Exome sequencing and functional analyses revealed CETN1 variants leads to impaired cell division and male fertility.

Human spermatogenesis requires an orchestrated expression of numerous genes in various germ cell subtypes. Therefore, the genetic landscape of male infertility is highly complex. Known genetic factors alone account for at least 15% of male infertility. However, ~40% of infertile men remain undiagnosed and are classified as idiopathic infertile men. We performed exome sequencing in 47 idiopathic infertile men (discovery cohort), followed by replication study (40 variants in 33 genes) in 844 infertile men and 709 controls using Sequenom MassARRAY® based genotyping. We report 17 variants in twelve genes that comprise both previously reported (DNAH8, DNAH17, FISP2 and SPEF2) and novel candidate genes (BRDT, CETN1, CATSPERD, GMCL1, SPATA6, TSSK4, TSKS and ZNF318) for male infertility. The latter have a strong biological nexus to human spermatogenesis and their respective mouse knockouts are concordant with human phenotypes. One candidate gene CETN1, identified in this study, was sequenced in another independent cohort of 840 infertile and 689 fertile men. Further, CETN1 variants were functionally characterized using biophysical and cell biology approaches. We demonstrate that CETN1 variant- p.Met72Thr leads to multipolar cells, fragmented nuclei during mitosis leading to cell death and show significantly perturbed ciliary disassembly dynamics. Whereas CETN1-5' UTR variant; rs367716858 leads to loss of a methylation site and increased reporter gene expression in vitro. We report a total of eight novel candidate genes identified by exome sequencing, which may have diagnostic relevance and can contribute to improved diagnostic workup and clinical management of male infertility.

TEX13B is essential for metabolic reprogramming during germ cell differentiation.

STUDY QUESTION: What is the functional significance of Tex13b in male germ cell development and differentiation? SUMMARY ANSWER: Tex13b regulates male germ cell differentiation by metabolic reprogramming during spermatogenesis. WHAT IS KNOWN ALREADY: Studies in mice and humans suggest that TEX13B is a transcription factor and is exclusively expressed in germ cells. STUDY DESIGN, SIZE, DURATION: We sequenced the coding regions of TEX13B in 628 infertile men and 427 ethnically matched fertile control men. Further, to identify the molecular function of Tex13b, we created a Tex13b knockout and conditional overexpression system in GC-1spg (hereafter, GC-1) cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: Our recent exome sequencing study identified novel candidate genes for male infertility. TEX13B was found to be one of the potential candidates, hence we explored the role of TEX13B in male infertility within a large infertile case-control cohort. We performed functional analyses of Tex13b in a GC-1 cell line using CRISPR-Cas9. We differentially labelled the cell proteins by stable isotope labelling of amino acids in cell culture (SILAC) and performed mass spectrometry-based whole-cell proteomics to identify the differential protein regulation in knockout cells compared to wild-type cells. We found that Tex13b knockout leads to downregulation of the OXPHOS complexes and upregulation of glycolysis genes, which was further validated by western blotting. These results were further confirmed by respirometry analysis in Tex13b knockout cells. Further, we also performed a conditional overexpression of TEX13B in GC-1 cells and studied the expression of OXPHOS complex proteins by western blotting. MAIN RESULTS AND THE ROLE OF CHANCE: We identified a rare variant, rs775429506 (p.Gly237Glu), exclusively in two non-obstructive-azoospermia (NOA) men, that may genetically predispose these men for infertility. Further, we demonstrated that Tex13b functions in the transcription regulation of OXPHOS complexes. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: We examined the function of Tex13b in GC-1 in vitro by knocking out and conditional overexpression, for understanding the function of Tex13b in germ cells. Unfortunately, this could not be replicated in either an animal model or in patient-derived tissue due to the non-availability of an animal model or patient's testis biopsies. WIDER IMPLICATIONS OF THE FINDINGS: This study identified that Tex13b plays an important role in male germ cell development and differentiation. The findings of this study would be useful for screening infertile males with spermatogenic failure and counselling them before the implementation of assisted reproduction technique(s). STUDY FUNDING/COMPETING INTEREST(S): Funding was provided by the Council of Scientific and Industrial Research (CSIR) under the network project (BSC0101 and MLP0113) and SERB, the Department of Science and Technology, Government of India (J C Bose Fellowship: JCB/2019/000027). The authors do not have any competing interest.

