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.

Molecular Role of Asn680Ser and Asp37Glu Missense Variants in Saudi Women with Female Infertility and Polycystic Ovarian Syndrome.

Female infertility (FI) is a global health issue. Polycystic ovary syndrome (PCOS) is a common cause of FI. The renalase gene (RNLS) is associated with FI and other human diseases. Based on the documented missense variants, rs6166 and rs2296545 single-nucleotide polymorphisms (SNPs) were not identified in Saudi women with FI and PCOS. This study aimed to investigate the molecular role of the two SNPs in Saudi women with FI and PCOS. In this cross-sectional study, 96 healthy controls, 96 women with FI, and 96 women with PCOS were recruited. DNA was isolated, and polymerase chain reactions and Sanger sequencing analysis were performed using rs6166 and rs2296545 SNPs. The data obtained from the three groups were used to perform statistical analyses based on genotype, allele frequencies, regression models, and ANOVA analysis. Both rs6166 and rs2296545 had no role in FI or PCOS in Saudi women. A predicted reason for non-association in Saudi women could be the role of elderly women in the controls compared with women with FI and PCOS. Moreover, age, weight, and body mass index were higher in the control group than the FI and PCOS groups. In conclusion, rs6166 and rs2296545 SNPs were not associated with FI or PCOS in Saudi women.

MicroRNA miR-125b can suppress ovarian granulosa cell functions: Interrelationships with FSH.

This study aimed to evaluate the involvement of miR-125b and its interrelationship with follicle-stimulating hormone (FSH) in the control of basic ovarian granulosa cell functions. The effect of miR-125b mimics on basic functions of porcine ovarian granulosa cells cultured with and without FSH, and the effect of FSH on the expression of endogenous miR-125b was examined. Expression levels of miR-125b, viability, proliferation (accumulation of PCNA and cyclin B1), apoptosis (accumulation of bax and caspase 3), the accumulation of FSH receptors (FSHR), steroid hormones, insulin-like growth factor I (IGF-I), oxytocin, and prostaglandin E2 release were analysed by reverse transcription-quantitative polymerase chain reaction, Trypan blue exclusion test, quantitative immunocytochemistry, and ELISA. Transfection of cells with miR-125b mimics inhibited cell viability, proliferation, apoptosis, the occurrence of FSHR, progesterone, testosterone, estradiol, and oxytocin release but stimulated prostaglandin E2 output. FSH promoted cell viability, proliferation, steroid hormones, IGF-I, oxytocin, and prostaglandin E2 output and reduced the expression of miR-125b and apoptosis. Furthermore, miR-125b mimics supported the effect of FSH on the release of estradiol, IGF-I, and prostaglandin E2, and inverted FSH influence on cell viability, proliferation, apoptosis, progesterone, and testosterone output. FSH supported both inhibitory and stimulatory action of miR-125b on ovarian cell functions. Present observations indicate that: miR-125b can be involved in the control of basic ovarian functions and that miR-125b and FSH are antagonists in their actions on ovarian cell functions. The ability of FSH to reduce miR-125b expression and the ability of miR-125b mimics to decrease the occurrence of FSHR and to modify FSH effects indicate the existence of the self-inhibiting FSH-miR-125b axis and that miR-125b can mediate the actions of FSH on ovarian cells.

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.

Effect of LHCGR and FSHR gene polymorphisms on fertility traits and milk yield of cross-bred dairy cows in Sri Lanka.

Single nucleotide polymorphism (SNP) analysis of fertility genes will be useful in improving the genetic potential of dairy cows. A population of 142 cross-bred dairy cows was screened for SNPs in bovine luteinizing hormone choriogonadotropin receptor (LHCGR) and bovine follicle stimulating hormone receptor (FSHR) genes. The effect of reported SNPs on selected fertility traits (average calving interval, average number of services per conception and age at first calving) and milk yield was determined. Altogether six SNPs were detected in animals screened under the present study. Among them, four SNPs (rs41256848, rs41256850, rs465790244, and rs45463781) were located in the exon 11 region of the LHCGR gene and two SNPs (rs43676359 and G-278-A (GU253337) were located in the five upstream region of the FSHR gene. Minor alleles identified for SNPs in the FSHR gene in the studied small population of cows differed from those reported in other research. In this study, only rs45463781 SNP at exon 11 of the LHCGR gene significantly affected the average milk yield in cross-bred cows. The nucleotide change from "G" to "A" negatively affected the average milk yield. Further investigations are needed to confirm the reported association with a larger sample size.

