genetic mechanisms that are presently known to be involved in ICH pathogenesis and provide a clinical overview of the 227 cases that have been collected by the collaborating centres of the Italian ICH Network.


2012 - Nursing assistance increases the efficiency of ultrasound-guided fine-needle aspiration biopsy (US-FNAB) in the management of thyroid nodules: the MoCyThy (Modena's Cytology of the Thyroid) DATABASE [Abstract in Rivista]
Belli, Serena; Carani, Cesare; Ansaloni, Anna; Katia, Cioni; Zirilli, Lucia; Marietta, Velasco; Simoni, Manuela; Madeo, Bruno; Rochira, Vincenzo
abstract

The study investigate the role of nursing assistance during ultrasound-guided fine-needle aspiration.


2012 - Prevalence of olfactory and other developmental anomalies in patients with central hypogonadotropic hypogonadism [Abstract in Rivista]
DELLA VALLE, Elisa; Vezzani, Silvia; Rochira, Vincenzo; Antonio, Granata; Carani, Cesare; Genovese, Elisabetta; Simoni, Manuela
abstract

Several congenital anomalies different from anosmia are frequent both in Kallmann Syndrome and other forms of Hypogonadotropic Hypogonadism


2012 - Report: a “silent” FSH-secreting adenoma [Abstract in Rivista]
Santi, Daniele; A. R. M., Granata; Rochira, Vincenzo; Simoni, Manuela; Carani, Cesare
abstract

Case-Report on FSH-secreting pituitary adenoma


2012 - Sphingosine kinase inhibition exerts both pro- and anti-atherogenic effects in low-density lipoprotein receptor-deficient (LDL-R-/-) mice. [Articolo su rivista]
Poti', Francesco; M., Bot; Costa, Sara; Bergonzini, Valeria; L., Maines; G., Varga; H., Freise; H., Robenek; Simoni, Manuela; Nofer, JERZY ROCH
abstract

Sphingosine 1-phosphate (S1P), a lysosphingolipid associated with high-density lipoprotein (HDL), contributes to the anti-atherogenic potential attributed to this lipoprotein. This study examined whether a reduction of S1P plasma levels affects atherosclerosis in a murine model of disease. LDL-R-/-mice on Western diet were given ABC294640, an inhibitor of sphingosine kinase (SphK) for 16 weeks. ABC294640 decreased plasma S1P by approximately 30%. However, ABC294640 failed to affect atherosclerotic lesion formation. Plasma triglycerides were reduced whereas total and HDL-cholesterol remained unchanged in course of ABC294640 treatment. ABC294640 increased plasma interleukin (IL)-12p70 and RANTES concentration as well as IL-12p70, RANTES and interferon (IFN)-γ production by peritoneal cells and this was paralleled by enhanced activity of peritoneal and spleen dendritic cells as evidenced by up-regulation of CD86 and MHC-II on CD11c+ cells. As a consequence, increased T-cell activation was noted in ABC294640-treated mice as indicated by enhanced CD4+ splenocyte proliferation, IFN-γ and IL-2 production, and CD69 expression. Concomitantly, however, ABC294640 treatment redistributed CD4+ and CD8+ cells from blood to lymphatic organs and reduced T-cell number within atherosclerotic lesions. In addition, plasma sVCAM-1, sICAM-1, and MCP-1 levels as well as in vivo leukocyte adhesion and CCL19-induced T-cell penetration into peritoneum were lower in ABC294640-treated animals. In vitro experiments demonstrated reduced VCAM-1 and ICAM-1 expression and lymphocyte adhesion to endothelial cells exposed to ABC294640. In conclusion, treatment with SphK inhibitor leads to both pro- and anti-atherogenic effects in LDL-R-/- mice. As a consequence, SphK inhibition fails to affect atherosclerosis despite significant S1P reduction in plasma.


2012 - Steroidogenesis in men with Klinefelter Syndrome (KS). [Abstract in Rivista]
Belli, Serena; E., Dall’Olio; Santi, Daniele; Rochira, Vincenzo; A. R., Granata; Carani, Cesare; Simoni, Manuela
abstract

Basal and stimulated sex steroid measurements with liquid mass spectrometry in men with Klinefelter Syndrome


2012 - Testosterone-induced prostate growth is blocked by co-and preadministration of norethisterone enanthate in castrated cynomolgus monkeys. [Articolo su rivista]
J., Wistuba; E., Nieschlag; A., Semjonow; R., Sandhove Klaverkamp; S., Friederichs Gromoll; M., Zitzmann; Simoni, Manuela; C. M., Luetjens CM
abstract

Introduction: Prostate size and function are regulated by testosterone. However, the progesterone receptor is ex- pressed in the primate prostate. Progestins affect the pros- tate by endocrine suppression, but can also act directly. Ex- amining the role of progestins, we studied the effects of norethisterone (NET) on testosterone undecanoate (TU)-in- duced prostate growth in castrated macaques. Materials and Methods: Two groups (n = 6 for each group) received TU every 9 weeks. Using a crossover setting, group I received norethisterone enanthate (NETE) 3 times at 3-week intervals, while group II received placebo. After 9 weeks, placebo was administered to group I, and group II received NETE. Results: In group II, the prostate grew under TU and placebo over the first period. In group I, coadministered with NETE, the in- crease was lower. After the crossover, prostates of animals previously treated with NETE did not increase to normal val- ues under placebo. Prostates of animals treated with TU and placebo in the first period shrank following NETE administra- tion after the crossover. The long half-life of NET can explainthe lack of a TU effect on animals coadministered with NETE after the crossover. Conclusions: Pre- and coadministration of NET reduces testosterone-induced prostate growth with possible implications for the treatment of benign prostate hyperplasia and hormonal male contraception.


2012 - The TRHR gene is associated to hypothalamo-pituitary sensitivity to levothyroxine in thyroidectomized patients [Abstract in Atti di Convegno]
Brigante, Giulia; Spaggiari, Giorgia; Cioni, K; Gnarini, Valentina; Pignatti, Elisa; Casarini, Livio; Marino, Marco; Tüttelmann, F; Carani, Cesare; Simoni, Manuela
abstract

Background: Patients thyroidectomized for thyroid cancer need variable doses of levothyroxine (LT4) to obtain TSH suppression. A predetermined thyroid function set-point for each individual has been hypothesized, suggesting a genetic influence in the regulation of pituitary-thyroid axis. We hypothesized of the TRHR gene could be associated with a different hypothalamo-pituitary sensitivity to the negative feedback of the thyroid hormones. Methods: We performed a case–control association study, enrolling 107 thyroidectomized patients, in follow-up for differentiated thyroid cancer, and 99 volunteer controls. Patients were evaluated first when TSH levels were suppressed (<0.1 mIU/l), by the lowest effective LT4 dose, and then when TSH was subsuppressed (0.1<TSH<0.5 mIU/l). We selected two SNPs of TRHR gene, rs3134105 and rs3110040, identified as informative markers, using the online database ‘HapMap’. We performed a frequency analysis of the mapped SNPs, followed by a linkage analysis using the HaploView software. Genotyping was performed using the High Resolution Melting technology. Results: The selected SNPs were in linkage disequilibrium. A significant difference between the three possible genotypes for rs3134105 was found for fT4/TSH ratio (P=0.03). Moreover, despite similar serum concentrations of fT3 and fT4 obtained by similar levothyroxine doses, carriers of at least one A allele of rs3134105 had significantly lower serum TSH levels (P=0.04) as well as higher fT3/TSH (P=0.05) and fT4/TSH ratios (P=0.02). Conclusions: We demonstrated an association between TSH and discrete alleles of the TRHR gene identified by the markers SNPs rs3134105 and rs3110040 in totally thyroidectomized patients with diagnosis of thyroid cancer under subsuppressive LT4 therapy. The TRHR gene is a determinant of hypothalamo-pituitary sensitivity to levothyroxine in such patients.


2012 - The Value of Repeated US-Guided Fine-Needle Aspirations (US-FNAB) in the Follow-Up of Thyroid Nodules: Preliminary Results from the MoCyThy (Modena's Cytology of the Thyroid) Database [Abstract in Rivista]
Rochira, Vincenzo; Ansaloni, Anna; Belli, Serena; Vezzani, Silvia; Diazzi, Chiara; Zirilli, Lucia; Simoni, Manuela; Carani, Cesare; Madeo, Bruno
abstract

Repeating ultrasound guided fine needle aspiration might be useful for detecting thyroid malinìgnant nodules


2012 - The cellular fate of CYP19A1 (aromatase) protein [Abstract in Rivista]
Pignatti, Elisa; Ferioli, Silvia; Carani, Cesare; Rochira, Vincenzo; Francesco, Potì; Simoni, Manuela
abstract

Le variazioni nell'attività enzimatica e nella degradazione dell'enzima indotte dalle mutazioni finora descritte del gene CYP19A1 sono state studiate in vitro mediante studi di transfezione e confrontate con l'enzima wild type.


2012 - Ultrasound-guided fine-needle aspiration biopsy (US-FNAB) of thyroid nodules: effects of the operator’s experience on adequacy of sampling: the MoCyThy (Modena’s Cytology of the Thyroid) DATABASE [Abstract in Rivista]
Vezzani, Silvia; Ansaloni, Anna; Zirilli, Lucia; Katia, Cioni; Diazzi, Chiara; Simoni, Manuela; Madeo, Bruno; Rochira, Vincenzo
abstract

Lo studio ha permesso di evidenziare come l'esperienza (in termini di anni e di numero di prestazioni eseguite) sia importante per i risultati dell'agoaspirato tiroideo ecoguidato dei noduli tiroidei


2011 - Bone mineral density and sex hormones in HIV-infected men [Abstract in Rivista]
Madeo, Bruno; Santi, Daniele; Orlando, Gabriella; Guaraldi, Giovanni; Fabio, Vescini; Simoni, Manuela; Carani, Cesare; Rochira, Vincenzo
abstract

In men with HIV bone mineral density depends on estrogens and not from testosterone as in non HIV-infected men.


2011 - Copy number variants in patients with severe oligozoospermia and Sertoli-cell-only syndrome. [Articolo su rivista]
Tüttelmann, F; Simoni, Manuela; Kliesch, S; Ledig, S; Dworniczak, B; Wieacker, P; Röpke, A.
abstract

A genetic origin is estimated in 30% of infertile men with the common phenotypes of oligo- or azoospermia, but the pathogenesis of spermatogenic failure remains frequently obscure. To determine the involvement of Copy Number Variants (CNVs) in the origin of male infertility, patients with idiopathic severe oligozoospermia (N = 89), Sertoli-cell-only syndrome (SCOS, N = 37)) and controls with normozoospermia (N = 100) were analysed by array-CGH using the 244A/400K array sets (Agilent Technologies). The mean number of CNVs and the amount of DNA gain/loss were comparable between all groups. Ten recurring CNVs were only found in patients with severe oligozoospermia, three only in SCOS and one CNV in both groups with spermatogenic failure but not in normozoospermic men. Sex-chromosomal, mostly private CNVs were significantly overrepresented in patients with SCOS. CNVs found several times in all groups were analysed in a case-control design and four additional candidate genes and two regions without known genes were associated with SCOS (P<1×10−3). In conclusion, by applying array-CGH to study male infertility for the first time, we provide a number of candidate genes possibly causing or being risk factors for the men's spermatogenic failure. The recurring, patient-specific and private, sex-chromosomal CNVs as well as those associated with SCOS are candidates for further, larger case-control and re-sequencing studies.


2011 - Effects of polymorphisms in gonadotropin and gonadotropin receptor genes on reproductive function. [Articolo su rivista]
Casarini, Livio; Pignatti, Elisa; Simoni, Manuela
abstract

Gonadotropins, the action of which is mediated at the level of their gonadal receptors, play a key role in sexual development, reproductive functions and in metabolism. The involvement of the gonadotropins and their receptor genotypes on reproductive function are widely studied. A large number of gonadotropins and their receptors gene polymorphisms are known, but the only one considerable as a clear, absolute genetic marker of reproductive features or disfunctions is the FSHR Asn680Ser polymorphism, since it modulates ovarian response to FSH. The aim of these studies would to be the prediction of the genetic causes of sex-related diseases to enable a customized clinical setting based on individual response of patients undergoing gonadotropin stimulation. In this review we discuss the latest information about the effects of polymorphisms of the gonadotropins and their receptor genes on reproductive functions of both male and female, and discuss their patho-physiological implications.


2011 - Effects of the FSH receptor gene polymorphism p.N680S on cAMP and steroid production in cultured primary human granulosa cells. [Articolo su rivista]
V., Nordhoff; B., Sonntag; D., von Tils; M., Götte; Schüring, A. N.; J., Gromoll; K., Redmann; Casarini, Livio; Simoni, Manuela
abstract

The study was designed to evaluate in vitro the cellular mechanisms of the single nucleotide polymorphism (SNP) p.N680S of the FSH receptor gene (FSHR) in human granulosa cells (GC) and included patients homozygous for the FSHR SNP (NN/SS) undergoing ovarian stimulation. GC were isolated during oocyte retrieval and cultured for 1–7 days. Basal oestradiol and progesterone concentrations were measured after short-term culture. The kinetics of cAMP, oestradiol and progesterone concentrations in response to various amounts of FSH were analysed in a 6–7 day culture. Basal oestradiol, but not progesterone, concentrations on day 1 of GC culture, were significantly higher in NN compared with SS (P = 0.045), but non-responsive to FSH stimulation. Immunofluorescence microscopy demonstrated the re-appearance of FSHR expression with increasing days in culture. Upon stimulation with FSH, GC cultured for 6–7 days displayed a dose-dependent increase of cAMP, oestradiol and progesterone but no difference in the EC50 values between both variants. Primary long-term GC cultures are a suitable system to study the effects of FSH in vitro. However, the experiments suggest that factors down-stream of progesterone production or external to GC might be involved in the clinically observed differences in an FSHR variant-mediated response to FSH


2011 - Inactivating mutations of CYP19A1 (aromatase) gene reduce the protein stability in vitro [Abstract in Rivista]
Pignatti, Elisa; Ferioli, Silvia; Simoni, Manuela; Carani, Cesare; Rochira, Vincenzo
abstract

Role of inactivating mutation of the aromatase gene on the protein structure, function and degradation


2011 - Molecular cloning and functional characterization of endogenous recombinant common marmoset monkey (Callithrix jacchus) follicle-stimulating hormone. [Articolo su rivista]
Müller, T; Hupfeld, T; Roessler, J; Simoni, Manuela; Gromoll, J; Behr, R.
abstract

BACKGROUND:Common marmoset monkeys (Callithrix jacchus) are readily used in biomedical research. However, superovulation for embryonic stem cell production and developmental research still remain difficult. Inexplicably, follicle-stimulating hormone (FSH) as key player in superovulation has to be administered in extremely high dosages in this non-human primate compared to human.METHODS:To evaluate whether marmoset FSH (cjFSH) is functionally more competent than its human homologue on the marmoset FSH receptor (cjFSHR), we established in vitro a homologous system characterizing homologous and recombinantly produced cjFSH.RESULTS:Upon stimulation of two cell lines stably expressing either the marmoset or the human FSH receptor (cj/hFSHR), respectively, the second messenger signaling of the activated receptor displayed no significant difference in ED(50) values. Thermostability of cjFSH was significantly prolonged by roughly 20% on both FSHRs.CONCLUSION:High FSH dosage in marmoset superovulation cannot be explained by enhanced biopotency of the natural animal's gonadotropin.


2011 - Pharmacogenetics of ovarian stimulation in the twenty-first century [Capitolo/Saggio]
Simoni, M.; Pugni, V.
abstract

Discovering the genetic variants associated with ovarian response to gonadotropins is an important step towards individualized pharmacogenetic protocols of ovarian stimulation. More than 90% of the genetic variability is caused by the presence of single nucleotide polymorphisms (SNPs). Pharmacogenetics is the science that describes the relationship between genetic variability and drug response and studies how to tailor pharmacological therapy to the genetic features of the individual patient and how to improve desired actions and minimize side effects. Different genes have been studied in relation to the characteristics of the normal ovarian cycle or different individual responses to controlled ovarian hyperstimulation (COH). The follicle stimulating hormone receptor gene (FSHR) is crucial in follicular maturation: inactivating FSHR mutations almost always leads to amenorrhea and activating mutations can cause a spontaneous ovarian hyperstimulation syndrome or predispose to iatrogenic ovarian hyperstimulation syndrome (OHSS).


2011 - Prevalence of secondary, primary, and compensated hypogonadism in HIV infected men in HAART era: a cross-sectional observational study [Abstract in Rivista]
Brigante, Giulia; Santi, Daniele; Zirilli, Lucia; Diazzi, Chiara; Orlando, Gabriella; Simoni, Manuela; Carani, Cesare; Guaraldi, Giovanni; Rochira, Vincenzo
abstract

The study investigates the prevalence of male hypogonadism among patients with HIV infection paying particular attention to the different types of hypogonadism


2011 - Two hormone for one receptors: dissecting out LH and hCG activity with an in vitro approach [Abstract in Atti di Convegno]
Casarini, Livio; LA MARCA, Antonio; Pignatti, Elisa; Simoni, Manuela
abstract

Introduction: LH and hCG act on the same receptor (LHCGR), have different half-lives and in vivo biopotency. It is not known whether they elicit the same cellular and molecular response. The aim of this study was to compare the kinetics of cAMP response to recombinant LH and hCG. Design: In COS-7 cells permanently expressing the human LHCGR (COS-7/LHCGR) we evaluated LH and hCG dose-response curves, by measuring total cAMP after 3 h of incubation. We then evaluated the time-course of intracellular cAMP production in the presence of ED50 doses of LH and hCG over 3 h. Finally we evaluated the long-term response to LH and hCG by exposing human primary granulosa lutein cells (hGLC) to ED50 doses over 12 h. All incubations were performed in the presence of IBMX. Results: In COS-7/LHCGR cells, we observed significantly different ED50 for LH (475.75±137.33 pM, mean±S.D.) and hCG (101.75±44.63 pM) (Mann–Whitney’s U-test, P=0.029; n=4). Maximal LH stimulation of intracellular cAMP, about 50 fold over control, reached a plateau in 10 min, while maximal hCG stimulation at similar levels was attained only after 1 h (Anova; P<0.05; n=3). In hGLC continuous exposure to LH and hCG resulted in a repetitive, pulsatile increase of intracellular cAMP with peaks every 3–4 h and significantly higher levels of stimulation in the presence of hCG vs LH (Anova; P<0.05; n=3). Conclusions: Equimolar concentrations of human recombinant LH and hCG result in significantly higher in vitro biopotency of hCG (about 5-fold). Equipotent concentration (ED50) of LH and hCG stimulate a faster response to LH within the first 3 h, but a quantitatively higher response to hCG over 12 h. hGLC respond to constant LH/hCG stimulation in a pulsatile fashion, suggesting a novel control of gonadotropins action at the receptor level.


2011 - Variation in CAG and GGN repeat lengths and CAG/GGN haplotype in androgen receptor gene polymorphism and prostate carcinoma in Nigerians. [Articolo su rivista]
O., Akinloye; J., Gromoll; Simoni, Manuela
abstract

Prostate cancer has become the most common cancer in Nigerian men. The growth of the prostate gland depends on circulating androgens and intracellular steroid signalling pathways. The effects of androgens are mediated through the androgen receptor (AR), a nuclear transcription factor encoded by the AR gene. The common polymorphisms, CAG and GGN repeats, in exon 1 of this gene have been implicated as possible risk factors. Thus far, existing supporting data are scanty and none are from sub-Saharan African populations. Therefore, this study investigates the possible association between AR polymorphism repeat length (CAG and GGN) and prostate cancer in Nigerians. A total of 261 subjects (70 with prostate cancer, 68 with benign prostate hyperplasia [BPH], 123 age-matched apparently normal subjects as controls) were studied. CAG and GGN repeats length were determined by fragment length analysis using GeneScan. The CAG repeat length in prostate cancer and in BPH compared to the controls was significantly different (P < 0.05) with reduce length of CAG repeats showing a significant odds ratio (OR) in both cases. However, this was not observed in GGN repeat length, which showed no significant difference between cases and controls (P > 0.05). CAG and GGN haplotype variation showed no significant difference between cases and controls (P > 0.05), except that the haplotypes CAG > or =21 and GGN < or =21 were more common in the control group. The results of this study, the first from sub-Saharan Africa, supports the hypothesis that reduced CAG repeat length is a risk factor for prostate cancer, and also suggests an association with BPH.


2010 - A common haplotype of protamine 1 and 2 genes is associated with higher sperm counts [Articolo su rivista]
Tüttelmann, F; Krenkova, P; Römer, S; Nestorovic, Ar; Ljujic, M; Stambergova, A; Macek jr, M; Macec, Srm; Nieschlag, E; Gromoll, J; Simoni, Manuela
abstract

Sperm chromatin compaction in the sperm head is achieved by histones being replaced by transition proteins and then protamines during spermatogenesis. Haploinsufficiency of the protamine 1 (PRM1) or PRM2 gene causes infertility in mice and sequence variants in PRM1 were associated with increased abnormal sperm morphology in infertile men. However, the published data remain inconclusive about a role of PRM1/2 variants in male infertility and their association with semen parameters. By full sequence analysis we assessed the frequency of sequence variations in PRM1 and PRM2 in groups of idiopathic patients with teratozoospermia and normal or reduced sperm concentration (N = 88 and 83, respectively) and in men with high percentage of normal sperm morphology and normal concentration (N = 77). Two rare (c.54G>A and c.102G>T) and one common SNP (c.230A>C) were identified in PRM1. In PRM2, some rare heterozygous mutations and the two common intronic SNPs 298G>C and 373C>A were detected. None of the PRM1/2 variants were associated with teratozoospermia or other semen parameters individually. However, significant linkage disequilibrium was detected between the common SNPs of PRM1 and PRM2 which formed haplotypes. The analysis of the pooled cohort revealed that homozygous carriers of the common haplotype ACC had a two-fold higher sperm concentration and count than men lacking this haplotype with sperm counts of heterozygotes for ACC being midway between the homozygotes. This markedly decreased sperm output might either be caused by spermatozoa lacking the ACC haplotype not being viable or these being negatively selected against. In addition, a significant deviation from Hardy-Weinberg-Equilibrium of these SNPs might indicate natural selection in favour of the ACC allele which lead to a higher sperm output and therefore better fertility. In conclusion, we describe for the first time an association of a common haplotype formed by PRM1 and PRM2 with sperm output in a large cohort of men.


2010 - Clinical experience with azoospermia: aetiology and chances for spermatozoa detection upon biopsy. [Articolo su rivista]
Tüttelmann, F; Werny, F; Cooper, Tg; Kliesch, S; Simoni, Manuela; Nieschlag, E.
abstract

Summary The clinical workup of the infertile male with azoospermia aims at determining the aetiology and estimating the chances of finding spermatozoa by testicular sperm extraction (TESE). To establish prognostic criteria, 1583 consecutive patients with azoospermia consulting the Centre of Reproductive Medicine and Andrology, Münster, a tertiary referral centre, between 1976 and 2009 comprising 9.8% of all patients providing a semen sample were included in this retrospective analysis. The frequencies of diagnoses were as follows: 21% genetic causes (14% Klinefelter syndrome, 1% other chromosomal aberrations, 2% Y-chromosomal microdeletions, 1% hypogonadotropic hypogonadism, 3% congenital bilateral absence of the vas deferens), 31% current or former maldescended testes, varicocele, urogenital infections, 15% malignancies, 11% obstructions, 7% endocrine or other chronic diseases and 12% idiopathic azoospermia. Receiver-operating characteristic curves for chances of finding spermatozoa by testicular biopsy were calculated for testicular volume, serum follicle-stimulating hormone (FSH) and the seminal markers alpha-glucosidase, fructose and zinc where these data were available (N = 283). Histograms of the seminal markers comparing data from men with obstructive azoospermia and normozoospermia visualize their discriminating power. Evidence-based threshold values for high chances of positive testicular biopsy serving as surrogate marker for TESE were derived from the subgroup of men with obstructive azoospermia for testicular volume (>/=21 mL), FSH (</=10 U/L) and seminal alpha-glucosidase (</=18 mU/ejaculate). Fructose and zinc could not predict the chances of finding spermatozoa upon biopsy. Based on these three parameters, positive biopsy and presumably TESE success can be quickly and reliably estimated in everyday practice with the colour-coded figures constructed from these data. As a seminal alpha-glucosidase reference limit of 18 mU/ejaculate can also be used to diagnose congenital bilateral absence of the vas deferens, alpha-glucosidase (rather than seminal fructose) should be determined as part of the clinical routine when counselling patients before testicular biopsy.


2010 - Cytogenetic and molecular genetic investigations [Capitolo/Saggio]
Simoni, M.; Wieacker, P.
abstract

Male infertility and hypogonadism may be caused by a genetic defect, which should be investigated by cytoge-netic or molecular genetic analysis. The main indications for genetic testing in andrology are azoospermia and severe oligozoospermia. In selected cases of hypogonadotropic hypogonadism and Kallmann syndrome, especially those with an evident familial component, mutation screening of the known genes may be indicated. Even if detection of a genetic alteration will not substantially change the treatment, genetic testing should be performed for two reasons: (1) to finalize a causal diagnosis, and (2) to assess the genetic risk for the offspring in case of successful treatment. © 2010 Springer-Verlag Berlin Heidelberg.


2010 - Diseases of the hypothalamus and the pituitary gland [Capitolo/Saggio]
Behre, H. M.; Nieschlag, E.; Partsch, C. -J.; Wieacker, P.; Simoni, M.
abstract

Among males Kallmann syndrome in its fully developed form has a prevalence of about 1 in 10,000. The prevalence in males is four times higher than in females (Seminara et al. 1998). Currently the genetic defect underlying Kallmann syndrome and IHH can be demonstrated in about half of the familial cases and in about 10% of the sporadic cases. The old denomination idio-pathic hypogonadotropic hypogonadism, which reflects ignorance of the causal defect, therefore appears obsolete. The acronym IHH should stand for isolated hypogonadotropic hypogonadism, referring to the inadequacy of gonadotropin secretion as the only defect. © 2010 Springer-Verlag Berlin Heidelberg.


2010 - Endocrine laboratory diagnosis [Capitolo/Saggio]
Simoni, M.; Nieschlag, E.
abstract

The evaluation of serum levels of LH and FSH in combination with testosterone provides important information to pinpoint the localization of hypogonad-ism, which is then decisive for adequate therapy. © 2010 Springer-Verlag Berlin Heidelberg.


2010 - Genetic screening for infertility: When should it be done? [Articolo su rivista]
Kara, Elda; Simoni, Manuela
abstract

Depending on the clinical findings, the infertile male patient needs genetic evaluation. Karyotype analysis and Y-chromosomal microdeletion screening should be performed in patients with azoospermia or severe oligozoospermia in order to rule out structural chromosomal abnormalities, Klinefelter syndrome and Y chromosome microdeletions. Infertile patients with obstructive azoospermia need cystic ibrosis transmembrane receptor gene screening, while in patients with hypogonadotropic hypogonadism mutation screening may be performed according to clinical features. All genetic analyses should be accompanied by expert counseling by a clinical genetist both in male and female patients. Primary amenorrhea should be investigated by karyotype analysis and selected mutation screening according to the patient's clinical features. Karyotype analyses and FMR1 gene screening is recommended in cases of POF. At present the infertility of patients with POF cannot be restored if the diagnosis is made after complete follicular depletion, but in some cases, early diagnosis by genetic investigation may instead lead to the advice of early conception or oocyte harvesting and preservation. In addition, the accumulation and annotation of array comparative genomic hybridization data might, in the near future, lead to the identiication of pathogenetic copy number variations and genes involved in POF. Karyotype analysis of both partners is recommended in all couples with recurrent pregnancy loss. No routine genetic test can be recommended so far in patients with PCOS. © 2010 Middle East Fertility Society. Production and Hosting by Elsevier B.V. All rights reserved.


2010 - Male 41, XXY* mice as a model for Klinefelter syndrome: Hyperactivation of Leydig cells [Articolo su rivista]
Wistuba, J; Luetjens, Cm; Stuckenborg, Jb; Poplinski, A; Werler, S; Dittmann, M; Damm, Os; Hamalainen, T; Simoni, Manuela; Gromoll, J.
abstract

Sex chromosome imbalance in males is linked to a supernumerary X chromosome, a condition resulting in Klinefelter syndrome (KS; 47, XXY). KS patients suffer from infertility, hypergonadotropic hypogonadism, and cognitive impairments. Mechanisms of KS pathophysiology are poorly understood and require further exploration using animal models. Therefore, we phenotypically characterized 41, XX(Y)* mice of different ages, evaluated observed germ cell loss, studied X-inactivation, and focused on the previously postulated impaired Leydig cell maturation and function as a possible cause of the underandrogenization seen in KS. Xist methylation analysis revealed normal X-chromosome inactivation similar to that seen in females. Germ cell loss was found to be complete and to occur during the peripubertal phase. Significantly elevated FSH and LH levels were persistent in 41, XX(Y)* mice of different ages. Although Leydig cell hyperplasia was prominent, isolated XX(Y)* Leydig cells showed a mature mRNA expression profile and a significantly higher transcriptional activity compared with controls. Stimulation of XX(Y)* Leydig cells in vitro by human chorionic gonadotropin indicated a mature LH receptor whose maximal response exceeded that of control Leydig cells. The hyperactivity of Leydig cells seen in XX(Y)* mice suggests that the changes in the endocrine milieu observed in KS is not due to impaired Leydig cell function. We suggest that the embedding of Leydig cells into the changed testicular environment in 41 XX(Y)* males as such influences their endocrine function.


2010 - Physiology of testicular function [Capitolo/Saggio]
Weinbauer, G. F.; Luetjens, C. M.; Simoni, M.; Nieschlag, E.
abstract

The testes produce the male gametes and the male sexual hormones (androgens). The term spermatogenesis describes and includes all the processes involved in the production of gametes, whereas steroidogenesis refers to the enzymatic reactions leading to the production of male steroid hormones. Spermatogenesis and steroido-genesis take place in two compartments morphologically and functionally distinguishable from each other. These are the tubular compartment, consisting of the seminiferous tubules (tubuli seminiferi) and the interstitial compartment (interstitium) between the seminiferous tubules (Figs. 2.1 and 2.2). Although anatomically separate, both compartments are closely connected with each other. For quantitatively and qualitatively normal production of sperm the integrity of both compartments is necessary. The function of the testis and thereby also the function of its compartments are governed by the hypothalamus and the pituitary gland (endocrine regulation). These endocrine effects are mediated and modulated at the testicular level by local control mechanisms (paracrine and autocrine factors). © 2010 Springer-Verlag Berlin Heidelberg.


