Poor Sperm Research Articles

Selenium efficiency in protecting sperm quality and testicular parameters of South African indigenous Zulu rams exposed to heat stress.

The study investigated selenium's (Se) efficiency in preserving South African Zulu rams' sperm quality and testicular parameters when they were exposed to heat stress. Indigenous Zulu rams (20) between 2 and 5 years old were allocated into four groups, namely the Se, testicular heat stress (THS), selenium plus testicular heat stress (SeTHS), and control. Each group comprised five rams; the groups were balanced according to the rams' body weight and scrotal circumference. The Se and SeTHS groups received sodium selenite orally bi-weekly for 5 months. To induce heat stress, testicular heat insulation bags were wrapped around the testes of the rams receiving the THS and SeTHS treatments for 49 days. Semen was collected from the rams weekly from the third month onward; the first two months were for Se & THS acclimatization. In addition, testicular measurements were taken bi-weekly. Analysis of variance was used to analyze the sperm quality data. Duncan's multiple range test was used to compare the groups' data for significant differences. The results showed that the Se-supplemented rams' scrotal circumference was smaller (p < 0.05) compared with the other groups. The Se, SeTHS, and control groups demonstrated similar total sperm motility; in contrast, the THS and SeTHS groups recorded low and high total sperm motility, respectively, compared with other treatment groups (p < 0.05). The semen from the rams that received THS without Se displayed a significantly higher number of immotile sperm cells (p < 0.05) and poor sperm quality, including total and progressive motility, and kinematic parameters when compared with other treatments, suggesting that Se protects sperm against THS. We concluded that selenium protected some sperm parameters (TSM, PSM, MV, VCL, VSL) of THS- treated rams while others did not improve (RV, NSM, C, STR).

The effect of male factors on embryo morphokinetics: a retrospective analysis of 2726 blastocysts.

Male infertility may influence fertilization rates, embryo morphology, and implantation rates in in vitro fertilization (IVF) cycles. Oocyte competence plays a major role in embryo development, but there is a limited understanding of the connection between sperm quality, embryo development, and morphokinetic parameters using donor oocytes. The study evaluated if sperm quality may influence the morphokinetic parameters in IVF cycles. A retrospective multicentric observational cohort study included 747 ICSI cycles using donor oocytes with fresh or frozen sperm. Embryos were cultured in time-lapse incubators until the blastocyst stage. The population was divided into three groups according to sperm concentration, as control group (> 16 mill/mL), severe oligospermia (0-5 mill/mL), and moderate oligospermia group (5-16 mill/mL). Morphokinetic analysis showed no difference in the time from the 2-cell to 6-cell stage of embryo development. A significant difference was observed on day 3 of embryo development, specifically at the 7-cell stage (t7), severe oligospermia 53.37 ± 9.81, moderate oligospermia 56.95 ± 9.78, and control 55.1 ± 8.85h post-insemination (hpi) (p = 0.024), and 8-cell stage (t8), severe oligospermia 55.41 ± 10.83, moderate oligospermia 61.86 ± 12.38 hpi (p < 0.001), and control 58.61 ± 11.33. Accordingly, the synchrony of the four cleavages going from 4 to 8 cells (s3) was found statistically different among the groups in the severe oligospermia 8.05 ± 9.99, moderate oligospermia 11.66 ± 11.04 hpi, and control 8.55 ± 8.58 (p = 0.009). Morphokinetic time ranges were obtained for t6, t7, t8, and s3 in order to identify the good-quality blastocysts. Poor sperm quality is associated with alterations in morphokinetic parameters on day 3 in IVF cycles with donor oocytes, underlining the important role of spermatozoa during embryo development.

Clinical Applications of Assisted Oocyte Activation in Couples with Various Reproductive Problems: A Systematic Review.

ICSI may face fertilization failure, prompting the use of assisted oocyte activation (AOA) techniques. While AOA is implemented in infertility clinics, its target patients and definitive application remain uncertain. This systematic review adheres to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines to assess reproductive outcomes in ICSI-AOA cycles compared to conventional ICSI and evaluate AOA effectiveness in various infertility disorders. A literature search encompassed PubMed, Web of Science, EMBASE, and Scopus databases until December 2023 for relevant English studies. Included studies compared ICSI-AOA with conventional ICSI in couples with prior fertilization failure, utilizing diverse AOA methods. Control groups consisted of sibling oocytes, previous cycles of the same couples, or couples undergoing conventional ICSI. Evaluated outcomes included fertilization, cleavage, embryo quality, implantation, pregnancy, and live birth rates. Article screening and data extraction were performed by two authors, with risk of bias assessed by another investigator. Out of 3088 initially identified articles, 30 studies were included, focusing on fertilization failure (n = 10), female infertility (n = 3), PLCζ defects (n = 4), poor sperm quality (n = 4), Globozoospermia (n = 4), and surgically retrieved sperm (n = 8). Most studies concluded that AOA could overcome fertilization failure, but success rates varied based on sperm-related or oocyte-related factors in ICSI-AOA cycles. Due to differences in patient inclusion criteria and sample sizes, most studies were not sufficiently similar for pooled analysis, limiting robust conclusions. There is insufficient evidence, particularly from randomized controlled trials (RCTs), to determine the efficacy or safety of ICSI-AOA as a treatment strategy. Registration number is PROSPERO, CRD42024551221.

