Identification of a Single Nucleotide Polymorphism of Vitamin D Receptor (VDR) and Vitamin D Binding Protein (VDBP) Gene and Its Dysregulated Pathway Through VDR-VDBP Interaction Network Analysis in Vitamin D-Deficient Infertile Females.

INTRODUCTION: The prevalence of female infertility in Pakistan is currently estimated at 22%, and emerging research suggests that vitamin D (VD) deficiency (VDD) may play a significant role in influencing female fertility. The focus of this study was to investigate the single nucleotide polymorphism (SNP) patterns within the VD binding protein (VDBP). The study aimed to explore dysregulated pathways and gene enrichment through an interaction network analysis, specifically focusing on the interplay between the VD receptor (VDR) and VDBP in females experiencing unexplained infertility (UI) coupled with VDD. METHODS: A cross-sectional study was conducted on VD-deficient, fertile, and UI female subjects. VDBP and VDR were assessed by enzyme-linked immunoassay and genotyping performed. FunRich (version 3.1.3; http://funrich.org/index.html) was employed for analysis of the identified proteins: VDR and VDBP and with their mapped gene datasets, gene enrichment, and protein-protein interaction (PPI) network. RESULTS: The mean VD and VDR values of infertile females were significantly lower than those of fertile females. VDBP in infertile females (median (IQR)): 296.05 (232.58-420.23)) was lower than that of fertile females (469.9 (269.57-875.55), (p=0.01)). On sequence analysis, a mutation rs 4588 SNP (Thr 436 Lys) was found in exon 11 of the VDBP gene of UI females, but no mutation in exons 8 and 9 of the VDR gene, with some insignificant intronic variants, was observed. The proteins such as plasma membrane estrogen receptor signaling pathway (p < 0.001), VDR, SMAD3, NCOR1, CREBBP, NCOA1, STAT1, GRB2, PPP2CA, TP53, and NCOA2 were enriched after biological pathway grouping when VDR was made the focused gene and directly interacting with VDBP. CONCLUSION: The females with UI exhibited significantly low VD, VDBP, and VDR. The plasma membrane estrogen receptor signaling pathway was enriched in VDD infertile females.

Genetic association of vitamin D receptor gene with female infertility.

BACKGROUND: Infertility is defined as failure to achieve a clinical pregnancy after 12 months of unprotected intercourse. It affects 15% of couples globally and 22% of couples within Pakistan. Female infertility can be caused by numerous genetic or environmental factors including hormone imbalances and exposure to chemicals or radiation. The prevalence of vitamin D deficiency among the adult population was reported to be 14-59% with a higher prevalence in Asian countries. Furthermore, the expression of Vitamin D receptor (VDR) can play a vital role in the reproductive organs of females. Hence, the aim of our present study was to check the association of VDR polymorphisms with infertile females. For this purpose, blood samples were collected for genotyping of four known VDR mutations [FokI (rs2228570), TaqI (rs731236), ApaI (rs7975232), and BsmI (rs1544410)] via PCR-based RFLP assay. RESULTS: Genotyping indicated that FokI, TaqI, and ApaI are associated with infertility (p = 0.004*, p = 0.013*, and p = 0.033*, respectively). However, BsmI did not show any significance. Multinomial regression analysis indicated that FokI heterozygous genotypes increase the risk of infertility by 2.5 times (hetero: OR = 2.5, 95%, p = 0.001*) as compared to wild type. Heterozygous genotypes of TaqI and ApaI were found to play a protective role and reduce the risk of infertility by 58 and 52%, respectively [TaqI: OR = 0.42, 95%, p = 0.004*, ApaI: OR = 0.48, 95%, p = 0.01*, respectively] as compared to wild type. Multinomial logistic regression analysis was also performed for allelic data as well. CONCLUSION: Thus, it could be summarized that among the studied polymorphisms of VDR, FokI SNP greatly increased the risk of infertility, while TaqI and ApaI genotypes protect from infertility. However, BsmI does not influence the risk of infertility in Pakistani females.

Unraveling the Genetic Associations of DENND1A (rs9696009) and ERBB4 (rs2178575) with Infertile Polycystic Ovary Syndrome Females in Pakistan.

