Apigetrin ameliorates doxorubicin prompted testicular damage: biochemical, spermatological and histological based study.

Doxorubicin (DOX) is a highly effective, commonly prescribed, potent anti-neoplastic drug that damages the testicular tissues and leads to infertility. Apigetrin (APG) is an important flavonoid that shows diverse biological activities. The present research was designed to evaluate the alleviative role of APG against DOX-induced testicular damages in rats. Forty-eight adult male albino rats were randomly distributed into 4 groups, control, DOX administered (3 mgkg-1), DOX + APG co-administered (3 mgkg-1 of DOX; 15 mgkg-1 of APG), and APG administered group (15 mgkg-1). Results of the current study indicated that DOX treatment significantly reduced the activities of superoxide dismutase (SOD), glutathione reductase (GSR), catalase (CAT) and glutathione peroxidase (GPx), while increasing the levels of malondialdehyde (MDA) and reactive oxygen species (ROS). DOX treatment also reduced the sperm count, viability, and motility. Moreover, DOX significantly increased the sperm morphological anomalies and reduced the levels of plasma testosterone, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The administration of DOX significantly increased the expressions of Bax and Caspase-3, as well as the levels of inflammatory markers. Additionally, DOX treatment significantly downregulated the expressions of steroidogenic enzymes (StAR, 3ß-HSD and 17ß-HSD) and Bcl-2. Furthermore, DOX administration provoked significant histopathological abnormalities in the testicular tissues. However, APG supplementation significantly reversed all the testicular damages due to its androgenic, anti-apoptotic, anti-oxidant and anti-inflammatory nature. Therefore, it is concluded that APG may prove a promising therapeutic agent to treat DOX-induced testicular damages.

Protective effect of didymin against 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin-induced reproductive toxicity in male rats.

PURPOSE: 2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin (TCDD) is one of the most potent environmental toxicants, which causes oxidative stress and adversely affects the male reproductive system. The current study aimed to evaluate the ameliorative role of didymin (DDM) against TCDD-induced testicular toxicity. METHODS: Forty-eight male Sprague-Dawley rats were divided into four equal groups (n=12). (i) Control group, (ii) TCDD-induced group was provided with 10 µg/kg/day of TCDD, (iii) TCDD + DDM group received 10 µg/kg/day of TCDD and 2 mg/kg/day of DDM, and (iv) DDM-treated group was administered with 2 mg/kg/day of DDM. After 56 days of treatment, biochemical, steroidogenic, hormonal, spermatogenic, apoptotic, and histopathological parameters were estimated. RESULTS: TCDD affected the biochemical profile by reducing the activities of antioxidant enzymes, while increasing the levels of malondialdehyde (MDA) and reactive oxygen species (ROS). Furthermore, it decreased the expressions of steroidogenic enzymes, 3ß-hydroxysteroid dehydrogenase (HSD), 17ß-HSD, steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (CYP11A1), and 17α-hydroxylase/17, 20-lyase (CYP17A1), as well as reduced the levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and plasma testosterone. Besides, epididymal sperm count, viability, and motility were decreased, while sperm morphological anomalies were increased. Moreover, TCDD altered the apoptotic profile by up-regulating the expressions of Bax and caspase-3, while downregulated the Bcl-2 expression. Additionally, histopathological damages were prompted due to TCDD administration. However, DDM restored all the TCDD-induced damages owing to its antioxidant, anti-apoptotic, and androgenic potential. CONCLUSION: Our data suggested that DDM might play its role as a therapeutic agent against TCDD-prompted testicular toxicity.

The Impact of Heavy Smoking on Male Infertility and Its Correlation with the Expression Levels of the PTPRN2 and PGAM5 Genes.

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.

Analysis of sperm chromatin packaging and reproductive biomarker to evaluate the consequence of advanced male age.

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 (<30 years), sperm SCD (23.2 ± 0.88%) was significantly (p = 0.01) lower as compared to male patients aged >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.

