Browsing by Author "Singh Rajender"

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Altered cord serum 25-hydroxyvitamin D signaling and placental inflammation is associated with pre-term birth
(Blackwell Publishing Ltd, 2020) Snehil Budhwar; Priyanka Verma; Rachna Verma; Shreshtha Gupta; Sangeeta Rai; Singh Rajender; Kiran Singh
Problem: Vitamin D is well-known for having anti-inflammatory and immunomodulatory properties. Impaired maternal vitamin D status has been known to increase the risk of adverse pregnancy outcomes like pre-term birth. The present study aims to evaluate the impact of fetal cord serum 25-hydroxyvitamin D-mediated signaling in mediating inflammatory responses in placenta during pre-term birth. Method of study: For the above purpose, cord serum 25 hydroxyvitamin D 25(OH)D were measured in term (n = 20) and pre-term (n = 20) born babies using ELISA. Vitamin D downstream signaling has also been checked in placenta (VDR, CYP27B1, cathelicidin LL37) along with expression of inflammatory markers (S100A8, HMGB1, TLR2, p-NF-kappaB) using Western blotting and immunohistochemistry. Pearson correlation model was used to do correlation study. Results: Compared with term born babies (59.31 ± 3.476), decline in cord serum 25(OH)D levels is observed in pre-term born babies (22.26 ± 1.083, P = <0.0001) that showed strong positive correlation with gestational age (r =.9368***) and birthweight (r =.9559***). On the other hand, vitamin D signaling markers were found to be downregulated and inflammatory markers were upregulated in placental tissue of pre-term born babies. Conclusion: Thus, our study demonstrated that insufficient cord 25(OH)D levels may disturb the homeostasis of inflammation in placenta. Altered cord serum 25(OH)D mediated anti-inflammatory signaling may be acting as trigger signals in modulating inflammatory responses in placenta and eliciting premature activation of spontaneous labor in pre-term birth. © 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Altered crosstalk of estradiol and progesterone with Myeloid-derived suppressor cells and Th1/Th2 cytokines in early miscarriage is associated with early breakdown of maternal-fetal tolerance
(Blackwell Publishing Ltd, 2019) Priyanka Verma; Rachna Verma; Rohini R. Nair; Snehil Budhwar; Anuradha Khanna; Nisha Rani Agrawal; Ruchi Sinha; Ruchi Birendra; Singh Rajender; Kiran Singh
Problem: Decline in myeloid-derived suppressor cells (MDSCs) and Th2 cytokines levels lead to early miscarriage (EM) but how the hormonal milieu of the body regulates MDSCs and Th1/Th2 cytokine balance is still a matter of investigation. Method of study: Peripheral blood and decidua samples were collected from 20 EM patients, and 20 healthy pregnant women opted for elective abortion. MDSCs and G-MDSCs levels were analyzed in peripheral blood mononuclear cells, and Th1/Th2 cytokines levels were determined in serum via flow cytometry. Estrogen (E2), Progesterone (P4), and Testosterone levels were measured via ELISA. Further, proliferation and apoptosis in decidual samples were checked via immunoblot/immunohistochemistry of estrogen receptor -α (ER-α), STAT-3/pSTAT-3, and caspase-3, respectively. Results: Our results clearly indicate that in EM patients; decline in E2 and P4 significantly correlates with decline in MDSCs, particularly with subtype granulocytic MDSCs (G-MDSCs) and skewness of the Th1/Th2 cytokines balance toward Th1 response. Downregulation of ER- α and increased caspase-3 expression in endometrium decidua signifies poor endometrial receptivity in EM. STAT-3 activation regulates proliferation, differentiation and suppressive potency of MDSCs. In decidua of EM, significantly lower expression of pSTAT-3 indicates that these processes pertaining to MDSCs are compromised. Conclusion: Altogether, this unfavorable systemic milieu may drive toward early breakdown of maternal-fetal tolerance in EM. Therefore, regulated crosstalk of E2, P4 with MDSCs and balanced Th1/Th2 cytokines is prerequisite for successful pregnancy. © 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Array-based DNA methylation profiling reveals peripheral blood differential methylation in male infertility
(Elsevier Inc., 2019) Saumya Sarkar; Kumar Mohanty Sujit; Vertika Singh; Rajesh Pandey; Sameer Trivedi; Kiran Singh; Gopal Gupta; Singh Rajender
Objective: To study peripheral blood DNA differential methylation in oligozoospermic infertile men in comparison with normozoospermic fertile controls. Design: Case-control study. Setting: Reproductive biology laboratory. Patients(s): Azoospermic and oligozoospermic infertile patients (n = 6) and normozoospermic fertile controls (n = 6) in the discovery phase, and oligo/asthenozoospermic infertile men (n = 11) and normozoospermic fertile controls (n = 10) in the validation phase. Intervention(s): Blood samples drawn from all participants, DNA isolation and methylation analysis. Main Outcome Measure(s): DNA methylation values analyzed using genomewide methylation 450K BeadChip array, followed by deep sequencing of selected regions for methylation analysis in the neighborhood regions of differentially methylated CpGs. Result(s): We