DNMT1 and DNMT3B gene variants and their association with endometriosis in South Indian women.

BACKGROUND: Endometriosis is a multifactorial estrogen dependent gynecological disease characterized by implantation of functional endometrial tissue at ectopic positions. Though this disease is benign, it is associated with an increased risk of malignant transformation. Epigenetic disruptions like aberrant DNA methylation, resulting changes in gene expression capacity, are important in tumor progression and malignant cellular transformation. Therefore, variation in genes involved in DNA methylation might lead to disease susceptibility. PURPOSE: To investigate the association between DNA methyl transferases (DNMT1 and DNMT3B) single nucleotide polymorphisms (SNPs) and the risk of endometriosis in South Indian women. METHODS: In the present study, we examined the genotypic and allele distribution of DNMT1 (rs10423341C/A, rs2228611G/Aandrs4804490C/A) and DNMT3B (rs1569686G/T) among the endometriosis patients (n = 150) and controls (n = 150). The genotypes were analyzed by polymerase chain reaction (PCR) and sequencing methods. Haplotype frequencies for multiple loci and the standardized disequilibrium coefficient (D') for pairwise linkage disequilibrium (LD) were surveyed by Haploview Software. RESULT: Significant increase in the frequencies of DNMT1 rs10423341 (P = 0.04601), rs2228611 (P = 0.00175) and DNMT3B rs1569686 (P = 0.033) genotypes and alleles was observed in patients compared to controls. In addition, the frequency of A/A/C (P = 0.0065) haplotype was significantly high in patients. But the DNMT1 (rs4804490) SNP did not show significant association with the disease. CONCLUSION: The DNMT1 and DNMT3B polymorphism may constitute an inheritable risk factor for endometriosis in South Indian women. To the best of our knowledge there is no reported study on the association of polymorphisms in DNMT1 and DNMT3B with endometriosis risk.

Histone deacetylase 1, Sirtuin 1, and Sirtuin 3 single-nucleotide polymorphisms and the risk of endometriosis in South Indian women.

The aim of the study was to investigate the association between Histone deacetylase 1 (HDAC1), Sirtuin1 (SIRT1), and Sirtuin3 (SIRT3) single-nucleotide polymorphisms (SNPs) and risk of endometriosis in South Indian women. A total of 300 subjects were recruited in this case-control study comprising 150 affected women and 150 women with no evidence of disease. All the subjects were of South Indian origin. The genotyping of HDAC1, SIRT1, and SIRT3 SNPs (rs1741981T/C, rs144124002A/G, and rs536715G/A) was carried out on DNA from subjects by PCR-RFLP and sequencing analysis. The genotype (p = .00782) and allele (p = .02561) frequencies of the HDAC1 rs1741981 polymorphism showed significant difference between cases and controls. In contrast, SIRT1 (rs144124002) and SIRT3 (rs536715) SNPs did not show significant association with the disease. The HDAC1 polymorphism may constitute a heritable risk factor for endometriosis in South Indian women. To date, there is no reported study on the association of polymorphisms in HDAC1, SIRT1, and SIRT3 with endometriosis risk. Impact StatementWhat is already known on this subject? Endometriosis is a benign gynaecological disease characterised by the implantation of functional endometrial tissue at ectopic positions, associated with an increased risk of malignant transformation. Epigenetic mechanisms are essential for normal development and maintenance of tissue-specific gene expression patterns. Histone modification, including deacetylation of lysine residues by HDACs, is a key epigenetic mechanism of gene expression regulation in endometriosis, therefore genetic variation in HDACs causing epigenetic control defects might lead to disease susceptibility.What do the results of this study add? Our study shows that the HDAC1 SNP is significantly associated with endometriosis in South Indian women, whereas the SNPs of SIRT1 and SIRT3 could not show any association with the disease.What are the implications of these findings for clinical practice and/or further research? The polymorphism of HDAC1 rs1741981 could be used as an important marker of genetic susceptibility to endometriosis development. Analysis of this SNP might help to identify patients at high risk for disease outcome.

