The relationship between primary ovarian insufficiency and gene variations: a prospective case-control study.

Around 70 percent of cases of Primary Ovarian Insufficiency (POI) etiology remain unexplained. The aim of our study is to contribute to the etiology and genetic background of POI. A total of 37 POI patients and 30 women in the reproductive period were included in this prospective, case-control study between August 2020 and December 2021. The women were examined for 36 genes with next-generation sequencing (NGS) panel. Gene variations were detected in 59.5 percent of the patients in the case group. FSHR p.S680N (rs6166, c.2039 G>A) and FSHR p.A307T (rs6165, c.919 G>A) gene variants, which are most frequently located in exon 10 of the FSHR gene, were detected in both groups. Although it was not found that these gene variants were significantly different between the groups, it was also found that they were significantly different in POI patients under 30 years of age and in those with a family history of POI. Variations were detected in 12 genes in POI patients. Two gene variants (FGFR1 [c.386A>C, rs765615419] and KISS1 [c.58 G>A, rs12998]) were detected in both groups, and the remaining gene variants were detected only in POI patients. No differences were detected between the groups in terms of gene variations. However, the gene variations detected only in POI patients may play a role in the etiology of POI.

Loss of Fshr Prevents Testicular Maturation in Atlantic Salmon (Salmo salar L.).

Early puberty poses a significant challenge for male Atlantic salmon in aquaculture due to its negative impact on growth and welfare. The regulation of puberty in vertebrates involves 2 key reproductive hormones: follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and their gonadal receptors. In male mice lacking FSH receptor, testes size is reduced, but fertility is maintained, while medaka and zebrafish with a disrupted fshr gene exhibit near normal testis size and fertility. In these fishes both Fsh and Lh are present during puberty and Lh may rescue fertility, while in salmonid fish only Fsh is present in the circulation during puberty. Using CRISPR-Cas9, we produced crispants with a high prevalence of fshr mutations at the target site, which remained fertile, although more than half showed a testis development deviating from wild-type (wt) males. Crossing out these F0 crispants to each other produced a viable F1 generation showing frameshift (fshr-/-) or in-frame mutations (fshrif/if). Nearly all wt males matured while all fshr-/- males remained immature with small testes containing A spermatogonia as the furthest developed germ cell type and prepubertal plasma androgen levels. Also, the pituitary transcript levels of gnrhr2bba and lhb, but not for fshb, were reduced in the fshr-/- males compared with maturing males. More than half of the fshrif/if mutant males showed no or a delayed maturation. In conclusion, Atlantic salmon show the unique characteristic that loss of Fshr function alone results in male infertility, offering new opportunities to control precocious puberty or fertility in salmon.

Tracking conformational transitions of the gonadotropin hormone receptors in a bilayer of (SDPC) poly-unsaturated lipids from all-atom molecular dynamics simulations.

Glycoprotein hormone receptors [thyrotropin (TSHR), luteinizing hormone/chorionic gonadotropin (LHCGR), and follicle stimulating hormone (FSHR) receptors] are rhodopsin-like G protein-coupled receptors. These receptors display common structural features including a prominent extracellular domain with leucine-rich repeats (LRR) stabilized by ß-sheets and a long and flexible loop known as the hinge region (HR), and a transmembrane (TM) domain with seven α-helices interconnected by intra- and extracellular loops. Binding of the ligand to the LRR resembles a hand coupling transversally to the α- and ß-subunits of the hormone, with the thumb being the HR. The structure of the FSH-FSHR complex suggests an activation mechanism in which Y335 at the HR binds into a pocket between the α- and ß-chains of the hormone, leading to an adjustment of the extracellular loops. In this study, we performed molecular dynamics (MD) simulations to identify the conformational changes of the FSHR and LHCGR. We set up a FSHR structure as predicted by AlphaFold (AF-P23945); for the LHCGR structure we took the cryo-electron microscopy structure for the active state (PDB:7FII) as initial coordinates. Specifically, the flexibility of the HR domain and the correlated motions of the LRR and TM domain were analyzed. From the conformational changes of the LRR, TM domain, and HR we explored the conformational landscape by means of MD trajectories in all-atom approximation, including a membrane of polyunsaturated phospholipids. The distances and procedures here defined may be useful to propose reaction coordinates to describe diverse processes, such as the active-to-inactive transition, and to identify intermediaries suited for allosteric regulation and biased binding to cellular transducers in a selective activation strategy.

