Sperm Chromatin Dispersion Research Articles

Cordycepin improves hyperactivation and acrosome reaction through adenosine receptors during human sperm capacitation in vitro.

Sperm capacitation is a prerequisite for natural or in vitro fertilization. After capacitation, sperm become hyperactivated and undergo an acrosome reaction, which helps them penetrate the oocyte. Cordycepin, a bioactive compound first isolated from Cordyceps militaris, is an adenosine analog with numerous physiological activities. However, its effects on sperm capacitation remain unclear. This study aims to elucidate the effects and mechanisms of cordycepin on human sperm capacitation. During in vitro capacitation culture, healthy human sperm were treated with cordycepin (20, 100, 500 µM). Sperm motility and hyperactivation were detected using a computer-assisted sperm analyzer. Sperm acrosome reaction was measured using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin. Sperm protein kinase A (PKA) activity was analyzed using an ELISA kit. The levels of sperm protein tyrosine phosphorylation were detected by western blotting. Sperm DNA damage was detected by a sperm chromatin dispersion assay. Reactive oxygen species (ROS) were measured using the fluorescence probe 2',7'-dichlorodihydrofluorescein diacetate. The expression and localization of adenosine receptors were analyzed by western blotting and immunofluorescence. The specific inhibitors of adenosine receptors were used to confirm their effects on cordycepin-induced sperm capacitation. Finally, molecular docking was performed to analyze the interaction between cordycepin and adenosine receptors. Cordycepin improved hyperactivated sperm motility, acrosome reaction, PKA activity, and protein tyrosine phosphorylation during capacitation while having no obvious effects on sperm ROS or DNA damage. Four adenosine receptor subtypes were expressed in human sperm, but their localizations differed. Inhibition of adenosine receptors significantly decreased cordycepin-induced sperm hyperactivation and the acrosome reaction. Molecular docking showed that cordycepin can bind to the four subtypes of adenosine receptors. Cordycepin may promote human sperm capacitation through adenosine receptor-mediated signaling pathways. These findings may be useful for assisted reproductive technology and animal breeding.

Application of Autologous Platelet-rich Plasma Exerts Cryoprotective Effects on Biological Characteristics of Human Oligoasthenoteratospermia Samples after Freezing and Thawing Procedures.

Platelet-rich plasma (PRP) is enriched with active biological components which showed proliferative and cytoprotective properties in healing different injuries in medicinal fields. This study was designed to assess the cryoprotective effects of autologous PRP on the quality of oligoasthenoteratospermia (OAT) samples during freezing and thawing procedure. The present study is an experimental research. Twenty OAT semen samples were obtained from individuals and prepared by discontinuous density - gradients technique DGC). The control group is sperm samples after DGC. After the procedure, the specimen was divided into four groups. The Freeze group which has no additive and the other three groups were cryopreserved with different concentrations of PRP (1×105/ μL, 0.5×105/μL and 0.25×105/μL). Autologous PRP was provided by each participant. After thawing, sperm parameters, DNA fragmentation by sperm chromatin dispersion test (SCD), protamine deficiency by (Chromomycin A3) CMA3 staining, acrosome integrity and malondialdehyde (MDA) level were evaluated. Cryopreservation resulted in a significant decrease in all factors compared to the control group. There were no significant changes in sperm count, morphology, non-progressive motility and acrosome reaction by adding PRP as a cryoprotectant in comparison with the freeze group. PRP at all three concentrations showed a significant increase in progressive motility (3.05 ± 2.01 vs. 14.05 ± 4.13, 12.35 ± 4.90 and 12.15 ± 9.65, P < 0.001) and viability (36.85 ± 10.25 vs. 47.85 ± 5.86, 51.30 ± 5.54 and 50.05 ± 5.67, P < 0.001) compared to the sperm samples without PRP. The percentage of immotile sperms decreased at all PRP concentrations compared to the freeze group. Moreover, PRP at 1×105/μL concentration showed cryoprotective effects on DNA fragmentation, protamine deficiency and MDA level compared to the other three concentrations. Cryopreservation and thawing procedures may exert adverse effects on biological factors of sperm samples. Therefore, adding PRP as cryoprotectant at all three concentrations especially 1×105/μL can be promising strategy to reduce adverse effects of cryopreservation on OAT samples.

Evaluation of Sperm DNA Fragmentation in Oligoasthenoteratozoospermia Patients Using Two Different Techniques: TUNEL and Sperm Chromatin Dispersion Assays

Background: Oligoasthenoteratozoospermia (OAT) is the most prevalent male infertility condition that is mainly caused by sperm DNA fragmentation (SDF). This study compared the sensitivity and effectiveness of two different approaches for analyzing SDF in patients with OAT: sperm chromatin dispersion (SCD) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Materials and Methods: In this study, which received ethical committee approval, participants were divided in to normal and OAT groups (n=20 for each). both TUNEL and SCD assays were used to analyze the sperm DNA fragmentation. And Malondialdehyde (MDA) levels was measured to determine levels of lipid peroxidation in the seminal plasma. Results: The TUNEL assay showed better ability to predict OAT patients than that of the SCD. For our patient population, the projected cut-off points for the DNA fragmentation index of 29% and 19% were reported using the TUNEL and SCD tests, respectively. Seminal levels of MDA were significantly higher in the OAT group (P=0.002) than that of control group. Conclusion: OAT patients showed higher MDA levels of seminal plasma and DNA fragmentation than the control group. Although sperm DNA fragmentation can be detected with high efficiency and sensitivity using both TUNEL and SCD assays, the TUNEL test was found to be a more accurate predictor for OAT patients.

