Browsing by Author "Kiran Singh"

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A functional single nucleotide polymorphism -1562c>t in the matrix metalloproteinase-9 promoter is associated with type 2 diabetes and diabetic foot ulcers
(2013) Kanhaiya Singh; Neeraj K. Agrawal; Sanjeev K. Gupta; Kiran Singh
Impaired neovascularization is the hallmark of type 2 diabetes, which results in various macro- and microvascular complications and the development of foot ulcerations later in life. Matrix metalloproteinases (MMPs) are the key enzymes which influence matrix remodeling. Here, we aim to investigate that whether single nucleotide polymorphism (SNP -1562C>T) (rs3918242) in the promoter region of MMP-9 gene, which alters the transcriptional activity of MMP-9 is associated with type 2 diabetes and diabetic foot ulcers (DFUs). This case-control study was composed of 730 individuals, out of which 463 patients were with type 2 diabetes mellitus (T2DM) and 267 were nondiabetic healthy controls (non-DM controls). T2DM patients were subclassified as 149 cases without any secondary complications (T2DMNSC), 110 with DFUs, 204 T2DM patients having one or the other secondary complications. Genotyping for -1562C>T SNP in MMP-9 gene was done by polymerase chain reaction-restriction fragment length polymorphism method and sequencing. SNP -1562C>T of MMP-9 gene showed a significant association with T2DM and DFU. The allele distribution differed significantly between patients and normal control group (odds ratio = 1.82, P =.00005, 95% confidence interval = 1.36-2.42 for T2DM vs control and odds ratio = 2.112, P =.00048, 95% confidence interval = 1.38-3.126 for DFU vs control) indicating strong association of SNP -1562C>T of MMP-9 gene with T2DM and DFU in an Indian population. SNP -1562C>T in the promoter of the MMP-9 gene results in increased expression at the level of the transcription. To the best of our knowledge, this is the first report that suggests that SNP -1562C>T in the promoter of the MMP-9 gene is associated with T2DM and DFU. An increased MMP-9 production from high expressing T allele may promote matrix degradation. © 2013 The Author(s).
A new rhodamine derivative as a single optical probe for the recognition of Cu2+ and Zn2+ ions
(Royal Society of Chemistry, 2015) Abhishek Rai; Niraj Kumari; Rohini Nair; Kiran Singh; Lallan Mishra
A bifunctional colorimetric and fluorescent chemosensor of type 3,5-dinitro-2-hydroxy benzaldehyde rhodamine hydrazone (RHDN) was synthesized by the condensation of 3,5-dinitro salicylaldehyde and rhodamine B hydrazide. It was characterized using spectroscopic techniques and single crystal studies. The chemosensor RHDN exhibited remarkably enhanced absorbance and colour changes from colourless to pink color on binding with Cu2+ ions. In contrast, Zn2+ ions were identified by their selective binding with RHDN showing OFF-ON type fluorescence, which changes from colorless to orange color in ultra violet-light. The absorbance and emission pattern of RHDN adduct separately with Cu2+ and Zn2+ ions were restored with the addition of an aqueous solution of disodium salt of ethylenediaminetetraacetic acid. Thus, RHDN was found to be very robust and reversible in its binding with Cu2+ and Zn2+ ions separately. © The Royal Society of Chemistry 2015.
