Follicle-stimulating Hormone Receptor Variants Research Articles

Identification and characterization of novel compound heterozygous variants in FSHR causing primary ovarian insufficiency with resistant ovary syndrome.

Primary ovarian insufficiency (POI) is among the foremost causes of women infertility due to premature partial or total loss of ovarian function. Resistant ovary syndrome (ROS) is a subtype of POI manifested as normal ovarian reserve but insensitive to gonadotropin stimulation. Inactivating variants of follicle-stimulating hormone receptor (FSHR), a class A G-protein coupled receptor, have been associated with POI and are inherited via an autosomal recessive pattern. In this study, we investigated the genetic causes of a primary infertility patient manifested as POI with ROS, and elucidated the structural and functional impact of variants of uncertain significance. Next-generation sequencing (NGS) combined with Sanger sequencing revealed novel compound heterozygous FSHR variants: c.1384G>C/p.Ala462Pro and c.1862C>T/p.Ala621Val, inherited from her father and mother, respectively. The two altered amino acid sequences, localized in the third and seventh transmembrane helix of FSHR, were predicted as deleterious by in silico prediction. In vitro experiments revealed that the p.Ala462Pro variant resulted in barely detectable levels of intracellular signaling both in cAMP-dependent CRE-reporter activity and ERK activation and displayed a severely reduced plasma membrane receptor expression. In contrast, the p.Ala621Val variant resulted in partial loss of receptor activation without disruption of cell surface expression. In conclusion, two unreported inactivating FSHR variants potentially responsible for POI with ROS were first identified. This study expands the current phenotypic and genotypic spectrum of POI.

Do follicle-stimulating hormone receptor polymorphisms in infertile men influence intracytoplasmic sperm injection outcomes? A prospective cohort study.

The follicle-stimulating hormone (FSH) and its receptor regulate the quantity and quality of spermatozoa production. Several studies have analyzed the effect of single nucleotide polymorphisms (SNPs) in exon 10 of the FSH receptor (FSHR) on basic semen parameters without yet reaching a firm consensus. The aim of this study was to evaluate the effect of p.Thr307Ala and p.Asn680Ser polymorphisms in exon 10 of the FSHR gene, in infertile men, on intracytoplasmic sperm injection (ICSI) outcomes. This study was conducted between March 2019 and February 2020 on infertile couples undergoing ICSI at Al Hadi Laboratory and Medical Center, Lebanon. Couples with severe infertility factors that may impair gametogenesis/embryogenesis (e.g. advanced maternal age, premature ovarian failure, underwent gonadotoxic treatments, etc.) were excluded from the study. Semen and blood samples were collected from infertile men on the day of oocyte collection. Infertile men (n = 173) were screened for FSHR variants using polymerase chain reaction-restriction fragment length polymorphism. Moreover, fertilization rates, embryo quality, and pregnancy outcomes were evaluated. Higher sperm concentrations were found in the p.Thr307Ala group than the p.Thr307Thr (P < 0.01) and p.Ala307Ala (P < 0.05) groups. Furthermore, fertilization rate was significantly lower in the p.Ala307Ala genotype than in the p.Thr307Thr genotype (P < 0.05). We showed that FSHR variants in infertile men undergoing ICSI could affect sperm concentration, motility, and fertilization rates. Therefore, it will be important to confirm these results in further studies using a larger sample size.

The N680S variant in the follicle-stimulating hormone receptor gene identifies hyperresponders to controlled ovarian stimulation.

ObjectiveTo study if the follicle-stimulating hormone receptor (FSHR) variant asparagine/serine in amino acid 680 (N680S) can predict hypersensitivity to gonadotropins in women undergoing assisted reproduction.Patients and methodsIn this retrospective study, 586 women undergoing their first in-vitro fertilisation treatment were enroled, and their FSHR N680S genetic variant was analysed. The main outcome measures were number of retrieved oocytes and any grade of ovarian hyperstimulation syndrome (OHSS). Experimental studies were performed on FSHR variants transfected into eukaryotic cells treated with 1–90 IU recombinant follicle-stimulating hormone. The receptors’ ability to induce a second messenger 3′,5′-cyclic AMP was measured.ResultsThe proportion of women who developed OHSS was 6% (n=36). None of the women who developed this condition had the homozygous serine variant. The N680S polymorphism in the FSHR was associated with the condition, Ptrend (genotype)=0.004 and Pallelic (alleles)=0.04. Mean oocyte number was 11±6 in women without OHSS and 16±8 in women who developed OHSS (P=0.001), despite exposure to lower total hormonal dose in the latter group. The odds ratio for developing OHSS in carriers of the asparagine allele was 1.7 (95% confidence interval: 1.025–2.839, P=0.04). A higher receptor activity in cells expressing asparagine compared with the serine was also evident at all concentrations of recombinant follicle-stimulating hormone used (P<0.05 for all).ConclusionThis study confirms previous findings regarding higher hormonal sensitivity in carriers of asparagine in the N680S position. These women are at higher risk for OHSS during in-vitro fertilisation. Genetic testing could identify those at highest risk to develop this adverse effect.

