The effect of polymorphisms in FSHR and FSHB genes on ovarian response: a prospective multicenter multinational study in Europe and Asia.

STUDY QUESTION: Does the presence of single nucleotide polymorphisms (SNPs) in the FSH receptor gene (FSHR) and/or FSH beta subunit-encoding gene (FSHB) influence ovarian response in predicted normal responders treated with rFSH? SUMMARY ANSWER: The presence of FSHR SNPs (rs6165, rs6166, rs1394205) has a statistically significant impact in ovarian response, although this effect is of minimal clinical relevance in predicted normal responders treated with a fixed dose of 150 IU rFSH. WHAT IS KNOWN ALREADY: Ovarian reserve markers have been a breakthrough in response prediction following ovarian stimulation. However, a significant percentage of patients show a disproportionate lower ovarian response, as compared with their actual ovarian reserve. Studies on pharmacogenetics have demonstrated a relationship between FSHR or FSHB genotyping and drug response, suggesting a potential effect of individual genetic variability on ovarian stimulation. However, evidence from these studies is inconsistent, due to the inclusion of patients with variable ovarian reserve, use of different starting gonadotropin doses, and allowance for dose adjustments during treatment. This highlights the necessity of a well-controlled prospective study in a homogenous population treated with the same fixed protocol. STUDY DESIGN, SIZE, DURATION: We conducted a multicenter multinational prospective study, including 368 patients from Vietnam, Belgium, and Spain (168 from Europe and 200 from Asia), from November 2016 until June 2019. All patients underwent ovarian stimulation followed by oocyte retrieval in an antagonist protocol with a fixed daily dose of 150 IU rFSH until triggering. Blood sampling and DNA extraction was performed prior to oocyte retrieval, followed by genotyping of four SNPs from FSHR (rs6165, rs6166, rs1394205) and FSHB (rs10835638). PARTICIPANTS/MATERIALS, SETTING, METHODS: Eligible were predicted normal responder women <38 years old undergoing their first or second ovarian stimulation cycle. Laboratory staff and clinicians were blinded to the clinical results and genotyping, respectively. The prevalence of hypo-responders, the number of oocytes retrieved, the follicular output rate (FORT), and the follicle to oocyte index (FOI) were compared between different FSHR and FSHB SNPs genotypes. MAIN RESULTS AND THE ROLE OF CHANCE: The prevalence of derived allele homozygous SNPs in the FSHR was rs6166 (genotype G/G) 15.8%, rs6165 (genotype G/G) 34.8%, and rs1394205 (genotype A/A) 14.1%, with significant differences between Caucasian and Asian women (P < 0.001). FSHB variant rs10835638 (c.-211 G>T) was very rare (0.5%). Genetic model analysis revealed that the presence of the G allele in FSHR variant rs6166 resulted in less oocytes retrieved when compared to the AA genotype (13.54 ± 0.46 vs 14.81 ± 0.61, estimated mean difference (EMD) -1.47 (95% CI -2.82 to -0.11)). In FSHR variant rs1394205, a significantly lower number of oocytes was retrieved in patients with an A allele when compared to G/G (13.33 ± 0.41 vs 15.06 ± 0.68, EMD -1.69 (95% CI -3.06 to -0.31)). A significantly higher prevalence of hypo-responders was found in patients with the genotype A/G for FSHR variant rs6166 (55.9%, n = 57) when compared to A/A (28.4%, n = 29), ORadj 1.87 (95% CI 1.08-3.24). No significant differences were found regarding the FORT across the genotypes for FSHR variants rs6166, rs6165, or rs1394205. Regarding the FOI, the presence of the G allele for FSHR variant rs6166 resulted in a lower FOI when compared to the A/A genotype, EMD -13.47 (95% CI -22.69 to -4.24). Regarding FSHR variant rs6165, a lower FOI was reported for genotype A/G (79.75 ± 3.35) when compared to genotype A/A (92.08 ± 6.23), EMD -13.81 (95% CI -25.41 to -2.21). LIMITATIONS, REASONS FOR CAUTION: The study was performed in relatively young women with normal ovarian reserve to eliminate biases related to age-related fertility decline; thus, caution is needed when extrapolating results to older populations. In addition, no analysis was performed for FSHB variant rs10835638 due to the very low prevalence of the genotype T/T (n = 2). WIDER IMPLICATIONS OF THE FINDINGS: Based on our results, genotyping FSHR SNPs rs6165, rs6166, rs1394205, and FSHB SNP rs10835638 prior to initiating an ovarian stimulation with rFSH in predicted normal responders should not be recommended, taking into account the minimal clinical impact of such information in this population. Future research may focus on other populations and other genes related to folliculogenesis or steroidogenesis. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by an unrestricted grant by Merck Sharp & Dohme (MSD). N.P.P. reports grants and/or personal fees from MSD, Merck Serono, Roche Diagnostics, Ferring International, Besins Healthcare, Gedeon Richter, Theramex, and Institut Biochimique SA (IBSA). N.L.V. and M.T.H. report consultancy and conference fees from Merck, Ferring, and MSD, outside the submitted work. P.D. has received honoraria for lecturing and/or research grants from MSD, Ferring International, and Merck. D.S. reports grants and/or personal fees from MSD, Ferring International, Merck Serono, Cook, and Gedeon Richter. A.R.N., B.A.M., C.S., J.M., L.H.L., P.Q.M.M., H.T., and S.G. report no conflict of interests. TRIAL REGISTRATION NUMBER: NCT03007043.

AMH mutations with reduced in vitro bioactivity are related to premature ovarian insufficiency.