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.

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.

Paeoniflorin Alleviates Cisplatin-Induced Diminished Ovarian Reserve by Restoring the Function of Ovarian Granulosa Cells via Activating FSHR/cAMP/PKA/CREB Signaling Pathway.

Paeoniflorin (PAE) is the main active compound of Radix Paeoniae Rubra (a valuable traditional Chinese medicine and a dietary supplement) and exerts beneficial effects on female reproductive function. However, the actions of PAE on diminished ovarian reserve (DOR, a very common ovarian function disorder) are still unclear. Herein, our study investigated the effect and potential mechanism of PAE on DOR by using cisplatin-induced DOR mice and functional impairment of estradiol (E2) synthesis of ovarian granulosa-like KGN cells. Our data show that PAE improved the estrous cycle, ovarian index, and serum hormones levels, including E2, and the number of antral follicles and corpora lutea in DOR mice. Further mechanism results reveal that PAE promoted aromatase expression (the key rate-limiting enzyme for E2 synthesis) and upregulated the FSHR/cAMP/PKA/CREB signaling pathway in the ovaries. Subsequently, PAE improved the levels of E2 and aromatase and activated the FSHR/cAMP/PKA/CREB signaling pathway in KGN cells, while these improving actions were inhibited by the siRNA-FSHR and FSHR antagonist treatments. In sum, PAE restored the function of E2 synthesis in ovarian granulosa cells to improve DOR by activating the FSHR/cAMP/PKA/CREB signaling pathway, which exhibited a new clue for the development of effective therapeutic agents for the treatment of DOR.

FSH receptor genotype and its influence on the results of donor ovarian stimulation using corifollitropin alfa.

RESEARCH QUESTION: Does the FSH receptor (FSHR) genotype influence the results of donor ovarian stimulation using corifollitropin alfa? DESIGN: A prospective cohort study was performed including 152 oocyte donor ovarian stimulations: group 1 (n = 80) using a single dose of 150 µg of corifollitropin alpha; and group 2 (n = 72) using in addition to corifollitropin alpha, continued stimulation using recombinant FSH 225 IU daily. Allelic discrimination was used to genotype the FSHR p.N680S polymorphism. Linear regression analysis was performed to study the differences between groups. RESULTS: No differences in clinical characteristics between genotypes were reported. Overall, the results of ovarian stimulation were better in oocyte donors with SN and NN genotypes compared with SS in terms of the number of retrieved oocytes (15.78 versus 10.83; P = 0.008) and retrieved metaphase II (MII) oocytes (12.34 versus 9.00; P = 0.032). Corresponding differences were also observed in group 1 for the number of retrieved oocytes (13.83 versus 7.50, P = 0.018) and retrieved MII oocytes (10.24 versus 5.42; P = 0.038). However, in group 2 no significant differences were found for oocytes retrieved (17.55 versus 13.06, P = 0.064) or MII oocytes (14.25 versus 11.39; P = 0.12). CONCLUSIONS: This study suggests that ovarian stimulation protocols with corifollitropin alfa in women with the SS genotypes could be associated with fewer oocytes and MII oocytes retrieved. Despite the fact that corifollitropin alfa has a longer half-life, the results for the SS genotype do not match those for the other genotypes, so other factors must be involved. Therefore, to tailor treatments, it would be advisable to genotype women at p.N680S of the FSHR.

Effects of different energy diets on DNA methylation and mRNA expression in follicle stimulating hormone receptor gene promoter region of Duolang sheep during estrus.