Polycystic Ovary Syndrome (PCOS) is a complex genetic disorder in reproductive-aged women which is associated with comorbidities of reproductive, metabolic, cardiovascular, endocrine, and psychological nature. PCOS is the most common cause of anovulatory infertility. Pathogenesis of PCOS involves strong interaction between environmental and genetic factors. Many Single-Nucleotide Polymorphisms (SNPs) have been associated with PCOS in different populations. Currently, very limited association studies of PCOS and infertility have been done on Pakistani population. The variants DENND1A rs9696009 and ERBB4 rs2178575 are significantly associated with PCOS in Chinese and European populations. These candidate genes regulate the production of androgen hormone, Anti-Mullerian Hormone (AMH), and luteinizing hormone. All these hormones are involved pathogenesis of PCOS and infertility. The aim of the study is to find an association of DENND1A rs9696009 and ERBB4 rs2178575 variants with PCOS in infertile Pakistani females. In this case-control study, 300 infertile females were recruited. The cases (n = 160) were infertile female diagnosed with PCOS (Rotterdam Criteria), and controls (n = 140) were infertile women with no evidence of PCOS. The genomic DNA was isolated, and genotyping was done by PCR-Restriction fragment length polymorphism and further validated by DNA Sanger Sequencing. The Chi-Square analysis showed rs2178575 (ERBB4) was significantly associated with infertility (χ2 = 10.282, p = 0.005852) while rs9696009 (DENND1A) did not show any significant association (χ2 = 3.10, p = 0.212036). Furthermore, multinomial logistic regression analysis was performed and revealed that rs2178575 (ERBB4) heterozygous genotypes (GA) and mutant genotypes (AA) decrease the risk of infertility by 0.541 times (OR = 0.541, 95% CI = 0.314-0.930, p = 0.026) and 0.416 times (OR = 0.416, 95% CI = 0.228-0.757, p = 0.004), respectively, compared to wild-type genotype (GG). The ERBB4 variant is significantly associated with PCOS infertile women and genetically indicated that ERBB4 (rs2178575) decreases the risk of infertility in females having PCOS.

SIRT1 and antioxidants in infertile females: Exploration of the role of vitamin D.

BACKGROUND: Deficiency of silent information regulator 1 (SIRT1) can trigger inflammation, mitochondrial malfunctioning, and apoptosis through the hypothalamic-pituitary-ovarian axis, producing poor quality oocytes, leading to infertility. Normal vitamin D (VD) levels promote SIRT1 activity required for optimal fertility, and low levels of either may result in fertility problems owing to cell-membrane de-stabilization, increased autophagy, DNA damage leading to increased reactive oxygen species and mitochondrial dysfunction. Therefore, in this study, we want to estimate the levels of VD, SIRT1 and antioxidants (MnSOD; manganese superoxide dismutase, GR; glutathione reductase, visfatin) and oxidants (adrenaline & cortisol) in individuals living with infertility and explore the association of VD with SIRT1 expression (levels), antioxidants, and oxidants contributing to infertility in women. The significance of this study is that it highlights the importance of maintaining optimal levels of VD for reproductive health in females. METHODS: This cross-sectional study included 342 (135 infertile and 207 fertile) female subjects. Serum levels of MnSOD, SIRT1, visfatin, GR, VD, adrenaline, and cortisol were analyzed by ELISA and were compared in fertile and infertile samples using the Mann Whitney U test. RESULTS: There were significantly high levels of VD, SIRT1, GR, MnSOD and visfatin in fertile female participants. However, mean adrenaline and cortisol levels were higher in infertile samples with a significant negative correlation with VD. A significant negative correlation of VD with MnSOD, SIRT1, visfatin and GR was observed (p <0.01). In VD subset groups, MnSOD levels were significantly high in VD sufficient groups however, adrenaline and cortisol levels were significantly high in groups suffering from VD deficiency. CONCLUSIONS: Deficiency of VD is associated with a decrease in SIRT1 and other antioxidants, which may deter natural reproductive functions leading to infertility. Further studies are required to determine the cause-effect relationship of VD deficiency on conception and interpretation of the involved mechanism.

