Sperm Count Research Articles

P-058 The role of sperm selection in improving embryo euploidy rate: a comparison between Swim-up and Microfluidic Sperm Selection

Abstract Study question Does Microfluidic Sperm Selection (MSS) improve the blastocysts’ development rate and the euploidy rate compared to Swim-up sperm selection? Summary answer MSS doesn’t improve the rate of blastocyst development and euploidy compared to Swim-up. Furthermore, in women age ≥ 37 years D6 aneuploid blastocyst rate increases What is known already Sperm defects have been shown to have a negative impact on embryo quality and development. MSS was introduced as an alternative to traditional centrifugation-based sperm preparation techniques to efficiently isolate highly motile and healthy spermatozoa. Centrifugation has been suggested to compromise membrane integrity, motility, mitochondrial energy production, and DNA structure. Less DNA fragmentation and ROS formation was observed in spermatozoa isolated with MSS compared to standard techniques. Our study aims to analyze whether MSS also improves biological outcomes. Study design, size, duration In a prospective study of 51 ICSI/PGT-A cycles performed between September 2022 and November 2023, freshly ejaculated semen sample was divided into two aliquots and processed by Swim-up and MSS. Split sperm sample was used to inject siblings’ mature oocytes (249 vs 274 oocytes in Swim-up vs MSS groups). Inclusion criteria: retrieved MII oocytes ≥8, oligoteratozoospermic and normozoospermic semen. Results were analyzed according to women’s age <37 years (21 cycles) and ≥37 years (30 cycles). Participants/materials, setting, methods Spermatozoa were selected using either conventional Swim-up or a microfluidics device (ZyMōtTM). Post treatment sperm parameters (sperm count; total sperm motility; progressive motility and hyperactivated motility) were examined for both. Embryo culture was performed in Irvine Continuous Culture-NX media until day5 or day6 and biopsy was performed at the blastocyst stage by next generation sequencing. Primary endpoint was fertilization rate, blastocyst development and usable blastocysts rate. Chi-square analysis was performed and p < 0.05 was considered significant Main results and the role of chance There is no significant differences overall between Swim-up and MSS in fertilization rate (73% vs 75%), blastocyst development rate (47% vs 54%) and ploidy rate: aneuploid (47% vs 50%), usable blastocyst (i.e. euploid+mosaics) (53% vs 50%). When considering female age, no significant difference is observed in the primary endpoints in the <37 years group. However, in women aged ≥37 years, the rate of blastocyst development is similar in the two sperm preparation groups, but a significant difference is observed in the timing of blastocyst development: The percentage of blastocysts formed on day 6 in the MSS group is significantly higher than in the Swim-up group (43% vs. 19% p < 0.05). In addition, the aneuploidy rate of blastocysts on day 6 is also significantly higher than in the Swim-up group (79% vs. 50% respectively, p < 0.05). The concentration of sperm recovered post-treatment was similar in Swim-up vs MSS (23 x106 vs 19 x106); total motility and progressive motility were similar in Swim-up compared to MSS (92.7% vs. 95.3% and 85% vs. 87% respectively). Only hyperactivated motility assessed with the SCASCOPE CASA System (Microptic) was significantly higher in MSS vs Swim-up 41% vs 4% respectively (p < 0.0005). Limitations, reasons for caution Our study included couples with different infertility diagnosis and semen parameters. Further studies with an increased number of patients may provide more comprehensive data to better evaluate biological and clinical outcomes to eventually suggest the use of MSS in laboratory routines Wider implications of the findings Recently, some studies reported that microfluidic-sorted sperm showed significantly less ROS formation and DNA fragmentation compared to those treated with conventional Swim-up method. Nevertheless, our data does not currently show the expected increase in embryo euploidy rate to justify the routine use of these expensive devices in male infertility treatment Trial registration number non applicabile

P-088 Bleomycin, etoposide and cisplatin (BEP) chemotherapy-induced testicular toxicity: ameliorative effect of sodium alginate by inhibiting oxidative stress, apoptosis and inflammation