Sperm chromatin dispersion assay reliability and assisted reproductive technology outcomes: Systematic review and meta-analysis.

Elevated sperm DNA fragmentation has potential implications for semen quality and fertility. The commonly used sperm chromatin dispersion test offers an indirect estimation but has limitations in terms of bias and variability. This study aimed to assess the reliability of the sperm chromatin dispersion assay for predicting assisted reproductive technology outcomes. This systematic review included studies published until December 2023 that adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. PubMed/MEDLINE, Scopus, and Google Scholar databases were searched. Various assisted reproductive technology outcomes in patients with high (≥30%) versus low (<30%) sperm DNA fragmentation were compared using a sperm chromatin dispersion assay and including a sub-analysis of intracytoplasmic sperm injection versus in vitro fertilization. A comprehensive meta-analysis software facilitated quantitative analysis with statistical comparisons between cases and controls. Interstudy heterogeneity was assessed, and sensitivity and publication bias tests were performed. Of the 199 abstracts assessed, 64 full-text articles were screened, and 44 articles were qualitatively synthesized. Fourteen articles representing 5346 participants were quantitatively analyzed. Using the sperm chromatin dispersion assay, elevated sperm DNA fragmentation was associated with lower fertilization and embryo cleavage rates. Notably, high sperm DNA fragmentation levels did not affect the clinical pregnancy, implantation, miscarriage, or live birth outcomes. Sub-analysis revealed lower fertilization, embryo cleavage, clinical pregnancy, live birth rates, and higher miscarriage rates in the intracytoplasmic sperm injection subgroup only. The sperm chromatin dispersion assay did not show significant differences in pregnancy or live birth rates between the high- and low-sperm DNA fragmentation groups. Noteworthy, high sperm DNA fragmentation was associated with worse assisted reproductive technology outcomes in the intracytoplasmic sperm injection group. Given the current quality of the evidence, affected by the experimental design and the absence of correction for female factors of infertility, clinicians should be wary of the assay's limited predictive power for pregnancy and live birth outcomes.

The association of sperm BAX and BCL-2 gene expression with reproductive outcome in Oligoasthenoteratozoospermia cases undergoing intracytoplasmic sperm injection: A case-control study.

The B-cell lymphoma 2 (BCL-2) protein is one of the members of the BCL-2 associated X (BAX) protein family that acts as an inducer of apoptosis. The present study aims to investigate the association between BAX and BCL-2 gene expression with reproductive outcome, in cases undergoing intracytoplasmic sperm injection. In this case-control study, 50 men were divided into healthy fertile and oligoasthenoteratozoospermic infertile men (n = 25/each). They were subjected to history taking, clinical examination, and semen analysis. Expression of BAX and BCL-2 genes were measured using real-time polymerase chain reaction. The DNA fragmentation index was measured using the sperm chromatin dispersion assay technique. Using World Health Organization criteria, sperm parameters were evaluated. Evaluation of apoptosis-related genes showed that oligoasthenoteratozoospermic significantly increased mRNA expression of BAX, and significantly decreased mRNA expression of BCL-2, when compared with control. Moreover, the BAX/BCL-2 ratio was significantly higher in oligoasthenoteratozoospermic compared to the normozoospermic group (p = 0.01). Also, this study showed that the BAX and BCL-2 genes expression had a significant correlation with sperm quality, and DNA fragmentation in the oligoasthenoteratozoospermic group (p = 0.01). The oligoasthenoteratozoospermic men, had a considerably lower proportion of fertilization rate and good-quality embryos at the cleavage stage than the normozoospermic subjects (p = 0.01). A significant correlation was observed between the expression of BAX and BCL-2 genes, fertilization, and embryo quality (p = 0.01). We concluded that the sperm BAX/BCL-2 ratio demonstrates a significant correlation with fertilization rate and embryo quality.

P-271 Deep sequencing of spermatozoal accessible chromatin reveals complex arrangements underlying spermatozoal chromatin packaging and fertilization

