Conventional Semen Research Articles

Impact of sperm sex sorting on sperm quality and in vitro embryo production in bovine

Increasing evidence suggests that environmental exposures can modify epigenetic marks in the germline, leading to the transmission of abnormal post-fertilization sperm epigenetic indicators and affecting embryonic development. Given the pivotal role of sperm cells in determining embryo quality, there is growing interest in understanding the potential effects of sperm sex sorting on embryo quality. This study aimed to investigate the impact of bovine sperm sexing on in vitro embryo production (IVP) and to associate molecular aspects of embryos analysis. Frozen semen samples from five Nellore bulls were used, with each bull contributing unsexed sperm (conventional semen – CV treatment) and female and male sexed sperm pooled after thawing (SX treatment). First, semen quality was assessed, including motility, morphology, acrosome integrity, and chromatin integrity to denaturation. Then, IVP was carried out, focusing on embryonic production and developmental kinetics. In the third experiment, embryo quality was evaluated by examining the gene expression of key markers (OCT4, NANOG, DNMT3A, TET1, and Fematrin-1) and the methylation pattern of the Satellite-1 and α-Satellite genes in blastocysts. Differences between CV and SX semen were only observed in motility, which was lower in SX compared with CV (P < 0.05). Although cleavage was similar, the SX groups showed lower blastocyst production than CV (P < 0.05). Of the genes evaluated, only NANOG showed high expression in the CV blastocysts compared with the SX blastocysts, but the methylation pattern revealed no differences. In conclusion, sex sorting markedly affects sperm motility and in vitro embryo production but showed no significant impact on embryo quality.

PSIII-6 Multi-sire artificial insemination of beef heifers in the Nebraska Sandhills effect on pregnancy rate compared with single-sire insemination

Abstract The objective of this study was to compare pregnancy outcomes resulting from artificial insemination (AI) with multi-sire semen (MS) compared with single-sire semen (SS) in beef heifers. Another study observed pregnancy and paternity outcomes to sexed MS without a control treatment. In the first study, unrelated ABS Global Angus bulls (n = 3) were selected for AI to 441 (Yr 1) and 391 (Yr 2) Angus crossbred heifers. Heifers were synchronized with the melengestrol acetate – prostaglandin F2α timed-AI protocol. Conventional semen, from each bull, were labeled A, B, or C. Remaining semen was pooled in equal amounts to form conventional MS straws. Heifers were AI using alternating treatment of MS or SS: A, B, or C with one-half of inseminated heifers receiving either MS or SS. The GLIMMIX procedure in SAS 9.4 analyzed responses to treatments using the BIN distribution option and reported as LSMEANS. Contrasts and estimates analyzed responses between treatments. Ultrasound determined SS pregnancy rate averaged 60% (SS-A = 64%, SS-B = 58%, SS-C = 58%) and MS average 62% (P = 0.63). Paternity was determined in 57 MS calves and confirmed in 49 SS calves. In Yr 1, Bull A sired 7%, bull B sired 56%, and bull C sired 37%. Although bull A was much less successful than bull B or C, the values are not significantly different due to the small sample size (P = 0.11 and P = 0.24, respectively). Few conclusions can be made from the ongoing study, but it is hypothesized MS may improve consistency of pregnancy rate by equilibrating semen quality in herds subjected to timed AI. In the observational study, multiple 5-way cross bulls born from the ranch herd were selected for AI of 951 ± 41 heifers for 3 yr. Heifers were synchronized using the previously mentioned protocol with split-time AI. Unequal amounts of semen were collected, pooled, and sexed to favor heifer progeny at an expected ratio of 9:1. Semen from 2 to 4 random bulls were pooled making 3 to 4 MS groups each year. Heifers exhibiting estrus were administered sexed MS, but there was no control treatment. The observed ultrasound determined average pregnancy rate of heifers administered sexed MS was 65% (Yr 1), 75% (Yr 2), and 72% (Yr 3). Previous literature suggests average pregnancy rate to AI with sexed SS is 53%. Paternity testing in Yr 2 found semen contribution mirrored paternity ratios within some groups but the sire group with 2 bulls found one sired 15% and the other sired 85%. This study exemplified a viable method for AI that requires further research. Further research into semen quality of a single sire, and pooled semen could lead to optimization of fertilization among individuals within a herd.

Use of a sperm-Hyaluronan binding assay for evaluation of sperm quality in dromedary camels

The objective of this study was to assess the ability of camel spermatozoa to bind in the Hyaluronan Binding Assay (HBA), to determine if conventional sperm quality parameters, in vitro fertilization capacity, and precursor of A-Kinase Anchoring Protein 4 (proAKAP4) values correlate with HBA results. The potential to predict post-thaw fertilization performance from HBA for fresh dromedary camel sperm was also evaluated. Semen samples were collected and assessed both fresh and post thawing, at 0 h and 1.5 h. Conventional semen analysis, HBA, and a proAKAP4 biomarker-test were used to validate sperm quality. A heterologous sperm penetration assay using zona pellucida-free goat oocytes was used to assess in vitro sperm fertilizing capacity. The results showed that dromedary camel spermatozoa bound to hyaluronan with no correlation between results from fresh samples and after thawing. Furthermore, the proAKAP4 test results showed a negative correlation with HBA at 0 h after thawing (r = - 0.62; P = 0.03). In the conventional analysis, only progressive motility (r = 0.65; P = 0.02) and straightness correlated with HBA for fresh semen (r = 0.69; P = 0.01). In the sperm penetration assay, a moderate but non-significant correlation was identified between fresh sperm HBA and penetration (r = 0.52; P = 0.07). In conclusion, results suggested that HBA can be used to assess camel sperm properties, but further investigation is needed to understand its correlation with other sperm quality parameters. The HBA score from fresh dromedary camel sperm was unable to predict post-thaw fertilization performance.