Abstract Study question Is there a potential for sodium alginate (NaAL) to alleviate the damage induced by BEP chemotherapy in testicular cells and the spermatogenesis process? Summary answer Administering NaAL at doses of 25 and 50 mg/kg demonstrates protective effects against BEP-evoked toxicity damage through the modulation of nitro-oxidative stress, inflammation, and apoptosis. What is known already Testicular tumours are a prevalent malignancy affecting young males aged 20 to 35 years. The globally accepted chemotherapy for testicular cancer involves administering the bleomycin, etoposide, and cisplatin (BEP) regimen in three to four cycles. Unfortunately, the BEP regimen poses a substantial risk of reproductive system toxicity, presenting a significant concern and one of the most challenging long-term complications for young cancer survivors. Sodium alginate (NaAL), an anionic polysaccharide abundant in nature, offers potential solutions with its inherent antioxidant, anti-inflammatory, anti-apoptotic, and anti-proliferative properties. Study design, size, duration Sixty adult male Wistar rats were randomly assigned to six groups (n = 10/group). Groups 1, 3, and 4 received a vehicle, 25 mg/kg, and 50 mg/kg of NaAL, respectively. The remaining groups underwent a three-week cycle of bleomycin, etoposide, and cisplatin (BEP) with or without NaAL. NaAL administration began one week before BEP initiation, continued on days 2, 9, and 16, and extended for one week post the BEP cycle. Participants/materials, setting, methods At the end of the study (day 42), various parameters, including body and testes weight, sperm parameters (count, motility, viability, and morphology), testosterone hormone levels, testicular histopathology, stereological parameters of the testes, and levels of malondialdehyde (MDA), nitric oxide (NO), and total antioxidant capacity (TAC), were assessed. Additionally, the expression of Bcl-2, Bax, caspase-3, p53, and TNF-α was evaluated using real-time PCR and immunohistochemistry. Main results and the role of chance NaAL treatment resulted in a significant improvement in both body weight and testis weight in animals injected with BEP compared to the BEP group (P < 0.05). Stereological procedures, QRT-PCR examination, and immunohistochemical (IHC) staining for quantitative testis analysis indicated that NaAL reversed the impaired spermatogenesis induced by BEP (P < 0.05). Additionally, NaAL restored the disturbance caused by BEP in sperm count, motility, viability, and morphology. The testosterone level significantly decreased in the BEP-treated group compared to the control group (P < 0.05). However, co-administration of 50 mg/kg of NaAL significantly increased the serum testosterone level (P < 0.05). Furthermore, NaAL improved the antioxidant status of the testis by increasing TAC and ameliorating MDA and NO levels. Notably, QRT-PCR examination revealed that NaAL treatment suppressed BEP-induced apoptosis and inflammation by modulating genes such as Bcl-2, Bax, Caspase-3, and p53 in the testis (P < 0.05). Co-administration of 25 and 50 mg/kg of NaAL with the BEP regimen significantly reduced the number of p53 and TNF-α-positive cells compared to the BEP group. Further, NaAL at low and high doses increased the expression of Bcl-2 protein in spermatogenic cells compared to the BEP-treated group. Limitations, reasons for caution Due to limitations in our laboratory resources, we were unable to pursue further studies to validate the mechanism of sodium alginate on specific targets using more advanced techniques such as transfection and transgenic methodologies. Wider implications of the findings These findings illuminate the potential clinical applications of sodium alginate, suggesting its promising role as a beneficial supplement in medical contexts, particularly in addressing fertility issues. Additionally, this antioxidant could accelerate the fertility recovery period in young patients undergoing the BEP chemotherapy regimen, ultimately enhancing their overall quality of life. Trial registration number N/A

O-305 Mapping the entire functionally active seminal microbiota and its association with semen quality parameters

Abstract Study question Does semen harbour functionally active microorganisms, and whether the microbial composition associate with semen quality parameters? Summary answer Seminal fluid harbours functionally alive microorganisms including bacteria, viruses, archaea, viroids and fungi whose composition and metabolic functions associate with semen quality parameters. What is known already Seminal fluid has been shown to possess different microbes, where different bacteria have been associated with spermatogenesis, seminal parameters and infertility. Nevertheless, previous results are inconclusive and the core microorganismal composition in semen is not determined and whether the identified bacterial DNA sequences refer to alive/functionally active microbes is not clear. Further, there is limited knowledge of the potential host-microbe interactions. Study design, size, duration In total 78 men with age between 18 and 45 years donated semen for the study, 59 men with good quality semen parameters and 19 men with low semen quality and diagnosed asthenospermia, oligoasthenozoospermia or oligoasthenoteratozoospermia comprised control and case groups, respectively. Participants/materials, setting, methods Semen quality parameters were analysed following WHO, 2021 guidelines. Seminal fluid underwent NextSeq total RNA sequencing, separating human and non-human sequences with Hisat2 aligner. Taxonomic classification of microorganisms was obtained using Kraken2. Microbial species and human RNA-Seq analyses, comparing control and infertility groups, utilized metagenomeSeq and edgeR R packages. Human and microbial pathways were annotated using MetaCyc, followed by GSEA analysis. The integration of metabolic pathways between host and microbes was investigated. Main results and the role of chance With our total RNAseq analysis, we mapped the entire alive microbiota composing of 6453 microorganisms within the seminal fluid samples. Microbes such as bacteria, fungi, viruses, viroids and archea were identified. When analysing microbiota associations with seminal parameters, 404 microbial species (Lactobacillus, Gardnerella, Prevotella, Atopobium, Pseudomonas among others) were significantly differently present among infertile men vs. good quality semen group. The host transcriptomic analysis identified 2569 differentially expressed genes in infertile vs. good quality semen groups, involved in sperm differentiation, spermatid development, reproduction, cilium movement and meiosis. Metabolic pathway analysis detected possible metabolic activity in the host-microbiota crosstalk in FAO-PWY: fatty acid & beta-oxidation, LIPASYN-PWY: phospholipases, PWY66-429: fatty acid biosynthesis, PWY-3781: aerobic respiration I pathways. Limitations, reasons for caution Bigger sample size of infertile men would be needed to validate the study findings. Wider implications of the findings We confirm the presence of active microbes in semen with implications in seminal quality parameters such as motility, sperm count and morphology. Our results contribute to better understanding of seminal microbiota dysregulation in infertility with broader implications for reproductive health, suggesting functional links between seminal microbiota and sperm parameters. Trial registration number NA

P-011 Maternal gestational methyl donor deficiency affects reproductive development and fertility of mice offspring