2010 - Prevalence and characterization of hypogonadism among men with human immunodeficiency virus infection: preliminary results [Abstract in Rivista]
Brigante, Giulia; Santi, Daniele; Zirilli, Lucia; Diazzi, Chiara; G., Orlando; Simoni, Manuela; Guaraldi, Giovanni; Carani, Cesare; Rochira, Vincenzo
abstract

Preliminary data on the prevalence and type of male hypogonadism in men with HIV infection


2009 - ANTI-MÜLLERIAN HORMONE IN OLIGO- COMPARED TO NORMOZOOSPERMIC MEN AND MEN WITH MALDESCENDED TESTES. [Articolo su rivista]
Tüttelmann, F; Dykstra, N; Themmen, Apn; Visser, Ja; Nieschlag, E; Simoni, Manuela
abstract

ObjectiveTo evaluate serum anti-Müllerian hormone (AMH) in well-characterized men with normal and reduced sperm concentration and in men with a history of or persistent maldescended testes as a possible clinical marker of male factor infertility and/or maldescended testes.DesignRetrospective analysis of 199 men selected from our database (Androbase).SettingThe university-based Institute of Reproductive Medicine.Patient(s)One hundred eight men with normal and 60 men with reduced sperm concentration without known cause of infertility and additionally 31 infertile men with current or former maldescended testes were evaluated.Intervention(s)Serum AMH was analyzed by an in-house ELISA.Main Outcome Measure(s)Hormone and semen parameters were compared and correlated with AMH.Result(s)No significant differences were found in AMH levels. Only in men with maldescended testes did AMH correlate negatively with FSH and positively with testicular volume and sperm concentration. No correlations between AMH and LH or testosterone (T) were found.Conclusion(s)Anti-Müllerian hormone serum levels are not significantly affected by impaired spermatogenesis in general but are correlated with spermatogenic parameters in men with current or former maldescended testes. Therefore, AMH measurement does not improve clinical routine diagnostics but should be evaluated further in patients with maldescended testes. Anti-Müllerian hormone might serve as a marker of Sertoli cell number, function, and/or maturation in these men.


2009 - Androgen receptor gene CAG and GGN polymorphisms in infertile Nigerian men. [Articolo su rivista]
Akinloye, O; Gromoll, J; Nieschlag, E; Simoni, Manuela
abstract

The human androgen receptor gene (AR) is an important regulator of male sexual development including spermatogenesis. Exon 1 of this gene encodes the N terminal domain, which controls transcriptional activity of the receptor and the two polymorphic repeats CAG and GGN. Many studies have reported association of the expanded CAG repeat length with male infertility, although this is still controversial. The GGN repeat, in contrast, has been less thoroughly studied. Thus far, only scanty studies have been reported from African populations and none from Nigeria. Therefore we have investigated the possible association between AR polymorphism repeats length (CAG and GGN) and reduced spermatogenesis in infertile Nigerian men (N=60) consisting of 20 non-obstructive azoospermic and 40 oligozoospermic subjects compared with controls with normozoospermia and proven evidence of fertility (N=38). In addition, 48 volunteers with normal spermatogenesis were recruited from a German population. CAG and GGN repeats length were determined by polymerase chain reaction (PCR) followed by electrophoresis on polyacrylamide gel and gene scan. The CAG and GGN repeats length of infertile compared to fertile populations were not significantly different (p>0.05). We found a unique AR GGN allele distribution with 20-23 GGN repeats predominant in the Nigerian study population. Our results show that CAG and GGN repeats polymorphisms are not a critical index of male infertility. While we do not find a relationship with CAG and GGN repeats haplotypes and male infertility, we report for the first time a unique and wider distribution of the GGN allele in the Nigerian population which is significantly different from the Caucasian population. The functional relevance of this variance to male fertility warrants in-depth elucidation.


2009 - Aromatase expression in peripheral blood leukocytes from adult and elderly female and male subjects [Abstract in Rivista]
Pignatti, Elisa; Rossi, Aldo; Scaltriti, Sara; Taliani, Erica; Rochira, Vincenzo; Simoni, Manuela; Carani, Cesare
abstract

Study of the expression of aromatase enzyme in peripheral blood leukocytes collected from adult and elderly female and male subjects. This suds highlights the importance of individual differences in aromatase expression as well as the differences related to different age.


2009 - Body Fat Content and Testosterone Pharmacokinetics Determine Gonadotropin suppression after intramuscular injections of testosterone preparations in normal men. [Articolo su rivista]
Kornmann, B; Nieschlag, E; Zitzmann, M; Gromoll, J; Simoni, Manuela; Von Eckardstein, S.
abstract

Interindividual differences in gonadotropin suppression achieved by a short and a long-acting intramuscular testosterone (T) preparation were studied to detect factors hindering complete suppression of gonadotropins as the prerequisite for effective male contraception. 40 healthy men received a single injection of testosterone propionate (TP) and four weeks later, two injections of 1000 mg testosterone undecanoate (TU) given six weeks apart. Following TU, decline of LH and FSH was consistent in 17.5% and almost absent in 25% of men. Men showing the most rapid and consistent decline in LH and FSH received a slightly higher dose per bodyweight of TU (13.1 +/- 0.6 vs.11.3 +/- 0.6 mg/kg; n.s.) and reached higher maximal concentrations of total T (40 +/- 4.8 vs. 18.4 +/- 2.4 nmol/L; p<0.001) and free T as well as estradiol. Men with high fat mass (mean +/- SEM: 10.3 +/- 1.5 vs. 23.2 +/- 6.4 kg) had a delayed increase in T levels and an impaired relative decline in LH (12 +/- 2% vs. 53 +/- 10%) and FSH (17 +/- 6%. vs. 70 +/- 25%) within the first two weeks after the first TU injection. We conclude that overweight reduces the chance of rapid and profound gonadotropin suppression during treatment with TU. Body weight needs to be considered to avoid failure of hormonal male contraception.


2009 - Deficit di GH in pazienti affetti da lipodistrofia HIV-correlata: dati preliminari sugli effetti del trattamento con r-hGH sulla composizione corporea [Abstract in Atti di Convegno]
Diazzi, Chiara; Brigante, Giulia; Orlando, Gabriella; Squillace, Nicola; Guaraldi, Giovanni; Carani, Cesare; Rochira, Vincenzo; Simoni, Manuela; Zirilli, Lucia
abstract

r-hGH treatment in HIV-infected patients with documented GH deficiency seems to improve fat distribution


2009 - Estrogeni nel maschio: una nuova fisiopatologia [Monografia/Trattato scientifico]
Rochira, Vincenzo; Scaltriti, Sara; Zirilli, Lucia; Simoni, Manuela; Carani, Cesare
abstract

The monograph deals with novel aspects of the pathophysiology of estrogens in men.


2009 - FSH receptor polymorphisms and ovarian function [Articolo su rivista]
Ochsenkühn, Robert; Von Schönfeldt, Viktoria; Simoni, Manuela
abstract

Follicle-stimulating hormone (FSH) is a key player in human reproduction. FSH activates the FSH receptor (FSHR) on granulosa cells in the ovary. The ovarian effects of endogenous and exogenous FSH can be modulated by polymorphisms of the FSHR gene. To date around 1,800 polymorphisms of the FSHR gene have been reported. This paper reviews the role of different polymorphisms for ovarian function, particularly in conjunction with the use of exogenous FSH in the course of controlled ovarian stimulation. There is currently only one polymorphism of the FSH receptor gene (codon 680) for which a sufficient number of studies have consistently identified a significant association to ovarian function. Polymorphisms of the FSHR gene may be used as markers to predict differences in FSHR function and ovarian response to FSH and may ultimately lead to stimulation protocols that are carefully personalized to each woman's individual needs.


2009 - Functional genetic polymorphisms and female reproductive disorders. Part II – Endometriosis. [Articolo su rivista]
Tempfer, Cb; Simoni, Manuela; Destenaves, B; Fauser, Bcjm
abstract

BACKGROUNDEndometriosis has a strong genetic component, and numerous genetic studies have been reported.METHODSWe have systematically reviewed these studies and included 114 in our final selection.RESULTSWe found no consistent evidence linking endometriosis with specific polymorphisms in genes encoding inflammatory mediators, proteins involved in sex steroid metabolism, vascular function and tissue remodelling. Although a number of polymorphisms have been associated with endometriosis in selected populations, the associations have not been independently confirmed, either because only single studies were carried out on these markers/genes or because other studies reported no association. The most solid evidence linking specific polymorphisms to endometriosis came from studies investigating glutathione-S-transferase, a phase II detoxification enzyme. Carriage of the GSTT1 null deletion variant showed consistent association with endometriosis with a 29% increased risk; however, it cannot be excluded that this result was due to publication bias, and this association should be independently confirmed in large-scale, well-designed case–control studies.CONCLUSIONSThe evidence of an association between genetic polymorphisms and endometriosis is weak. Carriage of the GSTT1 null deletion may moderately increase the risk of this disease. We suggest that the methodology of association studies should be improved in order to identify and validate associations in endometriosis.


2009 - Impaired recognition memory in male mice with a supernumerary X chromosome. [Articolo su rivista]
Lewejohann, L; Damm, O; Luetjens, M; Hämäläinen, ; Simoni, Manuela; Nieschlag, E; Gromoll, J; Wistuba, J.
abstract

Several aberrant chromosomal constellations are known in men. Of these the karyotype XXY (Klinefelter syndrome, KS) is the most common chromosomal disorder with a prevalence of about one in 800 live-born boys. KS is associated with hypogonadism and is suspected to cause variable physical, physiological and cognitive abnormalities. As a supernumerary X chromosome is also associated with infertility, sound animal models for KS are difficult to obtain. In this study, male mice with two X chromosomes (XXYlow asterisk) were derived from fathers carrying a structurally rearranged Y chromosome (Ylow asterisk) that resulted in physical attachment of a part of the Y chromosome to one X. These animals display certain physiological features that resemble closely those of human KS and can also be utilized to study X chromosomal imbalance and cognition. Therefore 15 XXYlow asterisk males and 15 XYlow asterisk controls were subjected to a battery of behavioral tests, including a general health check, analysis of spontaneous exploration and locomotor activity, measures for anxiety-related behavior and the “novel object task” to test memory performance. Physiologically, XYlow asterisk males did not differ from C57Bl/6 wild type mice carrying a normal Y chromosome, which provided a valid control group. All mice appeared healthy. XXYlow asterisk mice did not differ from their wild type littermates with respect to locomotion, exploration and anxiety-related behavior. XXYlow asterisk male mice, however, exhibited no significant recognition memory performance in contrast with wild type XYlow asterisk males that readily fulfilled a given task. These findings support the hypothesis that the presence of a supernumerary X in male mice influences cognitive abilities. We suggest that the altered endocrine state and/or changes in the dosage of X-linked genes in the XXYlow asterisk mouse model affect brain function, in particular those regions responsible for cognition and learning behavior.


2009 - New horizons for in vitro spermatogenesis? An update on novel three-dimensional culture systems as tools for meiotic and postmeiotic differentiation of testicular germ cells. [Articolo su rivista]
Stukenborg, Jb; Schlatt, S; Simoni, Manuela; Yeung, Ch; Elhija, Ma; Luetjens, Cm; Huleihel, M; Wistuba, J.
abstract

Culture and differentiation of male germ cells has been performed for various purposes in the past. To date, none of the studies aimed at in-vitro spermatogenesis have resulted in a sufficient number of mature gametes. Numerous studies have revealed worthy pieces of information, building up a body of information on conditions that are required to maintain and mature male germ cells in-vitro. In this review, we report on previously published and unpublished experiments addressing murine germ cell differentiation in three-dimensional in-vitro culture systems. In a systematic set of experiments, we examined the influence of two different matrices (soft agar and methylcellulose) as well as the need for gonadotropin support. For the first time, we demonstrate that pre-meiotic male germ cells (revealed by the absence of meiotic marker expression (e.g. Boule)) obtained from immature mice pass through meiosis in vitro. After several weeks of culture, we obtained morphologically normal spermatozoa embedded in the matrix substance. Complete maturation relied on support from somatic testicular cells and the presence of gonadotropins but appeared independent from the matrix in a three-dimensional culture environment. Further research efforts are required to reveal the applicability of this culture technique for human germ cells and the functionality of the spermatozoa for generating offspring.


2009 - Phenotypic variation within European carriers of the Y-chromosomal gr/gr deletion is independent of Y-chromosomal background. [Articolo su rivista]
Krausz, C; Giachini, C; Xue, Y; Obryan, Mk; Gromoll, J; RAJPERT DE MEYTS, E; Oliva, R; AKNIN SEIFER, I; Erdei, E; Jorgensen, N; Simoni, Manuela; Ballescà, Jl; Levy, R; Balercia, G; Piomboni, P; Nieschlag, E; Forti, G; Mclachlan, R; TYLER SMITH, C.
abstract

BackgroundPrevious studies have compared sperm phenotypes between men with partial [1] deletions within the AZFc region of the Y chromosome with non-carriers, with variable results. Here, we have investigated a separate question, the basis of the variation in sperm phenotype within gr/gr deletion carriers, which ranges from normozoospermia to azoospermia. Differences in the genes removed by independent gr/gr deletions, the occurrence of subsequent duplications or the presence of linked modifying variants elsewhere on the chromosome have been suggested as possible causal factors. We set out to test these possibilities in a large sample of gr/gr deletion carriers with known phenotypes spanning the complete range.ResultsWe assembled a collection of 169 men diagnosed with gr/gr deletions from six centres in Europe and one in Australia, and characterized the DAZ and CDY1 copies retained, the presence or absence of duplications and the Y-chromosomal haplogroup. Although our study had good power to detect factors that accounted for ≥5.5% of the variation in sperm concentration, no such factor was detected. A negative effect of gr/gr deletions followed by b2/b4 duplication was observed within the normospermic group, which remains to be further explored in a larger study population. Finally, we observed significant geographical differences in the frequency of different subtypes of gr/gr deletions which may have relevance for the interpretation of case control studies dealing with admixed populations.ConclusionsWe conclude that the phenotypic variation of gr/gr carriers in men of European origin is largely independent of the Y-chromosomal background.


2008 - Clinical consequences of microdeletions of the Y chromosome: the extended Münster experience. [Articolo su rivista]
Simoni, Manuela; Tüttelmann, F; Gromoll, J; Nieschlag, E.
abstract

A total of 3179 patients were screened for Y-chromosome microdeletions and 821 patients for partial AZFc deletions. Thirty-nine Y-chromosomal microdeletions were found (2.4% of men with <1 x 106/ml spermatozoa): two AZFa, two AZFb, one AZFbc, one partial AZFb, one partial AZFb+c and 32 AZFc (b2/b4). Partial AZFc deletions were found in 45 patients (5.5%), mostly gr/gr deletions (n = 28). In patients with AZFc deletion, azoospermia was found in 53.1% and sperm concentrations of mostly <0.1 x 106/ml were found in 46.9%. Semen analyses and FSH measurements showed no trend over time. Elongated spermatids were seen in 6/15 AZFc patients and bilateral Sertoli cell-only was found in 4/15. Testicular sperm extraction (TESE) was attempted in 10 patients and spermatozoa were found in six. Compared with infertile men matched by sperm concentration, no differences in hormonal and seminal parameters could be found in patients with AZFc or gr/gr deletions. It is concluded that: (i) frequency of AZF deletions in Germany is much lower than in other countries; (ii) AZFc deletions are associated with severe disturbances of spermatogenesis and TESE is not possible in half of these patients; (iii) AZFc and gr/ gr deletions are not associated with any clinical diagnostic parameter; (iv) and no trend is apparent over time.


2008 - Complete spermatogenesis in orthotopic but not in ectopic transplants of autologously grafted marmoset testicular tissue. [Articolo su rivista]
Luetjens, Cm; Stukenborg, Jb; Nieschlag, E; Simoni, Manuela; Wistuba, J.
abstract

Testicular grafting has the potential to become a method to preserve fertility in prepubertal boys undergoing cancer treatment. The possibility of successful germ cell maturation after autologous grafting should be proven preclinically in a nonhuman primate model. Therefore, in two experiments, we analyzed the potential of autologous testicular grafting in the marmoset model. A first experiment in immature and adult hemi-castrated monkeys addressed the question of whether full spermatogenesis in an ectopic graft could be achieved under a relatively normal endocrine milieu and whether the donor's age is of influence. A second experiment in castrated immature animals examined whether the transplantation site [ectopic (back skin) or orthotopic (scrotum)] influences spermatogenic progress and whether cryopreserved tissue can be successfully transplanted. Grafts were analyzed by histology, immunohistochemistry, and morphometry. Bioactive chorionic gonadotropin and serum testosterone were measured. In the adults, ectopic grafts degenerated, whereas in the immature animals, grafts survived at the spermatogonial level. In the castrates, none of the cryopreserved grafts survived, ectopic grafts were meiotically arrested, but orthotopic transplants completed spermatogenesis. Androgen and bioactive chorionic gonadotropin levels were not decisive for graft development. When ectopic and orthotopic transplantation sites were compared, the scrotum has a substantially lower temperature. Thus, the higher temperature at the ectopic transplantation site may contribute to spermatogenic arrest. Autologous grafting of nonhuman primate testicular tissues can result in complete spermatogenesis. Our findings indicate that transplantation site and developmental age of the tissue play a role more important than the endocrine milieu.


2008 - Current recommendations for genetic testing in male infertility [Articolo su rivista]
Tüttelmann, Frank; Simoni, Manuela
abstract

Abstract


2008 - Effects of tadalafil on Nocturnal Penile Tumescence and Rigidity in normal men: Randomized, Placebo-Controlled, Crossover Study [Abstract in Atti di Convegno]
Antonio R. M., Granata; Rochira, Vincenzo; DE MOLA, Giovanni; Guidi, Alessandro; Pugni, Valeria; Scaltriti, Sara; Simoni, Manuela; Carani, Cesare
abstract

This Randomized, Placebo-Controlled, Crossover Study evaluates the effects of tadalafil on Nocturnal Penile Tumescence and Rigidity in normal men.


2008 - FSH RECEPTOR AND DAZL GENE POLYMORPHISMS DO NOT AFFECT THE AGE AT MENOPAUSE. [Articolo su rivista]
Zerbetto, I; Gromoll, J; Luisi, S; Reis, Fm; Nieschlag, E; Simoni, Manuela; Petraglia, F.
abstract

ObjectiveTo evaluate whether single nucleotide FSH receptor and DAZL gene polymorphisms are associated with menarche and menopause timing.DesignProspective study.SettingSiena and Münster Universities.Patient(s)Physiologically menopausal women.Intervention(s)The presence of FSH receptor or DAZL gene polymorphisms was investigated. Blood samples were collected and polymorphisms evaluated in extracted genomic DNA.Main Outcome Measure(s)Menarche age, menopausal age, and total years of fertility were evaluated on the basis of FSH receptor (genotypes Asn/Asn, Asn/Ser, and Ser/Ser at codon 680) and DAZL gene.Result(s)The median age of menarche was 13 years in the Asn/Asn group and 12 years in the Asn/Ser and Ser/Ser groups. The median age at menopause was 50 years in the Asn/Asn and Asn/Ser groups and 51 years in the Ser/Ser group. The length of the fertile period was 37 years in the Asn/Asn group, 38 years in the Asn/Ser group, and 39 years in the Ser/Ser group. Regarding the DAZL polymorphism, A/A, A/G, and G/G had the same age at menarche, age at menopause, and length of fertile period.Conclusion(s)We found that genotype of FSH receptor or SNP of DAZL do not predict the age at natural menopause and duration of fertility in women. The presence of Asn680/Asn680 genotype is associated with a slightly delayed age at menarche.


2008 - Functional genetic polymorphisms and female reproductive disorders. Part I – Polycystic ovary syndrome and ovarian response. [Articolo su rivista]
Simoni, Manuela; Tempfer, Cb; Destenaves, B; Fauser, Bcjm
abstract

BACKGROUNDThe identification of polymorphisms associated with a disease can help to elucidate its pathogenesis, and this knowledge can be used to improve prognosis for women with a particular disorder, such as polycystic ovary syndrome (PCOS). Since an altered response to ovarian stimulation is also a characteristic of the disease, further knowledge about its aetiology could help in defining the parameters that determine the response of an individual to ovarian stimulation.METHODSPubMed and EMBASE databases were systematically searched for gene association studies published until the end of August 2007, using search criteria relevant to PCOS and ovarian response to stimulation. Data from additional papers identified through hand searches were also included; 139 publications were reviewed.RESULTSSeveral genes involved in ovarian function and metabolism are associated with increased susceptibility to PCOS, but none is strong enough to correlate alone with susceptibility to the disease, or response to therapy. A single-nucleotide polymorphism in exon 10 of the FSH receptor (FSHR) gene, FSHR p.N680S, was consistently identified as having a significant association with ovarian response to FSH.CONCLUSIONSNo consistent association between gene polymorphism and PCOS could be identified. The FSHR gene may play a significant role in the success of ovarian stimulation, and can be used as a marker to predict differences in FSHR function and ovarian response to FSH. Genotyping the FSHR p.N680S polymorphism may provide a means of identifying a population of poor responders before in vitro fertilization procedures are initiated.


2008 - KRÜPPEL-LIKE FACTOR 4 IS INVOLVED IN FUNCTIONAL DIFFERENTIATION OF TESTICULAR SERTOLI CELLS. [Articolo su rivista]
Godmann, M; Katzt, Jp; Guillou, F; Simoni, Manuela; Kaestner, Kh; Behr, R.
abstract

Krüppel-like factor 4 (KLF4) is a pleiotropic zinc finger transcription factor that regulates genes being involved in differentiation and cell-cycle control. Knock out studies revealed a critical function for KLF4 in the terminal differentiation of many epithelial cells. In testicular Sertoli cells, Klf4 is strongly inducible by the glycoprotein Follicle stimulating hormone (FSH). Since KLF4 is essential for postnatal survival in mice, we deleted Klf4 specifically in Sertoli cells using the Cre/loxP system. Importantly, around postnatal day 18, a critical period of terminal Sertoli cell differentiation, mutant seminiferous tubules exhibited a disorganized germinal epithelium and delayed lumen formation. The ultrastructural finding of highly vacuolized Sertoli cell cytoplasm and the identification of differentially expressed genes, which are known to play roles during vesicle transport and fusion or for maintenance of the differentiated cell state, suggest impaired apical secretion of the Sertoli cell. Interestingly, a high proportion of all identified genes was localized in a small subregion of chromosome 7 suggesting coordinated regulation. Intriguingly, adult mutant mice are fertile and show normal testicular morphology, although the testosterone levels are decreased. In summary, KLF4 plays a significant role for proper and timely Sertoli cell differentiation in pubertal mice.


2008 - Mutations in a novel, cryptic exon of the luteinizing hormone/chorionic gonadotropin receptor gene cause male pseudohermaphroditismus. [Articolo su rivista]
Kossack, N; Simoni, Manuela; RICHTER UNRUH, A; Themmen, A; Gromoll, J.
abstract

BackgroundMale pseudohermaphroditism, or Leydig cell hypoplasia (LCH), is an autosomal recessive disorder in individuals with a 46,XY karyotype, characterized by a predominantly female phenotype, a blind-ending vagina, absence of breast development, primary amenorrhea, and the presence of testicular structures. It is caused by mutations in the luteinizing hormone/chorionic gonadotropin receptor gene (LHCGR), which impair either LH/CG binding or signal transduction. However, molecular analysis has revealed that the LHCGR is apparently normal in about 50% of patients with the full clinical phenotype of LCH. We therefore searched the LHCGR for novel genomic elements causative for LCH.Methods and FindingsIn the present study we have identified a novel, primate-specific bona fide exon (exon 6A) within the LHCGR gene. It displays composite characteristics of an internal/terminal exon and possesses stop codons triggering nonsense-mediated mRNA decay (NMD) in LHCGR. Transcripts including exon 6A are physiologically highly expressed in human testes and granulosa cells, and result in an intracellular, truncated LHCGR protein of 209 amino acids. We sequenced exon 6A in 16 patients with unexplained LCH and detected mutations in three patients. Functional studies revealed a dramatic increase in the expression of the mutated internal exon 6A transcripts, indicating aberrant NMD. These altered ratios of LHCGR transcripts result in the generation of predominantly nonfunctional LHCGR isoforms, thereby preventing proper expression and functioning.ConclusionsThe identification and characterization of this novel exon not only identifies a new regulatory element within the genomic organization of LHCGR, but also points toward a complex network of receptor regulation, including events at the transcriptional level. These findings add to the molecular diagnostic tools for LCH and extend our understanding of the endocrine regulation of sexual differentiation.


2008 - Polymorphisms of the luteinizing hormone/chorionic gonadotropin receptor (LHCGR) gene: association with maldescended testes and male infertility. [Articolo su rivista]
Simoni, Manuela; Tüttelmann, F; Michel, C; Böckenfeld, Y; Nieschlag, E; Gromoll, J.
abstract

BACKGROUNDThe identification of polymorphisms associated with a disease can help to elucidate its pathogenesis, and this knowledge can be used to improve prognosis for women with a particular disorder, such as polycystic ovary syndrome (PCOS). Since an altered response to ovarian stimulation is also a characteristic of the disease, further knowledge about its aetiology could help in defining the parameters that determine the response of an individual to ovarian stimulation.METHODSPubMed and EMBASE databases were systematically searched for gene association studies published until the end of August 2007, using search criteria relevant to PCOS and ovarian response to stimulation. Data from additional papers identified through hand searches were also included; 139 publications were reviewed.RESULTSSeveral genes involved in ovarian function and metabolism are associated with increased susceptibility to PCOS, but none is strong enough to correlate alone with susceptibility to the disease, or response to therapy. A single-nucleotide polymorphism in exon 10 of the FSH receptor (FSHR) gene, FSHR p.N680S, was consistently identified as having a significant association with ovarian response to FSH.CONCLUSIONSNo consistent association between gene polymorphism and PCOS could be identified. The FSHR gene may play a significant role in the success of ovarian stimulation, and can be used as a marker to predict differences in FSHR function and ovarian response to FSH. Genotyping the FSHR p.N680S polymorphism may provide a means of identifying a population of poor responders before in vitro fertilization procedures are initiated.


2007 - AMH AND AMH TYPE II RECEPTOR POLYMORPHISMS ARE ASSOCIATED WITH FOLLICULAR PHASE ESTRADIOL LEVELS IN NORMO-OVULATORY WOMEN. [Articolo su rivista]
Kevenaar, Me; Themmen, Apn; Laven, Jse; Sonntag, B; Fong, Sl; Uitterlinden, Ag; DE JONG, Fh; Pols, Hap; Simoni, Manuela; Visser, Ja
abstract

BACKGROUND: In mice, anti-Müllerian hormone (AMH) inhibits primordial follicle recruitment and decreases FSH sensitivity. Little is known about the role of AMH in human ovarian physiology. We hypothesize that in women AMH has a similar role in ovarian function as in mice and investigated this using a genetic approach.METHODS: The association of the AMH Ile49Ser and the AMH type II receptor (AMHR2) –482 A > G polymorphisms with menstrual cycle characteristics was studied in a Dutch (n = 32) and a German (n = 21) cohort of normo-ovulatory women.RESULTS: Carriers of the AMH Ser49 allele had higher serum estradiol (E2) levels on menstrual cycle day 3 when compared with non-carriers in the Dutch cohort (P = 0.012) and in the combined Dutch and German cohort (P = 0.03). Carriers of the AMHR2 –482G allele also had higher follicular phase E2 levels when compared with non-carriers in the Dutch cohort (P = 0.028), the German cohort (P = 0.048) and hence also the combined cohort (P = 0.012). Women carrying both AMH Ser49 and AMHR2 –482G alleles had highest E2 levels (P = 0.001). For both polymorphisms no association with serum AMH or FSH levels was observed.CONCLUSIONS: Polymorphisms in the AMH and AMHR2 genes are associated with follicular phase E2 levels, suggesting a role for AMH in the regulation of FSH sensitivity in the human ovary.


2007 - Chorionic gonadotropin beta subunit gene expression in the marmoset pituitary is controlled by steroidogenic factor 1 (SF1), early growth response protein 1 (Egr1) and pituitary homeobox factor 1 (Pitx1). [Articolo su rivista]
Henke, A; Luetjens, Cm; Simoni, Manuela; Gromoll, J.
abstract

In most mammals, the gonads are under the control of the pituitary gonadotropins LH and FSH. However, in the common marmoset monkey Callithrix jacchus, no LH is detectable in the pituitary but chorionic gonadotropin (CG) instead, normally produced in the placenta. This study investigated the mechanism of CGß subunit activation in the pituitary and why humans do not express CG in the pituitary. 5'-Rapid amplification of cDNA ends, EMSA, and promoter-driven luciferase assays performed with the gonadotropic LßT2 cells showed that marmoset monkey CGß is GnRH responsive and activated similar to human LHß by the transcription factors steroidogenic factor 1 (SF1), early growth response protein 1 (Egr1), and pituitary homeobox factor 1 (Pitx1) and displayed a transcriptional start site 7 bp upstream of exon 1. In contrast, the human CGß promoter displayed in the binding elements for pituitary homeobox factor 1 and early growth response protein 1 three consensus sequence mismatches, leading to very low activity that could be drastically increased by mutation to the consensus sequences. Vice versa, marmoset CGß promoter activity was reduced after introduction of the human CGß mismatches. An in vivo study in pregnant marmoset monkeys showed that during pregnancy, there is no significant decrease of pituitary CG production, contrasting human LH down-regulation. In conclusion, pituitary CG production is lacking in humans due to the absence of appropriate DNA-binding elements, which are present in marmosets, thereby enabling GnRH activation of expression. However, during pregnancy of marmosets, pituitary CG expression is not inhibited.