A single-domain intrabody targeting the follicle-stimulating hormone receptor impacts FSH-induced G protein-dependent signalling.

Intracellular variable fragments of heavy-chain antibody from camelids (intra-VHH) have been successfully used as chaperones to solve the 3D structure of active G protein-coupled receptors bound to their transducers. However, their effect on signalling has been poorly explored, although they may provide a better understanding of the relationships between receptor conformation and activity. Here, we isolated and characterized iPRC1, the first intra-VHH recognizing a member of the large glycoprotein hormone receptor family, the follicle-stimulating hormone receptor (FSHR). This intra-VHH recognizes the FSHR third intracellular loop and decreases cAMP production in response to FSH, without altering Gαs recruitment. Hence, iPRC1 behaves as an allosteric modulator and provides a new tool to complete structure/activity studies performed thus far on this receptor.

Follicle-stimulating hormone orchestrates glucose-stimulated insulin secretion of pancreatic islets.

Follicle-stimulating hormone (FSH) is involved in mammalian reproduction via binding to FSH receptor (FSHR). However, several studies have found that FSH and FSHR play important roles in extragonadal tissue. Here, we identified the expression of FSHR in human and mouse pancreatic islet ß-cells. Blocking FSH signaling by Fshr knock-out led to impaired glucose tolerance owing to decreased insulin secretion, while high FSH levels caused insufficient insulin secretion as well. In vitro, we found that FSH orchestrated glucose-stimulated insulin secretion (GSIS) in a bell curve manner. Mechanistically, FSH primarily activates Gαs via FSHR, promoting the cAMP/protein kinase A (PKA) and calcium pathways to stimulate GSIS, whereas high FSH levels could activate Gαi to inhibit the cAMP/PKA pathway and the amplified effect on GSIS. Our results reveal the role of FSH in regulating pancreatic islet insulin secretion and provide avenues for future clinical investigation and therapeutic strategies for postmenopausal diabetes.

Combined Multiplexed Phage Display, High-Throughput Sequencing, and Functional Assays as a Platform for Identifying Modulatory VHHs Targeting the FSHR.

Developing modulatory antibodies against G protein-coupled receptors is challenging. In this study, we targeted the follicle-stimulating hormone receptor (FSHR), a significant regulator of reproduction, with variable domains of heavy chain-only antibodies (VHHs). We built two immune VHH libraries and submitted them to multiplexed phage display approaches. We used next-generation sequencing to identify 34 clusters of specifically enriched sequences that were functionally assessed in a primary screen based on a cAMP response element (CRE)-dependent reporter gene assay. In this assay, 23 VHHs displayed negative or positive modulation of FSH-induced responses, suggesting a high success rate of the multiplexed strategy. We then focused on the largest cluster identified (i.e., PRC1) that displayed positive modulation of FSH action. We demonstrated that PRC1 specifically binds to the human FSHR and human FSHR/FSH complex while potentiating FSH-induced cAMP production and Gs recruitment. We conclude that the improved selection strategy reported here is effective for rapidly identifying functionally active VHHs and could be adapted to target other challenging membrane receptors. This study also led to the identification of PRC1, the first potential positive modulator VHH reported for the human FSHR.

Membrane estrogen receptor and follicle-stimulating hormone receptor.

Follicle-stimulating hormone (FSH) and estrogens are fundamental to support reproductive functions. Beside the well-known FSH membrane receptor (FSHR), a G protein-coupled estrogen receptor (GPER) has been found, over the last two decades, in several tissues. It may trigger rapid, non-genomic responses of estradiol, activating proliferative and survival stimuli. The two receptors were co-characterized in the ovary, where they modulate different intracellular signaling cascades, according to the expression level and developmental stage of ovarian follicles. Moreover, they may physically interact to form heteromeric assemblies, suggestive of a new mode of action to regulate FSH-specific signals, and likely determining the follicular fate between atresia and dominance. The knowledge of FSH and estrogen membrane receptors provides a new, deeper level of comprehension of human reproduction.