Abstract Study question Is maternal methyl donor deficiency during gestation affects offspring’s reproductive health at adulthood? Summary answer Maternal gestational methyl donor deficiency affects reproductive development and fertility of F1 offspring and also leads to increased post implantation loss What is known already Reproductive disorders and their exact mechanisms are scarce in “The developmental origins of health and disease” (DOHaD) hypothesis which well explains the metabolic complications such as obesity, type 2 diabetes and hypertension that may take root during early development. The maternal nutritional environment during early development has important implications on offspring’s health later in life including reproductive health. The gestational period is of critical importance during which major epigenetic reprogramming occurs. The maternal methyl donor deficiency during gestational period may affect this process and epigenetic signatures of developing gametes and the fertility potential Study design, size, duration Pregnant female OCT4-GFP mice, fed on control chow diet (CCD) & 40% methyl-donors (Folate, Methionine & Vitamin B12) deficient diet (MDD) from gestation day (GD) 5 to GD 20. The gonadal development of F1 offsprings was studied on gestation day GD 18, post-natal day (PND) 22 and PND 60. Fertility study was done by mating adult F1 male offspring with adult female mice fed on standard diet Participants/materials, setting, methods Homocysteine levels in dams (F0) at GD18 were estimated by ELISA. Testosterone levels in F1 offsprings at PND 60 were estimated by ELISA. Furthermore, Serum glucose levels in F1 offsprings were measured at PND 60 and PND 75 by glucometer. Testicular histology, quantitative histomorphometry, immunofluorescence and TUNEL assay was performed at PND 60. Main results and the role of chance Fetal weight was reduced at GD13 and GD18 but postnatal body weight at PND 22 and 60 was not significantly affected in MDD group. Teratological defects were observed in pups of 40% MDD group as compared to CDD. Testosterone levels were significantly reduced in F1 offsprings of 40% MDD group. Glucose levels were similar in both the groups at PND 60 but at PND 75 it is significantly higher in case of F1 offsprings of 40% MDD. Maternal serum homocysteine levels and global DNA methylation levels of fetal gonads were affected. Testicular histology was significantly impaired in 40% MDD group at GD18, PND22 and PND60. Quantitative histomorphometry and immunofluorescence study showed the loss of spermatogonia and spermatocytes with significant increase in germ cells sloughing and seminiferous tubules atrophy. Apoptosis is also observed in seminiferous tubules. Copulation index, sperm count and motility was significantly reduced in case of 40% MDD group at adulthood. The F1 offspring of this group were sub-fertile with significant post implantation loss. Limitations, reasons for caution Although mice share ∼90% genome with humans, they may not resemble all aspect of physiology or pathology in humans hence it could limit the findings to extrapolate to humans. Wider implications of the findings The knowledge gained from this work could be helpful in highlighting the need of gestational micronutrient interventions specially methyl donors, which could help improving maternal and child health as well as reducing the future disease burden of reproductive disorders 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-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 &amp;gt;40%; LOW &amp;lt;40%) and Count (LOW &amp;lt;5 mil/ml; MEDIUM 5-15 mil/ml; HIGH &amp;gt;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 &amp;lt; 0.0001 and increasing aneuploidy rates (p &amp;lt; 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

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-651 Neither high nor low serum progesterone levels affect clinical outcomes in Artificially prepared-frozen embryo transfer cycles using intramuscular progesterone

Abstract Study question Does serum progesterone level on the day of frozen-thawed embryo-transfer (FET) correlate with the likelihood of achieving pregnancy in artificial FET cycles utilizing intramuscular progesterone? Summary answer Serum progesterone levels on the day of FET do not correlate with the likelihood of achieving a pregnancy in artificial FET cycles employing intramuscular progesterone. What is known already There is scientific evidence suggesting that serum progesterone levels under a specific cutoff level on the day of FET significantly decrease the clinical outcomes in patients under artificial endometrial preparation using micronized vaginal progesterone. However, no substantial evidence exists in patients under artificial endometrial preparation using intramuscular progesterone. Study design, size, duration This prospective cohort study was conducted from February to December 2022 and enrolled 353 patients undergoing FET in hormone replacement therapy (HRT) cycles using intramuscular progesterone until the day of embryo transfer. Participants/materials, setting, methods The study included women aged 21-40 years old undergoing FET, triple layer endometrium &amp;gt;7mm, and ≥2 high-quality embryos (Gardner’s grade 3/4/5/AA/AB/BA/BB) available. Exclusion criteria included patients with uncontrolled diabetes, hypertension, heart disease, known autoimmune diseases, uterine infertility factor, severe male factor infertility (total sperm count &amp;lt; 5 million/ml), and cases requiring surgically retrieved sperm for ICSI. Main results and the role of chance The cohort had an average age of 32.09 ± 4.19 years, BMI of 24.70 ± 3.07 kg/m², and endometrial thickness of 8.87 ± 1.06 mm on the day of embryo transfer. The mean serum progesterone level was 31.36 ± 13.78 ng/ml. Participants were categorized into quartiles based on serum progesterone levels. :Q1 (&amp;lt;21.7 ng/ml), Q2 (21.7-28.1 ng/ml), Q3 (28.2-40.0 ng/ml), and Q4 (≥40.0 ng/ml). No significant difference in Clinical Pregnancy Rate (CPR), Ongoing Pregnancy Rates (OPR), and first-trimester miscarriage rates were observed across quartiles. The OPRs according to serum progesterone quartiles were: Q1: 57.5%; Q2: 62.6%; Q3: 59.6%; Q4:62.8%, with no statistical difference among the groups. Binary logistic regression revealed no statistically significant differences in clinical pregnancy and ongoing odds among the quartiles. Limitations, reasons for caution Although a prospective study, its relatively small sample size may affect the results’ robustness and generalizability. Wider implications of the findings These findings suggest that when using intramuscular progesterone before embryo transfer in FET under HRT, serum progesterone testing may not be necessary before embryo transfer, as progesterone levels have no impact on clinical outcomes in this group of patients. Trial registration number CTRI/2022/11/046995

P-071 The dichotomy of micronutrients in male fertility: doses matter - striking the right balance for optimal reproductive health