Abstract Study question Could structural aspects of sperm DNA packaging contribute to fertilization and early embryo development? Summary answer Structural aspects of sperm DNA include complex patterns of repetitive-DNA and developmental gene packaging that could delineate a paternal impact on fertilization and early development. What is known already Genetics, epigenetics and transcriptomics of ejaculate human spermatozoa could have important effects on the establishment of embryonic chromatin architecture that may impact early fetal development. Spermatozoa are transcriptionally silent; however, a small fraction (∼5%-15%) of their chromatin escapes repackaging by protamines and remains in a more open nucleosomal conformation that may be poised for transcription. The sensitivity of this fraction to progressive salt/detergent disruption is likely assayed in sperm chromatin dispersion (SCD) or halo tests that are commonly used in assessments of sperm DNA fragmentation. Study design, size, duration Eleven healthy male donors and 9 women undergoing IVF treatment agreed to participate in this study. Eleven normozoospermic semen samples were donated by male donors, aged 25-39 years, who abstained from sexual activity for 3 days. Seven MII oocytes and 5 pronuclear zygotes were donated from 5 and 4 women, respectively, undergoing IVF treatment. The samples were collected within a time frame of 6 months. Female donors provided either MII oocytes or pronuclear zygotes. Participants/materials, setting, methods Sperm chromatin was processed by differential salt extraction tailored with endonuclease or micrococcal nuclease treatment and RNA isolated using established protocols. Oocytes and pronuclear zygotes were analyzed at the single cell level. DNA library construction employed the NEBNext Ultra approach (NEB) and RNA libraries the Ovation Single Cell (NuGEN) for Illumina. Bioinformatics analysis included FastQC, HISAT2, StringTie, GREAT, edgeR and other source-software. Public datasets were employed to further increase the statistical power. Main results and the role of chance The aim of our approach was to dissect the accessible sperm chromatin fraction, identifying regions of great importance that could contribute towards the formation of the zygote and/or early embryonic development. Differing extraction methods revealed complementary accessible and resistant fractions in sperm, with accessible regions being significantly enriched for chromatin important in very early embryonic development. These chromatin fractions contained various genomic features including promoter sequences, CpG-islands and CTCF-binding sites that could attract transcription binding factors in the paternally-derived chromatin. Accessible sperm chromatin also contains high levels of specific DNA repeat classes of retroviral origin, known to impact early gene expression and gene expression programs. Accessible sperm chromatin was strongly associated with developmental and regulatory signatures with Bonferroni P values ranging between 2.07x10-4 and 1.03x10-20, potentially revealing a role for paternal chromatin in the initial start-up of early zygotic development. We further investigated paternally- and maternally- derived transcripts and identified mono-allelically transcribed genes present in the zygotes, that were entirely absent from all seven oocyte transcriptomes, suggesting a paternal origin, and potentially important roles following oocyte fertilization. Limitations, reasons for caution Due to the restraints of obtaining human material, the study relied on a relatively small sample size. However, sequencing results showed quite homogeneous clustering in both DNA/RNA samples, with at least 5 biological replicates from at least 4 donors per group, potentially reducing the impact of low sample size. Wider implications of the findings A better understanding of differentially accessible sperm chromatin and residual RNA, could help support the discovery of biomarkers that can be used to select more competent spermatozoa for IVF cycles, resulting in higher success rates in couples undergoing ART. Trial registration number not applicable

P-012 Euploid and good-quality blastocysts using testicular sperm from men with high sperm DNA fragmentation (SDF), oligozoospermia, and previous failed ICSI with ejaculated sperm

Abstract Study question Are euploid rate and blastocyst quality improved when testicular sperm is used in patients with elevated SDF and poor semen parameters or/and previous ICSI failure? Summary answer The use of testicular spermatozoa in non-azoospermic males does preserve blastocyst ploidy while favoring the proportion of good-quality embryos available for the couple. What is known already Sperm extracted from the testicle has been proposed as a strategy to improve reproductive outcomes in couples with elevated DNA fragmentation in the ejaculate and with severe male infertility or previous ICSI failure. Different studies and meta-analyses show to increase pregnancy and live birth rates, and one of the reasons could be the lower genetic damage of spermatozoa retrieved from the testicle. However, there is little information on the effect on the quality of the embryos generated and their ploidy since the use of testicular sperm tends to have a higher rate of aneuploidy. Study design, size, duration This is a prospective, blinded, randomized, and controlled trial conducted in a private university-affiliated IVF center between October 2020 and June 2023. In each patient (n = 6), oocytes were randomly microinjected by sperm from the testis (T-ICSI) or ejaculate (E-ICSI) assigned in a 1:1 ratio. All males had an SDF&amp;gt;30% (measured by Sperm Chromatin dispersion (SCD) test), severe oligozoospermia, and/or a previous ICSI failure. Participants/materials, setting, methods Males underwent testicular sperm extraction (TESE) to obtain spermatozoa, which were frozen along with an ejaculated semen sample obtained previously on the same day. Women were &amp;lt;38 years old with good ovarian reserve (antral follicle count&amp;gt;10). SDF level was compared between both sources. The primary outcome was the euploid rate (among a total number of biopsied embryos). Secondary outcomes were fertilization rate, blastocyst rate, good-quality blastocyst rate, and ongoing pregnancy rate per embryo transfer (ET). Main results and the role of chance Mean ejaculated SDF was 44.83±16.6 (IC95% 27.5 -62.2), while mean women age was 31.8±2.9 (28.76-34.9). An average of 14.5±5.8 metaphase II oocytes were microinjected per patient. A total of 87 oocytes were randomized to the T-ICSI group (n = 45) or E-ICSI group (n = 42). There was a 14% difference between the SDF of testicular (44.7% (31.3-58.0) and ejaculated sperm (58.5% (37.6-79.4) (p = 0.2). The fertilization rate was slightly lower in the T-ICSI group (53.3% (38.1-68.1) compared to the E-ICSI group (61.9 (61.8-76.0) (p = 0.6). A total of 21 embryos reached the blastocyst stage, being the blastocyst arrival rate per correctly fertilized oocyte 41.7% (10/24) (22.8-63.1) for T-ICSI and 42.3% (11/26) (24.0-62.8) for E-ICSI (p = 1.0). Of them, a higher proportion of good-quality blastocysts was found in the T-ICSI (29.2% (13.4-51.6)) group than in the E-ICSI (19.2% (7.3-40.0)) group, although the difference was not statistically significant. In addition, the euploidy rate was comparable between the T-ICSI (60% (27.4-86.3)) and E-ICSI (63.6% (31.6-87.6)) groups (p = 1.0). Seven embryos were transferred in 6 ET, three in each study group. Only one of the ET in the E-ICSI group resulted in pregnancy (33.3% (1.8-87.5), while the three transfers in the T-ICSI group resulted in an ongoing pregnancy (100% (31.0-100)) (p = 1.0). Limitations, reasons for caution Despite being a randomized controlled trial, the number of subjects recruited is small; it is necessary to increase the number of cases to obtain results with greater statistical power. TESE is a technique that is not risk-free, so its performance should be evaluated in terms of its cost-benefit for patients. Wider implications of the findings The use of less genome-damaged testicular spermatozoa in non-azoospermic males seems to be a reliable strategy for couples with high ejaculated SDF and severe oligospermia and/or previous ICSI failures. However, although we have found an improvement, remains to be statistically confirmed with more cases. Trial registration number NCT04795440

