Abnormal Sperm Research Articles

Enhancing cryopreserved ram sperm quality at-80 °C with Spirulina platensis and Salvia verbenaca extracts.

This study was conducted in two steps to evaluate the influence of freezing methods and natural extracts on cryopreserved ram sperm quality. Initially, the research compared the effects of two freezing methods: liquid nitrogen (LN2) versus -80 °C, on post-thawed ram semen on total and progressive motilities and velocity parameters. Experiment I revealed no significant differences (P > 0.05) between the LN2 and -80 °C freezing methods, indicating similar effects on the analyzed parameters. Experiment II aimed to examine the influence of Spirulina platensis (SP) and Salvia verbenaca (SV) extracts added to egg yolk extender on cryopreserved sperm quality, utilizing the -80 °C freezing method. Various concentrations (1.25, 3.75, 6.25 and 8.75 µg*mL-1) of acetone (Ac-SP and Ac-SV) and hexanoic (Hex-SP), as well as methanolic (MeOH-SV) extracts, were added into the extender. A thorough assessment of post-thawed sperm quality parameters, encompassing motility, velocity parameters, viability, membrane integrity, abnormality and lipid peroxidation was conducted. The outcomes demonstrated that 1.25 and 3.75 g*mL-1 of Ac-SP and Hex-SP and 1.25 µg*mL-1 of AC-SV and MeOH-SV increased the post-thawed ram sperm quality. In conclusion, this study emphasizes the antioxidant properties of SP and SV extracts, highlighting their potential to protect cryopreserved sperm cells from oxidative stress at -80 °C.

P-039 Parthenogenetic chimeras may be the cause of the development of a case of 46, XX/46, XY chimeras complicated by severe oligozoospermia

Abstract Study question How did the 46,XX/46,XY chimeric case of severe oligozoospermia arise? Summary answer DNA analysis suggested the possibility of monogenetic chimerism as a mechanism for the development of this case. What is known already Sex-chromosome discordant chimerism in humans(XX/XY chimerism)is a rare chromosomal abnormality. Although the first case was described in 1962, its incidence is still unknown. The XX/XY chimera manifests variable genital phenotypes. The XX/XY chimera is classified into subtypes; tetragametic chimeras, parthenogenetic chimeras, androgenetic chimeras and sesquizygotic twinning chimeras. Genotyping of XX/XY chimeras is important no only to clarify its developmental mechanism but also for diagnosis and treatment. As a result, patients occasionally present with infertility. We present a case of a parthenogenetic chimera with a karyotype of XX/XY in relation to infertility. Study design, size, duration case research. We researched from July 2023 to December 2023. Participants/materials, setting, methods A 38-year-old man diagnosed with azoospermia by his previous physician presented to our clinic with primary infertility for 1 year and 6 months. At 28 years old, he presented with left testicular inflammation and subsequent atrophy. He has no sexual problems. He has an older sister and a younger sister, both married with children. Main results and the role of chance Physical findings: BH 163 cm, BW 53 kg, BMI 19.95, external genitalia: male. Semen analysis: severe oligozoospermia: mostly immobile sperm with some sperm moving slightly in situ. Morphology (Kurger strict criteria): no abnormal sperm morphology. Left testicular hypoplasia: USG: 12 ml right testis, 1 ml left, no other problems. Abdominal MRI: no abnormalities in the kidney to the bladder. No uterus or ovaries. Prostate and seminal vesicles normal; AMH 3.34ng/ml (0.77-14.5) Testosterone 6.55ng/ml (1.95-7.63), DHEA-S 199 μg/dl(106-464),LH 6.2, FSH 7.5, E2 32.5, PRL 12.43, blood type A, Rh+, RH-Hr:CcDee, HLA-A, B, C, DR: all undetermined, Y chromosome SRY (FISH): SRY signal numbers 0 and 1 were observed. Chromosome analysis (G band): 46, XX[22]/46, XY[8]. SNP microarray chromosome examination: presumptive parthenogenic chimerism Treatment: His wife underwent oocyte pick up and intracytoplasmic sperm injection(ICSI) was performed using his frozen sperm. One blastocyst was frozen, but one frozen-thawed blastocyst transfer during an HRT cycle failed to result in pregnancy. The use of assisted reproductive technology(ART) is currently ongoing. Limitations, reasons for caution DNA analysis of both parents was necessary to determine the cause of the problem, but the patient refused to have her parents tested. Wider implications of the findings By accumulating and studying a large number of cases like this one, it may be possible to correctly treat patients with 46, XX/46, XY chimeric infertility. Trial registration number not applicable

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<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 < 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 < 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

P-124 Machine learning scores reveal mosaic embryo quality as the key predictor for live births, outperforming mosaicism characteristics in ART success