A386G polymorphism of the DAZL gene is not associated with idiopathic male infertility in North India
(2009) Kiran Singh; Rajiva Raman
Background: Male infertility is a multifactorial disorder which affects approximately 10% of couples at childbearing age with substantial clinical and social impact. Genetic variation and environmental factors contribute to susceptibility to spermatogenic impairment in humans. The A386G (T54A) polymorphism of the autosomal gene, DAZL, has shown susceptibility to spermatogenic failure in Taiwanese population. However, no such association has been seen in infertile patients from Italy and South India. Aim: This study aims to find out the possible association between A386G (T54A) polymorphism of the autosomal gene, DAZL and idiopathic male infertility in patients from North India. DESIGN: Case-control study. Materials and Methods: The prevalence of A386G (T54A) polymorphism was determined in 165 idiopathic infertile azoo-/oligospermic patients and 200 fertile healthy control men. PCR-RFLP analysis was employed to determine the genotypes. PCR amplicons were subjected to restriction digestion with AluI as this mutation created a restriction site (AGCT), and separated on a 12% polyacrylamide gel. Results: Analysis of 165 idiopathic infertile azoo-/oligospermic and 200 fertile control men revealed only one case of the variant as a heterozygote in the control population. Single Nucleotide Polymorphism (SNP) was absent in the infertile patients. Conclusion: As in the report from Italy and South India, our results illustrate the rarity of this mutation. Apparently, this mutation is of recent origin and/or has poor selective value. Its preponderance in infertile patients from Taiwan (all heterozygotes) suggests a founder effect and also that its low selective value could be due to impaired spermatogenesis.
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
Association of FAS -1377 G>A and FAS -670 A>G functional polymorphisms of FAS gene of cell death pathway with recurrent early pregnancy loss risk
(2012) Rohini R. Nair; Anuradha Khanna; Kiran Singh
Apoptosis during the early stages of pregnancy enables the remodeling of the uterus for proper placentation. Apoptosis in the maternal activated cytotoxic T lymphocytes allows maternal immune tolerance to pregnancy and in glandular and stromal cells it helps with trophoblastic endometrial invasion. FAS gene is expressed at the maternal-fetal interface and is involved in the regulation of immune response and implantation. Altered FAS expression may result in altered apoptosis and ultimately affects both immune response and implantation. FAS -1377 G>A and FAS -670 A>G functional polymorphisms in the promoter region of FAS gene modulate its expression at transcriptional level. In a case-control study the contribution of FAS -1377 G>A and FAS -670 A>G polymorphisms to the risk of recurrent early pregnancy loss (REPL) was evaluated. DNA from 134 cases with a history of three or more REPL and 124 healthy controls with successful pregnancy outcomes were genotyped through PCR-RFLP. DNA sequencing was used to ascertain PCR-RFLP results. The genotype and allele frequencies for FAS -1377 G>A and FAS -670 A>G polymorphisms were compared in REPL and controls. FAS -1377 AA and AG genotypes were associated with an increased risk of REPL (OR, 3.25; 95%CI, 1.52-6.98 and OR, 2.62; 95%CI, 1.48-4.64, respectively), whereas FAS -670 genotypes conferred no risk. The -1377 AA/-670 GG genotypes combination of FAS polymorphisms showed highest risk (OR, 8.15; 95%CI, 2.75-25.81). Genotype combinations -1377 GA/-670 AA and -1377 GA/-670 AG were also statistically significant, suggestive of their role in REPL risk. © 2012 Elsevier Ireland Ltd.
Association of functional SNP-1562C > T in MMP9 promoter with proliferative diabetic retinopathy in north Indian type 2 diabetes mellitus patients
(Elsevier Inc., 2017) Kanhaiya Singh; Prabhjot Goyal; Manju Singh; Sujit Deshmukh; Divyesh Upadhyay; Sri Kant; Neeraj K. Agrawal; Sanjeev K. Gupta; Kiran Singh
Objective Retinal angiogenesis is a hallmark of diabetic retinopathy. Matrix Metalloproteinases (MMPs) are involved in degradation of extracellular matrix (ECM). Functional SNP-1562C > T in the promoter of the MMP-9 gene results increase in transcriptional activity. The present work was designed to evaluate the contribution of functional SNP-1562C > T of MMP-9 gene to the risk of proliferative diabetic retinopathy (PDR) in type 2 diabetes mellitus (T2DM) patients in north Indian Population. Methods This Case control study comprised of a total of 645 individuals in which 320 were T2DM patients out of which 73 had PDR, 98 had non- proliferative diabetic retinopathy (NPDR), 149 T2DM cases without any eye related disease (DM) and 325 non diabetic healthy individuals as controls (non DM controls). Genotyping for SNP-1562C > T of MMP-9 was done by polymerase chain reactions followed by restriction analyses with specific endonucleases (PCR-RFLP). DNA sequencing was used to ascertain PCR-RFLP results. Results T allele frequency in PDR patients was 32.1%, 20.4% in NPDR, 15.4% in DM and 13.7% in controls. Statistically significant difference was observed in both allele and genotype distribution between the PDR versus non-DM control group (p < 0.0001 by T allele; p = 0.002 by TT and p < 0.0001 by CT genotype). Conclusions The present study suggests that the functional SNP-1562C > T in the promoter of the MMP-9 gene could be regarded as a major risk factor for PDR as increased MMP-9 production from high expressing T allele may promote retinal angiogenesis. © 2017 Elsevier Inc.
