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.

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.

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.

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.

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.

Tobacco smoking and its impact on the expression level of sperm nuclear protein genes: H2BFWT, TNP1, TNP2, PRM1 and PRM2.

The aim of this current study was to investigate the influence of tobacco smoke on sperm quality determined by standard parameters, on sperm DNA maturity tested by chromomycin A3 (CMA3) staining, on sperm DNA fragmentation tested by TUNEL assay and on the transcript level of sperm nuclear proteins H2BFWT, PRM1, PRM2, TNP1 and TNP2 genes quantified by RT-PCR. One hundred forty-one (141) sperm samples (43 nonsmokers (G.1) and 98 heavy smokers (G.2)) of couples undergoing ICSI were enrolled in this study. In G2, a significant decrease in standard semen parameters in comparison with nonsmokers was shown (p < .01). In contrast, protamine deficiency (CMA3 positivity) and sperm DNA fragmentation (sDF) were significantly higher in G2 than in G1 (p < .01). Furthermore, the studied genes were differentially expressed (p < .01), down-regulated in the spermatozoa of G.2 compared to that of G.1 (fold change <0.5) and were significantly correlated between each other (p < .01). Moreover, in comparison with G1, the protamine mRNA ratio in G2 was significantly higher (p < .01). It can therefore be concluded that smoking alters mRNA expression levels of H2BFWT, TNP1, TNP2, PRM1 and PRM2 genes and the protamine mRNA ratio and consequently alters normal sperm function.

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.

Impact of the Vegan Diet on Sperm Quality and Sperm Oxidative Stress Values: A Preliminary Study.

BACKGROUND: Insufficient nutrition and inappropriate diet have been related to many diseases. Although the literature confirms the hypothesis that particular nutritional factors can influence the quality of semen, until today, there are no specific dietary recommendations created for infertile males. Since the male contribution to the fertility of a couple is crucial, it is of high importance to determine the dietary factors that can affect male fertility. AIM: The aim of the present study was to evaluate differences in sperm quality parameters, sperm oxidative stress values and sperm acrosome reaction between vegan diet consumers and non-vegans. SETTING AND DESIGN: Prospective study in a University Medical School. MATERIALS AND METHODS: The present study was undertaken to evaluate the sperm quality parameters of vegan diet consumers (10 males who had a strictly vegetable diet with no animal products) and compare them with non-vegans (10 males with no diet restrictions). Semen quality was assessed following the World Health Organization (2010) criteria. Acrosome and DNA integrity has been evaluated using the immunofluorescence technique. STATISTICAL ANALYSIS: All variables were analysed by IBM SPSS version 24. Mean differences among groups were compared by Mann-Whitney U-test. RESULTS: Obtained results showed that total sperm count (224.7 [117-369] vs. 119.7 [64.8-442.8]; P = 0.011) and the percentage of rapid progressively motile sperm were significantly higher in the vegan group compared with the non-vegan group (1 [0-7] vs. 17.5 [15-30]; P < 0.0001). Furthermore, the oxidation-reduction potential (0.4 [0.3-0.9] vs. 1.5 [0.6-2.8]; P < 0.0001) and the proportion of spermatozoon with DNA damage (14.7 [7-33.5] vs. 8.2 [3-19.5]; P = 0.05) were significantly higher in the non-vegan group in comparison to the vegan group. CONCLUSIONS: Results obtained in this study provide additional evidence about the favourable effect of a plant-based diet on sperm parameters. To confirm our preliminary findings, further studies including larger cohorts are warranted.

Correlation between total sperm count and sperm motility and pregnancy rate in couples undergoing intrauterine insemination.

The frequency and significance of sterility is increasing due to different socio-demographic factors in the industrialized countries. At the same time, the patients' demand for more natural and less invasive fertility treatments is increasing. The most common method used in subfertility is intrauterine insemination (IUI). Retrospectively, the data from the patients were analyzed, in which at least one insemination and a maximum of eight inseminations were performed in the last five years (observation period 01.01.2014-31.12.2018) at the Women's University Hospital Homburg. The primary endpoint was the onset of a clinical pregnancy. Clinical pregnancy was correlated with the partner's total sperm count (sperm density in millions), sperm concentration and motility during insemination. These three parameters were evaluated according the World Health Organization (WHO) 2010 guidelines. The results of the spermiograms were correlated with clinical pregnancy outcome. The data were examined for 138 women with sterility, in which a total of 345 inseminations were performed (median 2.5 per woman, range 8 inseminations). There was no correlation found between spermiogram parameters and pregnancy probability in any of the inseminations. After 5 inseminations no further pregnancy occurred. The present study showed no correlation between the conception probability of intrauterine insemination (IUI) and the total sperm count/concentration/motility. After the sixth IUI, we no longer found conceptions in our patient collective. Therefore, data from this study indicate that intrauterine inseminations can be performed at all severity levels of oligoasthenozoospermia. However, the treatment should be limited to five attempts.

Influence of variation in global sperm DNA methylation level on the expression level of protamine genes and human semen parameters.

