Spermatozoa Of Infertile Men Research Articles

Impact of human papillomavirus and coinfection with other sexually transmitted pathogens on male infertility.

This study primarily aimed to investigate the prevalence of human papillomavirus (HPV) and other common pathogens of sexually transmitted infections (STIs) in spermatozoa of infertile men and their effects on semen parameters. These pathogens included Ureaplasma urealyticum, Ureaplasma parvum, Chlamydia trachomatis, Mycoplasma genitalium, herpes simplex virus 2, Neisseria gonorrhoeae, Enterococcus faecalis, Streptococcus agalactiae, Pseudomonas aeruginosa, and Staphylococcus aureus. A total of 1951 men of infertile couples were recruited between 23 March 2023, and 17 May 2023, at the Department of Reproductive Medicine of The First People's Hospital of Yunnan Province (Kunming, China). Multiplex polymerase chain reaction and capillary electrophoresis were used for HPV genotyping. Polymerase chain reaction and electrophoresis were also used to detect the presence of other STIs. The overall prevalence of HPV infection was 12.4%. The top five prevalent HPV subtypes were types 56, 52, 43, 16, and 53 among those tested positive for HPV. Other common infections with high prevalence rates were Ureaplasma urealyticum (28.3%), Ureaplasma parvum (20.4%), and Enterococcus faecalis (9.5%). The prevalence rates of HPV coinfection with Ureaplasma urealyticum, Ureaplasma parvum, Chlamydia trachomatis, Mycoplasma genitalium, herpes simplex virus 2, Neisseria gonorrhoeae, Enterococcus faecalis, Streptococcus agalactiae, and Staphylococcus aureus were 24.8%, 25.4%, 10.6%, 6.4%, 2.4%, 7.9%, 5.9%, 0.9%, and 1.3%, respectively. The semen volume and total sperm count were greatly decreased by HPV infection alone. Coinfection with HPV and Ureaplasma urealyticum significantly reduced sperm motility and viability. Our study shows that coinfection with STIs is highly prevalent in the semen of infertile men and that coinfection with pathogens can seriously affect semen parameters, emphasizing the necessity of semen screening for STIs.

Diagnostic sensitivity and specificity of indicators of glutathione antioxidant system in spermatozoa of infertile men with different forms of pathospermia

Background. One of the most important antioxidant defence mechanism in spermatozoa is the gluthatione system which includes glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST) and reduced glutathione (GSH). It is promising to use ROC analysis, which allows to assessing the diagnostic sensitivity and specificity of indicators. Materials and methods. Infertile men were divided into 3 groups: patients with oligozoospermia (n = 30), asthenozoospermia (n = 34), and oligoasthenozoospermia (n = 22). To assess the diagnostic sensitivity and specificity of indicators, the values of the biomarkers were tested using the receiver operating characteristic (ROC) curve, and the area under it (AUC), the standard error (SE) and the 95% confidence interval (CI 95%) were calculated. Results. The ROC analysis showed that GP activity was characterized by excellent diagnostic significance for diagnosing both oligozoospermia and asthenozoospermia (the sensitivity was 100%, and specificity – 100%). The GR activity has moderate diagnostic significance, since the AUC is 0.654 (95% CI from 0.503 to 0.785, P = 0.0645) for oligozoospermic, the AUC is 0.612 (95% CI from 0.454 to 0.7555, P = 0.1979) for asthenozoospermic men. The analysis of the ROC curve revealed a good diagnostic value of GsT activity in sperm samples for the diagnosis of pathospermia (sensitivity of 75% and specificity of 80%). Simultaneously, it was shown that GSH content could not serve as valuable biomarkers for distinguishing patients with pathospermia from healthy controls, with an AUC of 0.615, corresponding to moderate diagnostic significance for oligozoospermia. Conclusion. The results of this study show that the cut-off points for the biomar­kers glutathione peroxidase and glutathione-S-transferase can be used to distinguish between patients with pathospermia and normozoospermia, and the parameters themselves can serve as valuable diagnostic biomarkers to distinguish patients with pathospermia from healthy controls, regardless of the causes of pathospermia. The value of these indicators below the cut-off point indicates the probable presence of pathology. Indicators of GR activity and GSH content have a low diagnostic value, which makes them unsuitable for use as laboratory tests for the diagnosis of male infertility.