Association of GSTT1 and GSTM1 polymorphisms with early pregnancy loss in an Indian population and a meta-analysis
(2013) Rohini R. Nair; Anuradha Khanna; Kiran Singh
Glutathione S-transferase theta 1 (GSTT1) and glutathione S-transferase Mu 1 (GSTM1) enzymes of the glutathione detoxification pathway protect the embryo from oxidative stress. This study investigated GSTT1 and GSTM1 in relation to their role in conferring genetic susceptibility to pregnancy loss. In a case-control study, 174 early pregnancy loss (EPL) patients, of which 130 were recurrent pregnancy loss (RPL) patients, and 180 healthy controls were investigated. Null genotypes of GSTT1 and GSTM1 were identified in duplex PCR reaction systems. Age-adjusted odds ratios (aOR) were calculated by logistic regression analysis. A meta-analysis was also conducted. The GSTT1 null genotype was significantly associated with EPL (aOR 4.47, P = 0.004) and RPL (aOR 4.39, P = 0.006). No significant association of the GSTM1 null genotype was found with RPL. In a meta-analysis study, the presence of the GSTM1 null genotype was shown to be a risk for RPL. The GSTT1 null genotype was not found to be a risk factor for pregnancy loss in the pooled population but its association with RPL was found in the Indian population. This study suggests that women carriers of GSTT1 and GSTM1 null genotypes are more often at genetic risk of pregnancy loss. Glutathione S-transferase theta 1 (GSTT1) and glutathione S-transferase mu 1 (GSTM1), enzymes of detoxification pathway, protect the embryo from oxidative stress. In the present study we have investigated GSTT1 and GSTM1 in relation to their role in conferring genetic susceptibility for early pregnancy loss (EPL) and recurrent pregnancy loss (RPL). Meta-analysis on the polymorphisms was conducted to support our findings that the presence of mutant genotypes at this site increases the risk of pregnancy loss. The GSTT1 null genotype was significantly associated with both EPL and RPL. In the meta-analysis, the overall result showed that the association between GSTM1 null genotype and risk for RPL was statistically significant. On comparing the GSTT1 studies, great heterogeneity was found between studies. A subgroup analysis was performed based on ethnicity. Our results showed a significantly increased risk with the GSTT1 null genotype in the Indian population, but no risk was found in the pooled population. In conclusion, the data of the present study clearly suggest that GSTT1 and GSTM1 polymorphisms are genetic risk factors for pregnancy loss in the study population. © 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
Association of increased S100A8 serum protein with early pregnancy loss
(2015) Rohini R. Nair; Anuradha Khanna; Kiran Singh
Problem: The contribution of systemic S100A8 protein in menstrual cycle, pregnancy, and early pregnancy loss (EPL) is not known. Altered expression of S100A8 in maternal decidua is associated with recurrent early pregnancy loss. The objective of this study was to investigate the systemic level of S100A8 in different phases of menstrual cycle, different trimester of pregnancy, and in EPL. Method of Study: Level of S100A8 was investigated in serum samples of the subjects through enzyme-linked immunosorbent assay (ELISA). Result and Conclusion: S100A8 levels were elevated during proliferative phase of menstural cycle. We found no statistical difference in S100A8 level in different trimester of pregnancy. S100A8 level was found to be significantly elevated in patients with EPL. This is the first study evaluating the systemic level of S100A8 predicting its role during menstural cycle and pregnancy. It opens a new perspective in which S100A8 can be used as a prognostic marker for EPL. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Association of interleukin 1 receptor antagonist (IL1RN) gene polymorphism with recurrent pregnancy loss risk in the North Indian Population and a meta-analysis