MTHFR-c 677C>T polymorphism and male infertility: An analysis in a cohort of Pakistani men / Polimorfismo MTHFR-c 677C>T e infertilidad masculina: un análisis en una cohorte de hombres pakistaníes

Objective: To analyze existence of an association between methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism with male infertility. Materials and methods: A case–control study was conducted from June 2017 to August 2018 in which 88 infertile and 40 fertile were recruited. Polymerase chain reaction (PCR) – restriction fragment length polymorphism (RFLP) assay was carried out to study the allelic frequency of C677T polymorphism. The differences in allelic and genotypic frequencies of C677T locus between fertile and infertile groups were evaluated by the Pearson chisquare test. A logistic regression model was used to calculate Odds ratios and 95% confidence intervals, p value<0.05 was considered significant. The Hardy–Weinberg equilibrium was tested using HWE software. Results: In infertile subjects, frequency distribution of CC allele was (60.2%), the CT allele was (30.7%) the TT allele was (9.1%) and in the fertile controls the frequency was CC allele (75%), CT allele (20%) and TT allele (5%) respectively. Analysis revealed MTHFR 677 CC genotype associated significantly with male infertility (p<.046, OR=2.385; 95% CI=1.014–5.608); Frequency of CT (30.7%) and TT (9.1) genotypes were higher in infertile men as compared to CT (20%) TT (5%) in fertile controls but statistically these were not significantly different (p=0.097; OR=0.455; CI=0.179–1.153 and p=0.431; OR=0.526; CI=0.107–2.599 respectively). Significant association of age and BMI with MTHFR genotypes and infertility was observed. Conclusion: Our results showed that MTHFR C677T polymorphism is not a risk factor for male infertility in our Pakistani population. (AU)

Objetivo: Observar el efecto del polimorfismo C677T en metilenetetrahidrofolato reductasa (MTHFR) en la infertilidad masculina. Materiales y métodos: Se realizó un estudio de casos y controles desde junio de 2017 hasta agosto de 2018 en el que se reclutaron 88 infértiles y 40 fértiles. Se llevó a cabo el ensayo reacción en cadena de la polimerasa (PCR) - polimorfismo de longitud de fragmento de restricción (RFLP) para estudiar la frecuencia alélica del polimorfismo C677T. La prueba de chi-cuadrado de Pearson se utilizó para estimar las diferencias en las frecuencias alélicas y genotípicas del locus C677T entre fértiles e infértiles. Los cocientes de probabilidad se obtuvieron mediante el análisis de regresión logística con intervalos de confianza del 95%, siendo significativo un valor de p<0,05. Se aplicó el equilibrio Hardy-Weinberg (HWE). Resultados: En sujetos infértiles, la distribución de frecuencia del alelo CC fue del 60,2%, la del alelo CT, del 30,7%, la del alelo TT, del 9,1%, y en los controles fértiles la frecuencia fue alelo CC fue del 75%, la del alelo CT, del 20%, y la del alelo TT, del 5%, respectivamente. El análisis reveló el genotipo CC MTHFR 677 asociado significativamente con infertilidad en los hombres (p<0,046, OR=2,385; IC95%: 1,014-5,608). La frecuencia de los genotipos CT (30,7%) y TT (9,1) fue mayor en hombres infértiles en comparación con CT (20%) y TT (5%) en controles fértiles, pero estadísticamente estos no fueron significativamente diferentes (p=0,097, OR=0,455; IC95%: 0,179-1,153, y p=0,431, OR=0,526; IC95%: 0,107-2,599, respectivamente). Se observó asociación significativa de edad e IMC con genotipos MTHFR e infertilidad. Conclusión: Nuestros resultados mostraron que el polimorfismo MTHFR C677T no está asociado con la infertilidad por factor masculino en nuestra población pakistaní. (AU)

MTHFR-c 677C>T polymorphism and male infertility: An analysis in a cohort of Pakistani men.