Novel differentially expressed male infertility-associated genes in sperm as prospective diagnostic biomarkers

Genetic defects in sperm are responsible for a great percentage of male infertility. Dysregulation of these genes directly influences sperm morphology, motility, and viability. Therefore, analyzing gene expression aberrancies is a must in male infertility. Microarray analysis is practically used for several aspects of male infertility, including the detection of differentially expressed genes and the identification of potential infertility biomarkers. We conducted a meta-analysis using microarray datasets, including the datasets containing sperm tissues from both healthy and infertile males. Seven datasets qualified for inclusion in this study and were then transformed into a single set of meta-data. For these genes, expression and diagnostic analyses were conducted. Additionally, enrichment analysis revealed the role and function of these genes in cellular processes. Six genes—S100Z, SLC2A2, IMPG1, HOXD12, RAPGEFL1, and DMBX1—were found to be significantly down-regulated in the sperm of infertile men. Notably, the expression of these genes was highly correlated in the sperm of these men. In addition, receiver operating curve analysis indicated that these genes may serve as useful biomarkers for infertility diagnosis. The role of these genes in transporting glucose, vitamins, and fructose as the sperm's primary fuel source was suggested by pathway analysis.

RELATIONSHIP BETWEEN SPERM DNA FRAGMENTATION AND EMBRYO DEVELOPMENT IN IVF PATIENTS

Infertility is a condition in married couples who have regular and adequate sexual intercourse for more than one year without using contraception, but have not had a pregnancy or offspring. Factors that cause infertility can be caused by men, women, or even both. Approximately 10% - 15% of couples of reproductive age experience infertility, with the contribution of the male factor, which is equal to 40% -50%. The analysis of embryo development and spermatozoa DNA fragmentation aims to analyze the function of spermatozoa in infertile men and to determine the relationship between sperm DNA fragmentation and embryo development in IVF patients. This study was an experimental study with a total of 30 samples of husband's patients who carried out the IVF program who met the researcher's inclusion criteria. The statistical test results obtained showed that there was a significant relationship between the DNA fragmentation index (DFI) (P<0.05), therefore it can be concluded that further research is needed so that many more things can be explored. of other causes of infertility.

Assessment of semen parameters after microsurgical varicocelectomy in men from infertile couples

Introduction. Varicocele is one of the frequently identified and corrected causes of male subfertility. However, varicocele correction results in improved sperm quality only in 60 – 70% of subfertile men. At the same time, it is not completely clear which semen parameters improve to a greater extent, and what is the proportion ofpatients with an improvement in parameters to normozoospermia.Objectives. To assess qualitative changes in semen after microsurgical varicocelectomy according to WHO 2021 Laboratory manual of semen analysis.Materials & methods. This study analysed qualitative changes in sperm in infertile men after varicocelectomy. All patients had a clinical varicocele, for which microsurgical varicocelectomy was performed. The study included 100 subfertile men with varicocele and abnormal sperm parameters.Results. In the postoperative period (after 6 months), the main semen parameters increased significantly. Also, the proportion of patients with isolated asthenozoospermia decreased by 22% (p < 0.05), the proportion of patients with oligoasthenozoospermia decreased by 14% (p < 0.05). The proportion of patients with normospermia was 35% (p < 0.0001). In patients with oligoasthenozoospermia significant improvement in sperm to normospermia was observed in 19% of cases.Conclusions. Varicocele repair leads to an improvement in the quality of the ejaculate in subfertile men with palpable varicocele and pathozoospermia, with the most significant improvement in sperm motility. However, only in a third of patients semen quality improved to normal levels. Additionally, varicocelectomy demonstrated high efficacy in patients with oligoasthenozoospermia.

Role of vitamin E and zinc sulfate in infertile men

Infertility affects 15-30% of couples that are trying to conceive, about half of these cases, male partner is the sole contributing factor. Infertility remains a controversial problem worldwide, male infertility caused by different anatomical defect like obstruction of seminal tube and neurological abnormalities, resulting in abnormal spermatogenesis which weakens the function of sperm.Follow-up study done for 60 infertile men with age range between 30-45 years. Data collected was carried out during the period from April 2019 to August 2019 in Bajii city which include seminal fluid analysis, determination of testosterone, prolactin, and T3 and T4 before and after treatment with vitamin E(800 I.U/daily) and zinc sulfate(50mg/daily) which last for three months , all data were presented as mean and standard deviation. In this study we exclude any disease or abnormal condition that related to this group.There are 60 infertile men participated in this study, age range between 30-45 years. In these cases we measured the seminal fluid analysis, determination of testosterone, prolactin, and T3 and T4 before and after treatment with vitamin E(800 I.U/daily) and zinc sulfate(50mg/daily) which last for three monthsAbout the seminal fluid analysis, there is a significant increase in number of sperm in infertile men after the treatment, and also there is a significant increase in activity of sperm after treatment. Regarding the seminal fluid analysis, there is a significant increase in number of sperm in infertile men after the treatment compared with this before treatment and also there is a significant increase in activity of sperm after treatment compared with this before treatment. About the percentage of non-motile sperm, there is a significant decrease in this percentage after treatment compared with this before treatment, also there is decrease in percentage of abnormal sperm shape after treatment compared with this before treatment, while there is a significant increase in percentage of normal sperm shape after treatment compared with this before treatment About the concentration of prolactin, there is none significant difference between its level after treatment and before treatment. Regarding the concentration of testosterone, there is a significant elevation in its concentration after the treatment compared with its concentration before treatment and About the concentration of T3 and T4, there is none significant difference in its concentration after treatment and before treatment respectively.

