Background: Low and middle-income countries are facing a rapid increase in obesity and overweight burden, particularly in urban settings. Being overweight in men is associated with infertility and a higher risk to have a low sperm count or no sperm in their ejaculate. Despite potential limitations, this is one of few studies conducted to determine the potential risk of paternal overweight on sperm standard parameters, sperm chromatin integrity and assisted conception outcome including fertilization, embryo quality, cleavage rate, reduce blastocyst development, implantation, and cumulative live birth rate (CLBR). Methods: A cross-sectional study of 750 infertile couples undergoing assisted reproduction technique at a single reproductive medicine center of Salma Kafeel Medical Centre Islamabad. Sperm from men undergoing ART were analyzed for chromatin integrity using sperm chromatin dispersion assay (SCD), Chromomycin A3 staining (CMA3), and toluidine blue (TB) staining, while other semen parameters were assessed on same day includes; standard semen parameters, reactive oxygen species (ROS), sperm deformity index (SDI), teratozoospermic index (TZI), and hypo-osmatic swelling test (HOST). Paternal body mass index (BMI) < 24.5–20 kg/m² served as the reference group, while the male patients with BMI > 24.5-30 kg/m² were considered to be overweight. Results: In the analysis of the percentage of spermatozoa with chromatin maturity (CMA3) and chromatin integrity (TB) was reduced significantly in overweight men (p < 0.01) compared with a reference group. Increase in paternal BMI correlate with the increase in sperm chromatin damage (SCD r = 0.282, TB r = 0.144, p < 0.05), immaturity (CMA3, r = 0.79, p < 0.05) and oxidative stress (ROS) (r = 0.282, p < 0.001). Peri-fertilization effects were increased in oocytes fertilization in couples with overweight men (FR = 67%) compared with normal-weight men (FR = 74.8%), similarly, after univariant regression paternal weight remain predictor of sperm chromatin maturity, successful fertilization and CLBR. In the embryo, developmental stage number of the embryo in cleavage was higher in normal weight men, while day 3 (D3) embryos, percent good quality embryo D3, and blastocyst formation rate were compared able between the groups. The paternal overweight group had significant (p < 0.001) increased neonatal birth weight (2952.14 ± 53.64gm; within normal range) when compared with the reference group (2577.24 ± 30.94gm) following assisted reproductive technology (ART). CLBR was higher (p < 0.05) in normal weight men compared to couples with overweight male partners. CLBR per embryo transfer and per 2PN was a statistically significant (p < 0.05) difference between the two groups. An inverse association was observed in the linear regression model between paternal BMI with fertilization rate and CLBR. Conclusion: The present study demonstrated the impact of paternal overweight on male reproductive health, as these patients had a higher percentage of immature sperm (CMA3) with impaired chromatin integrity (SCD, TB) in their semen and had decreased fertilization rate, CLBR following assisted reproductive treatments. The present study supports that paternal overweight should be regarded as one of the predictors for fertilization, CLBR and useful for counseling, to consider body mass index not only in women but also for men, in those couples opting for ART treatment, and warrant a poor reproductive outcome in overweight men.

Sperm separation plays a critical role in assisted reproductive technology. Based on migration, density gradient centrifugation and filtration, a properly selected sperm could help in increasing assisted reproductive outcomes in teratozoospermia (TZs). The current study aimed to assess the prognostic value of four sperm selection techniques: density gradient centrifugation (DGC), swim-up (SU), DGC-SU and DGC followed by magnetic-activated cell sorting (DGC-MACS). These were evaluated using spermatozoa functional parameters. A total of 385 infertile couples underwent the procedure of intracytoplasmic sperm injection (ICSI), with an isolated teratozoospermia in the male partner. Semen samples were prepared by using one of the mentioned sperm preparation techniques. The improvements in the percentage of normal mature spermatozoa, rate of fertilization, cleavage, pregnancy and the number of live births were assessed. The normal morphology, spermatozoa DNA fragmentation (SDF) and chromatin maturity checked by using chromomycin A3 (CMA3) with DGC-MACS preparation were better compared to the other three methods. Embryo cleavage, clinical pregnancy and implantation were better improved in the DGC-MACS than in the other tested techniques. The DGC-MACS technique helped in the selection of an increased percentage of normal viable and mature sperm with intact chromatin integrity in patients with teratozoospermia.