BACKGROUND: The aim of this study was to study the relationship between the methylation level of the promoter region of follicle-stimulating hormone receptor (FSHR) gene and the mRNA expression of Duolang sheep fed different energy diets. METHODS: In this study, polyembryo estrus Duolang sheep under different energy levels were selected as the experimental subjects. Dietary nutrient level reference (NY/T 816-2004), medium energy level was 10.88 MJ/day, high and low energy groups were increased and decreased by 15% on the basis of medium energy level, respectively 12.51 MJ/day, 9.25 MJ/day. Through RNA and DNA extraction, qPCR, bisulfitegenomicse-quencing PCR (BSP), sequence matching and other analysis of ovarian tissue of Duolang sheep. The difference of DNA methylation level and mRNA expression of FSHR gene during estrus in Duolang sheep fed with different energy diets was detected. RESULTS: The results showed the expression level of FSHR in high energy group was significantly higher than that in low energy group (P < 0.01), the expression level of FSHR in high energy group was significantly higher than that in medium energy group (P < 0.05), and the expression level of FSHR in medium energy group was significantly higher than that in low energy group (P < 0.05). In the target fragment of the promoter region of the FSHR gene, the methylation rate was 25% in the high energy group, 50% in the normal group, and 75% in the low energy group. CONCLUSIONS: This study revealed that different dietary energy levels had certain effects on the FSHR gene DNA methylation level and mRNA expression, and the expression level was negatively correlated with methylation level.

Differential expression of FSHR and LHR genes and proteins during development of rabbit ovarian follicles.

The development of an ovarian follicle is a complex process at the cellular and molecular level that is mainly regulated by follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR). To elucidate the contribution of these receptors to ovarian follicle development, it is necessary to determine their expression profiles during this biological process. Therefore, this study aimed to investigate the relationship between ovarian development pattern and the differential ovarian expression pattern of FSHR and LHR genes as well as proteins at different developmental stages. Ovaries were collected from 30 New Zealand rabbits at day 0 (birth), week 2 (neonate), week 4 (cub), week 16 (maturity), and day 18 pregnancy. Ovarian histology, and gene as well as protein expression were determined using light microscopy, real-time PCR and western blotting, respectively. The results showed that the expression levels of FSHR mRNA and protein increased coincidently with age and the growth of ovarian follicles. The levels of LHR mRNA and protein remained low from the day of birth until week 4 and became significantly higher by week 16 coinciding with appearance of growing and antral follicles, which have a defined thecal layer. FSHR gene and protein expression decreased with pregnancy, whereas LHR increased, reaching a peak level during pregnancy. It can be concluded that changes in FSHR and LHR gene and protein expression could be related to the growth and development of follicles, indicating the regulatory role for these receptors in rabbit folliculogenesis.

Follicle-stimulating hormone beta subunit and receptor variations in infertile men in Central Black Sea Region of Turkey.

Genetic variants affecting the interaction of FSH-FSHR may negatively affect the male reproductive potential. The aim of this case-control study was to evaluate FSHB c.-211G>T and FSHR c.2039A>G variants in a cohort of infertile men from Central Black Sea Region in Turkey. One hundred and nine infertile men and 50 proven fertile controls were enrolled in the study. Genotyping was assessed by RFLP. The genotype frequencies of FSHB -211G>T and FSHR 2039A>G showed significant variation between infertile and fertile groups (χ2 , p = 0.046, GG vs. GT+TT, and p = 0.008, AA vs. AG+GG). FSHB -211GG was found to be higher in patients with OAT compared to fertile controls (82.3% vs. 64.0%, χ2 , p = 0.028). The distribution of FSHR 2039A>G alleles was different between infertile and fertile men (χ2 , p = 0.005, total infertile vs. fertile groups, p = 0.019, OAT vs. NOA vs. fertile groups). Further analysis showed that the frequencies of FSHR 2039AA wild-type genotype were higher in the oligoasthenoteratozoospermic and non-obstructive azoospermic groups compared with the controls (χ2 , 39.3% vs. 17.0%, p = 0.012, and 37.5% vs. 17.0%, p = 0.025 respectively). Our results showed wild-types of FSHB -211G>T and FSHR 2039A>G variants may cause susceptibility to male infertility in the Central Black Sea Region of Turkey.

LINC00707 knockdown inhibits IL-1ß-induced apoptosis and extracellular matrix degradation of osteoarthritis chondrocytes by the miR-330-5p/FSHR axis.