CAG Expansion in Androgen Receptor Gene of Infertile Men in Erbil Governorate

Spermatogenesis and male phenotypic development during puberty are mainly done by androgen and their function is regulated by the androgen receptor (AR) gene. This gene has a polymorphism site in Exon1 which encode androgen receptor and have various length of CAG trinucleotide repeat which causes the production of polyglutamine chain in different length of the N-terminal domain of AR protein which reduces producing sperm by disrupting spermatogenesis.
 The aim is to determine the relation of infertility in male with the increased frequency of CAG repeats in the AR gene, and the correlation between CAG repetition and hormonal changes.
 The case-control research was carried out in the Immunogene center and IVF center in the maternity teaching hospital Erbil-Kurdistan region-Iraq. The convenience sample included 50 men, 30 infertile and 20 fertile over one year starting from March 2021 to March 2022. 
 Men with infertility had CAG repeats in their AR gene, ranging from (17-26) repeats, with a mean (21.3 ±0.16). In infertile men, CAG expansion was longer than the fertile men. The motility and normal morphology of sperm in infertile men have negative relation while sperm count and concentration have a positive relation with CAG expansion. The relation of hormones (Testosterone, LH, and FSH) with CAG repetition was statistically not significant.
 In conclusion, CAG expansion was longer in infertile (case) men compared with fertile (control) men. Polyglutamine effect on increasing sperm abnormal morphology and immotility which is the reason for infertility but statistically not significant and it will not affect hormonal assay in infertile men.

Altered DNA methylation in estrogen-responsive repetitive sequences of spermatozoa of infertile men with shortened anogenital distance

BackgroundIt has been suggested that antenatal exposure to environmental endocrine disruptors is responsible for adverse trends in male reproductive health, including male infertility, impaired semen quality, cryptorchidism and testicular cancer, a condition known as testicular dysgenesis syndrome. Anogenital distance (AGD) is an anthropomorphic measure of antenatal exposure to endocrine disruptors, with higher exposure levels leading to shortened AGD. We hypothesized that exposure to endocrine disruptors could lead to changes in DNA methylation during early embryonic development, which could then persist in the sperm of infertile men with shortened AGD.ResultsUsing fluorescence activated cell sorting based on staining with either YO-PRO-1 (YOPRO) or chromomycin-3 (CMA3), we isolated four sperm fractions from eleven infertile men with short AGD and ten healthy semen donors. We examined DNA methylation in these sorted spermatozoa using reduced representation bisulfite sequencing. We found that fractions of spermatozoa from infertile men stained with CMA3 or YOPRO were more likely to contain transposable elements harboring an estrogen receptor response element (ERE). Abnormal sperm (as judged by high CMA3 or YOPRO staining) from infertile men shows substantial hypomethylation in estrogenic Alu sequences. Conversely, normal sperm fractions (as judged by low CMA3 or YO-PRO-1 staining) of either healthy donors or infertile patients were more likely to contain hypermethylated Alu sequences with ERE.ConclusionsShortened AGD, as related to previous exposure to endocrine disruptors, and male infertility are accompanied by increased presence of hormonal response elements in the differentially methylated regulatory sequences of the genome of sperm fractions characterized by chromatin decondensation and apoptosis.

Sertoli cells require TDP-43 to support spermatogenesis†.