2007 - Gene polymorphisms and male infertility. A meta-analysis and literature review. [Articolo su rivista]
Tüttelmann, F; RAJPERT DE MEYTS, E; Nieschlag, E; Simoni, Manuela
abstract

Many genetic polymorphisms have been studied extensively to elucidate their role in the pathophysiology of male infertility. This article presents a review of the literature following a thorough search of PubMed, a compilation of meta-analyses of studies reporting an association with male fertility where the population(s) could be clearly identified as fertile and/or infertile, and a summary of all polymorphisms that have been investigated in single case-control studies to date. The meta-analyses revealed significant associations between polymorphism and male fertility only for AZF gr/gr deletions (OR 1.81, 1.46-2.24 CI, P < 0.00001) and MTHFR 677C→T (OR 1.39, 1.15-2.69 95% CI, P = 0.0006) but not for POLG, DAZL, USP26 or FSHR. The influence of CAG repeat length in AR remains open and debated. Genes encoding nuclear proteins (PRM1/2, TNP1/2) and ER1 are possible candidates for further examination, while the role of TAF7L remains unclear. Polymorphisms in 16 other genes have been investigated in single studies, but the results remain doubtful due to often small and heterogeneous cohorts and in the absence of independent replications. The genetic studies performed so far emphasize the complexity of male infertility as a presumably polygenetic trait amended by environmental, lifestyle or occupational factors.


2007 - Genetics of hypogonadotropic hypogonadism. [Articolo su rivista]
Simoni, Manuela; Nieschlag, E.
abstract

Background: Idiopathic hypogonadotropic hypogonadism (HH) results from a defect in the normal pulsatile secretion pattern of gonadotropin-releasing hormone (GnRH) from the hypothalamus. Clinically it can be categorized as one of two types: HH associated with anosmia, known as Kallmann syndrome, and isolated HH. The anatomical explanation for Kallmann syndrome stems from incomplete or total failure of GnRH-secreting neurons to migrate from the olfactory epithelium to their final destination in the mediobasal hypothalamus. Several genes are involved in the migration of the GnRH neurons. Conclusions: Mutations of the KAL1 gene, encoding for anosmin 1, and of the FGFR1 (or KAL2) gene, encoding for fibroblast growth factor receptor 1, can be found in familial cases of Kallmann syndrome. KAL1 mutations are responsible for X-linked recessive inheritance, and FGFR1 mutations are the autosomal dominant form. Moreover, mutations of the gonadotropin-releasing hormone receptor gene and G-protein-coupled receptor 54 gene are found in over 50% of familial cases of isolated HH with autosomal recessive inheritance.


2007 - Genomic checkpoints of exon 10 usage in the luteinizing hormone receptor type 1 and type 2. Mol Endocrinol [Articolo su rivista]
Gromoll, J; Lahrmann, L; Godmann, M; Müller, T; Stamm, S; Simoni, Manuela
abstract

Alternative splicing is a hallmark of glycoprotein hormone receptor gene regulation, but its molecular mechanism is unknown. The LH receptor (LHR) gene possesses 11 exons, but exon 10 is constitutively skipped in the New World monkey lineage (LHR type 2), whereas it is constitutively spliced in the human (LHR type 1). This study identifies the regulatory elements of exon 10 usage. Sequencing of genomic marmoset DNA revealed that the cryptic LHR exon 10 is highly homologous to exon 10 from other species and displays intact splice sites. Functional studies using a minigene approach excluded the contribution of intronic, marmoset-specific long interspersed nucleotide-1 elements to exon 10 skipping. Sequencing of the genomic regions surrounding exon 10 from several primate lineages, sequence comparisons including the human and mouse LHR gene, revealed the presence of unique nucleotides at 3'-intronic position –19 and –10 and at position +26 within exon 10 of the marmoset LHR. Exon trap experiments and in vitro mutagenesis of these nucleotides resulted in the identification of a composite regulatory element of splicing consisting of cis-acting elements represented by two polypyrimidine tracts and a trans-acting element within exon 10, which affect the secondary RNA structure. Changes within this complex resulted either in constitutive exon inclusion, constitutive skipping, or alternative splicing of exon 10. This work delineates the molecular pathway leading to intronization of exon 10 in the LHR type 2 and reveals, for the first time, the essential function of regulatory and structural elements involved in glycoprotein hormone receptor splicing.


2007 - LH splicing variants and gene expression in the marmoset monkey. [Articolo su rivista]
MICHEL C., GROMOLL J; Chandolia, R; Wistuba, J; Luetjens, Cm; Simoni, Manuela
abstract

In the marmoset monkey, the LHR type II, lacking exon 10, is the native receptor type. We characterised the LHR splicing pattern in marmoset testes and the adrenals during puberty and in pre- and postpubertal ovaries and quantified mRNA LHR expression in the testis. We detected 11 LHR splicing variants expressed at similar levels and generated by exon skipping and/or usage of cryptic splice sites. No preferred splicing variant during pubertal maturation was observed in both sexes. Testicular and adrenal LHR expression levels did not significantly change with age. However, a significant increase during pubertal maturation for the serum testosterone/LHR ratio indicated that testosterone secretion increases in the presence of constant LHR mRNA expression levels. We conclude that LHR splicing in the marmoset displays a homogenous pattern and that the main function of the LHR is established in the testis, reaching its highest efficiency during pubertal maturation.


2007 - Mutation analysis of the X-chromosome linked, testis-specific TAF7L gene in spermatogenic failure. [Articolo su rivista]
Akinloye, O; Gromoll, J; Callies, C; Nieschlag, E; Simoni, Manuela
abstract

The precise temporal and spatial expressions of specific transcription regulation factors (TRF) have long been considered essential for spermatogenesis. Recently, it has been speculated that mammals have evolved more specialised TRF genes. In the human, the TAF7L gene may be essential for maintenance of spermatogenesis. In this study, we investigated the possible role of the TAF7L gene located on the X chromosome in testicular function and spermatogenic failure. In a case-controlled retrospective study, we recruited 16 infertile males with consistent, nonobstructive azoospermia and with normal serum follicle-stimulating hormone (FSH) levels. Twenty age-matched men with normal spermatogenesis with the same ethnic background (Caucasian) were recruited as controls. Their genomic DNA was screened for sequence changes in the coding regions and part of the flanking introns of the TAF7L gene by direct sequencing. Amino acid sequence was compared with the NCBI standard sequence (BC043391). Semen analysis and hormone evaluation were performed. We observed six sequence variations in four patients, consisting of two point mutations, one each in exon 9 and 13 and one six-basepair deletion in exon 13 with concomitant changes in amino acid. One additional nucleotide exchange was observed in intron 8. Most of these changes were also found in eight controls with the exception of changes in exon 13. A meta-analysis including the present study and literature data suggests a possible association of the point mutation in exon 13 with infertility. There was no association or relationship with reproductive hormones. In conclusion, the sequence variants in the cDNA sequence observed are common polymorphisms. The changes in intron 8 appear novel. We report for the first time that most of the alterations are not associated with gonadal dysfunction, while the sequence variant in exon 13 may represent a risk factor for spermatogenic failure.


2007 - Polymorphism of the FSH receptor and ovarian response to FSH. [Articolo su rivista]
Wunsch, A; Sonntag, B; Simoni, Manuela
abstract

L'hormone folliculostimulante (FSH) joue un rôle crucial dans la reproduction humaine. Son récepteur (FSHR) est exclusivement localisé dans les cellules de la granulosa de l'ovaire ainsi que dans les cellules de Sertoli des testicules. Ils existent deux SNP dans l'exon 10 du gène du FSHR qui entraînent la formation de deux formes alléliques d'une fréquence quasiment similaire. Au codon 307, situé dans la region charnière, se trouve ou bien une thréonine (Thr) ou bien une alanine (Ala), tandis qu'en position 680, dans le domaine intracellulaire, se trouve une asparagine (Asn) ou une sérine (Ser). Des études cliniques ont bien montré que le polymorphisme p.N680S détermine la réponse ovarienne chez des patientes soumises à un traitement inducteur de l'ovulation. Les patientes portant l'allèle Ser680 nécessitent plus de FSH pour atteindre le même taux d'estradiol comparées aux patientes porteuses de l'allèle Asn680. Une étude analysant des femmes ayant un cycle menstruel normal a révélé que le génotype Ser680/Ser680 entraîne un taux élevé de la FSH et une prolongation du cycle mensuel. Le mécanisme moléculaire responsable de la « résistance » partielle du Ser680-FSHR par rapport à la FSH est inconnu jusqu'à présent. Des expériences futures devraient contribuer à notre compréhension concernant l'effet de la FSH sur la sélection et la dominance folliculaire, et ainsi, permettre des traitements sur mesure de l'infertilité et de la préservation de la fertilité.


2007 - Replacement of connexin43 by connexin26 in transgenic mice leads to gonadal dysfunction. [Articolo su rivista]
Winterhager, E; Pielensticker, N; Freyer, J; Ghanem, A; Schrickel, Jw; KIM J., S; Behr, R; Grümmer, R; Maass, K; Urschel, S; Lewalter, T; Tiemann, K; Simoni, Manuela; Willecke, K.
abstract

BackgroundIn order to further distinguish unique from general functions of connexin43, we have generated mice in which the coding region of connexin43 was replaced by that of connexin26.ResultsHeterozygous mothers showed impaired mammary gland development responsible for decreased lactation and early postnatal death of the pups which could be partially rescued by wild type foster mothers. Only about 17% of the homozygous connexin43 knock-in connexin26 mice instead of 25% expected according to Mendelian inheritance, were born and only 6% survived to day 21 post partum and longer. Neonatal and adult connexin43 knock-in connexin26 mice exhibited slowed ventricular conduction in their hearts, i.e. similar but delayed electrophysiological abnormalities as connexin43 deficient mice. Furthermore, connexin43 knock-in connexin26 male and female mice were infertile and exhibited hypotrophic gonads. In testes, tubuli seminiferi were developed and spermatogonia as well as some primary spermatocytes were present, but further differentiated stages of spermatogenesis were absent. Ovaries of female connexin43 knock-in connexin26 mice revealed only few follicles and the maturation of follicles was completely impaired.ConclusionThe impaired gametogenesis of homozygous males and females can explain their infertility.


2006 - CHANGES IN ENDOCRINE PROFILE AND REPRODUCTIVE ORGANS DURING PUBERTY IN THE MALE MARMOSET MONKEY. [Articolo su rivista]
Chandolia, Rk; Luetjens, Cm; Wistuba, J; Yeung, Ch; Nieschlag, E; Simoni, Manuela
abstract

Data on pubertal maturation in male marmoset, a model for human reproduction, are scant and conflicting. We collected data on novel parameters to characterize puberty. Twenty-five marmoset monkeys were assigned to five age groups by weeks (wk): 21 (pre-pubertal), 43 (onset of puberty), 52 (fully pubertal), 70 (mature), and 116 (fully adult). Serum and intratesticular testosterone and pituitary bioactive chorionic gonadotropin (bioCG) were measured. Testicular development was assessed by ultrasonography, histology, and flow cytometry. Three consecutive blood samples revealed extreme fluctuations in testosterone concentrations, suggesting an erratic secretion. Age-related changes in serum testosterone and pituitary bioCG concentrations were observed. Intratesticular androgens (ITAs) showed high fluctuations within groups at all ages and were high in some animals by 21 wk. Unexpectedly, no correlation between pituitary bioCG and serum testosterone or ITAs was found, but these parameters significantly correlated with testicular weight and volume. These observations were consistent a dependence on the testis growth on bioCG. Unfortunately, the low serum levels of bioCG were not measurable in this study. At 43 wk, the animals reached puberty. At 52 wk of age, animals attained maximum body and epididymal weights and qualitatively normal spermatogenesis, but testes continued growing, reaching a maximum of all parameters at 70 wk of age, without further major changes at the age of 116 wk. It is concluded that (1) gonadal activation is evident at wk 21, (2) the male marmoset reaches the pubertal threshold around 43 wk of age, attains qualitative parameters at 52 wk, matures further to sexual maturity at 70 wk, and (3) serum testosterone and ITAs are highly variable without any identifiable correlation with pituitary bioCG.


2006 - Elevated follicle-stimulating hormone levels and the chances for azoospermic men to become fathers after retrieval of elongated spermatids from cryopreserved testicular tissue [Articolo su rivista]
Zitzmann, M; Nordhoff, V; VON SCHÖNFELDT, V; NORDSIEK MENGEDE, A; Kliesch, S; Schüring, A; Leutjens, Cm; Kamischke, A; Cooper, Tg; Simoni, Manuela; Nieschlag, E.
abstract

OBJECTIVE: To assess individual chances for a live-born child in azoospermic men by performance of testicular sperm extraction (TESE) followed by intracytoplasmatic sperm injection (ICSI). DESIGN: A retrospective cohort study. SETTING: An academic fertility care center and research unit. PATIENT(S): Two hundred three couples who wished to have a child; all men had azoospermia. INTERVENTION(S): All men were operated for TESE; 112 men were found to have elongated spermatids (ES), and 209 ICSI cycles were performed in these men using cryopreserved tissue. MAIN OUTCOME MEASURE(S): Predictors for the chances to obtain live sperm and for probabilities of fertilization, clinical pregnancies, and live births. RESULT(S): Testicular volume, FSH, and inhibin B levels were predictors for the presence of ES. Intracytoplasmic sperm injection resulted in 23 pregnancies, leading to 20 live births. Despite the presence of ES and performance of ICSI in cases of FSH levels >or=20 IU/L, no pregnancy resulted in these men (n = 21). Receiver operating characteristics revealed FSH levels of >or=20 IU/L as cutoff for treatment success. The number of testicular tubuli containing ES served as a predictor for clinical pregnancy as well as for live birth. Cigarette smoking by the male partner exerted a significant negative influence on treatment success. CONCLUSION(S): The degree of completely maintained spermatogenesis within the biopsy appears to reflect intrinsic abilities of spermatozoa to induce normal embryo development. Charts based on regression models are presented for counseling patients before TESE; these explain chances of finding ES and probability of successful ICSI. Obtaining offspring is unlikely in cases of azoospermia and of FSH levels of >or=20 IU/L.


2006 - Follicular reserve and response to stimulation. Molecular genetic aspects [Articolo su rivista]
Sonntag, B.; Schuring, A.; Simoni, M.; Kiesel, L.
abstract

Ovarian response to stimulation therapy and the timing of follicular depletion vary substantially between individual patients. Both factors are of outstanding importance for the performance and outcome of infertility therapy. Besides established clinical markers, the analysis of genetic variants may be of use as a prognostic tool in the near future. Clinical studies on the p.N680S-polymorphism of the FSH receptor gene have demonstrated the homozygous Ser/Ser variant to be less sensitive to endogenous or exogenous FSH in terms of estradiol production. Prospective genotyping before stimulation therapy could therefore individualize the FSH dose to the patients' requirements and improve the balance between adequate ovarian response and unwanted side effects. Furthermore, the identification and analysis of other candidate genes could shed light on the variable timing of follicular reserve and depletion. © Springer Medizin Verlag 2006.


2006 - Hypogonadism in boys with Prader-Labhart-Willi syndrome. A specific disorder with hypothalamic and gonadal components. [Articolo su rivista]
Eiholzer, U; Lallemand, D; Rousson, V; Schlumpf, M; Gasser, T; Girard, J; Grüters, A; Simoni, Manuela
abstract

Context: The specific form of hypogonadism in Prader-Labhart-Willi syndrome (PWS), central or peripheral, remains unexplained.Objectives: The objectives of this study were to investigate the cause of hypogonadism in PWS and determine whether human chorionic gonadotropin (hCG) treatment can restore pubertal development.Design: This was a clinical follow-up study, divided into two samples, over a duration of 1.5 and 4.5 yr.Patients: Eight male infants and six peripubertal boys (age at start of observation, 0.06–0.93 and 8.1–10.8 yr, respectively) with genetically confirmed PWS were studied.Intervention: hCG (500–1500 U twice weekly) was given from age 13.5 yr to the present.Main Outcome Measures: Serum FSH, LH, inhibin B, and testosterone levels and pubertal development were the main outcome measures.Results: Infants with PWS presented normal LH (2.3 ± 0.7 U/liter) and testosterone (2.5 ± 0.9 nmol/liter) levels (mean ± SEM at 5 months) compared with the reference range. However, two thirds of the boys displayed cryptorchidism. Inhibin B levels were at the lowest level of the normal range and decreased significantly between infancy and puberty (at 13 yr, 72 ± 17 pg/ml), whereas FSH secretion increased (9.9 ± 2.6 U/liter). Pubertal maturation stopped at an average bone age of 13.9 yr. hCG therapy increased testosterone (11 ± 2 nmol/liter) and reduced FSH (at 16 yr, 1.1 ± 0.9 U/liter) levels. Testicular volume (5.6 ± 1 ml) and inhibin B (26.5 ± 11.9 pg/ml) remained low.Conclusion: Children with PWS display a specific form of combined hypothalamic (low LH) and peripheral (low inhibin B and high FSH) hypogonadism, suggesting a primary defect in Sertoli and/or germ cell maturation or an early germ cell loss. hCG therapy stimulates testosterone production and virilization.


2006 - Meiosis in autologous ectopic transplants of immature testicular tissue grafted to Callithrix jacchus. [Articolo su rivista]
Wistuba, J; Luetjens, Cm; Wesselmann, R; Nieschlag, E; Simoni, Manuela; Schlatt, S.
abstract

Grafting of immature testicular tissue provides a tool to examine testicular development and may offer a perspective for preservation of fertility in prepubertal patients. Successful xenografting in mice, resulting in mature spermatids, has been performed in several species but has failed with testicular tissues from the common marmoset, Callithrix jacchus. Previous data indicate that the hormonal milieu provided by the mouse host might cause this failure. We conducted autologous ectopic transplantation of testicular fragments under the back skin in newborn marmoset monkeys. Seventeen months after transplantation, we found viable transplants in 2 out of the 4 grafted animals. In the transplants, tubules developed up to a state intermediate between the pregraft situation and adult controls. Dividing spermatogonia and primary spermatocytes were present. Boule-like positivity and CDC25A negativity indicated that spermatogenesis was arrested at early meiosis. Immunohistochemistry revealed normal maturation of Sertoli cells, Leydig cells, and peritubular cells. Serum testosterone values were not restored to the normal range and bioactive chorionic gonadotropin levels increased to castrate levels. Meiotic arrest could have occurred in the grafts because of lack of sufficient testosterone or because of hyperthermia caused by the ectopic position of the grafts. We conclude that autologous transplants of immature testicular tissues in the marmoset can mature up to meiosis but that normal serum testosterone levels are not restored. Further studies have to be performed to overcome the meiotic arrest to explore the model further and to develop therapeutic options.


2006 - Tissue expression of the nuclear progesterone receptor in male non-human primates and men. [Articolo su rivista]
Luetjens, Cm; Didolkar, A; Kliesch, S; Paulus, W; Jeibmann, A; Böcker, W; Nieschlag, E; Simoni, Manuela
abstract

In females, progesterone is associated with reproductive functions. In males, its role and the expression of its genomic receptor are not very well understood. In attempts to achieve a hormonal male contraceptive method, gestagens are used to downregulate gonadotropin and sperm production. It is therefore essential to understand the mechanism of action of progesterone at the molecular level in males, especially in primates. This investigation was undertaken: (a) to determine whether the genomic progesterone receptor is expressed in males; and (b) to locate it in various organs that are potential targets of gestagens. Human tissues were obtained at surgery for benign prostatic hyperplasia or prostate cancer and at autopsy. Non-human primate tissues were obtained at autopsy. This study was performed by analyzing the genomic progesterone receptor by immunohistochemistry, Western blot and RT-PCR. The nuclear progesterone receptor was expressed in pituitary and hypothalamus of both monkeys and men. In the testis progesterone receptor expression was found in a few peritubular and interstitial cells, but not in germ cells. In addition, expression was detected in the epididymis, prostate and male mammary gland. Reverse transcriptase (RT)-PCR experiments indicated that progesterone receptor A and B are expressed in all tissues analyzed. These data exclude direct genomic effects of gestagens at the spermatogenic level but indicate that a male contraceptive based on gestagens might have some effects on other tissues, such as the epididymis, prostate and mammary gland.


2006 - Y-chromosomal microdeletions and partial deletions of the AZFc region in normozoospermic, severe oligozoospermic and azoospermic men from Sri Lanka. [Articolo su rivista]
Fernando, L; Gromoll, J; Weerasooriya, Tr; Nieschlag, E; Simoni, Manuela
abstract

Aim: To assess for the first time the occurrence of Y chromosomal microdeletions and partial deletions of the Azoospermia Factor c (AZFc) region in Sri Lankan men and to correlate them with clinical parameters.Methods: In a retrospective study, we analyzed 96 infertile men (78 with non-obstructive azoospermia) and 87 controls with normal spermatogenesis. AZFa, AZFb, AZFc and partial deletions within the AZFc region were analyzed by multiplex polymerase chain reaction (PCR) according to established protocols.Results: No AZFa, AZFb or AZFc deletions were found in the control group. Seven patients in the group of infertile men were found to have deletions as following: one AZFa, two AZFc, two AZFbc and two AZFabc. The relative distribution of these patterns was significantly different compared with that found in the German population. Extension analysis confirmed that the deletions occurred according to the current pathogenic model. gr/gr deletions were found to be equally present both in the patients (n= 4) and in the control group (n= 4). One b2/b3 deletion was found in the patient group.Conclusion: These results suggest that the frequency and pattern of microdeletions of the Y chromosome in Sri Lankan men are similar to those found in other populations and confirm that gr/gr deletions are not sufficient to cause spermatogenetic failure.


2005 - A common single nucleotide polymorphism in exon 10 of the human follicle-stimulating hormone receptor is a major determinant of length and hormonal dynamics of the menstrual cycle. [Articolo su rivista]
Greb, Rr; Grieshaber, K; Gromoll, J; Sonntag, B; Nieschlag, E; Kiesel, L; Simoni, Manuela
abstract

CONTEXT: FSH is essential for follicular maturation. Data from ovarian hyperstimulation cycles suggest that FSH action is attenuated by a frequent single nucleotide polymorphism of the FSH receptor gene exchanging Asn for Ser at codon 680. OBJECTIVE: We hypothesized that the FSH receptor genotype influences menstrual cycle dynamics. DESIGN: Menstrual cycle was monitored from the midluteal phase through ovulation until the consecutive menstruation. SETTING: The study was conducted at the University research center. SUBJECTS: Women homozygous for the Asn680 (n = 12) and Ser680 (n = 9) variants with normal menstrual cycles volunteered for the study. INTERVENTIONS: There were no interventions. MAIN OUTCOME MEASUREMENTS: Follicular growth, serum LH, FSH, estradiol, progesterone, inhibin A, inhibin B and antimullerian hormone were measured. RESULTS: During the luteo-follicular transition, serum levels of estradiol, progesterone, and inhibin A were significantly lower, and FSH started to rise earlier in the Ser680/Ser680 group. FSH levels were steadily and significantly higher, and the mean area under the FSH curve was 31% greater in this group (P < 0.002). No differences were observed in estradiol, inhibin B, and growth velocities of dominant follicles. The time from luteolysis to ovulation was significantly longer in women with the Ser680/Ser680 (13.6 +/- 1.01 d) compared with Asn680/Asn680 (11.3 +/- 0.61 d, P < 0.05) genotype with a significant difference in total menstrual cycle length (29.3 vs. 27.0 d, respectively; P < 0.05). CONCLUSIONS: The FSH receptor Ser680/Ser680 genotype is associated with higher ovarian threshold to FSH, decreased negative feedback of luteal secretion to the pituitary during the intercycle transition, and longer menstrual cycles.


2005 - ENDOGENOUS PROGESTERONE AS WELL AS THE EXOGENOUS PROGESTIN NORETHISTERNONE ENENTHATE ARE ASSOCIATED WITH A PRO-INFLAMMATORY PROFILE IN HEALTHY MEN. [Articolo su rivista]
Zitzmann, M; Erren, M; Kamischke, A; Simoni, Manuela; Nieschlag, E.
abstract

CONTEXT: Inflammatory processes are related to atherosclerosis. Identification of inflammation triggers may furnish new therapeutic pathways. In women, progestins can have a marked inflammatory capacity. OBJECTIVE AND DESIGN: We investigated the effects of progesterone in men within the setting of two independent trials. First, the relation of endogenous progesterone levels to inflammation markers was assessed in 67 healthy nonsmoking Caucasian men (age, 20-50 yr) on a cross-sectional basis. Second, in a longitudinal controlled trial (52 wk) involving 28 healthy men receiving i.m. medication, we determined the effects of an exogenous progestin (norethisterone enanthate 200 mg) in combination with a long-acting testosterone preparation (testosterone undecanoate 1000 mg) administered to avoid androgen deficiency caused by pituitary-hypothalamic feedback. Controls received testosterone plus placebo. RESULTS: In the cross-sectional study, progesterone levels were positively related to concentrations of IL-6 (r = 0.41; P < 0.001), C-reactive protein (r = 0.37; P = 0.007), soluble vascular cell adhesion molecule 1 (r = 0.28; P = 0.02), E-selectin (r = 0.45; P < 0.001), leptin (r = 0.42; P < 0.001), neutrophils (r = 0.62; P < 0.001), and serum protein fractions alpha-1 (r = 0.44; P < 0.001) and alpha-2 (r = 0.36; P = 0.002). During the pharmacological trial, the testosterone/progestin group exhibited a marked increase of IL-6 concentrations (P < 0.001), whereas these decreased in the testosterone/placebo group (P = 0.03). Antiinflammatory IL-10 levels decreased in the group receiving testosterone/progestin (P = 0.01) but did not change in the testosterone/placebo group. CONCLUSION: Progesterone concentrations correspond to an inflammatory profile in healthy men, and external progestins elicit a similar effect. Men receiving regimens for hormonal male contraception involving progestins should be monitored for inflammatory effects. Speculatively, testosterone treatment decreasing endogenous progesterone production may facilitate beneficial effects on inflammation profiles even in eugonadal men.


2005 - FSH receptor gene haplotype distribution in normozoospermic and azoospermic men. [Articolo su rivista]
Ahda, Y; Gromoll, J; Wunsch, A; Asatiani, K; Zitzmann, M; Nieschlag, E; Simoni, Manuela
abstract

The human follicle-stimulating hormone (FSH) receptor (FSHR) gene possesses single nucleotide polymorphisms (SNP) in exon 10, which influence serum FSH levels in women, but not in men. In the present study we extend our previous investigation and for the first time analyze a novel, common SNP at position -29 of the FSHR core promoter in men. The SNP in codon 680 was analyzed in 438 men with nonobstructive azoospermia and in 304 controls. The SNP in codon 307 and at position -29 was analyzed in 345 men with nonobstructive azoospermia and 186 controls. SNPs were determined by allelic discrimination. No significant difference in the frequency of the polymorphism at position 680 and serum FSH levels was found. At position -29 (A/G) the A-29 allele was less frequent than the G-29 allele both in controls (25% vs 75%) and in patients (30% vs 70%) (P not signficant). Together the three SNPs form four discrete haplotypes (A-Thr-Asn, G-Thr-Asn, A-Ala-Ser, and G-Ala-Ser) occurring in 10 combinations. A statistically significant difference in the allelic distribution between controls and azoospermic men was found (P < .05 by {chi}2 test). The A-Ala-Ser allele was more frequent in patients (9.1%) than in controls (5.4%), whereas the G-Thr-Asn allele was less frequent in patients (33.1%) than in controls (40.6%) (P < .01 by Fisher's exact test). No significant correlation between serum FSH levels and FSHR allele was found. We conclude that the FSHR haplotype does not associate with different serum FSH levels but it is differently distributed in normal and azoospermic men. The A-Ala-Ser and the G-Thr-Asn allele might represent genetic factors contributing to phenotypic expression of severe spermatogenetic impairment.


2005 - Genetic complexity of FSH receptor function. [Articolo su rivista]
Gromoll, J; Simoni, Manuela
abstract

The interaction between follicle-stimulating hormone (FSH) and the FSH receptor (FSHR) is essential for normal oogenesis and spermatogenesis. Recently, single-nucleotide polymorphisms (SNPs) have been assigned to the FSHR gene. These give rise to different FSHR haplotypes that modify the action of FSH. In women, FSH sensitivities during the menstrual cycle and different cycle lengths are observed, depending on the FSHR haplotype. Thus, SNPs of the FSHR determine the ovarian response and should, therefore, be considered in controlled ovarian hyperstimulation during assisted-reproduction techniques in women with normal ovarian function. In men, the impact of the FSHR SNPs is unclear. The genetic complexity of FSHR should be considered when studying FSH action. These SNPs are one of the first examples in which genetic changes contribute to fine-tuning the endocrine regulation of reproduction. A rational pharmacogenetic approach that combines FSH dose according to the FSHR haplotype is envisaged.


2005 - Glucagon-like peptide-1 reduces the pulsatile component of testosterone secretion in healthy males. [Articolo su rivista]
Jeibmann, A; Zahedi, S; Simoni, Manuela; Nieschlag, E; Byrne, Mm
abstract

Background Glucagon-like-peptide-1 (7–36) amide (GLP-1), a potent regulator of glucose homeostasis, has been implicated in the control of hypothalamic-pituitary function. In vivo it is a relevant neuroendocrine modulator of gonadotropin-releasing hormone release, suggesting its possible role as a metabolic signal to the reproductive system. The present study was undertaken to establish its effect on luteinizing hormone (LH) and testosterone secretion in nine healthy male volunteers.Materials and methods Each subject underwent an oral glucose tolerance test to establish LH, testosterone, and GLP-1 responses to glucose. Euglycaemic clamp experiments (6 h) were performed on two occasions with saline or with a constant infusion of GLP-1 (0·4 pmol kg−1 min −1). Blood samples were drawn at 10-min intervals to measure the pulsatile pattern of LH and testosterone secretion.Results Ingestion of oral glucose resulted in a reduction in plasma testosterone levels at 30 min compared with baseline (P < 0·004) despite unaltered LH levels (P = 0·5). Constant GLP-1 infusion resulted in no change in LH (P = 0·83), testosterone (P = 0·96), follicle stimulating hormone (FSH) (P = 0·86) and leptin levels (P = 0·3). Pulse analysis revealed no significant difference in the number (P = 0·1) or median absolute amplitude (P = 0·3) of the LH pulses. However, there was a significant decrease in the number (3·0 ± 0·6 vs. 1·3 ± 0·4; P < 0·05) and a tendency for increased duration of testosterone pulses (97·4 16·7 vs. 170 27·1 min; P = 0·06).Conclusion Oral glucose ingestion and intravenous GLP-1 infusion reduce the pulsatile component of testosterone secretion by a mechanism independent of LH release.