Prenatal cadmium exposure has inter-generational adverse effects on Sertoli cells through the follicle-stimulating hormone receptor pathway.

In brief: Exposure to cadmium (Cd) during pregnancy can potentially harm the reproductive system of male offspring. This article shows that pregnant woman should be protected from cadmium exposure. Abstract: Exposure to cadmium (Cd) during pregnancy can potentially harm the reproductive system of male offspring, although the full extent of its heritable effects remains partially unresolved. In this study, we examined the inter-generational impacts of Cd using a distinct male-lineage generational model. Pregnant Sprague-Dawley female rats (F0) were administered control or cadmium chloride (0.5, 1 and 2 mg/day) via intra-gastric administration from gestation day 1 to 20. Subsequently, the first filial generation (F1) male rats were mated with untreated females (not exposed to Cd) to produce the second filial generation (F2). Histopathological analysis of the F1 and F2 generations revealed abnormal testicular development, while ultrastructural examination indicated damage to Sertoli cells. Cd exposure also led to alterations in serum hormone levels (gonadotropin-releasing hormone, follicle-stimulating hormone) and reduced follicle-stimulating hormone receptor (FSHR) protein expression in Sertoli cells in the F1 generation. Furthermore, Cd affected the mRNA and protein expression of FSHR pathway factors and DNA methyltransferase, albeit with distinct patterns and inconsistencies observed between the F1 and F2 generations. Overall, our findings indicate that prenatal Cd exposure, using a male-lineage transmission model, can induce inter-generational effects on male reproduction, particularly by causing toxicity in Sertoli cells. This effect appears to be primarily mediated through disruptions in the FSHR pathway and changes in DNA methyltransferase activity in the male testes.

Oral follicle-stimulating hormone receptor agonist affects granulosa cells differently than recombinant human FSH.

OBJECTIVE: To determine whether TOP5300, a novel oral follicle-stimulating hormone (FSH) receptor (FSHR) allosteric agonist, elicits a different cellular response than recombinant human FSH (rh-FSH) in human granulosa cells from patients undergoing in vitro fertilization. DESIGN: Basic science research with a preclinical allosteric FSHR agonist. SETTING: University hospital. PATIENT(S): Patients with infertility at a single academic fertility clinic were recruited under an Institutional Review Board-approved protocol. Primary granulosa cell cultures were established for 41 patients, of whom 8 had normal ovarian reserve (NOR), 17 were of advanced reproductive age (ARA), 12 had a diagnosis of polycystic ovary syndrome (PCOS), and 4 had a combination of diagnoses, such as ARA and PCOS. INTERVENTION(S): Primary granulosa-lutein (GL) cell cultures were treated with rh-FSH, TOP5300, or vehicle. MAIN OUTCOME MEASURE(S): Estradiol (E2) production using enzyme-linked immunosorbent assay, steroid pathway gene expression of StAR and aromatase using quantitative polymerase chain reaction, and FSHR membrane localization using immunofluorescence were measured in human GL cells. RESULT(S): TOP5300 consistently stimulated E2 production among patients with NOR, ARA, and PCOS. Recombinant FSH was the more potent ligand in GL cells from patients with NOR but was ineffective in cells from patients with ARA or PCOS. The lowest level of FSHR plasma membrane localization was seen in patients with ARA, although FSHR localization was more abundant in cells from patients with PCOS; the highest levels were present in cells from patients with NOR. The localization of FSHR was not affected by TOP5300 relative to rh-FSH in any patient group. TOP5300 stimulated greater expression of StAR and CYP19A1 across cells from all patients with NOR, ARA, and PCOS combined, although rh-FSH was unable to stimulate StAR and aromatase (CYP19A1) expression in cells from patients with PCOS. TOP5300-induced expression of StAR and CYP19A1 mRNA among patients with ARA and NOR was consistently lower than that observed in cells from patients with PCOS. CONCLUSION(S): TOP5300 appears to stimulate E2 production and steroidogenic gene expression from GL cells more than rh-FSH in PCOS, relative to patients with ARA and NOR. It does not appear that localization of FSHR at cell membranes is a limiting step for TOP5300 or rh-FSH stimulation of steroidogenic gene expression and E2 production.