TAR DNA binding protein of 43 kD (TDP-43) is an evolutionarily conserved, ubiquitously expressed transcription factor and RNA-binding protein with major human health relevance. TDP-43 is present in Sertoli and germ cells of the testis and is aberrantly expressed in the sperm of infertile men. Sertoli cells play a key role in spermatogenesis by offering physical and nutritional support to male germ cells. The current study investigated the requirement of TDP-43 in Sertoli cells. Conditional knockout (cKO) of TDP-43 in mouse Sertoli cells caused failure of spermatogenesis and male subfertility. The cKO mice showed decreased testis weight, and low sperm count. Testis showed loss of germ cell layers, presence of vacuoles, and sloughing of round spermatids, suggesting loss of contact with Sertoli cells. Using a biotin tracer, we found that the blood-testis barrier (BTB) was disrupted as early as postnatal day 24 and worsened in adult cKO mice. We noted aberrant expression of the junction proteins connexin-43 (gap junction) and N-cadherin (ectoplasmic specialization). Oil Red O staining showed a decrease in lipid droplets (phagocytic function) in tubule cross-sections, Sertoli cells cytoplasm, and in the lumen of seminiferous tubules of cKO mice. Finally, qRT-PCR showed upregulation of genes involved in the formation and/or maintenance of Sertoli cell junctions as well as in the phagocytic pathway. Sertoli cells require TDP-43 for germ cell attachment, formation and maintenance of BTB, and phagocytic function, thus indicating an essential role for TDP-43 in the maintenance of spermatogenesis.

Proteomic and genetic dissection of testis-specific histone 2B in infertile men reveals its contribution to meiosis and sperm motility

To investigate testis-specific histone 2B (TSH2B) and its gene anomalies in infertile men. Case-control study. Basic science laboratory. Fertile and infertile men. Not applicable. The histone and protamine status of sperm was studied by aniline blue and chromomycin A3 staining, respectively. Testis-specific histone 2B, total H2B, and phosphorylated TSH2B (pTSH2B) were estimated by Western blot analysis. The frequency of genetic polymorphisms and rare variants in H2BC1 was studied by Sanger sequencing. Phosphosites on TSH2B in sperm were identified by reverse-phase high-performance liquid chromatography purification of TSH2B followed by mass spectrometric analysis. Aniline blue and chromomycin A3 staining revealed significantly higher histone retention and low protamine in sperm of infertile men. Sperm TSH2B and total H2B levels were significantly lower in oligozoospermic and oligoasthenozoospermic men (in both groups). The TSH2B levels were comparable in asthenozoospermic men; however, the pTSH2B level was significantly low. The H2BC1 gene sequencing identified 6 variants, of which 2 are rare variants (rs368672899 and rs544942090) and 4 (rs4711096, rs4712959, rs4712960 and rs4712961) are single nucleotide polymorphisms. Minor allele frequency of 5'-untranslated region variant rs4711096 was significantly lower in infertile men (OR = 0.65). The rare nonsynonymous variant, rs368672899, p.Ser5Pro was seen in 1 oligoasthenoteratozoospermic individual. Interestingly, mass spectrometric analysis identified a site on TSH2B to bear a phosphate group in the sperm of fertile men. Our study reveals a defect in the replacement of somatic histones with testis-specific variants in infertile men. Chromatin compaction positively correlates with sperm motility, which is suggestive of its utility in diagnostic semen analysis of infertile individuals. Our observations with TSH2B and its cognate gene in sperm of infertile men indicate an essential role for TSH2B in meiosis and its phosphorylation in sperm motility, respectively.

Effects of PRP Intratesticle Injection in Non-Obstructive Azoospermic Infertile Men in vivo on Spermatogenesis and Reproductive Hormonal Levels(FSH and LH)

Non-obstructive azoospermia (NOA) is the absence of sperm in infertile men ejaculate as a result of significant spermatogenesis defects or hormonal malfunction. PRP intratesticular injection may be employed in assisted reproductive technology as a therapeutic effect for this problem. The aim is to assess the level of spermatogenesis and reproductive hormones after PRP autologous intratesticular injection. 50 nonabstructive azoospermia infertile men participated in the current study. The patients were given an intra-testicular injection of platelet-rich plasma using a 1 ml syringe; FSH LH hormonal levels were estimated; if there was no sperm count, the patient should be prepared for a testicular biopsy by the expert doctor. Small number of sperm may be discovered during a biopsy while the testes are being examined in the lab. The therapeutic benefits were seen in 15 patients 3-4 months after using autologous platelet-rich plasma. The results showed that there was a highly significant difference in FSH and LH hormonal levels after PRP injection P(0.001). PRP can help non-obstructive azoospermic infertile males with their testis' structural and functional dysfunction.