Molecular dynamics simulation of the follicle-stimulating hormone receptor. Understanding the conformational dynamics of receptor variants at positions N680 and D408 from in silico analysis.

Follicle-stimulating hormone receptor (FSHR) is a G-protein coupled receptor (GPCR) and a prototype of the glycoprotein hormone receptors subfamily of GPCRs. Structural data of the FSHR ectodomain in complex with follicle-stimulating hormone suggests a “pull and lift” activation mechanism that triggers a conformational change on the seven α-helix transmembrane domain (TMD). To analyze the conformational changes of the FSHR TMD resulting from sequence variants associated with reproductive impairment in humans, we set up a computational approach combining helix modeling and molecular simulation methods to generate conformational ensembles of the receptor at room (300 K) and physiological (310 K) temperatures. We examined the receptor dynamics in an explicit membrane environment of polyunsaturated phospholipids and solvent water molecules. The analysis of the conformational dynamics of the functional (N680 and S680) and dysfunctional (mutations at D408) variants of the FSHR allowed us to validate the FSHR-TMD model. Functional variants display a concerted motion of flexible intracellular regions at TMD helices 5 and 6. Disruption of side chain interactions and conformational dynamics were detected upon mutation at D408 when replaced with alanine, arginine, or tyrosine. Dynamical network analysis confirmed that TMD helices 2 and 5 may share communication pathways in the functional FSHR variants, whereas no connectivity was detected in the dysfunctional mutants, indicating that the global dynamics of the FSHR was sensitive to mutations at amino acid residue 408, a key position apparently linked to misfolding and variable cell surface plasma membrane expression of FSHRs with distinct mutations at this position.

Leutinizing hormone/choriogonadotropin receptor and follicle stimulating hormone receptor gene variants and risk of recurrent pregnancy loss: A case control study

We investigated the association of luteinizing hormone/chorionic gonadotropin receptor (LHCGR) and follicle stimulating hormone receptor (FSHR) polymorphisms with recurrent pregnancy loss (RPL). This was a retrospective case-control study involving 308 women with RPL and 310 control women. FSHR and LHCGR genotyping was done by allelic exclusion method. Lower minor allele frequency (MAF) of LHCGR rs7371084, and rs4597581, and FSHR rs6166, and higher MAF of rs1007541, and rs2055571 FSHR variants were seen in RPL cases. Significantly lower frequencies of heterozygous LHCGR rs2293275, rs7371084, and rs4597581, and homozygous FSHR rs6166, and higher frequency of homozygous FSHR rs2055571 carriers were seen in RPL cases, after controlling for key covariates. LHCGR rs7371084 and rs4597581 were negatively associated with RPL in non-obese subjects. On the other hand, FSHR rs1007541 and rs2055571 were positively, while FSHR rs6166 was negatively associated with RPL in obese subjects. Limited linkage disequilibrium was seen only among FSHR variants; higher frequency of GTAAA and AAGGA, and lower frequency of GTGGG FSHR haplotypes was seen in RPL cases; the frequencies of remaining FSHR haplotypes, and all LHCGR haplotypes were similar between cases and controls. This is the first study to demonstrate association of novel LHCGR (rs7371084 and rs4597581) and FSHR (rs6166, rs1007541 and rs2055571) SNPs with RPL, particularly according to obesity.

Combined assessment of polymorphisms in the LHCGR and FSHR genes predict chance of pregnancy after in vitro fertilization.

