Variants in follicle-stimulating hormone receptor gene in infertile brazilian men and the correlation to FSH serum levels and sperm count

The aim of the study was to analyze the distribution of the follicle-stimulating hormone (FSH) receptor (FSHR) Ala307Thr and Asn680Ser polymorphisms in infertile Brazilian men and evaluate the possible role of these polymorphisms on the serum levels of FSH and in sperm count. A case-control study was performed comprising138 infertile men with nonobstructive azoospermia (n = 53) or severe oligozoospermia (n = 85), and 217 fertile men as controls. Genotyping of FSHR polymorphisms was performed by real-time polymerase chain reaction (PCR). The results were analyzed statistically and a P value <.05 was considered significant. According to the sperm count, relatively similar FSHR polymorphisms genotype and allele frequencies were found among the groups, and combined genotypes of 2 polymorphisms did not identify a haplotype associated with sperm count. Considering FSH serum level according to genotypes of the Ala307Thr and Asn680Ser polymorphisms individually, statistical analysis showed no difference among the groups. When the combined genotypes of the FSHR polymorphisms were compared to FSH serum levels, no difference was also found among the groups. In conclusion, the findings demonstrate that, in Brazilian population studied, genetic variations, Asn680Ser and Thr307Ala, of the FSHR gene are not correlated with serum FSH levels or sperm count in male infertility.     

Identification of a novel m.9588G > A missense mutation in the mitochondrial COIII gene in asthenozoospermic Tunisian infertile men

Purpose:

Infertility affects 10–15 % of the population, of which, approximately 40 % is due to male etiology consisting primarily of low sperm count (oligozoospermia) and/or abnormal sperm motility (asthenozoospermia). It has been demonstrated that mtDNA base substitutions can greatly influence semen quality.

Methods:

In the present study we performed a systematic sequence analysis of the mitochondrial cytochrome oxidase III (COIII) gene in 31 asthenozoospermic infertile men in comparaison to normozoospermic infertile men (n=33) and fertile men (n=150) from Tunisian population.

Results:

A novel m.9588G>A mutation was found in the mtDNA sperm’s in all asthenozoospermic patients and was absent in the normozoospermic and in fertile men. The m.9588G>A mutation substitutes a highly conserved Glutamate at position 128 to Lysine. In addition, PolyPhen-2 analysis predicted that this variant is “probably damaging”.     

The common variant N372H in BRCA2 gene may be associated with idiopathic male infertility with azoospermia or severe oligozoospermia

OBJECTIVE: To explore the possible association between the common single nucleotide polymorphism N372H in human breast cancer susceptibility gene 2 (BRCA2) and the idiopathic male infertility with azoospermia or severe oligozoospermia. STUDY DESIGN: The study included 240 infertile patients with idiopathic azoospermia or severe oligozoospermia and 250 fathered controls. The allele and genotype frequencies of the polymorphism N372H in BRCA2 gene were investigated in both patients and controls using denaturing high performance liquid chromatography analysis (DHPLC). RESULTS: The frequency of allele H of the polymorphism N372H in patients was significantly higher than that of the controls (23.5% versus 17.6%, OR = 1.49, 95% CI 1.06-1.97, P = 0.02) and the subjects bearing rare allele H (NH + HH) significantly increased in patients compared with controls (41.7% versus 32.4%, 95% CI 1.03-2.15, P = 0.03). CONCLUSION: The results of this study suggested that the polymorphism N372H in BRCA2 gene may be associated with idiopathic male infertility with azoospermia or severe oligozoospermia.     

Study of single nucleotide polymorphism (rs28368082) in SPO11 gene and its association with male infertility

Purpose

The present study is a case–control analysis of a SNP (rs28368082) in exon 7 of the SPO11 gene and its possible association with male infertility in three provinces of Iran. We also searched for genetic differences among populations.

Methods

Using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) analysis, we genotyped 113 infertile men and 50 fertile controls. Then, samples consisting SNP, as determined by PCR-RFLP, were genotyped by sequencing. The differences in genotype distributions between cases and fertile controls were examined using Chi-squared analysis. The genetic difference between individuals with mutated nucleotide was investigated by phylogenetic trees. Genetic difference among populations (provinces) was analyzed through ANOVA test, and homogeneity was investigated using STRUCTURE and K-means clustering analysis.