BACKGROUND: Osteoarthritis (OA) is a severe disabling condition that causes major health problems. The roles of long non-coding RNAs (lncRNAs) in regulating OA progression have been increasingly researched. Based on previously published microarray analysis, LINC00707 is upregulated in OA. This research was done to uncover the function of LINC00707 in IL-1ß-induced chondrocyte injury and its possible mechanisms. METHODS: LINC00707, miR-330-5p, and follicle-stimulating hormone receptor (FSHR) expression in OA cartilage tissues were assessed by RT-qPCR. Primary chondrocytes were isolated from OA tissues and treated with IL-1ß to establish an in vitro OA model. Under the indicated treatment, chondrocyte apoptosis, senescence, ECM degradation, and inflammation were determined using flow cytometry, TUNEL, SA-ß-Gal staining, and ELISA experiments, respectively. Interactions between gene were evaluated using Ago2 RIP and luciferase reporter assays. RESULTS: LINC00707 and FSHR were elevated, and miR-330-5p was reduced in cartilage tissues of OA patients and in IL-1ß-treated primary chondrocytes. Silencing LINC00707 hampered chondrocytes apoptosis, senescence, ECM degradation, and inflammation. LINC00707 acted as a ceRNA to regulate FSHR through controlling miR-330-5p availability. Additionally, both miR-330-5p depletion and FSHR overexpression diminished the effects of silencing LINC00707 in OA progression. CONCLUSIONS: Silencing LINC00707 mitigates chondrocyte injury in osteoarthritis via sponging miR-330-5p and inhibiting FSHR.

The N-Linked Glycosylation Site N191 Is Necessary for PKA Signal Transduction in Eel Follicle-Stimulating Hormone Receptor.

The follicle-stimulating hormone receptor (FSHR) contains several N-linked glycosylation sites in its extracellular region. We conducted the present study to determine whether conserved glycosylated sites in eel FSHR are necessary for cyclic adenosine monophosphate (cAMP) signal transduction. We used site-directed mutagenesis to induce four mutations (N120Q, N191Q, N272Q, and N288Q) in the N-linked glycosylation sites of eel FSHR. In the eel FSHR wild-type (wt), the cAMP response was gradually increased in a dose-dependent manner (0.01-1500 ng/mL), displaying a high response (approximately 57.5 nM/104 cells) at the Rmax level. Three mutants (N120Q, N272Q, and N288Q) showed a considerably decreased signal transduction as a result of high-ligand treatment, whereas one mutant (N191Q) exhibited a completely impaired signal transduction. The expression level of the N191Q mutant was only 9.2% relative to that of the eel FSHR-wt, indicating a negligible expression level. The expression levels of the N120Q and N272Q mutants were approximately 35.9% and 24% of the FSHG-wt, respectively. The N288Q mutant had an expression level similar to that of the eel FSHR-wt, despite the mostly impaired cAMP responsiveness. The loss of the cell surface agonist-receptor complexes was very rapid in the cells expressing eel FSHR-wt and FSHR-N288Q mutants. Specifically, the N191Q mutant was completely impaired by the loss of cell surface receptors, despite treatment with a high concentration of the agonist. Therefore, we suggest that the N191 site is necessary for cAMP signal transduction. This finding implies that the cAMP response, mediated by G proteins, is directly related to the loss of cell surface receptors as a result of high-agonist treatment.

TRIB3 regulates FSHR expression in human granulosa cells under high levels of free fatty acids.

