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2023 - Gene expression differences between small and large follicles [Abstract in Rivista]
Baschieri, Lara; Cimadomo, Danilo; Davolio, Federica; Sperduti, Samantha; Mascolo, Elisa; Paradiso, Elia; Lazzaretti, Clara; Perri, Carmela; Roy, Neena; Innocenti, Federica; Albricci, Laura; Rienzi, Laura; Vaiarelli, Alberto; Maria, Ubaldi Filippo; Simoni, Manuela; Casarini, Livio

2023 - In vitro evaluation of the impact of two differently glycosylated recombinant FSH on signal transduction [Abstract in Rivista]
Mascolo, Elisa; Baschieri, Lara; Roy, Neena; Lazzaretti, Clara; Paradiso, Elia; Perri, Carmela; Sperduti, Samantha; Simoni, Manuela; Casarini, Livio

2023 - Intracellular cGMP increase is not involved in thyroid cancer cell death [Articolo su rivista]
Alessandro, Sara D'; Paradiso, Elia; Lazzaretti, Clara; Sperduti, Samantha; Perri, Carmela; Antoniani, Francesco; Righi, Sara; Simoni, Manuela; Brigante, Giulia; Casarini, Livio

Introduction: Type 5 phosphodiesterase (PDE5) inhibitors (PDE5i) lead to intracellular cyclic-guanosine monophosphate (cGMP) increase and are used for clinical treatment of erectile dysfunction. Studies found that cGMP may up/downregulate the growth of certain endocrine tumor cells, suggesting that PDE5i could impact cancer risk. Aim: We evaluated if PDE5i may modulate thyroid cancer cell growth in vitro. Materials and methods: We used malignant (K1) and benign (Nthy-ori 3-1) thyroid cell lines, as well as the COS7 cells as a reference model. Cells were treated 0-24 h with the PDE5i vardenafil or the cGMP analog 8-br-cGMP (nM-μM range). cGMP levels and caspase 3 cleavage were evaluated by BRET, in cGMP or caspase 3 biosensor-expressing cells. Phosphorylation of the proliferation-associated extracellularly-regulated kinases 1 and 2 (ERK1/2) was evaluated by Western blotting, while nuclear fragmentation by DAPI staining. Cell viability was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results: Both vardenafil and 8-br-cGMP effectively induced dose-dependent cGMP BRET signals (p≤0.05) in all the cell lines. However, no differences in caspase 3 activation occurred comparing PDE5i-treated vs untreated cells, at all concentrations and time-points tested (p>0.05). These results match those obtained upon cell treatment with 8-br-cGMP, which failed in inducing caspase 3 cleavage in all the cell lines (p>0.05). Moreover, they reflect the lack of nuclear fragmentation. Interestingly, the modulation of intracellular cGMP levels with vardenafil or the analog did not impact cell viability of both malignant and benign thyroid tumor cell lines, nor the phosphorylation of ERK1/2 (p>0.05). Conclusions: This study demonstrates that increased cGMP levels are not linked to cell viability or death in K1 and Nthy-ori 3-1 cell lines, suggesting that PDE5i do not impact the growth of thyroid cancer cells. Since different results were previously published, further investigations are recommended to clarify the impact of PDE5i on thyroid cancer cells.

2023 - Lack of GPER-TSHR heteromers is a hallmark of thyroid cancer [Abstract in Rivista]
Perri, Carmela; D'Alessandro, Sara; Paradiso, Elia; Lazzaretti, Clara; Mascolo, Elisa; Baschieri, Lara; Roy, Neena; Sperduti, Samantha; Simoni, Manuela; Brigante, Giulia; Casarini, Livio

2023 - LH increases the response to FSH in granulosa-lutein cells from sub/poor-responder patients in vitro [Articolo su rivista]
Sperduti, Samantha; Paradiso, Elia; Anzivino, Claudia; Lazzaretti, Clara; Limoncella, Silvia; D'Alessandro, Sara; Roy, Neena; Reggianini, Francesca; Ferrari, Tommaso; Melli, Beatrice; La Sala, Giovanni Battista; Nicoli, Alessia; Daolio, Jessica; Villani, Maria Teresa; Tagliavini, Simonetta; Trenti, Tommaso; Potì, Francesco; Sandhowe, Reinhild; Centonze, Chiara; Lispi, Monica; Simoni, Manuela; Casarini, Livio