found 329 differentially methylated CpG spots, out of which 245 referred to the genes, representing 170 genes. Deep-sequencing analysis confirmed the methylation pattern suggested by 450K array. A thorough literature search suggested that 38 genes play roles in spermatogenesis (PDHA2, PARP12, FHIT, RPTOR, GSTM1, GSTM5, MAGI2, BCAN, DDB2, KDM4C, AGPAT3, CAMTA1, CCR6, CUX1, DNAH17, ELMO1, FNDC3B, GNRHR, HDAC4, IRS2, LIF, SMAD3, SOD3, TALDO1, TRIM27, GAA, PAX8, RNF39, HLA-C, HLA-DRB6), are testis enriched (NFATC1, NMNAT3, PIAS2, SRPK2, WDR36, WWP2), or show methylation differences between infertile cases and controls (PTPRN2, RPH3AL). Conclusion(s): We found a statistically significant correlation between peripheral blood DNA methylation and male infertility, raising the hope that epigenome-based blood markers can be used for screening male infertility risk. The study also identified new candidates for spermatogenesis and fertility. © 2019 American Society for Reproductive Medicine
AZF deletions in Indian populations: original study and meta-analyses
(Springer, 2020) Andrabi Syed Waseem; Vertika Singh; Girish Chandra Makker; Sameer Trivedi; Geetanjali Mishra; Kiran Singh; Singh Rajender
Purpose: To identify the frequency of Y chromosome microdeletions in Indian populations and to quantitatively estimate the significance of association between these deletions and male infertility. Methods: A total of 379 infertile males (302 azoospermic and 77 oligozoospermic infertile males) and 265 normozoospermic fertile males were evaluated for Y chromosome microdeletions (YCD) using PCR amplification and gel electrophoresis. Meta-analyses were performed on AZFa (2079 cases and 1217 controls), AZFb (2212 cases and 1267 controls), AZFc (4131 cases and 2008 controls), and AZFb+c (1573 cases and 942 controls) deletions data to quantitatively estimate the significance of association between these deletions and male infertility in Indian populations. Results: The results revealed that out of 379 infertile azoospermic and oligozoospermic males, 38 (10.02%) had AZF deletions. No deletion was found in control samples. The highest percentage of deletions was observed in the AZFc region, followed by AZFa and AZFb. Qualitative analysis showed that AZF deletions were present in 0.59 to 32.62% (average 13.48%) of infertile cases in Indian populations. Meta-analysis revealed a significant association of AZFa (OR = 6.74, p value = 0.001), AZFb (OR = 4.694, p value = 0.004), AZFc (OR = 13.575, p value = 0.000), and AZFb+c (OR = 5.946, p value = 0.018) deletions with male infertility. Conclusion: AZF deletions were seen in 10.02% of azoospermic and oligozoospermic cases with the highest frequency of AZFc deletions. Pooled analysis for all studies showed deletion frequency from 0.59 to 32.62% (average = 13.48%). Meta-analysis showed significant association of AZFa, AZFb, and AZFb+c deletions with male infertility. Analysis of Y chromosome microdeletions should be reckoned as an essential testing for diagnostic and therapeutic purposes. © 2020, Springer Science+Business Media, LLC, part of Springer Nature.
Azoospermic infertility is associated with altered expression of DNA repair genes
(Elsevier B.V., 2019) Vertika Singh; Deepika Jaiswal; Kanhaiya Singh; Sameer Trivedi; Neeraj K Agrawal; Gopal Gupta; Singh Rajender; Kiran Singh
Compelling evidence suggest that germs cells are predominantly sensitive to DNA damaging agents in comparison to other cells. High fidelity DNA repair in testicular cells thus becomes indispensable to preserve the genomic integrity for passing on to the progeny. Compromised DNA repair machinery in the testicular cells may result in impaired spermatogenesis and infertility. It remains unclear if the alterations in the expression of DNA repair genes correlate with azoospermia and male infertility. In the present study, 54 non-obstructive azoospermic infertile patients with hypospermatogenesis (HS, n = 26), maturation arrest (MA, n = 15), Sertoli cell only syndrome (SCOS, n = 13) and 14 controls with obstructive azoospermia, but normal spermatogenesis were recruited. Expression profiling of 84 DNA repair genes in testicular biopsy samples was performed using PCR array. Out of 84 genes, 27, 64 and 28 genes showed >5 fold down-regulation in the HS, MA and SCOS groups, respectively. On the basis of differential expression and their functional significance in spermatogenesis, ten genes (MSH2, BRIP1, CCNH, LIG4, MGMT, NTHL1, PMS1, DMC1, POLB and XPA) were selected for validation of transcript levels in a higher number of cases using RT-PCR, which corroborated the findings of array. Four genes (MSH2, LIG4, PMS1 and DMC1) were analyzed for protein levels using immunohistochemistry, which further validated the loss of DNA repair gene expression. Caspase-3 immunostaining showed that the loss of DNA repair correlated with increased testicular apoptosis in patients. Maturation arrest showed the highest apoptotic index with maximum number of downregulated genes. We conclude that the loss of DNA repair genes expression in testis correlates with increased apoptosis, azoospermia and infertility. © 2019 Elsevier B.V.