STUDY QUESTION: Could anti-Müllerian hormone (AMH) mutations be implicated in the development of idiopathic premature ovarian insufficiency (POI)? SUMMARY ANSWER: Three rare or unknown missense variants of the AMH gene were identified in a cohort of 55 POI patients; all three variants showed a drastically reduced in vitro bioactivity. WHAT IS KNOWN ALREADY: Genetic factors are implicated in 5-15% of cases of POI. However, only a few genes have been shown to be involved in its development. AMH inhibits the recruitment of primordial follicles in the ovary and defective or absent AMH leads to premature depletion of the primordial follicle pool in AMH null mice. STUDY DESIGN, SIZE, DURATION: The whole coding sequence and the exon-intron junction of the AMH gene was sequenced in a cohort of 55 POI patients recruited over a period of 8 years. The studied variants were also sequenced in 197 ethnically matched controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: POI was defined as amenorrhea of more than 4 months with increased FSH before the age of 40. Patients with POI resulting from radio- or chemotherapy, surgery, chromosomal anomalies or FMR1 gene pre-mutation were excluded from the study. Recombinant human wild-type (wt) and mutated AMH proteins were produced in HEK293 T cells. KK-1 cells transfected with the AMH receptor type 2 (AMHR2) and a BMP responsive element coupled to a luciferase reporter vector were stimulated with different concentrations of wt AMH and the three tested variants. MAIN RESULTS AND THE ROLE OF CHANCE: The whole coding sequence of the AMH gene could be performed and analyzed for 50 POI patients: 16 variants were found, including 6 missense variants from which 1 was unknown (R444H) and 2 were very rare (G264R and D288E). The variant D288E was also found in one of the patient's mother who also underwent POI at 32 years old. The stimulation of the AMHR2 assessed by the luciferase activity was drastically reduced for the three variants when compared with the wt AMH. LIMITATIONS, REASONS FOR CAUTION: The study is limited by a relatively small number of patients in the POI cohort. WIDER IMPLICATIONS OF THE FINDINGS: This is the first time that the bioactivity of AMH variants related to POI patients is tested in vitro. The functional study showed a drastic reduction of the protein activity for the three variants, supporting their contribution to the development of the ovarian insufficiency. The familial segregation further supports the implication of AMH in the development of POI. STUDY FUNDING/COMPETING INTERESTS: The study was performed thanks to funding from the 'Fondation Erasme'. No conflicts of interest are declared.

Pregnancy outcome after oocyte donation in patients with Turner's syndrome and partial X monosomy.

BACKGROUND: Fertility expectations for patients with Turner's syndrome (TS) have clearly changed in the last three decades. However, medical risks during pregnancy are supposed to be highly increased. The aim of the study was to assess clinical outcome and obstetrical complications in a series of patients with TS in an oocyte donor programme. METHODS: A retrospective study was carried out on 24 women with TS seeking a pregnancy in the Fertility Clinic of the Erasme Hospital from 1992 up until March 2011. RESULTS: Twenty-three patients with TS were included in an oocyte donation cycle. Forty-nine oocyte donation cycles were performed, which led to 45 fresh and 10 frozen-thawed embryo transfers. Altogether, 18 pregnancies were obtained, 10 deliveries (9 singletons and 1 pair of twins), 3 miscarriages and 5 biochemical pregnancies. The clinical pregnancy rate per transfer was 24.4% in fresh cycles and 20% in frozen replacement cycles. Complications of pregnancy occurred in 5 of 10 pregnancies (50%), which led to three premature deliveries because of pregnancy-induced hypertensive disorders. The mean birthweight (g) (±SD) for singletons and twins was 2728 ± 577 and 2335 ± 318, respectively. Four babies were below the 10th percentile. No cardiac complications were observed in any of the pregnant women. CONCLUSIONS: Pregnancy rates after oocyte donation in patients with TS are comparable with those previously published but a high risk of pregnancy hypertensive disorders and a high risk of low birthweight can be highlighted from our study. Strict inclusion criteria and single embryo transfer are necessary to minimize complications during pregnancy in this high-risk group.

Variants of the BMP15 gene in a cohort of patients with premature ovarian failure.

BACKGROUND: Bone morphogenetic protein 15 (BMP15) is an oocyte-derived growth factor acting as a major player in follicle differentiation in mammals. Mutations in the BMP15 gene, some of which lead to defective secretion of bioactive dimers, have been associated with premature ovarian failure (POF) in humans. METHODS: Fifty patients diagnosed with POF with a normal karyotype were included in the study. After DNA extraction and amplification by PCR, the entire coding sequence and intron-exon junctions of BMP15 gene were analysed in the cohort of POF patients and in a control group of 214 patients. RESULTS: Nine variants of the BMP15 gene including six missense substitutions and one insertion of three nucleotides were identified in the POF group. Three of them were previously described as single nucleotide polymorphisms and were also found in the control group. Two variants (H81R and G199R) have not been previously described and were not identified among controls but were not predicted to be deleterious. One variant (A180T) was identified among two POF cases, and also in two controls. One variant (F194S), predicted as potentially deleterious, was identified for the first time in a POF patient but also identified in one control. One variant (L148P), potentially deleterious, previously reported in POF patients, was identified for the first time among controls. The variant 788insTCT, previously identified among POF patients, probably has a low biological impact as it was also found in control patients and is a common polymorphism in sub-Saharan African populations. CONCLUSIONS: Various missense variants of the BMP15 gene were identified among patients with POF. For most variants, the impact of the amino-acid substitution on the protein structure and function was predicted to be low. The two variants predicted as potentially deleterious were also identified among controls and could be considered as rare polymorphisms. Although some of these variants could contribute to the development of POF in a complex manner, the demonstration of their role in the pathogenesis of POF requires additional functional studies.