An infertility problem is a complex issue that affects 15% approximately of couples worldwide. The current study was designed to evaluate if there is a variation in the status of global DNA methylation among the study groups and to assess their impact on the protamine expression level and human semen parameters. Totalling 200 semen samples were collected from men (50 proved fertile, 60 normospermia and 90 oligospermia) with an average age of 34.9 ± 4.3 years. The DNA and RNA were isolated from purified spermatozoa; then, ELISA and qPCR were applied to estimate the status of global sperm DNA methylation and protamine expression level respectively. Besides that, the sperm chromatin decondensation and sperm DNA fragmentation were assessed. A significant variation was found in the global sperm DNA methylation and the protamine 1 and protamine 2 expression level among the study groups (p ≤ .001). Down-regulation has been found in the protamine 1 and protamine 2 expression levels in the oligospermia group compared to the proved fertile group with fold change (0.001 and 0.0002 respectively). In conclusion, this study proposes that the alteration in global DNA methylation may influence the protamine expression level and may be lead to abnormalities in human semen parameters.

An additional marker for sperm DNA quality evaluation in spermatozoa of male partners of couples undergoing assisted reproduction technique (IVF/ICSI): Protamine ratio.

The aim of this study was to evaluate the relationship between the protamine ratio (P1/P2), DNA fragmentation of spermatozoa and protamine deficiency. Patients were grouped into fertile (G1; n = 151) and sub-fertile (G2; n = 121). DNA fragmentation in spermatozoa was analysed by a TUNEL assay (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling), and the protamination was determined by CMA3 staining, while Western blot was used to measure protamine P1 and P2. While sperm DNA fragmentation (SDF) and protamine ratio were significantly elevated in G2 compared with G1 (12.31 ± 7.01% vs. 17.5 ± 9.5%; p = .001) and (0.91 ± 0.43 vs. 0.75 ± 0.42; p = .003); respectively, the CMA3 positive showed no difference at all between G1 and G2. In G1, the CMA3 positive correlated negatively with the P1/P2 ratio and SDF (r = -.586, r = -.297; p = .001 respectively). In contrast, the protamine ratio correlated positively with SDF (r = .356; p = .001). In G2, no correlation was observed between CMA3 positive, SDF and the P1/P2 ratio but the P1/P2 ratio showed a positive correlation with SDF (r = .479; p = .001). In conclusion, the spermatozoa DNA deterioration was closely associated with abnormal protamination but showed an association with the protamine ratio, more than with CMA3 positive. Therefore, for the evaluation of DNA damage in spermatozoa, the P1/P2 ratio might act as an additional biomarker.

Influence of cryopreservation on the CATSPER2 and TEKT2 expression levels and protein levels in human spermatozoa.

This study designed to assess the expression level of CATSPER2 and TEKT2 and to evaluate the levels of CatSper2 and Tektin2 proteins in human spermatozoa before and after cryopreservation. One hundred and twenty semen samples were included in this study. All the samples were subjected to qPCR and Western blot analysis. The results showed a significant reduction in the expression levels of CATSPER2 and TEKT2 in the cryopreserved compared to the fresh samples (P = 0.0039 and P = 0.0166, respectively), and the results showed down-regulation in the expression level of CATSPER2 and TEKT2 genes between the study groups. Moreover, the protein levels of the CatSper2 and Tektin2 were lower in cryopreserved samples compared to fresh samples (P = 0.0001). In conclusion, the reduction in the proteins level and expression level of the CATSPER2 and TEKT2 in cryopreserved samples could be used as an indicator of sperm motility loss.

The relationship between preimplantation morphokinetics of human embryos and sex chromosome pattern.

The aim of this study was to determine the relationship between the gender of human embryos and chronological changes during the two pronuclear (2PN) embryonic stage and blastocyst formation on day five after injection using time-lapse imaging and preimplantation genetic testing. A total of 120 couples who underwent intracytoplasmic sperm injection with preimplantation genetic testing were included in the study. Only normal embryos (n = 416) were enrolled in this study. Time-lapse imaging data of male (n = 227) and female (n = 189) embryo progression was carried out to estimate the times of initial appearance of the 2PN stage, fading times and day five blastulation rates. The results revealed that the 2PN stage (fertilisation) was reached significantly earlier in female embryos (9.09 ± 1.31 h) than in male embryos (9.52 ± 1.48 h, p-value = 0.0044). Conversely, the fading time of 2PN was significantly faster in male embryos (22.13 ± 2.02) than in female embryos (24.16 ± 2.61, p-value < 0.001). The blastulation rate was significantly higher in female than male embryos (68.25% vs. 57.71%; p-value = 0.025).

Relationships between bacteriospermia, DNA integrity, nuclear protamine alteration, sperm quality and ICSI outcome.

The Aim of this study was to evaluate the effects of bacteriospermia on human sperm parameters, nuclear protamines, DNA integrity and ICSI outcome in patients enrolled for ICSI treatment. 84 unselected couples consulting in infertility and obstetrics clinic and enrolled for ICSI treatment were included in this study. The semen specimens were screened bacteriologically; semen and sperm parameters were also evaluated according to WHO guidelines. DNA integrity, protamines concentration and protamine deficiency were estimated by TUNEL assay, AU-PAGE and Chromomycin (CMA3) respectively. The results of this study revealed that 34.52% of studied semen samples were infected with bacteria. The isolated bacteria were identified as Staphylococcus aureus, Staph. epidermidis, Staph. haemolyticus, Escherichia coli, Enterococcus faecalis and Streptococcus agalactiae. Bacteriospermia had a significant (p < .010) negative effect on sperm parameters; concentration, motility, progressive motility and chromatin condensation. Moreover, high DNA fragmentation with low P1 and P2 concentrations were noticed in infected patients in comparison to non-infected patients but non-significant. Also, the fertilization rate decreased significantly (p < .05) with infected patients. IN CONCLUSION: bacteriospermia has significant negative effect on sperm quality and fertilization rate in patients who underwent ICSI treatment.