Evaluation the expression of insulin and insulin receptor-beta (IR-β) in sperm of infertile male with failed intracytoplasmic sperm injection (ICSI)

Introduction: Most infertility treatment centers are currently using semen analysis to differentiate between fertile and infertile individuals. Recent studies have evaluated the expression and secretion of independent insulin and related beta receptor (IR-β) in mammals’ ejaculated spermatozoa such as humans and pigs, and its effect on motility parameters capacitation and acrosomal reaction. Objectives: This study aimed to investigate the expression of insulin, IR-β and the parameters of sperm and DNA fragmentation index (DFI) in men with intracytoplasmic sperm injections (ICSI) failed. Patients and Methods: This study was conducted on 15 men with unknown infertility with a history of unsuccessful ICSI and 10 fertile men. After collecting samples, sperm parameters (concentration and motility), sperm DNA fragmentation and expression of insulin and IR-β genes were investigated. To assess DFI from SDFA kit and to review the expression, the genes were analyzed via RT-PCR method. Data were analyzed statistically by t-test and covariance tests. Results: Based on the findings of this research, the concentration and percentage of motility in infertile men with a history of unsuccessful ICSI was significantly lower than fertile individuals, moreover, the amount of DNA fragmentation significantly increased in infertile men with a history of unsuccessful ICSI compared to fertile men (P < 0.05). Insulin expression in the sperm of infertile men with a failed ICSI history was significantly lower than fertile men (P < 0.05); however, the IR-β expression rate was not significantly different between the two groups. Conclusion: Evaluation of insulin gene expression can be helpful as a good marker for identifying infertility with an unknown cause.

Contemporary Use of ICSI and Epigenetic Risks to Future Generations.

Since the birth of Louise Brown in 1978 via IVF, reproductive specialists have acquired enormous knowledge and refined several procedures, which are nowadays applied in assisted reproductive technology (ART). One of the most critical steps in this practice is the fertilization process. In the early days of IVF, a remarkable concern was the unpleasant outcomes of failed fertilization, overtaken by introducing intracytoplasmic sperm injection (ICSI), delineating a real breakthrough in modern ART. ICSI became standard practice and was soon used as the most common method to fertilize oocytes. It has been used for severe male factor infertility and non-male factors, such as unexplained infertility or advanced maternal age, without robust scientific evidence. However, applying ICSI blindly is not free of potential detrimental consequences since novel studies report possible health consequences to offspring. DNA methylation and epigenetic alterations in sperm cells of infertile men might help explain some of the adverse effects reported in ICSI studies on reproductive health in future generations. Collected data concerning the health of ICSI children over the past thirty years seems to support the notion that there might be an increased risk of epigenetic disorders, congenital malformations, chromosomal alterations, and subfertility in babies born following ICSI compared to naturally conceived children. However, it is still to be elucidated to what level these data are associated with the cause of infertility or the ICSI technique. This review provides an overview of epigenetic mechanisms and possible imprinting alterations following the use of ART, in particular ICSI. It also highlights the sperm contribution to embryo epigenetic regulation and the risks of in vitro culture conditions on epigenetic dysregulation. Lastly, it summarizes the literature concerning the possible epigenetic disorders in children born after ART.

Beneficial Effects of Antioxidants in Male Infertility Management: A Narrative Review

Background: Infertility, defined as the failure to conceive after one year of regular, unprotected intercourse, affects 50–80 million people worldwide. A male factor is involved in approximately 20–30% of cases. In the etiology of male infertility, the association between poor semen quality and oxidative stress (OS) is well known. High levels of reactive oxygen species (ROS) allow the oxidation of DNA, proteins, and lipids of sperm cells, modifying their vitality, motility, and morphology. Methods: To evaluate the effects of antioxidants on sperm in infertile men, we queried the MEDLINE database (via the PubMed interface) for published studies in the last 10 years (2011–2021). The following keywords were used: “infertility” and -“inositol”, -“alpha-lipoic acid”, -“zinc”, -“folate”, -“coenzyme Q10”, -“selenium”, and -“vitamin”. Results: Inositol regulates OS levels in sperm cells thanks to its role in mitochondrial reactions and is involved in several processes favoring sperm–oocyte interactions. Alpha-lipoic acid (ALA) reduces ROS damage and improves semen parameters in terms of spermatozoa’s motility, morphology, and count. Poor zinc nutrition may be related to low quality of sperm. Supplementation of folate plus zinc has a positive effect on the sperm concentration and morphology. Supplementation with CoQ10 increases sperm concentration, total and progressive motility. Selenium (Se) supplementation improves the overall semen quality and is related to a higher ejaculated volume. Among vitamins, only vitamin B12 shows a positive effect on semen quality; it increases sperm count and motility and reduces sperm DNA damage. Conclusions: In men showing low-quality semen, diet supplementation with antioxidants may improve the sperm quality by alleviating OS-induced sperm damage and enhancing hormone synthesis and spermatozoa concentration, motility, and morphology. Future clinical trials should be focused on the possible association of several antioxidants to take advantage of combined mechanisms of action.

