Oligoasthenoteratozoospermia Research Articles

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.

Loss-of-function variant in TDRD6 cause male infertility with severe oligo-astheno-teratozoospermia in human and mice.

Oligo-astheno-teratozoospermia (OAT) is a common cause of male infertility, but the genetic basis of most OAT cases is still unknown. Here, one homozygous loss-of-function (LOF) variant in TDRD6, c.G1825T/p.Gly609X, was identified in an infertile patient with severe OAT by whole-exome sequencing (WES) and Sanger confirmation. Furthermore, Tdrd6-mutant mice (p.Gly615X; equivalent to p.Gly609X in human TDRD6) were generated. Remarkably, the Tdrd6-mutated mice mimicked the severe OAT symptoms of the patient. In addition, the architecture of chromatoid bodies (CBs) were disrupted in round spermatids from Tdrd6-mutant mice, leading to blocked spermatogenesis in the round spermatids. The assembly of PIWIL1, TDRD1, TDRD7 and DDX25 in CBs was disturbed in the Tdrd6-mutant mice. Applying immunoprecipitation-mass spectrometry (IP-MS), we identified some TDRD6-interacting partners, including CB proteins TDRD7, MAEL and PCBP1. Moreover, we described the assisted reproductive technology (ART) outcomes of the infertile patient and his partner. Altogether, our findings provide necessary evidences to support the idea that the homozygous LOF variant in TDRD6 induces male infertility with severe OAT, suggesting that TDRD6 could be a useful genetic diagnostic target for male infertility.

Novel and recurrent hemizygous variants in BCORL1 cause oligoasthenoteratozoospermia by interfering transcription.

Oligoasthenoteratozoospermia (OAT) is a common cause of male infertility, of which the causes remain largely unknown. Recently, BCORL1 was identified as a contributor to male infertility from non-obstructive azoospermia (NOA) to OAT. To identify novel and hotspot variants in BCORL1 from infertile men with OAT and reveal their outcomes of assisted reproductive treatments (ARTs). Forty-six infertile men characterized by OAT were recruited from 2017 to 2022. Variants in OAT patients were identified by whole-exome sequencing (WES) and verified by Sanger sequencing. Papanicolaou staining was used for sperm morphology analysis. Pathogenicity of BCORL1 variants were analyzed by bioinformatics analysis, and further confirmed in vitro by using recombinant plasmids and cells. Meanwhile, ARTs were performed on these patients to investigate the appropriate clinical treatment strategy. We identified a novel hemizygous missense variant (NM_021946: c.G4171A; p.G1391R) and a recurrent variant (NM_021946: c.T2615G; p.V872G) in BCORL1 from four OAT patients. Notably, routine semen assessment and Papanicolaou staining revealed a special OAT phenotype of patients with BCORL1 variants, whose rare mature sperm characterized by acephalic and abnormal acrosome. Pathogenicity analysis showed the interaction between BCORL1 with histone deacetylases (HDACs) were disrupted after variance, accompanied with epigenetic alterations and finally the orderly transcriptions of spermatogenetic genes were interfering. Besides, clinical record presented the poor outcomes of ARTs in these patients with BCORL1 variants. Our findings further expand the variant spectrum of BCORL1 related to OAT, and provide new evidences that BCORL1 acts as an important transcriptional regulator, participating in epigenetic regulation and directing the expression of key genes throughout spermatogenesis. The outcomes of ARTs will facilitate the genetic counseling and clinical treatment of infertile men with BCORL1 variants in the future.

Phenotypic continuum and poor intracytoplasmic sperm injection intracytoplasmic sperm injection prognosis in patients harboring HENMT1 variants.

Small RNAs interacting with PIWI (piRNAs) play a crucial role in regulating transposable elements and translation during spermatogenesis and are essential in male germ cell development. Disruptions in the piRNA pathway typically lead to severe spermatogenic defects and thus male infertility. The HENMT1 gene is a key player in piRNAs primary biogenesis and dysfunction of HENMT1 protein in meiotic and haploid germ cells resulted in the loss of piRNA methylation, piRNA instability, and TE de-repression. Henmt1-knockout mice exhibit a severe oligo-astheno-teratozoospermia (OAT) phenotype, whereas patients with HENMT1 variants display more severe azoospermia phenotypes, ranging from meiotic arrest to hypospermatogenesis. Through whole-exome sequencing (WES) of infertile patient cohorts, we identified two new patients with variants in the HENMT1 gene presenting spermatozoa in their ejcaulate, providing us the opportunity to study spermatozoa from these patients. Investigate the spermatozoa of two patients harboring an HENMT1 variant to determine whether or not these scarce spermatozoa could be used with assisted reproductive technologies. HENMT1 variants identified by WES were validated through Sanger sequencing. Comprehensive semen analysis was conducted, and sperm cells were subjected to transmission electron microscopy for structural examination, in situ hybridization for aneuploidy assessment, and aniline blue staining for DNA compaction status. Subsequently, we assessed their suitability for in vitro fertilization using intracytoplasmic sperm injection (IVF-ICSI). Our investigations revealed a severe OAT phenotype similar to knockout mice, revealing altered sperm concentration, mobility, morphology, aneuploidy and nuclear compaction defects. Multiple IVF-ICSI attempts were also performed, but no live births were achieved. We confirm the crucial role of HENMT1 in spermatogenesis and highlight a phenotypic continuum associated with HENMT1 variants. Unfortunately, the clinical outcome of these genetic predispositions remains unfavorable, regardless of the patient's phenotype. The presence of spermatozoa is insufficient to anticipate ICSI pregnancy success in HENMT1 patients.