Results

According to the statistical analysis, the SNP was significantly associated with male infertility in all populations except oligozoospermic cases of the Center region. The phylogenetic trees showed partial genetic variation among the individuals, although ANOVA test showed no significant genetic difference between populations (provinces) for both azoospermic, and oligozoospermic cases. Eventually, we affirmed that individuals in the inclusive populations had genetic difference, but it was not statistically significant for dividing underlying populations to separate groups, so each population was homogenous.

Conclusion

Our study indicates that the mentioned polymorphism in SPO11 gene may be linked to the susceptibility of azoospermia and oligozoospermia male infertility in three provinces of Iran. Further studies are required to support obtained results. It finally should be noted that the possible association between a particular SNP and a specific disease completely depends on the underlying population.     

卵泡刺激素受体基因的点突变和单核苷酸多态性

卵泡刺激素受体(FSHR)是由FSHR基因编码的G蛋白耦联受体蛋白,由胞外区、跨膜区及胞内区3部分构成。胞外区与FSH特异性结合组成FSH/FSHR系统,在人类生殖过程中发挥着重要作用。FSHR基因上突变基因分为活性突变和失活突变2种,失活突变可能导致原发或继发性闭经、高促性腺激素性功能障碍、卵巢早衰及生精功能障碍等生殖疾病,活性突变主要与卵巢过度刺激综合征(OHSS)关系密切。FSHR基因的点突变出现概率非常小,大部分仅有1次报道,而大多数FSHR基因突变为单核苷酸多态性。卵巢和睾丸的正常发育及发挥功能均依赖于完整的FSHR介导,FSHR突变对两性生殖表型的影响存在着差异。     

Induction of infertility in adult male bonnet monkeys by immunization with phage-expressed peptides of the extracellular domain of FSH receptor

Active immunization of proven fertile adult male bonnet monkeys (Macaca radiata) with phage-expressed follicle-stimulating hormone receptor (FSHR)-specific peptides from the extracellular domain resulted in a progressive drop in sperm count with all animals becoming azoospermic by day 100. However, serum testosterone concentrations were unaltered during the entire course of study and animals exhibited normal mating behaviour. Breeding studies with proven fertile female monkeys revealed that all the immunized males were infertile. Following interruption of immunization on day 225, sperm counts returned to normal with restoration of fertility. These results indicate that infertility can be induced in adult male monkeys by interfering with the action of FSH using specific peptides of the extracellular domain of FSHR as antigens, without the risk of producing cross-reacting antibodies to the other glycoprotein hormones.     

Effects of the FSH receptor gene polymorphism p.N680S on cAMP and steroid production in cultured primary human granulosa cells

The study was designed to evaluate in vitro the cellular mechanisms of the single nucleotide polymorphism (SNP) p.N680S of the FSH receptor gene (FSHR) in human granulosa cells (GC) and included patients homozygous for the FSHR SNP (NN/SS) undergoing ovarian stimulation. GC were isolated during oocyte retrieval and cultured for 1–7 days. Basal oestradiol and progesterone concentrations were measured after short-term culture. The kinetics of cAMP, oestradiol and progesterone concentrations in response to various amounts of FSH were analysed in a 6–7 day culture. Basal oestradiol, but not progesterone, concentrations on day 1 of GC culture, were significantly higher in NN compared with SS (P = 0.045), but non-responsive to FSH stimulation. Immunofluorescence microscopy demonstrated the re-appearance of FSHR expression with increasing days in culture. Upon stimulation with FSH, GC cultured for 6–7 days displayed a dose-dependent increase of cAMP, oestradiol and progesterone but no difference in the EC50 values between both variants. Primary long-term GC cultures are a suitable system to study the effects of FSH in vitro. However, the experiments suggest that factors down-stream of progesterone production or external to GC might be involved in the clinically observed differences in an FSHR variant-mediated response to FSH.     