2005 - Noethisterone enanthate has neither direct effect on the testis nor on the epididymis: A study in adult male cynomolgus monkeys (Macaca fascicularis). [Articolo su rivista]
Junaidi, A; Luetjens, Mc; Wistuba, J; Kamischke, A; Yeung, Ch; Simoni, Manuela; Nieschlag, E.
abstract

Objective: Norethisterone enanthate (NETE) is evaluated in trials of hormonal male contraception. It has been speculated that progestins may exert their contraceptive effects not only by suppressing gonadotropins but also by direct effects on male organs. NETE was given to monkeys in which endogenous gonadotropin secretion was suppressed by a gonadotropin releasing hormone (GnRH) antagonist, and replaced by human follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hCG). If NETE has a direct effect on spermatogenesis and/or epididymal function, some changes in testicular histology, sperm motility and/or morphology should occur soon after exposure to NETE.Methods: Fifteen adult intact male monkeys were grouped and treated for a 38-day period. Group I received GnRH antagonist, FSH, hCG and NETE while group II received a regime identical to group I without NETE and group III received only NETE and vehicle. Ejaculates, body weight, testicular biopsies and volume, and hormones were evaluated.Results: There was a similar pattern of serum FSH and testosterone in groups I and II. Testicular volume and the proportion of tubuli exhibiting spermatids was significantly decreased in group III. There were no significant differences between group I and group II in any parameters measured. The forward progression of sperm was not affected by NETE treatment. The consistently low percentages of grade c sperm indicated no sign of hyperactivation. No changes in the gross morphology of the acrosome were detected.Conclusions: Short-term NETE treatment has neither a direct effect on the testis nor on the epididymis in this nonhuman primate model and its contraceptive effects appear to be exerted exclusively through gonadotropin suppression.


2005 - Partial deletions in the AZFc region of the Y chromosome occur in men with impaired as well as normal spermatogenesis. [Articolo su rivista]
Hucklenbroich, K; Gromoll, J; Heinrich, M; Hohoff, C; Nieschlag, E; Simoni, Manuela
abstract

BACKGROUND: Partial deletions of the AZFc region of the Y chromosome were reported to be a significant risk factor for oligo-/azoospermia. In this study, we assessed the occurrence and frequency of partial AZFc microdeletions in patients with spermatogenic failure and in controls with normal spermatogenesis. METHODS: In a retrospective study design, gr/gr, b1/b3 and b2/b3 deletions were analysed by multiplex PCR in 170 men with normal spermatogenesis and 348 men with non-obstructive oligo-/azoospermia. RESULTS: gr/gr deletions were found in 14 men with oligozoospermia or azoospermia (4.0%) and in three normozoospermic men (1.8%) (NS). b1/b3 deletions were found both in controls (n=1) and in patients (n=1). b2/b3 deletions were significantly more frequent in the normozoospermic (five out of 170) than in the oligo-/azoospermic men (two out of 348). Three novel partial AZFc deletion patterns were found in four oligo-/azoospermic men. No correlation with semen or other clinical parameters was found. CONCLUSIONS: The frequency of gr/gr deletions is not significantly increased in men with oligo-/azoospermia, indicating that they are not sufficient per se to cause spermatogenetic impairment and infertility. b1/b3 and b2/b3 deletions are probably irrelevant for spermatogenesis. Novel deletion patterns found exclusively in infertile men suggest that other, still unexplored partial deletions might contribute to spermatogenic failure.


2005 - Pharamkogenetik bei der ovariellen Stimulationstherapie. [Articolo su rivista]
Greb, Rr; Behre, Hm; Simoni, Manuela
abstract

not available


2005 - Pharmacogenetics in ovarian stimulation. Current concepts and future options. [Articolo su rivista]
Greb, Rr; Behre, Hm; Simoni, Manuela
abstract

Tailoring ovarian stimulation to the individual patient can be challenging because the ovarian response varies substantially between patients. Pharmacogenetics has emerged as a new area of research to improve the balance between desired and undesired actions of drugs, based upon the genetic predisposition of the individual patient. Clinical studies have demonstrated that the p.N680S polymorphism of the FSH-receptor gene determines the ovarian response to FSH stimulation in patients undergoing IVF. In homozygous Ser680/Ser680 type women, the FSH receptor appears to be more resistant to FSH action even in normal menstrual cycles. Therefore, genotyping of patients scheduled for ovarian stimulation could be an attractive tool to individualize FSH dosing according to genetic differences in ovarian sensitivity. More clinical studies are warranted to investigate the usefulness of genotyping for the p.N680S polymorphism as a routine diagnostic test before ovarian stimulation.


2005 - Pharmacokinetics and pharmacodynamics of injectable testosterone undecanoate in castrated cynomulgus monkeys (Macaca fascicularis) are independent of the vehicle. [Articolo su rivista]
Wistuba, J; Luetjens, Cm; Kamischke, A; GU Y., Q; Schlatt, S; Simoni, Manuela; Nieschlag, E.
abstract

Testosterone undecanoate (TU) dissolved in soybean oil was developed in China to improve the pharmacokinetics of this testosterone ester in comparison with TU in castor or tea seed oil. As a pre-clinical primate model, three groups of five castrated cynomolgus macaques received either a single intramuscular injection of 10 mg/kg bodyweight TU in soybean oil, in tea seed oil, or in castor oil (equals 6.3 mg pure T/kg bodyweight for all preparations). Testosterone, estradiol, luteinizing hormone, and follicle-stimulating hormone as well as prostate volume, body weight and ejaculate weight were evaluated. After injection supraphysiological testosterone levels were induced. There were no significant differences in the pharmacokinetics of the three TU preparations for testosterone and estradiol. The gonadotropin levels showed a high individual variation. Prostate volumes increased equally in all groups after administration and declined to castrate level afterwards. The results suggest that TU in soybean oil produces similar effects as TU in the other vehicles. This study in non-human primates provides no objection to testing of this new preparation in humans.


2005 - Pharmakogenetik bei der ovariellen stimulationstherapie [Articolo su rivista]
Greb, R. R.; Behre, H. M.; Simoni, M.
abstract

Tailoring controlled ovarian hyperstimulation (COH) to the individual patient can be challenging because the ovarian response varies substantially between patients. Pharmacogenetics has emerged as a new area of research to improve the balance between desired and undesired actions of drugs based upon the genetic predisposition of the individual patient. Clinical studies demonstrated that the p.N680S polymorphism of the FSH receptor gene determines the ovarian response to FSH stimulation in patients undergoing in vitro fertilisation. In homozygous Ser680/Ser680 type women the FSH receptor appears to be more resistant to FSH action even in normal menstrual cycles. Therefore, genotyping of patients scheduled for COH could be an attractive tool to individualise FSH dosing according to genetic differences in ovarian sensitivity. More clinical studies are warranted to investigate genotyping for the p.N680S polymorphism as a routine diagnostic test before COH.


2005 - Role of sequence variations of the GnRH receptor and GPR54 gene in male idiopathic hypogonadotropic hypogonadism. [Articolo su rivista]
Lanfranco, F; Gromoll, J; VON ECKARDSTEIN, S; Herding, Em; Nieschlag, E; Simoni, Manuela
abstract

OBJECTIVE: To determine the frequency of mutations of the gonadotropin-releasing hormone receptor (GnRHR) and of the G protein-coupled receptor 54 (GPR54) genes in normosmic idiopathic hypogonadotropic hypogonadism (IHH). METHODS: In a retrospective study we analyzed the GnRHR and the GPR54 genes of 45 IHH patients and 50 controls. Genomic DNA was amplified by PCR to obtain partially overlapping amplicons encompassing the exon-intron boundaries of the GnRHR and GPR54 genes and analyzed by single-stranded conformation polymorphism gel electrophoresis and/or DNA sequencing. RESULTS: One heterozygous R262Q mutation of the GnRHR gene was identified in one patient with familial IHH. The silent single-nucleotide polymorphism (SNP) 453C > T occurred at the same frequency in patients and controls. One patient with sporadic IHH and consanguineous parents showed a novel homozygous sequence variation of the GPR54 gene (1001_1002insC) resulting in an open reading frame shift and elongation of 43 amino acids with an increased number of proline residues in the intracellular receptor domain. This patient had delayed puberty, low testosterone (3.4 nmol/l), and low-normal LH and FSH levels responsive to GnRH. Pulsatile GnRH administration normalized testosterone levels and induced spermatogenesis sufficiently to induce a pregnancy with assisted reproduction. Two common SNPs in exon 1 and exon 5 of the GPR54 gene showed similar frequency distribution and hormonal profiles in IHH and controls. CONCLUSIONS: Mutations of the GnRHR and of the GPR54 gene are rare in IHH and should be investigated especially in cases with autosomal recessive transmission. Common SNPs of the GnRHR and GPR54 genes do not play any role in IHH.


2005 - Significance of a common single nucleotide polymorphism in exon of the follicle-stimulating hormone receptor gene for ovarian response to FSH: a pharmacogenetic approach to control ovarian hyperstimulation. [Articolo su rivista]
Behre, Hm; Greb, Rr; Mempel, A; Sonntag, B; Kiesel, L; Kaltwasser, P; Seliger, E; Röpke, F; Gromoll, J; Nieschlag, E; Simoni, Manuela
abstract

The p.N680S sequence variation of the follicle-stimulating hormone (FSH) receptor gene was previously shown to influence the ovarian response to FSH in normo-ovulatory women undergoing controlled ovarian hyperstimulation. In this prospective, randomized, controlled study, we tested whether the same daily dose of FSH results in lower levels of oestradiol in women homozygous for the p.N680S sequence variation, and whether the difference can be overcome by higher FSH doses. Women undergoing controlled ovarian hyperstimulation for in vitro fertilization or intracytoplasmic sperm injection and homozygous for the wild-type or for the p.N680S FSH receptor were randomly assigned to group I (Ser/Ser, n=24), receiving an FSH dose of 150 U/day, or group II (Ser/Ser, n=25), receiving an FSH dose of 225 U/day. In group III (Asn/Asn, n=44), the FSH dose was 150 U/day. Age and basal FSH levels were not different between groups. At ovulation induction, total FSH doses were comparable in group I (1631+/-96 U) and group III (1640+/-57 U) but significantly higher in group II (2421+/-112 U) (P<0.001). Peak oestradiol levels on the day of human chorionic gonadotrophin (hCG) administration were significantly lower in group I (5680+/-675 pmol/l) compared to group III (8679+/-804 pmol/l) (P=0.028). Increasing the FSH dose from 150 to 225 U/day overcame the lower oestradiol response in women with Ser/Ser (group II, 7804+/-983 pmol/l). In women undergoing controlled ovarian hyperstimulation, the p.N680S sequence variation results in lower oestradiol levels following FSH stimulation. This lower FSH receptor sensitivity can be overcome by higher FSH doses.


2005 - Single nucleotide polymorphisms in the promoter region influence the expression of the follicle-stimulating hormone receptor. [Articolo su rivista]
Wunsch, A; Ahda, Y; BANAZ YASAR, F; Sonntag, B; Nieschlag, E; Simoni, Manuela; Gromoll, J.
abstract

OBJECTIVE: To characterize novel single-nucleotide polymorphisms (SNPs) in the human FSH receptor (FSHR) promoter region. DESIGN: Retrospective and basic research study. SETTING: University hospital. PATIENTS: Women (202 from Germany and 55 from Indonesia) with male or tubal factor infertility undergoing controlled ovarian stimulation for IVF treatment. INTERVENTIONS: None. MAIN OUTCOME MEASURE(S): Frequency, distribution, and correlation with clinical data of the SNPs. Dual luciferase assays and electrophoretic mobility shift assays (EMSA). RESULT(S): We identified two SNPs and three mutations in the promoter region of the human FSHR which could be allocated to positions -29, -37, -114, -123, and -138 upstream of the translational initiation codon. One SNP showed a high incidence (-29: 44%, n = 202), but no correlation with basal FSH serum levels or ovarian response with the SNP at position -29 was found. Luciferase reporter assays, using pGL3 vector constructs, showed that mutations at positions -37 and -138 lead to significantly higher promoter activity. EMSA indicate that putative binding sites for transcription factors are affected by the SNPs. CONCLUSIONS: The newly identified SNPs do not seem to influence clinical parameters substantially, but modulate expression of the FSHR via changes in transcription factor binding sites.


2005 - Testosterone substitution with a new transdermal, hydroalcoholic gel applied to scrotal or non-scrotal skin: a multicentre trial. [Articolo su rivista]
Kühnert, B; Byrne, M; Simoni, Manuela; Kopcke, W; Gerss, J; Lemmnitz, G; Nieschlag, E.
abstract

OBJECTIVE: Testosterone-containing gels have improved testosterone substitution therapy, but they are associated with the risk of interpersonal transfer. Therefore, we tested a new hydroalcoholic 2.5% testosterone gel (TGW), which was removed by washing 10 min after administration. DESIGN: The gel was applied to scrotal or non-scrotal skin in comparison to two 2.5 mg Androderm patches in a randomised, three-arm, parallel-group, controlled multicentre trial over a period of 24 weeks. We included symptomatic hypogonadal men whose morning testosterone levels were <10 nmol/l. Either 1 g TGW was applied to scrotal skin (n = 54) or 5 g to non-scrotal skin (n = 56) once daily; the patch group (n = 52) applied two patches/day. Dose titration was allowed. RESULTS: Whereas serum testosterone levels and the pre-post changes of the areas under the curve of testosterone and free testosterone between weeks 0 and 24 indicated equivalent treatment success for the patch and scrotal groups, the dermal gel group was significantly superior to the other two groups. Questionnaires on sexual function, mood and quality of life did not differ significantly between study groups, nor were prostate volume, prostate-specific antigen (PSA) levels and prostate symptoms different. However, tolerability was much better in the gel groups than the patch group. CONCLUSION: Efficacy, safety and tolerability suggest TGW as a favourable treatment for hypogonadal patients.


2005 - The distribution of FSH receptor isoforms is related to basal FSH levels in subfertile women with normal menstrual cycles. [Articolo su rivista]
DE KONING, Ch; Benjamins, T; Harms, P; Homburg, R; VAN MONTFRANS, Jm; Gromoll, J; Simoni, Manuela; Lambalk, Cb
abstract

BACKGROUND: Recently a polymorphic variant of the FSH receptor in which amino acid asparagine (Asn) at position 680 is replaced by serine (Ser) was found. This is associated with higher FSH levels in the early follicular phase and an increased FSH requirement to obtain follicular response in IVF patients. The aim of our study was to test the hypothesis that this receptor isoform occurs more often in regularly menstruating subfertility patients with elevated basal FSH than in those with normal early follicular phase FSH. METHODS: A retrospective cohort study of 38 subfertility patients with a regular menstrual cycle and elevated FSH (&gt;10 IU/l) compared to 40 patients with normal early follicular phase FSH was carried out. DNA was analysed to determine the FSH receptor genotype. RESULTS: The N680S variant on one or both alleles of the FSH receptor gene was significantly more prevalent in patients with elevated FSH (P &lt; 0.05). The homozygous Asn/Asn variant at codon 680 was found in 45% of women with normal FSH and in 21% of women with elevated FSH. The homozygous Ser/Ser receptor variant was present in 12.5% of women with normal FSH and in 21% of patients with elevated FSH. Also the heterozygous combination of both variants Asn/Ser occurred more often in women with elevated FSH (58 versus 42.5%). CONCLUSIONS: The N680S sequence variation of the FSH receptor is found in &gt;75% of the cases with elevated basal FSH and suggests a higher FSH threshold.


2004 - EAA/EMQN best practice guidelines for molecular diagnosis of Y-chromosomal microdeletions. State of the art 2004. [Articolo su rivista]
Simoni, Manuela; Bakker, E; Krausz, C.
abstract

Microdeletions of the Y chromosome are the second most frequent genetic cause of spermatogenetic failure in infertile men after the Klinefelter syndrome. The molecular diagnosis of Y-chromosomal microdeletions is routinely performed in the workup of male infertility in men with azoospermia or severe oligozoospermia. Since 1999, the European Academy of Andrology (EAA) and the European Molecular Genetics Quality Network (EMQN) support the improvement of the quality of the diagnostic assays by publication of the laboratory guidelines for molecular diagnosis of Y-chromosomal microdeletions and by offering external quality assessment trials. The present revision of the 1999 laboratory guidelines summarizes the results of a 'Best Practice Meeting' held in Florence (Italy) in October 2003. The basic protocol for microdeletion screening suggested in the 1999 guidelines proved to be very accurate, sensitive and robust. In the light of the recent advance in the knowledge of the Y chromosome sequence and of the mechanism of microdeletion it was agreed that the basic 1999 protocol, based on two multiplex polymerase chain reactions each covering the three AZF regions, is still fully valid and appropriate for accurate diagnosis.


2004 - Epidermal growth factor receptor pathway substrate 8 (Eps8) expression in maturing testis. [Articolo su rivista]
Wunsch, A; Strothmann, K; Simoni, Manuela; Gromoll, J; Nieschlag, E; Luetjens, Cm
abstract

AIM: Although epidermal growth factor receptors are expressed in the testes, whether they signal through epidermal growth factor receptor pathway substrate 8 (Eps8) is unknown. Here we evaluated the expression pattern of Eps8 in the maturing testis. METHODS: The expression of Eps8 was analysed by Northern blotting, immunocytochemistry and Western blotting in primary Sertoli cell cultures and in testicular tissue of rodents. RESULTS: Eps8 is specifically expressed in gonocytes, Leydig and Sertoli cells of the neonatal rats and in Leydig and Sertoli cells of the adult rats and mice. Although gonocytes express Eps8, no signal was found in prepubertal or mature spermatogonia and the expression level of Eps8 in Sertoli cells increases with age. No regulation of Eps8 expression in primary immature rat Sertoli cells by Follicle stimulating hormone (FSH) was detected by Western blotting. CONCLUSION: Eps8 seems to be involved in the growth factor-controlled regulation of cell proliferation and differentiation in the seminiferous epithelium. Eps8 is a possible marker for gonocytes and in Sertoli cells it could be involved in crosstalk with other growth factor pathways.


2004 - Gene expression profiling of mouse Sertoli cell lines. [Articolo su rivista]
Strothmann, K; Simoni, Manuela; Mathur, P; Siakhamary, S; Nieschlag, E; Gromoll, J.
abstract

The proliferation and differentiation of Sertoli cells is regulated by follicle-stimulating hormone (FSH). The molecular events following FSH stimulation are only partially known. To investigate FSH action in Sertoli cells, we established two novel FSH-responsive mouse Sertoli-cell-derived lines expressing human wild-type (WT) FSH receptor (FSHR) or overexpressing mutated (Asp567Gly) constitutively active FSHR (MUT). Gene expression profiling with commercially available cDNA arrays, including 588 mouse genes, revealed 146 genes expressed in both cell lines. Compared with the expression pattern of WT cells, 20 genes were identified as being either up- or down-regulated (>two-fold) in the MUT cells. We observed a strong differential expression of factors involved in cellular proliferation, e.g. cyclin D2 (repressed to nearly undetectable levels), proliferating cell nuclear antigen (2.5-fold repression) and Eps-8 (six-fold repression), and in genes involved in cellular differentiation, e.g. cytokeratin-18 (13-fold induction). The cDNA array results for six representative genes were confirmed by Northern blotting, which also included the parental SK-11 cell line devoid of FSHR expression. We found no further acute FSH- or forskolin-induced change in expression levels after 3-h stimulations, suggesting that the observed differences between the two cell lines is a consequence of mild, chronically increased, cAMP production in MUT cells. These results provide a platform for the further investigation of selected candidate genes in primary cultures and/or in vivo.


2004 - Natural transmission of an AZFc Y-chromosomal microdeletion from father to his sons. [Articolo su rivista]
Kühnert, B; Kostova, E; Tschanter, P; Luetjens, Cm; Simoni, Manuela; Nieschlag, E.
abstract

Y-chromosomal microdeletions, associated with oligozoospermia or azoospermia, are usually de novo deletions in the affected patients. We report here the rare case of an affected father who transmitted a Y-chromosomal microdeletion to at least two of his three sons naturally and who also fathered a daughter. The extent of the deletion, which was determined with new STS-primers and covers 3.5 Mb, was identical in the father and his azoospermic sons. To determine any possibly modifying influence of other genes involved in spermatogenesis, we analysed two polymorphisms of the DAZL gene, the autosomal homologue of the deleted DAZ gene. DAZL and DAZ might be functionally related to each other. However, we found identical polymorphisms in exon 2 and 3 of the DAZL gene, in both father and his sons, corresponding to the most prevalent genotype in fertile men. Thus, other genes or environmental factors must modify spermatogenesis in men with identical Y-chromosomal microdeletions.


2004 - Serum adiponectine levels in hypogonadal males: influence of testosterone replacement therapy. [Articolo su rivista]
Lanfranco, F; Zitzmann, M; Simoni, Manuela; Nieschlag, E.
abstract

OBJECTIVE: Adiponectin is an adipocyte-specific secretory protein which exhibits antiatherogenic, anti-inflammatory and antidiabetic properties. We hypothesized that testosterone plays an important role in the regulation of its secretion in humans, as adiponectin concentrations are higher in women than in men and as testosterone administration is accompanied by a reduction in serum adiponectin in animals and by reduced protein secretion in cultured adipocytes. This study aimed to evaluate adiponectin levels in hypogonadal men prior to and during testosterone replacement therapy. SUBJECTS AND METHODS: In a retrospective study, adiponectin, total and free testosterone, oestradiol, SHBG, total cholesterol and triglyceride levels were evaluated in 31 hypogonadal men [HM; age, mean +/- SEM: 36.5 +/- 2.4 years; body mass index (BMI) 24.6 +/- 0.8 kg/m2] and 29 weight-matched eugonadal men (EM; age 30.8 +/- 1.5 years; BMI 23.4 +/- 0.6 kg/m2). In 13 HM (age 33.9 +/- 3.2 years; BMI 24.2 +/- 0.9 kg/m2) the same parameters were also evaluated after 6 months of testosterone replacement therapy. Correlation analysis between adiponectin and hormonal, biochemical and anthropometric parameters was performed in all subjects. RESULTS: Testosterone, free testosterone and oestradiol concentrations were significantly lower in HM than in EM (4.4 +/- 0.4 nmol/l, 78.4 +/- 10.9 pmol/l and 36.1 +/- 3.0 pmol/l, respectively, in HM vs. 21.9 +/- 0.7 nmol/l, 507.9 +/- 13.8 pmol/l and 65.2 +/- 1.8 pmol/l, respectively, in EM, P < 0.0001), while SHBG levels in HM were higher than in EM (54.4 +/- 7.5 vs. 30.9 +/- 2.2 nmol/l, P < 0.005). Serum adiponectin levels in HM were significantly higher than in EM (9.53 +/- 0.73 vs. 6.80 +/- 0.55 microg/ml, P < 0.01). Calculation of the Pearson coefficient showed that adiponectin levels in HM were not correlated with any of the anthropometric and hormonal parameters examined, but showed a significant negative correlation with serum triglycerides (r = -0.38, P < 0.05). Serum adiponectin levels were negatively correlated with body weight (r = -0.41, P < 0.05) in EM but not with other anthropometric, hormonal or biochemical parameters. Six months after initiation of testosterone replacement therapy, which increased testosterone and free testosterone levels to the normal range, adiponectin levels were significantly reduced in HM (6.37 +/- 0.93 vs. 9.26 +/- 1.01 microg/ml, P < 0.01) and similar to those recorded in EM. CONCLUSIONS: Compared to eugonadal subjects, hypogonadal men show higher adiponectin levels which are reduced by testosterone replacement therapy. This study indicates that testosterone exerts a regulatory role on adiponectin secretion in humans.


2004 - The carboxyterminal peptide of chorionic gonadotropin facilitates activation of the marmoset LH receptor. [Articolo su rivista]
Müller, T; Gromoll, J; Simula, A; Norman, R; SANDHOWE KLAVERKAMP, R; Simoni, Manuela
abstract

Luteinizing hormone (LH) and chorionic gonadotropin (CG) are heterodimeric glycoprotein hormones acting on the luteinizing hormone receptor (LHR). In the LHR, which is genomically encoded by eleven exons, exon 10 encodes for the hinge region and its elimination impairs LH action, while CG maintains normal activity. The two gonadotropins differ in the carboxyterminal peptide (CTP) present in CG but absent in LH. Since the marmoset monkey (Callithrix jacchus) LHR naturally lacks exon 10 (LHR type II), we generated two recombinant marmoset gonadotropin preparations, one consisting of the wild type CG and one of truncated CG lacking the CTP (CG (-CTP)). After calibration in a mouse Leydig cell bioassay against the WHO LH80/522 standard, the ED (50) of the CG preparation on a COS7 cell line permanently expressing the marmoset LHR was 4.25 +/- 0.21 IU/L (n = 3). Stimulation of the COS7 cell line with equipotent concentrations of CG and CG (-CTP) resulted in significantly different formation of cAMP (two-way ANOVA, p < 0.001). In particular, cAMP production stimulated by CG (-CTP) was 3 - 4 times lower compared to CG at the saturating CG concentration (8 IU/L). We conclude, supplementing one current model of LHR activation, that exon 10 might play a permissive role in releasing the constraint of the receptor upon hormone binding, resulting in receptor activation. We speculate that, when exon 10 is lacking, the CTP can overcome its absence and facilitates the opening of the receptor, resulting in normal activation.


2004 - The chorionic gonadotropin β subunit mRNA but not the luteinising hormone β subunit mRNA is expressed in thepituitary of the common marmoset (Callithrix jacchus). [Articolo su rivista]
Müller, T; Simoni, Manuela; Pekel, E; Luetjens, Cm; Chandolia, R; Amato, F; Norman, Rj; Gromoll, J.
abstract

The pituitary gonadotrophins LH and FSH are responsible for regulation of gametogenesis in the testis and ovary. Chorionic gonadotrophin (CG), a third closely related glycoprotein hormone derived by gene duplication of the LHbeta gene and secreted by the placenta in primates, is essential for the rescue of the corpus luteum and maintenance of pregnancy. We have recently shown that marmoset (m) CGbeta mRNA is highly expressed in the pituitary of the common marmoset (Callithrix jacchus) and that LH is less active than human CG in activating the human LH receptor lacking exon 10. To investigate further which gonadotrophin is the actual ligand of the LH receptor (LHR) of the marmoset monkey that naturally lacks exon 10, we identified and characterised the genomic organisation of the mLHbeta gene and its expression. Intergenic PCR amplification of the region encompassing the mLHbeta and the mCGbeta genes revealed that, surprisingly, mCGbeta is located 20 kbp upstream of the LHbeta gene, whereas in other species the intergenic distance is approximately 2-3 kbp. Sequence analysis of the mLHbeta coding region showed 70% identity to mCGbeta and 90% identity to human LHbeta at the amino acid level. Both gonadotrophin beta subunits are present at the genomic level, but RT-PCR of pituitary and placental total RNA using specific oligonucleotides for mCGbeta and mLHbeta showed high expression of mCGbeta mRNA in both tissues, whereas LHbeta was expressed neither in the pituitary nor in the placenta. Thus mLHbeta mRNA is lacking in the marmoset pituitary. Immunohistochemistry of marmoset pituitaries showed that mCG was confined to the gonadotrophes, and partly co-localised in cells stained positively for FSH. Western blot analysis confirmed the presence of mCG in the pituitary. Northern blot analysis using mCGbeta as a probe displayed one transcript of 0.7 kb in the pituitary and detected two transcripts of 1.1 kb and 2 kb in the marmoset placenta. Our results suggest that, in the common marmoset, CG is the only gonadotrophin with luteinising function that is present in the pituitary. We postulate that, owing to an unknown mutational event in evolution, expression of mLH was completely abolished, and CG - which, unlike LH, acts normally even when exon 10 is missing from the LHR - took over its function.


2003 - A new subclass of LH/CG receptor lacking exon 10 in the New World (Platyrrhini) lineage. [Articolo su rivista]
Gromoll, J; Wistuba, J; Terwort, N; Godmann, M; Müller, T; Simoni, Manuela
abstract

The luteinizing hormone receptor (LHR) plays an essential role as a mediator of LH and CG action during embryonic sexual differentiation and in gametogenesis. In a hypogonadal male patient, we recently demonstrated that a genomic deletion of exon 10, located in the hinge region of the extracellular domain, results in discrimination of LH and hCG action. In the common marmoset (Calltithrix jacchus), exon 10 of the LHR is naturally missing at the mRNA level. In order to investigate whether this is an isolated species-specific phenomenon, we performed a phylogenetic screening, searching for the presence of LHR exon 10 mRNA in a number of primate species representative for the major lineages of primate evolution. The expressed LHR region encompassing exon 10 was amplified from testicular tissue by RT-PCR, cloned, and sequenced. In addition, we performed Southern blot analysis of the LHR of selected New World and Old World primates. The results revealed that exon 10 mRNA is lacking in the complete New World monkey (Platyrrhini) lineage but is present in both more primitive and more advanced primates. However, exon 10 seems to be present at the genomic level, arguing for a splicing failure possibly due to a genomic mutation or the lack of appropriate splicing factors. Considering that, in the human, LH is far less active than hCG on the LHR lacking exon 10, we addressed the question whether the existence of such a receptor has any consequences on the dual hormone LH/CG system present in Platyrrhini. Using primers specific for the known marmoset CG beta cDNA, we amplified the CG beta subunit cDNA from male common marmoset pituitaries by RT-PCR, while LH beta could not be amplified, suggesting a possible physiological role of pituitary CG in this species. In conclusion, we demonstrated for the first time that the LH mRNA without exon10 is the natural wild-type LHR in the Platyrrhini lineage. We propose that this LHR represents a new subclass of receptors that should be named LHR type II. In addition, the high expression of CG beta in the marmoset pituitary suggests a physiological role of CG in the reproductive function of these primates beyond pregnancy.