What sperm parameters effect blastocyst formation and quality during ICSI with severe male infertility

Sperm-derived genetic material contributes half of the genome to the embryo, hence it’s crucial to investigate which sperm parameter influences blastocyst formation in the intracytoplasmic sperm injection (ICSI) cycles with severe male infertility. The retrospective study analyzed 296 ICSI cycles with severe oligoasthenoteratozoospermia (OAT) and 99 ICSI cycles with preimplantation genetic testing for aneuploidy (PGT-A). Following the correlation analysis, data stratifications were performed in the OAT ICSI subgroup. The results showed that the matching blastocyst in the OAT ICSI cycles had inferior sperm parameters. DFI and sperm morphology had an influence on the blastocyst formation rate and the high-quality blastocysts formation rate on Day6, but no significant effect on the blastocyst development on Day 5. The high-quality blastocysts formation rate and ratio of high-quality blastocyst on Day 6 were demonstrably better in the subgroup of the teratozoospermic morphology when DFI was within the normal range. In the case of the normal sperm morphology, no statistically significant difference was found in blastocyst development, although there were numerical differences within different DFI subgroups. It was concluded that the blastocyst quality and development declined with the decreased sperm qualities.

Effects of Testicular Platelet-Rich Plasma (PRP) Injection on Sperm Parameters in Men with Severe Oligoasthenoteratozoospermia (OAT): A Clinical Evaluation.

Severe oligoasthenoteratozoospermia (OAT), characterized by a reduced sperm count, motility, and altered morphology, presents a significant challenge in the field of male infertility. Platelet-rich plasma (PRP), renowned for its regenerative capabilities, emerges as a potential intervention for this condition. This study aims to explore the impact of PRP on male infertility, focusing specifically on individuals with severe OAT. The clinical trial study involved 88 infertile men diagnosed with OAT and devoid of underlying diseases. These participants were referred to the infertility center and subsequently divided into two cohorts: a control (44 individuals) and an intervention group (44 individuals). Patients in the intervention group received 2 cc of PRP in each testicle, prepared by centrifuging the patients autologous blood samples. Sperm parameters and DNA fragmentation index (DFI) of the patients were measured before and after the procedure. Statistical analysis used SPSS version 16 software, with a significance level set at less than 5%. The statistical analysis revealed a significant difference in concentration (11.32 ± 8.44 vs. 16.06 ± 15.16, P=0.030), progressive motility (8.86 ± 7.79 vs. 11.97 ± 11.82%, P=0.014) and DNA fragmentation (25.62 ± 12.84 vs. 17.23 ± 9.15%, P<0.001) between the control and intervention groups after PRP injection. However, no significant difference was found in normal morphology (1.63 ± 1.44 vs. 1.81 ± 3.68%, P=0.628) and volume (2.13 ± 0.82 vs. 2.24 ± 1.43, P=0.663) between the control and intervention groups after PRP injection. This study demonstrates the effectiveness of PRP treatment in increasing sperm concentration and motility, while also reducing sperm DNA fragmentation. However, further studies are needed to validate these findings (registration number: IRCT20220317054318N2).

O-194 Cumulative live birth and neonatal outcomes according to different male factors in IVF/ICSI cycles