Abstract Study question What is the impact of escalating doses of common water-soluble antioxidant nutrients on semen parameters and fertility in healthy (CD1) and stressed (Gpx5-/-) mice? Summary answer High-dose supplementation of previously “thought-to-be-safe” common water-soluble antioxidant nutrients can cause significant DNA damage/impaired fertility in healthy mice. What is known already In assisted conception, men increasingly take preconceptual antioxidant supplements (even if they are generally healthy) to enhance the likelihood of a successful pregnancy. Recent published reviews suggest 79% of male fertility supplements include ingredients at dosages exceeding Food Safety Authorities’ Recommended Dietary Allowances (RDAs). While many fertility experts question the efficacy of such supplements, most still carry the notion that “dietary ingredients”, regardless of the doses given, pose little safety concerns. Moreover, without established RDAs for many natural substances such as carnitines, supplement makers often subscribe to the “more is better” philosophy, potentially compromising safety in their product formulations. Study design, size, duration This study evaluates escalating doses of common water-soluble antioxidant nutrients (Vitamin C, Zinc, Folate, and Carnitines) on redox status, semen parameters, genetic integrity, and fertility in healthy (CD1) and stressed (Gpx5 -/-) mice over 28 days. The treated groups (n = 6) were compared against pooled controls (n = 24). We analyzed the doses of these ingredients in commercial fertility supplements against our new “safety profiles” to highlight potential overdosing risks and asses the broader safety implications. Participants/materials, setting, methods Experimental ingredient exposure dosages were based on current US-FDA RDA/I declarations, commercial popularity, and/or use in clinical fertility settings. Animal dosage conversion calculations were based on standard pharmaceutical methodologies. Assessments of male mouse redox status and reproductive function included general sperm parameters (count, motility, viability, acrosome integrity), DNA parameters [oxidation (8-OHdG staining), fragmentation (TUNEL), decondensation (Toluidine Blue)], and blood analysis (total antioxidant capacity, glutathione, malondialdehyde). Pregnancy outcomes were assessed in Carnitine fed, CD1 mice. Main results and the role of chance In CD1 mice, escalating doses of individual micronutrients had minimal effect on semen parameters, except for a significant sperm count increase at the highest Carnitine dose. However, there were considerable adverse effects on sperm DNA integrity and blood plasma redox state. The high-dose Carnitine group notably exhibited reduced pregnancy rates, in terms of both impregnation and total pups born, compared to controls. Yet, there were no differences in the number of pups born or resorptions, per pregnant female, between the high-dose Carnitine group and controls. In GPX5-/- mice, general semen parameters were mostly unaffected. Intriguingly, Carnitine positively influenced sperm DNA integrity in this model, underscoring the established link between micronutrient deficiency, oxidative stress, and semen quality. In the analysis of 34 random male fertility supplements, almost every product contained at least one ingredient at a dose that was harmful according to our study. Alarmingly, 88% had at least two such ingredients, 68% had three or more, and roughly 33% contained four or more at doses considered unsafe. Limitations, reasons for caution Carnitines’ pungent odor may have affected the mating efficiency of carnitine exposed CD1 males. Due to synergy of action, antioxidants administered individually, even at the same dose, may exert a different biochemical profile than when in combination with other ingredients (as in most commercial formulations). Wider implications of the findings The majority of male fertility supplements often combine aggressive doses of antioxidant nutrients. Our results highlight the potential dangers that may arise from their use and provide a clear warning to fertility experts against routinely recommending random, unsubstantiated supplements, especially in the absence of prior oxidative stress assessment. Trial registration number not applicable

P-064 Adolescent varicocele: clinical profile and impact of treatment

Abstract Study question What is the clinical presentation of adolescent varicocele and the impact of its repair on semen quality? Summary answer Adolescent varicocele usually presents as testicular pain and does not influence semen quality. Varicocele ligation does not impact postoperative semen results in this patient population. What is known already Varicocele is a common condition characterized by the abnormal dilation of the pampiniform plexus of veins resulting in testicular dysfunction and impaired semen quality. While the effect of varicocelectomy on the fertility potential of adult men has been commonly explored, its impact on semen parameters of adolescents is less commonly investigated. Study design, size, duration The charts of 2171 patients who underwent microsurgical subinguinal varicocelectomy for clinically palpable disease between 2011-2023 were retrospectively reviewed. Patients &amp;lt;19 years of age were included in the analysis. Participants/materials, setting, methods The patients’ medical charts were reviewed to include demographic and clinical characteristics; indication of surgery, body mass index, testis size, grade of clinical disease, vein diameter (assessed with scrotal ultrasound) and semen analysis results (WHO 2010). Data were analyzed using Wilcoxon Signed Ranked. Mean ± SD and median (IQR) were used to report normally and non-normally distributed data, respectively. A p value &amp;lt;0.05 was considered significant. Main results and the role of chance A total of 94 adolescent patients were included in the study. Their mean BMI was 22.14±5 Kg/m2, mean left testis size was 12.26 ± 3.05 cm3 and right testis size 13.26±3.94 cm3. The maximum vein diameter 4.7±1.04 mm and right vein diameter 2.21±0.83 mm. 83 patients underwent unilateral Microsurgical Varicocelectomy while 5 patients underwent bilateral Microsurgical Varicocelectomy. Left varicocele was of grades 3, 2 and 1 in 65, 18 and 11 patients, respectively. Surgery was indicated for testis size discrepancy in 36.2% of cases and on elective basis (prior to army recruitment) in 63.8% of cases. Table 1 presents the pre- and post-operative semen parameter results. Patients had normal pre-operative medians of sperm count, total motility and normal morphology. No significant post-operative changes were observed in the semen quality of adolescents. Limitations, reasons for caution Results were obtained from a relatively small sample size. However, varicocele is less commonly performed on adolescents necessitating further studies on this patient population. Wider implications of the findings Results of this study shed the light on the characteristics of adolescents presenting with varicocele. Trial registration number NA