STUDY QUESTION Does LH addition to FSH in vitro recover the human primary granulosa lutein cell (hGLC) sub/poor-response? SUMMARY ANSWER A picomolar concentration of LH may recover the FSH-induced cAMP and progesterone production of hGLC from sub/poor-responder women. WHAT is KNOWN ALREADY Clinical studies suggested that FSH and LH co-treatment may be beneficial for the ovarian response of sub/poor-responders undergoing ovarian stimulation during ART. STUDY DESIGN, SIZE, DURATION hGLC samples from 286 anonymous women undergoing oocyte retrieval for ART were collected from October 2017 to February 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS hGLCs from women undergoing ovarian stimulation during ART were blindly purified, cultured, genotyped and treated in vitro by increasing concentrations of FSH (nM) +/- 0.5 nM LH. cAMP and progesterone levels produced after 3 and 24 h, respectively, were measured. In vitro data were stratified a posteriori, according to the donors' ovarian response, into normo-, sub- and poor-responder groups and statistically compared. The effects of LH addition to FSH were compared with those obtained by FSH alone in all the groups as well. MAIN RESULTS AND THE ROLE of CHANCE hGLCs from normo-responders were shown to have higher sensitivity to FSH treatment than sub-/poor-responders in vitro. Equimolar FSH concentrations induced higher cAMP (about 2.5- to 4.2-fold), and progesterone plateau levels (1.2- to 2.1-fold), in cells from normo-responder women than those from sub-/poor-responders (ANOVA; P < 0.05). The addition of LH to the cell treatment significantly increased overall FSH efficacy, indicated by cAMP and progesterone levels, within all groups (P > 0.05). Interestingly, these in vitro endpoints, collected from the normo-responder group treated with FSH alone, were similar to those obtained in the sub-/poor-responder group under FSH + LH treatment. No different allele frequencies and FSH receptor (FSHR) gene expression levels between groups were found, excluding genetics of gonadotropin and their receptors as a factor linked to the normo-, sub- and poor-response. In conclusion, FSH elicits phenotype-specific ovarian lutein cell response. Most importantly, LH addition may fill the gap between cAMP and steroid production patterns between normo- and sub/poor-responders. LIMITATIONS, REASONS FOR CAUTION Although the number of experimental replicates is overall high for an in vitro study, clinical trials are required to demonstrate if the endpoints evaluated herein reflect parameters of successful ART. hGLC retrieved after ovarian stimulation may not fully reproduce the response to hormones of granulosa cells from the antral follicular stage. WIDER IMPLICATIONS of THE FINDINGS This in vitro assay may describe the individual response to personalize ART stimulation protocol, according to the normo-, sub- and poor-responder status. Moreover, this in vitro study supports the need to conduct optimally designed, randomized clinical trials exploring the personalized use of LH in assisted reproduction. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by Merck KGaA. M.L. and C.C. are employees of Merck KGaA or of the affiliate Merck Serono SpA. Other authors have no competing interests to declare.

2023 - Protein kinase B (Akt) blockade inhibits LH/hCG-mediated 17,20-lyase, but not 17[alpha]-hydroxylase activity of CYP17a1 in mouse Leydig cell steroidogenesis [Abstract in Rivista]
Paradiso, Elia; Lazzaretti, Clara; Sperduti, Samantha; Melli, Beatrice; Perri, Carmela; D'Alessandro, Sara; Baschieri, Lara; Mascolo, Elisa; Roy, Neena; Simoni, Manuela; Casarini, Livio

2023 - Protein kinase B (Akt) blockade inhibits LH/hCG-mediated 17,20-lyase, but not 17α-hydroxylase activity of Cyp17a1 in mouse Leydig cell steroidogenesis [Articolo su rivista]
Paradiso, Elia; Lazzaretti, Clara; Sperduti, Samantha; Melli, Beatrice; Trenti, Tommaso; Tagliavini, Simonetta; Roli, Laura; D'Achille, Fabio; Beltrán-Frutos, Ester; Simoni, Manuela; Casarini, Livio