Abstract Study question Is it possible to score the mosaic embryos in order to prioritise them according to their pregnancy potencial? Summary answer The machine-learning score underscores the crucial role of embryo quality over mosaicism, prioritizing high-quality, day-5 biopsied embryos for enhanced live birth outcomes in ART. What is known already After embryo biopsy and PGT-A analysis, embryos can be classified as euploid, aneuploid, or mosaic, wherein a combination of chromosomally normal and altered cells is detected. The transfer of mosaic embryos has been controversial, but currently, the recommendations of international scientific societies, including ESHRE, advocate for their transfer. In case of pregnancy, a prenatal test is advised to rule out any chromosomal aberration. The non-transfer of these embryos reduces ART success chances, as chromosomally normal children may be born from them. Study design, size, duration The study design is observational and retrospective. A total of 8346 embryos from 2583 PGT-A cycles were considered (January-2017 to January-2023). Only transferred mosaic embryos (n = 263) were included in the study. The trophectoderm biopsies of D5, D6, or D7 blastocysts were analyzed by NGS using the Illumina platform (VeriSeq Illumina®, San Diego, CA, USA). The biopsied embryos were vitrified and transferred in a subsequent cycle. Participants/materials, setting, methods The primary indications for PGT-A included advanced maternal age, abnormal sperm FISH, and recurrent miscarriage or implantation failure. Clinical outcomes, along with variables related to progenitors, embryos, biopsy, ovarian stimulation, and adjuvant treatments, were recorded in a database. Machine learning models were conducted using R statistical software (v.4.3.1), and the ‘Caret’ library was utilized to implement different algorithms. ‘Autoscore,’ as an interpretable scoring tool, combined machine learning and regression modeling. Main results and the role of chance The mean maternal and paternal ages were 34.95±6.38 and 39.13±6.74 years. Biopsies were predominantly performed on day 5 (61%). Additionally, 44% of embryos were of A quality, and 49% were of B quality. Regarding clinical outcomes for mosaic embryos, biochemical, clinical pregnancy, and live birth rates were 51%, 41%, and 36%, respectively. The features used to generate the score included those related to embryo quality, biopsy, and mosaicism. Through 10-fold cross-validation, the study identified top-ranking predictors maximizing the AUC of the predictive model. The highest weight in the score was assigned to embryo quality (42%) and day of biopsy (34%), with lower weights for factors related to mosaicism: monosomy and/or trisomy (18%) and mosaicism level (6%). The machine-learning score indicates that, in transferring embryos with mosaicism, priority should be given to high-quality embryos biopsied on day 5. The type and level of mosaicism have a minor impact on the embryo’s gestational capacity, becoming relevant only when prioritizing embryos of identical quality. In such cases, embryos with lower mosaicism degrees and no monosomy demonstrate higher implantation potential. Limitations, reasons for caution The inherent limitations of a retrospective analysis highlight the need for prospective randomized studies to confirm the validity and usefulness of the mosaic embryo transfer score. Wider implications of the findings A scoring system has been developed to prioritize the transfer of mosaic embryos. The crucial variables are associated with embryo quality and the day of biopsy, reflecting the progression of embryonic development. Conversely, variables related to mosaicism play a secondary role. Trial registration number Not applicable

P-092 PGT-A Evaluates Advanced Sperm Selection in Embryos from High Teratozoospermia Males; a prospective randomized controlled trial

Abstract Study question Can sperm selection techniques improve the reproductive outcomes of ICSI patients, particularly in males with high teratozoospermia index (TZI) and sperm DNA fragmentation (SDF)? Summary answer Sperm selection techniques significantly increase fertilization and blastocyst development rates in males with high TZI and SDF, achieving a euploidy rate comparable to normal cases. What is known already Abnormal sperm morphology negative effects begin at the fertilization and reach the blastocyst quality and euploidy status. Men with abnormal teratozoospermic index (TZI) exhibit high levels of chromosomal abnormalities and sperm DNA fragmentation (SDF). SDF adversely affects pre and post-implantation embryo development events. Blastocyst aneuploidy isn’t solely due to the oocyte; but also, sperm contribution should be taken into consideration. Previous studies reported that advanced sperm selection techniques (PICSI and MACS) can efficiently enhance reproductive outcomes in abnormal SDF ICSI cases. The efficiency of these techniques in improving ICSI outcomes of patients with high TZI plus SDF remains unexplored. Study design, size, duration A prospective randomized controlled trial included a total of 264 couples, divided into three groups: (1) Control-1 group: males having normal TZI and no sperm selection techniques were used (n = 111), (2) Control-2 group (NO-SS): males having high TZI and no sperm selection techniques were used (n = 63), and (3) Sperm selection (SS) group: males having high TZI and SDF, and sperm selection techniques were used (n = 90). The study started from January till October 2023. Participants/materials, setting, methods Eligibility: females had ≥5MII oocytes, males had abnormal (>1.8) TZI, and all blastocysts (≥4BB) had trophoectoderm biopsy. Cases were randomized on the ICSI day to (control-2) and (SS) groups, along with (control-1) with normal TZI for comparison. Females were categorized into 2 subgroups (<35, and ≥35 years). SS techniques included physiological ICSI and magnetic-activated cell sorting. PGT-A was done using Illumina platform (Miseq). Statistical analysis (SPSS) of pre-implantation embryo development and PGT-A results was done. Main results and the role of chance We found no significant differences between the female, male ages and number of mature oocytes between the studied groups. Limitations, reasons for caution The results included all pre-implantation embryo parameters but, subsequent post-implantation events like implantation, pregnancy, and miscarriage rates will add more data about the competency of the resulting embryos. A Small sample size of females aged from 36 to 40 years old. Wider implications of the findings Sperm selection techniques appear to be efficient in improving fertilization, blastocyst development rates, and euploidy rates in males with high TZI and sperm DNA fragmentation, suggesting potential benefits for couples undergoing infertility treatment. Trial registration number NCT06231589