OBJECTIVE: To analyze existence of an association between methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism with male infertility. MATERIALS AND METHODS: A case-control study was conducted from June 2017 to August 2018 in which 88 infertile and 40 fertile were recruited. Polymerase chain reaction (PCR) - restriction fragment length polymorphism (RFLP) assay was carried out to study the allelic frequency of C677T polymorphism. The differences in allelic and genotypic frequencies of C677T locus between fertile and infertile groups were evaluated by the Pearson chisquare test. A logistic regression model was used to calculate Odds ratios and 95% confidence intervals, p value<0.05 was considered significant. The Hardy-Weinberg equilibrium was tested using HWE software. RESULTS: In infertile subjects, frequency distribution of CC allele was (60.2%), the CT allele was (30.7%) the TT allele was (9.1%) and in the fertile controls the frequency was CC allele (75%), CT allele (20%) and TT allele (5%) respectively. Analysis revealed MTHFR 677 CC genotype associated significantly with male infertility (p<.046, OR=2.385; 95% CI=1.014-5.608); Frequency of CT (30.7%) and TT (9.1) genotypes were higher in infertile men as compared to CT (20%) TT (5%) in fertile controls but statistically these were not significantly different (p=0.097; OR=0.455; CI=0.179-1.153 and p=0.431; OR=0.526; CI=0.107-2.599 respectively). Significant association of age and BMI with MTHFR genotypes and infertility was observed. CONCLUSION: Our results showed that MTHFR C677T polymorphism is not a risk factor for male infertility in our Pakistani population.

Association of trinucleotide repeat polymorphisms CAG and GGC in exon 1 of the androgen receptor gene with male infertility: a cross-sectional study.

BACKGROUND: : Infertility is a global problem that brings about serious sexual and social consequences that strain the health sector and society. The expansion of CAG and GGC repeats in androgen receptor (AR) gene (Ensembl number ENSG00000169083) may lead to reduced fertility. Our objective was to determine the association of CAG and GGC repeats with altered sperm parameters in male infertile subjects. METHODS: This was a cross-sectional study conducted at Aga Khan University, Karachi, Pakistan. A total of 376 males were recruited, out of which group A (N = 208) and group B (N = 168) were comprised of subjects with normal and altered sperm parameters, respectively, from 18 to 60 years. The numbers of CAG and GGC repeats were determined by using PCR amplification and sequence analysis using the Molecular Evolutionary Genetic Analysis (MEGA) software version 6.0. Statistical analysis was performed using the SPSS version 20 and the P-value of <0.05 was considered significant. RESULTS: The mean androgen receptor gene CAG repeats were significantly longer in males with altered sperm parameters as compared to male subjects with normal sperm parameters (P < 0.001). There was no significant difference found for GGC repeats for subjects with altered sperm parameters. DISCUSSION: Longer CAG length corresponded to greater severity of spermatogenic defect and may lead to subfertility recommendations.

Male infertility: Role of vitamin D and oxidative stress markers.

Spermatozoa are vulnerable to oxidative stress because of their inherent reduced antioxidant defence and DNA repair mechanisms. Polyunsaturated fatty acids in sperm plasma membrane break down to cytotoxic lipid aldehyde, 4-Hydroxynonenal, whereas 3-Nitrotyrosine is generated by peroxynitrite induced tyrosine nitration. Both oxidative stress markers contribute to altered sperm function and infertility. Vitamin D, a membrane antioxidant, has a potential scavenger capacity. We compared oxidative stress markers and vitamin D in male subjects with normal and altered sperm parameters and explored association of these markers: 4-Hydroxynonenal and 3-Nitrotyrosine with Vitamin D. Higher 4-Hydroxynonenal levels in altered sperm parameter group and a negative correlation with sperm count, motility and morphology (p < 0.001) was observed. Vitamin D serum concentration in altered sperm parameters was less (p = 0.016) showing a significant positive correlation with sperm count and morphology. 4-Hydroxynonenal was significantly higher in altered sperm parameters showing negative correlation with vitamin D. Highest serum concentrations of 4-Hydroxynonenal were observed in vitamin D-deficient subjects. Significantly higher concentration of 4-Hydroxynonenal was estimated in altered sperm parameters of vitamin D sufficient group (p < 0.001). This suggests 4-Hydroxynonenal as an oxidative stress marker leading to altered sperm function and infertility with some association with vitamin D; needs to be explored.

Endometriosis, infertility and MicroRNA's: A review.