Can gonadotrophin receptor variants separately or in combination, be used for the prediction of pregnancy chances in in vitro fertilization (IVF) trials? The luteinizing hormone/human chorionic gonadotrophin receptor (LHCGR) variant N312S and the follicle-stimulating hormone receptor (FSHR) variant N680S can be utilized for the prediction of pregnancy chances in women undergoing IVF. The FSHR N680S polymorphism has been shown to affect the ovarian response in response to gonadotrophin treatment, while no information is currently available regarding variants of the LHCGR in this context. Cross-sectional study, duration from September 2010 to February 2015. Women undergoing IVF were consecutively enrolled and genetic variants compared between those who became pregnant and those who did not. The study was subsequently replicated in an independent sample. Granulosa cells from a subset of women were investigated regarding functionality of the genetic variants. Women undergoing IVF (n = 384) were enrolled in the study and genotyped. Clinical variables were retrieved from medical records. For replication, an additional group of n = 233 women was utilized. Granulosa cells from n = 135 women were isolated by flow cytometry, stimulated with Follitropin alpha or Menotropin, and the downstream targets 3',5'-cyclic adenosine monophosphate (cAMP) and inositol 1,4,5-trisphosphate (IP3) measured with enzyme-linked immunosorbent assay. Women homozygous for serine (S) in both polymorphisms displayed higher pregnancy rates than women homozygous asparagine (N) (OR = 14.4, 95% CI: [1.65, 126], P = 0.016). Higher pregnancy rates were also evident for women carrying LHCGR S312, regardless of FSHR variant (OR = 1.61, 95% CI: [1.13, 2.29], P = 0.008). These women required higher doses of FSH for follicle recruitment than women homozygous N (161 versus 148 IU, P = 0.030). When combining the study cohort with the replication cohort (n = 606), even stronger associations with pregnancy rates were noted for the combined genotypes (OR = 11.5, 95% CI: [1.86, 71.0], P = 0.009) and for women carrying LHCGR S312 (OR = 1.49, 95% CI: [1.14, 1.96], P = 0.004). A linear significant trend with pregnancy rate and increasing number of G alleles was also evident in the merged study population (OR = 1.34, 95% CI: [1.10, 1.64], P = 0.004). A lower cAMP response in granulosa cells was noted following Follitropin alpha stimulation for women homozygous N in both polymorphisms, compared with women with other genotypes (0.901 pmol cAMP/mg total protein versus 2.19 pmol cAMP/mg total protein, P = 0.035). Due to racial differences in LHCGR genotype distribution, these results may not be applicable for all populations. Despite that >250 000 cycles of gonadotrophin stimulations are performed annually worldwide prior to IVF, it has not been possible to predict neither the pregnancy outcome, nor the response to the hormone with accuracy. If LHCGR and FSHR variants are recognized as biomarkers for chance of pregnancy, more individualized and thereby more efficient treatment modalities can be developed. This work was supported by Interreg IV A, EU (grant 167158) and ALF governments grant (F2014/354). Merck-Serono (Darmstadt, Germany) supported the enrollment of the subjects. The authors declare no conflict of interest.

Leutinizing hormone/choriogonadotropin receptor and follicle stimulating hormone receptor gene variants in polycystic ovary syndrome.

Previous studies identified follicle-stimulating hormone receptor (FSHR) and luteinizing hormone/choriogonadotropin receptor (LHCGR) genes as polycystic ovary syndrome (PCOS) susceptibility loci, which was dependent on the racial/ethnic background of studied population. We investigated the association of genetic variants in FSHR and LHCGR with PCOS in Bahraini Arab women. A retrospective case-control study, involving 203 women with PCOS, and 211 age- and ethnically-matched control women. FSHR and LHCGR genotyping was done by allelic exclusion method (real-time PCR). Significantly lower frequencies of heterozygous LHCGR rs7371084 and FSHR rs11692782 genotype carriers were seen between women with PCOS vs. controls, and increased frequency of heterozygous homozygous LHCGR rs4953616 genotype carriers were detected between women with PCOS compared to control women. Limited linkage disequilibrium was noted among LHCGR and FSHR SNPs, and 2 blocks were constructed: the first (Block 1) spanning 61kb contained the six tested LHCGR SNPs, and the second (Block 2) spanning 298kb contained four of the five tested FSHR SNPs. Higher frequency of LHCGR GTCAAG haplotype was seen in women with PCOS compared to controls; the frequencies of the remaining LHCGR haplotypes, and all FSHR haplotypes were similar between cases and controls. This is the first study to confirm the association of novel LHCGR (rs7371084, rs4953616) and FSHR (rs11692782) SNPs with PCOS. The differential association of LHCGR and FSHR variants with PCOS confirms the racial/ethnic contribution to their association with PCOS.