Association of genetic variations in phosphatase and tensin homolog (PTEN) gene with polycystic ovary syndrome in South Indian women: a case control study.

PURPOSE: The purpose of the study was to investigate the association between gene phosphate and tensin homolog (PTEN) single nucleotide polymorphisms (SNPs) and risk of developing polycystic ovary syndrome (PCOS) in South Indian women. PTEN is one of the most important tumor suppressor genes that regulate cell proliferation, migration, and death. It is also involved in the maintenance of genome stability. PCOS is one of the most common endocrine disorders among women of reproductive age. It is a heterogeneous syndrome characterized by abnormal reproductive cycles, irregular ovulation, hormonal imbalance, hyperandrogenism, acne and hirsutism. RESEARCH QUESTION: What is the association status of PTEN SNPs with PCOS? METHODS: A total of 240 subjects were recruited in this case-control study comprising 110 patients with PCOS and 130 individuals without PCOS. All the subjects were of South Indian origin. The genotyping of PTEN SNPs (rs1903858 A/G, rs185262832G/A and rs10490920T/C) was carried out on DNA from subjects by polymerase chain reaction (PCR) and sequencing analysis. Haplotype frequencies for multiple loci and the standardized disequilibrium coefficient (D') for pairwise linkage disequilibrium (LD) were surveyed by Haploview Software. RESULTS: Our results showed significant increase in the frequencies of rs1903858 A/G (P = 0.0016), rs185262832 G/A (P = 0.0122) and rs10490920 T/C (P = 0.0234) genotypes and alleles in cases compared to controls. CONCLUSION: The PTEN (rs1903858A/G, rs185262832G/A and rs10490920T/C) gene polymorphisms may constitute an inheritable risk factor for PCOS in South Indian women.

Vitamin D receptor gene polymorphisms and risk of polycystic ovary syndrome in South Indian women.

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder of reproductive age women. Emerging evidence suggests that Vitamin D Receptor (VDR) might be a causal factor for characteristics associated with PCOS such as obesity and type 2 diabetes. Present study investigated association between VDR gene BsmI A/G (rs1544410), ApaI A/C (rs7975232) and TaqI T/C (rs731236) single nucleotide polymorphisms and PCOS risk in South Indian women. Genotyping of VDR gene SNPs was carried out in PCOS patients (n = 95) and controls (n = 130) by PCR-RFLP method and confirmed by sequencing analysis. Haplotype frequencies for multiple loci and the standardized disequilibrium coefficient (D') for pairwise linkage disequilibrium (LD) were assessed by Haploview software. Results showed significantly increased frequencies of BsmI G/G (p = .0197), ApaI C/C (p = .048), TaqI C/C (p = .044) genotypes and BsmI G (p = .0181), ApaI C (p = .0092), TaqI C (p = .0066) alleles in patients compared to controls. In addition, the frequency of the 'BsmI G, ApaI C, TaqI C' haplotype was also significantly elevated in patients (p = .0087). In conclusion, the VDR gene BsmI A/G ApaI A/C TaqI T/C and haplotype may constitute an inheritable risk factor for PCOS in South Indian women.

BRCA1 alterations are associated with endometriosis, but BRCA2 alterations show no detectable endometriosis risk: a study in Indian population.