Study on the changes of LHR, FSHR and AR with the development of testis cells in Hu sheep.

The process of testis development in mammals is accompanied by the proliferation and maturation of Sertoli, Leydig and germ cells. Spermatogenesis depends on hormone regulation, which must bind to a receptor to exert its biological effects. The changes in Hu sheep testis cell composition and FSHR, LHR and AR expression during different developmental stages are unclear (newborn, puberty and adulthood). To address this, using single-cell RNA sequencing, we analyzed testis cell composition and hormone receptor expression changes during three important developmental stages of Hu sheep. We observed significant changes in the composition of somatic and germ cells in different Hu sheep testis developmental stages. Furthermore, we analyzed the FSHR, LHR and AR distribution and expression changes at three important periods and verified them by qRT-PCR and immunofluorescence. Our results suggest that after birth, the proportion of germ cells increased gradually, peaking in adulthood; the proportion of Sertoli cells decreased gradually, reaching the lowest in adulthood; and the proportion of Leydig cells increased and then decreased, reaching the lowest in adulthood. In addition, FSHR, LHR and AR are mainly located in Sertoli, Leydig and germ cells. LHR and FSHR expression decreased with increasing age, while AR expression increased and then decreased with increasing age.

Polymorphisms in FSHR modulating susceptibility to polycystic ovary syndrome: an updated meta-analysis.

BACKGROUND: Two polymorphisms, rs6165 and rs6166 located in the intracellular domain of FSHR has been reported to affect folliculogenesis, steroidogenesis and oocyte maturation. Several studies have highlighted the role of FSHR polymorphisms in PCOS but the findings are conflicting. A meta-analysis was carried out to decipher the emerging perspectives. METHODOLOGY: A comprehensive literature search was made using PubMed, PCOSkb, and Google Scholar. New Ottawa Scale has been utilized to evaluate the quality of each article. To evaluate the strength of association under different genetic models of rs6165 and rs6166 polymorphisms, odds ratio with a 95% confidence interval (CI) was calculated. RESULTS: A total of 20 articles were selected for the present study. In pooled analysis and after the stratification by ethnicity, polymorphism rs6165 remains unrelated to the onset of PCOS. Besides, rs6166 exhibits significant protection in the Indian population under recessive, additive, and allele models (OR = 0.7, CI: 0.54-0.9, p = 0.006, OR = 0.65, CI: 0.48-0.89, p = 0.006, OR = 0.82, CI: 0.7-0.95, p = 0.01, respectively) and low to moderate risk in the Caucasian population under allele model (OR = 1.17, CI: 1.04-1.32, p = 0.01). CONCLUSION: This meta-analysis suggests that GG genotype of rs6166 provides protection against PCOS, in a population-specific manner.

Ala307Thr variation modulates FSHR structure and impairs its binding affinity for FSH: Implications in polycystic ovarian syndrome.

Follicle-stimulating hormone receptor (FSHR) belongs to the family of G-protein coupled receptors and acts as a cognate receptor for follicle-stimulating hormone (FSH). Among the various polymorphic changes reported in FSHR, rs6165 polymorphism leading to Ala307Thr variation in the extracellular domain of the FSHR (FSHRED ) is widely reported. Therefore we attempted to evaluate the functional implications of this variation by studying its effects on FSHRED structure as well as FSH binding. Our atomic-scale investigations reveal that the hinge region, a key hormone interaction site in the extracellular domain of Wt FSHR, exhibits significantly more flexibility compared with the variant structure. Moreover, the Wt receptor in complex with FSH was observed to form a pocket-like structure in its hinge region whereas such a structure was not detected in the variant. The study further reveals that the key residue, sTyr335, required for FSH recognition and FSHR activation, exhibits lower binding free energy in the variant structure as compared to the Wt. In conclusion, our results point out that Ala307Thr variation leads to structural and conformational anomalies in FSHRED which may alter its FSH binding and affect its activation.

Association between sequence variants in the FSHR gene and reproductive outcomes following IVF in predicted normoresponders.