Abstract Study question What are the impacts of different male factors on cumulative live birth and neonatal outcomes of IVF/ICSI cycles? Summary answer Severe male infertility factors may be negatively associated with fertilization and egg utilization, embryo development and cumulative live birth rate, but not with neonatal outcomes. What is known already Several studies have explored the effect of semen quality on IVF/ICSI clinical outcomes and have drawn contradictory conclusions because the semen quality of male partners could be fluctuated by many factors and there are many confounding factors from women partner. However, an all-round and definitive conclusion has not been reached yet, and clinicians have not yet determined whether men with abnormal semen parameters should be treated to improve their semen parameters and IVF clinical outcomes. Study design, size, duration The retrospective cohort study involved 6,580 males with abnormal sperm parameters and 17,254 males with normozoospermia who underwent their first IVF/ICSI cycle between January 2018 and September 2022 in the reproductive medicine center at a university hospital. They were divided into five different study groups: normozoospermia (N), moderate male factor (MMF), severe oligoasthenoteratozoospermia (OAT-S), azoospermia-husband (azoospermia-H), and azoospermia-donor (azoospermia-D). We compared fertilization, embryo development, cumulative live birth and neonatal outcome. Participants/materials, setting, methods Reproductive and neonatal outcomes of men with abnormal sperm parameters (MMF, OAT-S, azoospermia-H, and azoospermia-D) were studied through four propensity score matching (PSM) comparisons along with corresponding control groups(N) (match factors: including female age, BMI, Gn time, controlled ovarian stimulation protocol, number of oocytes obtained, endometrial thickness and duration of infertility). Fertilization outcomes were also compared stratified by IVF or ICSI. Main results and the role of chance The median ages of the females in the azoospermia, OAT-S, MMF, and N groups were 29, 30, 32 and 32 years old, respectively, which were comparable among groups after PSM. The fertilization rates were significantly reduced in OAT-S and azoospermia-H compared with N in ICSI cycles (67.05% and 65.05% versus 71.50%, p &amp;lt; 0.001, respectively). The oocyte utilization rates were also significantly declined in OAT-S and azoospermia-H compared with N in IVF/ICSI cycles (28.39% and 29.64% versus 34.83%, p &amp;lt; 0.001, respectively). Azoospermia-H group was divided into obstructive azoospermia (OA) and non-obstructive azoospermia (NOA). The conservative cumulative live birth rate (CLBR) in the OAT-S group with poor spermatogenic function decreased (63.6% vs. 72.9%, P &amp;lt; 0.001), and was also declined in the NOA group with the poorest spermatogenic function (62.4% vs. 72.9%, P &amp;lt; 0.001), compared with that in the OA group with mostly normal spermatogenic function. After binary multifactorial logistic regression, we found that conservative CLBR was lower in the OAT-S (aOR 0.675, 95%CI 0.536-0.850, P = 0.001) and NOA (aOR 0.574, 95%CI 0.384-0.857, P = 0.007) groups than in the OA group. No impact of sperm factor on obstetrical/perinatal outcomes was observed. In IVF/ICSI cycles, reproductive and neonatal outcomes were similar between MMF, azoospermia-D and normal semen groups. Limitations, reasons for caution The main limitation of this study is that it was a single-center, observational and retrospective study. However, the large sample size, use of PSMs and use of a multivariate regression model for a wide array of possible confounding factors rendered the conclusion relatively reliable. Wider implications of the findings This is the first study involving long-term outcomes (obstetrical and neonatal outcomes, cumulative pregnancy and live rates) from IVF/ICSI cycles for different male factors, which can help clinicians identify paternal influence in an all-round way. Trial registration number not applicable

P-056 NOA-SERA: combined autologous progenitor cells and platelet-derived growth factors for male factor infertility due to non-obstructive azoospermia and severe oligoasthenoteratozoospermia (OATS) – a case series

Abstract Study question Can NOA-SERA be utilizing autologous progenitor cells and platelet-derived growth factors,effectively address male factor infertility in non-obstructive azoospermia (NOA) and severeoligoasthenoteratozoospermia (OATS) Summary answer Intra-testicular NOA-SERA transplantation of autologous progenitor cells andgrowth factors shows promise in improving spermatogenesis and fertilization potential,presenting potential breakthrough for male factor infertility. What is known already Male infertility affects 10–15% of couples, with non-obstructive azoospermia (NOA)diagnosed in 60% of azoospermic men. Current interventions, including platelet-rich plasma and stem cells, show variable success. This study introduces a novel protocol, NOA-SERA, combining autologous mobilized progenitor cells and platelet-derived growth factors to restore spermatogenesis. Despite advancements in assisted reproduction, male factor infertility due to primary NOA remains challenging. This case series explores the safety and efficacy of NOA-SERA, avoiding in vitro breeding risks and emphasizing intratesticular transplantation’s safety and efficacy Study design, size, duration his case series (March 2019–November 2023) includes 7 male factor infertility patients,consisting of 4 non-obstructive azoospermic NOA, 1 cryptozoospermia and 2 severe oligoaesthenoteratospermic (OATS, strongly desirous of pregnancy with self-gamete. All patients received NOA-SERA following written consent. One-year follow-up included hormonal profiling, semen analysis, and testicular biopsy assessment Participants/materials, setting, methods Seven men, with normal karyotype, underwent NOA-SERA due to male factor infertility. This intervention involved intra-testicular transplantation of autologous CD34+/CD133+ enriched peripheral blood mononuclear stem cells (MPB-MNCs) combined with platelet derived growth factors. The procedure, avoiding in vitro breeding and manipulation, prioritized safety and efficacy. Follow-up for one year included hormonal profile, semen analysis, testicular biopsy assessments, clinical pregnancy (b-hCG), sustained implantation (&amp;gt;8 weeks), and live birth rates were followed. Main results and the role of chance Analysis of NOA-SERA treatment in this case series revealed positive outcomes in 70% of patients, showing increased testicular size, elevated testosterone, and reduced FSH levels. In our study, out of 4 NOA, 2 NOA remained NOA, 1 progressed to OATS, 1 progressed to normal semen analysis after 3 months. Couple conceived with intrauterine insemination (IUI) first pregnancy and they also had a subsequent natural conception. 1 Crypto progressed to OATS and 1 severe OATS remained the same and one progressed to OATS. Although no appearance of sperm in the ejaculate was observed, 2 patients displayed sperms in Testicular Sperm Aspiration (TESA) sufficient for ICSI. Couples undergoing IVF therapy post-biopsy achieved successful fertilization in two cases, leading to positive pregnancies and sustained implantation and livebirth of healthy full-term babies. Post-procedure FSH was slightly lower than pre-procedural levels, indicating hormonal improvement without observed therapy-related deterioration. The novelty lies in the intratesticular transplantation of mobilized purified autologous CD34+/CD133+ enriched MPBMNCs with platelet-derived growth factors, avoiding in vitro risks.” To summarise, 3 out of 7 achieved successful pregnancy, 2 NOA with ICSI, 1 NOA progressed to normal semen analysis and pregnancy with IUI Limitations, reasons for caution Despite interesting results, limitations include the self-controlled case series design and asmall patient cohort, lacking a comparison to a control group. Future studies should include more patients, stratified for age, aetiology, and other risk factors, to further elucidate this intervention’s mechanism of action Wider implications of the findings NOA-SERA demonstrates promise in enhancing spermatogenesis and fertilization potential, emphasizing necessity for larger randomized trials to ascertain clinical efficacy, particularly in subgroups of OATS&amp;NOA patients. NOA-SERA emerges as safe, cost-effective adjuvant for optimizing spermatogenesis and improving IVF success and pregnancy outcomes in male factor infertility. Trial registration number Not applicable