P-082 Use of TetraSOD® in male infertility: double-blind randomized placebo-controlled study

Abstract Study question Can the use of TetraSOD® (microalgae with high content of the antioxidant enzyme superoxide dismutase) improve semen quality and reduce oxidative stress in sperm? Summary answer The use of TetraSOD® reduces the oxidative stress of sperm, increasing the total sperm count in oligozoospermia and reducing the double-stranded DNA sperm fragmentation. What is known already Male factor is present in approximately 50% of the infertile couples, a large number of them of unknown or idiopathic origin. It has been demonstrated that semen quality is deteriorating over time, perhaps as a result of exposure to several environmental factors related with lifestyle that could impact both directly and indirectly on the complex process of spermatogenesis. Some of these bad habits of lifestyle increase the levels of oxidative stress in semen, which is associated with lower sperm concentration and motility. In this scenario, the use of antioxidants can be useful to increase sperm quality. Study design, size, duration Double-blind randomized placebo-controlled clinical study to assess the effect of 250 mg of TetraSOD® (microalgae with high content of the primary antioxidant enzyme superoxide dismutase). Eighty patients with idiopathic infertility showing asthenozoospermia, oligozoospermia or oligoasthenozoospermia were recruited and randomized to receive 250 mg of TetraSOD® (TSOD group) or placebo (Placebo group) for 3 months. Participants/materials, setting, methods Men with asthenozoospermia, oligozoospemia or aligoastenozoospermia and idiopatic infertility were recruited after informed consent signature. Patients were randomized into two groups. TetraSOD® group received 250 mg of TetraSOD®during 3 months, and Placebo group received capsules without TetraSOD® during the same period of time. Seminogram, double-strand sperm DNA fragmentation (COMET assay) and sORP (static oxidation- reduction potential) were studied at baseline (V0) and after 3 months (V3). Main results and the role of chance TetraSOD® consumption for three months elicited a statistically significant increase (Wilcoxon matched-pairs signed rank test; p=0.02) in the average number of sperms per ejaculate (from 17.06 x 106 to 41.94 x 106) in participants displaying oligozoospermia (&amp;lt;39 x 106 sperms) at V0. In contrast, the lower variation in the measured values in the Placebo group was not statistically significant. Regarding sORP, a statistically significant decrease (Wilcoxon matched-pairs signed rank test; p=0.0233) was observed in participants who displayed a value at V0 between 1.34-10. After TetraSOD® consumption, the average changed from 2.96 to 1.91 mv/106 sperm/ml from V0 to V3. However, not statistically significant changes were detected in the Placebo group between sORP values at V0 and V3. Moreover, in relation to double-stranded DNA breaks, a statistically significant decrease (Wilcoxon matched-pairs signed rank test; p=0.01) in the average percentages (from 31.81% to 25.93%) was measured after consumption of TetraSOD® at V3 in participants displaying a compromised redox status (sORP&amp;gt;1.34-10) at V0. No significant changes were found in the Placebo group. Limitations, reasons for caution The study was performed only in one ethnic group, so in future studies other ethnicities should be included. Wider implications of the findings TetraSOD® may improve male fertility because increases the number of sperms per ejaculate in oligozoospermia and reduces double-stranded DNA fragmentation in sperm when individuals display an altered redox status in semen. Such results might be related to the indirect antioxidant effect of TetraSOD®, reducing oxidative stress in seminal fluid. Trial registration number Not applicable

P-040 The characteristic of human seminal plasma extracellular vesicle (SPEVs) profiles with different sperm features in a large cohort of 309 Taiwanese men

Abstract Study question To investigate human seminal plasma extracellular vesicle profiles in different sperm characteristics and their potential clinical application. Summary answer Normozoospermic semen has significantly higher SPEV concentration than asthenozoospermic or oligozoospermic samples. Moreover, SPEV concentration was highly correlated with total motile sperm count. What is known already Seminal plasma extracellular vesicles (SPEVs) are cell-derived membrane structures that carry proteins, nucleic acid, and lipids, which can modify the constitution and biological function of sperm. However, the characteristics of SPEVs in different sperm features remained unclear. Study design, size, duration From January 2021 to September 2023, 309 men who visited the NCKUH andrology clinic agreed to collect semen samples for this study, which the Institutional Review Board has approved. Participants/materials, setting, methods Semen parameters were measured by the X1 CASA system (Bonraybio, Taiwan). SPEVs were isolated by size exclusion chromatography column and ExoQuick™. Next, the SPEVs’ concentration and size distribution were performed with nanoparticle tracking analysis. The Kruskal-Wallis test calculated comparison in multiple groups, and Dunn’s multiple comparison tests accessed group-group comparison. The Pearson correlation coefficient r of exosome concentration with individual sperm parameters was calculated. A p-value less than 0.05 was set as statistical significance. Main results and the role of chance Among them, 59 semen samples were presented with normozoospermia, 36 were oligozoospermia, 73 were asthenozoospermia, and 141 samples had no sperm in semen. Among these 141 azoospermic men, 89 men are characterized by non-obstructive azoospermia (NOA), while the other 52 men had a vasectomy. Our data showed that normozoospermic semen has significantly higher SPEV concentration than asthenozoospermic or oligozoospermic semen samples (26 x109 [IQR 17.25 x109-56.25 x109] versus 12 x109 [IQR 5.6 x109-19.5 x109] versus 15.5 x109 [IQR 8.575 x109-26.75 x109], p = 0.0001). Nevertheless, azoospermic semen has the lowest SPEV concentrations. The concentration of SPEVs from infertile men diagnosed with NOA is slightly lower than vasectomized men (3.9 x109 [IQR 1.875 x109-7.675 x109] versus 5.4 x109 [IQR 2.9 x109-8 x109], p = 0.0001). The Pearson r coefficient of SPEV concentration with sperm concentration, progressive motility, and total motile sperm count was 0.238, 0.303, and 0.451, respectively. (P &amp;lt; 0.0001) Limitations, reasons for caution Because our data were collected from Taiwanese men, it is not yet known whether SPEV profiles differ among races. Wider implications of the findings Human SPEV concentration and its bio-content may be a potential non-invasive biomarker to predict the male reproductive outcome. Trial registration number not applicable