Analyzing the Differential Impact of Semen Preparation Methods on the Outcomes of Assisted Reproductive Techniques.

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.

Mechanistic insight into the protective effects of fisetin against arsenic-induced reproductive toxicity in male rats.

Arsenic is one of the most hazardous environmental contaminants, which adversely affects the dynamics of male reproductive system. Fisetin (FIS) is a bioactive flavonoid, which is known to exert strong antioxidative effects. Therefore, the current research was planned to evaluate the alleviative efficacy of FIS against arsenic-induced reproductive damages. Forty-eight male albino rats were divided into 4 groups (n = 12), which were treated as follows: (1) Control, (2) Arsenic-intoxicated group (8 mg kg-1), (3) Arsenic + FIS-treated group (8 mg kg-1 + 10 mg kg-1), and (4) FIS-treated group (10 mgkg-1). After 56 days of treatment, the biochemical, lipidemic, steroidogenic, hormonal, spermatological, apoptotic and histoarchitectural profiles of rats were analyzed. Arsenic intoxication reduced the enzymatic activities of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GSR), in addition to glutathione (GSH) level. Conversely, the levels of thiobarbituric acid reactive substance (TBARS) and reactive oxygen species (ROS) were increased. Moreover, it escalated the level of low-density lipoprotein (LDL), triglycerides and total cholesterol, while declining the level of high-density lipoprotein (HDL). Furthermore, steroidogenic enzymes expressions, 3ß-hydroxysteroid dehydrogenase (HSD), 17ß-HSD, steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (CYP11A1) and 17α-hydroxylase/17, 20-lyase (CYP17A1), were found to be reduced, which brought down the level of testosterone. Besides, the levels of gonadotropins (LH and FSH) were decreased. Additionally, a decline in sperm mitochondrial membrane potential (MMP), motility, epididymal sperm count and hypo-osmotic swelling (HOS) coil-tailed sperms was observed, whereas the dead sperms and structural damages (head, midpiece and tail) of sperms were escalated. Moreover, arsenic exposure up-regulated the mRNA expressions of apoptotic markers, namely Bax and caspase-3, whereas lowered the expression of anti-apoptotic marker, Bcl-2. In addition, it induced histoarchitectural changes in testes of rats. However, FIS treatment resulted in remarkable improvements in testicular and sperm parameters. Therefore, it was inferred that FIS could serve as a therapeutic candidate against arsenic-generated male reproductive toxicity attributing to its anti-oxidant, anti-lipoperoxidative, anti-apoptotic, and androgenic efficacy.

Impact of tobacco smoking in association with H2BFWT, PRM1 and PRM2 genes variants on male infertility.

Tobacco's genotoxic components can cause a wide range of gene defects in spermatozoa such as single- or double-strand DNA breaks, cross-links, DNA-adducts, higher frequencies of aneuploidy and chromosomal abnormalities. The aim in this study was to determine the correlation between sperm quality determined by standard parameters, sperm DNA maturity tested by Chromomycin A3 (CMA3) staining, sperm DNA fragmentation tested by TUNEL assay and tobacco smoking in association with the single nucleotides polymorphisms (SNP) of three nuclear protein genes in spermatozoa (H2BFWT, PRM1 and PRM2). In this study, semen samples of 167 male patients were collected and divided into 54 non-smokers and 113 smokers. The target sequences in the extracted sperm DNA were amplified by PCR followed by Sanger sequencing. The results showed the presence of three variants: rs7885967, rs553509 and rs578953 in H2BFWT gene in the study population. Only one variant rs737008 was detected in PRM1 gene, and three variants were detected in the PRM2 gene: rs2070923, rs1646022 and rs424908. No significant association was observed between the concentration, progressive motility, morphology and the occurrence of H2BFWT, PRM1 and PRM2 SNPs. However, sperm parameters were significantly lower in heavy smokers compared to controls (p < 0.01) (sperm count: 46.00 vs. 78.50 mill/ml, progressive motility: 15.00% vs. 22.00%, and morphology 4.00% vs. 5.00%, respectively). Moreover, the heavy smoker individuals exhibited a considerable increase in CMA3 positivity and sDF compared to non-smokers (p < 0.01) (29.50% vs. 20.50% and 24.50% vs. 12.00%, respectively). In conclusion, smoking altered sperm parameters and sperm DNA integrity, but did not show a linkage with genetic variants in H2BFWT, and protamine genes (PRM1 and PRM2).