O-195 Decreased embryo developmental potential and lower cumulative pregnancy rate in males with multiple morphological abnormalities of the sperm flagella

Abstract Study question Is there any association between the multiple morphological abnormalities of the sperm flagella (MMAF) patients and the comprehensive outcomes of assisted reproductive technology (ART) cycles? Summary answer Patients with MMAF presented compromised ART outcomes, including lower embryo developmental potential and impaired clinical outcomes, compared with the cohort of oligoasthenospermia. What is known already Several studies have reported a positive intracytoplasmic sperm injection (ICSI) outcome in the MMAF cohort, and others have opposite opinions. The association between MMAF and ART outcomes is still controversial and open for debate. Furthermore, the neonatal outcomes and the information regarding the mutations of MMAF patients were incomplete. Study design, size, duration A retrospective cohort study of MMAF patients was performed at a reproductive medicine center in China between January 2014 and July 2022. Participants/materials, setting, methods Thirty-eight MMAF individuals were recruited based on a typical MMAF phenotype (abnormal sperm flagella, including short, absent, coiled, bent, and/or irregular flagella) and 131 couples with male infertility undergoing in vitro fertilization (IVF)/ICSI treatments over the same period were included as the control pool. Whole exome sequencing (WES) was performed to confirm the genetic pathogenesis of MMAF. Main results and the role of chance Pathogenic variants in known genes of DNAH1, DNAH11, CFAP43, FSIP2, and SPEF2 were identified in MMAF patients. The ART outcomes, including the fertilization rate, 2PN cleavage rate, blastocyst formation rate, and available blastocyte rate, were all significantly lower in the MMAF group than those in the control group (P<0.05). Moreover, the CLPR in the MMAF group was lower than that in the control group according to the embryo transfer times (68.6% vs. 86.5%, P=0.033) and complete cycles (66.7% vs. 84.2%, P=0.020), while no significant differences were found in the neonatal outcomes. Limitations, reasons for caution The sample size was limited due to the low incidence of MMAF and WES was not performed in all MMAF patients. Further exploration of the potential genetic mutations or underlying mechanisms in patients without identified mutations was needed. Moreover, long-term follow-up of neonates needs to be addressed in the future. Wider implications of the findings The current study identified the causative genetic mutations which were partly obligated to the pathogenesis of MMAF and presented the relationship between MMAF and compromised ART outcomes. These results may provide evidence support for clinicians in genetic counseling and clinical instruction in specific MMAF patients. Trial registration number the National Key Research & Development Program of China (2021YFC2700603)

P-510 The impact of the severe male factor in the egg donation cycles using preimplantation genetic test for aneuploidy

Abstract Study question Does severe male factor affect the euploidy rate and IVF outcomes in donor egg ICSI cycles using PGT-A? Summary answer Severe male factor affects the euploidy rate in egg donation cycles, however, no statistical differences were reported in live birth and cumulative live birth rate. What is known already Around 50% of couples seeking fertility treatment have male-factor infertility. Intracytoplasmic sperm injection (ICSI) relies on evaluating sperm morphology, but the direct relationship between semen quality and embryonic potential remains unclear. Evidence indicates that poor semen characteristics correlate with an increase in aneuploidy rate of spermatozoa. Therefore, pregenetic testing for aneuploidies (PGT-A) is recommended in cases of male factor infertility. The relationship between aneuploidy rates and abnormal sperm remains a debated topic. The use of the egg donation model offers a valuable chance to isolate the male-related factor and evaluate its influence on the occurrence of embryonic aneuploidies. Study design, size, duration This is a retrospective, observational, cohort study including all European IVI clinics. The study included egg donation cycles using pre-implantation genetic testing for aneuploidies (PGT-A) generated in IVI clinics from January 2017 until December 2023. 690 egg donation cycles were included in the study, N = 202 in the group with sperm < 5mil/ml, N = 102 in the sperm group with 5-15 mil/ml and N = 386 in the group with sperm >15 mil/ml. Participants/materials, setting, methods The study included women between 30-49 years undergoing egg-donor cycles, using fresh/frozen sperm Sperm samples were derived after 2-5 days of abstinence. The cycles of egg donation were divided into three groups according to sperm concentration, severe oligozoospermia (SO, < 5mil/ml), moderate oligozoospermia (MO, between 5-15 mil/ml), and control group(>15 mil/ml). Trophectoderm biopsy was performed on day 5 or 6 and subsequent analysis used the next sequencing generation (NGS). Single euploid blastocyst transfers were included. Main results and the role of chance The fertilization rate was decreased in SO (73.33 ± 16.19) than in the MO group (79.07 ±12.45) and control group (77.80 ±14.72) p = 0.0047. The number of blastocysts was higher in the control (5.80 ± 2.72) and MO (5.97± 2.78) than in the SO group (4.99 ± 2.40), p = <0.001. The number of euploid blastocysts was decreased in SO (3.21 ± 2.07) than in the control (4.09 ± 2.41) and MO group (4.05 ± 2.15), p = <0.001. The euploidy rate of SO decreased (63.32 ±26.39) compared to the control (69.67 ± 25.08) and MO group (68.55 ± 20.37), p = 0.012. The logistic regression model included possible confounders (male age, age donor, male BMI, sperm motility, sperm morphology and sperm concentration) that showed no effect on the euploidy rate. The pregnancy rate was similar across the groups 69.77% vs 66.05% vs 66.23%, p = 0.54. No statistical differences were reported for the clinical pregnancy rate 62.79% vs 61.73% vs 58.40%, p = 0.44, and the live birth rate 62.79% vs 61.73% vs 58.40%, p = 0.44. 48.05 vs 46.38% vs 42.64%, p = 0.37. No differences were reported in the miscarriage rate (p = 0.37) and CLBR (p = 0.26). Limitations, reasons for caution The retrospective nature of the study and the sample size represent the principal limitations of the study. The clinical reason for severe male factors was not considered in the study. Wider implications of the findings SO affects the quality affect the early embryonic potential through an increase in aneuploidy, despite no clinical effects.The use of PGT-A in case of severe male factor remains not justified in the egg donation.Future prospective multi-center studies are needed to highlight the effect of sperm quality on early embryonic potential. Trial registration number not applicable