Impact of sperm DNA fragmentation in couples with unexplained recurrent pregnancy loss: A cross-sectional study.

Recurrent pregnancy loss (RPL) has a multifactorial etiology, with a majority of cases remaining unexplained. To account for these unexplained cases, possible male factors are being explored. Conventional semen analysis lacks a qualitative assessment of sperms and information regarding sperm DNA integrity. Sperm DNA fragmentation (SDF) has diagnostic value in unexplained RPL, and it may account for a number of unexplained cases. Hence, we planned a study to explore and evaluate the impact of sperm DNA fragmentation in couples with unexplained recurrent pregnancy losses. Analytical cross-sectional study was conducted at a tertiary-level referral facility in India between August 2021 and July 2023. Participants (n = 70) were divided into two groups-male partners of couples with unexplained RPL (following spontaneous conceptions) (n = 35) and men with at least one previous live birth (spontaneous or following fertility treatments for female factor infertility such as ovulation induction or intrauterine insemination) as controls (n = 35). Neither of the two groups of couples recruited for this study had undergone ART as fertility treatment. Primary outcome assessed was mean DNA fragmentation index (DFI). Secondary outcomes included differences in semen parameters such as sperm concentration, progressive sperm motility and morphology, proportion of men with high (≥30%) and low DFI in the two groups, and the association between various semen parameters and DFI. Univariate logistic regression revealed that sperm DNA fragmentation was higher in men with unexplained RPL (30.0; IQR (interquartile range) 19.0, 46.0) as compared to controls (22.0; IQR 14.0, 30.0) although it was not statistically significant (OR, odds ratio, 1.02; 95% CI 1.0-1.1, p = 0.08). A higher proportion of men with unexplained RPL had DFI ≥30% compared to controls (54.2% vs. 25.7%; OR 3.43 (95% CI 1.2-9.4); p = 0.02). No statistically significant differences were observed in semen volume, sperm concentration, progressive motility, and morphology between the two groups. Sperm DNA fragmentation index also showed a weak but significant inverse relationship with sperm morphology (r = -0.336, p = 0.004). The current study did not show any significant difference in the mean sperm DNA fragmentation levels in male partners of couples with unexplained RPL compared to controls. However, a higher proportion of men with DFI ≥30% were observed in unexplained RPL population when compared to controls.

Buserelin Acetate Added to Boar Semen Enhances Litter Size in Gilts in Tropical Environments.

The use of exogenous hormones has long been of interest for improving reproductive performance in swine production. Enhancing litter size directly impacts the economic efficiency of pig production. Various strategies, including nutritional, genetic, and hormonal approaches, have been explored with varying degrees of success. Administering a gonadotropin-releasing hormone (GnRH) agonist, such as buserelin, at the onset of estrus can induce ovulation and reduce the variation in ovulation timing among sows. This study assessed the impact of GnRH agonist supplementation in boar semen doses on the litter size of inseminated gilts. The research was conducted on a commercial swine herd in northern Thailand. A total of 231 Landrace × Yorkshire crossbred gilts, aged 224.5 ± 16.2 days at the onset of estrus synchronization, participated in the experiment. The gilts' estrus was synchronized with oral altrenogest supplementation at a dosage of 20 mg/day for 18 days. After exhibiting standing estrus, the gilts were randomly divided into three groups. Control group: gilts were inseminated at 0 and 12 h post standing estrus onset with a conventional semen dose (n = 94). Treatment 1: similar to the control group, but with an added 5 µg (1.25 mL) of buserelin acetate to the boar semen dose during the first insemination (n = 71). Treatment 2: similar to the control group, but with 10 µg (2.5 mL) of buserelin acetate added to the boar semen dose during the first insemination (n = 66). All gilts were inseminated twice during their standing estrus using the intrauterine artificial insemination method. Each semen dose contained 3.0 × 109 motile sperm in 80 mL. The farrowing rate averaged 78.8% and did not significantly differ between the groups (p = 0.141). The total number of piglets born per litter in the treatment 2 group was greater than in the control group (14.0 ± 0.3 vs. 13.2 ± 0.3, respectively, p = 0.049), but was not significantly different from the treatment 1 group (13.3 ± 0.3, p = 0.154). Similarly, the number of live-born piglets in the treatment 2 group was greater than in the control and treatment 1 groups (13.2 ± 0.4 vs. 12.3 ± 0.3 and 12.0 ± 0.4, respectively, p < 0.05). Moreover, the live-born piglets' litter birth weight in the treatment 2 group was greater than in the control group (17.0 ± 0.4 vs. 15.6 ± 0.3 kg, respectively, p = 0.008) and the treatment 1 group (15.7 ± 0.4 kg, p = 0.025). In conclusion, adding a GnRH agonist to boar semen appears to enhance the litter size of gilts. Further research should focus on understanding the underlying mechanisms and determining the optimal dose and timing for GnRH agonist supplementation.