A lack of a definite correlation between male sub-fertility and single nucleotide polymorphisms in sperm mitochondrial genes MT-CO3, MT-ATP6 and MT-ATP8.

BACKGROUND: An inability of a man to conceive a potentially fertile woman after a year of unprotected intercourse is defined as male infertility. It is reported that 30-40% of males in their reproductive years have abnormalities in sperm production, either qualitatively or quantitatively, or both. However, genetic factors result in up to 15% of male infertility cases. The present study aimed to analyze the possible correlations between sub-fertility and polymorphisms in sperm mitochondrial CO3, ATP6 and ATP8 genes in sub-fertile men. METHODS AND RESULTS: For 67 sub-fertile and 44 fertile male samples, Sanger sequencing of selected mitochondrial DNA genes was done. A total of twelve SNPs in the MT-CO3 gene: rs2248727, rs7520428, rs3134801, rs9743, rs28358272, rs2853824, rs2856985, rs2854139, rs41347846, rs28380140, rs3902407, and 28,411,821, fourteen SNPs in the MT-ATP6: rs2001031, rs2000975, rs2298011, rs7520428, rs9645429, rs112660509, rs6650105, rs6594033, rs6594034, rs6594035, rs3020563, rs28358887, rs2096044, and rs9283154, and ten SNPs in the MT-ATP8: rs9285835, rs9285836, rs9283154, rs8179289, rs121434446, rs1116906, rs2153588, rs1116905, rs1116907, and rs3020563 were detected in the case and control groups at different nucleotide positions. Only the rs7520428 in the MT-CO3 and MT-ATP6 showed a statistically significant difference between sub-fertile and fertile groups in the genotype's and allele's frequency test (P < 0.0001 for both). CONCLUSION: The results of our study suggest that male sub-fertility is linked with rs7520428 SNP in MT-CO3 and MT-ATP6. The studied polymorphic variations in the MT-ATP8 gene, on the contrary, did not reveal any significant association with male sub-fertility.

Iridoid glycoside Aucubin protects against nonylphenol-induced testicular damage in male rats via modulation of steroidogenic and apoptotic signaling.

Aucubin (AU) is one of the widespread compounds belonging to the group of iridoid glycosides, which possesses numerous beneficial properties. Nonylphenol (NP), is a synthetic environmental toxicant that has the potential to cause male infertility through excessive production of reactive oxygen species. In the current study, the remedial potential of Aucubin was assessed against NP-generated testicular damage in male rats. Animals were distributed into four groups and treated for 56 days in this study. Control-group (0.1% DMSO + food), NP group (100 µg/kg), NP + AU group (100 µg/kg + 5 mg/kg) and AU group (5 mg/kg). NP exposure significantly (p < 0.05) reduced the activity of antioxidant enzymes i.e., glutathione reductase, catalase (CAT), superoxide dismutase, glutathione peroxidase (GPx), and total protein content (TPC), whereas the level of reactive oxygen species (ROS) and thiobarbituric acid reactive substances (TBARS) was enhanced substantially (p < 0.05). Treatment with AU substantially (p < 0.05) recovered activities of antioxidant enzymes, TPC, ROS, and TBARS levels. Moreover, decrease in the levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), plasma testosterone, sperm count, motility, sperm membrane integrity, and the number of spermatocytes of different stages along with the level of steroidogenic enzymes i.e., 17ß-hydroxysteroid dehydrogenase (17ß-HSD), 3ß-hydroxysteroid dehydrogenase (3ß-HSD), and B-cell lymphoma 2 (Bcl-2) by NP administration were recovered to control values by AU treatment. However, AU mitigated the sperm abnormalities (head/midpiece/tail), the number of dead sperms, and proapoptotic proteins i.e., Bcl-2 associated X protein (Bax), caspase-9, and caspase-3 that were increased by NP. Besides, AU treatment recovered the NP-induced potential histopathological alterations in the testicular tissues such as the height of epithelium, seminiferous tubules diameter as well as the height of tunica propria. Overall, NP-induced toxicity was effectively recuperated by the AU administration. These results indicate that AU might be considered as a potential protective agent against testicular damage. The observed protection may be due to its antioxidant, anti-apoptotic, anti-inflammatory and androgenic potential.