: Androgens are produced by adrenal and gonadal cells thanks to the action of specific enzymes. We investigated the role of protein kinase B (Akt) in the modulation of Δ4 steroidogenic enzymes' activity, in the mouse Leydig tumor cell line mLTC1. Cells were treated for 0-24 h with the 3 × 50% effective concentration of human luteinizing hormone (LH) and choriogonadotropin (hCG), in the presence and in the absence of the specific Akt inhibitor 3CAI. Cell signaling analysis was performed by bioluminescence resonance energy transfer (BRET) and Western blotting, while the expression of key target genes was investigated by real-time PCR. The synthesis of progesterone, 17α-hydroxy (OH)-progesterone and testosterone was measured by immunoassay. Control experiments for cell viability and caspase 3 activation were performed as well. We found that both hormones activated cAMP and downstream effectors, such as extracellularly-regulated kinase 1/2 (Erk1/2) and cAMP response element-binding protein (Creb), as well as Akt, and the transcription of Stard1, Hsd3b1, Cyp17a1 and Hsd17b3 genes, boosting the Δ4 steroidogenic pathway. Interestingly, Akt blockade decreased selectively Cyp17a1 expression levels, inhibiting its 17,20-lyase, but not the 17-hydroxylase activity. This effect is consistent with lower Cyp17a1 affinity to 17α-OH-progesterone than progesterone. As a result, cell treatment with 3CAI resulted in 17α-OH-progesterone accumulation at 16-24 h and decreased testosterone levels after 24 h. In conclusion, in the mouse Leydig cell line mLTC1, we found substantial Akt dependence of the 17,20-lyase activity and testosterone synthesis. Our results indicate that different intracellular pathways modulate selectively the dual activity of Cyp17a1.

2023 - Reprogramming of reproductive signals via human luteinizing hormone/choriogonadotropin receptor (LHCGR)/G protein-coupled estrogen receptor (GPER) heteromers [Abstract in Rivista]
Lazzaretti, Clara; Paradiso, Elia; Sperduti, Samantha; Sayers, Niamh; Pelagatti, Ginevra; D'Alessandro, Sara; Perri, Carmela; Baschieri, Lara; Mascolo, Elisa; Roy, Neena; Simoni, Manuela; Hanyaloglu, Aylin; Casarini, Livio

2023 - Short-Term Exposure to Bisphenol A Does Not Impact Gonadal Cell Steroidogenesis In Vitro [Articolo su rivista]
Roy, Neena; Lazzaretti, Clara; Paradiso, Elia; Capponi, Chiara; Ferrari, Tommaso; Reggianini, Francesca; Sperduti, Samantha; Baschieri, Lara; Mascolo, Elisa; Perri, Carmela; Varani, Manuela; Canu, Giulia; Trenti, Tommaso; Nicoli, Alessia; Morini, Daria; Iannotti, Francesca; Villani, Maria Teresa; Vicini, Elena; Simoni, Manuela; Casarini, Livio

: Bisphenol A (BPA) is a ubiquitous, synthetic chemical proven to induce reproductive disorders in both men and women. The available studies investigated the effects of BPA on male and female steroidogenesis following long-term exposure to the compound at relatively high environmental concentrations. However, the impact of short-term exposure to BPA on reproduction is poorly studied. We evaluated if 8 and 24 h exposure to 1 nM and 1 µM BPA perturbs luteinizing hormone/choriogonadotropin (LH/hCG)-mediated signalling in two steroidogenic cell models, i.e., the mouse tumour Leydig cell line mLTC1, and human primary granulosa lutein cells (hGLC). Cell signalling studies were performed using a homogeneous time-resolved fluorescence (HTRF) assay and Western blotting, while gene expression analysis was carried out using real-time PCR. Immunostainings and an immunoassay were used for intracellular protein expression and steroidogenesis analyses, respectively. The presence of BPA leads to no significant changes in gonadotropin-induced cAMP accumulation, alongside phosphorylation of downstream molecules, such as ERK1/2, CREB and p38 MAPK, in both the cell models. BPA did not impact STARD1, CYP11A1 and CYP19A1 gene expression in hGLC, nor Stard1 and Cyp17a1 expression in mLTC1 treated with LH/hCG. Additionally, the StAR protein expression was unchanged upon exposure to BPA. Progesterone and oestradiol levels in the culture medium, measured by hGLC, as well as the testosterone and progesterone levels in the culture medium, measured by mLTC1, did not change in the presence of BPA combined with LH/hCG. These data suggest that short-term exposure to environmental concentrations of BPA does not compromise the LH/hCG-induced steroidogenic potential of either human granulosa or mouse Leydig cells.