PURPOSE: To investigate the role of genetic variations and expression alterations of BRCA1 and BRCA2 genes in the pathophysiology of endometriosis. METHODS: A genetic association study was conducted in 573 endometriosis cases and 490 controls of Indian origin. We genotyped 13 selected promoter SNPs of BRCA1 gene and 2 selected promoter SNPs of BRCA2 gene by PCR-sequencing analysis. In addition, to better understand genetic contributions to the pathophysiology of endometriosis, the expression pattern of BRCA1 & 2 was analyzed in the eutopic endometria of endometriosis cases and controls by western-blot and immunohistochemical analysis. RESULTS: Our results revealed significant association between BRCA1 rs71361504 (-/GTT) SNP and endometriosis risk in Indian women (P < 0.0001), while the remaining SNPs of both BRCA1 & 2 genes showed no difference between cases and controls. Western-blot and immunohistochemical analysis revealed significantly decreased BRCA1 expression levels in eutopic endometria of patients compared with controls (P < 0.05). Furthermore, nuclear BRCA1 was frequently lost compared with cytoplasmic BRCA1 in eutopic endometria of patients. Expression of BRCA2 did not differ between patients and controls. CONCLUSIONS: BRCA1 rs71361504 SNP may modify the endometriosis risk in Indian women. In addition, decreased expression of BRCA1 may play an important role in the pathophysiology of endometriosis. The analysis of BRCA1 genetic variants and/or expression might help to identify patients at high risk for disease outcome.

Mutations in the PTEN tumor gene and risk of endometriosis: a case-control study.

STUDY QUESTION: Are mutations in the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene associated with endometriosis? SUMMARY ANSWER: Loss of heterozygosity (LOH) at the 10q23.3 locus, PTEN somatic mutations and changes in the levels and distribution of proteins in the PTEN-PI3K/Akt signal transduction pathway are associated with endometriosis. WHAT IS KNOWN ALREADY: Endometriosis has a strong genetic basis. Recent genome-wide association and linkage studies have reported a significant association of endometriosis with 7p15.2, 9p21 and 10q23-26 loci. PTEN, which maps to 10q23.3, acts as a tumor suppressor gene through the action of its phosphatase protein product, phosphatase and tensin homolog (PTEN). This phosphatase is involved in the regulation of the cell cycle, and mutations of PTEN are a step in the development of many cancers. STUDY DESIGN, SIZE, DURATION: A total of 1252 subjects of Indian origin (endometriosis patients = 752; controls = 500) were recruited to participate in this case-control study. Recruitment took place from 2001 to 2009 at Institute of Reproductive Medicine (IRM), Kolkata, India; Infertility Institute and Research Centre (IIRC), Secundrabad, India and Vasavi Medical and Research Centre, Hyderabad, India. PARTICIPANTS/MATERIALS, SETTING, METHODS: LOH on 10q, 9p and 7p was analyzed in analogous ectopic-eutopic endometria along with blood samples from 32 advanced stage endometriosis patients by PCR-GeneScan analysis. Genotyping of PTEN was carried out on genomic DNA of analogous ectopic-eutopic endometria (n = 32) as well as blood samples from 720 patients and 500 controls by PCR-sequencing analysis to explore somatic and germ-line mutations, respectively. The levels and distribution of PTEN, p-Akt, p-Bad and p27 were analyzed in the eutopic endometria of patients (n = 5) and controls (n = 5) using western-blot and immunohistochemistry. MAIN RESULTS AND THE ROLE OF CHANCE: PCR-GeneScan analysis revealed a higher LOH frequency at 10q23.3 (84.4%) compared with other loci analyzed, hence we focused our attention on PTEN. PCR-sequencing analysis revealed seven novel somatic mutations and 23 germ-line polymorphisms in patients. Among somatic mutations, a frame-shift insertion at 10:89692992-89692993 (in the functionally important N-terminal phosphatase domain of PTEN) occurred in 11 of the 32 ectopic endometria. Western-blot and immunohistochemical analysis revealed decreased PTEN and increased p-Akt and p-Bad levels in eutopic endometria of patients compared with controls (all comparisons, P < 0.0001). Furthermore, PTEN loss was more frequent in the nucleus than in the cytoplasm. Expression of p27 did not differ between patients and controls. LIMITATIONS, REASONS FOR CAUTION: Protein analysis was performed in eutopic endometrial samples from only a small number of patients and controls. In future investigations, a larger sample size should be used and the role of the other genes involved in the PTEN-PI3K/Akt signal transduction pathway should be analyzed. WIDER IMPLICATIONS OF THE FINDINGS: Our findings revealed a possible involvement of the PTEN-PI3K/Akt-Bad axis in the pathogenesis of endometriosis, which may facilitate the discovery of suitable pathway inhibitors for disease treatment. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Science & Engineering Research Board (SERB), India (Lr No: SR/FT/LS-188/2009) to BM. The authors have no competing interests to declare.