Follicle-stimulating hormone receptor gene polymorphism in chronic anovulatory women, with or without polycystic ovary syndrome: a cross-sectional study.

BackgroundPolymorphisms at codons 307 and 680 are the most commonly encountered allelic variants of the follicle-stimulating hormone receptor (FSHR) gene. Studies in Caucasians suggest that certain FSHR variants are more common in women with polycystic ovary syndrome (PCOS) than normal women. The objective of this study was to determine the distribution of FSHR gene polymorphisms at codons 307 and 680 in Thai women with chronic anovulation, without (121 women) and with PCOS (133 women), using 132 known fertile women as controls.MethodsDNA samples from peripheral blood lymphocytes were extracted and analyzed by polymerase chain reaction-restriction fragment length polymorphism.ResultsThe prevalence of Threonine307Threonine (TT), Threonine307Alanine (TA), and Alanine307Alanine (AA) genotypes at codon 307 was 53.0% (95% CI = 44.2-61.7%), 42.4% (95% CI = 34–51.3%), and 4.5% (95% CI = 1.9-10.1%) in controls; 52.6% (95% CI = 43.8-61.3%), 39.8% (95% CI = 31.6-48.7%), and 7.5% (95% CI = 3.9-13.7%) in PCOS women; and 50.4% (95% CI = 42.8-61.2%), 45.4% (95% CI = 34.9-53.1%), and 4.5% (95% CI = 1.5-9.6%) in anovulatory women without PCOS, respectively. The prevalence of Asparagine680Asparagine (NN), Asparagine680Serine (NS), and Serine680Serine (SS) genotypes at codon 680 was 54.5% (95% CI = 45.7-63.2%), 40.9% (95% CI = 32.5-49.8%), and 4.5% (95% CI = 1.9-10.1%) in controls; 51.9% (95% CI = 43.1-60.6%), 44.4% (95% CI = 35.8-53.2%), and 3.8% (95% CI = 1.4-9.0%) in PCOS women; and 47.9% (95% CI = 40.4-58.8%), 47.1% (95% CI = 36.5-54.7%), and 5.0% (95% CI = 2–10.9%) in anovulatory women without PCOS, respectively. The prevalence of FSHR gene polymorphisms at both codons were not statistically different among the three groups.ConclusionsIn Thai women, there was no association between the FSHR gene polymorphism at codons 307 and 680 and chronic anovulation.

Follicular expression of follicle stimulating hormone receptor variants in the ewe

BackgroundSeveral alternatively-spliced mRNA transcripts of the follicle stimulating hormone receptor (FSHR) have been identified in sheep, including FSHR-1 (G protein-coupled form), FSHR-2 (dominant negative form), and FSHR-3 (growth factor type-1 form). Our objective was to determine which of these variants is predominantly expressed in follicles collected from ewes at various times after estrus.MethodsSuffolk-cross ewes (n = 8) were allowed to come into estrus naturally and were euthanized 24 (n = 3), 36 (n = 3), or 48 (n = 2) hours after the onset of estrus. All visible follicles were measured, aspirated and pooled according to follicular diameter: small (<= 2.0 mm), medium (2.1-4.0 mm), large (4.1-6.0 mm), and preovulatory (> = 6.1 mm). Aspirated cells were separated from follicular fluid by centrifugation. Total RNA was extracted from cell pellets and reverse transcribed. The resulting cDNA was subjected to qPCR, using primer sets designed to amplify each variant specifically. Gene expression was normalized to that of beta–actin within samples, and compared by analysis of variance with the level of significant differences set at p < .05.ResultsRelative expression of FSHR-3 exceeded that of both FSHR-1 and FSHR-2 in medium follicles, and tended to be higher in small follicles (p = .09) regardless of time after onset of estrus, and thus results from different time points were pooled. Expression of FSHR-3 was greater than that of FSHR-2 and luteinizing hormone receptor (LHR) in small and medium follicles. Expression of LHR was greatest in preovulatory follicles.ConclusionsThese experiments show that in addition to the well characterized G protein-coupled form of the FSHR, alternatively spliced variants of the FSHR may participate in follicular dynamics during follicular waves of the sheep estrous cycle. Furthermore, these results indicate that an “alternatively” spliced form of the FSHR (FSHR-3) is the predominant form of the FSHR in the sheep.

