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Varicocele has been associated with reduced male reproductive potential. With the advances in biomolecular techniques, it has been possible to better understand the mechanisms involved in testicular damage provoked by varicocele. Current evidence suggests the central role of reactive oxygen species （ROS） and the resultant oxidative stress （OS） in the pathogenesis of varicocele-associated male subfertility although the mechanisms have not yet been fully described and it is likely to be multifactorial. Excessive ROS is associated with sperm DNA fragmentation, which may mediate the clinical manifestation of poor sperm function and fertilization outcome related to varicocele. Testing of ROS/OS and DNA fragmentation has the potential to provide additional diagnostic and prognostic information compared to conventional semen analysis and may guide therapeutic management strategies in individual patient.

Activation of caspase, externalization of phosphatidyl serine, change in the mitochondrial membrane potential, and DNA fragmentation are apoptosis markers found in human ejaculated spermatozoa. Also, reactive oxygen species (ROS) play a vital role in the different types of male infertility. In this review, data sources including Google Scholar, Scopus, PubMed, and Science Direct were searched for publications with no particular time restriction to get a holistic and comprehensive view of the research. Apoptosis regulates the male germ cells, correct function and development from the early embryonic stages of gonadal differentiation to fertilization. In addition to maintaining a reasonable ratio between the Sertoli and germ cells, apoptosis is one of the well-known quality control mechanisms in the testis. Also, high ROS levels cause a heightened and dysregulated apoptotic response. Apoptosis is one of the well-known mechanisms of quality control in the testis. Nevertheless, increased apoptosis may have adverse effects on sperm production. Recent studies have shown that ROS and the consequent oxidative stress play a crucial role in apoptosis. This review aims to assimilate and summarize recent findings on the apoptosis in male reproduction and fertility. Also, this review discusses the update on the role of ROS in normal sperm function to guide future research in this area. Key words: Fertility, Spermatogonia, Apoptosis, Reproduction, DNA fragmentation, DNA integrity, ROS.

Introduction Infertility affects one in every six couples in developed countries, and approximately 50% is of male origin. In 2021, sperm DNA fragmentation (SDF) testing became an evidence-based test for fertility evaluations depicting fertility more clearly than standard semen parameters. Therefore, we aimed to summarize the potential prognostic factors of a higher SDF. Methods We conducted a systematic search in three medical databases and included studies investigating any risk factors for SDF values. We calculated mean differences (MD) in SDF with 95% confidence interval (CI) for exposed and non-exposed individuals. Results We included 190 studies in our analysis. In the group of associated health conditions, varicocele (MD = 13.62%, CI: 9.39–17.84) and impaired glucose tolerance (MD = 13.75%, CI: 6.99–20.51) had the most significant increase in SDF. Among malignancies, testicular tumors had the highest impact, with a maximum of MD = 11.3% (CI: 7.84–14.76). Among infections, the overall effects of both Chlamydia and HPV were negligible. Of lifestyle factors, smoking had the most disruptive effect on SDF – an increase of 9.19% (CI: 4.33–14.06). Different periods of sexual abstinence did not show significant variations in SDF values. Age seemed to have a more drastic effect on SDF from age 50 onwards, with a mean difference of 12.58% (CI: 7.31–17.86). Pollution also had a detrimental effect – 9.68% (CI: 6.85–12.52). Conclusion Of the above risk factors, varicocele, impaired glucose tolerance, testicular tumors, smoking, pollution, and paternal age of over 50 were associated with the highest SDF. Trial registration CRD42021282533.