Comparison of two intravaginal progesterone-releasing devices in lactating Holstein cows synchronized with a 5-d GnRH-based timed-AI protocol

The objective of the study was to compare the effectiveness of CIDR vs. PRID-Delta devices for use in a 5-day Ovsynch protocol for TAI in lactating Holstein cows that were either not in estrus after the end of the voluntary waiting period or non-pregnant and not returning to estrus following the previous AI. Cows fitted with a collar-mounted automated activity monitoring system (Alta Cow Watch) were subjected to a standard 5-d Ovsynch protocol [100 μg of gonadorelin (GnRH) on Day 0 and 500 μg of cloprostenol on Days 5 and 6] and allocated randomly to receive either an intravaginal device containing 1.35 g (CIDR; n = 304) or 1.55 g (PRID ® DELTA; n = 304) of progesterone between Day 0 and 5. All cows received a second administration of GnRH at approximately 56 h and timed-AI (TAI) 72 h after intravaginal device removal. Inseminations were done using conventional frozen-thawed semen. Estrus events prior to TAI were recorded and transrectal ultrasonography was done on Day 0 to determine presence of a corpus luteum (CL) and 33 and 61 d post-TAI, respectively, to diagnose and confirm pregnancy. Cows had an average of 2.2 lactations, 124.3 days in milk, and a milk yield of 43.6 kg/d at enrollment. The overall percentage of cows with a CL at initiation of treatment was 68.8 % and did not differ between treatment groups. Cows with a CL had greater pregnancy per AI (P/AI) at 33 and 61 d post-TAI than cows without a CL (P < 0.01; 46.9 and 42.3 % vs. 32.1 and 27.4 %, respectively). The overall percentage of cows that expressed estrus prior to TAI was 24.8 % and did not differ between treatment groups; however, estrus expression prior to TAI affected P/AI at 33 and 61 d post-TAI (P < 0.01; 53.6 and 49.0 % vs. 38.5 and 33.9 % for those expressing or not expressing estrus, respectively). Pregnancy per AI at 33 d post-TAI tended to differ between treatment groups (P = 0.08; 46.1 vs. 38.5 % for PRID and CIDR groups, respectively) and P/AI at 61 d post-TAI was greater (P < 0.01) for PRID-treated cows (43.8 %) compared to CIDR-treated cows (31.6 %). Thus, PRID-treated cows had lower pregnancy loss than CIDR-treated cows (P < 0.01; 5.0 vs. 17.9 %). Also, treatment with a PRID tended (P = 0.08) to result in fewer twin pregnancies (7.9 vs. 14.5 % for PRID and CIDR treated cows, respectively). In conclusion, lactating dairy cows subjected to a 5-d Ovsynch TAI protocol plus a PRID-Delta had greater P/AI at 61 d post-TAI, lower pregnancy loss between 33 and 61 d post-TAI, and fewer twin pregnancies compared to cows subjected to a 5-d Ovsynch protocol plus a CIDR.

Reproductive outcomes in Holstein heifers synchronized with timed-AI protocols that provide for a lengthened proestrus.

This study compared reproductive outcomes among two protocols for synchronization of ovulation that provide for a lengthened proestrus with the conventional oestradiol-based protocol currently used for timed-AI (TAI). Holstein heifers (13-15 months) at one location were assigned randomly to one of three TAI protocols. Heifers (n = 150) in the 7-day oestradiol benzoate (EB) group received a progesterone device (Cue-Mate) and 2 mg EB on Day 0; 500 μg of cloprostenol (PGF) and Cue-Mate removal on Day 7; 1 mg of EB on Day 8 and TAI on Day 9 (54 h after Cue-Mate removal). Heifers (n = 150) in the 5-day CO-Synch (CO) group received a Cue-Mate and 100 μg of gonadotropin-releasing hormone (GnRH) on Day 2; Cue-Mate removal and PGF (twice, 12 h apart) on Day 7; and GnRH along with TAI on Day 10 (72 h after Cue-Mate removal). Heifers (n = 150) in the J-Synch (JS) group received a Cue-Mate and 2 mg of EB on Day 1; PGF and Cue-Mate removal on Day 7; GnRH and TAI on Day 10 (72 h after Cue-Mate removal). Heifers were inseminated by one technician with frozen-thawed conventional semen from one of four commercially available sires. Plasma progesterone (P4) concentrations (ng/mL) were determined at Cue-Mate removal and TAI. Ovarian ultrasonography was done in a subset of 217 heifers at the initiation of protocols, at Cue-Mate removal; TAI; and 7 days after TAI. Approximately, 28 and 50 days after TAI pregnancy status was determined by ultrasonography. Mean (±SEM) plasma P4 concentration at Cue-Mate removal was greater (p < .01) in CO (6.02 ± 0.2) and JS (6.51 ± 0.2) compared to EB heifers (4.53 ± 0.2). Mean (±SEM) plasma P4 concentration at TAI was lowest in the JS (0.28 ± 0.05), intermediate in CO (0.46 ± 0.02), and greatest in EB heifers (0.66 ± 0.05, p < .01). The diameter of the ovulatory follicle (mean ± SEM) was the smallest in the JS group compared to that in the CO and EB groups (15.8 ± 0.5; 13.9 ± 0.5; and 12.7 ± 0.5 mm for EB, CO and JS, respectively). More (p < .01) heifers in the JS group had their oestrous cycle synchronized (50.0, 78.8 and 82.4% for EB, CO and JS groups), and were pregnant at 28 (40.3, 51.3 and 63.3% for EB, CO and JS groups) and 50 days after TAI (32.6, 46.0 and 60.0% for EB, CO and JS groups). In summary, heifers subjected to the J-Synch TAI protocol had lower P4 at TAI, and better overall response to hormonal treatments, which resulted in increased P/AI at 28 and 50 days after TAI compared to those heifers subjected to either a 7-day EB protocol or a 5-day CO-synch protocol.

Laparoscopic artificial insemination of white-tailed deer with fresh sex-sorted semen

This case study describes the feasibility of using chilled, sex-sorted semen for laparoscopic artificial insemination (AI) in the captive white-tailed deer (Odocoileus virginianus) industry. Ejaculates from 3 bucks were collected, extended, and shipped overnight for flow cytometric sex-sorting. Sex-sorted semen was chilled and shipped back overnight. Synchronized does were bred using chilled, sex-sorted (n = 30, farm 1; n = 10, farm 2) or conventional frozen (n = 7, farm 2) semen by laparoscopic AI. Does were subsequently turned out with bucks. Percentage of bucks born via AI with sex-sorted semen was 85% (28/33) for farm 1 and 100% (16/16) for farm 2. Percentage of bucks born by natural cover was 50% (12/24) for farm 1 and percentage of bucks born by using either conventional semen or live cover was 56% (9/14) for farm 2. This case study demonstrated acceptable results using chilled, sex-sorted semen in white-tailed deer herds as a technique to manipulate the sex proportions per breeding season.