(Kluwer Academic Publishers, 2014) Rohini Ravindran Nair; Anuradha Khanna; Kiran Singh
An appropriate ratio of interleukin 1 beta to interleukin 1 receptor antagonist (IL1Ra) is required for successful pregnancy. Our objective was to study the genetic association between IL1RN variable numbers of tandem repeat (VNTR) polymorphism and recurrent pregnancy loss (RPL). To analyze the association between IL1RN VNTR allele and RPL, we investigated the IL1RN VNTR polymorphism in 136 RPL patients and in 200 healthy control women. Meta-analysis on this polymorphism was conducted to support our findings. PCR based approach was used to analyze IL1RN VNTR polymorphism and it was further confirmed by sequencing. Systematic review and meta-analysis was done using electronic database (Pub-Med, Google Scholar and Ovid) up to February 27, 2013. This meta-analysis was assessed by comprehensive meta-analysis software version 2. For meta-analysis 549 cases and 1,450 controls were included. The frequency of IL1RN genotype 2/2 was significantly higher in RPL compared to control group (AORs 3.10, 95 % CI 1.58-6.11, p = 0.001). The presence of rare allele also increased the risk of RPL significantly (ORs 1.63, 95 % CI 1.16-2.29, p = 0.004). The meta-analysis stratified by ethnicity showed that individuals with allele 2 had increased risk of RPL (OR 1.29, 95 % CI 1.04-1.61, p = 0.01), in Asians population by using fixed model. However the data of the present study clearly suggests that IL1RN VNTR polymorphism is a genetic risk factor for pregnancy loss in the study population. © 2014 Springer Science+Business Media.
Association of interleukin-1beta C + 3953T gene polymorphism with human male infertility
(2013) Deepika Jaiswal; Sameer Trivedi; Neeraj K. Agrawal; Rajendra Singh; Kiran Singh
Cytokines are involved in the regulation of spermatogenesis likely mediating the crosstalk among Sertoli and germ cells to facilitate germ cell movement across the seminiferous epithelium during cellular events such as germ cell differentiation. Members of the Interleukin-1 (IL-1) family are pleiotropic cytokines that are involved in inflammation, immunoregulation, and other homeostatic functions. Interleukin-1 alpha (IL-1α), IL-1β, and the IL-1 antagonistic molecule (IL-1 Ra) are present in the testis under normal homeostasis and they further increase upon infection/inflammation. In the present study we have examined the association of C + 3953T polymorphism of the human IL-1B gene with human male infertility. The case control study comprised of two groups: 222 infertile patients and 230 fertile healthy control men. Genotyping for SNP C + 3953T IL-1B was carried out by polymerase chain reaction followed by analysis with specific endonucleases (PCR-RFLP). DNA sequencing was used to validate the PCR-RFLP results. The genotype frequencies of the IL-1B Taq C/T polymorphism were compared between infertile men and controls. The frequency was significantly higher in asthenozoospermic patients compared to fertile control men (odds ratio = 10.4, CI: 2.50- 43.96, p = 0.001). The C + 3953T of the IL-1B gene is associated with male infertility risk in the asthenozoospermic patients from an Indian population. © 2013 Informa Healthcare USA, Inc.