In silico approach uncovers the shared genetic landscape of type 2 diabetes mellitus and asthenozoospermia

Asthenozoospermia (AZS) is one of the most common types of male infertility. Current evidence revealed that type 2 diabetes mellitus (T2DM) is closely associated with declining semen quality, especially for poor sperm motility. This study aimed to uncover the genetic interrelationships and important biomarkers between AZS and T2DM. Transcriptome data regarding AZS and T2DM were downloaded from the Gene Expression Omnibus (GEO) database. We performed GO and pathway analysis, and protein-protein interaction (PPI) network construction for T2DM-related differentially expressed genes (DMRGs). Moreover, we calculated receiver operator characteristic (ROC) curve and conducted external independent validation. Expression of hub DMRGs was assessed for patients using the qPCR method. MiRNA interaction and immune infiltration were subsequently characterized. A total of 554 overlapping DMRGs were identified between the AZS/T2DM and healthy groups. These overlapping DMRG participated in the DNA damage-, energy metabolism-, and immune-related biological pathways. Module function analysis discovered that the top three PPI modules were tightly correlated with DNA damage-related processes. After external validation in other independent datasets, two hub DMRGs (TBC1D12 and SCG5) were obtained. ROC analysis revealed that TBC1D12 and SCG5 had good diagnostic performance (area under the curve > 0.75). Immune infiltration profile showed that the level of T cell co-stimulation and CD8+_T_cells were negatively related to the hub DMRGs expression. Mirna interaction analysis showed 15 significant hub DMRGs-miRNA interactions. The qPCR results showed that expression of TBC1D12 and SCG5 were significantly different between sperm samples from diabetic patients with AZS and controls. The present study revealed molecular signatures and critical pathways between the AZS and T2DM, and identified two hub DMRGs of TBC1D12 and SCG5. The data would provide novel understandings of shared pathogenic mechanisms in T2DM-associated AZS.

Exploring the potential of selenium nanoparticles and fabricated selenium nanoparticles @vitamin C nanocomposite in mitigating nicotine-induced testicular toxicity in rats.

The tobacco epidemic signifies a major public health threat. Nicotine (NIC), a major active constituent in tobacco, impedes male fertility and semen quality. This work is implemented to explore the potential of selenium nanoparticles (SeNPs) and the newly fabricated SeNPs @vitamin C (SeNPs@VITC) nanocomposite in mitigating testicular toxicity induced by NIC. The six groups of 48 adult Wistar rats were designed as follows: the control group injected intraperitoneally with normal saline, the SeNPs group treated orally with 2mg/kg of SeNPs, the SeNPs@VITC nanocomposite group treated orally with 2mg/kg of SeNPs@VITC nanocomposite, the NIC group injected intraperitoneally with 1.25mL/kg of NIC, the NIC+ SeNPs group received SeNPs plus NIC, and the NIC+ SeNPs@VITC nanocomposite group received SeNPs@VITC nanocomposite plus NIC. Treatments were administered over a 28-day period. NIC treatment significantly caused poor sperm quality, decreased serum testosterone, increased follicle-stimulating hormone (FSH), luteinizing hormone (LH) concentrations, reduced hemoglobin levels, leukocytosis, disrupted testicular oxidant/antioxidant balance, and disorganized testicular structure. The construction of the novel SeNPs@VITC nanocomposite, compared to NIC plus SeNPs alone, demonstrated a more potent ameliorative effect on NIC-induced reproductive toxicity in adult rats. The SeNPs@VITC nanocomposite significantly increased sperm count, reduced the percentage of sperm head abnormalities, lowered both serum FSH and LH concentrations, and improved the hemoglobin response. Both SeNPs and SeNPs@VITC nanocomposite alleviated the testicular toxicity induced by NIC, but the SeNPs@VITC nanocomposite exhibited superior efficacy. The SeNPs@VITC nanocomposite could be employed to advance enhanced therapeutic strategies for addressing male infertility.

Distinct roles of Kif6 and Kif9 in mammalian ciliary trafficking and motility.

Ciliary beat and intraflagellar transport depend on dynein and kinesin motors. The kinesin-9 family members Kif6 and Kif9 are implicated in motile cilia motilities across protists and mammals. How they function and whether they act redundantly, however, remain unclear. Here, we show that Kif6 and Kif9 play distinct roles in mammals. Kif6 forms puncta that move bidirectionally along axonemes, whereas Kif9 appears to oscillate regionally on the ciliary central apparatus. Consistently, only Kif6 displays microtubule-based motor activity in vitro, and its ciliary localization requires its ATPase activity. Kif6 deficiency in mice disrupts coordinated ciliary beat across ependymal tissues and impairs cerebrospinal fluid flow, resulting in severe hydrocephalus and high mortality. Kif9 deficiency causes mild hydrocephalus without obviously affecting the ciliary beat or the lifespan. Kif6-/- and Kif9-/- males are infertile but exhibit oligozoospermia with poor sperm motility and defective forward motion of sperms, respectively. These results suggest Kif6 as a motor for cargo transport and Kif9 as a central apparatus regulator.

Potential for Offspring with Varicocele and Pcos

Infertility affects a significant portion of couples worldwide, with 30% of cases attributed to male factors, 30% to female factors, 30% to both partners, and 10% to unknown causes. This study focuses on two common causes of infertility: varicocele in men and Polycystic Ovary Syndrome (PCOS) in women. Varicocele, characterized by the enlargement of veins within the scrotum, disrupts spermatogenesis by increasing testicular temperature, leading to poor sperm quality. PCOS, affecting 5-10% of women of reproductive age, is marked by excessive androgen levels, irregular menstruation, and obesity, significantly raising the risk of infertility. Through case studies and literature review, this research highlights the potential for individuals with varicocele or PCOS to achieve pregnancy with appropriate treatment. The study aims to explore the specific examination and treatment processes required for these conditions and to provide insights into improving fertility outcomes for affected individuals.

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.