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 < 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 < 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 < 0.001), and was also declined in the NOA group with the poorest spermatogenic function (62.4% vs. 72.9%, P < 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-084 Exome sequencing reveals the novel genetic mutations associated with teratospermia

Abstract Study question This research aims to explore the molecular mechanisms of genetic variations in the small acrosome sperm subtype within teratospermia and its correlation with male infertility. Summary answer Novel gene mutations associated with teratospermia were identified, providing important insights into the genetic mechanisms underlying this condition. What is known already Male infertility is a substantial challenge in reproductive medicine, impacting a significant proportion of couples globally. Teratospermia, marked by abnormal sperm morphology, is a major factor in male reproductive disorders. Unraveling the genetic foundation of teratospermia is essential for comprehending the intricate molecular mechanisms governing male fertility. Study design, size, duration This study uses exome sequencing to investigate the genetic landscape of male infertility, particularly focusing on teratospermia. Through a comprehensive examination of the exome, our goal is to uncover novel gene mutations and understand the molecular mechanisms connecting genetic changes to abnormal sperm morphology and, consequently, male infertility. Participants/materials, setting, methods Sperm DNA was extracted from 55 patients with a confirmed diagnosis of teratospermia for this study. Subsequently, comprehensive exome sequencing was performed on the extracted sperm DNA to investigate potential genetic variations associated with teratospermia.Further functional studies were conducted to unveil the potential roles of mutated genes in the development of the reproductive system and spermogenesis. Main results and the role of chance Within the known genes associated with spermatogenic dysfunction, we identified one pathogenic/likely pathogenic variant and four variants of unknown significance, all of which have not been previously reported.Cell phenotype experiments revealed that mutations in CCDC62 and KCNU1 have specific effects on the proliferation and apoptosis of GC-2 cells. Limitations, reasons for caution Further molecular experiments and pathway mechanism studies are required to enhance our understanding of the observed effects and provide a more comprehensive interpretation of the findings. Wider implications of the findings This study unveils novel genes and pathways in teratospermia, informing diagnosis, prognosis, and potential treatments for male infertility. Exploring the genetic landscape enhances our comprehension of the complex interplay between genetics and male fertility, guiding personalized approaches in reproductive medicine. Trial registration number not applicable

P-527 Low sperm motility and elevated sperm DNA fragmentation index (DFI) are associated with increased rates of embryo aneuploidy in patients with advanced reproductive age