Study in 1790 Baltic men:FSHRAsn680Ser polymorphism affects total testes volume

Follicle-stimulating hormone receptor (FSHR) contains two common linked polymorphisms, Thr307Ala (rs6165) and Asn680Ser (rs6166), shown to modulate ovarian function in women. The effect on male fertility and reproductive parameters has been inconclusive. We studied FSHR Asn680Ser polymorphism in a large study group (n = 1790) from the Baltic countries. The population-based Baltic male cohort (Estonians, Latvians, Lithuanians; n = 1052) and Estonian oligo-/azoospermic (sperm concentration <20 × 106/mL) idiopathic infertile patients (n = 738) were genotyped for the FSHR Asn680Ser using PCR-RFLP. Genetic associations were tested using linear regression under additive model and results were combined in meta-analysis. No statistical difference was detected in allelic distribution of the FSHR Asn680Ser between the Baltic cohort and Estonian male infertility group. A consistent significant association was detected between the FSHR Ser680 allele and lower total testes volume in both, the Baltic cohort (p = 0.010, effect = −1.16 mL) and Estonian idiopathic infertility group (p = 0.007, effect = −1.77 mL). In meta-analysis, the statistical significance was enhanced (p = 0.000066, effect = −1.40 mL). Meta-analysis supported further associations with moderate effect between the FSHR Ser680 variant and higher serum FSH (p = 0.072), lower Inhibin B (p = 0.037) and total testosterone (p = 0.034). No statistically significant associations were identified with serum LH and estradiol, and sperm parameters. In conclusion, the study in 1790 Baltic men shows statistically highly significant association of the FSHR Asn680Ser with total testes volume and supportive association with serum reproductive hormone levels indicative to the functional effect of the alternative FSHR variants on male reproductive physiology.

Pharmacogenetics of follicle-stimulating hormone action

To review the current knowledge of genetic variants in the two genes affecting the individual responsiveness to follicle-stimulating hormone (FSH) action-the FSH beta-subunit (FSHB) and the FSH receptor (FSHR), as well as the pharmacogenetic ramifications of the findings. Four common variants in the FSHB and the FSHR genes were shown to exhibit significant effect on FSH action: linked FSHR variants Thr307Ala and Asn680Ser determining common receptor isoforms, and gene expression affecting polymorphisms FSHR -29G/A and FSHB -211G/T. In women, the FSHR Thr307Ala/Asn680Ser polymorphisms show consistent predictive value for estimating the most optimal recombinant FSH dosage in controlled ovarian hyperstimulation (COH). The same variants exhibit a potential for the pharmacogenetic assessment of the treatment of polycystic ovarian syndrome. The FSHR -29G/A variant was also shown to contribute to ovarian response to COH. Pilot studies have suggested the FSHB -211 TT homozygous oligozoospermic men with genetically determined low concentration of FSH, as potentially the best responders to FSH treatment; furthermore, modulation of this response by FSHR polymorphisms is possible. Genetic variants in FSHB and FSHR exhibit a potential for pharmacogenetic applications in selecting appropriate treatment options (timing and dosage) in male and female conditions requiring or benefiting from FSH therapy.

Impact of follicle stimulating hormone receptor variants in fertility

Genetic variation plays a crucial role in modification of normal or disease pathophysiology. Follicle stimulating hormone receptor (FSHR) signaling is necessary for normal development and function of the ovaries and testes. Here, we review the associations between FSHR polymorphisms and fertility or subfertility. FSHR polymorphisms consist of single nucleotide changes within the coding and regulatory regions and/or alternatively spliced products. Most of the investigations focused on two single nucleotide polymorphisms (SNPs) in the coding region of the receptor, which result in amino acid changes (p.307Thr/Ala, p.680Asn/Ser). In women, these SNPs were associated with variable response to ovarian stimulation with FSH during infertility treatment. Not all studies revealed an association, and those that did showed a small effect. Alternative splice variants of the receptor affecting the extracellular domain without causing a frameshift have been found in women undergoing ovarian stimulation, and in infertile men. Associations with polycystic ovary syndrome, premature ovarian failure, osteoporosis, and cancer found small effect. The identification of FSHR variants in a select infertility patient population has significant clinical implications in demonstrating a possible genetic cause to female infertility and improves our understanding of the genetic basis of infertility as a whole. Pharmacogenomics is a new field aiming to devise individualized treatments for disorders based on the genetic signature of the patients.