P-041 Impact of cigarette smoking on level of sperm reactive oxygen species in normozoospermic men

Abstract Study question Does cigarette smoking influence the levels of sperm reactive oxygen species (ROS) in men with normal semen routine parameters? Summary answer Cigarettes impact the sperm ROS of normozoospermic smokers, whose sperm ROS level is elevated when they smoke equal to or more than 10 cigarettes daily. What is known already Increasing evidence indicates that oxidative stress (OS) plays an independent role in the etiology of male infertility, with 30% to 80% of infertile men having elevated ROS levels in semen. Cigarette smoking leads to an imbalance between ROS and antioxidants in the semen of smokers, which may negatively affect semen quality and male fertility. However, few studies concern whether cigarette smoking would influence the sperm ROS levels in normozoospermic men. Study design, size, duration Data of semen analysis from January 2021 to December 2023 were involved. Men with normal semen routine parameters, including sperm concentration, total sperm number, sperm progressive motility, total motility and normal forms of sperm morphology, were included in the study. Sperm ROS levels of normozoospermic men were retrospectively analyzed. Men with varicocele and leucocytospermia were excluded. Participants/materials, setting, methods Sperm count and motility were analyzed by the computer-assisted sperm analysis (CASA) system, while sperm morphology was examined after the Diff-Quik rapid staining procedure. Sperm ROS was measured by flow cytometry using the probe DCFH-DA. Sperm ROS levels of smokers and non-smokers were compared by the Mann-Whitney U test. The Kruskal-Wallis H test was used for the comparison of sperm ROS levels among the control group and smoking subgroups. Main results and the role of chance A total of 2946 subjects were involved in the present study. The included subjects were divided into smokers (n = 730) and non-smokers (n = 2216) according to the clinical records. And the included smokers were subsequently divided into three subgroups according to the daily cigarettes taken, which were group A (1∼9 cigarettes/day, n = 184), group B (1∼19 cigarettes/day, n = 315), and group C (&amp;gt;20 cigarettes/day, n = 231). No statistically significant differences in age or BMI were found between smokers and non-smokers with normozoospermia. The sperm ROS level of the smoker was higher than that of the non-smoker [14.53% (14.84%) vs. 13.07% (12.09%), p = 0.001]. Compared to the control group (non-smoker), the sperm ROS levels of group B [13.07% (12.09%) vs. 15.44% (14.28%), p = 0.002] and group C [13.07% (12.09%) vs. 14.80% (14.31%)., p = 0.042] were significantly elevated. ROS levels in the control group and group A were comparable [13.07% (12.09%) vs. 13.46% (15.74%)., p = 0.391]. Limitations, reasons for caution It is a single-center study and the above results were from a retrospective analysis. Wider implications of the findings The above results indicate that cigarette smoking, although it would not deteriorate semen routine parameters, may increase the ROS level in sperm. Elevated ROS in sperm may lead to an imbalance of redox in sperm, which may contribute to male infertility. Trial registration number not applicable

P-130 Comparing head-first and tail-first injection techniques in guiding new embryologists for human oocyte Piezo-ICSI