Lead Acetate Provokes Poor Sperm Quality and Reduced Sperm Concentration in Male Wistars Rats

Background: Lead acetate, a toxic heavy metal, adversely affects male reproductive health in both humans and animals. However, its specific impact on sperm quality and concentration in Wistar rats remains understudied. Objectives: This study aimed to examine the effects of lead acetate on sperm quality and concentration in male Wistar rats, assessing motility, viability, total sperm count, and morphology. Additionally, it evaluated whether a recovery period could mitigate these adverse effects. Methods: Thirty male Wistar rats were divided into three groups of 10 each: Control (1 mL/kg BW distilled water), Lead Acetate (60 mg/kg BW orally for 28 days), and Recovery (28 days lead acetate followed by 28 days distilled water). Sperm samples were assessed for various parameters. Statistical analysis was performed using ANOVA with significance set at P &lt; 0.05, using GraphPad Prism 8. Results: The lead acetate-exposed group (Group 2) showed significantly reduced sperm motility, viability, and total sperm count compared to the control group (Group 1). Additionally, Group 2 exhibited a higher incidence of sperm head abnormalities (P &lt; 0.05). The recovery group (Group 3) demonstrated some improvement in sperm parameters compared to Group 2, but these improvements were not statistically significant. Conclusions: This study provides evidence that exposure to lead acetate has a substantial negative impact on the capacity of sperm to move, survive, and reproduce in male Wistar rats.

Influence of male body mass index on semen analysis parameters and in vitro fertilization outcomes.

Concurrent global increase of prevalence of obesity and male fertility implies link between overweight and obesity with male subfertility. This hypothesis is supported by numerous population-based epidemiological studies. Increase in body mass index (BMI) is associated with poor sperm quality in fertile, and more noticeable in infertile men. Nevertheless, some studies disprove damaging effect of BMI on semen quality. To examine the influence of men's BMI in infertile couples undergoing in vitro fertilization (IVF) on semen analysis parameters and IVF outcomes. Study encompassed all couples who underwent IVF at Gynecology and Obstetrics Clinic Narodni Front in Belgrade during 2018 and 2019. Exclusion criteria were azoospermia, conditions and diseases that could affect the semen analysis parameters (diabetes, malignant diseases treated with radiation and/or chemotherapy, trauma or surgery of the genital organs, mumps or undescended testicles in childhood). Evaluated semen analysis parameters included semen ejaculate volume, sperm pH, sperm count, sperm motility, and sperm morphology. IVF outcomes comprised total number of embryos, number and percentage of obtained good-quality embryos and clinical pregnancy rates. Based on BMI value, participants were divided into a group of underweight (Group 1), normally weight (Group 2), overweight (Group 3), and obese men (Group 4). After applying inclusion and exclusion criteria, 411 men (couples) were included in the analysis. The largest number of men were overweight, while the smallest belonged to the group of underweight participants. There are no significant differences in the semen analysis parameters between study groups. Correlation analysis shown weak and insignificant correlation between BMI and semen analysis parameters. The number and proportion of good quality embryos is significantly lower in overweight and obese study groups compared to normal weight and underweight groups (2.89, 2.91, 2.42, and 2.36, respectively, P = .041). The differences in other IVF outcomes: total number of embryos (3.61, 3.74, 3.21, and 3.37, respectively) and clinical pregnancy rates (41.26%, 43.09%, 42.78%, and 39.95%, respectively) between study groups were not significant (P > .05). BMI does not significantly affect semen analysis parameters, but a higher BMI is associated with a lower number and proportion of good quality embryos in IVF outcomes.

Poor Sperm Chromatin Condensation Is Associated with Cryopreservation-Induced DNA Fragmentation and Cell Death in Human Spermatozoa.

Background/Objectives: Semen cryopreservation is routinely performed in fertility clinics for a variety of reasons, including fertility preservation and storage of donor sperm, yet the freeze-thaw process leads to cellular damage via ice crystal formation, osmotic shock, and supraphysiological levels of oxidative stress. Sperm resistance to damage during the freeze-thaw process varies widely, yet the intrinsic factors associated with sperm cryotolerance are largely unknown. The study aimed to investigate whether poor chromatin condensation renders sperm vulnerable to DNA fragmentation and cell death induced by the freeze-thaw process. Methods: Participants (n = 51) from the general community who met the inclusion criteria collected a semen sample after 3-8 days of abstinence. Neat semen samples underwent traditional semen analysis, aniline blue (AB)-eosin staining for chromatin condensation, the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay for DNA fragmentation, and the Annexin V assay for apoptosis/necrosis, prior to being cryopreserved using the liquid nitrogen vapour method and stored at -196 °C. Stored samples were later thawed at room temperature and processed using density gradient centrifugation. Motile sperm concentration, DNA fragmentation and apoptosis/necrosis were analysed in post-thaw samples. Results: As indicated by a significant interaction effect in linear mixed models, an increased proportion of AB-positive sperm in the pre-freeze sample exacerbated the adverse effect of freezing on sperm DNA fragmentation (p = 0.004), late apoptosis (p = 0.007), and necrosis (p = 0.007). AB-staining was positively correlated with all three parameters in the post-thaw sample (all rs ≥ 0.424, all p < 0.01) and remained significant after adjusting for neat sperm concentration (all partial rs ≥ 0.493, all p < 0.01). Similarly, AB-staining was significantly correlated with the percentage point change in sperm DNA fragmentation (rs = 0.366, p = 0.014) and necrosis (rs = 0.403, p = 0.009), both of which remained significant after adjusting for neat sperm concentration (both partial rs ≥ 0.404, both p < 0.01), and borderline significantly correlated with percentage point change in late apoptosis (rs = 0.307, p = 0.051). Conclusions: Sperm with poorly condensed chromatin may be more susceptible to cellular damage during the freeze-thaw process, independent of pre-freeze sperm concentration. These findings may help to explain the intrinsic variation in sperm resistance to cryodamage within and between individuals that is poorly understood.