A deleted form of follicle stimulating hormone (FSH) receptor, found in women with low response to FSH, impairs the function of the normal receptor when co-expressed in vitro

OBJECTIVE: FSH mediates cyclic follicle growth and is widely used for controlled ovarian stimulation. The ovarian response to FSH is variable, and has been partly attributed to two common variants of the FSH receptor (FSHR). In addition, we have recently identified four abnormal FSHR splicing products in the cumulus cells of 37% (13 of 35) of infertile women tested [Gerasimova et al. JCEM 2010]. All alterations affected the extracellular part of the receptor. All patients with variant receptors also possessed normal FSHR. Our aim in this study was to determine the molecular mechanism of action of one of the mutant receptor forms. DESIGN: Investigations of cell lines expressing wild-type and mutant FSHR. MATERIALS AND METHODS: We established stable HEK293 cell lines expressing wild-type [FSHR(wt)] and splice-variant receptor [FSHR(del ex2)] under the control of the inducible Tet-off system. Intracellular cAMP, the first messenger produced upon FSH stimulation, was measured after the cells were stimulated with 0-1000 mIU/ml FSH for 12 h. Protein interactions were also tested by co-immunoprecipitation. Plasma membrane proteins were visualized using surface biotinylation. Subcellular localization was determined by immunofluorescence and fractionation in discontinuous sucrose gradients. RESULTS: In vitro expression of FSHR(del ex2) alone did not elicit a response to FSH, while co-expression of FSHR(del ex2) with FSHR(wt) resulted in a significant reduction of the wt activity when stimulated with FSH. Immunostaining and surface biotinylation showed that, unlike the FSHR(wt), less than half of the total amount of FSHR(del ex 2) protein is present on the cell surface, and a large amount of protein remains trapped in intracellular compartments. CONCLUSION: Our findings suggest that FSHR variants can contribute to abnormal response to stimulation in certain women undergoing IVF. The variant receptors require the presence of wt but can significantly alter the receptor's activity, resulting in less than optimal ovarian stimulation.

Two FSHR variants, haplotypes and meta-analysis in Chinese women with premature ovarian failure and polycystic ovary syndrome

In this study, two polymorphisms of follicle stimulating hormone receptor ( FSHR) gene were analysed in the case-control sample using 40 premature ovarian failure (POF) patients, 60 polycystic ovary syndrome (PCOS) patients and 92 healthy controls. All subjects were unrelated Han Chinese from Shanghai. No difference was observed on the allelic or genotypic distribution of FSHR gene polymorphisms between the groups. However, the two-marker haplotypes covering components Thr307Ala (rs6165) G and Asn680Ser (rs6166) A were observed to be significantly associated with PCOS ( p = 0.007, corrected p = 0.042). Meanwhile, a meta-analysis including our study (altogether six POF and eight PCOS studies) showed significant association between rs6166 marker and PCOS ( p < 0.05). The results suggest that FSH receptor might play a role in genetic susceptibility to PCOS. However, confirmatory studies in independent samples are needed.

Identification and In vitro Characterization of Follicle Stimulating Hormone (FSH) Receptor Variants Associated with Abnormal Ovarian Response to FSH

ABSTRACT Context Follicle stimulating hormone (FSH) mediates cyclic follicle growth and development and is widely used for controlled ovarian stimulation in women undergoing infertility treatment. The ovarian response of women to FSH is variable, ranging from poor response to ovarian hyperstimulation. Objective We investigated whether genetic alterations of the FSH receptor (FSHR) contribute to this variability. Design and Patients Our approach was to study women undergoing treatment with In Vitro Fertilization (IVF) falling into the edges of the normal distribution of ovarian response to FSH, with respect to age. Setting Yale Fertility Clinic. Methods We extracted RNA from cumulus cells surrounding the oocytes of women undergoing IVF and analyzed the FSHR mRNA by RT-PCR and sequencing. Results We identified four abnormal FSHR splicing products (3 exon deletions and 1 intron insertion) in the FSHR mRNA in 37% (13/35) of women tested. All alterations affected the extracellular ligand-binding portion of the receptor without causing a frameshift. When transfected in HEK293T cells, all four splicing variants showed markedly decreased cAMP activation compared to controls. Untransfected cells showed no response to FSH, while all the cell lines showed normal cAMP activation when treated with Forskolin, a non-receptor mediated cAMP stimulant. None of the normal or mutant forms showed any response to luteinizing or thyroid stimulating hormones. Conclusions Our findings strongly indicate FSHR variants as being an intrinsic genetic cause of some forms of infertility and identify a need for functional characterization of these variants and the investigation of more individualized ovarian stimulation protocols.