Abstract Study question Which technique is more effective for new embryologists in human oocyte Piezo-ICSI: Head-first or tail-first injection? Summary answer Tail-first injection is superior as it significantly increases fertilization rates without adversely affecting embryo development compared to head-first injection in Piezo-ICSI. What is known already ICSI is an essential technique in assisted reproductive technology. Recently, some researchers have reported that Piezo-ICSI is clinically more useful than Standard-ICSI. In most papers, Piezo-ICSI has shown significantly higher fertilization rates than Standard-ICSI. This is because, in Piezo-ICSI, it eliminates the need to aspirate the cytoplasm into the ICSI needle upon membrane breakage. After membrane breakage, the sperm is injected into the cytoplasm. A few papers evaluated the effect of sperm injection direction (head-first or tail-first) on ICSI results for human oocytes. However, little information is available to guide new embryologists on whether to use head-first or tail-first injection. Study design, size, duration During their Piezo-ICSI training, new embryologists were taught either head-first or tail-first injection techniques before commencing clinical procedures. Only the head part of the sperm was injected into the cytoplasm in the head-first group, and half of the whole sperm was injected in the tail-first group. This study divided the new embryologists into two groups (head-first or tail-first) and collected and retrospectively analyzed their first 200 oocyte ICSI results between January 2017 and November 2023. Participants/materials, setting, methods This study involved five new embryologists and a total of 1,000 oocytes. Two embryologists performed head-first injection for 400 oocytes, and three performed tail-first injection for 600 oocytes. The rates of degeneration, 2PN, 0PN, 1PN, ≥3PN, good quality day-3 embryo (≥7cell, ≤15%fragmemtation) per 2PN, blastocyst, and good quality blastocyst (better than BB with Gardner criteria) were compared. Statistical analysis was performed using either a t-test, Welch’s t-test, or Fisher’s exact test as appropriate. Main results and the role of chance The average ages of women and their husbands in the head-first and tail-first injection groups were 37.8 vs. 37.2 and 40.1 vs. 39.2, respectively, with average total motile sperm counts of 52.2 x106 vs. 52.6 x106. No significant difference was observed in these comparison items between the groups. Theoretical injected medium volume into the cytoplasm with sperm of the head-first and tail-first injection groups was 104μm3 vs. 520μm3. The 2PN rate of the tail-first injection group (88.3%; 530/600) was significantly higher (P = 0.0151) than head-first (82.8%; 331/400). The 0PN rate of the tail-first group (4.0%; 24/600) was significantly lower (P = 0.0007) than the head-first (9.5%; 38/400). The degeneration rates, 1PN rates, and ≥3PN rates of head-first and tail-first injection groups were 1.5% (6/400) vs. 1.3% (8/600), 3.5% (14/400) vs. 2.3% (14/600), and 2.8% (11/400) vs. 4.0% (24/600). No significant difference was observed in these comparison items between the groups. The good quality day-3 embryo per 2PN rates, blastocyst rates, and good quality blastocyst rates of head-first and tail-first injection groups were 64.7% (214/331) vs. 64.3% (341/530), 62.1% (192/309) vs. 62.7% (289/461), 35.3% (109/309) vs. 35.6% (164/461). No significant difference was observed in these comparison items between the groups. Limitations, reasons for caution The estimated volumes of medium injected into the cytoplasm for both head-first and tail-first injections were theoretical. We calculated the length of the sperm head and whole sperm as 6μm and 60μm. Some day-3 embryos were transferred or cryopreserved, so the blastocyst and good-quality blastocyst rates were reference ranges. Wider implications of the findings Despite the larger volume of medium injected with sperm, tail-first injection increases fertilization rates without adversely impacting embryo development compared to head-first injection. Therefore, the tail-first injection is better to guide the new embryologists for human oocytes Piezo-ICSI than the head-first injection. Trial registration number 17-0450221102

P-488 The importance of identifying and addressing sexual functioning in couples with unexplained infertility

Abstract Study question How is the sexual functioning of newly diagnosed unexplained infertile couples and which factors are associated? Summary answer At least 25% of couples are at risk of sexual dysfunction. Sexual functioning is associated with age, infertility duration, sperm quality, personal and relational wellbeing. What is known already A large proportion of unexplained infertile couples have the ability to conceive naturally over time. Sexual functioning is important for natural conception rates, but infertility is associated with reduced sexual functioning. Surprisingly, sexual functioning and its risk-factors are still understudied in unexplained infertility. Knowing the risk factors for reduced sexual functioning would enable fertility specialists to timely diagnose, prevent or treat sexual dysfunction, and in this way improve natural conception rates. The current literature is mainly based on individual-level data, neglecting the fact that observations, such as sexual functioning, arising from couples are not independent. Study design, size, duration Cross-sectional digital survey data and medical chart factors, which also served as the baseline assessment of a randomized controlled trial (Pleasure&amp;Pregnancy, trial registration number NTR5709), were subjected to a dyadic analysis followed by linear regression. A total of 700 heterosexual couples recently diagnosed with unexplained infertility in secondary or tertiary fertility clinics were addressed between 2016 and 2021. Participants/materials, setting, methods Heterosexual couples (female age 18-38 year) with unexplained infertility and who were able to have coitus were eligible. Sexual functioning was assessed with the Female Sexual Function Index (FSFI) and International Index of Erectile Function (IIEF). Determinants included demographic, lifestyle and diagnostic and wellbeing factors as assessed with the Hospital Anxiety and Depression Scale (HADS) and the Revised Dyadic Adjustment Scale (R-DAS). B represents the impact on sexual functioning when a factor changes 1 unit. Main results and the role of chance A total of 581 (83%) women and 478 (68%) men filled out questionnaires. Survey data and medical chart factors were available for 451 couples. About one in four women (24.3%) and one in fourteen men (7.3%) were at risk of respectively female sexual dysfunction and erectile dysfunction after fertility work-up. Couples had a mean coital frequency of 7 times per month (SD 2.7). Higher female and male sexual desire (B: 0.04, p&amp;lt;0.01 and B: 0.02, p&amp;lt;0.02) and satisfaction (B: 0.03, p&amp;lt;0.01 and B: 0.06, p&amp;lt;0.01), but not orgasm were significantly associated with increased coital frequency. Own age (B: -0.64), anxiety (B: -9.47), depression (B: -7.61) and relationship distress (B: -8.97), and partners’ relationship distress (B: -5.08) and total motility sperm count (B: -4.88) were associated with lower female sexual functioning. Own age (B: -0.17), anxiety (B: -5.03), depression (B: -3.65), relationship distress (B: -5.77), and partners’ age (B: -0.46) and relationship distress (B: -3.14) and couples’ duration of infertility (B: -0.24) were associated with lower male sexual functioning. Limitations, reasons for caution The study is prone to selection bias given the inclusion of couples seeking medical help, consenting to a RCT and were having coitus. Not all previous identified determinants of sexual functioning were studied due to factors as missing data, low case numbers or not being assessed in the Pleasure&amp;Pregnancy RCT. Wider implications of the findings Clinicians advising couples with unexplained infertility to continue natural conception, need to be aware that at least 25% of couples are at risk of sexual dysfunction. Clinicians should consider risk factors of reduced sexual functioning, take a sexual anamnesis and advice sexual counselling and treatment if indicated. Trial registration number Baseline assessment of RCT (Pleasure&amp;Pregnancy) with trial registration number NTR5709