A novel murine model mimicking male genital Neisseria species infection using Neisseria musculi†.

With ~78 million cases yearly, the sexually transmitted bacterium Neisseria gonorrhoeae is an urgent threat to global public health due to continued emergence of antimicrobial resistance. In the male reproductive tract, untreated infections may cause permanent damage, poor sperm quality, and subsequently subfertility. Currently, few animal models exist for N. gonorrhoeae infection, which has strict human tropism, and available models have limited translatability to human disease. The absence of appropriate models inhibits the development of vital new diagnostics and treatments. However, the discovery of Neisseria musculi, a mouse oral cavity bacterium, offers much promise. This bacterium has already been used to develop an oral Neisseria infection model, but the feasibility of establishing urogenital gonococcal models is unexplored. We inoculated mice via the intrapenile route with N. musculi. We assessed bacterial burden throughout the male reproductive tract, the systemic and tissue-specific immune response 2-weeks postinfection, and the effect of infection on sperm health. Neisseria musculi was found in penis (2/5) and vas deferens (3/5) tissues. Infection altered immune cell counts: CD19+ (spleen, lymph node, penis), F4/80+ (spleen, lymph node, epididymus), and Gr1+ (penis) compared with noninfected mice. This culminated in sperm from infected mice having poor viability, motility, and morphology. We hypothesize that in the absence of testis infection, infection and inflammation in other reproductive is sufficient to damage sperm quality. Many results herein are consistent with outcomes of gonorrhoea infection, indicating the potential of this model as a tool for enhancing the understanding of Neisseria infections of the human male reproductive tract.

Post-cooling sperm processing can rescue sperm quality of cooled-stored stallion semen

Poor sperm quality in cooled-shipped semen has been related to subpar fertility in horses. Therefore, this study aimed to evaluate the ability of post-cooling sperm processing to improve sperm parameters of cooled-stored stallion semen for artificial insemination. For all experiments, ejaculates were collected, processed, and diluted in skimmed milk-based (SM) medium and stored at 5 °C/24h. In all experiments an aliquot of unprocessed cooled semen was used as a control. In the first experiment (Exp 1.), cooled-stored semen from 16 stallions (n = 32) was processed by SpermFilter or centrifugation (600×g/10min) and resuspended in an egg yolk-based freezing medium containing permeating cryoprotectants (EY-C) for cryopreservation. Sperm recovery and motility parameters were immediately assessed after sperm resuspension in both groups and compared with unprocessed (Unp) samples. In Exp 2., cooled semen samples from six stallions (n = 18) were processed using SpermFilter and resuspended in SM or EY-C. Motility parameters and plasma membrane integrity were assessed in all groups (Unp, SM, and EY-C). In Exp 3, cooled semen from four stallions (n = 20) was processed by SpermFilter, resuspended in SM, EY-C, or egg yolk-based medium without cryoprotectants (EY-nC); and submitted to a thermoresistance test (37 °C/3h). Motility parameters, plasma membrane integrity and stability, mitochondrial membrane potential, mitochondrial superoxide generation, and DNA fragmentation index were evaluated in all groups. Finally, in Exp 4, 39 estrous cycles of 11 mares were inseminated with unprocessed (n = 6) cooled-stored semen or semen cooled at 5 °C/24h and then processed by SpermFilter and resuspended in SM (n = 5), EY-C (n = 11), EY-nC (n = 11), or centrifuged and resuspended in EY-C (n = 6). Overall, semen processing and resuspension in EY mediums (EY-C and EY-nC) improved sperm parameters compared with those of unprocessed semen (P < 0.05). Centrifugation (91 ± 5 %) recovered more sperm than SpermFilter (84 ± 9 %; P < 0.05). Sperm resuspended in EY-nC maintained better sperm parameters throughout the thermoresistance test than those in the other groups (P < 0.05). The fertility rates were similar between all groups (P > 0.05). In conclusion, processing and resuspension in EY medium can improve sperm parameters in post-cooled-stored stallion semen.

P-013 A potential therapeutic approach of intermittent fasting on sperm viscosity

