Mutation Analysis of NR5A1 Encoding Steroidogenic Factor 1 in 77 Patients with 46, XY Disorders of Sex Development (DSD) Including Hypospadias

BACKGROUND: Mutations of the NR5A1 gene encoding steroidogenic factor-1 have been reported in association with a wide spectrum of 46,XY DSD (Disorder of Sex Development) phenotypes including severe forms of hypospadias. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated the frequency of NR5A1 gene mutations in a large series of patients presenting with 46,XY DSD and hypospadias. Based on their clinical presentation 77 patients were classified either as complete or partial gonadal dysgenesis (uterus seen at genitography and/or surgery, n = 11), ambiguous external genitalia without uterus (n = 33) or hypospadias (n = 33). We identified heterozygous NR5A1 mutations in 4 cases of ambiguous external genitalia without uterus (12.1%; p.Trp279Arg, pArg39Pro, c.390delG, c140_141insCACG) and a de novo missense mutation in one case with distal hypospadias (3%; p.Arg313Cys). Mutant proteins showed reduced transactivation activity and mutants p.Arg39Pro and p.Arg313Cys did not synergize with the GATA4 cofactor to stimulate reporter gene activity, although they retained their ability to physically interact with the GATA4 protein. CONCLUSIONS/SIGNIFICANCE: Mutations in NR5A1 were observed in 5/77 (6.5%) cases of 46,XY DSD including hypospadias. Excluding the cases of 46,XY gonadal dysgenesis the incidence of NR5A1 mutations was 5/66 (7.6%). An individual with isolated distal hypopadias carried a de novo heterozygous missense mutation, thus extending the range of phenotypes associated with NR5A1 mutations and suggesting that this group of patients should be screened for NR5A1 mutations

Impact des polymorphismes génétiques sur les chances individuelles des patientes prises en charge en AMP

L aide médicale à la procréation constitue aujourd hui le premier traitement pour les infertilités, mais malgré de nombreux progrès enregistrés, les taux de succès restent autour de 30% par cycle. La réponse ovarienne aux gonadotrophines et l implantation embryonnaire demeurent très variables entre les patientes et très difficile à prévoir. Notre travail a porté sur l étude de l impact des polymorphismes génétiques sur la réponse ovarienne et sur l implantation embryonnaire. Sur les 13 polymorphismes génétiques analysés, le résultat de la stimulation ovarienne, objectivés par le nombre d ovocytes recueillis, varie en fonction du génotype de 4 polymorphismes : FSHR-Asn680Ser, p53-Arg72Pro, ESR2+1730-G/A et AMH-Ile49Ser. Les taux d implantation embryonnaire et de grossesse varient quand à eux en fonction du génotype de 2 polymorphismes : VEGF+405-G/C et TNFa-308-GA individuellement ou associés. A coté de l approche gène candidat, nous avons réalisé une étude whole génome sur l impact des CNPs sur l implantation embryonnaire. Des résultats préliminaires montrent l impact de 2 CNV sur l implantation embryonnaire contenant 2 gènes BTNL3 etGSTT1. Au total, nos résultats montrent que l étude des facteurs génétiques de prédisposition àl infertilité pourrait nous permettre d adapter la prise en charge des patientes en AMP.Medical assistance for procreation is now the first treatment for infertility, but in spite of various advances, success rates remain around 30% per cycle. Ovarian response to gonadotropins and embryo implantation are variable between patients and very difficult to predict. Our work focused on studying the influence of genetic polymorphisms on ovarian response and embryo implantation. Of the 13 polymorphisms analyzed, the result of ovarian stimulation, objectified by the number of oocytes collected, varies with the genotype 4 polymorphisms: FSHR-Asn680Ser, p53-Arg72Pro, ESR2 +1730- G / A and AMH-Ile49Ser. Rates of embryo implantation and pregnancy vary with the genotype of two polymorphisms : VEGF+405-G/C and TNFa-308-GA individually or combined. Besides the candidate gene approach, we performed a whole genome study on the impact of CNPs on embryo implantation. Preliminary results show the impact of two CNVs on two genes containing embryo implantation BTNL3 andGSTT1. Altogether, our results show that the study of genetic factors predisposing to infertility could allow us to tailor the care of patients in AMP.VERSAILLES-BU Sciences et IUT (786462101) / SudocSudocFranceF

Genetic polymorphisms influence the ovarian response to rFSH stimulation in patients undergoing in vitro fertilization programs with ICSI.