P-086 Consequences of elevated levels of DNA fragmentation and its influence on pregnancy – A multicentric cohort analysis

Abstract Study question Does an abnormal DNA Fragmentation Index impact the clinical outcomes of patients undergoing IVF treatment? Summary answer Statistical differences were seen in biochemical, implantation, clinical, and ongoing pregnancy rate in sperms with high and low DFI scores with autologous and donated oocytes. What is known already Sperm chromatin integrity is associated with embryo development and clinical outcomes of an IVF cycle. DNA Fragmentation Index (DFI) is a diagnostic tool for the detection of spermatozoa with damaged chromosomes in the ejaculate. DNA fragmentation is a significant cause of day 3 embryo arrest and the development of poor-quality embryos. The interventions associated with elevated levels of DFI are ICSI, lifestyle changes and antioxidant therapies. However, the published data regarding the potential of embryo development with high DFI is limited to the sample size thus, encouraging a need for more research in this area. Study design, size, duration The study is population-based and included 768 male patients who underwent DFI score detection across 44 centers at Indira IVF Hospital Private Limited from January 2023 to September 2023. The abstinence period for all the patients was recorded during the DFI detection. After the DFI assessment patients were classified into normal DFI score ( &amp;lt; =15%) (n = 203) and abnormal DFI score (&amp;gt;15%) (n = 565). Participants/materials, setting, methods The retrospective cohort analysis was performed on our databank on the male patients regardless of their age (21-50), ethnicity, and medical history. DFI was calculated using a Sperm Chromatin Dispersion assay. Patients were categorized into two groups: Normal DFI ( &amp;lt; =15%) and Abnormal DFI (&amp;gt;15%). The characteristics of population along with clinical outcomes were evaluated after transferring embryos fertilized with normal DFI sperm and abnormal DFI sperm. The data was analyzed and estimated by SPSS software. Main results and the role of chance Our analyses discovered that embryos fertilized with sperms of normal DFI score have significantly enhanced the likelihood of implantation compared with embryos fertilized with sperms of abnormal DFI (50.27% v/s 44.40%; p = 0.047). Simultaneously, early pregnancy was evaluated by determining the values of beta-HCG in blood, also known as biochemical pregnancy rate (BPR). The embryos fertilized with sperms of normal DFI score significantly enhanced the BPR compared with embryos fertilised with sperms of abnormal DFI (67% v/s 59.1%; p = 0.048). Clinical Pregnancy Rate (CPR) defined as presence of a heartbeat of gestational sac observed during the 6-week scan. Furthermore, it was evaluated that the CPR showed a significant enhancement in embryos fertilized with sperms of normal DFI score in comparison with embryos fertilized with sperms of abnormal DFI (65.5% v/s 57.9%; p = 0.034). However, it was noticed that male DFI score had no impact on the miscarriage rate of embryos inseminated with normal DFI score and abnormal DFI score (7.52% v/s 7.34%; p = 0.687). Moreover, the pregnancy was tracked, and it was revealed that embryos inseminated with sperms of normal DFI score significantly enhanced the ongoing pregnancy till 12 weeks compared with embryos inseminated with sperms of abnormal DFI score (60.6% v/s 53.6%; p = 0.039). Limitations, reasons for caution Embryos are known to have a self-correct mechanism to correct the presence of mosaicism in the embryos. Hence, the oocyte quality also affects the outcomes of the IVF cycle. Therefore, using a high grade of the oocyte for the patients with abnormal DFI score can be useful. Wider implications of the findings Our data suggest that abnormal DFI have an impact on the clinical outcomes of an IVF cycle. Therefore, examining the levels of DNA Fragmentation is utmost imperative for a successful pregnancy. Trial registration number not applicable

Revalidation of DNA Fragmentation Analyses for Human Sperm-Measurement Principles, Comparative Standards, Calibration Curve, Required Sensitivity, and Eligibility Criteria for Test Sperm.