Ambient air pollution decreased normal fertilization rate via the mediation of seminal prosaposin

ObjectiveThis study focuses on the association between seminal concentration of prosaposin and ambient air pollutants and whether the association affects the normal fertilization rate in vitro fertilization (IVF) treatment. MethodsThe cohort of 323 couple participants aged 22–46 was recruited from Jan. 2013 to Jun. 2018. At enrollment, resident address information was obtained and semen parameters of male counterparts were evaluated according to WHO criteria. We used inverse distance weighting interpolation to estimate the levels of ambient pollutants (SO2, O3, CO, NO2, PM2.5, and PM10) in the surrounding area. The exposure of each participant was estimated based on the data gathered from air quality monitoring stations and their home address over various periods (0–9, 10–14, and 0–90 days) before semen sampling. The generalized linear regression model (GLM) and the Bayesian kernel machine regression (BKMR) were used to analyze the associations between pollutants, semen parameters, prosaposin, and normal fertilization. Additionally, the mediating effect of prosaposin and semen parameters on the link between pollutants and normal fertilization was investigated. ResultsGLM and BKMR showed exposure to ambient air pollutants was all associated with the concentration of seminal prosaposin, among them, O3 and CO were also associated with normal fertilization (-0.10, 95 %CI: −0.13, −0.06; −26.43, 95 %CI: −33.79, −19.07). Among the semen parameters, only the concentration of prosaposin and total motile sperm count (TMC) was associated with normal fertilization (0.059, 95 %CI: 0.047, 0.071; 0.016, 95 %CI: 0.012, 0.020). Mediation analysis showed that prosaposin played a stronger mediating role than TMC in the relationship between short-term exposure to O3 and fertilization (66.83 %, P<0.001 versus 3.05 %, P>0.05). ConclusionSeminal plasma prosaposin showed a stronger meditating effect reflect the correlation between ambient air pollutants and normal fertilization rate than conventional semen parameters, which may be used as one of the indicators between pollution and fertilization in IVF.

Association between spectrum of mycotoxins and semen quality: A cross-sectional study in Beijing, China

Exposure to mycotoxins is unavoidable in daily life through ingestion, dermal, and inhalation routes. Toxicological studies found that exposure to mycotoxins might affect male reproductive function. However, there is still a lack of population evidence. We aimed to assess the association of individual and joint exposure to spectrum of mycotoxins with semen quality. The present study included 192 participants in Beijing, China. We measured conventional semen parameters and assessed semen quality. Sixty-seven traditional or emerging mycotoxins were determined to describe the spectrum of mycotoxins. The participants were widely exposed to multiple mycotoxins, and nearly half were simultaneously exposed to more than six mycotoxins. After adjusting potential confounders, logistic regression indicated that the number and concentration of plasma mycotoxin were correlated to the risk of low semen quality. Plasma beauvericin and citrinin concentrations were associated with lower semen quality. The least absolute shrinkage and selection operator regression showed similar results to logistic regression. Quantile-based g-computation and Bayesian kernel machine regression models found that the mixture of mycotoxins was harmful to semen quality, especially in sperm motility. In conclusion, both individual and mixture of mycotoxin exposure were correlated with lower semen quality. More regulations and measures should be taken to reduce mycotoxin contamination.

O-196 Men who fathered embryos with poor pre-implantation development rates exhibit an increased frequency of sperm with high DNA accessibility

Abstract Study question Do differing levels of DNA accessibility in sperm affect the ability of embryos generated by ART to undergo pre-implantation development? Summary answer Individuals whose sperm generated embryos that failed to efficiently form blastocysts tend to have more sperm with high levels of DNA accessibility. What is known already Chromatin is a complex composed of nucleic acids and proteins that serves to condense and regulate the genome in all eukaryotic cells. Sperm chromatin is packaged into a structure with higher density than that found in precursor germ cells or somatic cells. Samples from individuals with decreased normal semen parameters have previously been shown to possess an increased frequency of sperm with aberrant chromatin packaging. However, existing methodologies for measuring sperm chromatin packaging are indirect and non-quantitative. Study design, size, duration Prospective cohort study, registered January 2020. We adapted an enzymatic methodology for measuring DNA accessibility known as NicE-view (Nicking Endonuclease-mediated visualization), for use in human sperm. DNA accessibility was quantified in three millions of individual sperm from semen obtained from 60 individuals. Both total sperm and sperm selected for motility via swim-up were analyzed for each participant. Participants/materials, setting, methods Participants sought treatment for infertility at Universitätsspital Basel and possessed normospermic levels in conventional semen analysis. Participant partners had normal endocrine parameters and ovulation response to hormonal stimulation. 20 couples obtained pregnancies without the need for ART, while 40 individuals who were treated using ART and divided into two groups differing in pre-implantation development rates: (1) 20 with blastocyst development of &amp;gt; 50% of all zygotes and (2) 20 produced 0-1 blastocyst following embryo culture. Main results and the role of chance We quantified DNA accessibility and the presence of single stranded DNA nicks in more than 3 million sperm derived from 117 samples. We found that while DNA nicks were only present in a small fraction of the sperm analyzed, DNA accessibility levels varied dramatically with the data indicating a bimodal distribution. At a single sperm level, DNA accessibility and the presence of single stranded DNA damage both correlated with nuclear size. We found that DNA nicking was more abundant in total sperm samples compared to sperm selected for motility via swim-up preparation. In contrast, sperm with high levels of DNA accessibility were more abundant following swim-up, perhaps resulting from capacitation. We observed that individuals with a history of low blastocyst development were more likely to have high frequencies of sperm harboring DNA nicks and high DNA accessibility. We also measured increased frequencies of sperm with high DNA accessibility to negatively correlate with conventional semen parameter values. Limitations, reasons for caution Our study cohort consists of only 60 individuals. Variation is also observed between individuals within each participant category, so further analysis with increased participant numbers is required to strengthen these conclusions. Wider implications of the findings Our results suggest measuring DNA accessibility in sperm from normospermic individuals provide additional information about the efficiency of pre-implantation development following ART. Future work examining the sources of increased DNA accessibility in sperm will also be interesting to identify possible treatments for such individuals. Trial registration number SNCTP000003654 | NCT04256668