RESEARCH QUESTION: Is there an association between FSHR sequence variants and reproductive outcomes following IVF in predicted normoresponders? DESIGN: Multicentre prospective cohort study conducted from November 2016 to June 2019 in Vietnam, Belgium and Spain including patients aged <38 years, and undergoing IVF with a predicted normal response with fixed-dose 150 IU rFSH in an antagonist protocol. Genotyping was performed for three FSHR (c.919A>G, c.2039A>G, c.-29G>A) and one FSHB sequence variants (c.-211G>T). Clinical pregnancy rate (CPR), live birth rate (LBR) and miscarriage rate in the first embryo transfer and cumulative live birth rate (CLBR) were compared between the different genotypes. RESULTS: A total of 351 patients underwent at least one embryo transfer. Genetic model analysis that adjusted for patient age, body mass index, ethnicity, type of embryo transfer, embryo stage and number of top-quality embryos transferred revealed a higher CPR for homozygous patients for the variant allele G of c.919A>G when compared to patients with genotype AA (60.3% versus 46.3%, adjusted odds ratio [ORadj] 1.96, 95% confidence interval [CI] 1.09-3.53). Also, c.919A>G genotypes AG and GG presented a higher CPR and LBR when compared with genotype AA (59.1% versus 46.3%, ORadj 1.80, 95% CI 1.08-3.00, and 51.3% versus 39.0%, ORadj 1.69, 95% CI 1.01-2.80, respectively). Cox regression models revealed a statistically significantly lower CLBR for c.2039A>G genotype GG in the codominant model (hazard ratio [HR] 0.66, 95% CI 0.43-0.99). CONCLUSION: These results demonstrate a previously unreported association between variant c.919A>G genotype GG and higher CPR and LBR in infertile patients and reinforce a potential role for genetic background in predicting the reproductive prognosis following IVF.

Exploring the expression and potential function of follicle stimulating hormone receptor in extragonadal cells related to abdominal aortic aneurysm.

INTRODUCTION: Follicle stimulating hormone (FSH) is identified to play a role in postmenopausal disease and hypothesized to affect abdominal aortic aneurysm (AAA) onset/progression in postmenopausal women. We aimed to detect FSHR gene expression in AAA tissue and cell types involved in AAA formation. METHODS: FSH stimulation of human umbilical cord endothelial cells (HUVECs), smooth muscle cells (HUCs) and PMA-differentiated macrophages to assess gene expression of FSHR and various markers. Human macrophages activated with various stimuli were assessed for FSHR gene expression. AAA dataset, AAA tissue samples and AAA-derived smooth muscle cells (SMC) obtained from elderly female donors were assessed for FSHR gene expression. AAA-SMCs were stimulated with FSH to assess its effect on gene expression. Lastly, oxidized low-density-lipoprotein (ox-LDL) uptake and abundance of cell surface protein markers were assessed by flow cytometry after FSH stimulation of human monocytes. RESULTS: FSH stimulation showed similar levels of gene expression in HUVECs and HUCs. Only ACTA2 was downregulated in HUCs. In PMA-differentiated macrophages, gene expression of inflammation markers was unchanged after FSH stimulation. FSHR gene expression was found to be low in the AAA datasets. Female AAA-SMCs show occasional FSHR gene expression at a very low level, yet stimulation with FSH did not affect gene expression of SMC- or inflammation markers. FSH stimulation did not impact ox-LDL uptake or alter cell surface protein expression in monocytes. While FSHR gene expression was detected in human testis tissue, it was below quantification level in all other investigated cell types, even upon activation of macrophages with various stimuli. CONCLUSION: Despite previous reports, we did not detect FSHR gene expression in various extragonadal cell types, except in occasional female AAA-SMCs. No clear effect on cell activation was observed upon FSH stimulation in any cell type. Our data suggest that a direct effect of FSH in AAA-related extragonadal cells is unlikely to influence AAA.

Cytogenetic screening of chromosomal abnormalities and genetic analysis of FSH receptor Ala307Thr and Ser680Asn genes in amenorrheic patients.