Abstract Study question Does intermittent fasting affect sperm viscosity and motility? Summary answer The obtained results demonstrate that intermittent fasting significantly improves sperm viscosity and motility, and might be considered as a potential therapeutic approach for sperm hyperviscosity. What is known already Numerous factors influence sperm quality, functionality, and, consequently, male fertility. In the last decade, nutritional deficiency and lifestyle, have been grouped as popular factors that affect human sperm function and fertilization processes. Semen hyperviscosity which occurs in 12-29% of ejaculates is another condition that can contribute to male subfertility, as it is associated with changes in the chemical and physical characteristics of semen. Additionally, semen hyperviscosity contributes as well to poor sperm motility. Based on the aforementioned facts, focusing on identifying factors to enhance sperm viscosity and motility is crucial for improving IVF outcomes. Study design, size, duration The present study was based on semen analyses of 60 samples from 30 IVF patients. Following the purpose of the study and inclusion criteria, all 30 patients were diagnosed with abnormal sperm viscosity. Out of this number, 13 patients had asthenozoospermia, 4 patients were diagnosed with asthenoteratozoospermia, 7 patients were diagnosed with teratozoospermia and 6 patients had normozoospermia. All patients followed 4 days of sexual abstinence. All specimens were analyzed within 1 hour of collection. Participants/materials, setting, methods Sperm analysis including sperm viscosity was processed according to the WHO laboratory manual sperm criteria (Sixth edition 2021). For the study purpose, all the participants followed an intermittent fasting period of 14-16 hours for 30 days. Samples were analyzed twice, the first time before fasting and the second time after 30 days of fasting. Most of the patients did not consume food from 6 pm until 8 or 10 am. Main results and the role of chance The average age of the patients included in the study was 37.53 ± 4.6 years, while the BMI ranged from 21.26 to 32.28 kg/m2. Abnormal viscosity (hyperviscosity) was diagnosed when the drop formed a thread more than 2 cm long, while normal liquefied ejaculate fell as small discrete drops. The obtained results indicated that sperm parameters were improved after intermittent fasting. The most significant difference was observed in semen viscosity where out of the 30 patients who were diagnosed with abnormal semen viscosity after 30 days of intermittent fasting only 9 patients still had viscose semen (p&amp;lt;0.0001). Additionally, a significant difference was revealed in sperm motility before and after the fasting period as well(33.73±12.15 vs 44.10 ±11.54; respectively p &amp;lt; 0.001). Among the 17 subjects initially diagnosed with abnormal motility, following the implementation of intermittent fasting, 11 demonstrated a sperm improvement and were diagnosed with normal motility. The difference in sperm viability (52.8± 7.71 vs 61.3±8.37; respectively p &amp;lt; 0.01) and Ph values before and after fasting as well were statistically significant. On the other side, although sperm concentration (Mil/ml) was increased after fasting, the difference was not statistically significant (39.03±19.52 vs 41.53±19.19; respectively p = 0.92). Additionally, intermittent fasting did not affect sperm morphology either. Limitations, reasons for caution The limitation of the presented study was a relatively small patient cohort. Further research should link intermittent fasting with IVF outcomes. Wider implications of the findings Until today there are no clear dietary guidelines created for infertile males. As far as we know this is the first research, that includes a comparison of sperm parameters, based on intermittent fasting, and the obtained results confirm that intermittent fasting can be used as a potential therapeutic approach. Trial registration number not applicable

O-308 Is AI the future of sperm quality assessment? A comparative study of AI-enhanced and conventional semen analysis systems

Abstract Study question How does the accuracy of an artificial intelligence (AI)-enhanced semen analysis system compare to conventional semi-automated methods when assessing semen quality? Summary answer Compared to conventional semi-automated methods for sperm quality assessment, the AI-enhanced system tends to overestimate progressive sperm motility and normal sperm morphology. What is known already Male factor infertility, specifically poor sperm quality, accounts for over 40% of all infertility cases. Accurate semen analysis is thus a critical step in guiding appropriate treatment strategies in male reproductive health. Past decades have witnessed the development of semi-automated systems for semen analysis, overcoming the limitations of traditional microscopy. While these systems offer precise data on kinetics and concentration, manual intervention remains necessary for assessing certain parameters, potentially affecting reproducibility. AI-based systems have been recently introduced into clinical practice, promising more objective and standardized analysis. Yet, the extent to which these methods surpass conventional approaches remains to be established. Study design, size, duration This is a single-centre, prospective, consecutive, paired study, conducted from February to September 2022. The study included 84 sperm samples from patients undergoing medically assisted reproduction. Participants/materials, setting, methods All sperm samples were analyzed using the AI-enhanced automated system (LENSHooke X1 Pro, Bonraybio®, Fertil Ibérica) and conventional methods: CASA semi-automatized device (ISAS, Proiser), Diff-Quik staining for manual morphology assessment, and pH test strips. We measured and compared several semen parameters, including morphology (% normal sperm), concentration (million/mL), motility (% progressive, % non-progressive, % total motility), and pH, using both approaches. Spearman and Kruskal-Wallis tests were used for statistical analyses. Main results and the role of chance We observed significant differences among several semen parameters when comparing the AI-enhanced system and conventional approaches. Mean normal sperm morphology, as assessed by the AI-enhanced automated device, was significantly higher than that evaluated by conventional microscopy (4.62% versus 3.07%, p &amp;lt; 0.00001). We also observed significant differences for motility and pH parameters (57.98 versus 25.66, p = 0.0001 and 7.86 versus 8.38, p = 0.0001, respectively). Correlations between variables assessed using both methods also varied. Sperm concentration showed a strong correlation between the AI-enhanced and conventional systems (Spearmann’s Rho=0.828), however a weak correlation was observed for total and progressive motility, as well as the percent of immotile sperm (69.54% versus 28.98% R2=0.541; 57.98% versus 25.66% R2=0.566, and 30.45% versus 47.12%, R2=0.541, respectively). There was no significant correlation for non-progressive motility between the two methods (6.53% versus 11.60%, R2=0.157). Limitations, reasons for caution This study exclusively compared two analysis methods among numerous AI-enhanced systems available on the market. Given the rapidly evolving landscape of AI technologies, caution is warranted when generalizing these findings to other AI models, as their performance may vary. Wider implications of the findings Observed differences between the AI-enhanced system and conventional methods require careful consideration. While fully automated semen analysis systems are designed to reduce subjectivity, extensive comparative research is crucial. A comprehensive market assessment will be essential for safeguarding patient treatment outcomes and advancing AI capabilities in semen analysis. Trial registration number not applicable

P-254 Optimizing clinical outcomes in failed ICSI Donor cycles: evaluating the efficacy of Assisted Oocyte Activation