The review aims to explore circulating small non- coding regulatory Ribonucleic Acids (miRNA) as biomarkers of endometriosis; a reproductive age group disorder. miRNA are linked with genetic, epigenetic and angiogenic factors, hormones, cytokines, chemokines, oxidative stress (OS) markers, mediators of inflammation, hypoxia, angiogenesis and altered immune system contributing to the pathogenesis of endometriosis. Hormonal imbalance occurs by decreased levels of miRNAs -23a and miRNAs -23b and increase in miRNAs -:135a, 135b, 29c and 194-3p. Angiogenesis by vascular endothelial growth factor is attributed to increased miRNAs -126, miRNAs -210, miRNAs -21, miRNAs -199a-5p and miRNAs 20A. OS upregulates miRNAs -302a by increased levels of Tumor Necrosis factor (TNF)-α, TNF- ß and Interleukin -1ß. Upregulation of miRNAs -199a and miRNAs -16 promotes inflammation and cell proliferation in the endometriotic lesions. The gold standard to diagnose endometriosis is laparoscopy, yet miRNA can be validated as diagnostic tool for detection, progression and prevention of endometriosis in large, independent cohorts of women, with and without endometriosis.

Suggested role of silent information regulator 1 (SIRT1) gene in female infertility: A cross-sectional study in Pakistan.

AIM & OBJECTIVE: Silent information regulator 1 (SIRT1) gene stimulates the expression of antioxidants and repairs damaged cells. It affects the mitochondrial activity within the oocytes to overcome the oxidant stress. We aimed to assess an association of SIRT1 polymorphism (Tag SNPs rs10509291 and rs12778366) with fertility, and assess serum levels of follicle stimulating hormone (FSH), luteinizing hormone (LH), oestradiol, progesterone, manganese superoxide (MnSOD) and SIRT1. MATERIAL AND METHODS: In this cross-sectional study, 207 fertile and 135 infertile subjects between the ages of 18-45 years were recruited. Polymerase chain reaction (PCR) was performed; products were electrophoresed in a 2% agarose gel. Descriptive analysis of continuous variables was expressed as mean ± standard deviation. Mann-Whitney test was performed for comparison of groups, P value <.001 was considered significant. Single Nucleotide Polymorphism (SNP) data were analysed by applying chi-squared statistics. RESULTS: All subjects were age matched (P = .896). SIRT1 levels were significantly lower in infertile females when compared with fertile subjects (P < .001). AA (rs10509291) and CC (rs12778366) variant frequency was higher in the infertile than fertile subjects (P < .01). Similarly, the frequency of A allele (rs10509291) and C allele (rs12778366) was higher in infertile subjects (P < .001). Infertile females (29%) showed existence of SNP rs10509291 while 49% demonstrated genetic variation of rs12778366. MnSOD and SIRT1 levels were found to be lower in these subjects. CONCLUSION: The presence of SIRT1 genetic variants (rs10509291 and rs12778366) apparently disturbs the expression of SIRT1 deteriorating mitochondrial antioxidant function within the oocytes, instigating oxidative stress within. Their probable effect on modulating oocyte maturation may be the cause of infertility in females.

Estimation of folic acid/micro nutrients levels; Does it reflect sperm parameters.

OBJECTIVE: To study the impact of vitamin B12, folic acid (FA) and methylmalonic acid (MMA) on sperm parameters; count, motility and morphology leading to male fertility. METHODS: The cross-sectional study comprised of one hundred and eighty-six subjects with normal sperm parameters (fertile) and 88 subjects with abnormal sperm parameters labelled as "infertile" from a sample population of Karachi, Pakistan. Vitamin B12, FA and MMA levels in serum were analysed by enzyme linked immune sorbent assay. Unadjusted and adjusted prevalence ratio with their 95% CI were reported by using cox regression algorithm to assess the association of Vitamin B12, FA and MMA and other factors with male Infertility. Unadjusted and adjusted beta coefficients with 95% CI were reported by using linear regression analysis for assessing relationship of Vitamin B12, FA and MMA and other factors with semen parameters (count, motility and morphology); P value of <.05 was considered significant. RESULTS: It was declared that with every 1 unit increase in vitamin B12, FA and MMA the prevalence of infertility was decreased by 1%, 17% and 74%, respectively. Multivariate analysis revealed that vitamin B12, FA and MMA had a significant association with total sperm count, motility and morphology. The sperm parameters were also affected by increase in; LH, BMI and body fat %. There was a significant positive correlation of; LH with Vitamin B12 and FA (0.423 < 0.001, 0.338 < 0.001) and testosterone with vitamin B12 and FA (0.326 < 0.001, 0.291 < 0.001), respectively. CONCLUSION: All the studied micronutrients; Vitamin B12, FA and MMA had a positive effect on sperm parameters; count, motility and morphology and the associated reproductive hormones which explains their role on reproductive functions required to acquire fertility.