The VEGF +405 G>C 5' untranslated region polymorphism and risk of PCOS: a study in the South Indian Women.

PURPOSE: The aim of this study was to investigate the association between two common single nucleotide polymorphisms (SNPs) in the vascular endothelial growth factor (VEGF) gene (-460C/T and +405G/C) and polycystic ovary syndrome (PCOS) risk in south Indian women. METHODS: This study involves clinically confirmed PCOS patients (n = 126) and non-PCOS controls (n = 130) of south Indian origin (Dravidian linguistic group). Genotyping of the VEGF gene -460C/T and +405G/C SNPs were performed by PCR and sequencing analysis. Haplotype frequencies for multiple loci and the standardized disequilibrium coefficient (D') for pairwise linkage disequilibrium (LD) were assessed by Haploview Software. RESULTS: The frequencies of +405G/G genotype (P = 0.03) and +405G alleles (P = 0.006) were significantly higher in patients compared to controls. Whereas the genotype and allele frequencies of -460C/T SNP were not significantly different between patients and controls. In addition, LD analysis revealed no significant difference between patients and controls. CONCLUSION: Our findings suggest that the VEGF +405G/C polymorphism may constitute an inheritable risk factor for PCOS in south Indian women.

An interleukin-6 gene promoter polymorphism is associated with polycystic ovary syndrome in South Indian women.

PURPOSE: Polycystic ovary syndrome (PCOS) is a most common endocrine disorder of reproductive age women. Interleukin-6 is involved in the pathophysiological characteristics associated with polycystic ovary syndrome (PCOS). The-174 G/C IL-6 gene promoter region single nucleotide polymorphism (SNP) may influence or modulate gene function and/or transcriptional efficiency. The current study was aimed to evaluate the association between IL-6 gene -174 G/C promoter polymorphism and Polycystic Ovary Syndrome in South Indian women. METHODS: In the present study, we examined the genotypic and allele distribution among the PCOS patients (n = 104) and controls (n = 156). The genotypes of IL-6 -174 G/C SNP were analyzed by polymerase chain reaction (PCR) and sequencing analysis. The allele frequency and genotype distributions of cases and controls were analyzed using Fisher's exact test. RESULTS: The genotype frequencies observed among the 104 cases and 156 controls were G/G 66.3 % and 49.4 %, G/C 29.8 % and 46.8 %, and C/C 3.8 % and 3.8 % (OR: 1.6226, CI: 1.0574-2.4899). The G and C allele frequencies were 81.25 % and 72.8 %, and 18.75 % and 27.2 %, respectively. The genotype and allele distribution revealed significant differences between PCOS patients and controls (all P values < 0.05). CONCLUSION: Our findings showed a significant statistical association between IL-6 -174 G/C SNP and PCOS risk in South Indian women. The 'G' allele frequency influences significantly higher in PCOS patients than controls. However, the exact mechanism by which 'G' allele frequency influence PCOS patients is yet to be determined.

Association of E-cadherin single nucleotide polymorphisms with the increased risk of endometriosis in Indian women.

The objective of the present study was to investigate the association between gene E-cadherin single nucleotide polymorphisms (SNPs) and risk of developing endometriosis in Indian women and to evaluate the role of E-cadherin expression in the pathophysiology of endometriosis. A genetic association study was conducted in 715 endometriosis cases and 500 controls of Indian origin. We genotyped -160 C/A, +54 C/T and -347 G/GA SNPs of gene E-cadherin by PCR-sequencing and PCR-restriction fragment length polymorphism techniques. Haplotype frequencies for multiple loci and the standardized disequilibrium coefficient (D') for pair-wise linkage disequilibrium (LD) were assessed by Haploview Software. In addition, to better understand genetic contributions to the pathophysiology of endometriosis, the expression pattern of E-cadherin in the endometrium of women with and without endometriosis was analyzed by western blot and immunohistochemical analysis. The frequencies of -347GA/GA (P = 0.026) and -160A/A (P = 0.0019) genotypes and -347G/-160A/+54C (P = 0.007) and -347GA/-160A/+54C (P < 0.0001) haplotypes were significantly different between patients and controls. Strong LD was observed between -347G/GA and -160C/A loci (D' = 0.64) when compared with -347G/GA and +54C/T (D' = 0.585) or -160C/A and +54C/T (D' = 0.05) loci in cases. Furthermore, increased membranous E-cadherin expression was observed in cases than in controls. The expression seems to be genotype dependent. In conclusion, the E-cadherin -347GA/GA and -160A/A genotypes and -347GA/-160A/+54C and -347G/-160A/+54C haplotypes may jointly modify the risk of endometriosis in Indian women. In addition, the differential expression of E-cadherin may play an important role in pathogenesis of endometriosis.