P-721 Exploring the impact of geographical variation: A retrospective study examining semen phenotypes within a cohort of 19,612 men attending a fertility center in the UAE

Abstract Study question What are the epidemiological trends of semen phenotypes and their association with geographic regions among men seeking fertility treatment in the UAE? Summary answer Following covariate adjustment, UAE Nationals attending a fertility center displayed lower incidence of oligozoospermia and higher prevalence of asthenozoospermia and teratozoospermia compared to other nationalities. What is known already Conventional semen analysis remains the gold standard for diagnosing and managing male infertility. It is well documented that semen is heterogenous and can be influenced by diverse factors, including but not limited to race, ethnicity, sociocultural background, seasonality, and geographical region. Although higher infertility prevalence has been reported in regions like North Africa and the Middle East, existing estimates rely largely on data from female partners of infertile couples and studies dating back to 1988. This underscores a significant data paucity in the Middle East. Study design, size, duration In this retrospective study, the anthropometric data (age, weight, height, body mass index, nationality) and semen parameters (semen volume, semen pH, sperm concentration, total sperm count (TSC), total motility (TM), progressive motility (PM), normal morphology, viability) of 19,612 men (32,664 samples) who attended a fertility center in the UAE between 2011 and 2022 were assessed. Ethical approvals for this study were obtained from appropriate bodies (REC/2022/P27 and MBRU IRB-2021-12). Participants/materials, setting, methods Data from patients were categorized into different geographical regions (Sub-Saharan Africa, North Africa, North America, Central America, South America, Asia, Europe, Middle East, and Oceania) based on nationality. Patients were further grouped as MENA (Middle East and North Africa) and non-MENA, while data from UAE Nationals (UAE) were also compared to Other Nationalities (Global). Unadjusted analysis was performed using SPSS. While covariate adjustment and propensity score analyses were performed using R package and STATA. Main results and the role of chance All semen parameters, remained within normal WHO reference values when stratifying according to geographical regions, MENA and Non-MENA as well as UAE and Global. Interestingly, MENA displayed a significant reduction in semen volume (p &amp;lt; 0.0001) and sperm concentration (p = 0.017), and an increase in PM (p &amp;lt; 0.00001). Similarly, PM was higher in UAE versus Global. To address skewed data distribution between UAE and Global, we employed propensity score analysis and logistic regression models, with semen parameters categorized according to WHO lower reference limits. Following covariate adjustment, all models demonstrated no significant difference in PM and TM between the two subgroups. However, logistic regression indicated a non-significant increase (4%) in the prevalence of asthenozoospermia in UAE compared to Global. Categorical analysis indicated that the Global samples had significantly lower TSC compared to the UAE group (24.6% vs. 26.3%, p&amp;lt;.001). Modelling adjusted for propensity score and UAE Nationality further suggested that UAE were 13% less likely to have lower TSC compared to Global (p&amp;lt;.001). About 58% of UAE samples exhibited teratozoospermia, while it was 56% for Global. After adjusting for confounders, analysis revealed a significantly higher prevalence (12%) of teratozoospermia among UAE compared to Global, with a potential range of 9%-16% in the long term. Limitations, reasons for caution A limitation of the current study is data distribution skewness. Of the 32,664 samples from 19,612 patients (113 countries), 21,649 samples are from UAE Nationals. However, logistic regression, propensity score adjustment accounting for Nationality, propensity score matching (1:2 ratio), and inverse probability weighting were applied to mitigate this bias. Wider implications of the findings Paucity of data on male fertility in the UAE and MENA region does not allow for accurate estimation of male infertility. Owed to its sample size and statistical robustness, this study provides evidence addressing this gap, offering insights into interpretation of global data on reproductive health. Trial registration number not applicable

P-061 Effect of Varicocelectomy on fertility potential and pregnancy outcome: results of a large cohort from a tertiary medical center