Abstract Study question Does Assisted Oocyte Activation (AOA) confer benefits to couples undergoing donor ICSI cycles with a history of poor clinical outcomes and poor sperm quality? Summary answer AOA demonstrates efficacy in enhancing reproductive outcomes in donor ICSI cycles, in cases where oocyte activation deficiency (OAD) is attributed to a sperm factor. What is known already Oocyte activation is a spatial-temporal process caused by a series of intracellular calcium oscillations (Ca2+)triggered by the entry of sperm into the oocyte. Insufficient Ca2+ oscillations, primarily attributed to the absence of sperm-specific phospholipase C zeta (PLCζ) can result in OAD that leads to unsuccessful fertilization and poor embryo growth. Ionomycin and calcimycin, are the most common activating agents used to restart meiosis by raising intercellular Ca2+. As far as our knowledge extends, there aren’t studies reporting clinical outcomes on the use of AOA in patients who undergo cycles both without and with this treatment in donor oocytes. Study design, size, duration This study includes 38 couples undergoing ICSI cycles with donor oocytes from January 2020 to December 2023. After a previous cycle with a fertilization rate of &amp;lt; 50% and poor clinical outcomes, these couples underwent a subsequent attempt with AOA. Maternal age of recipients was 42±4.7 and 42±5 years in cycles without and with AOA (P=NS). Fertilization, blastocyst quality, implantation, clinical pregnancy rates (CPR) and pregnancy loss were compared between the cycles with and without AOA. Participants/materials, setting, methods Oocytes were exposed to a ready-to-use solution of Ca2+-ionophore A23187 (GM508 Cult-Active, Gynemed, Germany) for 15 min after sperm injection. Embryos were cultured in a time-lapse technology (Geri®) andevaluated according to the Gardner blastocyst grading system. Embryo transfer was performed on Day 5 or Day 6. Vitrified and fresh ejaculated sperm samples were used. T-test and Fisher’s Exact test were used for statistical analysis. P-value was considered statistically significant at a threshold of &amp;lt; 0.05. Main results and the role of chance A total of 38 men underwent ICSI cycles alongside their female partners, utilizing donor oocytes. The men’s average semen parameters were as follows: concentration of 15.3±25 x 106/mL, motility of 20.1±15%, and 2.0±1% morphology. In the initial 41 cycles without the AOA procedure, an average and standard deviation of 5.8±3 donor oocytes were injected, resulting in a fertilization rate of 33.9% (81/239) and a blastocyst rate of 30.8% (25/81). Among these cryopreserved embryos, 72% (18/25) were identified as top-quality, characterized by A and/or B quality of both inner cell mass and trophectoderm. Couples (N = 13) who underwent a transfer had an implantation rate of 19% (4/21), with CPR of 28.5% (4/14), which resulted in a pregnancy loss of 75% (3/4). Subsequently, these couples underwent 47 ICSI cycles with AOA, injecting an average of 5±1.7 donor oocytes (P=NS). This led to a significantly higher fertilization rate of 74.2% (176/237, P &amp;lt; 0.0001) and a blastocyst rate of 51.7% (91/176, P &amp;lt; 0.01), with 61% (56/91) identified as top-quality embryos (P=NS). Couples (N = 31) who underwent a transfer had a notable increase in implantation rate to 52% (25/48, P &amp;lt; 0.05), accompanied by a significant rise in CPR, reaching 71.4% (25/35, P &amp;lt; 0.01). Importantly, pregnancy loss decreased to 26% (6/23, P=NS). Limitations, reasons for caution The number of patients included in this study is limited. Although we excluded that OAD could be oocyte-related by using donor gamete, PLCζ or mouse oocyte activation tests were not performed to confirm sperm-related OAD. All patients, diagnosed with teratozoospermia, likely exhibit abnormalities causing PLCζlocalization issues or its absence. Wider implications of the findings In our study, sperm’s inability to generate accurate calcium signaling pathways correlates with poor clinical outcomes, even with donor oocytes. Consequently, for couples facing Oocyte Activation Deficiency attributed to sperm factors, implementing the AOA procedure significantly improves fertilization, blastocyst formation, implantation, and clinical pregnancy rates. Trial registration number na

O-198 The decline in semen quality parameters enhances mosaicism in trophectoderm biopsy of human blastocysts

Abstract Study question Do poor sperm parameters impact chromosomal analysis compared to sperm with normal parameters? Summary answer Fertilization with more than two abnormal patterns, including severe oligoasthenoteratozoospermia sperm, may increase the rate of embryo mosaicism. What is known already Sperm cells constitute the genetic half of the embryo, and the male factor plays a crucial role in embryo chromosome integrity. While PGT-A is widely used to enhance IVF outcomes, the influence of male factors on embryo genetics remains underexplored. We aimed to determine the effect of semen profile on the outcome of PGT-A cycles performed at the blastocyst stage. Study design, size, duration A single-center retrospective study conducted between January 2019 and July 2023 analyzed 609 blastocysts with Next Generation Sequencing (NGS) of trophectoderm (TE) biopsies. Four study groups were formed based on sperm parameters: normozoospermia (N), moderate male factor (MMF), severe oligoasthenoteratozoospermia (s-OAT), obstructive azoospermia (OA). Participants/materials, setting, methods Patients treated with a minimal ovarian stimulation protocol. PGT-A cycles were divided into three paternal age groups. OUTCOMES: Euploidy, aneuploidy, mosaicism rates; blast formation time; good-quality blastocyst rates. METHODS: Multivariate logistic analysis, Chi-squared tests, and STATA 12.1 statistical software. Main results and the role of chance In the 609 TE biopsied blastocysts, euploidy rate was 49.9%, aneuploidy rate was 31.5%, and mosaicism rate was 18.6%. Paternal age ≥43 increased aneuploidy risk compared with &amp;lt;35 (40.1% vs 26.1%, p &amp;lt; 0.05). The euploidy rate and aneuploidy rate were no different in MMF, s-OAT, and OA compared with N (44.8%, 40.6%, and 42.1% vs. 54.2%; 36.8%, 25.0%, and 31.6% vs. 30.5%). Mosaicism rates were significantly difference between sperm parameter groups (N 17.1%, MMF 18.4%, s-OAT 34.4% and OA 26.3%, p &amp;lt; 0.05). In logistic regression analysis adjusted for confounders, severe oligoasthenoteratozoospermia showed 2.5 times higher mosaicism rate (aOR 2.58; 95% CI 1.08-5.08, p = 0.027). The good-quality blastocyst rate was significantly reduced in the OA compared with N, MMF, and s-OAT (21.1% vs 43.4%, 39.2% and 37.5%, p &amp;lt; 0.05). No impact of semen parameters on blastocyst formation time and vitrification time outcomes was observed. Limitations, reasons for caution Retrospective, single-center study. Excludes DNA fragmentation index and TESE results, impacting sperm quality evaluation. Wider implications of the findings Severe oligoasthenoteratozoospermia is positively associated with mosaicism rate. Poor sperm parameters lead to mosaic embryos. Couples undergoing PGT-A for male factors should discuss potential outcomes. Patients with abnormal semen parameters should consider pretreatment for improved semen quality. Results useful for genetic counseling and clinical decision-making. Trial registration number None