Changes in vitamin E levels as a marker of female infertility.

OBJECTIVE: To study the impact of Vitamin E (VE) levels of follicular fluid (FF) on oocyte competence, embryo development and pregnancy outcome in patients after intra cytoplasmic sperm injection (ICSI). METHODS: It was a cross-sectional study conducted in Islamabad Clinic Serving Infertile Couples in which follicular fluid of 137 females booked for ICSI, was obtained during oocyte retrieval, centrifuged and stored for analysis. VE levels in FF were analyzed by enzyme linked immune sorbent assay. Receiver Operating Curve (ROC)was used to demarcate VE levels required for acquiring pregnancy. Generalized linear model using log binomial regression was applied to see the effect of VE on pregnancy, the effect of VE on oocyte and embryo parameters was assessed by linear regression; all p-values less than 0.05 were considered statistically significant. RESULTS: ROC suggested 5.49 (unit) as the cutoff value of VE in the pregnancy group, with 72.9% area under the curve. Ninety-one females comprised Group I with VE > 5.49, whereas forty six females formed Group II with VE < 5.49. Follicular fluid VE levels were significantly high in 39 (28.5%) females who compromised pregnancy group. Chances of pregnancy increased to 4% with an increase in VE levels (p-value 0.01). VE gave significant positive relationship with all oocyte (retrieved, mature and fertilized) parameters, cleavage of embryo till its differentiation to blastocysts (p<0.01). CONCLUSIONS: Adequate amount of VE in follicular fluid enhances the possibility of maturation of oocytes which resulted in better reproductive outcome after ICSI.

Association of trinucleotide repeat polymorphisms CAG and GGC in exon 1 of the Androgen Receptor gene with male infertility: a cross-sectional study.

BACKGROUND: Infertility is a global problem that brings with serious sexual and social consequences that strain the health sector and society. The expansion of CAG & GGC repeats in androgen receptor (AR) gene Ensembl number; ENSG00000169083 may leads to reduced fertility. Our objective was to determine association of CAG and GGC repeats with altered sperm parameters in male infertile subjects. MATERIALS AND METHODS: It was a cross sectional study conducted at Aga Khan University, Karachi Pakistan. A total of 376 males were recruited, out of which group A (N=208) and group B (N=168) comprised of subjects with normal and altered sperm parameters respectively from 18 to 60 years. Numbers of CAG and GGC repeats were determined by using PCR amplification and sequence analysis using Molecular Evolutionary Genetic Analysis (MEGA) software version 6.0. Statistical analysis was done using SPSS version 20 and p-value of < 0.05 were considered significant. RESULTS: The mean androgen receptor gene CAG repeats were significantly longer in males with altered sperm parameters as compared to male subjects with normal sperm parameters (p< 0.001). There was no significant difference found for GGC repeats for subjects with altered sperm parameters. CONCLUSIONS: Longer CAG length corresponded to greater severity of spermatogenic defect and may lead to subfertility recommendations.

Role of oxidative stress and altered thyroid hormones in unexplained infertility.