(1) Background: Double-strand breaks (DSBs) in a single nucleus are usually measured using the sperm chromatin structure assay (SCSA), sperm chromatin dispersion (SCD) test, and comet assay (CA). Mono-dimensional single-cell pulsed-field gel electrophoresis (1D-SCPFGE) and angle-modulated two- dimensional single-cell pulsed-field gel electrophoresis (2D-SCPFGE) were developed to observe DNA fragmentation in separated motile sperm. (2) Methods: Comparative standards, calibration curves, required sensitivity levels, and eligibility criteria for test sperm were set up to validate the measurement principles of these tests. (3) Results: The conventional methods overlooked the interference of nucleoproteins in their measurements. In-gel proteolysis improves the measurement accuracies of 1D- and 2D-SCPFGE. Naked DNA is suitable for comparative standards and test specimens. Moreover, several dysfunctions that might induce DNA damage are observed in the separated motile sperm. Overall, the discussion highlights the need to revisit the conventional univariable analyses based on the SCSA, SCD test, and CA. (4) Conclusions: Human infertility is a complex syndrome, and the aim of quality control in intracytoplasmic sperm injection is to identify the underlying dysfunctions remaining in the separated motile sperm that render them ineligible for injection. Multivariable analyses with special consideration to confounding factors are necessary in future cohort studies.

Pentoxifylline treatment as a safe method for selecting viable testicular spermatozoa before cryopreservation of a small numbers of spermatozoa in azoospermia individuals.

Single sperm cryopreservation (SSC) is a specific technique especially used in individuals with small numbers of sperm who suffered from non-obstructive azoospermia (NOA). Testicular specimens possess poor motility and low population of viable spermatozoa. Therefore, sperm selection methods such as applying pentoxifylline (PTX) may improve motility in these cases. The main aim of this study was to evaluate the protective effects of PTX on testicular spermatozoa before and after performing SSC. Thirty testicular samples were obtained from men with azoospermia. This study was conducted in two phases. Phase 1 evaluated the effect of PTX for sperm selection before SSC. Twenty testicular samples were divided to two experimental groups: SSC without (I) and with PTX treatment (II). For PTX treatment spermatozoa were incubated with PTX at 37°C for 30 min and only motile spermatozoa were selected for SSC. In phase 2, ten testicular samples were cryopreserved with SSC and warming procedure was carried out in droplet with and without PTX. Motility and viability rates, morphology by motile sperm organelle morphology examination (MSOME), DNA fragmentation by sperm chromatin dispersion test (SCD) and mitochondrial membrane potential (MMP) were evaluated. In phase 1, post warm motility rate was higher in PTX exposed group compared to the unexposed group (25.6 ± 8.13 vs. 0.85 ± 2.1) (p > 0.00). Recovery rate, viability and morphology were not significantly different between groups. DNA integrity and MMP were also similar between both groups. In phase 2 although motility increased in PTX group compared to without PTX group (29.30 ± 12.73 vs. 1.90 ± 2.64) (p > 0.00), the viability rate was not different (70.40 ± 12.12 vs. 65.30 ± 11.87). All above mentioned parameters were similar between the two SSC groups. Supplementation of testicular spermatozoa with PTX before cryopreservation increases motility and did not have adverse effects on viability, morphology, DNA integrity and MMP. PTX could be used as sperm selection method before single sperm cryopreservation, but PTX could not maintain motile the most of viable testicular sperms.

Антиоксидантная терапия в коррекции ДНК-фрагментации сперматозоидов.

Introduction. The progressive deterioration of reproductive function, observed throughout the world in recent decades, is an extremely alarming trend, while the incidence of male infertility is increasing. The universal pathogenetic mechanism of spermatogenesis disorders, which underlies almost all forms of male infertility, is oxidative stress of sperm. It has various manifestations, the essence of which, as a rule, boils down to the formation of mitochondrial dysfunction and fragmentation of sperm DNA. It has been found that consuming antioxidants and microelements helps increase the chances of conception for subfertile couples, and also reduces the risk of reproductive losses. Of greatest and justifiable interest are medicinal complexes designed to reduce the level of oxidative stress and sperm DNA fragmentation. The purpose of the study was to evaluate the effect of antioxidant therapy on the degree of sperm DNA fragmentation in men suffering from infertility. Materials and methods. The study involved 42 men aged 25 to 37 years with an established diagnosis of male infertility who were treated at the Center for Family Health and Reproduction. The patients were divided into 2 groups, comparable according to the main criteria assessed. Patients in the control group (12 people, 29%) received standard antioxidant therapy. Patients of the main group (30 people, 71%) received the BESTFertil-DHA® complex. The course of treatment for patients in both groups was 3 months. Control examinations were carried out at 1.5 and 3 months from the start of therapy. Spermogram data and assessment of the degree of DNA fragmentation in sperm using the SCD (Sperm chromatin dispersion test) method were used as the main criterion for assessing the effectiveness of therapy. Results. After three months of taking BESTFertil-DHA®, in 16 patients (53%), the degree of sperm DNA fragmentation decreased to less than 15%. Against this background, 5 couples became pregnant naturally, and 4 couples became pregnant as a result of the in vitro fertilization procedure. The average level of sperm DNA fragmentation was less than 13% (p &lt; 0.05). A decrease in the level of DNA fragmentation was noted in 53% of patients after 1.5 months of taking BESTFertil-DHA®, which is especially important when preparing patients for assisted reproductive technology programs. In patients taking BESTFertil-DHA®, the total progressive motility of spermatozoa «A+B» increased by 28%, and the concentration of spermatozoa in 1 ml of ejaculate – by 61%. In the control group, the degree of sperm DNA fragmentation did not undergo statistically significant changes during treatment. Conclusion. BESTFertil-DHA® statistically significantly helps to reduce the degree of DNA fragmentation in sperm, increase the number of actively motile forms of sperm and reduce the degree of teratozoospermia.