O-018 Utilisation of second successive ejaculate as a way to overcome poor semen parameters for couples who wish to undergo conventional-IVF

Abstract Study question Can a second successive-ejaculate (SSE) be an option for patients with poor sperm parameters (PSPs) on ovum pick-up (OPU)-day who prefer to undergo conventional-IVF (c-IVF)? Summary answer SSE can be a valid option for patients with PSPs who do not wish to undergo ICSI as an assisted reproductive technology (ART) treatment. What is known already ART is applied when first-line treatment for infertility (timed intercourse or intrauterine insemination) is unsuccessful. The male partner is requested to follow ejaculatory abstinence, which according to the WHO guidelines is 2-7 days, while ESHRE recommends 3-4 days. Among these couples there are cases that require ICSI due to semen analysis with PSPs on the day of OPU. However, some couples are reluctant to undergo ICSI and prefer c-IVF as a treatment method. For those cases, we request a second semen sample on the same day (time interval: 1-4 h) and c-IVF is performed using the two ejaculates combined. Study design, size, duration This is a single-centre retrospective study employing 212 semen from male partners with PSPs in the first semen collection on the OPU-day and subsequently, an SSE was requested in order to perform c-IVF according to patients’ wishes. The data were collected from May 2006 to October 2023 in our clinic (SSE-group). The Control group included 271 semen samples from men where c-IVF was conducted using one ejaculate from January to December 2021 in our clinic. Participants/materials, setting, methods The sperm parameters were analysed according to the WHO manual for the examination and processing of human semen and compared between the first and second ejaculates in the SSE-group. Fertilisation rate (FR), percentage of clinically utilised embryos and clinical pregnancy rate were compared between the SSE-group and the Control group. The SSE-group had 254 ART-cycles and the mean maternal age was 36.8±4.9y.o. The Control group included 432 ART-cycles and the mean maternal age was 38.1±5.2y.o. Main results and the role of chance In SSE-group the semen volume of the first ejaculate was significantly higher compared to the second ejaculate (P&amp;lt; 0.0001). The sperm concentration and sperm motility were significantly higher in the second ejaculate compared to the first one (P&amp;lt;0.0001). In the same fashion, the total-motile sperm-count was significantly higher in the second ejaculate compared to the first (P=0.01). All the semen parameters in the Control group were significantly higher compared to both first and second ejaculates from the SSE-group (P&amp;lt;0.00001). The normal FR was significantly higher in the Control group compared to the SSE–group (P&amp;lt;0.01). On the other hand, there was no significant difference in the percentage of embryos that could be utilized (cryopreserved + freshly-transferred) between the two groups (P=0.218). Pregnancy rate for fresh embryo transfers (ETs) was significantly higher in the SSE-group compared to the Control (P=0.005), while there was no significant difference between the two groups for pregnancy rate for frozen/thawed ETs (P=0.728). The main strength of our study is that the utilisation of the SSE combined with the first ejaculate did not decrease the pregnancy rate for the couples with PSPs that wished to follow c-IVF as their treatment and it could actually provide an effective option for them. Limitations, reasons for caution SSE may improve the sperm parameters and give the opportunity to infertile couples to undergo c-IVF according to their wish. However, since the FR was lower in the SSE-group, it is of paramount importance to provide sufficient explanation to those couples and obtain their consent when requesting an SSE. Wider implications of the findings Overall, SSE had positive effect to a large number of parameters evaluated in this study. Additionally, the rate of utilised embryos was similar between the two groups. Two successive-ejaculates combined can be employed without any negative impact for couples with PSPs on the OPU-day that are reluctant to undergo ICSI. Trial registration number not applicable