OBJECTIVE: To explore the link between altered thyroid profile and oxidative stress marker in females with unexplained infertility. METHODS: The cross-sectional case-control study was carried out at the Islamabad Clinic Serving Infertile Couples, Islamabad, Pakistan, from June 2016 to August 2017, and comprised women aged 18-40 years regardless of ethnic background who were divided into two groups; those with unexplained infertility were the cases, while fertile women acted as the controls. Serum was analysed for triiodothyronine, thyroxine and thyroid stimulating hormone as well as for oxidative stress markers including manganese superoxide dismutase, glutathione reductase and adrenaline using enzymelinked immunosorbent assay. Data was analysed using SPSS 19. RESULTS: Of the 88 subjects, there were 44(50%) in each of the two groups. There was no significant difference in terms of thyroids markers except thyroxine and thyroid stimulating hormone (p<0.05). There were significant differences in terms of oxidative stress markers between the groups (p<0.05). A significant positive correlation of thyroid stimulating hormone was observed with manganese superoxide dismutase and adrenaline (p<0.05) with a weak non significant association of glutathione reductase (p>0.05). CONCLUSIONS: Increased thyroxine levels in females with unexplained infertility was associated with decrease in the serum levels of antioxidants.

Association of polymorphism c.-124G>A and c.-16 C>T in the promoter region of human INHA gene with altered sperm parameters; A pilot study.

OBJECTIVE: The objective of this was to demonstrate the association of Inhibin α (INHα) c.-124G>A and INHα-c.-16 C>T polymorphisms with altered sperm parameters in a selected male population of Karachi, Pakistan. STUDY DESIGN & SETTINGS: In this pilot study, male subjects were stratified on the basis of the WHO criteria for altered sperm parameters; 83 (cases-altered sperm parameters) and 30 (controls-normal sperm parameters) subjects were included for analysis of INHα-c.124G>A polymorphism and 88 (cases) and 38 (controls) were analysed for INHα -c-16 C>T polymorphism. Genotyping of INHα-c.-124G>A and INHα-c.-16 C>T was performed by PCR-RFLP, genotype distribution in Hardy-Weinberg equilibrium was evaluated by binary logistic regression model. RESULTS: For the c.-124G>A polymorphism in INHα gene, frequency of the three major genotypes in controls was: GG: 80.0%, GA: 20.0% and AA: 0% and in cases was: GG: 59.0%, GA: 30.2% and AA: 10.8%. The GG genotype was significantly associated with male infertility (P < .045, OR = 2.776, 95% CI = 1.025-7.513) while the GA genotype was not significantly associated with infertility (P < .290 OR = 0.580, 95% CI = 0.211-1.593). Frequency of mutant AA genotype was 10.8% in cases (altered sperm parameters) and absent (0%) in normal sperm parameter (controls). The frequencies of three major genotypes CC, CT and TT did not show any significant difference between cases and controls (P > .05). CONCLUSION: The results from our study exhibited a significant association of c.-124G>A polymorphism in the INHα gene promoter region with male infertility in the Pakistani population. A significant association of c.-16 C>T polymorphism with male infertility, however, was not observed. Further large-scale studies should be conducted to confirm this association.

Oxidative stress and male infertility: a cross sectional study.

OBJECTIVE: To compare stress markers and antioxidants in fertile and infertile males, and to explore their effects on reproductive hormones and fertility. METHODS: The cross-sectional case-control study was conducted from July 2017 to July 2018 at the Islamabad Clinic Serving Infertile Couples, Islamabad, Pakistan, and comprised male subjects aged 25-55 years. Infertile subjects were the cases, while healthy fertile males acted as the controls. Stress hormones cortisol and adrenaline and antioxidants glutathione peroxidase and superoxide dismutase were measured using enzyme-linked immunosorbent assay. Data was analysed using SPSS 22. RESULTS: Of the 376 subjects, 241(64%) were cases and 135(36%) were controls. Median cortisol, adrenaline, superoxide dismutase and glutathione levels were significantly higher among the cases compared to te controls (p<0.05). Follicle stimulating hormone and luteinizing hormone levels were higher in cases compared to the controls (p=0.05). Mean testosterone level was higher among the controls than the cases (p<0.001). After adjusting for other covariates, every increase of 7 units in cortisol increased the prevalence of infertility by 3% (p=0.001). There was significant interaction between luteinizing hormone and testosterone in the final model (p<0.05). CONCLUSIONS: Stress together with decrease in antioxidants was found to play a significant role in reducing the fertilising potential of male infertile subjects.