Abstract Study question Is there a correlation between the paternal age, sperm characteristics, and the prevalence of chromosomal abnormalities in embryos generated through in vitro fertilization (IVF)? Summary answer A correlation exists between advanced paternal age, suboptimal sperm parameters, and elevated rates of embryo aneuploidy in individuals of advanced reproductive age. What is known already The exact mechanisms underlying the relationship between paternal age, sperm quality and embryo ploidy, although a subject of considerable research in assisted reproduction, are not fully elucidated. Some reports, including our group’s, suggest that paternal age may influence sperm quality and segmental embryo aneuploidy, while others suggest a potential association between high DFI(DNA Fragmentation Index) and an increased likelihood of embryo aneuploidy. Studies also show that sperm with optimal motility and fertilization competence are more likely to contribute to the development of euploid embryos, while data on the effect of abnormal sperm parameters on embryo ploidy is scarce and conflicting. Study design, size, duration This was a retrospective cohort study performed in the Reproductive Genetics Department, CReATe Fertility Centre, Toronto, Canada, between January 2019-December 2023. A total of 7628 blastocysts from 1512 cycles (1392 patients) were analyzed using high resolution NGS PGT-A. Sperm motility, concentration and DFI data, as well as, IVF cycle parameter data were collected. In addition, age of gamete providers and any relevant clinical, laboratory and demographic data were obtained for statistical analysis. Participants/materials, setting, methods Embryos were allocated into two groups by oocyte-provider age: OP-≤33 years and OP-≥34 years. Each group was divided into two sub-groups by paternal age: SP-≤39 years (n = 4570); SP-≥40 years (n = 3058). Multiple logistic/ordinal regressions were conducted to evaluate Segmental abnormalities and Segmental mosaicism between these age groups in relation to sperm DFI (GOOD ≤15%; FAIR 15-30%; POOR ≥30%), Motility (HIGH >40%; LOW <40%) and Count (LOW <5 mil/ml; MEDIUM 5-15 mil/ml; HIGH >15 mil/ml). Main results and the role of chance PGT-A analysis of 7628 embryos showed 51.5% euploidy, 36.7% aneuploidy and 11.8% mosaicism. In the OP-≤33 group, there were no significant differences between the two paternal age groups for euploidy, aneuploidy or mosaicism rates. In the OP-≥34 group, decreasing euploidy rates (p < 0.0001 and increasing aneuploidy rates (p < 0.0001) were observed between the two paternal age groups, while mosaicism rates were not different, an expected oocyte age-effect result. No significant differences in aneuploidy rates were observed for each of the sperm parameters (DFI, motility, count) in the groups of the OP-≤33 with SP-≤39, OP-≤33 with SP-≥40 and OP-≥34 with SP-≤39. In the group of OP-≥34 with SP-≥40, in patients with LOW motility vs. HIGH motility the aneuploidy rates were higher (56.8% vs.50.6%, p = 0.005) and euploidy rates were lower (33.8% vs.38.4%, p = 0.03). Each sperm parameter in each age group was further analyzed for chromosomal segmental abnormalities and segmental mosaicism. Higher rates of segmental abnormalities were seen in LOW motility groups with POOR-DFI compared to GOOD DFI in OP-≤33 with SP-≤39 (3.2% vs. 7.4%, p = 0.03). Limitations, reasons for caution The main limitation was the retrospective nature of this study; however, multiple regression analyses were conducted to limit potential bias by adjusting for sperm and oocyte characteristics. In addition, PGT-A analysis cannot provide information on origin of the aneuploidy and all conclusions are deduced from indirect associations to paternal age. Wider implications of the findings The identified associations could stimulate further research into exploring the molecular/cellular processes involved in fertilization, early embryonic development, and the potential impact of sperm-related factors on the chromosomal stability of developing embryos. The broader implications of these findings extend to clinical practice, patient counseling, technological advancements, and ethical considerations. Trial registration number not applicable

Association of urinary metal element with semen quality: a cross-sectional study from Eastern China.

The effect of metallic elements on semen quality remains controversial, with limited evidence on the effects of metal mixtures. We conducted a study involving 338 participants from multiple centers in Eastern China, measuring 17 urinary metals and semen quality parameters. Our analysis used various statistical models, including multivariate logistic and linear regression, Bayesian Kernel Machine Regression, and weighted quantile sum models, to examine the associations between metal levels and semen quality. Logistic regression showed that higher urinary lead was associated with increased risk of abnormal sperm concentration (OR = 1.86, p = 0.021), arsenic to higher abnormal progressive motility risk (OR = 1.49, p = 0.027), and antimony to greater abnormal total motility risk (OR = 1.37, p = 0.018). Conversely, tin was negatively correlated with the risk of abnormal progressive motility (OR = 0.76, p = 0.012) and total motility (OR = 0.74, p = 0.003), respectively. Moreover, the linear models showed an inverse association between barium and sperm count, even after adjusting for other metals (β = - 0.32, p < 0.001). Additionally, the WQS models showed that the metal mixture may increase the risk of abnormal total motility (βWQS = 0.55, p = 0.046). In conclusion, semen quality may be adversely affected by exposure to metals such as arsenic, barium, lead, and antimony. The combined effect of the metal mixture appears to be particularly impaired total motility.

P-090 Is preimplantation genetic testing for aneuploidy (PGT-A) necessary in the treatment strategy for ICSI with testicular sperm?