2003 - Absence of exon 10 of the human LH receptor impairs LH but not hCG action [Articolo su rivista]
Müller, T; Gromoll, J; Simoni, Manuela
abstract

The LH receptor (LHR) mediates the actions of LH and human chorionic gonadotropin (hCG). In vivo data showed that deletion of exon 10 does not affect hCG action, whereas LH action is impaired. To investigate the role of exon 10 in LH/hCG action in vitro we created stable COS-7 cells expressing the LHR with (wt) or without (-ex10) exon 10. Binding experiments showed that the affinities of LH and hCG to the LHR wt and -ex10 were similar. Stimulation of wt with hCG or LH resulted in increased cAMP. cAMP production was significantly impaired in -ex10 stimulated with LH. This response was not altered by pertussis toxin, excluding that Gi becomes activated in LHR -ex10. In desensitization experiments, intracellular cAMP of LHR wt and -ex10 declined to approximately 30%. No difference in intracellular cAMP was detected between LHR wt or -ex10 after recovery and restimulation with hCG or LH. These experiments show that impaired cAMP production of LHR -ex10 stimulated with LH is not due to anomalous receptor coupling or desensitization. We conclude that although exon 10 of the LHR plays no role in ligand binding, it is important for receptor activation by LH by a mechanism probably involving extracellular conformational changes.


2003 - CAG repeat lenght in the androgen receptor gene affects the risk of male infertility. [Articolo su rivista]
Asatiani, K; VON ECKARDSTEIN, S; Simoni, Manuela; Gromoll, J; Nieschlag, E.
abstract

During recent years several studies have suggested that a slight increase in the number of CAG repeat sequences in exon 1 of the androgen receptor gene causes idiopathic oligozoospermia. We tested whether CAG repeats are more numerous in men with idiopathic infertility compared to those with known causes of oligozoospermia. CAG repeats were analysed in a consecutive sample of 217 infertile men covering a wide range of diagnoses and sperm counts. Data were compared with those of a control group of 131 normozoospermic men including 62 fathers. CAG repeats (x +/- SD) did not differ between idiopathically (21.4 +/- 2.9) and non-idiopathically infertile men (21.6 +/- 2.8) or normozoospermic men of unproven fertility (20.6 +/- 3.0). Only fathers had significantly fewer repeats (19.4 +/- 3.1; p < 0.001). Different from controls, no correlation between CAG repeats and any semen parameter existed in patients. Comparison of our and published studies showed that odds ratios for infertility in men with CAG repeat length in the upper quartile of the normal range increased when the controls were selected by proven fertility. We conclude that more numerous CAG repeats do not directly cause oligozoospermia and propose that men with longer CAG repeats might be more prone to develop infertility in response to any pathogen/epigenetic factors.


2003 - Follicle-stimuating hormone receptor polymorphism in women with normogonadotropic anovulatory infertility. [Articolo su rivista]
Laven, Jse; Mulders, Agmgj; Suryandary, Da; Gromoll, J; Nieschlag, E; Fauser, Bcjm; Simoni, Manuela
abstract

OBJECTIVE: To assess the incidence of different FSH receptor genotypes in normogonadotropic anovulatory infertile women (World Health Organization class II) and normo-ovulatory controls and to correlate these genotypes with baseline characteristics and ovarian responsiveness during ovulation induction. DESIGN: Cross-sectional study. SETTING: University hospital. PATIENT(S): Thirty normo-ovulatory controls and 148 normogonadotropic anovulatory infertile women. INTERVENTION(S): All participants underwent a standardized evaluation that included cycle history, body mass index measurement, and transvaginal ultrasonography of ovaries. Fasting blood samples were obtained for endocrine evaluation. Ovarian responsiveness to FSH in normogonadotropic anovulatory infertile women was assessed during ovulation induction, and DNA was analyzed to determine the FSH receptor genotype. MAIN OUTCOME MEASURE(S): Prevalence of FSH receptor polymorphisms, baseline serum FSH levels, amount of FSH administered, duration of stimulation, and ovarian response dose. RESULT(S): The Thr/Thr 307 genotype was significantly less prevalent (52% vs. 23%) and the Ser/Ser 680 polymorphism was significantly more prevalent (40% vs. 16%) in patients compared with controls. Normogonadotropic anovulatory infertile women with the Ser/Ser 680 polymorphism presented with higher median FSH serum levels (5.2 IU/L [range, 2.4-9.7 IU/L]) than did those with the Asn/Asn 680 (4.6 IU/L [range, 1.4-5.8 IU/L) and Asn/Ser 680 (4.5 IU/L [range, 1.8-9.7 IU/L) variants. However, ovarian responsiveness to FSH was similar among anovulatory women with the various polymorphisms. CONCLUSION(S): Normogonadotropic anovulatory infertile patients have a different FSH receptor genotype than do normo-ovulatory controls. Although this characteristic is associated with increased baseline FSH serum levels, altered ovarian sensitivity to exogenous FSH during ovulation induction could not be established.


2003 - Targeted expression of human FSH receptor Asp567Gly mutant mRNA in testis of transgenic mice: role of the human FSH receptor promoter. [Articolo su rivista]
Nordhoff, V; Gromoll, J; Foppiani, L; Luetjens, Cm; Schlatt, S; Kostova, E; Huhtaniemi, I; Nieschlag, E; Simoni, Manuela
abstract

AIM: To specifically express the Asp567Gly human follicle-stimulating hormone receptor (FSHR) under the control of its promoter to evaluate the phenotypic consequences in the presence of normal pituitary function. METHODS: We produced transgenic mice overexpressing the Asp567Gly human FSHR under the control of a 1.5kb 5'-flanking region fragment of its promoter. RESULTS: Mice were phenotypically normal and fertile. In males, mRNA could be detected in the testis and the brain, indicating that the 1.5kb promoter fragment drives expression not only in the gonads. The testis weight/body weight ratio and the testosterone levels in transgenic and non-transgenic littermates were similar. By in situ hybridisation we found that the transgenic FSHR was highly expressed in Sertoli cells, spermatocytes and round spermatids. However, a radioligand receptor assay failed to show a significant difference in total FSHR binding sites in testis homogenates of transgenic and wild type animals, suggesting that the transgenic FSHR is probably not translated into functional receptor protein. CONCLUSION: A 1.5kb 5'-region of the human FSHR drives mRNA expression of the transgene in the testis but leads to ectopic expression in germ cells and in the brain. No phenotypic consequences could be documented due to the lack of protein expression.


2002 - An activated human follicle-stimulating hormone (FSH) receptor stimulates FSH-like activity in gonadotrophin-deficient transgenic mice. [Articolo su rivista]
M, Haywood; N, Tymchenko; J, Spalivero; A, Koch; M, Jimenez; J, Gromoll; Simoni, M.; V, Nordhoff; Dj, Handelsman; Cm, Allan
abstract

FSH mediates its testicular actions via a specific Sertoli cell G protein-coupled receptor. We created a novel transgenic model to investigate a mutant human FSH receptor (FSHR(+)) containing a single amino acid substitution (Asp567Gly) equivalent to activating mutations in related glycoprotein hormone receptors. To examine the ligand-independent gonadal actions of FSHR(+), the rat androgen-binding protein gene promoter was used to direct FSHR(+) transgene expression to Sertoli cells of gonadotropin-deficient hypogonadal (hpg) mice. Both normal and hpg mouse testes expressed FSHR(+) mRNA. Testis weights of transgenic FSHR(+) hpg mice were increased approximately 2-fold relative to hpg controls (P &lt; 0.02) and contained mature Sertoli cells and postmeiotic germ cells absent in controls, revealing FSHR(+)-initiated autonomous FSH-like testicular activity. Isolated transgenic Sertoli cells had significantly higher basal ( approximately 2-fold) and FSH-stimulated ( approximately 50%) cAMP levels compared with controls, demonstrating constitutive signaling and cell-surface expression of FSHR(+), respectively. Transgenic FSHR(+) also elevated testosterone production in hpg testes, in the absence of circulating LH (or FSH), and it was not expressed functionally on steroidogenic cells, suggesting a paracrine effect mediated by Sertoli cells. The FSHR(+) response was additive with a maximal testosterone dose on hpg testicular development, demonstrating FSHR(+) activity independent of androgen-specific actions. The FSHR(+) response was male specific as ovarian expression of FSHR(+) had no effect on hpg ovary size. These findings reveal transgenic FSHR(+) stimulated a constitutive FSH-like Sertoli cell response in gonadotropin-deficient testes, and pathways that induced LH-independent testicular steroidogenesis. This novel transgenic paradigm provides a unique approach to investigate the in vivo actions of mutated activating gonadotropin receptors.


2002 - CAG repeat length in the androgen receptor gene and gonadotrophin suppression influence the effectiveness of hormonal male contraception [Articolo su rivista]
Eckardstein, S. V.; Schmidt, A.; Kamischke, A.; Simoni, M.; Gromoll, J.; Nieschlag, E.
abstract

OBJECTIVE: Nonuniformity in suppression of spermatogenesis induced by various hormones or hormone combinations has impeded the development of an effective hormonal male contraceptive. The basis for this heterogeneity in response remained unresolved to date; however, the presence of ethnic differences points to an involvement of genetic factors. PATIENTS AND MEASUREMENTS: In a retrospective analysis we investigated the impact of a CAG repeat polymorphism in the androgen receptor and polymorphic sites in the oestrogen and FSH receptor genes on spermatogenic suppression in 85 Caucasian men treated with different regimens of hormonal contraception. RESULTS: Failure to reduce sperm concentrations below 3 million/ml was significantly associated with insufficient suppression of gonadotrophins. The extent of gonadotrophin suppression was not explained by any polymorphism but was primarily pharmacological, resulting from addition of gestagens to testosterone. When LH and FSH suppression was rapid and persistent none of the polymorphisms studied explained why some men failed to achieve azoospermia. In cases with incomplete gonadotrophin suppression the chances of becoming azoospermic were 2.5 times higher in men having more than 22 CAG repeats. CONCLUSIONS: In summary, our analysis shows that in a subset of men, effective hormonal male contraception can be achieved even in the absence of complete gonadotrophin suppression.


2002 - CAG repeat length in the androgen receptor gene and gonadotrophin suppression influence the effectiveness of hormonal male contraception. Clin Endocrinol [Articolo su rivista]
VON ECKARDSTEIN, S; Schmidt, A; Kamischke, A; Simoni, Manuela; Gromoll, J; Nieschlag, E.
abstract

OBJECTIVE: Nonuniformity in suppression of spermatogenesis induced by various hormones or hormone combinations has impeded the development of an effective hormonal male contraceptive. The basis for this heterogeneity in response remained unresolved to date; however, the presence of ethnic differences points to an involvement of genetic factors. PATIENTS AND MEASUREMENTS: In a retrospective analysis we investigated the impact of a CAG repeat polymorphism in the androgen receptor and polymorphic sites in the oestrogen and FSH receptor genes on spermatogenic suppression in 85 Caucasian men treated with different regimens of hormonal contraception. RESULTS: Failure to reduce sperm concentrations below 3 million/ml was significantly associated with insufficient suppression of gonadotrophins. The extent of gonadotrophin suppression was not explained by any polymorphism but was primarily pharmacological, resulting from addition of gestagens to testosterone. When LH and FSH suppression was rapid and persistent none of the polymorphisms studied explained why some men failed to achieve azoospermia. In cases with incomplete gonadotrophin suppression the chances of becoming azoospermic were 2.5 times higher in men having more than 22 CAG repeats. CONCLUSIONS: In summary, our analysis shows that in a subset of men, effective hormonal male contraception can be achieved even in the absence of complete gonadotrophin suppression.


2002 - Distribution and function of FSH receptor genetic variants in normal men. [Articolo su rivista]
Asatiani, K; Gromoll, J; VON ECKARDSTEIN, S; Zitzmann, M; Nieschlag, E; Simoni, Manuela
abstract

Follicle stimulating hormone (FSH) plays a key role in the maintenance of qualitatively and quantitatively normal spermatogenesis. It controls gamete development through Sertoli cells, via binding to its receptor. The influence and importance of FSH receptor (FSHR) variants on Sertoli cell function is not completely understood and remains to be investigated. In this retrospective study, we explored the impact and action of two distinct FSHR isoforms, Thr307/Asn680 and Ala307/Ser680, in a large group of men. This investigation includes 288 normal healthy men, 86 of whom were proven fathers previously studied, and 202 were newly recruited subjects. The FSHR polymorphism at position 680 was analyzed in the whole group, while position 307 was investigated in 150 subjects, both of them by single-stranded conformation polymorphism (SSCP) gel electrophoresis. The distribution frequency for position 680 was 29% for the Asn/Asn, 52% for the Asn-Ser, 19% for the Ser-Ser variant, and for position 307, 27% for the Thr-Thr, 55% for the Ala-Thr, 18% for the Ala-Ala, respectively. Polymorphism combinations that were different from Thr307/Asn680 - Ala307/Ser680 were found in four subjects. When subjects were grouped according to genotype at position 680, no significant differences between basal FSH, testosterone, inhibin B levels and semen parameters were found. This clinical finding demonstrates that, differently from females, in whom a significant correlation between FSHR polymorphism and basal FSH levels was found, the FSHR genotype has no influence on clinical parameters in males.


2002 - Isoforms and single nucleotide polymorphisms of the follicle-stimulating hormone receptor gene: implications for human reproduction. [Articolo su rivista]
Simoni, Manuela; Nieschlag, E; Gromoll, J.
abstract

The FSH receptor shows three single nucleotide polymorphisms (SNPs), one in the promoter and two in exon 10. In addition, the FSH receptor mRNA undergoes extensive alternative splicing. While no physiological role for the SNP in the promoter and for alternative spliced isoforms has been demonstrated so far, the SNPs in exon 10 result in four discrete allelic variants characterized by the amino acid combinations Thr307-Asn680, Ala307-Ser680, Ala307-Asn680 and Thr307-Ser680. Several studies have demonstrated that the first two allelic variants are very frequent (approximately 60 and 40% respectively) in the Caucasian population. The rarer Ala307-Asn680 and Thr307-Ser680 variants are much less frequent (<5%) in the Chinese. In males the FSH receptor variants are not related to testicular volume, serum FSH or serum inhibin B levels. The two most common receptor variants transiently transfected in COS-7 cells displayed similar functional characteristics. Frequency distribution of the two polymorphisms in normal women and patients with polycystic ovarian syndrome or premature ovarian failure is still under investigation. The homozygous Ala307-Ser680 variant seems to be associated with significantly higher basal serum FSH levels and with a higher amount of FSH required for ovarian stimulation in women undergoing assisted reproduction. This suggests that the FSH receptor genotype can influence the ovarian response to FSH stimulation. The presence of SNPs in the FSH receptor gene capable of modifying FSH action paves the way for future patient-tailored, genotype-based hormone therapies.


2002 - Maintenance of spermatogenesis in hypogonadotropic hypogonadal men with hCG alone. [Articolo su rivista]
Depenbusch, M; VON ECKARDSTEIN, M; Simoni, Manuela; Nieschlag, E.
abstract

OBJECTIVE: It is generally accepted that both gonadotropins LH and FSH are necessary for initiation and maintenance of spermatogenesis. We investigated the relative importance of FSH for the maintenance of spermatogenesis in hypogonadotropic men. SUBJECTS AND METHODS: 13 patients with gonadotropin deficiency due to idiopathic hypogonadotropic hypogonadism (IHH), Kallmann syndrome or pituitary insufficiency were analyzed retrospectively. They had been treated with gonadotropin-releasing hormone (GnRH) (n=1) or human chorionic gonadotropin/human menopausal gonadotropin (hCG/hMG) (n=12) for induction of spermatogenesis. After successful induction of spermatogenesis they were treated with hCG alone for maintenance of secondary sex characteristics and in order to check whether sperm production could be maintained by hCG alone. Serum LH, FSH and testosterone levels, semen parameters and testicular Volume were determined every three to six Months. RESULTS: After spermatogenesis had been successfully induced by treatment with GnRH or hCG/hMG, hCG treatment alone continued for 3-24 Months. After 12 Months under hCG alone, sperm counts decreased gradually but remained present in all patients except one who became azoospermic. Testicular Volume decreased only slightly and reached 87% of the Volume achieved with hCG/hMG. During treatment with hCG alone, FSH and LH levels were suppressed to below the detection limit of the assay. CONCLUSION: Once spermatogenesis is induced in patients with secondary hypogonadism by GnRH or hCG/hMG treatment, it can be maintained in most of the patients qualitatively by hCG alone, in the absence of FSH, for extended periods. However, the decreasing sperm counts indicate that FSH is essential for maintenance of quantitatively normal spermatogenesis.


2002 - Manifestations of Y-chromosomal deletions in the human testis: a morphometrical and immunocytochemical evaluation. [Articolo su rivista]
Luetjens, Cm; Gromoll, J; Engelhardt, M; VON ECKARDSTEIN, S; Bergmann, M; Nieschlag, E; Simoni, Manuela
abstract

BACKGROUND: Deletions of the AZF (azoospermia factor) subregions on the Y chromosome are accompanied by a diverse spectrum of spermatogenic disturbances ranging from hypospermatogenesis to total depletion of germ cells causing infertility. The AZF region encodes gene products which are candidates for the genetic control of spermatogenesis. Although it is known which genes are involved, a general principle of cause and effect cannot yet be deciphered and the deletion type has non-uniform histological phenotypes. METHODS AND RESULTS: We analysed morphological parameters of testicular biopsies from 17 patients diagnosed for Y chromosome microdeletions. As control groups we analysed testes from patients with idiopathic Sertoli cell-only (SCO) syndrome (n = 11), mixed atrophy (n = 10) and complete spermatogenesis (n = 11). A detailed genetic analysis on the extension of the observed microdeletions revealed similar breakpoints in the distal and proximal region of the AZFc region, indicating a common mechanism of homologous recombination for such deletions, as has been suggested before. Morphometric parameters such as the diameter of the tubules, lumen, thickness of the lamina propria and height of the tubule epithelia were investigated. The diameter of the tubules from patients with microdeletions was found to be significantly smaller compared with patients with mixed atrophy. Considering also the size of the tubules, lumen and epithelia, a Y-chromosomal microdeletion represents an intermediate state between an idiopathic SCO and normal spermatogenesis. The immunohistochemical analysis of six different Sertoli cell markers, cytokeratin 18, vimentin, inhibin {alpha} subunit, 14-3-3 {theta}, FSH receptor and androgen receptor, revealed no impact of AZF deletion on the specific expression pattern of these genes. CONCLUSIONS: Our results suggest that, notwithstanding the deletion of a common region in the AZFc region, microdeletions of the Y chromosome lead to an intermediate status between idiopathic SCO and complete spermatogenesis, resulting in a heterogeneous histological profile regardless of the seminiferous activity. The Sertoli cell function seems not to be altered.


2002 - NATURAL TRANSMISSION OF A PARTIAL AZFB DELETION OF THE Y CHROMOSOME OVER THREE GENERATIONS. [Articolo su rivista]
Rolf, C; Gromoll, J; Simoni, Manuela; Nieschlag, E.
abstract

The natural transmission of microdeletions of the Y chromosome is occasionally reported in the literature. Here we describe the natural transmission of a partial AZFb deletion over three generations. PCR amplification of several sequence tagged site markers in the three AZF regions of the Y chromosome was carried out in a patient with oligoasthenoteratozoospermia, his father and his naturally conceived son. The deletion was confirmed by Southern blotting. The propositum, his father and his son showed a probably identical, partial deletion of the distal part of the AZFb region, involving sY130 and sY143. The deletion was confirmed by Southern blotting using the sY130 probe. Partial AZFb microdeletions can be associated with moderate oligozoospermia allowing natural conception and therefore natural transmission of this genetic anomaly. Further studies are needed to define the pathogenetic significance of microdeletions involving sY130 and sY143.


2002 - Prize winners of the 2002 VFS spring meeting: Follicle-stimulating hormone receptor polymorphisms in patients with normogonadotropic anovulation (WHO II) [Articolo su rivista]
Laven, J.; Mulders, A.; Suryandari, D.; Gromoll, J.; Simoni, M.; Nieschlag, E.; Fauser, B.
abstract


2002 - Proceedings of the 7th Andrology Symposium. Treatment of male infertility - Viewpoints, controversies, perspectives. Giessen, Germany, 17 November 2001 [Articolo su rivista]
Schill, W. -B.; Schuppe, H. -C.; Weid, W.; Manning, M.; Schirren, C.; Adamopoulos, D. A.; Beutel, M. E.; Comhaire, F.; El Garem, Y.; Glander, H. -J.; Haidl, G.; Hargreave, T. B.; Ghosh, C.; Jungwirth, A.; Kleinstein, J.; Kohn, F. -M.; Krause, W.; Bohring, C.; Nieschlag, E.; Simoni, M.; Gromoll, J.; Ochsendorf, F. R.; Pflieger-Bruss, S.; Blocher, S.; Monsees, T. K.; Schlatt, S.; Schroeder-Printzen, I.; Weidner, W.; Ludwig, M.; Diemer, T.
abstract


2002 - Self-adjusted postmenopausal hormone replacement therapy: effects on biological and immunological profile of follicle-stimulating hormone and correlation to climateric symptoms. [Articolo su rivista]
Vihtamäki, T; Simoni, Manuela; Tuimala, R; Nieschlag, E; Vihko, Kk
abstract

OBJECTIVE: The purpose of the present study was to evaluate the hormonal profile of patients of postmenopausal age during estrogen replacement therapy (ERT) with special reference to the serum levels of biologically active FSH (B-FSH) in a self-adjusted ERT model. DESIGN: The hormonal values found have been correlated to climateric symptoms reported by the patients (scored by the Kupperman menopausal index (KI)). METHODS: B-FSH was measured using an assay based on a cell system transfected permanently with FSH receptor cDNA. All women (n=32) applied estradiol percutaneously using 1 mg estradiol-17beta (E(2)) as an initial dose and were encouraged to increase the daily dose until they felt comfortable according to a specific scheme. Twelve of the 32 women were hysterectomized and treated, accordingly, with ERT only; 20 women received megestrol acetate monthly for 10 days. RESULTS: The initial average KI was 30 (range 10-54). A high degree of correlation (r=0.83; P<0.001) was observed between B-FSH and immunologically active FSH (I-FSH). Serum I-FSH and E(2) correlated negatively (r=-0.21; P<0.001); similarly, a negative correlation (r=-0.15; P<0.01) was observed between serum B-FSH and E(2) levels. Serum I-FSH and KI showed modest but significant positive correlation (r=0.13; P<0.01); a somewhat higher degree of correlation (r=0.19; P<0.005) was observed when B-FSH and KI were compared. E(2) showed positive correlation to serum sex-hormone binding globulin levels (r=0.22; P<0.001). CONCLUSIONS: This study shows that the transdermal self-adjusted hormone replacement therapy (HRT) model introduced is suitable for studies on endocrine changes during postmenopausal ERT. The finding of poor correlation between serum E(2) levels and KI emphasizes the importance of hormonal measurements during postmenopausal HRT.


2001 - Follicle-stimulating hormone receptor and twinning. [Articolo su rivista]
Gromoll, J; Simoni, Manuela
abstract

not available


2001 - Inhibin B in male reproduction: Pathophysiology and clinical relevance. [Articolo su rivista]
Meachem, S; Nieschlag, E; Simoni, Manuela
abstract

The recent availability of specific inhibin assays has demonstrated that inhibin B is the relevant circulating inhibin form in the human male. Inhibin B is a dimer of an alpha and a betaB subunit. It is produced exclusively by the testis, predominantly by the Sertoli cells in the prepubertal testis, while the site of production in the adult is still controversial. Inhibin B controls FSH secretion via a negative feedback mechanism. In the adult, inhibin B production depends both on FSH and on spermatogenic status, but it is not known in which way germ cells contribute to inhibin B production. The regulation of inhibin B production changes during life. There is an inhibin B peak in serum shortly after birth only partly correlated with an increase in serum FSH, probably reflecting the proliferating activity of the Sertoli cells during this phase of life. Afterwards, inhibin B levels decrease and remain low until puberty, when they rise again, first as a consequence of FSH stimulation and then as a result of the combined regulation by FSH and the ongoing spermatogenesis. In the adult, serum inhibin B shows a clear diurnal variation closely related to that of testosterone. The administration of FSH increases the secretion of inhibin B in normal men, but is much more pronounced in males with secondary hypogonadism. The treatment of infertile men with FSH, however, does not result in an unequivocal inhibin B increase. There is a clear inverse relationship between serum inhibin B and FSH in the adult. Serum inhibin B levels are strongly positively correlated with testicular volume and sperm counts. In infertile patients, inhibin B decreases and FSH increases. In general, there is very good correlation with the degree of spermatogenetic damage, with the arrest at the earlier stages having the lowest inhibin B levels. However, for unknown reasons, there are cases of Sertoli-cell-only syndrome with normal inhibin B levels. Inhibin B and FSH together are a more sensitive and specific marker for spermatogenesis than either one alone. However, the inhibin B concentrations are not a reliable predictor of the presence of sperm in biopsy samples for testicular sperm extraction. Suppression of spermatogenesis with testosterone and gestagens leads to a partial reduction of inhibin B in serum but it is never completely suppressed. In contrast, testicular irradiation in monkeys or humans leads to a rapid and dramatic decrease of inhibin B, which becomes undetectable when germ cells are completely absent. In summary, although inhibin B is a valuable index of spermatogenesis, the measurement of serum inhibin B levels is still of limited clinical relevance for individual patients.


2001 - Inverse correlation between sperm concentration and number of androgen receptor CAG repeats in normal men. [Articolo su rivista]
VON ECKARDSTEIN, S; Syska, A; Gromoll, J; Kamischke, A; Simoni, Manuela; Nieschlag, E.
abstract

Androgens are essential for the maintenance of normal spermatogenesis. Androgen action is mediated by the androgen receptor (AR), which in the testis is expressed by Leydig, peritubular, and Sertoli cells. The fact that sperm numbers range from 20 up to 300 million/mL in normal men without any indication of changed endocrine parameters led us to assume that genetic variability of transduction of androgen signaling could be important. We therefore compared the variable number of CAG repeats in the AR with sperm concentrations in men with normal ejaculate parameters (62 fathers and 69 volunteers participating in clinical trials). In multivariate analysis CAG repeat length did not differ between the volunteers (19.4 +/- 3.1) and the fathers (20.6 +/- 3.0), but was significantly correlated to sperm concentrations with a coefficient of -0.25. When compared with a group of infertile men with (n = 14) or without (n = 30) a family history of infertility, no such correlation was found. These results indicate that men with short CAG repeats have the highest sperm output within the normal fertile population. Polymorphisms of the AR contribute to the efficiency of spermatogenesis in normal men, but do not play a predominant role in male infertility.


2001 - Is inhibin B a pharmacodynamic parameter for FSH in normal men? [Articolo su rivista]
Kamischke, A; Simoni, Manuela; Schrameyer, K; Nieschlag, E.
abstract

OBJECTIVE: This study aims to investigate the pharmacodynamic effect of FSH on inhibin B serum levels in normal men in order to elucidate the physiological regulation of inhibin B secretion in more detail. DESIGN AND METHODS: Injections of 3000 IU recombinant, human FSH (rhFSH) were followed by single-blinded injections of placebo, 1000 and 2000 IU rhFSH spaced by at least 28 days between injections. RESULTS: After injection of 3000 IU rhFSH, inhibin B values were significantly elevated above baseline for 24, 96 and 120 h (maximal increase after 96 h, mean +/- s.e.m. 303+/-18 pg/ml). Injection of 2000 IU rhFSH led to a significant increase in inhibin B (maximum mean +/- s.e.m. 318+/-20 pg/ml) from 24 to 120 h. Injection of 1000 IU rhFSH led to a significant increase in inhibin B after 96 h (maximum mean +/- s.e.m. 300+/-16 pg/ml). The inhibin B areas under the curve after injection of 2000 and 3000 IU rhFSH were significantly higher than those following the placebo and 1000 IU rhFSH. In the 12 fertile men investigated, at baseline a strong diurnal rhythm of inhibin B parallel to that of testosterone was observed. CONCLUSIONS: Serum inhibin B can be considered only a partial pharmacodynamic parameter of FSH in vivo, since the integrity of the spermatogenic process appears to be a second fundamental component in the regulation of its secretion from the testis.


2001 - Molecular Diagnosis of Y-chromosomal microdeletions in Europe: state of the art and quality control. [Articolo su rivista]
Simoni, Manuela
abstract

The polymerase chain reaction (PCR) screening of microdeletions of the Y chromosome has become an important diagnostic step in the work-up of male infertility. However, there is no agreement about how this diagnosis should be performed. There are suggestions that the large variation in deletion frequency reported in the literature could be due to the various selection criteria of the patients analysed, although methodological aspects may play a role as well. As for other genetic diseases, molecular diagnosis of Y chromosome microdeletions should be controlled by adopting strict internal quality control measures and by participating in external quality assessment schemes. Such an external quality assessment project is presently being organized jointly by the European Academy of Andrology and the European Molecular Genetics Quality Network. Three preliminary trials have given a state-of-the-art picture of the diagnostic performance in various European laboratories, showing an overall rate of misdiagnosis of ~5% for both AZFb and AZFc regions, and providing data useful in the generation of guidelines for the molecular diagnosis of Y chromosome microdeletions.


2001 - Mutations of gonadotropins and their receptors [Mutationen der gonadotropine und gonadotropinrezeptoren] [Articolo su rivista]
Gromoll, J.; Nieschlag, E.; Simoni, M.
abstract

not available


2001 - Prevalence of Y chromosome microdeletions in infertile men consulting a tertiary care medical centre: the Münster experience. [Articolo su rivista]
Maurer, B; Gromoll, J; Simoni, Manuela; Nieschlag, E.
abstract

The long arm of the human Y chromosome is required for male fertility. Microdeletions in three different regions of the human Y chromosome, designated AZFa, AZFb and AZFc, respectively, are frequently associated with male infertility. The varying frequency of Y microdeletions found in cohorts of infertile men (0.4-55.5%) is probably related to the criteria by which the patients are selected. We report the diagnosis of Y chromosomal microdeletion in a total of 1,470 men who attended our infertility clinic, the largest sample of infertile patients to have been analysed to date. This cohort consists of three populations. The first subgroup comprises 228 selected patients with severely impaired spermatogenesis. Since microdeletions had also been reported in patients with less severe defects in spermatogenesis, we then intended to define the deletion frequency in unselected patients (population II: 378 patients). Population III comprises 864 prospectively selected patients and intracytoplasmic sperm injection candidates. Altogether, 19 patients with microdeletions were found (1.3%). The microdeletion frequencies in populations I, II and III were 3.5%, 0.3% and 1.2%, respectively. Our study helps to define a subgroup of infertile men at risk of Y chromosomal microdeletions, and strongly supports the recommendation that Y microdeletion analysis should be limited to azoospermic and severely oligozoospermic men and candidates for intracytoplasmic sperm injection.