Sperm Chromatin Dispersion Test Detects Sperm DNA Fragmentation Mainly Associated with Unviable Spermatozoa and Underestimates the Values with Respect to TUNEL Assay.

Several clinical laboratories assess sperm DNA fragmentation (sDF) in addition to semen analysis in male infertility diagnosis. Among tests evaluating sDF, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) and SCD (Sperm Chromatin Dispersion) are widely used. Our lab developed a modified version of TUNEL (TUNEL/PI) able to distinguish two sperm populations (PI Brighter and PI Dimmer) differently associated with sperm viability and reproductive outcomes. The aim of this study was to compare sDF levels detected by SCD and TUNEL/PI in the semen samples from 71 male subjects attending our Andrology Laboratory. Our results demonstrate that SCD is less sensitive in determining sDF compared to TUNEL/PI. The statistically significant positive correlation found between sDF evaluated by SCD and PI Dimmer (consisting of all dead spermatozoa) suggests that SCD mainly detects sDF in unviable spermatozoa. We confirmed that most spermatozoa detected by SCD are unviable by performing SCD after incubation in hypo-osmotic medium to discriminate viable and unviable cells in 52 samples. Such results might explain the lower ability of this test in discriminating couples having successful ART outcomes demonstrated in published metanalyses. Overall, our results indicate that SCD is less sensitive in evaluating sDF for diagnostic purposes.

Evaluation of Sperm DNA Fragmentation Using Halosperm Technique after the Freezing-Thawing Process in Men: A Study on the Validation of the SCD Protocol.

DNA fragmentation index (DFI) enhances routine semen analysis by providing valuable insights into male reproductive potential. Utilizing Halosperm test, a sperm chromatin dispersion (SCD) assay based on induced condensation. The purpose of this study was to assess sperm DNA damage both before and after freezing. By following the specified kit instructions, an attempt was made to validate the SCD test protocol, with a particular emphasis on the implications of sperm freezing on its DNA integrity. In total, 380 fresh human semen samples from normozoospermic patients were frozen at -20°C for 10 days, using SCD cryopreservation reagent. Routine semen analysis and DNA fragmentation index (DFI) were determined for each sample before freezing and after thawing. Semen morphology and sperm DFI were compared before and after freezing/thawing process. There was a significant decrease in sperm normal morphology after thawing (9.31±2.42% vs. 7.1±1.53%, p<0.05, respectively). The sperm head, midpiece, and tail defect rate increased after freezing at -20°C. Moreover, DFI was significantly higher after thawing compared to before freezing (20.71±1.61% before freezing vs. 29.1±0.21% after thawing with p<0.001). Cryoconservation of semen samples at -20°C for 10 days using SCD cryopreservation reagent seems to damage sperm morphology, resulting in a reduction in sperm DNA integrity. The measurement of DFI on a fresh sample remains the most reliable technique for obtaining accurate results.

A loss-of-function variant in ZCWPW1 causes human male infertility with sperm head defect and high DNA fragmentation.

Male infertility is a global health issue. The more causative genes related to human male infertility should be further explored. The essential role of Zcwpw1 in male mouse fertility has been established and the role of ZCWPW1 in human reproduction needs further investigation to verify. An infertile man with oligoasthenoteratozoospermia phenotype and his parents were recruited from West China Second University Hospital, Sichuan University. A total of 200 healthy Han Chinese volunteers without any evidence of infertility were recruited as normal controls, while an additional 150 infertile individuals were included to assess the prevalence of ZCWPW1 variants in a sporadic male sterile population. The causative gene variant was identified by Whole-exome sequencing and Sanger sequencing. The phenotype of the oligoasthenoteratozoospermia was determined by Papanicolaou staining, immunofluorescence staining and electron microscope. In-vitro experiments, western blot and in-silicon analysis were applied to assess the pathogenicity of the identified variant. Additionally, we examined the influence of the variant on the DNA fragmentation and DNA repair capability by Sperm Chromatin Dispersion and Neutral Comet Assay. The proband exhibits a phenotype of oligoasthenoteratozoospermia, his spermatozoa show head defects by semen examination, Papanicolaou staining and electron microscope assays. Whole-exome sequencing and Sanger sequencing found the proband carries a homozygous ZCWPW1 variant (c.1064C > T, p. P355L). Immunofluorescence analysis shows a significant decrease in ZCWPW1 expression in the proband's sperm. By exogenous expression with ZCWPW1 mutant plasmid in vitro, the obvious declined expression of ZCWPW1 with the mutation is validated in HEK293T. After being treated by hydroxyurea, MUT-ZCWPW1 transfected cells and empty vector transfected cells have a higher level of γ-H2AX, increased tail DNA and reduced H3K9ac level than WT-ZCWPW1 transfected cells. Furthermore, the Sperm Chromatin Dispersion assay revealed the proband's spermatozoa have high DNA fragmentation. It is the first report that a novel homozygous missense mutation in ZCWPW1 caused human male infertility with sperm head defects and high DNA fragmentation. This finding enriches the gene variant spectrum and etiology of oligoasthenoteratozoospermia.