Abstract Study question Does the combination of testicular sperm extraction (TESE) and ICSI increase the risk of embryo aneuploidy? Summary answer Embryo aneuploidy occurrence is not increased when using testicular sperm and is not affected by the etiology of azoospermia. What is known already TESE-ICSI, especially in patients with non-obstructive azoospermia (NOA), has been reported to result in poor outcomes and difficulty in obtaining good blastocysts, which may be due to immature sperm retrieved by TESE. In addition, patients who require TESE have been shown to be at particularly high risk of chromosomally abnormal sperm. These factors may increase the risk of embryo aneuploidy in TESE-ICSI and contribute to poor outcomes. However, few reports have investigated the results of PGT-A following TESE-ICSI. This study was therefore designed to investigate whether using testicular sperm is more likely to result in embryo aneuploidy. Study design, size, duration This retrospective study included 81 couples and 197 TESE-ICSI cycles undergoing PGT-A (average maternal age: 38.1±5.4 years) between September 2016 and November 2023. Of the total cycles, 94 cycles were case of obstructive azoospermia (OA) and 53 cycles were cases of NOA. The etiologies of NOA included were unexplained (22 cycles), Klinefelter’s syndrome (KS) (10 cycles), post-chemotherapy (3 cycles), post-orchiopexy (11 cycles), and microdeletion of azoospermia factor (AZF) c on the Y chromosome (7 cycles). Participants/materials, setting, methods ICSI outcomes were compared between different etiologies (OA vs. NOA). A total of 356 blastocysts acquired were analyzed using PGT-A with next-generation sequencing (NGS). The PGT-A results were classified according to maternal age (&amp;lt;38, 38-41, &amp;gt;41 years) and paternal age (&amp;lt;40, ≥40 years). In addition, TESE-ICSI cycles were limited to the maternal age of &amp;lt; 38 years to remove the effect of maternal factors, the euploidy rates were compared between different etiologies or morphological grade. Main results and the role of chance The rates of fertilization and blastocyst formation for TESE-ICSI were significantly lower in NOA than OA (47.4% vs. 61.6%, P &amp;lt; 0.001 and 37.3% vs. 50.7%, P &amp;lt; 0.01, respectively). The rate of good-quality blastocyst (16.6% vs. 21.2%) was also lower, but not statistically significant. The euploidy rate of all evaluated TESE-ICSI embryos decreased with increasing maternal age (52.9% in &amp;lt; 38, 29.3% in 38-41, 10.0% in &amp;gt; 41 years), but was not affected by the paternal age (40.6% in &amp;lt; 40, 42.5% in ≥ 40 years). For cycles with the maternal age of &amp;lt; 38 years, the euploidy rate was not significantly different between OA and NOA patients (55.3% vs. 49.3%). In addition, the association between embryo euploidy and morphological grade (using Gardner’s classification, ≥BB for good-quality vs. &amp;lt;BB for poor-quality embryos) was evaluated, and the euploidy rate was significantly higher in good-quality embryos compared to poor-quality embryos (60.3% vs. 33.3%, P &amp;lt; 0.01). These results indicate the frequency of embryo aneuploidy in TESE-ICSI is comparable to commonly used ejaculated sperm. Furthermore, it suggests that embryo aneuploidy is dependent on maternal age or morphological grade of embryo, but not on paternal age or the etiology of azoospermia. Limitations, reasons for caution Patients with chromosomal abnormalities, i.e. KS, could not be evaluated due to the small number of cases and patients with chromosomal translocation were not included in this study. Therefore, the effects of male factors in NOA on embryo aneuploidy require extended investigation. Wider implications of the findings In TESE-ICSI, especially in the case of NOA, it is difficult to obtain good quality embryos due to poor ICSI outcomes. However, if good quality embryos are obtained, the euploidy rate is high and there is no need to perform PGT-A on the sole basis of using testicular sperm. Trial registration number not applicable

Health risk and germ cell toxicity of five commercially available sachet waters in Nigeria: a public health concern.

Sachet water is the most common form of portable water commercially available in Nigeria. Using the murine sperm count and sperm abnormality assay, the germ cell toxicity of five common commercially available sachet waters in Nigeria was assessed in this study. The levels of hormones such as Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH) and Total Testosterone (TT); and activities of catalase (CAT), alanine aminotransferase (ALT), superoxide dismutase (SOD), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) were evaluated. The heavy metal and physicochemical parameters of the sachet waters were also analyzed. Healthy male mice were allowed to freely drink the sachet waters for 35days after which they were sacrificed. The findings indicated that the concentrations of some heavy metals (As, Cr, and Cd) in the sachet waters exceeded the limit by regulatory organizations. The data of the total carcinogenic risk (TCR) and total non-carcinogenic risk (THQ) of some heavy metals associated with the ingestion of sachet water for adults and children showed that the values exceeded the acceptable threshold, and thus, is indicative of a high non-carcinogenic and carcinogenic risks. The data of the sperm abnormality assay showed that in the exposed mice, the five sachet waters induced a statistically significant (P<0.05) increase in abnormal sperm cells and a significantly lower mean sperm count. Additionally noted were changes in the serum activities of TT, FSH, ALP, AST, ALT, and LH. Thus, the sachet waters studied contained agents that can induce reproductive toxicity in exposed humans. This is of public health importance and calls for immediate action by regulatory bodies.

Clusterin expression and distribution in spermatozoa as predictor of male fertility.

Clusterin (CLU), one of the main glycoproteins in mammalian semen and the male reproductive tract, plays a role in spermatogenesis and sperm maturation. Given the poor reliability of classic seminal studies in determining male-fertilizing capacity and the differences in CLU abundance between normal and abnormal spermatozoa, we investigated the potential value of mRNA-CLU levels and protein distribution in spermatozoa as markers of sperm quality and predictors of male fertility. This multicenter study included 90 patients undergoing in vitro fertilization (IVF) treatment with their partners, and a control group of 36 fertile males with normal seminograms. We assessed the relationship between IVF treatment outcomes, seminogram variables, mRNA-CLU levels byquantitative real-time-PCR and CLU distribution by immunostaining in spermatozoa. Our study reveals CLU staining in the acrosome (p = 0.002, OR 14.8, 95% CI: 2.7-79.3) and mRNA-CLU levels (p = 0.005, OR 10.85, 95% CI: 2.0-57.4) as independent risk factors for pregnancy failure, irrespective of traditional seminogram variables. Additionally, our results suggest that CLU, and specially its secreted isoform, constitutes a component of the protein pool that human spermatozoa can produce during its maturation process, exhibiting a variable abundance and distribution in spermatozoa from fertile men compared to those in patients with altered seminograms and infertile patients with normal seminograms. Our study is the first to identify mRNA-CLU levels and CLU immunostaining in the spermatozoa acrosome as independent risk factors for pregnancy failure, with distribution patterns correlating with sperm maturity and seminogram alterations.