2000 - Male hypogonadism caused by a homozygous deletion of exon 10 of the luteinizing hormone (LH) receptor: differential action of human chorionic gonadotropin and LH. [Articolo su rivista]
Gromoll, J; Eiholzer, U; Nieschlag, E; Simoni, Manuela
abstract

We report the unique case of a patient with Leydig cell hypoplasia (LCH) type II caused by a genomic deletion resulting in the complete absence of exon 10 of the LH receptor (LHR). The patient presented at the age of 18 yr with retarded pubertal development, small testicles, and delayed bone maturation. LH was highly elevated, with very low serum testosterone levels. Genetic analysis revealed a homozygous deletion of approximately 5 kbp encompassing exon 10 of the LHR gene. Screening of family members demonstrated heterozygosity for the deletion, indicating autosomal recessive inheritance. At the time of examination, the patient displayed nearly normal male phenotype, but lacked pubertal development and was hypogonadal. Obviously, fetal male development sustained by hCG was normal, whereas LH action, important for pubertal development, was impaired. A hCG stimulation test induced testosterone biosynthesis and secretion within the normal range. Subsequently, hCG treatment was continued, resulting in an increase in testicular volume and the appearance of spermatozoa in the ejaculate after 16 weeks of treatment (5.3 million/mL). Despite highly elevated endogenous LH serum levels, the response to hCG indicates a possible dual mechanism of hormone binding and signal transduction for hCG and LH on a LHR that lacks exon 10. Furthermore, this patient represents the clinical counterpart of the normal male marmoset monkey (Callithrix jacchus), in which the expressed LHR lacks exon 10 in toto. This case provides important clinical insights about the possible role of exon 10 of the LHR in discriminating between LH and hCG actions.


2000 - Ovarian response to FSH stimulation depends on the FSH receptor genotype. [Articolo su rivista]
PEREZ MAYORGA, M; Gromoll, J; Behre, Hm; Gassner, C; Nieschlag, E; Simoni, Manuela
abstract

Because the ovarian response to FSH stimulation in assisted reproduction is variable, ranging from hyporesponse to hyperresponse, with the possible complication of ovarian hyperstimulation, it would be of great benefit to predict the response of the patients to FSH. To date, no clear-cut predictors of ovarian responsiveness to FSH have been identified. In this study, we investigated the role of two distinct FSH receptor (FSHR) variants, Thr307/Asn680 and Ala307/Ser680, in the response to FSH in women undergoing controlled ovarian stimulation.The FSHR polymorphism at position 680 was analyzed by restriction-fragment-length polymorphism in 161 ovulatory women below the age of 40 yr. With reference to the couple, infertility has been diagnosed as being attributable to male causes (76%), tubal factor (11%), or both (13%). The distribution was 29% for the Asn/Asn, 45% for the Asn/Ser, and 26% for the Ser/Ser FSHR variant. Peak estradiol levels, number of preovulatory follicles, and number of retrieved oocytes were similar in the 3 groups. However, basal FSH levels were significantly different among the 3 groups (6.4 ± 0.4 IU/L, 7.9 ± 0.3 IU/L, and 8.3 ± 0.6 IU/L for the Asn/Asn, Asn/Ser, and Ser/Ser groups, respectively, P < 0.01). The number of FSH ampoules required for successful stimulation was significantly different among the 3 groups (31.8 ± 2.4, 40.7 ± 2.3, and 46.8 ± 5.0 for the Asn/Asn, Asn/Ser, and Ser/Ser groups, respectively, P < 0.05). According to multiple linear regression analysis, the number of ampoules needed could be predicted from a linear combination of both the type of polymorphism and basal FSH levels (P < 0.001).These clinical findings demonstrate that the ovarian response to FSH stimulation depends on the FSHR genotype.


2000 - Physiologie der Hodenfunktion. [Capitolo/Saggio]
Weinbauer, G. F; Gromoll, J; Simoni, Manuela; Nieschlag, E.
abstract


2000 - Physiology of the testicular function. [Capitolo/Saggio]
Weinbauer, G. F; Gromoll, J; Simoni, Manuela; Nieschlag, E.
abstract


2000 - Y chromosome deletion screening in infertile men. [Articolo su rivista]
Maurer, B; Simoni, Manuela
abstract

Molecular analysis of Y-chromosomal microdeletions is routinely performed in the work-up of male infertility, in order to establish a diagnosis and for genetic counseling of the couple, since such microdeletions are transmitted to the male offspring. The review of published data shows that microdeletions are relatively common in patients with azoospermia or severe oligozoospermia, with wide variations in the reported deletion frequency depending mainly on the selection criteria. In general, patients with proximal deletions, involving the AZFa and/or the AZFb region show severe defects of spermatogenesis with a high prevalence of Sertoli cell only syndrome, while deletions of the distal AZFb and of the AZFc region can be compatible with residual spermatogenesis. Microdeletions have been only sporadically found in normozoospermic patients. For the time being the molecular analysis of microdeletions of the Y chromosome is indicated in infertile patients with sperm concentration <5 x 10(6)/ml and in men undergoing assisted reproduction techniques, since the genetic defect and, most probably, the related infertility problem will be transmitted to the sons.


1999 - Clinical trial of transdermal testosterone and oral levonorgestrel for male contraception. [Articolo su rivista]
Büchter, D; VON ECKARDSTEIN, S; VON ECKARDSTEIN, A; Kamischke, A; Simoni, Manuela; BEHRE H., M; Nieschlag, E.
abstract

Approaches to hormonal male contraception are predominantly based on injectable testosterone (T) application. As most users would prefer an injection-independent modality, this study was designed to develop a self-applicable hormonal male contraceptive regimen by combining transdermal T with an oral gestagen. Eleven healthy men (23–40 yr old) were treated with oral levonorgestrel and transdermal T for 24 weeks. T was applied daily as a transdermal patch to be worn on the trunk. Levonorgestrel was taken orally at a dose of 250 µg daily up to week 12, followed by 500 µg to week 24 in those volunteers who had not become azoospermic by that time. Within 24 weeks, 2 of 11 volunteers had become azoospermic, and 3 of 11 showed sperm concentrations below 3 million/mL. The sperm concentrations of the remaining volunteers declined, but failed to reach the limit considered compatible with contraception by WHO. Treatment resulted in suppression of LH, FSH, and sex hormone-binding globulin, whereby the volunteers with lower sperm concentrations showed more pronounced suppression than the others. Mean T concentrations remained within the lower limit of normal and on occasions were below this level. There were no complaints of hypoandrogenism. Although mean levels of low density lipoprotein cholesterol, apolipoprotein B, as well as basal and postprandial insulin increased, high density lipoprotein cholesterol and apolipoprotein A-I decreased during the treatment phase. Changes in lipid parameters were normalized within 3 weeks after cessation of medication. Although only 5 of 11 volunteers reached the target sperm counts (<3 million/mL), the study shows that a self-applicable hormonal male contraceptive could be developed.


1999 - Cloning and expression of cynomolgus monkey (Macaca fascicularis) gonadotropins luteinizing hormone and follicle-stimulating hormone and identification of two polymorphic sites in the luteinizing hormone β subunit. [Articolo su rivista]
Schmidt, A; Gromoll, J; Weinbauer, Gf; GALLA H. J., Chappel; S, ; Simoni, Manuela
abstract

The genes encoding the cynomolgus monkey gonadotropin subunits, alpha, follicle-stimulating hormone (FSH) beta and luteinizing hormone (LH) beta, were cloned by reverse transcriptase polymerase chain reaction (RT-PCR) using pituitary RNA. The predicted amino acid sequences displayed 82, 96 and 87% identity to human subunit sequences, respectively. Northern blot hybridization of monkey tissues revealed pituitary specific transcripts of 1.0 and 0.6 kb for the alpha and LHbeta subunit, respectively, and two bands of 1.8 and 0.65 kb for the FSHbeta. Upon sequencing LHbeta cDNAs from different monkeys, two polymorphic sites were detected, resulting in the amino acid transitions Ser32Thr and His60Arg. Restriction analysis revealed different homo- and heterozygous combinations of the polymorphic sites indicating linkage dysequilibrium. Transient co-expression of the alpha subunit together with the FSHbeta or LHbeta subunit in COS7 and CHO cells resulted in secretion of in vitro bioactive hormones. This work represents a further step towards production of recombinant monkey LH and FSH which can be used in a homologous experimental setting in the cynomolgus monkey.


1999 - Constitutively active mutations of G protein-coupled receptors: the case of the human luteinizing hormone and follicle-stimulating hormone receptors. [Articolo su rivista]
Nordhoff, V; Gromoll, J; Simoni, Manuela
abstract

Activating mutations of the luteinizing hormone receptor (LHR) and the follicle-stimulating hormone receptor (FSHR) have been known for several years. These activating mutations permanently stimulate, in the absence of their cognate ligand, the receptor signaling pathways. In the case of the LHR, the induced chronic stimulation causes sporadic and familial pseudoprecocious puberty, a phenotype observed only in males. The absence of a female phenotype is probably due to the requirement for FSH in the induction of LHR expression. For the FSHR, one activating mutation was found in a patient with normal spermatogenesis without detectable gonadotropins. Whether activating mutations of the gonadotropin receptors are involved in tumor development is not yet clear. Activating mutations of the FSHR were supposedly involved but not found in ovarian tumors. For the LHR, only one patient with a seminoma and an activating mutation was described. The different occurrence of activating mutations of the LHR compared to the FSHR is surprising, since the two genes are adjacently located on chromosome 2 and should therefore be affected by a similar mutation rate. It might well be that mutations occur with the same frequency, but that activating mutations of the FSHR do not result in any particular phenotype.


1999 - Estrogen resistance and aromatase deficiency [Capitolo/Saggio]
Simoni, Manuela; Rochira, Vincenzo; Marco Faustini, Fustini; Carani, Cesare
abstract

The chapter deals with the aspects of estrogen resistance and aromatase deficiency. In particular the role of serum estrogens in the control of gonadotropin feedback is reviewed.


1999 - Inhibin B is a more sensitive marker of spermatogenic damage than FSH in the irradiated non-human primate model. [Articolo su rivista]
Foppiani, L; Schlatt, S; Simoni, Manuela; Weinbauer, Gf; HACKER KLOM, U; Nieschlag, E.
abstract

This study evaluated the effect of bilateral testicular irradiation (2 Gy) on reproductive hormones, testicular volume (TV) and sperm parameters in six adult cynomolgus monkeys. Hormone levels (FSH, inhibin B and testosterone (T)) were determined to find the most valuable endocrine marker of irradiation-induced damage. All parameters were analysed at weekly intervals for 14 weeks. Histological evaluation of both testes was performed at week 14 after irradiation when one monkey was castrated and at week 27 when the remaining five monkeys were bilaterally biopsied. A decrease in body weight, TV (30% of the pre-treatment size) and sperm count was observed after irradiation. Severe oligozoospermia was achieved throughout the study but azoospermia was recorded only occasionally. Histological evaluation revealed a heterogeneous picture with patchy arrangement of seminiferous tubules containing advanced germ cell types. An increase (P<0.05) in FSH levels and, to a lesser degree also in T levels, occurred several weeks after irradiation. Inhibin B levels showed a sharp decline (P<0.001) as soon as 1 week after irradiation. FSH and inhibin B did not return to baseline levels during the observation period. A negative correlation was found between FSH and inhibin B values (r=-0.35, P<0.001). Inhibin B correlated positively with testis volume (r=0.73, P<0.001) and sperm counts (r=0.55, P<0.01). In conclusion, this study shows that inhibin B represents an early and more sensitive marker of testicular damage than FSH. Furthermore, the rapid fall of inhibin B after irradiation suggests that this hormone is a direct parameter of premeiotic germ cell proliferation.


1999 - Laboratory guidelines for molecular diagnosis of Y-chromosomal microdeletions. [Articolo su rivista]
Simoni, Manuela; Bakker, E; MATTHIJS G, EURLINGS M. C. M.; Moro, E; Müller, Cr; Vogt, Ph
abstract

not available


1999 - Mutation screening and isoform prevalence of the follicle stimulating hormone receptor gene in women with premature ovarian failure, resistant ovary syndrome and polycystic ovary syndrome. [Articolo su rivista]
Conway, Gs; Conway, E; Walker, C; Höppner, W; Gromoll, J; Simoni, Manuela
abstract

OBJECTIVE: To determine whether mutations in the FSH receptor gene are associated with premature ovarian failure (POF) or resistant ovary syndrome (ROS) in women in the UK. To determine whether an allelic variant of the FSH receptor gene affects fertility parameters in women with polycystic ovary syndrome (PCOS). DESIGN: A mutation screen using DNA from women with POF and ROS. Restriction digest of amplified DNA from women with POF, ROS, PCOS and controls to determine allelic variant status. Fertility parameters were compared between allelic variant subgroups of women with PCOS. PATIENTS: The study population comprised 49 women with POF, 5 with ROS, 93 with PCOS and 51 controls. MEASUREMENTS: In women with PCOS, fertility and menstrual status was recorded and serum FSH and ovarian volume were measured. RESULTS: No mutation of the FSH receptor gene was found in women with POF or ROS. The allelic variant Thr307/Ser680 was found to be similarly prevalent in all study groups. The Thr307/Ser680 variant was found to have no phenotype in terms of fertility parameters in women with PCOS. CONCLUSIONS: Mutations of the FSH receptor gene are rare in women with premature ovarian failure or resistant ovary syndrome in the UK. Polymorphisms of the FSH receptor gene do not appear to have pathophysiological significance with regard to ovarian function.


1999 - Mutational analysis of the follicle-stimulating hormone (FSH) receptor in normal and infertile men: identification and characterization of two discrete FSH receptor isoforms. [Articolo su rivista]
Simoni, Manuela; Gromoll, J; Höppner, W; Kamischke, A; Krafft, T; Stähle, D; Nieschlag, E.
abstract

In a search for pathophysiological causes of idiopathic male infertility we investigated the occurrence of mutations of the FSH receptor in 48 men with this disorder. The entire FSH receptor gene was analyzed by single stranded conformation polymorphism analysis (SSCP). A heterozygous point mutation without functional consequences, exchanging Val to Ala in codon 341, was found in one patient. SSCP analysis led to the identification of 2 polymorphisms in exon 10 associated in 2 discrete FSH receptor allelic variants, i.e. Thr307-Asn680 and Ala307-Ser680. The frequency and distribution of the two allelic variants was further analyzed in 86 proven fathers and 75 infertile men by SSCP (codon 307) and restriction fragment length polymorphism (codon 680). The 2 receptor isoforms showed similar Mendelian distribution in proven fathers and in infertile men. Serum FSH, inhibin B, and combined testicular volume did not differ between subjects with different receptor isoforms. Binding studies in transiently transfected COS-7 cells showed similar binding affinity for the two receptor variants. Moreover, the Ala307-Ser680 and the Thr307-Asn680 FSH receptors responded in vitro to FSH with comparable cAMP production. These data suggest that different isoforms of the FSH receptor with similar functional properties exist in normal and infertile men. We conclude that mutations of the FSH receptor or the FSH receptor genotype do not play a pathogenic role in male idiopathic infertility. The possibility that different FSH isoforms might interact differently with the 2 receptor variants remains to be investigated.


1999 - Repeated intramuscolar injections of testosterone undecanoate for substitution therapy in hypogonadal men. [Articolo su rivista]
Nieschlag, E; Büchter, D; VON ECKARDSTEIN, S; Abshagen, K; Simoni, Manuela; Behre, Hm
abstract

OBJECTIVETo investigate the suitability of intramuscular testosterone undecanoate (TU) injections for substitution therapy in hypogonadal men. STUDY DESIGNClinical, open-label, non-randomized trial of 13 hypogonadal men receiving 4 intramuscular injections of 1000 mg TU in 4-ml castor oil at 6-week intervals. General wellbeing, sexual parameters, clinical chemistry, hormone levels, prostate size and prostate-specific antigen (PSA) were evaluated over 24 weeks and compared with baseline values. RESULTSTestosterone serum levels were never found below the lower limit of normal and only briefly after the 3rd and 4th injection above the upper limit of normal, while peak and trough values increased over the 24-week observation period. Oestradiol and dihydrotestosterone followed this pattern, not exceeding the normal limits. No serious side-effects were noted. Slight increases in body weight, haemoglobin, haematocrit, prostate volume and PSA, suppression of gonadotrophins as well as increased ejaculation frequency occurred as signs of adequate testosterone substitution. CONCLUSIONTestosterone undecanoate is well tolerated by the patients. The injection intervals can be extended even beyond the 6-week periods chosen in the present study. Altogether, intramuscular testosterone undecanoate appears to be well suited for long-term substitution therapy in hypogonadism and hormonal male contraception.


1999 - Role of FSH in male gonadal function. [Articolo su rivista]
Simoni, Manuela; Weinbauer, Gf; Gromoll, J; Nieschlag, E.
abstract

The production of male gametes depends on the concerted action of the two gonadotropins FSH and LH on the testis. The action of LH is mediated through the production of testosterone by the Leydig cells. Since male germ cells possess neither FSH nor androgen receptors, the action of FSH and testosterone occurs through the Sertoli cells. Although the precise function of these two hormones remains elusive, the existing evidence suggest that both FSH and testosterone are able to stimulate all phases of spermatogenesis. In the male FSH is required for the determination of Sertoli cell number, and for induction and maintenance of normal sperm production. The crucial role of FSH in male gonadal function has been clearly illustrated by the discovery of a patient with an activating mutation of the FSH receptor. This patient had been hypophysectomized because of a pituitary tumor and, under testosterone substitution was unexpectedly fertile in spite of undetectable serum gonadotropin levels and had fathered three children. In this patient we could demonstrate a heterozygous activating mutation of the FSH receptor which resulted in cAMP production independent of FSH stimulation. This finding represents the first description of an activating mutation of the FSH receptor and demonstrates that FSH alone maintains spermatogenesis in man. On the other hand, the effects of the lack of FSH action are unclear. Among five men with a homozygous inactivating mutation of the FSH receptor only one was infertile and spermatogenesis was variably affected in the others. However, serum inhibin B values in these men were not completely suppressed and serum FSH levels were only moderately elevated, indicating the possibility that FSH receptor function was not completely abolished by the mutation. Elimination of FSH action is a prerequisite to suppress completely spermatogenesis for contraceptive purposes, while administration of both LH and FSH is necessary to induce sperm production in patients with hypogonadotropic hypogonadism. Experimental immunization of male monkeys against FSH markedly reduced germ cell proliferation and even induced infertility. At the cellular level, FSH stimulates the cAMP-dependent activation of protein kinase A in Sertoli cells, but the molecular mechanism of FSH action is poorly understood. In the primate, the gonadotropin withdrawal achieved by administration of a GnRH antagonist leads to a premeiotic arrest of germ cell proliferation, probably due to inhibition of the mitotic division of A-pale spermatogonia. Therefore, FSH might be the prime inducer of spermatogonial proliferation, while the successive maturation process could proceed independently of FSH. In summary, clinical and experimental evidence support the concept of an irreplaceble role of FSH in the primate. Only the combination of FSH and testosterone, however, supports a qualitatively and quantitatively fully normal spermatogenesis.


1999 - Role of FSH in the regulation of spermatogenesis: clinical aspects. [Articolo su rivista]
Nieschlag, E; Simoni, Manuela; Gromoll, J; Weinbauer, Gf
abstract

not available


1999 - Serum inhibin B in combination with serum FSH is a more sensitive marker than serum FSH alone of impaired spermatogenesis in men, but cannot predict the presence of sperm in testicular tissue samples. [Articolo su rivista]
VON ECKARDSTEIN, S; Simoni, Manuela; Bergmann, M; Weinbauer, Gf; Gassner, P; Schäpers, A; Nieschlag, E.
abstract

The measurement of serum FSH is useful in the diagnostic workup of the infertile male, but fails to predict the presence of sperm in testicular tissue. We investigated whether inhibin B reflects testicular morphology and the presence of sperm more accurately than FSH. Serum inhibin B and gonadotropin levels were determined in 91 infertile men undergoing diagnostic bilateral testicular biopsy. In 52 of the 91 patients multiple samples were taken for testicular sperm extraction (TESE). Inhibin B levels were (mean ± SEM) 238 ± 32 pg/mL in men with normal spermatogenesis (n = 9), 102 ± 18 pg/mL in men with spermatogenetic arrest (n = 15), 98 ± 16 pg/mL in hypospermatogenesis (n = 23), 41 ± 6 pg/mL in focal Sertoli cell-only syndrome (SCO; n = 26), and 27 ± 8 pg/mL in complete SCO (n = 18). The percentage of SCO tubuli was more strongly correlated to serum inhibin B (r = -0.58; P < 0.01) than to FSH (r = 0.34; P < 0.05). Similarly, the percentage of tubules with elongated spermatids was significantly (P < 0.05) more strongly correlated to serum inhibin B (r = 0.65; P < 0.01) than to FSH (r = -0.4; P < 0.01). Thus, inhibin B is slightly more sensitive than FSH as an index of the spermatogenic status. Neither FSH nor inhibin B alone, however, could predict the type of spermatogenetic damage exactly. The combination of FSH and inhibin B had high diagnostic sensitivity (88%) and specificity (83%) for the presence of elongated spermatids in testicular biopsies. Sperm could be retrieved in 34 (65%) of the TESE patients. The combination of inhibin B and FSH measurement showed a sensitivity of 75% and a specificity of 73% when identifying patients in whom sperm could possibly be retrieved by TESE. We conclude that although the measurement of serum inhibin B improves the sensitivity of predictive tests for the presence of sperm in histology or for TESE, this parameter cannot accurately predict TESE outcome.


1999 - Transmission of a Y-chromosomal deletion involving DAZ and CDY genes from father to son through ICSI. [Articolo su rivista]
Kamischke, A; Gromoll, J; Simoni, Manuela; Behre, Hm; Nieschlag, E.
abstract

The transmission of a deleted in azoospermia (DAZ) deletion from a severely oligozoospermic patient to his son following intracytoplasmic sperm injection (ICSI) treatment is reported. The case report highlights the fertilizing capacity of spermatozoa carrying Y chromosome deletions in patients treated with ICSI and stresses the importance of genetic counselling in severe male infertility.


1998 - A mutation in the first transmembrane domain of the lutropin receptor causes male precocious puberty. [Articolo su rivista]
Gromoll, J; PARTSCH C., J; Simoni, Manuela; Nordhoff, V; Sippel, Wg; Nieschlag, E; Saxena, Bb
abstract

We describe a patient with onset of puberty at the age of 5 yr, characterized by accelerated growth, enlargement of genitalia, pubarche, and serum hormone levels compatible with noncentral precocious puberty. Exon 11 of the LH receptor gene was amplified from genomic DNA by PCR and directly sequenced. We identified a heterozygous C to T base change at nucleotide position 1126, exchanging codon 373 from Ala to Val in the first transmembrane domain. The LH receptor sequence of the parents was normal. The mutated receptor displayed an up to 7.5-fold increase in basal cAMP production compared to that of the wild-type receptor in transiently transfected COS-7 cells. Treatment of the patient with ketoconazole resulted in inconsistent suppression of serum testosterone levels. At the age of 9.1 yr, central activation of the hypothalamic-pituitary-gonadal axis occurred. Additional treatment with a GnRH agonist led to complete suppression of testosterone secretion. This is the first description of constitutive activation of the LH receptor in the first transmembrane segment. It suggests the involvement of the first transmembrane helix in signal transduction and provides further insight into the structural organization of the seven transmembrane domains of the glycoprotein hormone receptor proteins.


1998 - Current status of the molecular diagnosis of Y-chromosomal microdeletions in the work-up of male infertility. Initiative for international quality control [Articolo su rivista]
Simoni, Manuela; Kamischke, A; Nieschlag, E.
abstract

not available


1998 - Molecular pathophysiology and clinics of the gonadotropin receptor defects. [Relazione in Atti di Convegno]
Simoni, Manuela; Gromoll, J; Nieschlag, E.
abstract

The gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) bind specific receptors, members of the G protein-coupled receptor superfamily. Mutations of gonadotropin receptors are classified into activating (constitutively active or gain-of-function mutations) and inactivating (loss-of-function mutations). Activating mutations of the LH receptor have been described in familial and sporadic forms of male-limited pseudoprecocious puberty, whereas they do not appear to have any particular phenotype in females. The only activating mutation of the FSH receptor described to date was found in a hypophysectomized man who was fertile despite undetectable serum gonadotropin levels; the effects of constitutive FSH receptor activity in the context of normal pituitary function are not known. Homozygous inactivating mutations of the LH and FSH receptor invariably lead to amenorrhea in genotypical female subjects. In males, inactivation of the LH receptor in its more severe form results in a clinical picture similar to the syndrome of complete androgen resistance, but milder forms of hypoandrogenization have been described as well. In males, homozygous inactivation of the FSH receptor can also be associated with infertility. Finally, polymorphic variants of the FSH receptor are present in the normal population.


1998 - Mutations of the G protein-coupled receptors of the hypothalamo-pituitary-gonadal axis. Where do we stand? [Articolo su rivista]
Simoni, Manuela
abstract

not available


1998 - Recombinant human FSH for treatment of male idiopathic infertility: a randomized, double-blind, placebo-controlled, clinical trial. [Articolo su rivista]
Kamischke, A; Behre, Hm; Bergmann, M; Simoni, Manuela; Schäfer, A; Nieschlag, E.
abstract

To examine the role of recombinant human follicle stimulating hormone (rhFSH) in male idiopathic infertility a randomized, double-blind, placebo-controlled study was performed. Of 211 patients screened, 67 were finally included. After two pre-examinations, patients were randomized and treated for 12 weeks, either with 150 IU rhFSH or with placebo. Examinations (physical examination, scrotal ultrasonography, semen analysis, hormone measurements, and in 31 patients electron microscopy (EM) of spermatozoa were performed 6 and 12 weeks after treatment initiation and 6 and 12 weeks after completion of treatment. Pregnancies were recorded for a further 3 months after the last examination. Of the 67 patients included in the study, 34 treated and 31 placebo patients could be analysed. In the treated group, FSH was elevated compared to baseline values (P < 0.001). At the end of treatment testicular volume in the treated group was increased compared to placebo (P < 0.05) and baseline (P < 0.001). Apart from an increase in sperm motility (P < 0.05) in the placebo group and in sperm DNA condensation (P < 0.001) in the treated group no significant changes were observed in semen parameters. Two spontaneous pregnancies in partners of men in the treated group and none in the placebo group occurred. However, two pregnancies occurred in partners of men in the placebo group induced by intrauterine insemination or intracytoplasmic sperm injection. In conclusion, at the chosen dose and duration, rhFSH did not lead to an improvement of conventional or EM sperm parameters nor to an increase in pregnancy rates. However, the increased testicular volume and sperm DNA condensation give reason for further investigations.


1998 - Testosterone: action, deficiency, substitution. [Articolo su rivista]
Büchter, D; Kamischke, A; Ochsenkühn, R; Rolf, C; Simoni, Manuela; von Eckardstein, S; Nieschlag, E.
abstract

not available


1998 - The EAA international quality control programme for Y-chromosomal microdeletions. [Articolo su rivista]
Simoni, Manuela
abstract

not available


1997 - Does the gonadotropic axis play a causal role in pathogenesis of Sertoli cell only syndrome? [Articolo su rivista]
Leifke, E; Simoni, Manuela; Kamischke, A; Gromoll, J; Bergmann, M; Nieschlag, E.
abstract

Infertile men with Sertoli-cell-only syndrome (SCO) have highly elevated serum FSH immunoactivity related to the degree of histological damage. The activity that serum FSH exerts at the target site depends on its glycosylation pattern and FSH receptor (FSHR) function. Either could be impaired, leading to failure of spermatogenesis. The aim of the present investigation was to study bioactivity and the glycosylation pattern of serum FSH and the occurrence of mutations in the FSH receptor in infertile patients with SCO compared to normal men. Blood was taken from 19 patients with bilateral testicular focal or complete SCO and eight normozoospermic controls. FSH bioactivity in serum was measured using an in-vitro FSH bioassay based on recombinant rat FSHR. The glycosylation pattern of serum FSH was determined by concanavalin A chromatography. Inhibin B was determined in serum using a recently available assay. Genomic DNA extracted from blood lymphocytes was amplified by PCR using primers specific for the FSHR and screened by single-stranded conformation polymorphism gel electrophoresis. Men with SCO showed significantly higher FSH in-vitro bioactivity (34.9 +/- 5.0 IU/l) than controls (9.6 +/- 0.8 IU/l: p < 0.01), as well as significantly elevated FSH immunoactivity (14.9 +/- 1.7 IU/l) compared to controls (3.1 +/- 0.5; p < 0.01). Immunoactivity of serum FSH was correlated with in-vitro bioactivity (r = 0.9; p < 0.001) and was related to the degree of testicular damage (proportion of SCO-tubules) (ANOVA: p < 0.001) and total testicular volume (r = -0.76; p < 0.01). An inverse relationship between serum FSH and inhibin B levels (r = -0.93; p < 0.001) was found. In the serum of SCO patients a slight increase in less glycosylated FSH isoforms was found (6.7 +/- 0.6% versus 3.6 +/- 0.3%; p < 0.05). No mutations of the FSHR were observed in SCO patients. We conclude that the spermatogenic failure observed in infertile patients with SCO histology and elevated FSH serum levels can be explained neither by a change in FSH bioactivity nor by mutations in the FSHR. The slight change in the FSH glycosylation pattern is probably related to higher hormonal secretion rates in SCO patients. The inverse relationship between serum FSH and inhibin B points to an intact endocrine testicular-pituitary circuit responsible for the compensatory increase of FSH in SCO.