Decline in seminal quality in Indian men over the last 37 years
(BioMed Central Ltd., 2018) Priyanka Mishra; Mahendra Pal Singh Negi; Mukesh Srivastava; Kiran Singh; Singh Rajender
Background: Since the first report of a decline in semen quality in 1974, there have been several reports of similar declines across populations. Despite some scattered reports of declining semen quality in the Indian sub-continent, comprehensive studies analyzing semen quality over the last few decades have not been undertaken. We undertook the present study to investigate the temporal trend in semen parameters in Indian populations over a period of 37 years (1979-2016). Methods: Publications providing semen analysis details for fertile and infertile men from the Indian sub-continent were collected by a thorough literature search. Semen quality data for 6466 normal fertile or presumptive normal men (from 119 studies/data sets) and 7020 infertile men (from 63 studies/data sets) published between 1979 and 2016 were retrieved. We undertook systematic review and quantitative analysis of mean sperm count, motility, normal morphology and other available parameters. Data were analyzed to estimate semen parameters reference values for Indian men and to assess temporal trends in infertile, fertile and all subjects. Results: Seminal quality shows a decreasing temporal trend and the decrease is higher in infertile than fertile males. In pooled analysis for all individuals, significant (p < 0.05 or < 0.001) declines in sperm concentration and normal morphology are observed; however, isolated analysis for each group shows declines without statistical significance. The mean (± SD) semen volume, sperm concentration, total motility, rapid linear progressive motility, normal sperm morphology and sperm viability for Indian fertile men are 2.88 ± 0.77 ml, 81.08 ± 29.21 million/ml, 66.37 ± 10.95%, 52.64 ± 15.78%, 56.68 ± 20.23% and 72.63 ± 8.31%, respectively, whereas in infertile these are 3.07 ± 1.27 ml, 37.94 ± 26.41 million/ml, 40.22 ± 13.76%, 26.79 ± 15.47%, 36.41 ± 21.66% and 55.25 ± 11.99%, respectively. The mean seminal parameter values were significantly lower (p < 0.001) in infertile as compared to fertile men, except semen volume. Conclusions: Semen parameters in Indian men have declined with time and the deterioration is quantitatively higher in the infertile group. The study also provides reference values for semen parameters in Indian men. © 2018 The Author(s).
Duplications in 19p13.3 are associated with male infertility
(Springer New York LLC, 2019) Vertika Singh; Renu Bala; Arijit Chakraborty; Singh Rajender; Sameer Trivedi; Kiran Singh
Purpose: To identify genomic imbalances and candidate loci in idiopathic male infertility. Methods: Affymetrix CytoScan 750K Array was used to analyze genomic imbalances and candidate loci in 34 idiopathic infertile cases of different phenotypes (hypo-spermatogenesis, n = 8; maturation arrest, n = 7; and Sertoli cell-only syndrome, n = 13, severe oligozoospermia, n = 6, and 10 normozoospermic fertile men). Ten ethnically matched controls were screened for comparison. Results: The cytogenetic array analysis detected a genomic gain at the 19p13.3 region in 9 (26.47%) cases, with the highest frequency in patients with Sertoli cell-only syndrome (SCOS) (38%). Its complete absence in the control group suggests its likely pathogenic nature. In addition to Y-classical, micro, and partial deletions, the duplication in 19p13.3 could serve as a unique biomarker for evaluation of infertility risk. The common region across the individuals harboring the duplication identified STK11, ATP5D, MIDN, CIRBP, and EFNA2 genes which make them strong candidates for further investigations. The largest duplicated region identified in this study displayed a major network of 7 genes, viz., CIRBP, FSTL3, GPX4, GAMT, KISS1R, STK11, and PCSK4, associated with reproductive system development and function. The role of chance was ruled out by screening of ethnically matched controls. Conclusion: The result clearly indicates the significance of 19p13.3 duplication in infertile men with severe testicular phenotypes. The present study underlines the utility and significance of whole genomic analysis in the cases of male infertility which goes undiagnosed due to limitations in the conventional cytogenetic techniques and for identifying genes that are essential for spermatogenesis. © 2019, Springer Science+Business Media, LLC, part of Springer Nature.