BACKGROUND: Granulosa cells (GCs) in cumulus oophorus highly express follicle stimulating hormone receptor (FSHR), which is the most important mediator of both estradiol synthesis and oocyte maturation. Obese women have elevated free fatty acids (FFAs) levels in their follicular fluids and decreased FSHR expression in GCs, which is related to an altered protein kinase B/glycogen synthase kinase 3ß (Akt/GSK3ß) signaling pathway. Such FFA increases accompany 3-fold rises in pseudokinase 3 (TRIB3) expression and reduce the Akt phosphorylation status in both the human liver and in insulinoma cell lines. Therefore, in a high FFA environment, we determined if TRIB3 mediates regulation of FSHR via the Akt/GSK3ß signaling pathway in human GCs. METHODS: GCs from women undergoing in vitro fertilization were collected and designated as high and low FFAs cohorts based on their follicular fluid FFA content. GCs with low FFA levels and a human granulosa-like tumor (KGN) cell line were exposed to palmitic acid (PA), which is a dominate FFA follicular fluid constituent. The effects were assessed of this substitution on the Akt/GSK3ß signaling pathway activity as well as the expressions of TRIB3 and FSHR at both the gene and protein levels by qPCR, Western blot and immunofluorescence staining analyses. Meanwhile, the individual effects of TRIB3 knockdown in KGN cells and p-AKT inhibitors were compared to determine the mechanisms of FFA-induced FSHR downregulation. RESULTS: The average FSH dose consuming per oocyte (FSH dose/oocyte) was elevated and Top embryo quality ratio was decreased in women with high levels of FFAs in their follicular fluid. In these women, the GC TRIB3 and ATF4 protein expression levels were upregulated which was accompanied by FSHR downregulation. Such upregulation was confirmed based on corresponding increases in their gene expression levels. On the other hand, the levels of p-Akt decreased while p-GSK3ß increased in the GCs. Moreover, TRIB3 knockdown reversed declines in FSHR expression and estradiol (E2) production in KGN cells treated with PA, which also resulted in increased p-Akt levels and declines in the p-GSK3ß level. In contrast, treatment of TRIB3-knockdown cells with an inhibitor of p-Akt (Ser473) resulted in rises in the levels of both p-GSK3ß as well as FSHR expression whereas E2 synthesis fell. CONCLUSIONS: During exposure to a high FFA content, TRIB3 can reduce FSHR expression through stimulation of the Akt/GSK3ß pathway in human GCs. This response may contribute to inducing oocyte maturation.

Associations of FSHR and LHCGR gene variants with ovarian reserve and clinical pregnancy rates.

RESEARCH QUESTION: Are there any associations between the variants of FSHR c.2039 G>A (p. Ser680Asn, rs6166) and LHCGR c.935A>G (p. Asn312Ser, rs2293275) and ovarian reserve, ovarian response, clinical pregnancy rate and POSEIDON group? DESIGN: A total of 210 infertile women were enrolled in this prospective study. The gene variants were analysed by the Sanger method. The clinical parameters were analysed based on genotypes. RESULTS: The frequency of heterozygous and homozygous G allele for FSHR c.2039 G>A in the low prognosis group was significantly higher than that in other response groups (P = 0.034); there was no significant association between LHCGR c.935 A>G and ovarian response. Moreover, the serum anti-Müllerian hormone (AMH) concentration, antral follicle count (AFC), oocytes retrieved, metaphase II (MII) oocytes and two-pronuclear (2PN) oocytes in patients with AG genotype for FSHR c.2039 G>A were significantly lower than those with AA genotype. The serum LH concentrations and clinical pregnancy rate of fresh embryo transfer in patients with GG genotype for LHCGR c.935 A>G were significantly higher than that of the AG genotype. In POSEIDON analysis, the low prognosis women with AA genotype for FSHR c.2039 G>A were more likely to appear in subgroup 1 (P = 0.038). CONCLUSION: The FSHR c.2039 G>A variant has a significant beneficial influence on ovarian reserve and ovarian response. The LHCGR c.935 A>G variant is associated with increased clinical pregnancy rate of fresh embryo transfer in infertile women. In addition, the low prognosis women with AA genotype for FSHR c.2039 G>A tend to show better ovarian reserve and prognosis.

Variants Ala307Ala and Ser680Ser of 307 and 680 FSHr polymorphisms negatively influence on assisted reproductive techniques outcome and determine high probability of non-pregnancy in Caucasian patients.