P-023 Gene expression in human sperm as biomarkers and tools for the assessment of male infertility

Abstract Study question Can gene expression as AURKA, HDAC4, and CARHSP1 be used for the diagnosis of sperm quality exploring male infertility? Summary answer The comprehensive outcome of gene expression involving AURKA, HDAC4, and CARHSP1 serves as a key to classifying sperm and predicting their functionality. What is known already Diagnostic male infertility is largely based on semen parameters according to the World Health Organization (WHO) reference values. While these parameters assess sperm quality, they do not provide insights into sperm quality and have a limited predictive value on natural fecundity, fertilization rates, and assisted reproductive technology (ART) outcomes. Study design, size, duration This prospective study investigates the gene expression involved in mitosis, epigenetic regulation, and early embryo development. From February to June 2023, we included all men aged from 20 to 60 years having sperm parameters analysis in our unit center. A total of 277 semen samples were collected, 129 (46.4%) exhibiting three normal sperm parameters according to WHO criteria. Among these, 32 samples showed oligo-astheno-teratozoospermia (OAT), while 116 samples had 1 or 2 abnormal parameters. Participants/materials, setting, methods The mRNA expression levels of three candidate genes (AURKA, HDAC4, and CARHSP1) were quantified in fresh ejaculate semen using real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The thresholds for the spermatozoa function index (SFI) were established by assessing the combined expression of these target genes, considering the morphology and the capability to reach a blastocyst stage. The sperm were ranked according to the concentration (million/ml), motility (%) and morphology (%). Main results and the role of chance We assess the quality of spermatozoa for three biomarkers by analyzing their mRNA expression levels. Receiver operator characteristics curves (ROC) indicate a combined index value expression. This model establishes an index threshold &amp;gt; 320 for an “over-index value,” distinguishing spermatozoa with a normal capacity, an index threshold &amp;lt; 290 for an “under-index value,” and 290 &amp;lt; index &amp;lt; 320 for an “intermediate-index value”. Among the 277, we find 112 (40.4%) have an over-expression index; 161 (58.1%) have an under-expressed index and 4 (1.4%) have an intermediate-index. Interestingly, among the 129 samples with the three normal WHO parameters, we show that 82 (63.5%) have an over-index value, 44 (34.1%) have an under-index value, and 3 (2.3%) display an intermediate-index value. In addition, all the 32 OAT samples have an under-index. These findings suggest that the OAT sperm population implies a potential epigenetic network connected with our three target genes. Furthermore, even in cases with normal sperm parameters, there may be an under-expressed index, indicating lower functionality in terms of fertilization and early embryo development. Limitations, reasons for caution The present method was validated only on ejaculate semen in men free of any chronic disease and any anti-oxidant treatment. This prospective study requires further validation in the largest population. Wider implications of the findings This study’s results could highlight understanding a chaotic early embryo development in ART and used to explore male infertility; particularly in unexplained infertility. Trial registration number not applicable

