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.

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.

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.

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.

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.

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.

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 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.