Frequency distribution of polymorphisms in the FSH receptor gene in infertility patients of different ethnicity

Studies on the frequency distribution of follicle-stimulating hormone receptor (FSHR) polymorphisms report conflicting results. It has been suggested that ethnicity might influence these outcomes. Therefore, the aim of this study was to determine the frequency distribution of FSHR polymorphisms at position 680 of exon 10 within a large group of women with fertility problems from different ethnic backgrounds. A total of 1771 women of different ethnic origin (Caucasian, Asian, Hindustani, Creole and Mediterranean) were studied. FSHR single-nucleotide polymorphisms at codon 680 of exon 10 were determined by restriction fragment length polymorphism of amplicons generated by polymerase chain reaction. Genotypes were compared with serum FSH concentrations and between different ethnic groups. A significantly lower number of Asians (10.5%) were found to have the Ser680Ser receptor variant compared with Caucasians (21.5%) and Mediterraneans (22.3%) (P = 0.010). FSH concentrations did not differ between the various ethnic groups, or the different FSHR polymorphisms. In conclusion, the Ser680Ser receptor variant is less common in the Asian subgroup compared with Caucasians and Mediterraneans. This indicates that, when comparing allelic frequency distributions of the FSHR polymorphism variants, ethnic background should be accounted for. FSH concentrations did not differ between FSHR polymorphisms or between ethnic groups.     

Mutations in the follicle-stimulating hormone-beta (FSH beta) and FSH receptor genes in mice and humans

Follicle-stimulating hormone (FSH), a dimeric glycoprotein synthesized in the anterior pituitary gland, is important for the production of sex steroids and gametes. FSH-beta (FSH beta) and FSH receptor (FSHR) knockout mice display impaired ovarian follicular development and infertility in females and small testes, oligospermia, and fertility in males. Humans with FSH beta gene mutations tend to have a more severe phenotype than those with FSHR gene mutations, although infertility and varying degrees of impaired sex steroid production occur in both types of mutations. Data from human and mouse mutations in the FSH beta and FSHR genes suggest that FSH is necessary for normal pubertal development and fertility in males and females.     

Mutations in KISS1 are not responsible for idiopathic hypogonadotropic hypogonadism in Chinese patients

PurposeTo investigate whether mutations in the KISS1 gene are present in 170 Chinese patients with idiopathic hypogonadotropic hypogonadism (IHH).MethodsMutational screening of the KISS1 gene was performed in 170 Chinese patients with IHH (133 male cases and 37 female cases) and 187 matched controls (94 males and 93 females).ResultsTwo known single-nucleotide polymorphisms (SNP), c. 58G > A in exon 1 and c. 242C > G in exon 2, were identified. However, no difference of genotype and allelic frequencies between cases and controls was observed.ConclusionsThe results suggest that mutations in the coding sequence of KISS1 are not common in patients with IHH in this Chinese population.     

Association between single-nucleotide polymorphisms of DNMT3L and infertility with azoospermia in Chinese men

The gene for DNA methyltransferase 3-like protein (DNMT3L) is essential for normal spermatogenesis and may be involved with spermatogenetic impairment and male infertility. To explore the possible association between the DNMT3L gene and male infertility, this study investigated allele, genotype and haplotype frequencies of three single nucleotide polymorphism (SNP) loci, rs2070565, rs2276248 and rs7354779, of DNMT3L in 233 infertile patients with azoospermia and 249 fertile controls from a population of Chinese men using polymerase chain reaction/restriction fragment length polymorphism. Results showed that the frequencies of allele A (20.6% versus 14.9%; P = 0.022) and the allele A carrier (GA + AA; 37.8% versus 28.1%; P = 0.027) in azoospermic patients were significantly higher than those in controls at the rs2070565 locus. The haplotype AAA frequency was significantly higher (18.1% versus 12.4%; P = 0.02) while the haplotype GAA frequency was significantly lower (53.2% versus 62.1%; P = 0.007) in infertile patients compared with fertile controls. These results indicated that SNP rs2070565, as well as haplotypes AAA and GAA, may be associated with male infertility and suggest that DNMT3L may contribute to azoospermia susceptibility in humans.     