P-098 The proteome of extracellular vesicle cargo from human seminal plasma

Abstract Study question Could Extracellular Vesicles be employed as a new tool to characterize the male fertility potential? Summary answer Extracellular Vesicles cargo is involved in mechanisms strictly linked to reproductive processes. What is known already Extracellular vesicle contain a plethora of bioactive molecules, such as lipids, DNA and RNA, mainly proteins, which are delivered to target cells to elicit a specific response. This multivarious EV functionality is accomplished by the transfer of their content to the sperm cell, followed by either downregulating or upregulating a series of proteins expressed throughout different sites of sperm cell structure. Despite various studies exploring the proteomic composition of these vesicles, our comprehension of their specific influence on sperm maturation, motility, and the attainment of fertilizing capacity is currently restricted. Study design, size, duration Semen samples were collected from subjects undergoing semen analysis at the UOSA PMA, Siena University Hospital, Italy. All participants were of Caucasian origin and signed a written informed consent to be enrolled in this study. The median age of the patients was 36.7 years (range: 20-45 years); the BMI ranged between 18 and 25 (normal weight). Patients were divided on three groups based on seminal parameters (WHO, 2021) into: Normozoospermic (NORMO); OligoAsthenoTeratozoospermic (OAT); Azoospermic (AZO). Participants/materials, setting, methods EVs from human semen samples were isolated following an in-house optimized protocol using a chemical precipitation reagent ev-GAG®. Samples were subjected to Gel-free and gel-based proteomic analysis. Comprehensive analysis of the vesicles involved acquiring a detailed proteomic profile through the use of 2-D electrophoresis and mass spectrometry. Subsequent inquiries included functional Omics analysis, utilizing bioinformatic tools like Metacore and STRING. We have combined and compared results from several proteomic approaches, both gel-free and gel-based ones. Main results and the role of chance A total of 1133 proteins were identified in NORMO EV samples from the Gel-free approach by LC-MS/MS. These proteins are involved in numerous functions important for reproduction activity. GO enrichment analysis identified involvement of pathways such as cytoskeleton regulation, cell adhesion, cell cycle, development and neurophysiology, proteostasis mechanisms, immune response, and inflammation, reproduction as progesterone signalling and spermatogenesis, protein translation, and signal transduction. 2DE analysis extrapolated a total of 80 differentially abundant spots among the three conditions. Biological Process analysis report that highly abundant proteins in NORMO EVs were significantly involved in chaperone-mediated protein complex assembly, autophagy and in protein folding, in negative regulation of intrinsic apoptotic signaling pathway, in positive regulation of protein- containing complex assembly, positive regulation of tau-protein kinase activity, in regulation of apoptotic processes, in protein stabilization and in regulation of protein ubiquitination. Biological processes related to the three group of proteins suggested that in OAT and even more in AZO EVs, the altered molecular cargo is involved in mechanism strictly linked to reproductive processes, protein folding, inflammation, immune responses, proteolysis, signal transduction. All these mechanisms, in healthy conditions, cooperate for a successful pregnancy. Limitations, reasons for caution To enhance characterization, it is essential to conduct a confirmatory investigation focused on the distinct group of different EVs such as epididymosomes and prostasomes, aiming to offer more precise insight. Wider implications of the findings EVs released along the male reproductive tract, specifically in seminal fluid, have been shown to play a role in sperm maturation. They have the potential to serve as an attractive diagnostic and therapeutic tool in the realm of human reproduction, particularly for issues related to male fertility and infertility. Trial registration number NA

The Effects of Varicocelectomy on Sperm DNA Fragmentation and Conventional Semen Parameters in Men with Severe Oligoasthenoteratozoospermia: A Prospective Study.

The dilation and torsion of testicular veins in the plexus pampiniformis causes Varicocele, which is a surgically repairable cause of male infertility. This study assessed the impact of varicocelectomy on semen characteristics, total motile sperm count (TMSC) and sperm DNA integrity in patients with severe oligoasthenoteratozoospermia (OAT). In this prospective study, semen samples of 360 men with severe OAT who underwent varicocelectomy according to World Health Organization (WHO) criteria 2021 were studied (pre-operatively and at 6, 12, and 18 months post-operatively). The average age of our patients was 38.5 years. The mean spermatozoa concentration was found to be 1.60 ± 0.83 million/ml pre-operatively, while the mean post-operative concentration was 5.17 ± 1.23 million/ml at 6 months, 8.32 ± 0.98 million/ml at 12 months, and 13.51 ± 1.48 million/ml at 18 months (P<0.0001). The mean percentage of A+B motile spermatozoa was 2.92 ± 1.17% pre-operatively, 6.10 ± 1.51% at six months, 9.58 ± 1.49% at 12 months and 13.92 ± 1.88% at 18 months postoperatively (P<0.0001). The mean Modified David's morphology score was 3.80 ± 1.43% pre-operatively, 5.95 ± 1.23% at 6 months, 7.94 ± 1.18% at 12 months, and 10.82 ± 1.91% at 18 months post-operatively (P<0.0001). The mean of total motile sperm count (TMSC) was statistically improved after varicocelectomy (P<0.001). The mean of DNA fragmentation index (DFI) of the spermatozoa was 31.40 ± 0.52% pre-operatively, and post-operatively at 28.20 ± 0.32% at 6 months, 25.90 ± 0.31% at 12 months and 20.50 ± 0.40% at 18 months (P<0.001). Varicocelectomy was associated with significant improvement of sperm parameters and DNA fragmentation resulting in significant improvement of spermatogenesis quality. We believe that universalization in the routinely used sperm dispersion chromatin (SDC) test could be beneficial in the treatment of infertility.