Environment, Lifestyle, and Female Infertility
(Springer Science and Business Media Deutschland GmbH, 2021) Renu Bala; Vertika Singh; Singh Rajender; Kiran Singh
Lifestyle factors, which include the practices we adopt in our daily life, have a significant role in shaping our overall health. These lifestyle choices are mainly centered on personal preferences and our surrounding social environment. In addition to lifestyle factors, we continuously interact with our environment, which impacts physiology. Several factors have been claimed to affect women’s fertility; lifestyle-related factors, in particular, have received great attention in the last decade. Due to societal and professional pressure, childbearing age in women has gradually shifted to the 30s. Delayed age of childbearing along with modern lifestyle offers a wider window of opportunity for various lifestyle and genetic perturbations to penetrate to affect fertility. While clinical studies have strengthened a direct correlation between lifestyle, environment, and female reproductive health; experimental studies on animal models have investigated their mechanism of action. In most instances, these factors target the neuroendocrine pathways, resulting in metabolic derangements. This review aims to dissect the plausible interconnection of lifestyle and environmental factors with various neuroendocrine pathways and to discuss how it can affect the female physiology in the long-term, resulting in reproductive incompetence. © 2020, Society for Reproductive Investigation.
Excess iodine impairs spermatogenesis by inducing oxidative stress and perturbing the blood testis barrier
(Elsevier Inc., 2020) Arijit Chakraborty; Vertika Singh; Kiran Singh; Singh Rajender
Approximately 2 billion people worldwide are susceptible to iodine deficiency. Iodine deficiency has largely been tackled by iodine fortification in salt; however indiscriminate use of iodine raises the risk of iodine toxicity. In this study, we aimed to investigate the molecular mechanisms underlying adverse effect of excess iodine on spermatogenesis. Sprague Dawley (SD) rats were orally administered with 0.7 mg potassium iodide (KI)/100 g Bw and 3.5 mg potassium iodide (KI)/100 g Bw for a period of 60 days. This resulted in significant loss of sperm count and motility. Molecular investigations provided evidence for the generation of oxidative stress with high SOD levels, reduced Nrf2, HO-1 and increased NF-kB and Follistatin. Further investigations showed increased apoptosis evidenced by reduced expression of anti-apoptotic (BCL-2, Survivin), increased expression of pro-apoptotic (Bid, Bax) markers, and increased expression of p53 and other modulators/effectors of apoptosis (cytochrome c, cleaved PARP, caspase3 and caspase9). Analysis of the blood testis barrier proteins showed reduced expression of tight junction (JAM-A, Tricellulin), ectoplasmic specialization (Integrin- β1), adherens junction (N-Cadherin, E-cadherin, β-catenin) proteins, and reduced expression of other junction protein coding genes (Claudin1, Claudin 5, Occludin, ZO-1, Testin, Fibronectin, CAR-F). Focal adhesion kinase (FAK) and key regulators of spermatogenesis (c-Kit receptor, androgen receptor) were also parallelly decreased. Further investigation showed reduced expression of germ cell proliferation and differentiation markers (PCNA, Cyclin D1, c-Kit, Cdk-4). These findings collectively explain the loss of spermatogenesis under excess iodine conditions. In conclusion, excess iodine causes loss of spermatogenesis by inducing oxidative stress and disrupting the blood testis barrier and cytoskeleton. © 2020
Genome-wide differential methylation analyses identifies methylation signatures of male infertility
(Oxford University Press, 2018) Kumar Mohanty Sujit; Saumya Sarkar; Vertika Singh; Rajesh Pandey; Neeraj Kumar Agrawal; Sameer Trivedi; Kiran Singh; Gopal Gupta; Singh Rajender
STUDY QUESTION: Do methylation changes in sperm DNA correlate with infertility? STUDY ANSWER: Loss of spermatogenesis and fertility was correlated with 1680 differentially-methylated CpGs (DMCs) across 1052 genes. WHAT IS KNOWN ALREADY: Methylation changes in a number of genes have been correlated with reduced sperm count and motility. STUDY DESIGN, SIZE, DURATION: This case-control study used spermatozoal DNA from 38 oligo-/oligoastheno-zoospermic infertile patients and 26 normozoospermic fertile men. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Genome-wide methylation analysis was undertaken using 450 K BeadChip on spermatozoal DNA from six infertile and six fertile men to identify DMCs. This was followed by deep sequencing of spermatozoal DNA from 32 infertile patients and 20 fertile controls. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 1680 DMCs were identified, out of which 1436 were hypermethylated and 244 were hypomethylated. Classification of DMCs according to the genes identified BCAN, CTNNA3, DLGAP2, GATA3, MAGI2 and TP73 among imprinted genes, SPATA5, SPATA7, SPATA16 and SPATA22 among spermatogenesis-associated genes, KDM4C and JMJD1C, EZH2 and HDAC4 among genes which regulate methylation and gene expression, HLA-C, HLA-DRB6 and HLA-DQA1 among complementation and immune response genes, and CRISPLD1, LPHN3 and CPEB2 among other genes. Genes showing significant differential methylation in deep sequencing, i.e. HOXB1, GATA3, EBF3, BCAN and TCERG1L, are strong candidates for further investigations. The role of chance was ruled out by deep sequencing of select genes. LARGE-SCALE DATA: N/A. LIMITATIONS, REASON FOR CAUTION: Genome-wide analyses are fairly accurate, but may not be exactly validated in replication studies across all DMCs. We used the 't' test in the genome-wide methylation analysis, whereas other tests could provide a more robust and powerful analysis. WIDER IMPLICATIONS OF THE FINDINGS: DMCs can serve as markers for inclusion in infertility screening panels, particularly those in the genes showing differential methylation consistent with previous studies. The genes validated by deep sequencing are strong candidates for investigations of their roles in spermatogenesis.STUDY FUNDING/COMPETING INTEREST(S): The study was funded by the Council of Scientific and Industrial Research (CSIR), Govt. of India with grant number BSC0101 awarded to Rajender Singh. None of the authors has any competing interest to declare. © The Author(s) 2018. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
Gr/gr deletions on Y-chromosome correlate with male infertility: An original study, meta-analyses, and trial sequential analyses
(Nature Publishing Group, 2016) Sandeep Kumar Bansal; Deepika Jaiswal; Nishi Gupta; Kiran Singh; Rima Dada; Satya Narayan Sankhwar; Gopal Gupta; Singh Rajender
We analyzed the AZFc region of the Y-chromosome for complete (b2/b4) and distinct partial deletions (gr/gr, b1/b3, b2/b3) in 822 infertile and 225 proven fertile men. We observed complete AZFc deletions in 0.97% and partial deletions in 6.20% of the cases. Among partial deletions, the frequency of gr/gr deletions was the highest (5.84%). The comparison of partial deletion data between cases and controls suggested a significant association of the gr/gr deletions with infertility (P = 0.0004); however, the other partial deletions did not correlate with infertility. In cohort analysis, men with gr/gr deletions had a relatively poor sperm count (54.20 ± 57.45 million/ml) in comparison to those without deletions (72.49 ± 60.06), though the difference was not statistically significant (p = 0.071). Meta-analysis also suggested that gr/gr deletions are significantly associated with male infertility risk (OR = 1.821, 95% CI = 1.39-2.37, p = 0.000). We also performed trial sequential analyses that strengthened the evidence for an overall significant association of gr/gr deletions with the risk of male infertility. Another meta-analysis suggested a significant association of the gr/gr deletions with low sperm count. In conclusion, the gr/gr deletions show a strong correlation with male infertility risk and low sperm count, particularly in the Caucasian populations.
High frequencies of Non Allelic Homologous Recombination (NAHR) events at the AZF loci and male infertility risk in Indian men
(Nature Publishing Group, 2019) Deepa Selvi Rani; Singh Rajender; Kadupu Pavani; Gyaneshwer Chaubey; Avinash A. Rasalkar; Nalini J. Gupta; Mamta Deendayal; Baidyanath Chakravarty; Kumarasamy Thangaraj
Deletions in the AZoospermia Factor (AZF) regions (spermatogenesis loci) on the human Y chromosome are reported as one of the most common causes of severe testiculopathy and spermatogenic defects leading to male infertility, yet not much data is available for Indian infertile men. Therefore, we screened for AZF region deletions in 973 infertile men consisting of 771 azoospermia, 105 oligozoospermia and 97 oligoteratozoospermia cases, along with 587 fertile normozoospermic men. The deletion screening was carried out using AZF-specific markers: STSs (Sequence Tagged Sites), SNVs (Single Nucleotide Variations), PCR-RFLP (Polymerase Chain Reaction - Restriction Fragment Length Polymorphism) analysis of STS amplicons, DNA sequencing and Southern hybridization techniques. Our study revealed deletion events in a total of 29.4% of infertile Indian men. Of these, non-allelic homologous recombination (NAHR) events accounted for 25.8%, which included 3.5% AZFb deletions, 2.3% AZFbc deletions, 6.9% complete AZFc deletions, and 13.1% partial AZFc deletions. We observed 3.2% AZFa deletions and a rare long AZFabc region deletion in 0.5% azoospermic men. This study illustrates how the ethnicity, endogamy and long-time geographical isolation of Indian populations might have played a major role in the high frequencies of deletion events. © 2019, The Author(s).