The influence of paternal overweight on sperm chromatin integrity, fertilization rate and pregnancy outcome among males attending fertility clinic for IVF/ICSI treatment.

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.

A Systematic Review of the Impact of Mitochondrial Variations on Male Infertility.

According to current estimates, infertility affects one in four couples trying to conceive. Primary or secondary infertility can be due either to both partners or only to the man or the woman. Up to 15% of infertility cases in men can be attributed to genetic factors that can lead to irreversible partial or complete spermatogenic arrest. The increased use of assisted reproductive technology (ART) has provided not only insights into the causes of male infertility but also afforded a diagnostic tool to detect and manage this condition among couples. Genes control a variety of physiological attributes, such as the hypothalamic-pituitary-gonadal axis, development, and germ cell differentiation. In the era of ART, it is important to understand the genetic basis of infertility so as to provide the most tailored therapy and counseling to couples. Genetic factors involved in male infertility can be chromosome abnormalities or single-gene disorders, mitochondrial DNA (mtDNA) mutations, Y-chromosome deletions, multifactorial disorders, imprinting disorders, or endocrine disorders of genetic origin. In this review, we discuss the role of mitochondria and the mitochondrial genome as an indicator of sperm quality and fertility.

Impact of heavy alcohol consumption and cigarette smoking on sperm DNA integrity.

The purposes of the presents study were to investigate the impact of alcohol consumption and cigarette smoking on semen parameters and sperm DNA quality, as well as to determine whether tobacco smoking, or alcohol consumption causes more deterioration of sperm quality. Two hundred and eleven semen samples of men were included in this study. Four groups were studied: heavy smokers (N = 48), heavy drinkers (N = 52), non-smokers (n = 70), and non-drinkers (n = 41). Semen parameters were determined according to WHO guidelines, protamine deficiency assessed by chromomycin (CMA3) staining, and sperm DNA fragmentation (sDF) evaluated by TUNEL assay. Sperm parameters were significantly higher in non-smokers versus smokers and in non-drinkers versus drinkers (p < 0.005). However, protamine deficiency and sDF were significantly lower in non-smokers versus smokers and in non-drinkers versus drinkers (p < 0.0001). No significant difference in the semen analysis parameters was observed between heavy smokers and heavy drinkers (semen volume: 3.20 ± 1.43 vs. 2.81 ± 1.56 ml, semen count: 65.75 ± 31.32 vs. 53.51 ± 32.67 mill/ml, total motility: 24.27 ± 8.18 vs. 23.75 ± 1.75%, sperm vitality: 36.15 ± 18.57 vs. 34.62 ± 16.65%, functional integrity: 41.56 ± 18.57 vs. 45.96 ± 17.98% and the morphologically normal spermatozoa: 28.77 ± 11.82 vs. 27.06 ± 13.13%, respectively). However, protamine deficiency was significantly higher among drinkers than smokers (37.03 ± 9.75 vs. 33.27 ± 8.56%, p = 0.020). The sDF was also significantly higher among drinkers than smokers (22.37 ± 7.60 vs. 15.55 ± 3.33%, p < 0.0001). Thus, cigarette smoking, and heavy alcohol intake can deteriorate sperm quality. However, alcohol consumption deteriorates sperm maturity and damages DNA integrity at significantly higher rates than cigarette smoking.