Objective: Amenorrhea is a rare reproductive medical condition defined by the absence of menstruation during puberty or later life. This study aims to establish the frequency and pattern of chromosomal abnormalities (CA) in both primary amenorrhea (PA) and secondary amenorrhea (SA), and further to detect the genetic changes in exon 10 at nucleotide positions 919 and 2039 of the genotypes Thr307Ala, and Asn680Ser, respectively. Design settings and patients: This cross-sectional study was conducted on a sample of seventy amenorrhoeic women according to the Helsinki declaration rules of medical ethics, as divided into 40 (57.14%) with PA and 30 (42.86%) with SA, and 30 healthy women with normal menstruation as the control. The chromosomal karyotyping was performed according to the ISCN, 2020. PCR products were submitted to RFLP and Sanger sequencing for women with normal karyotype and high FSH serum levels. Results: The classical Turner Syndrome was the most common CA in PA, followed by isochromosome X [46, Xi(X)(q10)], mosaicism of Turner and isochromosome X [45, X /46, Xi(X)(q10)], sex reversal (46, XY) and (46, XX,-3,+der3,-19,del 19 p). Abnormal SA cases were characterized by mosaicism Turner syndrome (45,X/46,XX) and (46,XX,-3,+der3,X,+derX). The homozygous genotypes AA and GG of Ala307Thr (rs6165) in the FSHR gene are most common in PA, while the homozygous genotype AA is more common in SA. GG and AG genotypes of Ser680Asn (rs6166) are more frequent in Iraqi patients with PA and SA compared to the healthy control women. Both PCR-RFLP and Sanger sequencing indicated a marked matching between genotypes. Conclusions: The study emphasizes the need for cytogenetic analysis to determine the genetic basis of PA and SA. Further, genotyping for women with normal karyotype and high FSH serum concentrations via PCR-RFLP should be considered for the precise diagnosis and development of appropriate management of and counselling for these patients.

Genotyping of cows by LHCGR, FSHR loci, and determination of the level of ovulation depending on the expression of the studied genes.

Background: Genotyping offers a promising avenue for identifying the healthy reproductive system in cows. The healthy reproductive system in cows is determined by measuring the level of ovulation and by identifying the type polymorphism of specific genes. Aim: The aim of the article is to explore how polymorphism of follicle stimulating hormone Receptor (FSHR) and luteinizing hormone/choriogonadotropin receptor (LHCGR) genes affect the reproduction trait of Holstein cows. Methods: Here we define a reproducible protocol to genotype and identify the polymorphism in specific genes from the extracted DNA of cows. Results: The results of genotyping showed that the only C allele (CC genotype) was observed in 100% of cows at the LHCGR locus, and three genotypes were observed at the FSHR locus (CC-67.74%, CG-9.03%, GG-23.22%). In cows with the CC genotype at the FSHR locus, the hormone concentration during ovulation was 1.1-2.5 ng/ml, which is within the physiological range for healthy reproduction. Conclusion: Cows with the CC genotype at the FSHR locus have a healthy course of the ovulation process, therefore good reproduction.

Effects of Follicle-Stimulating Hormone on Human Sperm Motility In Vitro.

To evaluate whether the follicle-stimulating hormone (FSH) receptor (FSHR) is expressed in human spermatozoa and the effects of FSH incubation on sperm function. Twenty-four Caucasian men were recruited. Thirteen patients had asthenozoospermia, and the remaining 11 had normal sperm parameters (controls). After confirming FSHR expression, spermatozoa from patients and controls were incubated with increasing concentrations of human purified FSH (hpFSH) to reassess FSHR expression and localization and to evaluate progressive and total sperm motility, the mitochondrial membrane potential, and protein kinase B (AKT) 473 and 308 phosphorylation. FSHR is expressed in the post-acrosomal segment, neck, midpiece, and tail of human spermatozoa. Its localization does not differ between patients and controls. Incubation with hpFSH at a concentration of 30 mIU/mL appeared to increase FSHR expression mainly in patients. Incubation of human spermatozoa with hpFSH overall resulted in an overall deterioration of both progressive and total motility in patients and controls and worse mitochondrial function only in controls. Finally, incubation with FSH increased AKT473/tubulin phosphorylation to a greater extent than AKT308. FSHR is expressed in the post-acrosomal region, neck, midpiece, and tail of human spermatozoa. Contrary to a previous study, we report a negative effect of FSH on sperm motility and mitochondrial function. FSH also activates the AKT473 signaling pathway.