SPERMATIC DNA FRAGMENTATION INDEX OF INFERTILE MEN: INTEREST IN THE CHOICE OF PMA TECHNIQUE PRELIMINARY STUDY

Introduction: Infertility constitutes a real public health problem. In men, the examinations involved in the choice of the PMA technique are sometimes insufficient in relation to the real procreation potential of the spermatozoa which could be detected by the sperm DNA fragmentation test. This preliminary study aimed to highlight the benefit of taking into account the sperm DNA fragmentation test in the choice of the proposed PMA technique. Material and Methods: This was a preliminary descriptive cross-sectional study carried out in a private spermiology laboratory in Abidjan. It included 52 patients who came for consultation for infertility and who gave their written consent to participate in the study. Spermogram, spermocytogram, migration survival (TMS) test and DNA fragmentation index were studied. The DNA fragmentation index (DFI) was determined by the Sperm Chromatin Dispersion (SCD) technique. Results: The majority of patients were aged between 35 and 45 years (47%) with an average age of 45 years (+/-8). The most common spermogram abnormalities were mobility abnormalities. 52% of patients had a high fragmentation index. A high fragmentation index was found in a patient with normal sperm standard parameters. Likewise, 8 patients for whom intrauterine insemination had been recommended according to the TMS result had a fragmentation index greater than 30%. Conclusion: The first-line request for the fragmentation index in the exploration of male infertility must be studied since it could take into account the integrity of the genetic material, a limiting factor in the current tests used for the choice of the PMA technique.

Seminal Oxidative Stress and Sperm DNA Fragmentation in Men from Couples with Infertility or Unexplained Recurrent Pregnancy Loss

(1) Background: This case–control study examined whether men from couples with unexplained recurrent pregnancy loss (RPL) or infertility exhibited higher seminal oxidative stress (OS) and sperm DNA fragmentation (SDF) compared to fertile controls. (2) Methods: The study included 30 participants from each group: unexplained RPL, unexplained infertility, and proven fertility. Data were collected at Aalborg University Hospital tertiary RPL and fertility treatment clinics (Aalborg, Denmark), excluding couples with mixed conditions for homogeneity. Semen samples were analyzed using computer-aided sperm analysis (CASA) for concentration, motility, and morphology. SDF was assessed via a CASA-based sperm chromatin dispersion test. OS was measured as static oxidation-reduction potential (sORP). (3) Results: The results showed no significant OS differences between groups. The RPL group had significantly lower SDF levels than the control group. A significant positive correlation between SDF and OS was observed in the infertility group. Overall, this study did not find significant differences in OS levels between men from couples with unexplained RPL or infertility and fertile controls, while SDF levels were lower in the RPL group compared to controls. (4) Conclusion: In conclusion, despite the existing literature suggesting that OS and SDF are negative prognostic factors, our findings suggest they may not be reliable diagnostic markers for RPL and infertility.

Improving human sperm motility via red and near-infrared laser irradiation: in-vitro study

Improved sperm motility is necessary for successful sperm passage through the female genital system, efficacious fertilization, and a greater probability of pregnancy. By stimulating the mitochondrial respiratory chain, low-level laser photobiomodulation has been shown to increase sperm motility and velocity. The respiratory chain in mitochondria is the primary site of action for cytochrome c oxidase because it can absorb light in the visible and infrared ranges. The present study aimed to investigate the effects of red laser 650 nm, near infrared laser (NIR) 980 nm, and combination of both on human spermatozoa motility and DNA integrity at different doses. An in-vitro controlled trial was performed in Al Zahraa university hospital laboratory using thirty fresh human semen specimens. Samples were exposed to red laser 650 nm, near infrared laser (NIR) 980 nm, and combination of both for various irradiation times. Sperm motility for the test and control aliquots was assessed as recommended in the manual of WHO-2021. Sperm chromatin integrity was evaluated using the Sperm Chromatin Structure Assay. Results revealed almost 70%, 80% and 100% increase in the total motility after 3 min of the 650-nm, 980-nm and the combined laser irradiation, respectively. Additionally, the Sperm Chromatin Dispersion assay was carried out on sperm heads utilizing human sperm DNA fragmentation, demonstrating that none of the three laser types had any discernible effects.Graphical abstract

Gaussian clustering and quantification of the sperm chromatin dispersion test using convolutional neural networks.