Protamines and DNA integrity as a biomarkers of sperm quality and assisted conception outcome.

Present research aim was to identify functional tests in semen associated with DNA damage and chromatin maturity (protamination) which predict the outcome in assisted reproduction. Couples were grouped according to male partner semen parameters, into normozoospermia (NZs), severe male factor (SMF) and mild male factor (MMF). DNA fragmentation index (DFI) in spermatozoa was analysed by sperms chromatin dispersion (SCD), sperm chromatin structure assay (SCSA) and acridine orange testing (AOT). Chromomycin A3 (CMA3) and toluidine blue (TB) staining to measure sperm chromatin maturity (CM). DFI and chromatin decondensation were significantly lower in N compared to male factor categories (MMF and SMF). Aneuploidy embryos were significantly higher in couples with male factor infertility (MMF and SMF). A positive correlation was observed between fertilization rate (FR) and live birth rate (LBR) with sperm concentration, motility, vitality, normal sperm morphology and negative correlation between sperm DFI and sperm CM. No correlation was observed between embryo aneuploidy and sperm DFI or CM. Lower percentage of spermatozoa chromatin integrity are associated with low fertilization and live birth rate. Male factor infertility, due to impaired semen parameters and chromatin defects could be regarded in future as an indication of IVF/ICSI, and predictor of assisted reproductive techniques outcome.

Association between the single nucleotide variants of the mitochondrial cytochrome B gene (MT-CYB) and the male infertility.

BACKGROUND: Idiopathic male infertility can be attributed to genetic predispositions that affect sperm performance and function. Genetic alterations in the mitochondrial DNA (mtDNA) have been linked to certain types of male infertility and abnormal sperm function. Mutations in the mitochondrial cytochrome B (MT-CYB) gene might lead to some deficiencies in mitochondrial function. Thus, in the current study, we aimed to investigate the effect of mutations in the MT-CYB gene on sperm motility and male infertility. METHODS AND RESULTS: Semen specimens were collected from 111 men where 67 men were subfertile and 44 were fertile. QIAamp DNA Mini Kit and REPLI-g Mitochondrial DNA Kit from QIAGEN were used to isolate and amplify the mitochondrial DNA. Followed by PCR and Sanger sequencing for the target sequence in the MT-CYP gene. Sequencing of the MT-CYB gene revealed a total of thirteen single nucleotide polymorphisms (SNPs). Eight SNPs were non-synonymous variant (missense variant) including: rs2853508, rs28357685, rs41518645, rs2853507, rs28357376, rs35070048, rs2853506, and rs28660155. While five SNPs were Synonymous variant: rs527236194, rs28357373, rs28357369, rs41504845, and rs2854124. Among these SNPs, three variants showed a significant difference in the frequency of the genotypes between subfertile and fertile groups: rs527236194 (T15784C) (P = 0.0005), rs28357373 (T15629C) (P = 0.0439), and rs41504845 (C15833T) (P = 0.0038). Moreover, two SNPs showed a significant association between allelic frequencies of rs527236194 (T15784C) (P = 0.0014) and rs41504845 (C15833T) (P = 0.0147) and male subfertility. CONCLUSION: The current study showed a significant association between the MT-CYB gene polymorphisms and the development of male infertility. In particular, rs527236194, rs28357373 and rs41504845 variants were found to be the most related to the subfertility group. Further studies on larger and other populations are required to reveal the exact role of this gene in the development of male infertility. In addition, functional studies will be helpful to elucidate the molecular impact of the MT-CYP polymorphisms on mitochondrial function.

Elevated seminal protein carbonyl concentration is correlated with asthenozoospermia and affects adversely the laboratory intracytoplasmic sperm injection (ICSI) outcomes.