Single nucleotide polymorphisms in Renalase and KCNQ1 genes and female infertility: A cross-sectional study in Pakistan.

A global increase in the incidence of subfertility is observed, and research suggests strong genetic influences that might restrict fertility directly or indirectly. It therefore becomes important to rule out the existence of genetic causes and counsel infertile couples before offering "Advanced Infertility Treatment Techniques." This cross-sectional study aimed to explore the association of KCNQ1 (rs2237895) and Renalase (rs2576178 and rs10887800) single nucleotide polymorphisms with different causes of infertility by analysing 508 fertile and 164 infertile women. Gene variant (AC/CC) of KCNQ1 rs2237895 showed a slight difference in the endometriosis group compared to the fertile group (p = .049), with the C allele showing a significant association with infertility overall (OR = 1.42 [1.100-1.833]; p < .0069). The variant AG/GG of Renalase rs2576178 was significantly associated with overall infertility (OR = 2.266; p < .001), with a strong G allele association with unexplained infertility OR = 2.796 (p = .002) that remained significant after adjusting for age and body mass index. Similarly, Renalase rs10887800 AG/GG and G allele showed significant association with both infertility due to polycystic ovarian syndrome and unexplained infertility. Expression of single nucleotide polymorphism rs2237895 and rs2576178 in both KCNQ1 and Renalase genes might be responsible for altering reproductive potential, hence leading to infertility in women.

Kisspeptin and attributes of infertile males and females: A cross-sectional study in a subset of Pakistani population.

Kisspeptin, a peptide hormone, plays a pivotal role in fertility and neuroendocrine regulation of hypothalamo-pituitary-gonadal axis. Increased kisspeptin and reproductive hormones are responsible for fertility in male and females. This study aimed to explore the role of kisspeptin on hypothalamo-pituitary-gonadal axis by comparing the levels of kisspeptin in fertile and infertile subjects and identifying single nucleotide polymorphisms (SNPs) of KISS1 gene in exon 2 and exon 3 of infertile male and female cohorts. A cross-sectional study was carried out on 80 males (44 infertile and 36 fertile) and 88 females (44 in each group). Significantly high levels of kisspeptin (KP), follicle-stimulating hormone (FSH), luteinizing hormone and testosterone were observed in fertile male and female subjects except low FSH levels in comparison with infertile female subjects. One polymorphism in exon 2 (E1225K [G/A 3673]) and three in exon 3 (P1945A [C/G 5833]; Insertion of T at 6075; G2026G [C/G 6078]) in infertile group were detected, with low KP and hormonal levels. Male subjects had abnormal sperm parameters and unsuccessful attempt of intracytoplasmic sperm injection in females. Expression of SNP in exon 2 and exon 3 of KISS1 could be responsible for alteration in release of reproductive hormones and gonadal functions, hence causing infertility.

Cytokines and endometrial receptivity after intracytoplasmic sperm injection - A cohort study at Islamabad.

OBJECTIVE: To evaluate the role of tumour necrosis factor alpha and interleukin-6 in conception after assisted reproductive treatment. METHODS: The longitudinal cohort study was conducted from August 2014 to May 2015 and comprised patients undergoing intracytoplasmic sperm injection at the Islamabad Clinic Serving Infertile Couples, Islamabad, Pakistan. During the procedure, endometrial thickness was measured on the day of ovulation induction and serum tumour necrosis factor alpha along with interleukin-6 were measured on the embryo transfer day. Subjects were divided into groups, with non-pregnant females in Group A and those with confirmed foetal cardiac activity in Group B and non-pregnant females were divided into two groups. The difference between the groups and the association of cytokines with endometrial thickness were measured. . RESULTS: Of the 131 subjects, 79(60%) were in Group A and 52(40%) in Group-B. The mean level of interleukin-6 was 49.65±3.04 in Group B and 104.14±76.03 in Group A, with significant inverse correlation with endometrial thickness (p<0.001). The level of tumour necrosis factor alpha were significantly higher in Group-B compared to Group-A (p<0.001). CONCLUSIONS: The pro-inflammatory cytokine, tumour necrosis factor alpha, takes part in preparation of endometrial lining for implantation of embryo in assisted reproductive treatment procedures.