Possible Impact of Human β-defensin 1 on sperm motility in infertile men with abnormal sperm parameters

Human β-defensins and interleukins may be auxiliary in sperm maturation. This cross-sectional study aimed to evaluate the expression of Human β-defensins 1 and 2, interleukins (ILs)− 10 and -18 genes in sperm, as well as seminal plasma levels of these two cytokines in subfertile men with different types of sperm abnormalities compared to those with normozoospermic men. Participants were separated into two experimental groups: the control group (n = 25) and the group with sperm abnormalities (SA) (n = 45). SA participants were further subdivided into the following groups with n = 15 individuals each: Teratozoospermia (T), Asthenoteratozoospermia (AT), and Oligoasthenoteratozoospermia (OAT) groups. The quantitative real-time polymerase chain reaction was used to quantify the mRNA levels of hBDs 1 and 2, IL-10, and IL-18 in sperm. The seminal plasma concentrations of IL-10 and IL-18 were measured by using the enzyme-linked immunosorbent assay technique. The mRNA expression of hBD-1 and IL-10 showed a significant decrease in the OAT compared to the controls (P < 0.0001 and P = 0.02, respectively). The lowest seminal plasma concentration of IL-10 belonged to the OAT (P = 0.04). ROC curve analysis showed a sensitivity, specificity, and cutoff value of 82.35%, 86.67%, and 0.63 for hBD-1 levels, respectively. A positive and significant correlation was found between hBD-1 expression and sperm motility and IL-10 expression rate and normal sperm morphology.Therefore, hBD-1 could be considered as the alternative biomaterial to pre-treatments of infertile men with abnormal sperm parameters, specifically OAT men, which led to improving the assisted reproduction success rate.

Beneficial effects of oral antioxidant supplementation on semen quality parameters, reproductive hormones, and sperm DNA integrity in men with idiopathic oligoasthenoteratozoospermia.

Recently, oral antioxidants in combined forms have been used to treat men with idiopathic infertility. This study aimed to evaluate the effects of treatment with vitamin C, vitamin E, selenium, zinc, arginine, L-carnitine, and coenzyme Q10 on sperm quality parameters, DNA integrity, reproductive hormones, and pregnancy rates in men with infertility and idiopathic oligoasthenoteratozoospermia (OAT). A prospective study was conducted on 420 men with infertility and idiopathic OAT who took an oral supplement of antioxidant SP-Power tablets twice daily for 6 months. Semen quality, reproductive hormones, and the DNA fragmentation index (DFI) were evaluated at baseline and at 3 and 6 months after supplementation, using the World Health Organization 2021 guidelines. No significant difference was observed in volume or the percentage of typical morphology during treatment. A significant improvement in sperm concentration was observed after supplementation (8.67±1.41, 12.17±1.91, and 19.01±0.86 at baseline, 3, and 6 months respectively, p<0.01). The total motility, progressive motility, and total motile sperm count also increased significantly (p<0.01), whereas the DFI decreased after 6 months. There was an increase in normal FSH levels and testosterone levels after 6 months of supplementation of antioxidant SP-Power but these differences were not statistically significant (p=not significant and p=0.06, respectively). Supplementation with SP-Power tablets improved sperm quality parameters, sperm DFI, some reproductive hormones, and pregnancy rates in men with infertility and idiopathic OAT, which could be attributed to the supplement's synergistic antioxidant action. Further studies are needed to determine the effects of supplementation on oxidative stress markers.

Molecular Study on Y Chromosome Microdeletion in Male Infertility: A Cross-Sectional Design in Indonesian Men

Y chromosome microdeletions (YCMs) are one kind of genetic disorder that contributes to male infertility. This study aims to determine the profile of YCMs in the infertile male population in Indonesia. This cross-sectional study was conducted by identifying YCMs testing data on 49 infertile male patients identified with azoospermia and oligoasthenoteratozoospermia (OAT) based on their sperm analysis, who visited andrology polyclinics in several hospitals in Yogyakarta Province between March 2021 to August 2022. Study participants underwent YCMs testing at the Molecular Medicine and Therapy Research Laboratory, Muhammadiyah University of Yogyakarta, Indonesia, using the Polymerase Chain Reaction (PCR) method according to the procedures established by the laboratory. Four out of 49 (8.2%) participants were identified to have YCMs with deletions in the Azoospermia Factor C (AZFc) subregion. Two participants identified with YCMs had cryptozoospermia in their sperm analysis. Hormonal examination showed variable results in 4 participants, including hypergonadotropic, hypogonadism, and normogonad. All participants in the study identified with YCMs showed a deletion in the AZFc subregion. This type of deletion is different from previous studies in Indonesia, so broad examinations of infertile male patients are required to figure out the deletion profile in a larger population of Indonesian sterile males.