Abstract Study question Does varicocelectomy improve semen parameters, hormones &amp; pregnancy outcomes in infertile men with clinical varicocele? Summary answer In infertile men with clinical varicocele, sperm count, motility, and DNA fragmentation improved significantly after varicocelectomy. Pregnancy outcomes improved for both spontaneous and assisted reproduction. What is known already Infertility is estimated to impact 15% of couples attempting to conceive worldwide. Varicocele has been identified as the most common surgically correctable cause of male infertility. Despite the significant role of varicocele in the pathogenesis of male infertility, the impact of varicocele repair (VR) on conventional semen parameters &amp; pregnancy outcomes remains controversial. Few systematic reviews and meta-analyses (SRMAs) evaluated the impact of VR on sperm concentration, total motility, and progressive motility, mostly using a before-after analytic approach with fewer studies reporting on pregnancy outcomes. Study design, size, duration This was a retrospective study of 2171 patients presenting with infertility &amp; clinical varicocele between 2014 and 2024. Participants/materials, setting, methods Exclusion criteria were prior medications, medical illnesses causing infertility, or endocrine disorders. All participants underwent sub-inguinal microsurgical varicocelectomy. Data regarding patient demographics, clinical findings, semen analysis, serum hormone levels &amp; pregnancy outcomes were collected pre and post-operatively. Variables were examined before &amp; after varicocelectomy. X2 test and Kruskal-Wallis test were used to analyze categorical and numerical values, respectively. A p value &amp;lt; 0.05 was considered statically significant. Main results and the role of chance A 2171 participants were included. The mean age ± SD of the study population was 33.65 ± 8.54 years. Their mean right testicular size, left testicular size, right vein size, and left vein size were 14.38 ± 4.56,13.46 ± 3.49, 2.47±0.79 &amp; 3.99 ±1.33 respectively. When subdividing them by grade of varicocele, on the right side, grades 1, 2, and 3 were detected in 692 (31.9%), 491 (22.6%), and 63 (2.9%), respectively. While on the left side, grades 1, 2, and 3 varicoceles were detected in 303 (13.9%), 999 (46%) &amp; 867 (39.9%), respectively. Indication for surgery was pain (37%, primary infertility (42.9%), secondary infertility (18.3%), and others (3.6%). The majority performed unilateral varicocelectomy (1776 {81.8%}), while 396 (18.2%) underwent bilateral varicocelectomy. Varicocelectomy significantly improved sperm count, total &amp; progressive motility &amp; DNA fragmentation. Pregnancy outcomes improved postoperatively for both spontaneous (32.3%) &amp; assisted reproduction (25.2%). No significant change was found in hormones postoperatively. Limitations, reasons for caution Retrospective study, single center experience. Wider implications of the findings It is important in patients’ counselling &amp; choose the best management for this group of patients. Trial registration number NA

P-066 A multi-bioassay approach to identify male gamete fertilization dysfunction

Abstract Study question Can a multi-bioassay approach identify a specific male gamete dysfunction often overlooked by a standard semen analysis? Summary answer In cases of persistent fertilization failure, additional testing may assist in pinpointing underlying factors contributing to compromised sperm embryo developmental competence and tailor treatment. What is known already Conventional semen analysis, while informative, ineffectively captures the complete spectrum of male gamete dysfunction. Despite adequate sperm concentration and motility, some couples may still experience fertilization failure with Intracytoplasmic Sperm Injection (ICSI). This highlights the complexity of male infertility, that extends beyond standard parameters. In addressing sperm-related fertilization challenges, various assisted oocyte activation (AOA) methods have been proposed to trigger intracellular Ca2+ release, leading to calcium spikes and meiosis initiation. Study design, size, duration In the past 10 months, we identified couples (n = 50) with a history of poor ICSI fertilization, exhibiting suboptimal head morphology. Screening for acrosomal dysfunction involved assessing the presence of PLCxand related gene mutations. We measured the degree of chromatin compaction by histone-to-protamine ratio and evaluated sperm chromatin fragmentation (SCF). To correct for eventual acrosomal dysfunction, couples were counseled to undergo subsequent ICSI cycles with AOA. Clinical outcome was compared between historical and treatment cycles. Participants/materials, setting, methods Fifty men with a history of poor ICSI fertilization were included. A standard semen analysis was conducted according to WHO guidelines. The presence of PLCx was measured through immunofluorescent staining (normal threshold, ≥ 30%), corroborated by gene mutation detection though DNA-seq. Sperm histone-to-protamine ratio was evaluated by Aniline Blue (normal threshold, &amp;lt;15% histone retention). SCF was assessed utilizing the TUNEL assay (normal threshold, ≤15%). AOA was performed using ionomycin or recombinant PLCx protein. Main results and the role of chance Fifty men experienced a poor clinical outcome following ICSI with a fertilization of 7.4% (48/647), embryo implantation of 6.6% (1/15), clinical pregnancy of 6.6% (1/15), and pregnancy loss rate of 100% (1/1). Their semen analysis, interestingly, revealed the following semen parameters: 3.0±1 mL volume, 50.0±20 x106/mL concentration, 41.4±21% motility, and 1.9±1% normal morphology. Nonetheless, PLCx content was diminished, with values ranging from 2-27%. Genomic assessment confirmed mutations in PLCx, DPY19L2, SPATA16 and PICK1 genes, all implicated in sperm nuclear remodeling and acrosome formation. An elevated histone content at 29.7% was positively correlated with an increased SCF at 18.0% (R2=0.6, P&amp;lt;0.001). In subsequent cycles employing AOA, the normal fertilization increased to 42.3% (305/721), the embryo implantation reached 13.0% (9/69), the clinical pregnancy rose to 21.1% (8/38), and the delivery rate was 21.1% (8/38), without any pregnancy loss. (P&amp;lt;0.001). Limitations, reasons for caution While this study affirms the effectiveness of a multifaceted assessment in pinpointing specific sperm dysfunctions and customizing treatment, it is still a retrospective observation requiring independent validation in a larger population. Wider implications of the findings Semen analysis alone fails to address specific aspects of male gamete dysfunction. Incorporating additional bioassays to assess chromatin structure, integrity, and acrosomal function offers valuable insights for fertility management in tailoring personalized treatments. This comprehensive approach enhances the understanding of male reproductive health, maximizing the potential for successful clinical outcome. 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-060 When is it the sperm’s fault? The incidence of sperm-related poor fertilization after ICSI in 13,859 matched oocyte donation cycles