Male factors may be present in up to 50–70% of infertile couples and the prevalence of male infertility accounts for 20–30% of infertility cases. Understanding the mechanisms and causes behind male infertility remains a challenge, but new diagnostic tools such as DNA fragmentation might aid in cases where the routine semen analysis is insufficient. DNA fragmentation, which refers to damages or breaks of the genetic material of the spermatozoa, is considered one of the main causes of male infertility due to impaired functional capability of sperm. The aim of the present narrative review is to investigate and enlighten the potential correlation between DNA fragmentation and male infertility parameters such as the seminal profile and the reproductive outcomes. Comprehensive research in PubMed/Medline and Scopus databases was conducted and 28 studies were included in the present review. Fourteen studies provided data regarding the impact of DNA fragmentation and seminal parameters and showed a correlation of significantly lower sperm count, lower concentration, motility, and abnormal morphology with an increased DNA fragmentation index (DFI). Similarly, 15 studies provided data regarding the impact of DFI on reproductive outcomes. Two studies showed higher aneuploidy rates with higher DFI values, and seven studies showed significantly lower pregnancy rates and live birth rates with higher DFI values. Ultimately, the studies included in this review highlight, collectively, the importance of measuring sperm DFI in the assessment of male infertility. Further studies are needed to explore the effectiveness of interventions aiming to reduce DFI levels.

Abstract Background Sperm DNA integrity is crucial for normal fertilization, implantation, and embryo development. Several assays are available to assess sperm DNA fragmentation but are limited by high price, complicated processes, and low accuracy. Methods We developed a secondary amplification detection system based on terminal deoxynucleotidyl transferase and endonuclease IV, which could efficiently measure the number of 3'-OH (equivalent to the number of breakpoints). We applied this detection system in single stranded DNA with standard concentrations to obtain the standard curve. We then broke the double stranded genomic DNA by ultrasound and enzyme digestion and used the detection system to monitor the increase of DNA breakpoints. Finally, we used this method to measure the mean number of sperm DNA breakpoints (MDB) in 80 sperm samples. Results We successfully measured the number of 3'-OH in single stranded DNA with standard concentration and obtained the standard curve. The linear range for the number of DNA breakpoints was from 0.1 nM to 15 nM. The detection method was successfully validated on λ DNA and 80 human sperm samples. The results of real clinical samples revealed that the mean number of DNA breakpoints (MDB) had a stronger relevance with the sperm motility and clinical pregnancy outcomes than the commonly used parameter of DNA fragmentation index (DFI). Conclusion We have developed a straight-forward method for direct measurement of the mean number of DNA breakpoints in sperms. The method has advantages of short time-consumption, simple operation, high analytical sensitivity, and low requirement for instrumentation, which makes it conducive to clinical application. The proposed new parameter (MDB) could be a more direct, accurate and clinically significant indicator for evaluating the sperm DNA integrity.

Plasma DNA obtained from a pregnant woman was sequenced to a depth of 270× haploid genome coverage. Comparing the maternal plasma DNA sequencing data with the parental genomic DNA data and using a series of bioinformatics filters, fetal de novo mutations were detected at a sensitivity of 85% and a positive predictive value of 74%. These results represent a 169-fold improvement in the positive predictive value over previous attempts. Improvements in the interpretation of the sequence information of every base position in the genome allowed us to interrogate the maternal inheritance of the fetus for 618,271 of 656,676 (94.2%) heterozygous SNPs within the maternal genome. The fetal genotype at each of these sites was deduced individually, unlike previously, where the inheritance was determined for a collection of sites within a haplotype. These results represent a 90-fold enhancement in the resolution in determining the fetus’s maternal inheritance. Selected genomic locations were more likely to be found at the ends of plasma DNA molecules. We found that a subset of such preferred ends exhibited selectivity for fetal- or maternal-derived DNA in maternal plasma. The ratio of the number of maternal plasma DNA molecules with fetal preferred ends to those with maternal preferred ends showed a correlation with the fetal DNA fraction. Finally, this second generation approach for noninvasive fetal whole-genome analysis was validated in a pregnancy diagnosed with cardiofaciocutaneous syndrome with maternal plasma DNA sequenced to 195× coverage. The causative de novo BRAF mutation was successfully detected through the maternal plasma DNA analysis.