Characterizing the consistency of motion of spermatozoa through nanoscale motion tracing

To demonstrate nanoscale motion tracing of spermatozoa and present analysis of the motion traces to characterize the consistency of motion of spermatozoa as a complement to progressive motility analysis. Anonymized sperm samples were videographed under a quantitative phase microscope, followed by generating and analyzing superresolution motion traces of individual spermatozoa. Not applicable. Centrifuged human sperm samples. Not applicable. Precision of motion trace of individual sperms, presence of a helical pattern in the motion trace, mean and standard deviations of helical periods and radii of sperm motion traces, speed of progression. Spatially sensitive quantitative phase imaging with a superresolution computational technique MUltiple SIgnal Classification ALgorithm allowed achieving motion precision of 340 nm using ×10, 0.25 numerical aperture lens whereas the diffraction-limited resolution at this setting was 1,320 nm. The motion traces thus derived facilitated new kinematic features of sperm, namely the statistics of helix period and radii per sperm. Through the analysis, 47 sperms with a speed >25 μm/s were randomly selected from the same healthy donor semen sample, it is seen that the kinematic features did not correlate with the speed of the sperms. In addition, it is noted that spermatozoa may experience changes in the periodicity and radius of the helical path over time. Further, some very fast sperms (e.g., >70 μm/s) may demonstrate irregular motion and need further investigation. Presented computational analysis can be used directly for sperm samples from both fertility patients with normal and abnormal sperm cell conditions. We note that MUltiple SIgnal Classification ALgorithm is an image analysis technique that may vaguely fall under the machine learning category, but the conventional metrics for reporting found in Enhancing the QUAlity and Transparency Of health Research network do not apply. Alternative suitable metrics are reported, and bias is avoided through random selection of regions for analysis. Detailed methods are included for reproducibility. Kinematic features derived from nanoscale motion traces of spermatozoa contain information complementary to the speed of the sperms, allowing further distinction among the progressively motile sperms. Some highly progressive spermatozoa may have irregular motion patterns, and whether irregularity of motion indicates poor quality regarding artificial insemination needs further investigation. The presented technique can be generalized for sperm analysis for a variety of fertility conditions.

The effect of hydroxy-selenomethionine on the productive and reproductive performance of old broiler breeders.

Selenium (Se) is a rare essential element that plays a vital role in the health and performance of animals. By interfering in the production of antioxidant enzymes such as glutathione peroxidase, thioredoxin reductase and methionine sulfoxide, Se plays a role in reducing the effects of oxidative stress and animal performance. This study aimed to investigate the effect of hydroxy-selenomethionine (OH-SeMet) in the diet of broiler breeder and old broiler breeder roosters on productive performance, reproduction and sperm quality parameters. For this purpose, 260 broiler breeders of the Ross 308 strain were used in a completely randomized design with four treatments and five replications (13 hens and one rooster in each replication). Experimental treatments included: (1) a basal diet without OH-SeMet (T1:control), (2) a broiler breeder diet without OH-SeMet and a rooster diet containing 0.1mg/kg OH-SeMet (T2), (3) broiler breeder diet containing 0.1mg/kg OH-SeMet and rooster diet without OH-SeMet (T3) and (4) broiler breeder and rooster diet contained 0.1mg/kg OH-SeMet (T4). The results showed that T3 and T4 treatments improved egg production, egg weight, egg mass and feed conversion ratio (FCR) compared to the control treatment (p<0.05). The fertility and hatchability percentages of T4 and T2 treatments increased compared to T1 and T3 treatments (p<0.05). The rate of embryonic losses in T1 was higher than in other treatments. However, grade one chickens were higher in T4 than in other treatments (p<0.05). Total motility and viability of sperms were significantly higher in T2 and T4 treatments than in T1 and T3 treatments. The sperm abnormality percentage and sperm MDA concentration decreased in T2 and T4 treatments. Therefore, using OH-SeMet may be a practical approach to help old broiler breeders' production and reproduction performance.

Spermocytogram: Comparison Between the Papanicolaou Staining and Modified May Grünwald Giemsa Staining

Introduction: The research and distribution of sperm abnormalities by the spermocytogram are an essential examination in the diagnosis of male infertility. The standard Papanicolaou staining method, recommended by the World Health Organization, for performing a spermocytogram, is too expensive in Congo. The work aimed to look for abnormalities in sperm forms by two comparative methods: Papanicolaou staining and modified May Grünwald Giemsa staining (less expensive), set up in our laboratories.Materials and methods: A series of one hundred spermocytograms were performed by two staining methods: the Papanicolaou method and the modified May Grünwald Giemsa method. The results of two techniques were compared qualitatively and quantitatively.Results: The mean detection of sperm form abnormalities was higher by the Papanicolaou method. However, the precision of the kind of abnormalities highlighted by the two techniques was superimposable.Conclusion: Despite the superiority of the Papanicolaou staining method, which is a reference technique of the spermocytogram, the modified May Grünwald Giemsa staining method can be a first-line technique in the male diagnosis of infertility, especially in the deep Congo. It is efficient, reliable, inexpensive and easily achievable.