Smoking has been linked to male infertility by affecting the sperm epigenome and genome. In this study, we aimed to determine possible changes in the transcript levels of PGAM5 (the phosphoglycerate mutase family member 5), PTPRN2 (protein tyrosine phosphatase, N2-type receptor), and TYRO3 (tyrosine protein kinase receptor) in heavy smokers compared to non-smokers, and to investigate their association with the fundamental sperm parameters. In total, 118 sperm samples (63 heavy-smokers (G1) and 55 non-smokers (G2)) were included in this study. A semen analysis was performed according to the WHO guidelines. After a total RNA extraction, RT-PCR was used to quantify the transcript levels of the studied genes. In G1, a significant decrease in the standard semen parameters in comparison to the non-smokers was shown (p < 0.05). Moreover, PGAM5 and PTPRN2 were differentially expressed (p ≤ 0.03 and p ≤ 0.01, respectively) and downregulated in the spermatozoa of G1 compared to G2. In contrast, no difference was observed for TYRO3 (p ≤ 0.3). In G1, the mRNA expression level of the studied genes was correlated negatively with motility, sperm count, normal form, vitality, and sperm membrane integrity (p < 0.05). Therefore, smoking may affect gene expression and male fertility by altering the DNA methylation patterns in the genes associated with fertility and sperm quality, including PGAM5, PTPRN2, and TYRO3.

In this study, the semen parameters, sperm chromatin integrity, antioxidant enzyme levels, and reproductive hormone levels of subfertile male subjects from Pakistan were assessed in relation to their age. Data on the demographic characteristics of the 750 study participants, including their general health, body mass index (BMI), and reproductive status, were collected from subfertile men from Pakistan. Semen and blood were collected to determine standard semen parameters, sperm chromatin dispersion (Halosperm-SCD), sperm chromatin integrity using toluidine blue (TB) staining, sperm chromatin maturity using chromomycin A3 (CMA3+) staining, and reproductive hormone (FSH, LH, prolactin and testosterone levels). The patients were divided into three groups according to their age: Group 1 included male subjects aged 30 years or less (n= 90), Group 2 included male subjects between the ages of 31 and 40 years (n= 330), and Group 3 included male subjects over 40 years of age (n= 330). Conventional semen parameters, reactive oxygen species (ROS), superoxide dismutase (SOD), guaiacol peroxidase (GPX), catalase (CAT), and lipid peroxidation (MDA) did not statistically (p> 0.05) differ with increasing male age or between different age groups. When compared to younger men (40 years (26.6 ± 0.6%). The concentration of LH, FSH, and testosterone levels were comparable between the groups (p> 0.05), while a significant (p= 0.04) increase in sperm chromatin immaturity CMA3+ (30 ± 0.71%) was observed in the old age group (>40 years) compared to the <30-year group (26.6 ± 1.03%). A positive association was observed between advanced male age and sperm chromatin dispersion (SCD) (r= 0.124,p= 0.001) and decondensation (CMA3+) (r= 0.1,p= 0.009). Despite potential limitations, this study has been carried out with extensive information on the potential risk of male age on sperm integrity. The present study demonstrated the impact of male age on male reproductive health, as these patients had a higher percentage of sperm chromatin damage (SCD) in their semen. Sperm DNA damage assessment will help in the evaluation and diagnosis of the underlying cause of poor fertility and can help clinicians in selecting the right treatment options. Male age is one of the factors that have an impact on the decline in male fertility. As a result, it is preferable for patients receiving assisted reproductive technology to be younger.

Abnormalities in sperm nuclei and chromatin can interfere with normal fertilization, embryonic development, implantation, and pregnancy. We aimed to study the impact of H2BFWT gene variants in sperm DNA on ICSI outcomes in couples undergoing ART treatment. One hundred and nineteen partners were divided into pregnant (G1) and non-pregnant (G2) groups. After semen analysis, complete DNA was extracted from purified sperm samples. The sequence of the H2BFWT gene was amplified by PCR and then subjected to Sanger sequencing. The results showed that there are three mutations in this gene: rs7885967, rs553509, and rs578953. Significant differences were shown in the distribution of alternative and reference alleles between G1 and G2 (p = 0.0004 and p = 0.0020, respectively) for rs553509 and rs578953. However, there was no association between these SNPs and the studied parameters. This study is the first to shed light on the connection between H2BFWT gene variants in sperm DNA and pregnancy after ICSI therapy. This is a pilot study, so further investigations about these gene variants at the transcriptional and translational levels will help to determine its functional consequences and to clarify the mechanism of how pregnancy can be affected by sperm DNA.