DNA methylation is one of the most important epigenetic modifications in the regulation of gene transcription. The current gold standard to study this modification is bisulfite sequencing. Although multiple commercial bisulfite treatment kits provide good conversion efficiencies, DNA loss and especially DNA fragmentation remain troublesome. This hampers DNA methylation profiling of long DNA sequences. Here, we explored the performance of twelve commercial bisulfite kits by an in-depth comparison of DNA fragmentation using gel electrophoresis, qPCR and digital PCR, DNA recovery by spectroscopic measurements and digital PCR and conversion efficiency by next generation sequencing. The results show a clear performance difference between the bisulfite kits, and depending on the specific goal of the study, the most appropriate kit might differ. Moreover, we demonstrated that digital PCR is a valuable method to monitor both DNA fragmentation as well as DNA recovery after bisulfite treatment.

Codeine, a 3-methylmorphine, and other related opioids have been implicated in androgen suppression, although the associated mechanisms remain unclear. Therefore, the objective of the current study was to elucidate the in vivo molecular mechanisms underlying codeine-induced androgen suppression. This study made use of twenty-one healthy male rabbits, distributed into three groups randomly, control and codeine-treated groups. The control had 1ml of normal saline daily p.o. The codeine-treated groups received either 4mg/kg b.w of codeine or 10mg/kg b.w of codeine p.o. for six weeks. Reproductive hormonal profile, testicular weight, testicular enzymes, oxidative and inflammatory parameters, testicular DNA fragmentation, histological examination and apoptosis marker were evaluated to examine the effects of codeine use. Oral administration of codeine resulted in testicular atrophy and alterations in testicular histomorphology, elevated testicular enzymes, and suppression of circulatory and intra-testicular testosterone. These changes were associated with a marked rise in oxidative markers and decline in the activities of testicular enzymatic antioxidants, as well as oxidative DNA damage, inflammatory response, testicular DNA fragmentation, and caspase-dependent apoptosis (p<0.05). In conclusion, chronic codeine use resulted in testicular degeneration and testosterone suppression, which is attributable to TNF-α/nitric oxide-/oxidative stress-mediated caspase-dependent apoptotic testicular cell death and loss of testicular function.

Semen analysis is the cornerstone of male fertility evaluation with WHO guidelines providing the basis for procedural standardization and reference values worldwide. The first WHO manual was published in 1980, and five editions have been subsequently released over the last four decades. The 6th Edition was published in July 2021. In this review, we identify the key changes of this 6th Edition. Additionally, we evaluate the utility of this 6th Edition in clinical practice using SWOT (strengths, weaknesses, opportunities, and threats) analysis. This new Edition has made the analysis of basic semen parameters more robust, taking into account the criticisms and grey areas of the previous editions. The tests assessing sperm DNA fragmentation and seminal oxidative stress are well-described. The main novelty is that this latest edition abandons the notion of reference thresholds, suggesting instead to replace them with “decision limits”. While this seems attractive, no decision limits are proposed for either basic semen parameters, or for extended or advanced parameters. This critical review of the 6th Edition of the WHO laboratory manual combined with a SWOT analysis summarizes the changes and novelties present in this new Edition and provides an in-depth analysis that could help its global use in the coming years.

Infertility is a prevalent condition affecting an estimated 70 million people globally. The World Health Organization estimates that 9% of couples worldwide struggle with fertility issues and that male factor contributes to 50% of the issues. Male infertility has a variety of causes, ranging from genetic mutations to lifestyle choices to medical illnesses or medications. Recent studies examining DNA fragmentation, capacitation, and advanced paternal age have shed light on previously unknown topics. The role of conventional male reproductive surgeries aimed at improving or addressing male factor infertility, such as varicocelectomy and testicular sperm extraction, have recently been studied in an attempt to expand their narrow indications. Despite advances in the understanding of male infertility, idiopathic sperm abnormalities still account for about 30% of male infertility. With current and future efforts examining the molecular and genetic factors responsible for spermatogenesis and fertilization, we may be better able to understand etiologies of male factor infertility and thus improve outcomes for our patients.