INTRODUCTION: Obtaining an adequate number of high-quality oocytes is a major challenge in controlled ovarian hyperstimulation (COH). To date, a range of hormonal and clinical parameters have been used to optimize COH but none have significant predictive value. This variability could be due to the genetic predispositions of single-nucleotide polymorphisms (SNPs). Here, we assessed the individual and combined impacts of thirteen SNPs that reportedly influence the outcome of in vitro fertilisation (IVF) on the ovarian response to rFSH stimulation for patients undergoing intracytoplasmic sperm injection program (ICSI). RESULTS: Univariate analysis revealed that only FSHR, ESR2 and p53 SNPs influenced the number of mature oocytes. The association was statistically significant for FSHR (p=0.0047) and ESR2 (0.0017) in the overall study population and for FSHR (p=0.0009) and p53 (p=0.0048) in subgroup that was more homogeneous in terms of clinical variables. After Bonferroni correction and a multivariate analysis, only the differences for FSHR and ESR2 polymorphisms were still statistically significant. In a multilocus analysis, only the FSHR and AMH SNP combination significantly influenced oocyte numbers in both population (pA), ESR1(-397T>C), BMP15(-9C>G), MTHFR1(677C>T), MTHFR2(1298A>C), HLA-G(-725C>G), VEGF(+405G>C), TNFα(-308A>G), AMHR(-482 A>G), PAI-1 (4 G/5 G), multiplex PCR assay was designed to genotype women undergoing ICSI program. We analyzed the overall study population (n=427) and a subgroup with homogeneous characteristics (n=112)

Associations between Individual and Combined Polymorphisms of the TNF and VEGF Genes and the Embryo Implantation Rate in Patients Undergoing <i>In Vitro</i> Fertilization (IVF) Programs

<div><p>Background</p><p>A multiple pregnancy is now considered to be the most common adverse outcome associated with <i>in vitro</i> fertilization (IVF). As a consequence, the identification of women with the best chances of embryo implantation is a challenge in IVF program, in which the objective is to offer elective single-embryo transfer (eSET) without decreasing the pregnancy rate. To date, a range of hormonal and clinical parameters have been used to optimize eSET but none have significant predictive value. This variability could be due to genetic predispositions related to single-nucleotide polymorphisms (SNPs). Here, we assessed the individual and combined impacts of thirteen SNPs that reportedly influence the outcome of in vitro fertilisation (IVF) on the embryo implantation rate for patients undergoing intracytoplasmic sperm injection program (ICSI).</p><p>Materials and Methods</p><p>A 13 gene polymorphisms: FSHR(Asn680Ser), p53(Arg72Pro), AMH(Ile49Ser), ESR2(+1730G>A), ESR1(−397T>C), BMP15(−9C>G), MTHFR1(677C>T), MTHFR2(1298A>C), HLA-G(−725C>G), VEGF(+405G>C), TNFα(−308A>G), AMHR(−482A>G), PAI-1(4G/5G), multiplex PCR assay was designed to genotype women undergoing ICSI program. We analyzed the total patients population (n = 428) and a subgroup with homogeneous characteristics (n = 112).</p><p>Results</p><p>Only the VEGF(+405G>C) and TNFα(−308A>G) polymorphisms impacted fertilization, embryo implantation and pregnancy rates. Moreover, the combined VEGF+405.GG and TNFα-308.AG or AA genotype occurred significantly more frequently in women with high implantation potential. In contrast, the VEGF+405.CC and TNFα-308.GG combination was associated with a low implantation rate.</p><p>Conclusion</p><p>We identified associations between VEGF(+405G>C) and TNFα(−308A>G) polymorphisms (when considered singly or as combinations) and the embryo implantation rate. These associations may be predictive of embryo implantation and could help to define populations in which elective single-embryo transfer should be recommended (or, conversely, ruled out). However, the mechanism underlying the function of these polymorphisms in embryo implantation remains to be determined and the associations observed here must be confirmed in a larger, more heterogeneous cohort.</p></div

TNFα/VEGF alleles in the selected subgroup: description and ART results.

a<p>: TNFAA+AG-VEGF GG+GC vs. TNFGG-VEGF GG+GC.</p>b<p>: TNFAA+AG-VEGF.GG+GC vs. TNFGG-VEGFCC.</p>c<p>: TNFGG-VEGF GG+GC vs. TNFGG-VEGFCC.</p><p>TNFα/VEGF alleles in the selected subgroup: description and ART results.</p

VEGF alleles in the total patient population: description and ART results.

<p>VEGF alleles in the total patient population: description and ART results.</p

VEGF alleles in the selected subgroup: description and ART results.

<p>VEGF alleles in the selected subgroup: description and ART results.</p

Electrophoregram profile with the 13 polymorphisms genotyped.

<p>The first and second lines show alleles fluorescently tagged with FAM and Hex dyes, respectively. The last electropherogram is the RO×500 size standard.</p

Primer design for the selected SNPs.

<p>Primer design for the selected SNPs.</p