P-096 Seminal microbiome and its possible association with poor sperm quality

Abstract Study question Does seminal microbiome differ between men with good and poor sperm quality parameters? Summary answer Men with poor sperm quality values presented a higher microbial richness, while after rigorous contamination controlling no specific bacteria associated with sperm quality parameters. What is known already Infertility represents a major health concern affecting 1 in every 6 couples. Approximately 50% of infertility cases are attributed to male factor, where seminal quality is crucial. Recent studies have brought to light the role of the microbiome in sperm quality and function. Changes in microbial communities can impact reproductive functions, as these microorganisms may affect spermatozoa by releasing specific molecules or influencing direct adhesion. Since current evidence in the field is inconclusive, as the studies are performed on small sample sizes and lack proper positive and negative controls, there is a need for rigorous studies on bigger cohorts. Study design, size, duration This cross-sectional study included 115 men (age= 33.6±9.6 years; BMI= 25.0±3.5). Semen samples assessment was performed between March 2019 and July 2021 at the sperm biobank and University Hospital. The study was approved by the regional Ethics Committee. Participants/materials, setting, methods The study population was categorized into two seminal quality groups based on WHO reference values. Accordingly, 66 men presented good sperm quality while 49 poor sperm quality. Sperm concentration and progressive motility parameters were assessed in semen. Seminal microbiome was analyzed using 16 rRNA gene sequencing. For rigorous contamination controlling, negative controls together with in silico decontamination (MicroDecon package) were applied. Microbial diversity and composition were assessed including age and BMI as confounders. Main results and the role of chance After rigorous decontamination analysis including negative controls, 774 microorganisms were identified in the semen. Ninety-one bacteria were detected as contaminants. Next, a filter of 30% prevalence was applied to identify the core seminal microbiome, which resulted in a total of 36 bacterial genera in the semen in our cohort of men. Bacteria such as Actinomyces, Finegoldia, Campylobacter, Anaerococcus and Peptoniphilus were the the most prevalent among seminal samples. Men with poor sperm quality presented a richer seminal microbiome compared to good sperm quality group (α-diversity p-value=0.022), while β-diversity did not show a significant microbial dissimilarity between groups (R2=0.012, p-value&amp;gt;0.05). Differential abundance analysis on the core microbiome detected increased abundances of Veillonella (logFC=1.836), Gemella (logFC=1.612) and a member of Pasteurellaceae family (logFC=1.628) in men with lower sperm quality (all p-values&amp;lt;0.05). However, differences did not remain significant after multiple comparison correction (FDR p-values&amp;gt;0.05). Limitations, reasons for caution This study reported specific microbial alterations in men with poor sperm quality parameters, which should be investigated further considering different life-style factors such as smoking and diet. Wider implications of the findings With our rigorous study protocol, we see that although seminal microbiome could have some potential implications on seminal quality, the core microbiome does not seem to associate with seminal parameters. More research is needed, but it could be there has been previously overinterpretation of the microbiome associations with seminal parameters. Trial registration number not applicable

P-016 Prevalence of high oxidative stress and poor sperm DNA fragmentation levels in different groups of patients

Abstract Study question What is the prevalence of patients with high oxidation-reduction potential (ORP) and high sperm DNA fragmentation (SDF) in different patient groups with and without varicocele? Summary answer A large percentage of normozoospermic patients and patients who are normally considered of manageable in assisted reproduction show high SDF and/or high ORP. What is known already Standard semen analysis, even according to WHO criteria, is very limited in predicting male fertility potential. Oxidative stress is widely recognized as a major contributor to male infertility. Therefore, sperm DNA fragmentation (SDF) is increasingly recommended as additional test in diagnostics. High levels of oxidative stress and SDF have also been reported in patients with varicocele. Yet, SDF and oxidative stress tests are not routinely tested in ART laboratories. Apart from the cost and/or complexity of these tests, a reason for the lack of usage is the lack of knowledge of the prevalence. Study design, size, duration This is a retrospective analysis of data obtained from 2,946 patients attending the Department of Urology, Hamad Medical Corporation, Doha, Qatar, for fertility diagnosis from January to December 2018. For this analysis only patients (n = 773) who had a full data set for sperm DNA fragmentation (SDF) and seminal oxidative stress as measured by means of the oxidation-reduction potential (ORP) were included. Participants/materials, setting, methods Apart from SDF and ORP, standard semen parameters (ejaculate volume, sperm concentration, total and progressive motility, and normal sperm morphology), and the presence of varicocele were determined. Semen parameters were determined according to WHO 2010 criteria. SDF was tested using the Halosperm G2 test (Halotech DNA SL, Madrid, Spain), while ORP was determined using the MiOXSYS® (Aytu BioScience, Englewood, CO). Patients were categorized as normo-, oligo-, astheno-, and teratozoospermic. Main results and the role of chance The mean sperm concentration of the patients was 33.5 × 106 sperm/mL with a mean total motility of 50.3%. The overall percentage of patients with high (&amp;gt;20% SDF) SDF was 57.4% and those with high (≥1.34 mV/106 sperm/mL) ORP 63.4%. Varicocele was diagnosed in 14.0% of the patients. In normozoospermic patients, 26.8% had high SDF, whereas in the non-normozoospermic group this was 59.8% (P = 0.0108). In non-oligozoospermic (53.7%), non-asthenozoospermic (34.7%) and non-teratozoospermic (43.4%) groups, SDF was significantly higher. High ORP values were recorded in 35.7% of normozoospermic, 53.4% non-oligozoospermic, 47.2% of non-asthenozoospermic, and 49.7% of teratozoospermic patients. The difference between the prevalence of high ORP in the normozoospermic (35.7%) and the non-normozoospermic group is significant (P = 0.0063). When both tests (high SDF or high ORP) were considered, 50.0% of normozoospermic, 76.9% of non-oligozoospermic, 59.7% of non-asthenozoospermic, and 71.2% of non-teratozoospermic patients showed seminal pathology. High SDF was detected in 59.3% of varicocele patients, while 50.0% of varicocele patients had high ORP values. In patients without varicocele, 57.1%, 65.6%, and 82.7% had high SDF, high ORP, and high SDF or high ORP, respectively. These percentages were significantly (P &amp;lt; 0.0003) higher than for the corresponding normal groups. Limitations, reasons for caution Although the overall sample size of 773 is reasonably high, after sub-grouping the sample size is much smaller. Since this study only analyzed the SDF and ORP in patients attending and andrological outpatient clinic, the results could be different if patients in IVF and/or ICSI treatment cycles were analyzed. Wider implications of the findings This study highlights that complementation of semen analysis with SDF and oxidative stress testing will identify a large percentage of patients who would benefit from andrological treatment before assisted reproductive techniques (ART). This approach would result in better chances of these patients for successful ART. Trial registration number not applicable