2023 - Spontaneous and iatrogenic ovarian hyperstimulation syndrome in the absence of FSHR mutations: a case report of two unexpected cases [Articolo su rivista]
Daolio, J.; Sperduti, S.; Casarini, L.; Falbo, A.; Materazzo, C.; Aguzzoli, L.; Villani, M. T.

Background: Ovarian hyperstimulation syndrome (OHSS) is a complication of controlled ovarian hyperstimulation (COH). It is a potentially life-threatening condition that usually occurs either after human chorionic gonadotropins (hCG) administration in susceptible patients or as a result of an implanting pregnancy, regardless of whether it was achieved by natural conception or infertility treatments. Despite many years of clinical experience regarding the adoption of preventive measures and the identification of patients at high risk, the pathophysiology of OHSS is poorly understood and no reliable predictive risk factors have been identified. Cases presentation: We report about two unexpected cases of OHSS following infertility treatments, occurring after freeze-all strategy with embryo cryopreservation approaches. The first case developed spontaneous OHSS (sOHSS), despite efforts to prevent its manifestation by a segmentation approach, including frozen embryo replacement cycle. The second case developed a late form of iatrogenic OHSS (iOHSS), even though the absence of any risk factors. No mutations in the follicle-stimulating hormone (FSH) receptor (FSHR)-encoding gene were detected, suggesting that the high levels of hCG due to the twin implanting pregnancies could be the only triggering factor of OHSS outbreak. Conclusion: Freeze-all strategy with embryo cryopreservation cannot entirely prevent the development of OHSS, which may occur in its spontaneous form independently from the FSHR genotype. Although OHSS remains a rare event, all infertile patients requiring ovulation induction or controlled ovarian stimulation (COS) may be at potential risk of OHSS, either in the presence or in the absence of risk factors. We suggest closely monitoring cases of pregnancy following infertility treatments in order to provide early diagnosis and adopt the conservative management.

2022 - Luteinizing hormone (LH)- and choriogonadotropin (hCG)-induced internalization of the receptor (LHCGR) is responsible for hormone-specific signaling [Abstract in Rivista]
Paradiso, Elia; Lazzaretti, Clara; D'Alessandro, Sara; Sperduti, Samantha; Roy, Neena; Mascolo, Elisa; Baschieri, Lara; Anzivino, Claudia; Simoni, Manuela; Casarini, Livio

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.

(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 - 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

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.

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.

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 - 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.

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.

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.

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 - 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.

Molecular Biology; Female Reproductive Endocrinology; Endocrine Regulation

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.

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.

2019 - Abacavir, nevirapine, and ritonavir modulate intracellular calcium levels without affecting GHRH-mediated growth hormone secretion in somatotropic cells in vitro [Articolo su rivista]
Brigante, G; Riccetti, L; Lazzaretti, C; Rofrano, L; Sperduti, S; Potì, F; Diazzi, C; Prodam, F; Guaraldi, G; Lania, Ag; Rochira, V; Casarini, L

Growth Hormone (GH) deficiency is frequent in HIV-infected patients treated with antiretroviral therapy. We treated GH3 cells with antiretrovirals (nevirapine, ritonavir or abacavir sulfate; 100 pM-1 mM range), after transfection with human growth hormone releasing hormone (GHRH) receptor cDNA. Cells viability, intracellular cAMP, phosphorylation of CREB and calcium increase, GH production and secretion were evaluated both in basal condition and after GHRH, using MTT, bioluminescence resonance energy transfer, western blotting and ELISA. Antiretroviral treatment did not affect GHRH 50% effective dose (EC50) calculated for 30-min intracellular cAMP increase (Mann-Whitney's U test; p ≥ 0.05; n = 4) nor 15-min CREB phosphorylation. The kinetics of GHRH-mediated, rapid intracellular calcium increase was perturbed by pre-incubation with drugs, while GHRH failed to induce the ion increase in ritonavir pre-treated cells (ANOVA; p < 0.05; n = 3). Antiretrovirals did not impact 24-h intracellular and extracellular GH levels (ANOVA; p ≥ 0.05; n = 3). We demonstrated the association between antiretrovirals and intracellular calcium increase, without consequences on somatotrope cells viability and GH synthesis. Overall, these results suggest that antiretrovirals may not directly impact on GH axis in HIV-infected patients.