1997 - Effect of testosterone and estradiol in a man with aromatase deficiency [Articolo su rivista]
Carani, Cesare; K., Qin; Simoni, Manuela; FAUSTINI FUSTINI, Marco; S., Serpente; J., Boyd; Ks, Korach; Er, Simpson
abstract

not available


1997 - Genetic and molecular diagnosis of male infertility [Articolo su rivista]
Nieschlag, E.; Meschede, D.; Gromoll, J.; Simoni, M.
abstract


1997 - Molecular pathophysiology of the pituitary-gonadal axis. [Relazione in Atti di Convegno]
Simoni, Manuela; Gromoll, J; Hoppner, W; Nieschlag, E.
abstract

Mutations of gonadotropin beta subunits or gonadotropin receptors are involved in some reproductive diseases leading to alterations of pubertal maturation or infertility. Homozygous inactivation of LH results in absence of pubertal maturation and hypogonadism in the male, whereas inactivation of FSH causes primary amenorrhea in females. Mutations of the gonadotropin receptors are classified into activating (the receptor is also active in the absence of the hormone: gain-of-function mutations) and inactivating types (the receptor is not properly processed and/or the hormone cannot bind: loss-of-function mutations). Activating mutations of the LH receptor have been described in familiar and sporadic forms of male-limited pseudoprecocious puberty, whereas they do not express any phenotype in females. The only activating mutation of the FSH receptor described to date was found in a hypophysectomized man who was fertile despite undetectable serum gonadotropin levels; the effects of constitutive FSH receptor activity occurring with normal pituitary function are not known. Homozygous inactivations of the LH and FSH receptor invariably lead to amenorrhea in genotypically female subjects. In males, inactivation of the LH receptor in its more severe form results in a clinical picture similar to the syndrome of complete androgen resistance, but milder forms of hypoandrogenization have been described as well. The clinical consequences of homozygous inactivation of the FSH receptor in males are associated with subfertility. Finally, polymorphic variants of both the gonadotropin LH and the FSH receptor are present in the normal population.


1997 - Screening for deletions of the Y chromosome involving the DAZ (Deleted in Azoospermia) gene in azoospermia and severe oligozoospermia. [Articolo su rivista]
Simoni, Manuela; Gromoll, J; Dworniczak, B; Rolf, C; Abshagen, K; Kamischke, A; Carani, Cesare; Meschede, D; Behre, Hm; Horst, J; Nieschlag, E.
abstract

OBJECTIVE: To evaluate the occurrence and prevalence of microdeletions of the Y chromosome involving the DAZ (Deleted in AZoospermia) gene in patients with azoospermia or severe oligozoospermia. DESIGN: Controlled clinical study. SETTING: University infertility clinic. PATIENT(S): Infertile men (n = 168) with nonobstructive, idiopathic azoospermia or severe oligozoospermia and normal LH. The control group consisted of proven fathers (n = 86). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Semen analysis; polymerase chain reaction amplification of the loci sY84, sY143, sY254, and sY255; serum FSH, LH, and T; testicular volume. RESULT(S): Deletions involving the sY254 and sY255 DAZ loci were found in three azoospermic patients and two men with sperm concentration < 1 x 10(6)/mL. Serum FSH was elevated in four patients and was normal in one. All five patients had decreased testicular volumes compared with controls. No deletions involving the sY84 and sY143 loci were found. The four loci were amplified normally in the control group. CONCLUSION(S): The estimated frequency of deletions involving the DAZ locus is 3% in azoospermic-severely oligozoospermic men consulting an infertility clinic. Polymerase chain reaction amplification of the DAZ locus is useful for the diagnosis of microdeletions of the Y chromosome. Deletions involving more proximal regions of the Y chromosome seem to be rare.


1997 - Strong association between leptin and testosterone levels in men. Clin Endocrinol [Articolo su rivista]
Behre, Hm; Simoni, Manuela; Nieschlag, E.
abstract

OBJECTIVE: Leptin serves as a hormonal signal linking food intake and energy expenditure to fat mass. As significant effects of testosterone administration on body composition and adipose tissue have been described recently, we examined a possible association between serum levels of leptin and testosterone which has not been reported so far. SUBJECTS: Three groups comprising a total of 58 adult age-matched males were included in this cross-sectional analysis by computer-assisted clinical database selection. Group 1 (n = 22) consisted of untreated hypogonadal patients with testosterone serum levels lower than 7 nmol/l. The inclusion criterion for Groups II (n = 20) and III (n = 16) was a serum testosterone level higher than 30 nmol/l. Group II involved hypogonadal patients under effective androgen substitution therapy; Group III comprised males without any endocrine disorder attending our clinic. MEASUREMENTS: Morning blood samples were taken for determination of serum levels of leptin, testosterone, oestradiol and sex-hormone binding globulin (SHBG). Serum levels of leptin were measured by homologous radioimmunoassay, the other hormones by standard immunoassays. RESULTS: No significant differences in serum leptin, serum testosterone, and body mass index (BMI) were detected between Group II (3.7 +/- 1.5 micrograms/l, 40.3 +/- 7.6 nmol/ l, 24.4 +/- 4.9 kg/m2, respectively (mean +/- SD)), and Group III (4.0 +/- 2.0 micrograms/l, 35.9 +/- 6.5 nmol/l, 24.8 +/- 2.6 kg/m2, respectively). However, hypogonadal patients of Group I who were selected for low testosterone serum levels (4.3 +/- 1.7 nmol/l) had significantly higher leptin serum levels of 18.8 +/- 9.7 micrograms/l (P < 0.001) and significantly higher BMI of 30.0 +/- 6.6 kg/m2 (P < 0.001). Multiple linear regression analysis revealed a significant independent association of leptin with testosterone serum levels (partial correlation coefficient = -0.84; P < 0.001) and with BMI (partial correlation coefficient = 0.44; P = 0.001), whereas serum levels of oestradiol and SHBG had no additional influence. CONCLUSIONS: This study demonstrates a close association between serum levels of testosterone and leptin in males which has not been described previously. Serum testosterone levels could be an important contributor to the known gender difference in serum leptin levels which can be found even after correction for body composition. These findings might be of clinical relevance for testosterone substitution therapy of hypogonadal as well as ageing men.


1997 - Strong association between serum levels of leptin and testosterone in men [Articolo su rivista]
Behre, H. M.; Simoni, M.; Nieschlag, E.
abstract

OBJECTIVE: Leptin serves as a hormonal signal linking food intake and energy expenditure to fat mass. As significant effects of testosterone administration on body composition and adipose tissue have been described recently, we examined a possible association between serum levels of leptin and testosterone which has not been reported so far. SUBJECTS: Three groups comprising a total of 58 adult age-matched males were included in this cross- sectional analysis by computer-assisted clinical database selection. Group 1 (n=22) consisted of untreated hypogonadal patients with testosterone serum levels lower than 7 nmol/l. The inclusion criterion for Groups II (n=20) and III (n=16) was a serum testosterone level higher than 30 nmol/l. Group II involved hypogonadal patients under effective androgen substitution therapy; Group Ill comprised males without any endocrine disorder attending our clinic. MEASUREMENTS: Morning blood samples were taken for determination of serum levels of leptin, testosterone, oestradiol and sex-hormone binding globulin (SHBG). Serum levels of leptin were measured by homologous radioimmunoassay, the other hormones by standard immunoassays. RESULTS: No significant differences in serum leptin, serum testosterone, and body mass index (BMI) were detected between Group II (3.7 ± 1.5 μg/l, 40.3 ± 7.6 nmol/l, 24.4 ± 4.9 kg/m2, respectively (mean ± SD)), and Group III (4.0 ± 2.0 μg/l, 35.9 ± 6.5 nmol/l, 24.8 ± 2.6 kg/m2, respectively). However, hypogonadal patients of Group I who were selected for low testosterone serum levels (4.3 ± 1.7 nmol/l) had significantly higher leptin serum levels of 18.8 ± 9.7 μg/l (P &lt; 0.001) and significantly higher BMI of 30.0 ± 6.6 kg/m2 (P &lt; 0.001). Multiple linear regression analysis revealed a significant independent association of leptin with testosterone serum levels (partial correlation coefficient =-0.84; P &lt; 0.001) and with BMI (partial correlation coefficient = 0.44; P = 0.001), whereas serum levels of oestradiol and SHBG had no additional influence. CONCLUSIONS: This study demonstrates a close association between serum levels of testosterone and leptin in males which has not been described previously. Serum testosterone levels could be an important contributor to the known gender difference in serum leptin levels which can be found even after correction for body composition. These findings might be of clinical relevance for testosterone substitution therapy of hypogonadal as well as ageing men.


1997 - The follicle-stimulating hormone receptor: biochemistry, molecular biology and pathophysiology. [Articolo su rivista]
Simoni, Manuela; Gromoll, J; Nieschlag, E.
abstract

not available


1997 - cynDAZ: a cynomolgus monkey homologue of the human deleted-in-azoospermia (DAZ) gene. [Articolo su rivista]
Carani, Cesare; Gromoll, J; Brinkworth, Mh; Simoni, Manuela; Weinbauer, Gf; Nieschlag, E.
abstract

A gene on the human Y chromosome, specifically deleted in azoospermic patients (DAZ: deleted in azoospermia), and a DAZ homologue (DAZH) on human chromosome 3, have been recently described. In the present work we report the isolation and characterization of the corresponding DAZH gene of the cynomolgus monkey (Macaca fascicularis), which we have named cynDAZLA (cynomolgus DAZ-like autosomal). Reverse transcription-polymerase chain reaction was used to amplify the monkey DAZ homologue, and sequence analysis revealed an open reading frame of 888 bp encoding 295 amino acids. Northern blot hybridization of different tissues to a probe derived from the cynDAZLA cDNA detected a transcript of 3.5 kb that, in the male, was expressed only in the testis. Comparison of the cynDAZLA sequence to autosomal DAZ homologues from human, mouse and Drosophila showed two RNA-recognition motifs (RRM) and the presence of only one DAZ consensus repeat compared with the seven repeats found in the human DAZ gene on the Y chromosome. The homology of the cynDAZLA cDNA compared with the human DAZH and mouse dazla cDNAs is 97.97 and 87.46% respectively. The identification of the monkey cynDAZLA enables further studies regarding the putative functions of DAZH, such as onset of expression and hormonal dependence of this gene.


1996 - An activating mutation of the follicle-stimulating hormone receptor autonomously sustains spermatogenesis in a hypophysectomized man. [Articolo su rivista]
Gromoll, J; Simoni, Manuela; Nieschlag, E.
abstract

As both gonadotropins, LH and FSH, are required for normal spermatogenesis, patients with pituitary insufficiency need hCG plus human menopausal gonadotropin therapy to induce spermatogenesis and establish fertility. In a patient hypophysectomized because of a pituitary tumor, who, despite undetectable serum gonadotropin levels, had normal testis volume and semen parameters and fathered three children under testosterone substitution alone, we hypothesized an activating mutation of the FSH receptor. Exon 10 of the FSH receptor gene was amplified from genomic DNA by PCR, screened by single stranded conformation polymorphism gel electrophoresis, and sequenced. We identified a heterozygous A-->G base change at nucleotide position 1700, leading to an Asp-->Gly transition in codon 567 in the third intracytoplasmatic loop. COS-7 cells transiently transfected with the mutated receptor displayed a 1.5-fold increase in basal cAMP production compared to wild-type receptor, indicating that this mutation leads to ligand-independent constitutive activation of the FSH receptor. We conclude that this activating mutation of the FSH receptor, the first ever described, autonomously sustains spermatogenesis in the absence of gonadotropins.


1996 - Der Stellenwert rekombinanter Gonadotropine in der Behandlung des unerfüllten Kinderwunsches. [Relazione in Atti di Convegno]
Breckwoldt, M; DE GEYTER, Ch; Schneider, Hpg; Simoni, Manuela; Strowitzki, T.
abstract

not available


1996 - Effects of gonadotropin-releasing hormone on bioactivity of follicle-stimulating hormone (FSH), and microstructure of FSH, luteinizing hormone and sex hormone-binding globulin in a testosterone-based contraceptive trial: evaluation of responders and nonresponders. [Articolo su rivista]
Simoni, Manuela; Peters, J; Behre, Hm; Kliesch, S; Leifke, E; Nieschlag, E.
abstract

Only a proportion of normal men participating in testosterone-based contraceptive trials develop azoospermia (responders). This study analyzed whether serum follicle-stimulating hormone (FSH), luteinizing hormone (LH) and sex hormone-binding globulin (SHBG) are qualitatively different between responders and non-responders. Determination of in vitro bioactive FSH after stimulation with gonadotropin-releasing hormone (GnRH) and analysis of molecular heterogeneity of serum FSH. LH and SHBG was carried out by chromatofocusing and concanavalin-A affinity chromatography in eight men who had participated in a previous contraceptive study with testosterone buciclate. Blood was withdrawn at 15-min intervals on two basal occasions and 30, 45 and 60 min after iv administration of GnRH (100 µg). Pools of sera were separated by chromatofocusing in the pH range 3–6 and by lectin chromatography on concanavalin A. Immunoreactive FSH, LH and SHBG were assayed in the eluates. Bioactive FSH was analyzed by the rat Sertoli cell bioassay. Serum bioactive FSH increased after GnRH stimulation, without significant differences between responders and non-responders. The chromatofocusing profiles of serum FSH showed a significant shift towards the less acidic region after GnRH. The isoform distribution was similar in responders and non-responders. No significant differences were found in the relative proportion of FSH, LH and SHBG retained by concanavalin A. It is concluded that the extent of suppression of sperm production by androgen administration cannot be foreseen either on the basis of the response of bioactive FSH to GnRH administration or from the glycosylation pattern of serum FSH, LH and SHBG.


1996 - Functional and clinical consequences of mutations in the FSH receptor. Mol Cell Endocrinol [Relazione in Atti di Convegno]
Gromoll, J; Simoni, Manuela; Nordhoff, V; Behre, Hm; DE GEYTER, C; Nieschlag, E.
abstract

The follicle-stimulating hormone (FSH) is essential for normal gametogenesis. In females FSH is required for ovarian development and follicle maturation whereas in males FSH determines Sertoli cell number and quantitatively and qualitatively normal spermatogenesis. FSH action is mediated by a G-protein coupled receptor expressed solely in granulosa and Sertoli cells. The FSH-receptor (FSHR) gene is localized on chromosome 2 p21 and spans a region of 54 kb. It consists of ten exons; exon one to nine encode the large extracellular domain and the transmembrane domain is comprised of exon ten. Mutations in the FSHR gene could severely affect gametogenesis and result in infertility. Therefore screening programs have been initiated, in which patients with disturbed fertility were searched for mutations in the FSHR gene. Several Finnish families were identified displaying an inherited pattern of ovarian dysgenesis, a disease leading to streaky underdeveloped ovaries and primary amenorrhea. By genetic linkage the locus of the genetic defect was confined to chromosome 2 p21. Analysis of the FSHR gene resulted in the identification of a mutation (Ala189Val) homozygous in all affected females. Functional studies revealed that the mutation affects the proper protein folding and thereby inactivates the receptor. In a male patient hypophysectomized because of a pituitary tumor, who despite undetectable serum gonadotropins had normal semen parameters, we hypothesized an activating mutation of the FSHR. Screening of exon ten of the FSHR gene resulted in the identification of a Asp567Gly transition in the third intracytoplasmatic loop. Functional studies resulted in a 1.5-fold increase in basal cAMP production compared to wild type FSHR, indicating that the heterozygous mutation leads to a ligand-independent constitutive activation of the FSHR. This patient provides an exceptional model of nature defining the role of FSH in human spermatogenesis. Mutations of the FSHR might have differential effects in each gender. For example activating mutations have not been described in women, therefore it is not clear whether the constitutive activity of the receptor could disturb normal follicular development resulting in certain infertility.


1996 - Monitoring the transfection effciency of the human follicle-stimulating hormone receptor cDNA in COS-7 cells: evaluation of the growth hormone transient gene expression assay system. [Articolo su rivista]
Simoni, Manuela; Gromoll, J.
abstract

The human growth hormone (GH) transient gene expression assay system is frequently used to monitor transfection efficiency in transient transfection experiments. In this paper, we analyzed the suitability of the GH reporter gene to monitor transfection efficiency in COS-7 cells of an expression vector carrying the cDNA for the normal and mutated human follicle-stimulating hormone receptor (FSHR). The FSHR cDNA was cloned in the pSG5 expression vector and mutagenized (Ala307-->Thr) by oligonucleotide-mediated, site-directed mutagenesis. The expression plasmid pXGH5, carrying the structural gene for human GH, was used to monitor transfection efficiency. Different concentrations of pXGH5 and pSG5 containing normal or mutated FSHR cDNA were transfected in COS-7 cells by lipofection. The results showed: 1) The expression of pXGH5 was constant within individual experiments, but only in culture wells cotransfected with the same type of FSHR construct. On the contrary, the GH values normalized by the cell densities changed consistently depending on the type of FSHR construct. 2) The expression of the GH plasmid was influenced by type and concentration of the cotransfected plasmid. 3) The expression of pXGH5 cotransfected with the same FSHR construct was quite variable between experiments, without any relationship to the type of FSHR construct. These data show that the GH secretion is not a good parameter to monitor the transfection efficiency of the FSHR in pSG5 in COS-7 cells. Nor are other parameters such as semiquantitative mRNA determination or ligand binding to the transfected receptor ideal when mutations resulting in changes in receptor function are expected.


1996 - Physiologie der Hodenfunktion. [Capitolo/Saggio]
Weinbauer, G. F; Gromoll, J; Simoni, Manuela; Nieschlag, E.
abstract


1996 - Une mutation activatrice du récepteur de la FSH induit la spermatogenèse chez un homme hypophysectomisé [Articolo su rivista]
Gromoll, J.; Simoni, M.; Nieschlag, E.
abstract


1995 - FSH in therapy: Physiological basis, new preparations and clinical use [Articolo su rivista]
Simoni, M.; Nieschlag, E.
abstract


1995 - Potential of testosterone buciclate for male contraception: endocrine differences between responders and non-responders. [Articolo su rivista]
Behre, Hm; Baus, St; Kliesch, S; Keck, Ch; Simoni, Manuela; Nieschlag, E.
abstract

Suppression of serum LH and FSH, by testosterone (T) alone or in combination with other agents, has proved to be the most promising approach to male contraception. T enanthate, the only androgen preparation tested in male contraceptive efficacy trials so far, must be injected every week due to its short terminal elimination half-life of 4.5 days and leads to supraphysiological T serum levels. A new T ester synthesized under WHO and NIH auspices, testosterone buciclate (TB), showed a favorable pharmacokinetic profile, with a terminal half- life of 29.5 days when tested in hypogonadal men. Here we describe the results of the first clinical trial with TB for male contraception. After two control examinations, normal healthy male volunteers were given a single im injection of 600 mg TB (group I; n = 4) and 1200 mg TB (group II; n = 8) on day 0. Follow-up examinations were performed every 2 weeks up to week 32. In both groups mean serum T levels remained in the normal physiological range throughout the study course. Serum levels of dihydrotestosterone (DHT) showed a dose- and time- dependent increase, with serum levels slightly above the normal range in group II for several weeks and a maximal concentration of 3.8 +/- 0.5 nmol/L (mean +/- SE) in week 6. No suppression of spermatogenesis to oligozoospermia was observed in group I. However, in group II, spermatogenesis was suppressed to azoospermia in three of eight volunteers in week 10 that persisted up to weeks 14, 20, and 22, respectively. In these three men, LH and FSH were suppressed by TB injections to the respective assay detection limits, whereas in the other five subjects, mean serum levels were only decreased to values near the lower normal limit for LH and FSH, respectively. In addition, throughout the study course, a significant difference in serum sex hormone-binding globulin was detected between the responders (mean values, 21.2-26.4 nmol/L) and nonresponders (mean values, 36.2-46.3 nmol/L). Serum levels of LH as well as total and free T at baseline and after TB injection were lower in the responders than in the nonresponders. Both subgroups showed similar increases in serum LH and FSH after GnRH stimulation. In a newly introduced GnRH antagonist suppression test, serum LH and T were decreased to significantly lower levels in the responders. These results indicate a different hormonal equilibrium and probably different susceptibility to feedback regulation of the responders compared to the nonresponders


1994 - In vitro Sertoli cell bioassay of follicle-stimulating hormone (FSH): serum from different animal species alters the morphology of rat Sertoli cells without affecting their response to FSH. [Articolo su rivista]
Simoni, Manuela; Schuhmann, B; Weinbauer, Gf
abstract

Follicle-stimulating hormone (FSH) stimulates aromatase activity of immature rat Sertoli cells cultured in vitro in the presence of an androgen substrate, a phenomenon that can be used to measure FSH bioactivity. This paper analyzes the effects of sera from various animal species on both aromatase activity and morphological aspects of cultured rat Sertoli cells. Treatment of Sertoli cells with increasing concentrations of serum from humans, monkeys, rats, hamsters, and mice gave a dose-dependent stimulation of estradiol production parallel to the response obtained with FSH standard. Serum from these species and from rabbits also had a characteristic morphological effect on the cells, to produce a fibroblast-like aspect and clumping. This effect was dose-dependent, was increased by the addition of FSH, and was eliminated by heating the sera at 56° for 30 min prior to incubation with the cells. Clumping did not interfere with the aromatase activity of Sertoli cells and did not cause the cellular response to deviate from parallelism with the standard curve. Heat treatment of FSH standard and serum samples did not significantly change the aromatase activity. FSH bioactivity could be measured accurately in intact and castrated monkeys. It is concluded that the Sertoli cell aromatase bioassay can be applied directly to the measurement of serum bioactive FSH in several animal species without any preliminary treatment of the serum samples.


1994 - In vitro bioassay for human FSH based on L cells transfected with recombinant rat FSH receptor: validation of a model system. [Articolo su rivista]
Gudermann, T; Brockmann, H; Simoni, Manuela; Gromoll, J; Nieschlag, E.
abstract

FSH plays a central role in normal reproductive function, i.e. control of follicular maturation in the female and initiation and maintenance of spermatogenesis in the male. The effects of FSH are mediated by its interaction with a specific receptor that belongs to the superfamily of guanine nucleotide-binding protein-coupled receptors. Due to the microheterogeneity of gonadotropins, measurement of immunoreactivity does not necessarily reflect their bioactivity. Mutations in gonadotropin beta-subunits, which affect bioreactivity and/or immunoreactivity of gonadotropins, have been described as causes of infertility, thus highlighting the need for rapid and convenient methods to measure bioactivity. To establish a model system for recombinant in vitro bioassays for FSH that would obviate the use of live animals, we developed a strategy for efficient expression of the rat FSH receptor (FSHR) in L cells. A cell line, FSHR 7/12, was developed that bound [125I]FSH with high affinity (Kd 1.42 nM) and responded to human FSH with an increase in cAMP accumulation. Untreated human serum was found to have an unspecific inhibitory effect on cAMP formation. This effect could be thoroughly avoided by mild heating (10 min at 56 C) of serum samples before addition to cells without detectable loss of FSH immunoactivity or bioactivity. Studies on the hormone-sensitive adenylyl cyclase system of transformed FSHR 7/12 cells and of the parental Ltk- cells showed that the cellular response to FSH was highly specific. Using a parallel line assay design, FSHR 7/12 cells were used to validate a novel recombinant in vitro bioassay relying on intracellular cAMP accumulation as a readout system. Up to 10% of serum could be added to the incubation buffer without leading to nonparallelism to the standard curve. When 70 serum samples of male patients attending an infertility clinic were analyzed, the novel assay system displayed high sensitivity and a close correlation (r > 0.8; P < 0.01) to the established rat Sertoli cell aromatase bioassay and to a highly specific fluoroimmunoassay. When sera of 25 normal menstruating women were analyzed for FSH bioactivity at different stages of the menstrual cycle, a midcycle FSH peak followed by a decline in the late luteal phase could be discerned. The analysis of 26 serum samples of postmenopausal women revealed a close correlation between FSH values obtained by the novel in vitro bioassay and by a fluoroimmunoassay (r = 0.90; P < 0.01). Thus, the present in vitro bioassay represents a sensitive, rapid, and convenient model system to measure bioactive FSH in human serum.


1994 - Pharmacokinetics and pharmacodynamics of recombinant and urinary human FSH in the male monkey (Macaca fascicularis). J Endocrinol [Articolo su rivista]
Weinbauer, Gf; Simoni, Manuela; Hutchison, J; Nieschlag, E.
abstract

A dose-finding study was performed in adult male monkeys (Macaca fascicularis) to evaluate the pharmacokinetics and pharmacodynamics of a recombinant human FSH preparation (rhFSH). Groups of five monkeys were randomly assigned to receive single i.m. injections of 0·9% (w/v) NaCl (diluent), 6, 12 or 24 IU rhFSH/kg or 24 IU urinary human FSH/kg (uhFSH). The doses were based on an in vivo ovarian weight gain assay. Blood samples were collected 24 h before and immediately prior to injections, and 4, 8, 12, 24, 72 and 96 h after injections for determination of serum levels of immunoactive FSH by fluoroimmunoassay, bioactive FSH by an in vitro Sertoli cell assay, and inhibin and testosterone by radioimmunoassay. Inhibin was chosen as a marker for in vivo hFSH activity, since the secretion of inhibin in male monkeys is under the control of FSH. Administration of hFSH resulted in dose-related increases in serum hFSH concentrations. rhFSH and uhFSH exhibited similar pharmacokinetics. Comparable findings were obtained when serum samples were analysed for in vitro FSH bioactivity. Maximum serum hFSH levels were obtained 4–6 h after administration and the elimination half-life of hFSH was on average 18–22 h. The serum pharmacokinetics of rhFSH were linear within the dose range explored. Baseline inhibin concentrations varied significantly between groups. However, when the changes in inhibin concentrations were normalized to the baseline values (per cent change, area under curve and maximum inhibin level), a dose-dependent stimulatory effect of rhFSH on serum inhibin was evident. This effect attained statistical significance with doses of 24 IU rhFSH/kg and 24 IU uhFSH/kg, and the serum inhibin responses to rhFSH and uhFSH were not significantly different. No significant differences were observed with regard to the serum concentrations of testosterone between the diluentand hFSH-treated groups. It was concluded that rhFSH is bioactive in terms of stimulating testicular inhibin production in the male monkey and that the pharmacokinetic properties of rhFSH and uhFSH are similar.


1994 - Polymorphism of human pituitary FSH: analysis of immunoreactivity and in-vitro bioactivity of different molecular species. [Articolo su rivista]
Simoni, Manuela; Jockenövel, F; Nieschlag, E.
abstract

Follicle-stimulating hormone is known to be highly heterogeneous in serum and in the pituitary. In the present study, we have partially separated different molecular species of human pituitary FSH and characterized their immunoreactivity and in vitro bioactivity. Pooled extracts of male (n=15) and female (n=9) human pituitary glands were chromatographed on a column of Sephacryl S-200 and FSH-containing fractions were fractionated by chromatofocusing in the pH range 4–6. FSH was measured in the individual fractions by an in vitro bioassay, based on the FSH-dependent aromatase activity of immature rat Sertoli cells, and by the following methods based on commercial kits: radioimmunoassay (RIA), immunofluorimetric assay (IFMA), immunoradiometric assay (IRMA), immunoenzymometric assay (IEMA). In each assay, the kit standard, calibrated against the 2nd International Reference Preparation (IRP) 78/549, and the International Standard (IS) 83/575 were run in parallel. The relative potencies of the kit standards in terms of IS 83/575 were: IFMA 3·08, IRMA 1·62, RIA 2·42, IEMA 1·45 and bioassay 1·14. After chromatofocusing, pituitary FSH eluted mostly in fractions with pH{approx}4·5, without sex-related differences. In both sexes {approx}25% of bioactive material showed a pI<4 and eluted with 1 M NaCl. Although the same IS 83/575 was used in the various assays, the profiles of immunoreactive FSH were significantly different. The highest intermethod variability was observed in the case of male pituitary FSH. The relative biopotency of the different molecular species of FSH did not appear to change according to their pI but, rather, varied with the assay method and the standard. In terms of 2nd IRP 78/549 the activity profiles were similar but not identical to those obtained in terms of IS 83/575 and the ratios between the two values (IS:IRP ratios) were significantly different among the methods. No significant correlation was found between IS:IRP ratios and FSH concentrations or pH. These data suggest that: (1) all the methods have different affinities for standard and unknown and/or for different molecular isoforms of FSH; (2) overall, they perform more accurately when assaying female pituitary FSH, perhaps because of a closer resemblance between standard and unknown; (3) the variability of the IS:IRP ratios indicates a different affinity of the antibodies for IS 83/575 and the kit standard, highlighting the importance of the molecular composition of the reference preparations for the final result; and (4) the results do not support the commonly accepted concept of a higher in vitro biopotency of less acidic species of pituitary FSH.


1994 - Pulsatile subcutaneous versus bolus intramuscolar gonadotropin administration after pituitary suppression with a long-acting GnRH analogue: a controlled prospective study. [Articolo su rivista]
DE GEYTER, C; DE GEYTER, M; Simoni, Manuela; Castro, E; BALS PRATSCH, M; Nieschlag, E; Schneider, Hpg
abstract

The potential advantages of pulsatile s.c. administration instead of daily bolus i.m.administration of human urinary gonadotrophin preparations were tested after the administration of a long-acting gonadotrophin-releasing hormone (GnRH) analogue within a programme for in-vitro fertilization (IVF) and embryo transfer. First, the pharmacokinetic properties of human urinary gonadotrophins were analysed with immunological and biological methods, both during bolus i.m. injections and during pulsatile s.c. administration. Second, a prospective randomized controlled study was performed in 75 patients undergoing IVF/embryo transfer in whom the effects of pulsatile s.c. administration were compared with the effects of single daily bolus i.m. injections of the same gonadotrophin preparation. The results showed that neither method of gonadotrophin administration induced measurable changes in the serum concentration of luteinizing hormone (LH). Both oestradiol and andro-stenedione concentrations were slightly lower during pulsatile s.c. gonadotrophin administration, suggesting that this method of gonadotrophin administration results in less LH occupying the ovarian LH receptors. Pulsatile s.c. gonadotrophin administration resembles a continuous infusion of follicle-stimulating hormone (FSH). Significant fluctuations in the serum concentrations of FSH were observed during single daily bolus i.m. administration of human urinary gonadotrophins, but the pregnancy rate of IVF/embryo transfer per cycle after pulsatile s.c. administration was not significantly better than after the daily bolus i.m. injection of gonadotrophins (42.1 versus 37.2%). It is concluded that pulsatile s.c. administration of gonadotrophins instead of single daily injections does not improve the pregnancy rate in IVF/embryo transfer


1994 - Transgenic animals in male reproductive research. [Articolo su rivista]
Simoni, Manuela
abstract

Although transgenic mouse technology has already been widely used for the study of gene function and regulation in many areas of biomedicine, it has been applied only sporadically to the investigation of testicular function. Nevertheless, the contribution of this experimental approach to the understanding of male reproduction is considerable, not least because of the frequency of infertility in transgenic mice. Transgenic mice can be produced by microinjection of DNA constructs in the male pronucleus of fertilized eggs that are then retransferred into the oviducts of pseudopregnant females and allowed to develop to term. A proportion of the offspring have the foreign DNA sequences permamently integrated into the genome and thus become transgenic. In this way it is possible to obtain either the over-expression of genes, which can be targeted to the testis using testis-specific promoters, or to effect interruption of the functional integrity of genes by insertional mutagenesis. The regulation of gene expression in vivo can be studied by producing transgenic mice where the transgene is composed of the regulatory sequences of a gene of interest driving the expression of a reporter gene. Specific genes can be "knocked out" by homologous recombination. This article reviews the contribution of the transgenic approach to the following areas of male reproduction: the identification of factors involved in sex determination and development of the reproductive tract; the study of the function and expression of genes important for spermatogenesis and male reproduction; the identification of genes involved in spermatogenesis and of genomic sequences directing the expression of a transgene in the testis; the study of the function of specific reproductive tissues or cells in vivo; oncogenesis in reproductive tissues; the creation of cell lines suitable for in vitro studies; gene therapy.