A hemizygous loss-of-function variant in BCORL1 is associated with male infertility and oligoasthenoteratozoospermia.

Oligoasthenoteratozoospermia (OAT) is a common type of male infertility; however, its genetic causes remain largely unknown. Some of the genetic determinants of OAT are gene defects affecting spermatogenesis. BCORL1 (BCL6 corepressor like 1) is a transcriptional corepressor that exhibits the OAT phenotype in a knockout mouse model. A hemizygous missense variant of BCORL1 (c.2615T > G:p.Val872Gly) was reported in an infertile male patient with non-obstructive azoospermia (NOA). Nevertheless, the correlation between BCORL1 variants and OAT in humans remains unknown. In this study, we used whole-exome sequencing to identify a novel hemizygous nonsense variant of BCORL1 (c.1564G > T:p.Glu522*) in a male patient with OAT from a Han Chinese family. Functional analysis showed that the variant produced a truncated protein with altered cellular localization and a dysfunctional interaction with SKP1 (S-phase kinase-associated protein 1). Further population screening identified four BCORL1 missense variants in subjects with both OAT (1 of 325, 0.31%) and NOA (4 of 355, 1.13%), but no pathogenic BCORL1 variants among 362 fertile subjects. In conclusion, our findings indicate that BCORL1 is a potential candidate gene in the pathogenesis of OAT and NOA, expanded its disease spectrum and suggested that BCORL1 may play a role in spermatogenesis by interacting with SKP1.

Bi-allelic variants in chromatoid body protein TDRD6 cause spermiogenesis defects and severe oligoasthenoteratozoospermia in humans

BackgroundThe association between the TDRD6 variants and human infertility remains unclear, as only one homozygous missense variant of TDRD6 was found to be associated with oligoasthenoteratozoospermia (OAT).MethodsWhole-exome sequencing and Sanger...

Moxibustion prevents tripterygium glycoside-induced oligoasthenoteratozoospermia in rats via reduced oxidative stress and modulation of the Nrf2/HO-1 signaling pathway.

Oligoasthenoteratozoospermia (OAT) decreases male fertility, seriously affecting the production of offspring. This study clarified the preventive impact of different moxibustion frequencies on OAT and selected the optimal frequency to elucidate the underlying mechanism. An OAT rat model was constructed by gavage of tripterygium glycosides (TGS) suspension. Daily moxibustion (DM) or alternate-day moxibustion (ADM) was administered on the day of TGS suspension administration. Finally, we selected DM for further study based on sperm quality and DNA fragmentation index, testicular and epididymal morphology, and reproductive hormone level results. Subsequently, the oxidative stress (OS) status was evaluated by observing the OS indices levels; malondialdehyde (MDA), 8-hydroxy-deoxyguanosine (8-OHdG), total antioxidant capacity (T-AOC), and total superoxide dismutase (T-SOD) in testicular tissue using colorimetry and enzyme-linked immunosorbent assay. Furthermore, heme oxygenase 1 (HO-1) and nuclear factor erythropoietin-2-related factor 2 (Nrf2) were evaluated using Western blotting. Immunohistochemistry was employed to locate and assess the expression of HO-1 and Nrf2 protein, while quantitative real-time polymerase chain reaction was utilized to detect their mRNA expression. MDA and 8-OHdG levels decreased following DM treatment, while T-SOD and T-AOC increased, suggesting that DM may prevent TGS-induced OAT in rats by decreasing OS in the testis. Furthermore, protein and mRNA expression of Nrf2 and HO-1 in the testis were elevated, indicating that DM may reduce OS by activating the signaling pathway of Nrf2/HO-1. Therefore, DM could prevent OAT in rats via the Nrf2/HO-1 pathway, thereby presenting a promising therapeutic approach against OAT.

The therapeutic effect and metabolic mechanism analysis of Guilingji on idiopathic oligo-asthenoteratozoospermia