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.

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 - 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

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.

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 - Inferring biallelism of two FSH receptor mutations associated with spontaneous ovarian hyperstimulation syndrome by evaluating FSH, LH and HCG cross-activity [Articolo su rivista]
Lazzaretti, C.; Riccetti, L.; Sperduti, S.; Anzivino, C.; Brigante, G.; De Pascali, F.; Poti, F.; Rovei, V.; Restagno, G.; Mari, C.; Lussiana, C.; Benedetto, C.; Revelli, A.; Casarini, L.

Research question: What is the cumulative effect of two follicle-stimulating hormone receptor (FSHR) mutations in spontaneous ovarian hyperstimulation syndrome (sOHSS) pathogenesis? Are these mutations in the mono- or biallelic state? Design: Two FSHR mutations were found in a pregnant patient affected by sOHSS with no predisposing conditions. While the p.Asn106His mutation is novel, the p.Ser128Tyr mutation has been associated with sOHSS previously. The patient's FSHR gene was analysed by Sanger sequencing, and FSHR cDNAs carrying a single or both point mutations were created by mutagenesis in vitro. cAMP activation by recombinant FSH, luteinizing hormone (LH), human chorionic gonadotropin (HCG) and thyroid-stimulating hormone (TSH) was evaluated in transfected HEK293 cells by bioluminescence resonance energy transfer. Results: All mutations decreased the 50% effective concentration of FSH calculated for cAMP (P < 0.05, n = 6), resulting in two- to 10-fold lower ligand potency. TSH failed to induce an FSHR-mediated increase in intracellular cAMP, while LH was approximately four-fold more potent than HCG in p.Ser128Tyr FSHR-expressing HEK293 cells despite lower cAMP plateau levels (P < 0.05, n = 5). The p.Ser128Tyr FSHR mutation was found to be responsible for an LH-/HCG-induced increase in cAMP when it was in the biallelic heterozygous state with p.Asn106His, but no increase in cAMP was induced in the monoallelic state. Conclusion: In-vitro data support that, in pregnant patients with sOHSS, the two FSHR mutations have an opposing effect on the pathogenesis of sOHSS and are in the biallelic heterozygous form, allowing HCG to induce a p.Ser128Tyr FSHR-mediated increase in cAMP.

2018 - Influence of APOE and RNF219 on Behavioral and Cognitive Features of Female Patients Affected by Mild Cognitive Impairment or Alzheimer's Disease [Articolo su rivista]
Mosca, Alessandra; Sperduti, Samantha; Pop, Viorela; Ciavardelli, Domenico; Granzotto, Alberto; Punzi, Miriam; Stuppia, Liborio; Gatta, Valentina; Assogna, Francesca; Banaj, Nerisa; Piras, Fabrizio; Piras, Federica; Caltagirone, Carlo; Spalletta, Gianfranco; Sensi, Stefano L

The risk for Alzheimer's disease (AD) is associated with the presence of the 4 allele of Apolipoprotein E (APOE) gene and, recently, with a novel genetic variant of the RNF219 gene. This study aimed at evaluating interactions between APOE-4 and RNF219/G variants in the modulation of behavioral and cognitive features of two cohorts of patients suffering from mild cognitive impairment (MCI) or AD. We enrolled a total of 173 female MCI or AD patients (83 MCI; 90 AD). Subjects were screened with a comprehensive set of neuropsychological evaluations and genotyped for the APOE and RNF219 polymorphic variants. Analysis of covariance was performed to assess the main and interaction effects of APOE and RNF219 genotypes on the cognitive and behavioral scores. The analysis revealed that the simultaneous presence of APOE-4 and RNF219/G variants results in significant effects on specific neuropsychiatric scores in MCI and AD patients. In MCI patients, RNF219 and APOE variants worked together to impact the levels of anxiety negatively. Similarly, in AD patients, the RNF219 variants were found to be associated with increased anxiety levels. Our data indicate a novel synergistic activity APOE and RNF219 in the modulation of behavioral traits of female MCI and AD patients.

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

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.