Elevated concentrations of reactive oxygen species (ROS) in the semen can lead to oxidative protein damage as they react with the amino acids' side chains in the protein, leading to the generation of carbonyl groups. This study aimed to investigate the effect of protein carbonyl (PC) concentration on sperm motility and the laboratory intracytoplasmic sperm injection (ICSI) outcomes. A total of 150 couples from the ICSI cycle were enrolled in this study and were divided into three groups (G) according to the PC concentration as following, G1 included samples with PC concentrations <0.65 nmol/mg, G2 included samples with 0.65≤PC≤2.23 nmol/mg and G3 included samples with PC>2.23 (nmol/mg). PC concentrations were measured in all semen samples, and the laboratory ICSI outcomes were evaluated for all injected oocytes. The Kruskal-Wallis p-values for the differences in the medians of sperm motility, fertilisation rate, embryo cleavage score and embryo quality score were <0.05. Furthermore, Dunn's post hoc test showed a significant difference between all groups, p-values <0.05, except for the medians of embryo quality score between G2 and G3. In conclusion, our results showed that sperm motility and laboratory ICSI outcomes are affected negatively by higher concentrations of PC in the semen.

Lack of association between single polymorphic variants of the mitochondrial nicotinamide adenine dinucleotide dehydrogenase 3, and 4L (MT-ND3 and MT-ND4L) and male infertility.

Male infertility is a multifactorial condition associated with different genetic abnormalities in at least 15%-30% of cases. The purpose of this study was to identify suspected correlations between infertility and polymorphisms in mitochondrial NADH dehydrogenase subunits 3 and 4L (MT-ND3 and MT-ND4L) in subfertile male spermatozoa. Sanger sequencing of the mitochondrial DNA target genes was performed on 68 subfertile and 44 fertile males. Eight single nucleotide polymorphisms (SNPs) in MT-ND3 (rs2853826, rs28435660, rs193302927, rs28358278, rs41467651, rs3899188, rs28358277 and rs28673954) and seven SNPs in MT-ND4L (rs28358280, rs28358281, rs28358279, rs2853487, rs2853488, rs193302933 and rs28532881) were detected and genotyped. The genotypes and allele frequencies of the study population have shown a lack of statistically significant association between MT-ND3 and MT-ND4L SNPs and male infertility. However, no statistically significant association was found between the asthenozoospermia, oligozoospermia, teratozoospermia, asthenoteratozoospermia, oligoasthenoteratozoospermia and oligoteratozoospermia subgroups of subfertile males. However, rs28358278 genotype of the MT-ND3 gene was reported in the subfertile group but not in the fertile group, which implies a possible role of this SNP in male infertility. In conclusion, the investigated polymorphic variants in the MT-ND3 and MT-ND4L genes did not show any significant association with the occurrence of male infertility. Further studies are required to evaluate these findings. Moreover, the subfertile individuals who exhibit a polymorphism at rs28358278 require further monitoring and evaluation.

Mitochondrial nicotinamide adenine dinucleotide hydride dehydrogenase (NADH) subunit 4 (MTND4) polymorphisms and their association with male infertility.

PURPOSE: The purpose of the present study was to determine the relationship between infertility and the polymorphisms of mitochondrial NADH dehydrogenase subunit 4 (MTND4) by spermatozoa analysis in fertile and subfertile men. METHODS: Samples were divided into 68 subfertile men (case group) and 44 fertile men (control group). After semen analysis, samples were purified. The whole genome was extracted using a QIAamp DNA Mini Kit and the mitochondrial DNA was amplified by using the REPLI-g Mitochondrial DNA Kit. Polymerase chain reaction (PCR) was used to amplify the MT-ND4 gene. Then, samples were purified and sequenced using the Sanger method. RESULTS: Twenty-five single-nucleotide polymorphisms (SNPs) were identified in the MTND4 gene. The genotype frequencies of the study population showed a statistically significant association between rs2853495 G>A (Gly320Gly) and male infertility (P = 0.0351). Similarly, the allele frequency test showed that rs2853495 G>A (Gly320Gly) and rs869096886 A>G (Leu164Leu) were significantly associated with male infertility (adjusted OR = 2.616, 95% CI = 1.374-4.983, P = 0.002; adjusted OR = 2.237, 95% CI = 1.245-4.017, P = 0.007, respectively). CONCLUSION: In conclusion, our findings suggested that male infertility was correlated with rs2853495 and rs869096886 SNPs in MTND4.