Characteristic of spermogram parameters in men with reproductive pathology in age-related aspect

Introduction. The prevalence of andrological diseases among adolescents and young adults resulting in lowered reproductive potential has been noted to progressively increase. At the same time, the number of couples starting to manage reproductive issues after 35–40 years of age highlighting the onset of male androgen deficiency continues to rise. Therefore, the analysis of spermogram as the key element in assessing male reproductive potential is better to conduct at different age periods of man's life.Aim: to compare spermogram parameters in different age groups of patients with reproductive pathology.Materials and Мethods. The analysis of spermograms in adolescents with left-sided grade II–III varicocele aged 17 years and in infertile males aged 22–48 years was performed. Semen analysis was conducted in accordance with the standards of the 5 th edition of the World Health Organization and included the following parameters: semen volume (ml), sperm concentration (million/ml), total sperm count (million), acidity, viscosity, progressive motility, total motility, viability, morphology, detected mucus, leukocytes, amyloid bodies, lecithin grains as well as sperm aggregation and agglutination. The stained preparations were used to assess the morphology of spermatozoa and spermatogenesis cells. According to the spermogram data obtained, the following conclusions were drawn: normozoospermia, oligozoospermia, asthenozoospermia, teratozoospermia. Statistical analysis was performed by using Statistica 10.0 software (StatSoft Inc., USA). The normality distribution was assessed using the χ2 test. Quantitative parameters were presented as arithmetic means and standard deviations (M ± SD). Assessing significance of differences was performed by using the Student's t-test, whereas inter-parameter correlation relations were analyzed by using the linear Pearson's correlation coefficient. A significance level between inter-group parameters was set at p < 0.05.Results. It was found that adolescents with varicocele vs. adult men had significantly decreased ejaculate volume. In particular, the average ejaculate volume in adolescents and adult men was 2.32 ± 1.22 ml and 3.50 ± 1.44 ml, respectively, so that the larger number of young patients were noted to have ejaculate volume below 1.5 ml. Compared to young subjects, aged patients had decreased sperm concentration (35.88 ± 25.74 versus 72.20 ± 49.32 million/ml) and total sperm count (120.58 ± 91.72 versus 173.07 ± 163.92 million). Young patients were found to have significantly superior data in all categories of sperm motility, whereas infertile men were diagnosed with impaired sperm motility. In particular, adolescents were featured with the average number of spermatozoa displaying fast and slow translational movement comprising 17.12 ± 11.04 % and 29.30 ± 12.29 %, respectively, the proportion of progressive motility spermatozoa was 46.20 ± 19.82 %. In contrast, similar parameters in adult men were 5.10 ± 6.36 %, 19.80 ± 9.61 %, and 24.95 ± 11.23 %, respectively. In infertile men prevalence of lacked spermatozoa with rapid forward movement was 46 (46.0 %), in adolescents – 8 (8.6%), whereas rate of immotile spermatozoa in infertile men, on average, accounted for 53.10 ± 14.56 %, in adolescents – 34.40 ± 21.83 %. In addition, adolescents with varicocele had significantly fewer spermatozoa with normal morphology – 14.14 ± 8.06 % (in adult men – 30.08 ± 17.94 %), there were more abundant defects in the sperm head – 58.01 ± 12.43 % (in men – 48.83 ± 18.95 %) and flagella – 17.24 ± 6.31 % (in men – 10.29 ± 6.21 %). The data obtained showed that adolescents were more often diagnosed with normozoospermia – in 49 (52.7 %) cases, in infertile men – in 12 (12.0 %) cases, whereas in aged men asthenozoospermia was detected in 82 (82.0 %) cases, in adolescents – 5 (5.4 %) cases.Conclusion. The abnormalities in the spermogram revealed in adolescents may be associated with unestablished spermatogenesis. Normozoospermia more common in adolescents with varicocele may evidence about preserved reproductive potential. Impaired sperm motility in aged patients seems to be related to the formation of oxidative stress and damage to spermatozoa by reactive oxygen species due to combined age-related changes, cumulation of the negative effects of environmental and lifestyle factors, as well as comorbidities.

Altered circadian clock gene expression in the sperm of infertile men with asthenozoospermia.