Abstract Study question What is the true incidence and impact of male factor-related poor fertilization in ICSI cycles when analyzed using matched pairs of high quality donor oocytes? Summary answer In ICSI cycles with donor oocytes, male factor-related poor fertilization occurs in 3.3% of cases and contributes to 86.7% of all poor (&amp;lt;30%) fertilization outcomes. What is known already Opting for oocyte donation is a complex decision for couples, involving significant financial and long-term implications. This decision is typically prompted by presumed female infertility as male fertility diagnostics remain limited. Although ICSI is a highly effective treatment, cases of poor or complete fertilization failure can still occur, even when sperm parameters appear normal. This suggests the presence of unidentified male-related factors. However, the precise incidence of such factors remains poorly understood. This study aims to address this gap, offering a unique perspective, by employing a matched oocyte donation model to investigate the influence of sperm-related factors on fertilization outcomes. Study design, size, duration This retrospective cohort study analyzed 14,928 oocyte donation ICSI cycles, derived from 7,519 ovarian stimulations (OS) of 2,963 unique oocyte donors. The data were from a single center from January 2015 to December 2022. Sibling oocytes from the same OS were utilized for at least two different recipients (n = 13,859), facilitating comparisons under varying paternal conditions. Participants/materials, setting, methods Cases from the same oocyte lot with poor (&amp;lt;30%) and good (&amp;gt;65%) fertilization rates were matched to isolate sperm-related factors influencing fertilization success. Paired t-tests were used to compare outcomes. Ordinary least squares regression was used to isolate variables associated with poor fertilization, such as oocyte status (fresh versus vitrified), sperm origin (partner versus donor) and the presence of severe male factor (defined as a concentration of &amp;lt; 1 million/mL and/or &amp;lt;1% progressive motility). Main results and the role of chance The mean fertilization rate was 73.0% across all cycles analyzed. The incidence of poor fertilization (≤30%) was found to be 3.8% across the entire cohort (532 out of 13,859 cycles). Of these, 86.7% (461/532) cases using the same oocyte lot could be matched to cases with high fertilization rates (&amp;gt;65%) in other recipient cycles (n = 1,441), indicating that poor fertilization was male-related. We observed comparable numbers of inseminated oocytes between matched cases with high and low fertilization (7.23 ± 1.32 and 7.42 ± 1.42, respectively, p = 0.011), while the mean number of fertilized oocytes (1.44 ± 0.77 versus 6.12 ± 1.36, p = &amp;lt;0.001) and mean fertilization rates (20% versus 83%, p &amp;lt; 0.001) differed significantly. Notably, oocyte vitrification and the presence of a severe male factor negatively affected fertilization outcomes (coeff. -0.0898, p &amp;lt; 0.001 and coeff. -0.1500, p &amp;lt; 0.001, respectively). Nevertheless, the majority of poor fertilization cases (92.7%) occurred in the absence of these factors, underscoring the limitations of conventional semen diagnosis tests as predictors of poor fertilization. Across the entire cohort male factor alone accounted for 3.3% (461/13,859) of cycles with &amp;lt;30% fertilization rates. Furthermore, sperm-related factors were the primary cause of poor fertilization outcomes in oocyte donation cycles, accounting for 86.7% of cases. Limitations, reasons for caution Certain variables were not analyzed due to the retrospective nature and extended timeframe of the study. The matched oocyte model may overlook certain non-male factor-related instances of poor fertilization, such as procedural or technical issues. Wider implications of the findings This extensive analysis emphasizes the clinical relevance of male factor in fertilization outcomes, highlighting the need for improved semen diagnosis. It also indicates the importance of considering male-related factors in treatment decisions and shifting the focus from female-centric to more balanced fertility evaluations. Trial registration number Not applicable

P-049 Identification of human spermatozoa with zona pellucida-binding ability using deep learning

Abstract Study question Can a deep learning model be used for identification of fertilization-competent human spermatozoa based on their distinctive morphological features? Summary answer Our results validated the clinical significance of image analysis of sperm morphology using deep learning to identify high-quality spermatozoa with zona pellucida (ZP)-binding ability. What is known already Sperm morphology is manually evaluated as part of the conventional semen analysis in clinical settings. However, the clinical predictive power of sperm morphology remains debatable due to its lack of association with the fertilization potential of spermatozoa. Human zona pellucida (ZP)-binding ability is a measurable, independent sperm parameter associated with in vitro fertilization (IVF) and pregnancy outcome. Supervised deep learning is an advanced branch to systematically identify the underlying relationship between the input images and designated output. A well-trained model with high generalization ability can generate plausible predictions for unseen images collected under the same condition as the training datasets. Study design, size, duration Human spermatozoa and oocytes were obtained with written consents from the donors attending our assisted reproduction program. Acrosome intact, ZP-bound spermatozoa were recovered after 30 min of gamete coincubation. Unbound spermatozoa were collected from clinical samples of spermatozoa with defective ZP-binding ability leading to fertilization failure following conventional insemination. Sperm images of infertile men (n = 165) with fertilization rates including 0-40% (low), 41-70% (intermediate) and 71-100% (high) following conventional insemination were collected for validation. Participants/materials, setting, methods Diff-Quik stained images of spermatozoa were processed by the K-means clustering algorithm to generate grey-scaled images of individual sperm heads. All sperm images of clinical samples were subjected an additional image conversion step using Cycle Generative Adversarial Network (CycleGAN) to ensure that their microenvironments are similar to those of the laboratory samples. A VGG 13 model was fine-tuned to classify a total of 1,083 images of ZP-bound and unbound spermatozoa over 50 epochs. Main results and the role of chance The newly fine-tuned VGG-13 model demonstrated excellent performance in classifying the ZP-bound and unbound spermatozoa, as reflected by the high sensitivity of 97.6%, specificity of 96.0%, and accuracy of 96.4%. It had a high discriminative power to distinguish between ZP-bound and unbound spermatozoa with an area under the receiver operating characteristic (ROC) curve (AUC) of 0.992. Saliency maps indicated that the model focused on the sperm head and mid-pieces in all images, with a primary emphasis on the anterior regions (43.0%±5.0%). The model was further validated on a vast collection of over 40,000 sperm images collected from the aforementioned fertilization groups. Our results revealed that the high fertilization rate group had a significantly higher percentage of predicted ZP-binding ability than the low fertilization rate group, as determined by the model (16.8%±7.2 vs. 3.2%±1.4; p &amp;lt; 0.05). By analyzing sperm images from infertile men (n = 113) in the high and low fertilization rates groups, a clinical threshold of 4.8% was identified using ROS curve (AUC:0.970) and Youden’s index, which can be used to distinguish between ZP-bound and unbound spermatozoa of clinical samples with a specificity of 93.8% and a sensitivity of 90.9%. Limitations, reasons for caution Our deep learning model is only applicable to diff-quik stained images of spermatozoa captured at high magnification. Further investigation is needed to examine its classification performance on images captured by different platforms and settings, ensuring consistent and reliable prediction accuracy regardless of varying degrees of image quality. Wider implications of the findings Our newly fine-tuned model can be used to evaluate sperm fertilizing potential, irrespective of the parameters evaluated in conventional semen analysis and to identify normospermic patients with defective ZP-binding ability who may benefit from intracytoplasmic sperm injection (ICSI) to improve their fertilization outcome. Trial registration number not applicable