The Ala307Thr polymorphism of the follicle-stimulating hormone receptor (FSHR) gene is associated with the dose of recombinant FSH received during IVF/ICSI treatment.

OBJECTIVE: Follicle-stimulating hormone (FSH) is essential for folliculogenesis, acting through the follicle-stimulating hormone receptor (FSHR) that is present on the membrane of granulosa cells. Polymorphisms in the FSHR gene may lead to an altered pattern of receptor expression on the cell surface or to changes in affinity for FSH. The aim of this prospective study was to detect any association between the follicle-stimulating hormone receptor (FSHR) gene Ala307Thr polymorphism (rs6165) and ovarian reserve, ovarian response or clinical results in IVF/ICSI treatment. METHODS: This prospective cohort study included 450 women who underwent IVF/ICSI cycles. DNA was extracted from peripheral blood, and the Ala307Thr FSHR polymorphism (rs6165) was genotyped using the TaqMan SNP genotyping assay. Participants were divided into three groups according to their Ala307Thr FSHR genotype: Thr/Thr (n:141), Thr/Ala (n=213) and Ala/Ala (n=96). The results were tested for associations with age, anti-Mullerian hormone (AMH) levels, antral follicle count (AFC), total dose of r-FSH, follicle size, number of retrieved oocytes, and clinical outcome of IVF/ICSI cycles. The statistical analyses were performed using Fisher's exact test and the Kruskal‒Wallis test. RESULTS: An association between the genotype of the FSHR (Ala307Thr) polymorphism and the dose of r-FSH was observed. Patients with the Ala/Ala genotype received a higher r-FSH dose than patients with the Ala/Thr (p=0.0002) and Thr/Thr (p=0.02) genotypes. No other correlation was observed. CONCLUSION: The Ala/Ala genotype was associated with the use of higher doses of recombinant FSH (r-FSH), suggesting that homozygosis of this allelic variant (Ala) provides lower sensitivity to r-FSH.

FSHR activation through small molecule modulators: Mechanistic insights from MD simulations.

Follicle-stimulating hormone receptor (FSHR) is a glycoprotein hormone receptor that plays a vital role in reproduction, cancer progression and osteoporosis. Owing to its therapeutic importance, several small molecule modulators have been identified by researchers through high throughput studies that usually include virtual screening of chemical libraries followed by in vitro validation through radio-ligand binding assays, cAMP accumulation and luciferase-based luminescence assays. The binding site of these modulators and structural changes that accompany modulator binding remains elusive. Here, we address these aspects through molecular docking and MD simulations on well-studied FSHR modulators and comparing the domain motions between agonist/FSH bound and antagonist bound FSHR structures. It was observed that agonist and antagonist modulators bind to the same site, but interact with distinct residues in transmembrane domain(TMD). FSHR(TMD) residues Ile522, Ala595, Ile602 and Val604 were found to interact only with agonist. Notably, these residues are conserved in the close homolog luteinizing hormone/choriogonadotropin receptor (LHCGR) and participate in interaction with its agonist Org43553. We observed distinctly prominent domain motions and conformational changes in TM helices 3, 4 and 6 for agonist bound FSHR structure. These structural changes have also been reported for LHCGR, and few GPCR members suggesting an important and well conserved mechanism of GPHR activation that could be exploited for design of novel modulators.

Undissociable chemically cross-linked and single-chain gonadotropins.

Undissociable gonadotropins can be obtained either by chemical cross-linking of the natural heterodimeric hormones or by expressing recombinant single-chain molecules through the fusion of their α and ß polypeptide sequences. These undissociable hormones are not more active than their natural heterodimeric counterparts indicating that the ß-subunit seatbelt embracing the α-subunit ensures the αß heterodimer stability in physiological conditions. The main interests of single-chain gonadotropins are that 1/only one single plasmid is required to produce an active recombinant hormone, 2/the two subunits' domains are constantly present in equal amounts and 3/they remain in close proximity even at low concentration for forming the hormone bioactive 3D structure. These undissociable gonadotropins have been shown to exhibit excellent stability and activity but they have not yet been commercialized probably because of immunogenicity risks and cost of production. Nevertheless, they might be used as a basis for the development of chemically simpler and cheaper ligands of LH and FSH receptors.