Male infertility is a complex multifactorial pathological condition, and asthenozoospermia (AZS) is one of the most common causes. Current evidence suggests the underlying role of the circadian clock on male fertility. This study aims to evaluate the expression levels of five principal clock genes in the sperm and their correlations with the sperm parameters in male infertility. We determined the expression profiles of BMAL1, CLOCK, CRY1, PER1, and PER2 in the sperm of infertile men with AZS (n=38) and healthy fertile men (n=40) using quantitative real-time PCR. Then we performed comprehensive association analyses on the clock gene levels and the sperm parameters, including progressive and total motility, concentration, and normal morphology of the sperm. Our results showed that the expression levels of five clock genes (BMAL1, CLOCK, CRY1, PER1, and PER2) are significantly decreased in the sperm of the infertile men with AZS as compared with that of healthy fertile men (P< 0.01). All five clock gene levels are associated with the percentage of progressive/total sperm motility (r= 0.546/0.589~0.677/0.695, P< 0.01). We also discovered that a combination of BMAL1, CLOCK, CRY1, PER1, and PER2 could reach a high diagnostic performance (areas under the curves, 92%) for infertility with AZS. This study first reports that sperm BMAL1, CLOCK, CRY1, PER1, and PER2 levels are altered in AZS and may be molecular markers for male infertility with AZS. These findings indicate the possibility of stabilizing circadian rhythmicity through therapeutic intervention on clock genes to prevent and treat infertility.

Association among sperm chromatin condensation, sperm DNA fragmentation and 8-OHdG in seminal plasma and semen parameters in infertile men with oligoasthenoteratozoospermia.

The present study aimed to investigate the clinical role of standard sperm diagnosis parameters (sperm concentration, motility, morphology) as well as aniline blue staining of histones, 8-OHdG, TUNEL assay were performed on semen samples in infertile men with oligoasthenoteratozoospermia (OAT). Thirty-two infertile and ten proven fertile men were included in the study. Chromatin condensation sperm in infertile men was significantly lower compared to the fertile men (p<0.0001). Age, sperm concentration, morphology and motility were significantly negatively correlated with chromatin condensation (p<0.05). However, no significant correlations among the chromatin condensation, SDF and sperm DNA damage were detected in terms of 8-OHdG concentration.

Beneficial Role of Platelet –Rich Plasma (PRP) combined with Glass Wool Filtration for Best Recovery of Spermatozoa in Infertile Men

Background: Platelet –Rich Plasma (PRP) is a novel therapeutic agent used in multiple medical fields and one of these fields is the reproduction for best spermatozoa preparation and activation for upgrading activity and motility of the spermatozoa and filtered with glass wool filtration to eliminate any round cells and leukocytes from the sample.&#x0D; Objectives: The aim of this research is to study some sperm characteristics in two groups, the normozoospermic infertile men and th asthenozoospermic infertile group before and after activation with Glass wool Filtration and PRP and compare between both.&#x0D; Patients and Methods: In this study 60 infertile men were enrolled and divided into 2 groups,15 normozoospermic infertile men, and 45 ashenozoospermic infertile men during their attendance to the Infertility Clinic in High Institute for Infertility Diagnosis and Assisted Reproductive Technologies Al-Nahrain University. The collected semen samples, and seminal fluid analysis were assessed, each semen sample was divided into 3 tubes and labelled as the 1st tube which was before activation, the 2nd tube was after glass wool filtration activation technique and the 3rd tube was for semen activated by adding PRP to the medium used for glass wool filtration activation.&#x0D; Results: Both techniques showed increased and improved sperm motility, but the PRP was superior to the glass wool alone in upgrading Sperm Grade A Motility %, Sperm Grade B Motility % and decreasing Sperm Grade C Motility %, and Sperm Grade D Motility (Immotile Sperm).&#x0D; Conclusion: The PRP was significantly effective in improving the sperm activity and upgrading sperm motility more than Glass Wool Filtration technique.