IntroductionGuilingji, a famous traditional Chinese medicine (TCM) formula, has been used to combat aging and male sexual dysfunction in China for centuries. To date, there has been little evidence-based clinical research on the use of Guilingji to treat idiopathic oligo-asthenoteratozoospermia (OAT), and the therapeutic mechanism from a metabolic perspective needs to be investigated further. MethodsThis was a multicenter, double-blind, randomized controlled clinical study of 240 patients with idiopathic OAT recruited from four hospitals between January 2020 and January 2022. Patients were randomly assigned in a 1꞉1 ratio to receive oral Guilingji capsules or placebo for 12 weeks. The total progressive motile sperm count (TPMSC) was considered the primary outcome, and the other sperm parameters, seminal plasma parameters and serum hormones were considered the secondary outcome. A nontargeted metabolomics analysis of serum from OAT patients before and after Guilingji administration was performed by HPLC–MS to identify key metabolites. Furthermore, we used a rat model to show spermatogenesis phenotypes to validate the effect of the key metabolites screened from the patients. ResultsAt weeks 4, 8 and 12, TPMSC and other sperm parameters were significantly improved in the Guilingji group compared with the placebo group (P < 0.05 for all comparisons). At week 4, superoxide dismutase (SOD) and acrosomal enzyme activity of seminal plasma were significantly elevated in the Guilingji group compared with the placebo group, while reactive oxygen species (ROS) levels were significantly reduced (P < 0.05). Lactate dehydrogenase-X (LDHX) levels appeared to be significantly increased after 12 weeks continuous medication compared with Placebo group (P = 0.032). The metabolomics analysis of serum from OAT patients before and after Guilingji administration showed that the glucose-6-phosphate (G6P) concentration in patients' serum was significantly elevated after Guilingji treatment. Compared to the control, when Kidney-Yang deficiency model rats were treated with Guilingji or its key intermediate metabolite G6P, their sperm concentration and spermatozoic activity were improved similarly, and their structural damage of rat's testicular and epididymal tissues were recovered. ConclusionThis study provided valuable clinical evidence for the utility of Guilingji as a treatment for OAT. These findings thus demonstrate that G6P is involved in the therapeutic mechanism of Guilingji in OAT treatment based on clinical and rat intervention studies.

Homozygous CCDC146 mutation causes oligoasthenoteratozoospermia in humans and mice.

Infertility represents a significant health concern, with sperm quantity and quality being crucial determinants of male fertility. Oligoasthenoteratozoospermia (OAT) is characterized by reduced sperm motility, lower sperm concentration, and morphological abnormalities in sperm heads and flagella. Although variants in several genes have been implicated in OAT, its genetic etiologies and pathogenetic mechanisms remain inadequately understood. In this study, we identified a homozygous nonsense mutation (c.916C>T, p.Arg306*) in the coiled-coil domain containing 146 ( CCDC146) gene in an infertile male patient with OAT. This mutation resulted in the production of a truncated CCDC146 protein (amino acids 1-305), retaining only two out of five coiled-coil domains. To validate the pathogenicity of the CCDC146 mutation, we generated a mouse model ( Ccdc146 mut/mut ) with a similar mutation to that of the patient. Consistently, the Ccdc146 mut/mut mice exhibited infertility, characterized by significantly reduced sperm counts, diminished motility, and multiple defects in sperm heads and flagella. Furthermore, the levels of axonemal proteins, including DNAH17, DNAH1, and SPAG6, were significantly reduced in the sperm of Ccdc146 mut/mut mice. Additionally, both human and mouse CCDC146 interacted with intraflagellar transport protein 20 (IFT20), but this interaction was lost in the mutated versions, leading to the degradation of IFT20. This study identified a novel deleterious homozygous nonsense mutation in CCDC146 that causes male infertility, potentially by disrupting axonemal protein transportation. These findings offer valuable insights for genetic counseling and understanding the mechanisms underlying CCDC146 mutant-associated infertility in human males.

#241 : Impact of Antioxidants in Improving Semen Parameters Like Count, Motility and DNA Fragmentation in Sub-Fertile Males: A Randomized, Double Blind, Placebo Controlled Clinical Trial

Background and Aims: Male infertilities are solely responsible for 20-30% of infertility cases. Oxidative damage of sperm DNA is positively linked with oligoasthenoteratozoospermia (OAT), and male infertility. The antioxidants are being explored worldwide to combat OAT, sperm DNA fragmentation and reactive oxygen species. The objective of the study was to assess the effectiveness of an antioxidant blend in improving sperm count, semen parameters and reducing DNA fragmentation index (DFI) in sub-fertile males. Method: A prospective, double-blind, randomized, placebo-controlled trial was conducted in 300 sub-fertile males (25–45 years) from ten study sites in India. Subjects were randomized in either the antioxidant blend treatment group or placebo group. We assessed changes in sperm count, motility, normal morphology, semen volume, and percent DFI before and after treatment (90 days). To further stratify data on different criteria post hoc analysis was performed. Statistical analysis was performed using SPSS 10.0 software. Results: There were improvements in sperm count, semen volume, sperm motility, and sperm normal morphology in the treatment group. There was improvement in sperm count in severe oligospermia subjects (sperm count &lt; 5 million/mL, 5–10 million/mL, 10.1–15 million/mL), and high-extremely higher baseline DFI (20–30%, 31–40% and above 40%), as per post hoc analysis. There was no premature discontinuation and adverse events were reported during the study, indicating safety and well-tolerability of treatment. Conclusion: Study results confirmed the well-researched fact of antioxidants being effective to reduce oxidative stress and thus improve sperm DNA integrity and also improved semen parameters particularly in males aged 40 and above.