Association between FSHR and ESR1 gene variants and ovarian response to gonadotropin in Egyptian women undergoing ICSI treatment.

The unpredictable variability in patients' responses to gonadotropins represents one of the most intractable IVF treatment problems. Identifying the genetic variants associated with ovarian responses to gonadotropins is an important step towards developing individualised pharmacogenetics protocols for ovarian stimulation. The purpose of the current study was to evaluate correlations between FSHR rs6165, FSHR rs616, and ESR1 rs2234693 gene variants and the degree of ovarian response to gonadotropin in Egyptian women undergoing ICSI treatment. Two hundred and eighty Egyptian women (mean age of 20-35) undergoing ICSI treatment were enrolled in a cross-sectional study conducted between January 2017 and May 2019. The women were classified into three groups based on ovarian response: normal responders (retrieved oocytes = 4-15) (n = 80), poor responders (retrieved oocytes < 4) (n = 92), and high responders (retrieved oocytes> 15) (n = 108). Genomic DNA was extracted from blood samples, and PCR and DNA sequencing were performed to identify genetic variations in the different study groups. FSHR and ESR1 genetic variants were then compared in normal, poor, and high responders. DNA sequencing results showed significant differences in the frequencies of FSHR rs6166 and ESR1 rs2234693 genotypes in poor responders compared with normal responders (P ≤ 0.001 and P ≤ 0.001, respectively). In contrast, no significant differences in the frequencies of FSHR rs6166, FSHR rs6165, or ESR1 rs2234693 genotypes were observed in high responders compared with normal responders (P ≤ 0.074, P ≤ 0.353, and P ≤ 0.060, respectively). These results suggest that FSHR and ESR1 gene variants could predict the degree of ovarian response to Controlled ovarian hyperstimulation in Egyptian women.

Impact of Mitochondrial Genetic Variants in ND1, ND2, ND5, and ND6 Genes on Sperm Motility and Intracytoplasmic Sperm Injection (ICSI) Outcomes.

Sperm mitochondrial dysfunction causes the generation of an insufficient amount of energy needed for sperm motility. This will affect sperm fertilization capacity, and thus, most asthenozoospermic men usually require assisted reproductive techniques. The etiology of asthenozoospermia remains largely unknown. The current study aimed to investigate the effect of mitochondrial genetic variants on sperm motility and intracytoplasmic sperm injection (ICSI) outcomes. A total of 150 couples from the ICSI cycle were enrolled in this study. One hundred five of the male partners were asthenozoospermic patients, and they were subdivided into three groups according to their percentage of sperm motility, while forty-five of the male partners were normozoospermic. Genetic variants were screened using direct Sanger's sequencing in four mitochondrial genes (nicotinamide adenine dinucleotide hydrogen (NADH) dehydrogenase 1 (ND1), NADH dehydrogenase 2 (ND2), NADH dehydrogenase 5 (ND5), and NADH dehydrogenase 6 (ND6)). We identified three significant variants: 13708G>A (rs28359178) in ND5, 4216T>C (rs1599988) in ND1, and a novel 12506T>A in ND5 with P values 0.006, 0.036, and 0.013, respectively. The medians of sperm motility, fertilization rate, embryo cleavage score, and embryo quality score were significantly different between men showing 4216T>C, 12506T>A, 13708G>A and wild type, Mann-Whitney P values for the differences in the medians were < 0.05 in all of them. The results from this study suggest that 13708G>A, 12506T>A, and 4216 T>C variants in sperm mitochondrial DNA negatively affect sperm motility and ICSI outcomes.