Sperm DNA fragmentation is a sign of sperm nuclear damage. The sperm chromatin dispersion (SCD) test is a reliable and economical method for the evaluation of DNA fragmentation. However, the cut-off value for differentiation of DNA fragmented sperms is fixed at 1/3 with limited statistical justification, making the SCD test a semi-quantitative method that gives user-dependent results. We construct a collection of deep neural networks to automate the evaluation of bright-field images for SCD tests. The model can detect valid sperm nuclei and their locations from the input images captured with a 20× objective and predict the geometric parameters of the halo ring. We construct an annotated dataset consisting of N = 3120 images. The ResNet 18 based network reaches an average precision (AP50) of 91.3%, a true positive rate of 96.67%, and a true negative rate of 96.72%. The distribution of relative halo radii is fit to the multi-peak Gaussian function (p > 0.99). DNA fragmentation is regarded as those with a relative halo radius 1.6 standard deviations smaller than the mean of a normal cluster. In conclusion, we have established a deep neural network based model for the automation and quantification of the SCD test that is ready for clinical application. The DNA fragmentation index is determined using Gaussian clustering, reflecting the natural distribution of halo geometry and is more tolerable to disturbances and sample conditions, which we believe will greatly improve the clinical significance of the SCD test.

Effects of Varicocele Repair on Sperm DNA Fragmentation and Seminal Malondialdehyde Levels in Infertile Men with Clinical Varicocele: A Systematic Review and Meta-Analysis.

Varicoceles can be a source of elevated seminal oxidative stress (OS) and sperm DNA fragmentation (SDF). However, it remains unclear whether varicocele repair (VR) could reduce these parameters. This systematic review and meta-analysis (SRMA) aims to investigate the impact of VR on SDF and seminal malondialdehyde (MDA). A literature search was performed in Scopus, PubMed, Ovid, Embase, and Cochrane databases. This SRMA included randomized controlled trials and observational studies reporting the pre- and postoperative levels of SDF and seminal OS in infertile men with clinical varicocele that underwent VR. Subgroup analyses included techniques of VR and SDF testing. The effect size was expressed as standardized mean difference (SMD). Out of 1,632 abstracts assessed for eligibility, 29 studies with 1,491 infertile men were included. The analysis showed a significant reduction in SDF after VR, compared to preoperative values (SMD -1.125, 95% confidence interval [CI] -1.410, -0.840; p<0.0001) with high inter-study heterogeneity (I²=90.965%). Reduction in SDF was evident with microsurgical technique and non-microsurgical inguinal approaches (SMD -1.014, 95% CI -1.263, -0.765; p<0.0001, and SMD -1.495, 95% CI -2.116, -0.873; p<0.0001), respectively. Reduction in SDF was significant irrespective of testing was done by sperm chromatin dispersion (SMD -2.197, 95% CI -3.187, -1.207; p<0.0001), sperm chromatin structure assay (SMD -0.857, 95% CI -1.156, -0.559; p<0.0001) or TUNEL (SMD -1.599, 95% CI -2.478, -0.719; p<0.0001). A significant decrease in seminal MDA levels was observed following VR (SMD -2.450, 95% CI -3.903 to -0.997, p=0.001) with high inter-study heterogeneity (I²=93.7%). Using pre- and post-intervention data, this SRMA indicates a significant reduction in SDF and seminal MDA levels in infertile men with clinical varicocele treated with VR. These findings may have important implications for the future management of this selected group of infertile patients.

The relationship between DNA fragmentation and the intensity of morphologically abnormal human spermatozoa

Objective: To determine the relationship between teratozoospermia and sperm DNA fragmentation (SDF) in the human ejaculate. Methods: This retrospective study included 100 normozoospermic men as a control cohort (abnormal forms &gt;14%), 210 patients with a high level of abnormal forms (≤4%) and 65 patients presenting with a moderate level of abnormal forms (&gt;4% to ≤14%) based on the World Health Organization definitions. Sperm morphology was assessed using bright field microscopy. Sperm DNA fragmentation was assessed using the sperm chromatin dispersion assay. Non-parametric analyses were conducted to examine the relationship between abnormal sperm morphology and sperm DNA fragmentation; receiver operating characteristic (ROC) analyses were conducted to assess sensitivity and specificity of this relationship. Results: A correlation analysis revealed that the higher the proportion of abnormal spermatozoa in the ejaculate, the higher the level of SDF (Spearman's Rho = -0.230; P&lt;0.001). Significant differences in the proportion of SDF were found when all cohorts were compared (P&lt;0.001); these significant differences were also retained when the different cohorts were compared pairwise. ROC analysis showed a moderate but significant predictive value for SDF to differentiate patients with different levels of teratozoospemia. Conclusions: Although analysis of a more continuous range of values for teratozoospermia would help further clarify any causal relationship with SDF, there is clearly a synergistic or coincident affiliation between these variables that needs to be acknowledged by the clinician when interpreting the spermiogram.