P–529 Whole-genome methylation analysis of testicular germ cells from cryptozoospermic men points to recurrent and functionally relevant DNA methylation changes

Abstract Study question Do DNA methylation changes occur in testicular germ cells (TGCs) from patients with impaired spermatogenesis? Summary answer TGCs from men with cryptozoospermia exhibit altered DNA methylation levels at several genomic regions, many of which are associated with genes involved in spermatogenesis. What is known already In the last 15 years, several studies have described DNA methylation changes in sperm of infertile men. More recently, using whole genome bisulfite sequencing (WGBS) we were able to refute these findings by demonstrating that somatic DNA contamination and genetic variation confound methylation studies in swim-up purified sperm of severely oligozoospermic men. However, it remains unknown whether altered DNA methylation plays a role during the development of the germ cells in the testes of these patients. Study design, size, duration For identifying DNA methylation differences associated with impaired spermatogenesis, we compared the TGC methylomes of men with cryptozoospermia (CZ) and men with obstructive azoospermia (n = 4 each), who had normal spermatogenesis and served as controls (CTR). Study participants were selected among an age-matched cohort of 24 CTR and 10 CZ. The selection was based on similar composition of the TGC suspension evaluated by ploidy analysis and absence of somatic DNA. Participants/materials, setting, methods TGCs were isolated from biopsies after short-term cell culture. Presence of somatic DNA was evaluated by analyzing the DNA methylation levels of H19, MEST, DDX4 and XIST. WGBS was performed at ∼14× coverage. Bioinformatic tools were used to compare global DNA methylation levels, identify differentially methylated regions (DMRs) and functionally annotate the DMRs. Single-cell RNA sequencing (scRNA-seq) was used to associate the DNA methylation changes to gene expression. Main results and the role of chance We could not identify any difference in the global DNA methylation level or at imprinted regions between CZ and CTR samples. However, using stringent filters to identify group-specific methylation differences, we detected 271 DMRs, 238 of which were hypermethylated in CZ (binominal test, p &amp;lt; 2.2 × 10–16). The DMRs are associated with 132 genes, 61 of which are known to be differentially expressed at various stages of spermatogenesis according to scRNA-seq studies. Almost all of the DMRs associated with the 61 genes are hypermethylated in CZ (63/67, p = 1.107 × 10–14). As assessed by scRNA-seq, 13 DMR-associated genes, which were mainly expressed during meiosis and spermiogenesis, show a significantly different pattern of expression in CZ patients. In four of these genes, the promoter was hypermethylated in CZ men, which correlates with a lower expression level in these patients. In the other nine genes, most of which downregulated in CZ, germ cell-specific enhancers may be affected. Limitations, reasons for caution The small sample size constitutes a limitation of this study. Furthermore, even though the cellular composition of samples was similar by ploidy analysis, we cannot rule out that the observed DNA methylation changes might be due to differences in the relative proportion of different germ cell types. Wider implications of the findings: Impaired spermatogenesis is associated with DNA methylation changes in testicular germ cells at functionally relevant regions of the genome, which points to an important role of DNA methylation in normal spermatogenesis. The DNA methylation changes may contribute to premature abortion of spermatogenesis and therefore not appear in mature sperm. Trial registration number N/A

Assessment of Atg7 and LC3II/LC3, as The Markers of Autophagy, in Sperm of Infertile Men with Globozoospermia: A Case-Control Study.

Objective Assessment of relationship between LC3II/LC3 and Autophagy-related 7 (Atg7) proteins, as markers of autophagy, as well as evaluating the sperm parameters and DNA fragmentation in spermatozoa of infertile men with globozoospermia. Materials and Methods In this case-control study, 10 semen samples from infertile men with globozoospermia and 10 fertile individuals were collected, and the sperm parameters, sperm DNA fragmentation, and main autophagy markers (Atg7 and LC3II/LC3) were assessed according to World Health Organization (WHO) criteria, TUNEL assay, and western blot technique, respectively. Results The mean of sperm concentration and motility were significantly lower, while the percentage of abnormal spermatozoa and DNA fragmentation were significantly higher in infertile men with globozoospermia compared to fertile individuals (P<0.01). Unlike the relative expression of LC3II/LC3 that did not significantly differ between the two groups, the relative expression of ATG7 was significantly higher in infertile men with globozoospermia compared to fertile individuals (P<0.05). There was a significantly negative correlation between the sperm concentration (r=-0.679; P=0.005) and motility (r=-0.64; P=0.01) with the expression of ATG7, while a significantly positive association was founf between the percentage of DNA fragmentation and expression of ATG7 (0.841; P =0.018). ConclusionThe increased expression of ATG7 and unaltered expression of LC3II/LC3 may indicate that the autophagy pathway is initiated but not completely executed in spermatozoa of individuals with globozoospermia. A significant correlation of ATG7 expression with increased sperm DNA fragmentation, reduced sperm concentration, and sperm motility may associate with the activation of a compensatory mechanism for promoting deficient spermatozoa to undergo cell death by the autophagy pathway. Therfore, this pathway could act as a double-edged sword that, at the physiological level, is involved in acrosome biogenesis, while, at the pathological level, such as globozoospermia, could act as a compensatory mechanism.