Expression of Follicle-Stimulating Hormone Receptor Variants During the Estrous Cycle of the Ewe.

Several splice-variants of the follicle stimulating hormone receptor (FSHR) have been identified in sheep. These include a G protein-coupled (FSHR-1), a putative dominant negative (FSHR-2), and a growth factor type-I (FSHR-3) form. These variants likely couple to different signaling pathways and may be involved in FSH responsiveness at different stages of follicular development. However, with the exception of FSHR-1, the roles of variant forms of the FSHR during follicular development in the ewe are unknown. Thus, our objective was to examine expression patterns of variant forms of the FSHR at different times during the estrous cycle and in different sized follicles in the ewe. In Exp. 1, 8 cycling Suffolk-cross ewes were euthanized 24 (n=3), 36 (n=3) and 48 (n=2) hr after the onset of estrus. In Exp. 2, 7 anestrous Suffolk-cross ewes received CIDRs for 14d, followed by treatment (n=3) with PMSG (500IU), or no PMSG (n=4; CIDR-only). Ewes were euthanized 24 (n=4; 2 CIDR-only, 2 PMSG-treated) or 36 (n=3; 2 CIDR-only, 1 PMSG-treated) hr after CIDR removal. Ovaries were harvested from each ewe and all visible follicles were aspirated. Granulosa cells (GC) were isolated from follicular fluid; each isolate was pooled according to follicular diameter: small (≤ 2.0 mm), medium (2.1-4.0 mm), large (4.1-6.0 mm) or preovulatory (≥ 6.1 mm). Total RNA was extracted from GC and reverse-transcribed. The resulting cDNA was subjected to qPCR using primers designed to amplify each FSHR variant specifically. For Exp. 1, FSHR-3 was the most highly expressed (P≤0.05) variant in small and medium follicles. Expression of FSHR-2 and FSHR-3 was higher (P≤0.05) in medium follicles compared to small and preovulatory follicles. Expression of FSHR-1 was not significantly different between follicle size classes, but was higher (P≤0.05) than FSHR-2 in small follicles. No differences in FSHR variant expression were found at different times after onset of estrus, nor were FSHR variants expressed at significantly different levels in preovulatory follicles. For Exp. 2, application of CIDRs during anestrous appeared to cause slight differences in expression patterns of FSHR variants. The most highly expressed variant (P≤0.05) in small and medium follicles was still FSHR-3; however, FSHR-1 expression was higher (P≤0.05) in large versus small and medium follicles, while no differences were found in experiment 1 for FSHR-1 in different sized follicles. Thus, progesterone treatment during anestrous may enhance expression of the G protein-coupled form of the FSHR as follicles develop. FSHR-2 was expressed at the lowest (P≤0.05) levels of any variant, but was higher (P≤0.05) in large compared to medium and small follicles. No significant differences in FSHR variant expression were found within large follicles. This is the first study to examine expression patterns of FSHR-1, FSHR-2, and FSHR-3 during development of follicles in the mature sheep ovary. Our results indicate that FSHR-3 is the predominant variant of the FSHR expressed by GC during the ewe estrous cycle, and thus FSH may have important effects on follicular development through other forms of its receptor in addition to the G protein-coupled form. (poster)

Association of Follicle Stimulating Hormone (FSH) Receptor Gene Polymorphisms With Outcome of Controlled Ovarian Stimulation in Patients Undergoing IVF