Effects of beef semen and beef embryo strategies on profitability: Economics of using beef semen and beef in vitro–produced embryo transfer in Jersey herds

Dairy herds have adopted sexed semen (SS) and beef semen (BS) to control heifer inventory and increase calf sales revenue. Beef in vitro-produced embryo transfer (beef IVP-ET) may be an alternative to increase calf sales revenue. Additionally, raising those Jersey-beef crossbred or pure beef animals in a dairy system may be a new source of revenue. We aimed to evaluate the effects of breed strategies combining dairy conventional semen (CS), SS, BS, and beef IVP-ET on herd dynamics and profitability with strategies of marketing those animals at 1 d old or raising them to a weight of 180 kg. A Markov chain model was developed to maximize the profitability of Jersey herds by changing the number of dairy heifers sold at birth and the culling rate of cows in their third parity or greater. The model presents inputs on the reproductive and productive performance of heifers and cows over time. The last year's data (year 10, steady state) was used to calculate accrual operational cost and revenue per cow per year. We varied the breeding strategy by breeding order and parities, the embryo transfer cost ($85 or $170), the pure beef calf market price ($200 or $300), and the marketing strategy. A total of 8 scenarios plus a default scenario were simulated. Overall, the proportion of SS use was 47.3 ± 0.6% (mean ± SD). For the scenarios replacing all CS breedings with BS breedings, the percentage of BS used was 52.3 ± 0.6%. When beef IVP-ET was used, the percentages of BS and beef IVP-ET used were 22.4 ± 0.1% and 31.0 ± 0.1%, respectively. We observed that when we compared the combination of BS and dairy SS with the default scenario, the production of purebred Jersey male calves was reduced by 83.5%, and profit per cow per year was increased from $113.50 to $203.30. When a beef IVP-ET cost of $85 per transfer was used (scenarios 2 and 3), profit per cow per year was $145.50 and $176.20 for pure beef calf prices of $200 and $300, respectively. In scenario 4, with a beef IVP-ET cost of $170, the lowest profit ($52.90 per cow per year) was found when marketing 1-d-old pure beef calves at $200. The highest profit was achieved for the scenario of raising the Jersey-beef crossbred animals to 180 kg ($232.90, scenario 6), followed by scenario 7 ($222.90, using the combination of BS, dairy SS, and beef IVP-ET) with an embryo transfer cost of $85. Under the current market conditions, combining SS and BS in the reproductive program was a feasible economic opportunity for Jersey herds, yielding the highest net return. The adoption of beef IVP-ET in a reproductive program can potentially increase profit per cow per year, but its profitability will depend on the beef IVP-ET pregnancy cost, the pure beef market price, calf performance, and herd reproductive performance. In conclusion, raising the Jersey-beef crossbred calves may be a profitable strategy, and dairy producers need to evaluate the best option to invest in, as it will take extra risk to produce high-quality animals.

Effect of treatment of chronic hepatitis c virus patients with direct-acting anti-retroviral drugs on semen and hormonal parameters

Objective: Hepatitis C virus (HCV) infection is known to influence the seminal and hormonal parameters of infected men. This study was performed to assess the effects of HCV clearance using direct-acting antiviral (DAA) agents on semen and hormonal parameters. Methods: A total of 50 patients with chronic HCV were enrolled, and conventional semen analysis was performed according to World Health Organization guidelines. Basal levels of total testosterone, free testosterone (FT), follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), prolactin, and sex hormone-binding globulin (SHBG) were assessed before and 3 months after treatment with DAAs.Results: Following DAA treatment, statistically significant increases were observed in sperm motility and the proportion of grade A sperm. Additionally, the percentage of abnormal forms was significantly decreased after treatment (p=0.000). However, no significant differences were observed in semen volume, concentration, or total sperm count. Sex hormone analysis of patients after DAA treatment revealed significant increases in FT, LH, and FSH levels, along with significant decreases in SHBG, prolactin, and E2 levels.Conclusion: Following HCV clearance, we noted an improvement in sperm motility and an increase in the percentage of sperm with normal morphology. Treatment with DAAs was also associated with increased levels of FT and LH, along with decreased levels of SHBG, prolactin, and E2.

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

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