High Level of APOA1 in Blood and Maternal Fetal Interface Is Associated With Early Miscarriage
(SAGE Publications Inc., 2019) Priyanka Verma; Rohini R. Nair; Suchita Singh; Singh Rajender; Anuradha Khanna; Rajesh K. Jha; Kiran Singh
Early miscarriage (EM) is one of the most devastating obstetrical complications globally affecting the quality of women’s life. In the present study, we aimed to identify proteins that correlate with and could act as biomarkers for EM. We performed 2-dimensional gel electrophoresis in chorionic villi samples followed by mass spectrometry for identification of differential protein expression with EM. Proteomic studies detected a total 124 protein spots, out of which 83 spots were differentially expressed between EM and controls in chorionic villi samples. Matrix assisted laser desorbtion/ionization-time of flight (MALDI-TOF) mass spectrometry analysis revealed Apolipoprotein A1 (APOA1) to be the most upregulated protein in the EM group that was validated by Western blotting and Enzyme-linked immunosorbent assay (ELISA). We found low but not statistically significant level of APOA1 on 21st day of menstruation in comparison to the 7th day. APOA1 level was observed to be the lowest in the first trimester. Hence, this study suggests that low APOA1 expression is critical in establishing pregnancy and elevated APOA1 expression in chorionic villi correlates with EM. Similar observation in serum samples suggests its potential as a marker for the risk of EM. © The Author(s) 2018.
Hyperhomocysteinemia and low vitamin B12 are associated with the risk of early pregnancy loss: A clinical study and meta-analyses
(Elsevier Inc., 2021) Renu Bala; Rachna Verma; Priyanka Verma; Vertika Singh; Namrata Yadav; Singh Rajender; Nisha Rani Agrawal; Kiran Singh
One-carbon metabolism is crucial for the maintenance of healthy pregnancy and alterations in this pathway have been associated with various pregnancy-related complications. Therefore, the present study was conducted to test the hypothesis that the altered folic acid, vitamin B12 and homocysteine levels are associated with the risk of early pregnancy loss (EPL). Plasma folic acid, vitamin B12 and homocysteine levels were analyzed in 83 females with EPL and 70 healthy pregnant females in their first trimester. Further, meta-analyses of folic acid, vitamin B12 and homocysteine were also performed involving various eligible studies. Results from our case-control study and meta-analysis showed that folic acid deficiency is not associated with the risk of EPL. On the other hand, low vitamin B12 and hyperhomocysteinemia were individually found to be significant risk factors for EPL in the present study (P < .01, P < .05, respectively) and meta-analysis as well (P < .001, P < .05, respectively). Vitamin B12 deficiency in combination with hyperhomocysteinemia was a more serious risk factor for EPL (Odds Ratio = 4.98, P = 0.002). Therefore, we conclude that vitamin B12 deficiency and elevated homocysteine levels are independent risk factors for EPL, and of higher risk when combined. The assessment of vitamin B12 and homocysteine levels may serve as a good screening marker for EPL risk. © 2021 Elsevier Inc.
Increased DNA methylation in the spermatogenesis-associated (SPATA) genes correlates with infertility
(Blackwell Publishing Ltd, 2020) Kumar Mohanty Sujit; Vertika Singh; Sameer Trivedi; Kiran Singh; Gopal Gupta; Singh Rajender
Background: Spermatogenesis-associated (SPATA) family of genes plays important roles in spermatogenesis, sperm maturation or fertilization. The knockout studies in mice have demonstrated that SPATA genes are crucial for fertility. Gene expression and genetic polymorphism studies have further suggested their correlation with infertility; however, methylation analysis of SPATA genes in human male infertility has not yet been undertaken. Objectives: To analyze the methylation status of SPATA4, SPATA5 and SPATA6 genes in oligozoospermic male infertility. Materials and methods: In the present study, we have analyzed DNA methylation pattern in the promoter regions of SPATA4, SPATA5 and SPATA6 genes in oligozoospermic patients and compared it with normozoospermic fertile controls. Semen samples were obtained from 30 oligozoospermic infertile and 19 normozoospermic fertile controls, and DNA methylation levels of the target gene promoters were analyzed by amplicon based deep sequencing methylation analysis using MiSeq. Results: SPATA4 (P < 0.0008), SPATA5 (P = 0.009) and SPATA6 (Promoter, P < 0.0005; Exon 1, P = 0.0128) genes were significantly hypermethylated in oligozoospermic patients in comparison to controls. This is the first study reporting a higher methylation in the promoters of SPATA4, SPATA5 and SPATA6 in oligozoospermic infertile individuals in comparison to the normozoospermic fertile controls. Discussion: Altered methylation of SPATA genes would affect pathways involved in sperm production or affect various processes linked to sperm fertility. Conclusion: In conclusion, hypermethylation in the SPATA4, SPATA5 and SPATA6 genes correlates with oligozoospermic infertility. © 2019 American Society of Andrology and European Academy of Andrology