Mutation analysis of the follicle-stimulating hormone receptor gene in girls with gonadotropin-independent precocious puberty resulting from autonomous cystic ovaries

OBJECTIVE: To search for germline activating mutations of the FSH receptor in girls with gonadotropin-independent precocious puberty. DESIGN: Molecular studies in human tissue. SETTING: Four girls with polycystic ovaries and gonadotropin-independent isosexual precocious puberty without clinical and molecular features of McCune-Albright syndrome. INTERVENTION(S): Peripheral blood was used for DNA extraction. The alpha-subunit of the Gs gene and the entire exon 10 of FSH receptor gene were amplified by polymerase chain reaction (PCR). Gs-alpha mutations characteristic of McCune-Albright syndrome were excluded by denaturating gradient gel electrophoresis (DGGE) and allele-specific PCR. Exon 10 of the FSH receptor gene was analyzed by DGGE and direct sequencing. MAIN OUTCOME MEASURE(S): Results of DGGE and direct sequencing. RESULT(S): No germline activating mutations were detected in exon 10 of our patients. Instead, two previously described polymorphisms were found, leading to the substitution of alanine for threonine at position 307 and of serine for asparagine at position 680 of the FSH receptor molecule. CONCLUSION(S): Germline activating mutations were not found in exon 10 of the FSHR gene in any of our patients. Further studies, preferably in ovarian tissue, will be required to exclude the presence of somatic activating mutations of the FSH receptor in these patients.     

A novel mutation in exon8 of the follicle-stimulating hormone receptor in a woman with primary amenorrhea

The FSH receptor (FSHR) gene mutation are rare, but recently have been detected in several cases with primary amenorrhea. We report a 25-year-old female who had primary amenorrhea, moderately developed secondary sex characteristics and normal sized ovaries with small antral follicles. Her ovaries reacted slightly to clomiphene citrate therapy; they did not react to the ordinary dose of human menopausal gonadotropin (hMG) (150 IU/day × 9 days), but did react to high-dose hMG (300 IU/day × 6 days) treatment. These ovarian responses to hMG stimulation suggested an FSHR dysfunction of her ovaries. We extracted genomic DNA and analysed FSHR gene sequence after we obtained the written informed consent from the patient under the approval by the Ethics Committee of Yamaguchi Grand Medical Centre and the Yamaguchi University School of Medicine. Direct sequencing revealed a heterozygous mutation 662T?>G resulting in the substitution of valine for glycine at position 221 in exon8 of the FSHR extracellular domain, which was also confirmed by the PCR-RFLP method. The sequencing results also showed two SNPs, 919G?>A (Ala307Thr) and 2039G?>A (Ser680Asn), in exon10. A novel mutation in exon8 of FSHR was identified in a woman with primary amenorrhea whose ovaries reacted to high-dose hMG treatment.     

Association of common SNP rs1136410 in PARP1 gene with the susceptibility to male infertility with oligospermia

Purpose

This study aims to explore possible associations between polymorphisms of common SNP rs1136410 and rS1805405 in PARP1 gene and male infertility with spermatogenesis impairment.

Methods

The polymorphic distributions of SNP rs1136410 and rS1805405 were investigated by polymerase chain reaction and restriction fragment length polymorphism analysis in a Chinese cohort including 371 infertile patients with idiopathic azoospermia or oligospermia and 231 controls.

Results

Significant differences in the frequencies of allele and genotype of SNP rs1136410 were observed between patients with oligospermia and controls. The allele C (46.3 % vs. 36.4 %, P = 0.003) and genotype CC (22.6 % vs. 13.4 %, P = 0.014) significantly increased, whereas genotype TT (30 % vs. 40.7 %, P = 0.021) significantly decreased in patients with oligospermia compared with controls at this SNP locus.

Conclusions

These results indicated that genotype CC of SNP rs1136410 may increase the risk of oligosoermia and genotype TT of rs1136410 may have some protective effect from oligospermia, suggesting that the polymorphism of SNP rs1136410 in PARP1 gene may modify the susceptibility to male infertility with oligospermia.     