Controlled ovarian hyperstimulation (COH) using FSH is a widely used treatment in most assisted reproduction techniques. FSH triggers the maturation of follicles and plays a pivotal role in the recruitment of the dominant follicle. These important actions of FSH are mediated by the FSH receptor. In this study, we evaluated the association of FSH receptor gene polymorphisms with outcome of COH in patients undergoing IVF. Analysis of genetic screening data and clinical outcome Genomic DNA was extracted from peripheral blood of 202 women, including 141 ones were undergoing COH. We investigated the frequency of FSH receptor variants, Thr307Ala (T/A) and Asn680Ser (N/S) polymorphisms by using polymerase chain reactions and restriction fragment length polymorphism analysis. Their clinical outcome such as basal FSH level, estradiol level at administration of hCG, dosage of FSH treated, number of retrieved oocytes and pregnancy rates were compared related to their genotypes. In a population of 202 Korean women, the frequency of TT/NN, TA/NS and AA/SS for the variant Thr307Al a and Asn680Ser was 50.0%, 36.6% and 9.9%, respectively. The frequency of the unlinked genotypes, TA/NN, TT/NS, AA/NN and AA/NS were 2.0%, 0.5%, 0.5% and 0.5%, respectively. There was no significant difference in the basal FSH level, dosage of FSH treated and pregnancy rate among the COH-IVF patients with different genotypes. However, the proportion of good responder, which is higher estradiol level (> 2,000 pmol/L) at administration of hCG and number of retrieved oocytes (> 10 oocytes), in AA/SS genotype (81.8% and 63.6%) was higher than those of TT/NN (37.3% and 46.7%) and TA/NS (38.1% and 41.8%). We found that Korean AA/SS genotype of FSH receptor was good responder of FSH treatment in COH for IVF. This finding is contrast to the reports with other ethnic populations. Our data should be substantiated by the further studies of FSH receptor genotyping in the Korean population.

Follicle‐stimulating hormone receptor polymorphisms in a population of infertile women

There are two known polymorphisms of clinical relevance in the follicle-stimulating hormone (FSH) receptor exon 10, alanine or threonine at position 307, and asparagine or serine at position 680, giving rise to two discrete allelic variants: Thr307/Asn680 and Ala307/Ser680. At position 680, three FSH receptor variants are possible: Asn/Asn, Asn/Ser, and Ser/Ser. We hypothesized an association between FSH receptor polymorphisms and ovarian reserve. FSH receptor polymorphisms at position 680 were studied in a population of 68 infertile women. We used serum FSH level at cycle day 3 as a screening for ovarian reserve. DNA was extracted from peripheral leukocytes in whole blood by using PCR and DNA sequencing in order to determine the type of FSH receptor. The distribution of FSH receptor variants was Asn/Asn (AA) 35%, Asn/Ser (AS) 24%, and Ser/Ser (SS) 41%. In women with normal ovarian reserve, FSH levels at cycle day 3 were 5.6 +/- 1.9 (AA group), 6.7 +/- 1.3 (AS group), and 5.7 +/- 1.7 (SS group) (non-significant). Corresponding FSH levels at cycle day 10 were 6.9 +/- 1.9, 6.3 +/- 1.7, and 8.3 +/- 2.8 (P < 0.01, AA and AS vs. SS group). In the SS group, FSH levels at cycle day 10 were significantly higher than they were at cycle day 3 (P < 0.05). The results show that Ser/Ser-680 predominates in the studied infertile population. Furthermore, women with normal ovarian reserve and the Ser/Ser FSH receptor variant had significantly higher FSH levels, compared to women with Asn/Asn and Asn/Ser variants. FSH receptor genotyping may, thus, be interesting as an adjunct indicator of ovarian reserve for infertile women undergoing assisted reproduction, and may be helpful in the determination of the starting dosage of FSH in in vitro fertilization.

Association of basal follicle stimulating hormone (FSH) levels with FSH receptor variants in subfertile women with normal menstrual cycle: implications for ovarian sensitivity to FSH

Objective: Recently a frequently occurring polymorphic variant of the FSH receptor gene has been described in which the amino acid asparagine (Asn) is replaced by serine (Ser) at position 680. The Ser variant is associated with higher FSH levels in the early follicular phase and more FSH is needed to obtain normal follicular response in ovulatory patients undergoing IVF treatment.(Perez Mayorga et al. J Clin Endocrinol Metab 85: 3365–3369, 2000). Aim of our study was to test whether this receptor variant occurs more often in regularly menstruating patients with elevated basal FSH, a condition which is associated with a higher ovarian FSH threshold (de Koning et al Hum Reprod 16 abstract book 1: 207, 2001).