Interleukin-17 gene polymorphisms and the risk of early miscarriage: A case-control study and meta-analysis
(Elsevier B.V., 2018) Priyanka Verma; Rohini R. Nair; Snehil Budhwar; Vertika Singh; Renu Bala; Anuradha Khanna; Nisha R. Agarwal; Punam Rai; Singh Rajender; Kiran Singh
Previous reports clearly suggest that IL-17 have important role in development of systemic and peripheral inflammation in early miscarriage (EM). In the present study, we have investigated the association between genetic variants in IL-17A, IL-17F and susceptibility to EM. We recruited 135 EM patients and 150 controls and used PCR-RFLP method for genotyping the polymorphisms of IL-17A, rs4711998 (−832 A/G), rs8193036 (−692C/T) and IL-17F rs763780 (7488 T/C). No significant difference was observed for all the three polymorphic sites between the EM patients and control group in terms of genotypic (rs4711998, χ2 = 1.95, p = 0.37; rs8193036, χ2 = 1.91, p = 0.38; rs763780, χ2 = 2.45, p = 0.29), and allelic frequencies (rs4711998, OR = 1.19, 95% CI = 0.84 to 1.67, p = 0.35; rs8193036,OR = 1.18, 95% CI = 0.58 to 2.06, p = 0.75; rs763780, OR = 1.5, 95% CI = 0.93 to 2.71, p = 0.11). Further, meta-analysis of IL-17F (rs763780) variant with EM also revealed non-significant association of IL-17F (rs763780) variant with EM in the presence of mutant genotype (CC) via random effect model (p = 0.70, OR = 1.30, 95% CI =0.33–5.11). © 2018
Is MTHFR 677 C>T polymorphism clinically important in Polycystic Ovarian Syndrome (PCOS)? A case-control study, meta-analysis and trial sequential analysis
(Public Library of Science, 2016) S. Justin Carlus; Saumya Sarkar; Sandeep Kumar Bansal; Vertika Singh; Kiran Singh; Rajesh Kumar Jha; Nirmala Sadasivam; Sri Revathy Sadasivam; P.S. Gireesha; Kumarasamy Thangaraj; Singh Rajender
Background: Optimum efficiency of the folate pathway is considered essential for adequate ovarian function. 677 C>T substitution in the 5, 10-methylene tertrahydrofolatereductase (MTHFR) gene compromises activity of the MTHFR enzyme by about 50%. The significance of correlation between 677C>T substitution and PCOS remains dubious due to the low power of published studies. Methods and Results: We analyzed MTHFR 677 C>T site in ethnically two different PCOS case-control groups (total 261 cases and 256 controls) from India. The data analysis revealed a lack of association between this polymorphism and PCOS [OR = 1.11 (95%CI = 0.71-1.72), P = 0.66]. Group-wise analysis on the basis of ethnicity also revealed no association in any of the ethnic groups [Indo-Europeans, P = 1; Dravidians, P = 0.70]. Homocysteine levels did not differ significantly between cases (15.51 μmol/L, SD = 2.89) and controls (15.89 μmol/L, SD = 2.23). We also undertook a meta-analysis on 960 cases and 1028 controls, which suggested a significant association of the substitution with PCOS in the dominant model of analysis (OR = 1.47 (95%CI = 1.04-2.09), P = 0.032]. Trial sequential analysis corroborated findings of the traditional meta-analysis. However, we found that the conclusions of meta-analysis were strongly influenced by studies that deviated from the Hardy Weinberg equilibrium. A careful investigation of each study and a trial sequential analysis suggested that 677 C>T substitution holds no clinical significance in PCOS in most of the populations. Conclusion: In conclusion, MTHFR 677 C>T polymorphism does not affect PCOS risk in India. The association seen in the meta-analysis is due to an outlier study and studies showing deviation from the Hardy Weinberg equilibrium. © 2016 Carlus et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
L712V mutation in the androgen receptor gene causes complete androgen insensitivity syndrome due to severe loss of androgen function
(2013) Singh Rajender; Nalini J. Gupta; Baidyanath Chakrabarty; Lalji Singh; Kumarasamy Thangaraj
Inability to respond to the circulating androgens is named as androgen insensitivity syndrome (AIS). Mutations in the androgen receptor (AR) gene are the most common cause of AIS. A cause and effect relationship between some of these mutations and the AIS phenotype has been proven by in vitro studies. Several other mutations have been identified, but need to be functionally validated for pathogenicity. Screening of the AR mutations upon presumptive diagnosis of AIS is recommended. We analyzed a case of complete androgen insensitivity syndrome (CAIS) for mutations in the AR gene. Sequencing of the entire coding region revealed C > G mutation (CTT-GTT) at codon 712 (position according to the NCBI database) in exon 4 of the gene, resulting in replacement of leucine with valine in the ligand-binding domain of the AR protein. No incidence of this mutation was observed in 230 normal male individuals analyzed for comparison. In vitro androgen binding and transactivation assays using mutant clone showed approximately 71% loss of ligand binding and about 76% loss of transactivation function. We conclude that CAIS in this individual was due to L712V substitution in the androgen receptor protein. © 2013 Elsevier Ltd. All rights reserved.