Association between FSHR polymorphisms and polycystic ovary syndrome among Chinese women in north China

Purpose

Polycystic ovary syndrome (PCOS) is a common endocrine disorder disease among women in reproductive-age. Since follicle stimulating hormone (FSH) exerts important biological functions, the association between PCOS and FSH receptor (FSHR) polymorphisms attracts wide attention. The aim of this study was to evaluate whether polymorphisms of FSHR at 307 and 680 codons are associated with PCOS patients in China.

Methods

Patients with PCOS (n = 215) and controls (n = 205) were recruited from Shanxi Province in north China. They are Han ethnics. Genomic DNA was isolated from the venous blood. The Ala307Thr and Ser680Asn polymorphisms of FSHR were analyzed by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) and direct DNA sequencing.

Results

The distributions of genotype and allele of Ala307Thr and Ser680Asn polymorphisms of FSHR were not statistically different between the PCOS patients and the controls. Analysis of the frequency of FSHR polymorphisms showed no statistical difference among the PCOS patients with different obesity standards. Although there were no statistical differences in the most of the endocrine parameters including LH, LH/FSH, E2, P and T as well as the clinical pregnancy rate, there were significant differences in the levels of FSH and PRL among PCOS patients carrying different genotypes of Ala307Thr and Ser680Asn polymorphisms.

Conclusion

The Ala307Thr and Ser680Asn polymorphisms of FSHR are not associated with PCOS in Han ethnic Chinese women in north China. The FSHR polymorphisms was related to the levels of FSH and PRL but not other PCOS-associated endocrine hormones as well as clinical pregnancy rate in PCOS patients of Han Chinese ethnical population.     

MTHFR C677T polymorphism associates with unexplained infertile male factors

PURPOSE: To determine whether 5,10-methylenetetrahydrofolate reductase (MTHFR C677T and A1298C) genotype is associated with male infertility. METHODS: Analysis of cytogenetic, Y chromosomal microdeletion assay (Yq), and the C677T and A1298C polymorphisms of the MTHFR gene by pyrosequencing and PCR-Restriction Fragment Length Polymorphism (RFLP) method. SAS 8.1 assessed the statistical risk of MTHFR genotype. RESULTS: The homozygous (T/T) C677T polymorphism of the MTHFR gene was present at a statistically high significance in unexplained infertile men with normal karyotype, instead at no significance in explained infertile men with chromosomal abnormality or Y chromosome deletion. There was no statistically significance of A1298C variation in infertile males. CONCLUSIONS: The MTHFR 677TT genotype may be a genetic risk factor for male infertility, especially with severe OAT and non-obstructive azoospermia in unexplained infertile males.     

Single-nucleotide polymorphism rs 175080 in the MLH3 gene and its relation to male infertility

PurposeMLH3, a MutL homolog protein in mammals playing a role in DNA mismatch repair, is associated with spermatogenesis and male infertility. The purpose of the present study was to investigate the association of the single-nucleotide polymorphism (SNP), rs 175080 in the MLH3 gene, with sperm parameters in a Greek population.MethodsThe study included 300 men of couples undergoing in vitro fertilization/intracytoplasmic sperm injection-embryo transfer (IVF/ICSI-ET) treatments (years 2011–2013). Genomic DNA was extracted from 300 peripheral blood samples, and conventional quantitative real-time PCR was performed for genotyping. Of them, 122 were from men used as “controls” and 178 from men used as “cases.” Allocation to the two groups was based on sperm concentrations (≥15 and <15 million/ml, respectively). Serum FSH, LH, estradiol, testosterone, and prolactin concentrations as well as sperm parameters were compared between three genotypes (GG, GA, and AA). Furthermore, the frequencies of these three genotypes were compared between “cases” and “controls.”ResultsAnthropometric parameters and hormonal values did not differ significantly between the three genotypes. Significantly lower sperm concentrations were found in men with the AA genotype as compared to men with the GG and GA genotypes (p < 0.001). The AA genotype had the lower progressive motility values as compared to the other two genotypes (p < 0.05). Also, there was a significantly different distribution of the frequencies of the three genotypes between “cases” and “controls” (p < 0.001).ConclusionsIt is suggested that the studied SNP in the MLH3 gene may be linked to oligozoospermia in Caucasian men of a certain area.