Association of maternal and fetal MTHFR A1298C polymorphism with the risk of pregnancy loss: A study of an Indian population and a meta-analysis
(Elsevier Inc., 2013) Rohini R. Nair; Anuradha Khanna; Rajender Singh; Kiran Singh
Objective: To study the genetic association between methylenetetrahydrofolate reductase (MTHFR) A1298 polymorphism and recurrent pregnancy loss (RPL). Design: Prospective case-control study, systematic review, and meta-analysis using an electronic database up to July 27, 2012. Setting: Meta-analysis of four studies on RPL and three studies on spontaneously aborted embryos, including the present study. Patient(s): A total of 129 RPL patients and 202 healthy control women with successful pregnancy were analyzed including 40 spontaneously aborted embryos and 40 aborted embryos as control samples. For meta-analysis, 1,080 case and 709 control subjects were included of RPL and 375 case and 384 control samples of spontaneously aborted embryos. Intervention(s): Blood was collected by peripheral venous punctures, and spontaneously aborted embryos were collected by curettage or manual vacuum aspiration. Meta-analysis was done on the basis of heterogeneity of the studies. Main Outcome Measure(s): Genotyping was done by polymerase chain reaction (PCR)-restriction-fragment- length polymorphism (RFLP). DNA sequencing was used to ascertain PCR-RFLP results. Age-adjusted odds ratios were calculated by logistic regression analysis. Meta-analysis on this polymorphism was conducted to support our findings. Result(s): We found that presence of rare allele "C" and heterozygous and rare homozygous genotypes significantly increased the risk of RPL. No significant change in the fetal MTHFR A1298C genotype frequency was observed, regardless of chromosomal integrity. Meta-analysis of A1298C polymorphism on both RPL and in spontaneously aborted embryos showed significantly increased risk in the carriers of AC and CC genotypes. Conclusion(s): The data of the present study clearly suggests that MTHFR A1298C polymorphism is a genetic risk factor for pregnancy loss.
Association of the IL1RN Gene VNTR Polymorphism with Human Male Infertility
(2012) Deepika Jaiswal; Sameer Trivedi; Rajendra Singh; Rima Dada; Kiran Singh
Interleukin-1 (IL-1) is a regulatory cytokine that plays an important role in the maintenance of the immune environment of the testis, regulation of junction dynamics and cell differentiation during spermatogenesis. Members of the IL-1 family are pleiotropic cytokines that are involved in inflammation, immunoregulation and other homeostatic functions in the body. IL-1α, IL-1β, and the IL-1 receptor antagonistic molecule (IL-1 Ra) are expressed in the testis under normal homeostasis and they further increase upon infection/inflammation. In the present study we have examined the association of Variable Number Tandem Repeats (VNTR) polymorphism of the Interleukin-1 receptor antagonist gene (IL1RN) with human male infertility. The case-control study comprised of two groups: 331 idiopathic infertile patients and 358 fertile healthy men. The study indicates risk of IL1RN2 variant with male infertility (OR: 1.43, CI: 1.1546 to 1.7804, P = 0.001). To our best knowledge, this is the first report that links IL1RN VNTR polymorphism with human male infertility. © 2012 Jaiswal et al.
Association of the patterns of global DNA methylation and expression analysis of DNA methyltransferases in impaired spermatogenic patients
(Elsevier (Singapore) Pte Ltd, 2015) Deepika Jaiswal; Sameer Trivedi; Neeraj K. Agrawal; Kiran Singh
Objective: To analyze global DNA methylation along with DNA methyltransferases (DNMTs) expression at transcript level in patients with impaired spermatogenesis to dissect its role in pathophysiology of human male infertility. Method: The content of Global methylated cytosine (mC) was determined using ELISA system (Imprint Methylated DNA Quantification Kit, Sigma-Aldrich) in 31 testicular biopsies showing impaired spermatogenesis and 8 with normal spermatogenesis. Real-time reverse transcription-polymerase chain reaction was done to analyze DNMTs (DNMT1, DNMT3A, DNMT3B and DNMT3L) mRNA levels in biopsies with different testicular phenotypes. Results: There was significant increase in levels of global methylation in different impaired testicular phenotypes as compared to normal. Expression analysis revealed significantly increased expression of DNMT1 and its positive correlation with global DNA methylation. Conclusion: In conclusion, increased levels of global methylation in impaired cases might be the one of the contributing factors for aberrant gene expression in infertile patients. © 2015 The Authors.