PURPOSE: To determine the influence of different genotypes of Ala307Thr and Asn680Ser FSHr polymorphisms on controlled ovarian stimulation (COS) outcome and pregnancy. METHODS: This study collected blood and physiological and clinical parameters of 517 Caucasian patients (Statistical power ≥ 80%) that underwent COS treatment. Genotypes of Ala307Thr and Asn680Ser polymorphisms were determined using PCR amplification followed by Bsu36I and BsrI digestion, respectively. RESULTS: Ala307Ala and Ser680Ser genotypes associated to worse parameters of COS outcome (preovulatory follicles P = 0.05, in both), justifying their lower pregnancy rate than Non-Ala307Ala, P = 0.01 and Non-Ser680Ser, P = 0.004, respectively or together, (P = 0.003). Within the Non-Ala307Ala group, Thr307Thr genotype showed higher number of fertilized oocytes (P = 0.04) and embryos (P = 0.01) than Non-Thr307Thr, but no influence on pregnancy rate. Ala307Ala and Ser680Ser patients doubled probability of non-pregnancy than Non-Ala307Ala (odds ratio = 2.0) and Non-Ser680Ser (odds ratio = 2.11), respectively. Ala307Ala and Ser680Ser genotypes tend to appear together (P < 0.0001), which increases the probability of non-pregnancy. CONCLUSIONS: Ala307Ala and Ser680Ser genotypes of 307 and 680 FSHr polymorphisms associate to worse COS outcome than its respective Non-Ala307Ala and Non-Ser680Ser. Within the Non-Ala307Ala genotypes, Thr307Thr, although shows higher Fertilized Oocytes and Embryos, do not influence on pregnancy rate. Ala307Ala and Ser680Ser genotypes double the probability of Non-Pregnancy than their respective Non-Ala307Ala and Non-Ser680Ser genotypes. Furthermore, the strong tendency of these genotypes to appear together worsens the probability of pregnancy in these patients.

Relative transcript abundance in porcine cumulus cells collected from different sized follicles.

Crosstalk between the oocyte and surrounding cumulus cells (CCs) is essential for the production of competent oocytes. Previous studies have analysed the relative transcript abundance in oocytes derived from small (SF: <3 mm diameter)- and medium-sized (MF: 3-6 mm diameter) follicles to determine the potential use of SF-derived oocytes in assisted reproductive technologies (ART). The aim of this study was to examine the relative transcript abundance of CCs obtained from cumulus-oocyte complexes (COCs) derived from SF and MF. Nine genes were selected according to their importance for developmental competence: AT-rich interaction domain 1B (ARID1B), bone morphogenic protein receptor 2 (BMPR2), CD44, follicle-stimulating hormone receptor (FSHR), follistatin (FST), inhibin beta-A (INHBA), luteinizing hormone receptor (LHR), nuclear receptor subfamily 2 group F member 6 (NR2F6) and vascular endothelial growth factor A (VEGFA). The expression of these genes was analysed by RT-qPCR. The results pointed to significant differences in five genes, and the relative transcript abundance of SF-derived CCs was lower in the case of INHBA, but higher in FSHR, FST, LHR and NR2F6 compared with MF-derived CCs. We provide information of gene activity in the porcine CCs from different sized follicles, thus improving our understanding of oocyte biology and providing new markers that identify viable and competent oocytes.

Pharmacological Characterization of Low Molecular Weight Biased Agonists at the Follicle Stimulating Hormone Receptor.

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

A genome-wide association study identifies FSHR rs2300441 associated with follicle-stimulating hormone levels.

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) play critical roles in female reproduction, while the underlying genetic basis is poorly understood. Genome-wide association studies (GWASs) of FSH and LH levels were conducted in 2590 Chinese females including 1882 polycystic ovary syndrome (PCOS) cases and 708 controls. GWAS for FSH level identified multiple variants at FSHR showing genome-wide significance with the top variant (rs2300441) located in the intron of FSHR. The A allele of rs2300441 led to a reduced level of FSH in the PCOS group (ß = -.43, P = 6.70 × 10-14 ) as well as in the control group (ß = -.35, P = 6.52 × 10-4 ). In the combined sample, this association was enhanced after adjusting for the PCOS status (before: ß = -.38, P = 1.77 × 10-13 ; after: ß = -.42, P = 3.33 × 10-16 ), suggesting the genetic effect is independent of the PCOS status. The rs2300441 explained sevenfold higher proportion of the FSH variance than the total variance explained by the two previously reported FSHR missense variants (rs2300441 R2 = 1.40% vs rs6166 R2 = 0.17%, rs6165 R2 = 0.03%). GWAS for LH did not identify any genome-wide significant associations. In conclusion, we identified genome-wide significant association between variants in FSHR and circulating FSH first, with the top associated variant rs2300441 might be a primary contributor at the population level.