1993 - A search for circulating immunoglobulins blocking follicle-stimulating hormone action in male idiopathic infertility. [Articolo su rivista]
Simoni, Manuela; Paschke, R; Nieschlag, E.
abstract

In this study, patients with idiopathic infertility were investigated for the presence of circulating antibodies which interfered with follicle-stimulating hormone (FSH) activity. A retrospective search for autoantibodies was undertaken using single plasma samples obtained from 29 infertile men with azoo/oligo/asthenoteratozoospermia and eight controls with normozoospermia and normal blood FSH levels. Plasma levels of immunoactive FSH, bioactive FSH, immunoactive luteinizing hormone and testosterone were measured in the individual samples. Plasma immunoglobulin G (IgG) was isolated using protein-A chromatography and tested at different dose levels for its ability to inhibit and/or stimulate basal and FSH-induced aromatase activity in immature rat Sertoli cells in vitro. In the first set of trials, IgGs isolated from three infertile patients showed apparent inhibition of FSH-stimulated aromatase activity, two showed further stimulation and five showed irregular fluctuations beyond the normal range of stimulation. When reanalysed at different doses, none of the IgG fractions exhibited abnormal interference with FSH action. It is concluded that, unlike many other endocrine disorders characterized by autoimmunity, the occurrence of autoantibodies of the IgG class blocking FSH action in male infertility is improbable.


1993 - Biological and immunological properties of the International Standard for FSH 83/575: isoelectrofocusing profile and comparison with other FSH praparations. [Articolo su rivista]
Simoni, Manuela; Jockenhövel, F; Nieschlag, E.
abstract

The new international standard for FSH, IS 83/575, has been analyzed, after isoelectric focusing separation, by Sertoli cell in vitro bioassay, radioligand receptor assay and two highly specific immunometric assays. Its molecular composition was then compared with the isoelectric focusing profiles obtained from the fractionation of the reference preparation 2nd IRP 78/549 and from pools of human male and female pituitary extracts and male and female sera. The results showed that > 80% of immunoreactive and bioactive FSH in the IS 83/575 has a pI value < 4, while such very acidic material was represented much less in the other FSH preparations tested. All the immunoreactive material contained in the IS 83/575 was shown to be capable of receptor binding and bioactivity in vitro. A generally good correspondence between IEF profiles obtained by bioassay and by immunofluorimetric assay was evident in the case of IS 83/575, 2nd IRP 78/549 and pituitary extracts, although the profiles recorded by immunofluorimetric assay were rather smooth and more isoforms were detected by bioassay. A striking discrepancy between immunoreactive FSH and bioactive FSH was observed after isoelectric focusing fractionation of the serum pools, in which some bioactive material was not detected by immunofluorimetric assay and some of the immunoreactive FSH peaks were devoid of bioactivity, indicating that serum contains inhibitors of FSH action and that immunometric assays based on monoclonal antibodies may miss some bioactive FSH isoforms.


1993 - Changes in seasonality of birth rates in Germany from 1951 to 1990. [Articolo su rivista]
Lerchl, A; Simoni, Manuela; Nieschlag, E.
abstract

PIP: Annual birth rates have been found to be affected up to 10-20% by seasonal variation. Variations affecting birth patterns are attributed to biological and social factors: annual rhythms of climatic conditions, or the photoperiod and varying sexual activity. A recent study placed greater priority on biological factors as responsible for seasonal variation: annual variations in sperm quality, serum levels of luteinizing hormone and testosterone in men, and ovulation variations in women by time of day. Seasonal variations in birth rates were found in Munster, Germany, between 1890-99, 1965-74, and 1981-90. Further examination was made to ascertain whether the variations pertained to the entire country of Germany. Data were obtained on monthly birth rates from 1951 to 1990 from the states of the former West Germany. Annual means were calculated and the monthly deviations from the mean computed (centered moving average with a window size of 13 months), as well as monthly means within 5-year intervals. Yearly cosine functions with 2 harmonics were also fitted to the data. The results showed maximum births in the first half of the year between 1951-75 and later months in the following years until stabilization after 1981. The amplitudes of the rhythms declined from 1972 until stabilizing and increasing after 1978. The annual patterns were smooth and stable with peaks in February and March, and a "shoulder" peak in September, which conforms to long suspected seasonal conceptions in December. The annual rhythms appear to support biologic seasonal variation until 1970. The shift after 1970 to conceptions in the winter months for Germany has also been observed with US data since 1950, and in southern hemisphere countries. The explanation may be environmental changes. The workplace environment has replaced the photoperiod influences and temperature fluctuations, but the shift by 6 months would not support this deseasonalization hypothesis. It is suggested that social reasons have affected the shift, such as the enactment of the liberal abortion law in 1976 and the pregnant woman employment protection act of 1968.


1993 - Effects of antiandrogens and ethane dimethane sulphonate (EDS) on gene expression, free subunits, bioactivity and secretion of pituitary gonadotrophins in male rats. [Articolo su rivista]
Gromoll, J; Weinbauer, Gf; Simoni, Manuela; Nieschlag, E.
abstract

In the male rat, testosterone has been shown to regulate gonadotrophin synthesis and secretion under experimental conditions such as castration or gonadotrophin-releasing hormone (GnRH) antagonist with or without testosterone. The present study aims at clarifying the effects of non-steroidal antiandrogens, Casodex and flutamide, and ethane dimethane sulphonate (EDS) on the regulation of gonadotrophin synthesis and secretion. To enable a direct comparison within this study to expected effects of testosterone, a GnRH antagonist-treated group and a castrated group were included. The gene expression of the subunits was correlated with changes in the pituitary and plasma content of immunoreactive luteinizing hormone (LH) and follicle-stimulating hormone (FSH), free subunits and pituitary content of in vitro bioactive LH and FSH. Groups of ten male rats each received the following treatments for 7 days: (1) vehicle; (2) castration; (3) EDS (75 mg/kg); (4) GnRH antagonist (Cetrorelix 250 μg/kg/day), (5) Casodex (20 mg/kg/day) or (6) flutamide (20 mg/kg/day).The effectiveness of testosterone deprivation was demonstrated by the reduction of weight in androgen-dependent organs such as epididymides and seminal vesicles in the treated groups. Treatment with flutamide, EDS or castration significantly increased (p < 0.05) serum levels of LH, FSH and α-subunit, whereas serum gonadotrophin levels were decreased in the GnRH antagonist-treated group. α-Subunit mRNA levels were elevated in the castrated, EDS and flutamide group and LH-β mRNA levels were increased in the castrated and EDS group. FSH-β mRNA levels were increased in the castrated group and decreased in the GnRH antagonist group, but remained unchanged in the flutamide and EDS group. In the flutamide and EDS group an overall decrease of pituitary content of free FSH-β subunit, immunoreactive FSH and bioactive FSH was observed after 7 days of treatment. This effect was not seen for LH. Casodex neither changed mRNA levels nor serum levels of the gonadotrophins, although the pituitary content of free LH-β subunit and bioactive LH and, in addition, testosterone serum levels were elevated.In summary: (1) Selective testosterone blockade/deprivation induced by flutamide and EDS uncoupled the gene expression of FSH-β subunit and secretion of FSH in vivo; (2) antiandrogens and EDS did not affect FSH-β mRNA levels; (3) in the EDS- and flutamide-treated group pituitary free FSH-β, immunoreactive and bioactive FSH were depleted; (4) Casodex seems to have a specific effect on pituitary LH bioactivity and free LH-β subunit, without altering FSH.These findings might indicate that testosterone exerts divergent effects at the pituitary level, namely increasing FSH-β mRNA availability and decreasing pituitary responsiveness to GnRH, thereby lowering FSH secretion. Furthermore, a steady-state level of FSH-β mRNA is maintained by GnRH and testicular factors other than testosterone. Treatment with antiandrogens might provide a useful tool to study the regulation of synthesis and secretion of FSH in vivo.


1993 - Molecular composition of two different batches of urofollitropin: analysis by immunofluorimetric assay, radioligand receptor assay and in vitro bioassay. [Articolo su rivista]
Simoni, Manuela; Weinbauer, Gf; Nieschlag, E.
abstract

The molecular heterogeneity of two different batches of commercially available urofolitropin was analyzed after fractionation by isoelectric focusing (IEF). FSH was measured before and after IEF by a highly specific time-resolved immunofluorimetric assay (IFMA), by a radioligand receptor assay (RRA) employing a preparation of calf testis FSH receptors, and by the in vitro bioassay based on FSH-dependent aromatase stimulation in immature rat Sertoli cells. An overall good correspondence between the results obtained with the three different methods was observed. However, the RRA and the in vitro bioassay appeared to be more suitable than the IFMA in resolving individual FSH isoforms. The mean isoelectric points of the two FSH preparations analyzed were slightly different, due to different molecular composition. These differences, however, seem too minute to be considered as cause of the different pharmacokinetics of FSH described in the literature or to explain the inconsistent therapeutical results seen in patients treated with FSH of urinary origin.


1993 - Molecular heterogeneity of serum follicle-stimulating hormone in hypogonadal patients before and during androgen replacement therapy and in normal men. [Articolo su rivista]
Harsch, Ia; Simoni, Manuela; Nieschlag, E.
abstract

OBJECTIVE: The present study was performed to characterize the molecular heterogeneity of serum FSH in normal males and to investigate the possible influence of testosterone on serum FSH in androgen-deficient men before and during testosterone administration. DESIGN AND PATIENTS: Serum samples were taken at 10-minute intervals between 0730 and 0830 h from nine healthy, eugonadal men and from eight men with primary hypogonadism (Klinefelter's syndrome). In the hypogonadal patients, sampling was performed before treatment (n = 8), 4-5 days after the first and the third injection of 250 mg testosterone enanthate given intramuscularly at three-weekly intervals (n = 6), as well as 3 months after the onset of therapy (n = 3). Sampling was repeated 7 days apart in two of the nine healthy volunteers. MEASUREMENTS: Aliquots from the individual serum samples were pooled and fractionated by chromatofocusing in the pH range 6-3. Immunoreactive FSH was measured by immunofluorometric assay (IFMA) in each fraction and the individual serum samples. In each serum pool, bioactive FSH was determined by in-vitro bioassay (rat Sertoli cell aromatase bioassay), testosterone by RIA and LH by IFMA. RESULTS: After grouping the percentage of immunoreactive FSH recovered in the individual fractions into intervals of 0.5 pH units, significant differences between controls and patients were observed in the pH regions 4-4.5, 5.5-6 and 6-6.5. No statistically significant changes in the isoform distribution of FSH were detected during therapy in the Klinefelter patients. A high degree of variability, which did not follow a common pattern, was observed in the isoform distribution of FSH within the same individuals, both in the hypogonadal patients during treatment and in the two normal men whose blood samples were taken on two different occasions. CONCLUSIONS: Serum FSH is highly heterogeneous in normal and hypogonadal men. There is a spontaneous intra-individual variability in the relative abundance of the different FSH isoforms in serum that may most probably be related to metabolic deglycosylation of FSH. Minor but significant differences in the molecular heterogeneity of serum FSH could be demonstrated in Klinefelter patients compared to normal men. These differences are not modified by administration of testosterone enanthate at doses achieving normal androgenization, suggesting that factors different from testosterone may modulate FSH pleomorphism.


1992 - Circadian rhythm of plasma testosterone in men with idiopathic hypogonatropic hypogonadism before and during pulsatile administration of gonadotropin-releasing hormone. [Articolo su rivista]
Simoni, Manuela; Montanini, V; FAUSTINI FUSTINI, M; DEL RIO, G; Cioni, K; Marrama, P.
abstract

OBJECTIVE: The aim was to investigate whether a pulsatile discharge of LH from the pituitary is necessary to achieve the circadian secretion of testosterone. DESIGN: The daily rhythm of the androgen has been studied in patients with idiopathic hypogonadotrophic hypogonadism (IHH) both in the absence of therapy and during pulsatile administration of gonadotrophin releasing hormone (GnRH). PATIENTS: Six patients with IHH and ten normal subjects were analysed. Blood sampling was performed at 2-hourly intervals, for 24 hours. The IHH patients then received synthetic GnRH i.v. at the rate of one pulse every 2 hours (10 micrograms/pulse). On day 11 of treatment, blood samples were taken for the rhythm analysis every 2 hours, for 24 hours. MEASUREMENTS: Plasma testosterone and LH were measured in the individual samples by radioimmunoassay. Evaluation of the rhythm was performed by cosinor analysis. RESULTS: A significant circadian rhythm of plasma testosterone was statistically validated in the normal subjects, whereas no rhythm was detected in the IHH patients in the absence of therapy. On day 11 of GnRH pulsatile administration the IHH patients showed normal testosterone levels and a statistically significant circadian rhythm of the androgen was evident, with acrophase between 0700 and 0800 h. Moreover, the amplitude, acrophase and mesor of testosterone rhythm in IHH patients in the course of treatment were statistically indistinguishable from the corresponding values in the normal subjects. Plasma LH did not show statistically significant circadian variations, either in the control group or in the IHH patients before or during therapy. CONCLUSIONS: We conclude that a physiological circadian rhythm of plasma testosterone can be obtained, in IHH men, by treatment with GnRH. Since the pulsatile administration of exogenous GnRH at constant doses induced a circadian rhythm in testosterone and no daily variations in LH were evident, we suggest that, although a pulsatile secretion of LH is probably necessary for the synchronization of the circadian rhythm with acrophase in the morning, the testosterone variations might be the results of a local testicular modulation of LH action.


1992 - Microheterogeneity of pituitary FSH in male rats: differential effects of chronic androgen deprivation induced by castration or androgen blockade. [Articolo su rivista]
Simoni, Manuela; Weinbauer, Gf; Chandolia, Rk; Nieschlag, E.
abstract

Testicular androgens are known to influence not only the secretion but also the bioactivity and molecular composition of pituitary FSH. In the present study, we investigated the effects of chronic androgen blockade and castration on the molecular heterogeneity of the gonadotrophin. Groups of male adult rats (five animals per group) received one of the following treatments: vehicle, the non-steroidal anti-androgens casodex (20 mg/kg per day) or flutamide (20 mg/kg per day), or castration. After 8 weeks, the animals were killed and individual pituitary homogenates fractionated by isoelectric focusing (IEF) on sucrose density gradients in the pH range 2·5–8. FSH was measured by radioimmunoassay (RIA) in the individual fractions and by in-vitro bioassay (Sertoli cell aromatase bioassay) in pools of fractions which were combined according to pH intervals of 0·5 units. Bioactive and immunoreactive FSH were also measured in sera and unfractionated pituitary extracts. Testosterone and inhibin were assayed in sera by RIA.A significant increase in serum immunoreactive and bioactive FSH was demonstrated in flutamide-treated and castrated animals, whereas the pituitary content of bioactive FSH remained unchanged in the four groups. Serum testosterone and inhibin were undetectable in castrated animals and significantly increased in those treated with flutamide. By RIA, the IEF profiles of the flutamide-treated and castrated rats showed a significant reduction of the FSH isoforms with 3·5<pI<4, with a significant increase in the isoforms with pI>4 only in the castrated group. By bioassay, there was a significant decrease in the isoforms with 3·5<pI<4 in both casodex- and flutamide-treated animals, with no significant differences between the two groups. Castration caused a further significant shift in the relative distribution of FSH isoforms towards the less acidic components, with a significant increase in the isoforms with 5<pI<5·5 not attained by androgen blockade alone.These results suggest that the effects of long-lasting castration on pituitary FSH heterogeneity cannot be entirely reproduced by the androgen blockade. Since inhibin, eliminated by castration but not by androgen blockade, is a major regulator of FSH in the male rat, we speculate that it might not only influence FSH secretion but also modulate its qualitative properties.


1992 - Report of the 7th european workshop on molecular and cellular endocrinology of the testis [Articolo su rivista]
Nieschlag, E.; Behre, H. M.; Weinbauer, G. F.; Gudermann, Th.; Simoni, M.; Spiteri-Grech, J.; Cooper, T. G.
abstract


1992 - Report on 8the 7th European Workshop on Molecular and Cellular Endocrinology of the testis. [Articolo su rivista]
Nieschlag, E; Behre, Hm; Weinbauer, G; Guderman, Th; Simoni, Manuela; SPITERI GRECH, J; Cooper, Tg
abstract

not applicable


1991 - Comparative effects of chronic administration of the non-steroidal antiandrogens flutamide and casodex on the reproductive system of the adult male rat. [Articolo su rivista]
Chandolia, Rk; Weinbauer, Gf; Simoni, Manuela; Behre, Hm; Nieschlag, E.
abstract

The effects of chronic blockade of androgen action by the antiandrogens flutamide and Casodex on serum and pituitary concentrations of LH and FSH, serum and testicular androgen levels, reproductive organ weights, and on spermatogenesis were compared in the adult rat. Animals were treated for 3 and 8 weeks with vehicle, Casodex (20 mg.kg-1.(day)-1, flutamide (20 mg.kg-1.(day)-1) and GnRH antagonist (150 micrograms/day, Detirelix). Treatment with GnRH antagonist suppressed gonadotropin and testosterone production, reduced the weights of testes, epididymides and seminal vesicles, and inhibited germ cell development. Flutamide administration markedly elevated serum and pituitary levels of gonadotropins as well as serum and testicular androgen concentrations. Casodex-induced elevation of gonadotropin concentrations was less pronounced and serum and testicular levels of androgens did not change significantly. The reduction of seminal vesicle weights was similar after Casodex and GnRH antagonist treatment, whereas flutamide was less effective. Testicular weight and spermatogenesis (assessed by light microscopical and flow-cytometric analysis) remained unaffected by Casodex and flutamide. It is concluded, that 1. Casodex, in contrast to flutamide, is a peripherally selective antiandrogen, and 2. Casodex influences release of gonadotropins into circulation less than flutamide. Therefore this antiandrogen might be useful clinically for selectively blocking androgen actions in the accessory sex glands.


1991 - In vitro bioassays for follicle-stimulating hormone: methods and clinical applications. [Articolo su rivista]
Simoni, Manuela; Nieschlag, E.
abstract

not applicable


1991 - Serum bioactive follicle-stimulating hormone-like activity in human pregnancy is a methodological artefact. [Articolo su rivista]
Simoni, Manuela; Khan, Sa; Nieschlag, E.
abstract

Serum from pregnant women has been shown to contain both FSH-like and FSH antagonistic activities when measured by an in vitro bioassay based upon FSH-dependent aromatase activity of immature rat Sertoli cells. In the present study we further tested the hypothesis that the FSH-like bioactivity of pregnancy serum was due to an authentic aromatase stimulator. A potent inhibitor of aromatase which completely blocked the FSH action on Sertoli cells had no effect on the bioactivity of pregnancy serum. Experiments using conversion of tritiated testosterone to estradiol showed that the factor did not stimulate aromatase activity in rat Sertoli cells. After incubation with pregnancy serum, equally high amounts of estradiol were measurable in the medium in both the absence and presence of Sertoli cells. The activity was almost completely lost after charcoal treatment or ether extraction of the serum and was shown to probably be due to the release of endogenous estrogens by the carrier proteins in the incubation medium of the Sertoli cell assay. These data suggest that the FSH-like bioactivity in serum from pregnant women is an artifact due to nonspecific interference(s) in the bioassay.


1991 - Terapia della pubertà precoce vera idiopatica. Descrizione di un caso clinico. [Articolo su rivista]
Cioni, K; FAUSTINI FUSTINI, Marco; Simoni, Manuela; Frasoldati, A.
abstract

non disponibile


1990 - Circannual rhythm of plasma thyrotropin in middle-aged and old euthyroid subjects [Articolo su rivista]
Simoni, M; Velardo, A; Montanini, V; Faustini Fustini, M; Seghedoni, S; Marrama, P
abstract

The circannual rhythm of plasma thyrotropin (TSH) was evaluated in 8,310 euthyroid, serially independent, young, middle-aged and old men and women. A statistically significant circannual rhythm of plasma TSH was validated, by the mean group-cosinor method, in the middle-aged and old men and women (p less than 0.05), with acrophase in December, whereas the young subjects did not show any rhythm. No significant correlation was found between TSH plasma levels and free thyroxine (fT4) or ambient temperature in any group. Moreover, plasma fT4 did not show seasonal variations.


1989 - Stimulatory and inhibitory influences of serum from pregnant women on aromatase activity of immature rat Sertoli cells. [Articolo su rivista]
Simoni, Manuela; Khan, Sa; DE GEYTER, Ch; Nieschlag, E.
abstract

Effects of serum from pregnant women on basal and FSH (or cAMP) stimulated aromatase activity of immature rat Sertoli cells in primary culture were studied. Pregnancy serum caused a dose-dependent stimulation of Sertoli cell aromatase activity and the response curves were parallel to those obtained with human FSH. This stimulatory (FSH-like) activity increased progressively during pregnancy, with a sharp drop immediately after delivery. However, the FSH-like bioactivity was not associated with immunoreactive FSH when a specific radioimmunoassay was employed. On the other hand, serum from pregnant women caused a dose-dependent inhibition of FSH and dibutyryl-cAMP-stimulated aromatase activity. These data suggest that human pregnancy serum contains factor(s) which may stimulate basal aromatase activity of Sertoli cells and may inhibit FSH-induced aromatase activity. These factors, most probably of placental origin, may play a role in the regulation of estrogen production during gestation.


1988 - Risposta dell'LH plasmatico immunoreattivo e biologicamente attivo all'LHRH test in pazienti con sindrome di Turner prima e dopo trattamento estrogenico. [Articolo su rivista]
FAUSTINI FUSTINI, M; Pagliani, U; Pantaleoni, M; Galetti, S; Marrama, D; Simoni, Manuela; Cioni, K.
abstract

not applicable


1988 - Ritmo circadiano del testosterone, sex hormone binding globulin e proteine plasmatiche in giovani adulti ed anziani di sesso maschile. [Articolo su rivista]
Simoni, Manuela; FAUSTINI FUSTINI, M; Mariani, M; Pantaleoni, M; Galetti, S; Cioni, K.
abstract

not applicable


1988 - Twenty-four-hour pattern of plasma SHBG, total proteins and testosterone in young and elderly men. [Articolo su rivista]
Simoni, Manuela; Baraldi, E; Baraghini, Gf; Boraldi, V; Roli, L; Seghedoni, S; Velardo, A; Montanini, V.
abstract

not applicable


1987 - Age-related changes in plasma dehydroepiandrosterone sulphate, cortisol, testosterone and free testosterone circadian rhythms in adult men. [Articolo su rivista]
Montanini, Vanna; Simoni, Manuela; Chiossi, G; Baraghini, Gf; Velardo, Antonino; Baraldi, E; Marrama, P.
abstract

The circadian rhythms of serum luteinizing hormone, follicle-stimulating hormone, testosterone (T), free testosterone (fT), sex hormone-binding globulin (SHBG), oestradiol, cortisol and dehydroepiandrosterone sulphate (DHA-s) have been investigated in 5 normal male adults and 6 elderly men. Circadian rhythms were detected statistically significant (p less than 0.05) by population mean cosinor analysis, for T, fT, cortisol and DHA-s in the young group. In the elderly population, serum cortisol showed a clear circadian rhythm, although with some phase modification, whereas DHA-s secretion lost its circadian rhythmicity. This demonstrates that ageing differently affects the two major adrenal functions, glucocorticoid and androgenic; further, the data suggest that an independent adrenal androgen-regulating system could be selectively impaired in the older subjects. In the elderly group the loss of T circadian rhythm was confirmed, but a statistically significant circadian rhythm of fT was recorded. It was characterized by a marked phase advance and not related with the SHBG modifications found in elderly men. This finding leads us to reconsider the role of fT, which appears more sensitive than total T, in studying circadian rhythm of gonadal androgen secretion.


1987 - Biologically active luteinizing hormone (LH) in Klinefelter's syndrome: response to gonadotropin releasing hormone (GnRH) and effects of testosterone undecanoate. [Articolo su rivista]
Montanini, V; Celani, Mf; Carani, C; Cioni, K; Simoni, Manuela; Baraghini, Gf; Marrama, P.
abstract

not applicable


1987 - Subnormal prolactin responsiveness to thyrotropin releasing hormone (TRH) in women with primary empty sella syndrome. [Articolo su rivista]
Celani, Mf; Giambuzzi, G; Simoni, Manuela; Montanini, V.
abstract

Basal prolactin (PRL) levels and PRL responsiveness to thyrotropin-releasing hormone (TRH) were studied in 10 women with primary empty sella (PES) syndrome (mean age 38.2 yr). Hyperprolactinemia (34 to 72 ng/ml) was found in 5 patients (hyperprolactinemic PES, H-PES), whereas 5 patients showed normal (9.5 to 19 ng/ml) PRL levels (normoprolactinemic PES, N-PES). The results were compared with those obtained in 10 healthy women (mean age 32.8 yr, PRL = 7 to 15 ng/ml) and in 8 women with a PRL-secreting pituitary microadenoma (MA) (mean age 37.5 yr, PRL = 39 to 85 ng/ml). The mean basal levels of PRL were significantly higher in patients with H-PES (50.8 +/- 13.2 ng/ml) or MA (64.0 +/- 18.3 ng/ml) than in the control group (10.9 +/- 2.6 ng/ml, p less than 0.02) and in the patients with N-PES (13.9 +/- 3.7 ng/ml, p less than 0.02). In contrast, the relative maximum response (RMR) of PRL to TRH (peak PRL/basal PRL) was significantly lower in the patients with PES (both H-PES and N-PES) or MA (1.4 +/- 0.4, 2.3 +/- 0.7 and 1.2 +/- 0.2, respectively) than in the control subjects (3.6 +/- 1.1; p less than 0.02, less than 0.05 and less than 0.02, respectively). Our results show that the pituitary responsiveness to the acute stimulation with TRH is significantly decreased both in patients with a PRL-secreting pituitary MA and in those with PES. Therefore, the clinical value of the TRH test in distinguishing the PES syndromes from prolactinomas seems to be questionable.


1986 - Daily rhythmicity of testosterone production by "in vitro" LH stimulated mouse Leydig cells. [Articolo su rivista]
Montanini, V; Simoni, Manuela; Syed, B; Chiossi, G; Bertoldi, G; Giardino, L; Baraghini, Gf
abstract

not applicable


1985 - Overestimation of plasma androstenedione in hypercholesterolemic samples. [Articolo su rivista]
Zini, D; Baraghini, Gf; Piccinini, D; Simoni, Manuela; Bolelli, Gf; Zirilli, E; Marrama, P.
abstract

not applicable


1985 - Secrezione pulsatile della gonadotropina LH nel maschio anziano. [Articolo su rivista]
Simoni, Manuela; Chiossi, G; Celani, Mf; Cavani, D; Laganà, Al; Diazzi, G; Sarti, G; Montanini, Vanna
abstract

not available


1985 - Secrezione surrenalica circadiana di soggetti talassemici in diversa situazione trasfusionale. [Articolo su rivista]
Rota, C; Montanini, V; Massolo, F; Celani, Mf; Messori, A; Simoni, Manuela
abstract

not applicable


1984 - Determinazione del testosterone libero plasmatico mediante ultrafiltrazione e successivo dosaggio radioimmunologico. [Articolo su rivista]
Baraghini, Gf; Simoni, Manuela; Zini, D; Seghedoni, S; Marrama, P.
abstract

not applicable


1984 - Further studies on the effects of heroin addiction on the hypothalamic-pituitary-gonadal function in man. [Articolo su rivista]
M. F., Celani MF; Carani, Cesare; V., Montanini; G. F., Baraghini; D., Zini; Simoni, Manuela; C., Ferretti; P., Marrama
abstract

The effects of chronic heroin addiction on LH biological and immunological activity, as well as on total and free testosterone concentrations, were investigated in 8 active young male addicts. The results were compared with those obtained in 33 normal young men. In addition, the effects of naloxone (N) administration on LH bio- and immuno-potency were studied in 3 normal men. LH biological activity (bLH) was assessed by a specific and sensitive "in vitro" bioassay, based upon testosterone production by mechanically dispersed mouse Leydig cell preparations. Double antibody radioimmunoassay methods were employed to assess serum levels of immunoreactive LH (iLH), total testosterone (T) and morphine (M). Free testosterone (FT) concentrations were determined by RIA after an ultrafiltration procedure. Mean basal values of bLH, iLH, T and LH bio/immuno (b/i) ratio observed in heroin addicts were similar to those obtained in the control group. In contrast, serum levels of FT and the mean FT/T ratio were significantly reduced in heroin addicts. A significant decrease of LH b/i ratio was observed during N infusion in the normal subjects.


1984 - L'uso dell'ultrafiltrazione nella determinazione degli steroidi liberi plasmatici. [Articolo su rivista]
Simoni, Manuela; Seghedoni, S; Zini, D; Baraghini, Gf
abstract

not applicable