Association of toll-like receptor 4 polymorphisms with diabetic foot ulcers and application of artificial neural network in DFU risk assessment in type 2 diabetes patients
(2013) Kiran Singh; Kanhaiya Singh; Vivek Kumar Singh; Neeraj K. Agrawal; Sanjeev K. Gupta
The Toll-Like receptor 4 (TLR4) plays an important role in immunity, tissue repair, and regeneration. The objective of the present work was to evaluate the association of TLR4 single nucleotide polymorphisms (SNPs) rs4986790, rs4986791, rs11536858 (merged into rs10759931), rs1927911, and rs1927914 with increased diabetic foot ulcer (DFU) risk in patients with type 2 diabetes mellitus (T2DM). PCR-RFLP was used for genotyping TLR4 SNPs in 125 T2DM patients with DFU and 130 controls. The haplotypes and linkage disequilibrium between the SNPs were determined using Haploview software. Multivariate linear regression (MLR) and artificial neural network (ANN) modeling was done to observe their predictability for the risk of DFU in T2DM patients. Risk genotypes of all SNPs except rs1927914 were significantly associated with DFU. Haplotype ACATC (P value = 9.3E-5) showed strong association with DFU risk. Two haplotypes ATATC (P value = 0.0119) and ATGTT (P value = 0.0087) were found to be protective against DFU. In conclusion TLR4 SNPs and their haplotypes may increase the risk of impairment of wound healing in T2DM patients. ANN model (83%) is found to be better than the MLR model (76%) and can be used as a tool for the DFU risk assessment in T2DM patients. © 2013 Kanhaiya Singh et al.
Association of variant rs7903146 (C/T) single nucleotide polymorphism of TCF7L2 gene with impairment in wound healing among north indian type 2 diabetes population: A case-control study
(2013) Kiran Singh; Kanhaiya Singh; Neeraj K. Agrawal; Sanjeev K. Gupta
The variants of transcription factor 7-like 2 (TCF7L2) gene have been shown to be associated with type 2 diabetes mellitus (T2DM) and its several secondary complications. Here, we aimed to examine the possible role of one of the common variant of this gene, rs7903146 (C/T), with impairment of wound healing in cases with T2DM. A total of 750 individuals, including 322 patients with T2DM and 120 patients with diabetic foot ulcers (DFUs) and 308 controls, were analyzed for rs7903146 variant of the TCF7L2 gene. Genotyping was done by polymerase chain reaction-restriction fragment length polymorphism. For rs7903146 variant, TT genotype frequency in patients with DFU was 10.8% and in controls was 5.2%. Risk genotype (TT) frequencies showed statistically significant difference between the DFU patients versus non-DM control group (odds ratio = 2.44, P =.037, 95% confidence interval = 1.05-5.64) compared with nonrisk genotype (CC + CT). In the present study, a positive significant association between DFU and the TT genotype of rs7903146 (C/T) variant of TCF7L2 gene was found. © The Author(s) 2013.
Autosomal genes in male infertility
(Springer Singapore, 2017) Vertika Singh; Sandeep Kumar Bansal; Rajender Singh; Kiran Singh
Spermatogenesis is driven by the master genes present on the Y chromosome. These driver genes need support from numerous other genes spread across the genome for a number of actions such as energy metabolism, cell death and apoptosis, protein turnover, synthesis of new proteins and garbage disposal. Preliminary studies on infertility focused on the Y chromosome genes due to their primary and indispensable role in spermatogenesis. A number of other studies on human infertility and mouse knockouts have identified several spermatogenically important genes present on chromosomes other than X and Y. For some of these genes, molecular pathways they participate in have also been worked out. This chapter summarizes the genes present on the autosomes that facilitate the process of spermatogenesis and fertility. © Springer Nature Singapore Pte Ltd. 2017. All rights reserved.
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.