Does Choosing Microfluidics for Sperm Sorting Offer an Advantage to Improve Clinical Pregnancies in Donor Egg Recipients?

Background: Microfluidics (MF), an advanced sperm sorting technology results in the extraction of spermatozoa with higher DNA integrity and lower DNA damage compared to existing conventional sperm sorting methods. Aims: The aim of the present study is to assess the efficiency of MF and to isolate the best spermatozoa for intracytoplasmic sperm injection (ICSI) over the density gradient (DG) technique. Study Setting and Design: We recruited couples who choose the oocyte donation programme for this study to eliminate confounding factors associated with oocyte quality. Materials and Methods: Sperm was processed by MF (n = 180) and DG (n = 151). ICSI was performed and positive pregnancy, miscarriage and clinical pregnancy rates were compared. Statistical Analysis: All variables were analysed using Graph Pad Prism 5. The unpaired two-tailed t-test was used to assess the significance. A value of P < 0.05 was considered statistically significant. Results: There was no significant difference in pregnancy rates between the groups. However, a clear demarcation is seen in terms of clinical pregnancy rates, where the DG group achieved higher clinical pregnancies (91.7%) compared to the MF group (80.7%). Further, we compared miscarriage rates and biochemical pregnancies, and found a significantly higher miscarriage and biochemical pregnancy rate in the MF group (14.5% and 4%, respectively) compared to the DG group (6% and 1%, respectively). Conclusions: Based on the available literature, we anticipated a higher clinical pregnancy rate with MF compared with conventional processing. Our results show MF does not have any add-on positive effect on clinical pregnancy rate.

Genetic Variants of VDR and PGC-1α Are Not Associated with the Risk of Endometriosis in Indian Women.

An aberrant immunologic mechanism and mitochondrial biogenesis have been suggested to be involved in the pathogenesis of endometriosis. Genetic alterations in the vitamin D receptor (VDR) gene and peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α) may lead to important defects in gene activation, which principally affect immune function and normal mitochondrial function. Therefore, we hypothesized a possible role of VDR and PGC-1α genes in the pathogenesis of endometriosis and analyzed the association of genetic variants ApaI A/C (rs7975232) and TaqI T/C (rs731236) of VDR and rs8192678 (G/A), rs13131226 (T/C), and rs2970856 (T/C) of PGC-1α gene. This study included a total of 425 reproductive-age women (cases = 200 and controls = 225). Detection of VDR and PGC-1α gene polymorphism was performed using polymerase chain reaction-restriction fragment length polymorphism and sequencing analysis. The chi-square test was used to compare allele and genotype frequencies between groups, and a p-value of <0.05 was considered statistically significant. The genotype and allele distribution of both the gene polymorphisms did not show statistically significant association with endometriosis. Our result indicated ApaI A and TaqI T of VDR and GTT of PGC-1α gene as the most common haplotype in Indian women. The data suggest that VDR and PGC-1α gene polymorphisms did not play an important role in the pathogenesis of endometriosis in Indian women studied.

Germline study of AR gene of Indian women with ovarian failure.