Seminal characteristics and efficacy of egg yolk free semen extender on cryopreservation of Murrah buffalo bull semen

The experiment was undertaken to study the efficacy of egg yolk free semen extender on cryopreservation of buffalo semen. A total of 24 semen ejaculates from Murrah bulls (5 to 6 years) maintained at Central Semen Station, Anjora, Durg, Chhattisgarh were divided into two aliquots and were diluted using Tris-fructose-egg yolk-glycerol (TFYG) and egg yolk free commercial extender (EYFCE, AndroMed). The overall average Murrah bull fresh semen volume (mL), sperm concentration (million/mL), initial progressive motility (%), live sperm (%), abnormal spermatozoa (%), intact acrosome (%) and HOS reactive sperm (%) were 4.90±0.49, 1286.92±100.23, 75.00±0.67, 81.45±0.61, 10.70±0.51, 80.16±0.43 and 68.83±0.40, respectively. There was significant difference between bulls in semen volume (P&lt;0.01), individual motility (P&lt;0.01) and total sperm abnormalities (P&lt;0.05). The semen extended with TFYG and EYFC extenders were cryopreserved in french mini-straw (0.25 mL) using programmable bio-freezer. The mean values of post thaw motility (%), live sperm (%), abnormal sperm (%), intact acrosome (%), HOST (%) of Murrah bull semen extended with TFYG and EYFCE were 45.83±0.77 vs 49.16±1.33, 57.41±0.54 vs 58.62±0.73, 12.54±0.46 vs 12.45±0.63, 69.62±0.82 vs 71.45±0.64 and 56.45±0.52 vs 57.37±0.55, respectively. Post thaw motility, live spermatozoa and intact acrosome were significantly higher (P&lt;0.01) in frozen thawed semen extended with as EYFCE compared to TFYG in Murrah bull semen. It is concluded that EYFCE results in improvement in post thaw semen characteristics viz. post thaw motility, live sperm percent and intact acrosome.

Effect of tocotrienol administration on reproductive efficiency and testicular tissue in obesity induced male rats

Obesity is a major public health problem for all age groups across the world. Diet, rather than hereditary alterations, is thought to have a significant role in the obesity pandemic. Obesity can considerably harm male reproductive health, resulting in lower libido, erectile dysfunction, and subfertility or infertility. Tocotrienols (T3s) are among the compounds that have metabolic effects due to their nutritional properties as a food supplement. The study aimed to identify the role of tocotrienol in ameliorating the risks of a high-fat diet on some reproductive hormones and efficiency. The eighteen male adult rats were randomly assigned to three separate sets of six each. The control group was fed a low-fat diet (LF ten percent kcal from fat), the high-fat diet (HFD) group was fed a high-fat diet (HF sixty percent kcal from fat), and the high-fat diet with tocotrienol (HFDT) group was fed a high-fat diet administered tocotrienol (60 mg/kg BW) dissolved in olive oil (1 mL/kg) for 12 weeks. LH hormone showed a significant decrease in concentration for the HFD and HFDT groups when compared with the control group. Tocotrienol supplement introduced its ability to improve motility and account for dead and abnormal sperm values. The histopathological examination of testes in rats fed a high-fat diet and supplemented with tocotrienol revealed normal spermatogenesis. The tocotrienol supplement ameliorated the deleterious effect of obesity in the experiment and appeared significantly less than the control group.

Selection based on the Breeding Soundness Evaluation is associated with the improvement of the reproductive quality of young Nellore bulls

Breeding soundness evaluation (BSE) is the best methodology to estimate the fertility potential of future bulls and performing indirect selection for their fertility. However, the outcome of the BSE is influenced by several factors, including genetics, environment, and BSE guidelines. Herein, in this retrospective study, our first aim was to characterize the reasons for failure in 46,566 BSE from 2-year-old beef Bos indicus bulls (Nellore) born from 1997 to 2018. Our second aim was to determine whether or not BSE was associated with reproductive potential improvement of the bulls over the years. Due to changes in the BSE criteria, we used the same dataset, but only bulls born from 2002 to 2018 were included resulting in 35,856 BSE. For the second aim, the effect of the year and farm were included in the model of the multivariate logistic regression. We also determined if the main reasons for BSE failure decreased over time. Bulls were classified as approved (satisfactory potential breeders and qualified for natural breeding service) and not approved (deferred and unsatisfactory potential breeders). The reasons for BSE failure in Nellore bulls were poor semen quality (53.1 %) and physical defects (46.9 %), with the main physical defect being testis abnormalities (19.7 %). The overall percentage of bulls approved each year was 87.1 %, with no improvement over the years of study. However, the percentage of approved bulls at the first BSE increased over the years (P < 0.05). This increase was evident by a reduction in the difference between the overall percentage of the bulls approved vs the percentage of bulls approved at the first BSE. Furthermore, there was an increase in the percentage of bulls classified as satisfactory potential breeders in the BSE and an evident decrease in the percentage of bulls qualified only for natural breeding service (P < 0.05). In addition, an increase of the scrotal circumference (SC) of the herd was found (P < 0.05). These results indicate the overall quality of the bulls improved over the years. To associate and identify the main sperm abnormalities, 3461 not approved bulls were clustered. The most frequent defects were strongly coiled tail spermatozoa, proximal droplets, and acrosomal defects. Overall, there was no change in the frequency of bulls not approved by the sperm morphology nor the frequency of the main sperm abnormalities over the years. Nevertheless, the frequency of the defects remained very low, implying they were controlled. Additionally, abnormalities in the testis decreased over the years and was associated with the increase in the SC of the herd and a decrease of culled bulls due to low SC. In conclusion, this study demonstrates that there is an association between implementation and use of BSE with improvements in the reproductive quality of future generation bulls.