MTHFR 1298A>C Substitution is a Strong Candidate for Analysis in Recurrent Pregnancy Loss: Evidence from 14,289 Subjects
(Springer Science and Business Media Deutschland GmbH, 2022) Poonam Mehta; Rahul Vishvkarma; Kiran Singh; Singh Rajender
We undertook meta-analyses on MTHFR 1298A>C substitution for critically evaluating its association with recurrent pregnancy loss (RPL). MTHFR genotype data for 5888 cases and 8401 controls from 39 studies were pooled to perform this meta-analyses. Genotype data were screened, scrutinized, pooled, analysed and subjected to sensitivity analysis to carefully evaluate the association between MTHFR 1298A>C and recurrent pregnancy loss. Genetic associations were sought using dominant, recessive and co-dominant models of genetic testing with odds ratio and 95% Confidence interval (CI) as the effect measures. Further analyses were undertaken by classifying the studies into Caucasian and East Asian sub-groups. Genetic heterogeneity was tested before pooling the data across studies. For assessing publication bias, Egger’s intercept test was undertaken. We found a significant association of 1298A>C substitution with increased risk of RPL in the dominant (P=0.000; OR = 1.58; 95% CI =1.25–1.99) as well as recessive (P=0.000; OR = 1.66; 95% CI =1.25–2.20) models. In sub-group analysis, we observed a significant association of the polymorphism with RPL in the Caucasian populations using dominant (P=0.000; OR = 1.98; 95% CI =1.42–2.76) and recessive (P=0.000; OR = 2.20; 95% CI =1.49–3.24) models. However, this substitution showed no association with RPL in the East Asian populations (P=0.149; OR = 1.187; 95% CI =0.94–1.50). MTHFR 1298A>C substitution shows association with the risk of recurrent pregnancy loss. The association is in a population-specific manner with the substitution being a strong risk factor only in the Caucasian populations. © 2021, Society for Reproductive Investigation.
SNPs in ERCC1, ERCC2, and XRCC1 genes of the DNA repair pathway and risk of male infertility in the Asian populations: association study, meta-analysis, and trial sequential analysis
(Springer New York LLC, 2019) Vertika Singh; Sandeep Kumar Bansal; D.V.S. Sudhakar; Neelabh; Arijit Chakraborty; Sameer Trivedi; Gopal Gupta; Kumarasamy Thangaraj; Singh Rajender; Kiran Singh
Purpose: We investigated if substitutions in the ERCC1, ERCC2, and XRCC1 genes of the DNA repair pathway correlate with non-obstructive azoospermia and male infertility. Methods: A total of 548 azoospermic infertile males and 410 fertile controls were genotyped for XRCC1 399A > G, 280G > A, and ERCC1 C > A 3′ UTR and 541 azoospermic infertile males and 416 fertile controls were genotyped for ERCC2 751A > C using iPLEX Gold Assay. Meta-analyses were performed on XRCC1 399A > G (1022 cases and 1004 controls), ERCC1 C > A 3′ UTR (879 cases and 1059 controls), and ERCC2 751A > C (914 cases and 850 controls) polymorphisms to quantitatively estimate the significance of the association between these polymorphisms and the risk of infertility. Results: Statistically significant association between ERCC2 751A > C SNP and male infertility was found using the codominant model (p = 0.03). Results of meta-analysis suggested a lack of correlation with male infertility risk, which could be due to pooling of studies from different ethnic populations. Due to limited the number of studies, a stratified analysis for different ethnic groups could not be performed. Conclusion (s): In conclusion, AA genotype of 751A > C SNP in ERCC2 correlated with a higher risk of male infertility and may contribute to an increased risk of azoospermia and male infertility in Indian men. © 2018, Springer Science+Business Media, LLC, part of Springer Nature.