OBJECTIVE: Present study was designed for carrying out the mutational analysis of the entire Androgen receptor (AR) gene including two microsatellite (CAG)n, (GGN)n, promoter region in cases of premature ovarian failure (POF) and primary amenorrhea (PA). DESIGN: Previous reports of AR knockout mice model showed POF phenotype, this draws an attention on the role of AR gene in the aetiology of POF for the case-control association studies in POF samples (n = 133), PA samples (n = 63) and control samples (n = 200). RESULTS: We identified six mutations including four novel mutations, i.e. c.636G > A, c.1885 + 9C > A, c.1948A > G, c.1972C > A, and two previously reported mutations, i.e. c.639G > A, c.2319-78T > G. Repeat length variation was noted in the two microsatellite regions CAG and GGN, located in the coding region of exon 1 at the N-terminal region of the AR gene. The CAG repeat length was homogeneously distributed with the same frequency and no association among all cases and controls. The GGN repeat showed a significant association among the SS and SL allele with p = 0.0231 and p = 0.0476, respectively, among the POF/control samples. CONCLUSIONS: Thus, AR gene mutations may play a role in the genetic cause of POF. Identification of the underlying genetic alteration of the AR gene is important for a proper diagnosis of POF subjects.

Proteomics-based study on asthenozoospermia: differential expression of proteasome alpha complex.

With a view to understand the molecular basis of sperm motility, we have tried to establish the human sperm proteome by two-dimensional PAGE MALDI MS/MS analysis. We report identification of 75 different proteins in the human spermatozoa. Comparative proteome analysis was carried out for asthenozoospermic and normozoospermic patients to understand the molecular basis of sperm motility. Analysis revealed eight proteins (including one unidentified) with altered intensity between the groups. Differential proteins distributed into three functional groups: 'energy and metabolism' (triose-phosphate isomerase, glycerol kinase 2, testis specific isoform and succinyl-CoA:3-ketoacid co-enzyme A transferase 1, mitochondrial precursor); 'movement and organization' (tubulin beta 2C and tektin 1) and 'protein turnover, folding and stress response' (proteasome alpha 3 subunit and heat shock-related 70 kDa protein 2). It was interesting to note that although the proteins falling in the functional group of 'energy and metabolism' are higher in the asthenozoospermic patients, the other two functional groups contain proteins, which are higher in the normozoospermic samples. Validation of results carried out for proteasome alpha 3 subunit by immunoblotting and confocal microscopy, confirmed significant changes in intensity of proteasome alpha 3 subunit in asthenozoospermic samples when compared with normozoospermic controls. Significant positive correlation too was found between proteasome alpha 3 subunit levels and rapid, linear progressive motility of the spermatozoa. In our understanding, this data would contribute appreciably to the presently limited information available about the proteins implicated in human sperm motility.

Genes governing premature ovarian failure.

Premature ovarian failure (POF) is unexplained amenorrhoea (>6 months), increased FSH (>20 IU/l) and LH occurring before 40 years. Several genes are reported as having significance in POF, including genes governing regulation of the hypothalamic-pituitary-ovarian axis, but their role in ovarian physiology is not known. Deletions or translocations in Xq arm have been found to be associated with POF, assuming presence of ovarian-related genes but ovary-related function of these genes is unclear. Several researchers have suggested specific loci on Xq critical region, POF1 and POF2 and genes DIA, FMR1 and FMR2. The understanding of ovarian physiology, its regulation and genes involved is important to explain the causes of POF. Some genes coordinate development of germ cell to primordial stage, e.g. GDF9, BMP15 and NGF, while others regulate development of further stages, such as FSH and LH. Mutation in these genes may lead to female infertility and are likely to be candidate genes for POF. Recently, association between blepharophimosis-ptosis-epicanthus inversus syndrome type 1 and POF has emerged as a possibility. Galactosaemia is also shown to be important in POF due to toxic effects of accumulated galactose or downstream products. Thus, understanding the role of several genes can be used for the appropriate genetic diagnosis, research and in the clinical practice of POF.

Impact of mitochondrial DNA copy number and displacement loop alterations on polycystic ovary syndrome risk in south Indian women.

Sequencing of mitochondrial displacement-loop (D-loop) of polycystic ovary syndrome (PCOS) patients and (n=118) and controls (n=114) of south Indian origin showed significant association of D310 (P=0.042) and A189G (P=0.018) SNPs with PCOS. qRT-PCR analysis revealed significantly diminished mtDNA copy number in PCOS patients compared to controls (P=0.038). Furthermore, mtDNA copy number was significantly lower in PCOS cases carrying D310 and 189G alleles when compared to non-carriers (P=0.001 and 0.006 respectively). The D310 carriers also showed significantly elevated LH/FSH ratio (P=0.026). In conclusion, mtDNA D-loop and copy number alterations may constitute an inheritable risk factor for PCOS in south Indian women.

Novel NR5A1 Pathogenic Variants Cause Phenotypic Heterogeneity in 46,XY Disorders of Sex Development.

Steroidogenic factor 1 (NR5A1/SF1) is a key transcription factor that is known to regulate the development of adrenal glands and gonads and is also involved in steroidogenesis. Several pathogenic NR5A1 variants have been reported to cause 46,XY disorders of sex development (DSD), with varying clinical phenotypes ranging from hypospadias to complete gonadal dysgenesis. Most often, the primary cause of DSD is due to variants in gene(s) related to gonadal development or the steroidogenic pathway. In the present study, we have analyzed 64 cases of 46,XY DSD for pathogenic NR5A1 variants. We report a total of 3 pathogenic variants of which 2 were novel (p.Gly22Ser and p.Ser143Asn) and 1 was already known (p.Ser32Asn). Functional studies have revealed that the 2 mutations p.Gly22Ser and p.Ser32Asn could significantly affect DNA binding and transactivation abilities. Further, these mutant proteins showed nuclear localization with aggregate formation. The third mutation, p.Ser143Asn, showed unspeckled nuclear localization and normal DNA binding, but the ability of transcriptional activation was significantly reduced. In conclusion, we recommend screening for NR5A1 pathogenic variants in individuals with features of 46,XY DSD for better diagnosis and management.

Short communication: Y chromosome microchimerism in female peripheral blood.

Male DNA of recognized fetal origin can be detected in the maternal circulation many years after delivery. It is referred to as fetal microchimerism, and is thus a possible explanation for the existence of low-level Y chromosome mosaicisms. Employing the nested polymerase chain reaction (PCR) technique, Y-specific markers were investigated in 13 cases with abnormal sex chromosome and 31 normal women. Sex-determining region Y (SRY) sequences were detected in normal women with a male child, which reflects the existence of fetal progenitor cells in the maternal circulation. This was completely absent in normal women with a female child. Individuals with the Y chromosome showed amplification for Y-specific markers. Microchimerism of Y was noted in Turner phenotype cytogenetically investigated with marker chromosome, and in an individual with XX karyotype. False positive amplifications are possible in nested PCR reactions, but the same could also be true for routine PCR. However, in the absence of any identifiable factor that could contribute to the recurrence of spurious PCR amplifications, cases of therapeutic importance must be tested at least five times. In such situations, DNA from an additional tissue should also be used for nested PCR.

The endothelial nitric oxide synthase Glu298Asp polymorphism is not a risk factor for endometriosis in south Indian women.

OBJECTIVE: To investigate whether the eNOS gene influences the risk of developing endometriosis in south Indian women. STUDY DESIGN: The single nucleotide polymorphism, Glu298Asp, in exon7 of the eNOS gene was tested for association in a case-control study of 232 affected women and 210 women with no evidence of disease. All the women were infertile, ascertained from the same infertility clinic. The genotype frequencies of the polymorphism were compared, using polymerase chain reaction and sequencing analysis. The localization and expression of eNOS in the eutopic endometrium of five cases and four controls was also analyzed using immunohistochemistry and western blotting. RESULTS: No statistically significant differences were observed in the genotype distributions and allele frequencies (p=0.3) between the cases and controls according to codominant, dominant and recessive genotype models. The localization and expression of this protein were similar in the endometrium of cases and controls. CONCLUSION: In the present study we could neither observe a difference in the eNOS expression nor establish an association between the eNOS Glu298Asp exon 7 polymorphism in south Indian women with endometriosis.