O-322 What is the number of IVF cycles needed to produce at least one euploid blastocyst in patients of all ages with varied AMH levels

Abstract Study question How many cycles are needed to get at least one blastocyst in patients of different AMH levels and varied ages? Summary answer Age and AMH determine the number of cycles, which ranges from 1 cycle in young patients up to 10 cycles in over-44-year-old patients. What is known already AMH secretion by ovarian granulosa cells initiates during the 36th week of gestation in pre-antral and antral follicles. This persists throughout a woman’s reproductive lifespan. AMH serves as a biomarker for assessing ovarian reserve and forecasting the ovarian response to stimulation. AMH is strongly associated with the quantity of antral follicles and the production of oocytes. AMH can be utilised to determine the initial dosage of FSH in an IVF cycle, as well as serving as a quantitative indicator of the number of oocytes produced. Study design, size, duration In this retrospective cohort study, data was collected from 16,417 ICSI cycles with Preimplantation Testing for Aneuploidy (PGT-A) conducted at three referral fertility centers from Jan 2019 to Dec 2023. We excluded cycles without serum AMH, endocrinopathies or recurrent pregnancy loss, males with total sperm count of less than 5 million and total motility less than 30%, and those with parental chromosomal abnormalities. Participants/materials, setting, methods Patients were stratified into three different categories of AMH (ng/ml), low (&amp;lt; 1.1 ng/mL), intermediate (&amp;gt;1.1 to &amp;lt; 3.0 ng/mL) and high (&amp;gt; 3.0 ng/mL). Each category was subdivided into 6 age sub-categories as follow (&amp;lt; 35), (35 to &amp;lt; 38), (38 to &amp;lt; 40), (40 to &amp;lt; 42), (42 to &amp;lt; 44), (&amp;gt; 44). All of the cycles underwent blastocyst stage biopsy and PGT-A utilising next generation sequencing (NGS). Main results and the role of chance Euploidy rate was statistically insignificant for all AMH levels in the same age group. However, euploid embryo numbers vary significantly. Euploid embryos numbers are shown in low, medium, and high AMH by age group. In the (&amp;lt;35) years was 1.9±1.5, 2.9±2.3, and 4.7±3.5, p &amp;lt; 0.0001. In the (35 to &amp;gt; 38) years was 1.3±0.8, 2.1±1.8, and 3.2±2.6, p &amp;lt; 0.0001. In the (38 to &amp;gt; 40) years was 0.9±0.8, 1.7±1.4, and 2.6±1.7, p &amp;lt; 0.0001. In the (40 to &amp;gt; 42) years was 0.6±0.5, 0.9±0.7, and 1.5±1.4, p &amp;lt; 0.0001. In the (42 to &amp;gt; 44) year was 0.26±0.2, 0.49±0.38, and 0.6±53, p &amp;lt; 0.0001. In the (≥44) years was 0.10±0.10, 0.23±0.21, and 0.40±0.37, p = 0.0170. We calculated the number of cycles needed to produce at least one grade BB or higher euploid blastocyst from the mean number of euploid embryos per cycle: The number of cycles is presented in low, medium, and high AMH. In the (&amp;lt;35) and (35 to &amp;gt; 38), 1 cycle is needed. In (38 to &amp;gt; 40) years, 2 cycles, 1 cycle and 1 cycle. In the (40-42) group 2 cycles, 2 cycles and 1 cycle. In the (42 to &amp;lt; 44) group 4 cycles, 3 cycles and 2 cycles. In the (≥44) group 10 cycles, 5 cycles and 3 cycles. Limitations, reasons for caution The retrospective nature of the study is the main limitation. Wider implications of the findings The study offers valuable insights for patient counselling regarding the required number of cycles to achieve euploid embryos, as well as for financial budgeting for both patients and insurance companies. Trial registration number Not applicable

P-099 The development of a novel sperm washing medium, enriched with serotonin as the active constituent, with the objective of enhancing sperm motility and viability

Abstract Study question Is it possible to develop a novel product that increases the success rate of treating male infertility through assisted reproductive techniques? Summary answer Sperm washing media, aiming to improve sperm quality, enhance sperm motility, and generally support male reproductive health in the field of assisted reproductive techniques. What is known already Infertility impacts 15% of all couples globally, with 50% of infertility cases attributed solely to male factors. Reduced semen motility is identified as a primary factor directly associated with infertility. The utilization of supplementation to enhance overall sperm quality has gained increasing popularity worldwide. The sperm preparation process in Assisted Reproductive Technology (ART) is crucial for successful fertilization, with sperm washing mediums playing a critical role in enhancing efficiency. Sperm-washing media is a specialized solution utilized in assisted reproductive technologies such as intrauterine insemination and in vitro fertilization to remove the seminal fluid and debris for sperm to undergo capacitation. Study design, size, duration In this study, 200 normozoospermic men were included, and their semen samples were analyzed over an 18-month period following the guidelines outlined by the World Health Organization (2010) to assess the efficacy of serotonin, selenium, zinc, L-Carnitine, and Coenzyme Q10 supplementation on sperm motility, viability, ROS and DNA damage. Participants/materials, setting, methods Sperm samples obtained from 200 volunteer men undergoing infertility assessments were subdivided into three samples.The sperm samples underwent preparation using the swim-up technique. The study involved the examination of samples in three distinct washing media: (1) Origio, (2) Irvine Scientific, (3) a prototype (serotonin, selenium, zinc, L-carnitine, and Coenzyme Q10). Each medium was analyzed for motility, apoptosis, reactive oxygen species (ROS), and DNA damage. Main results and the role of chance It is found that mean basal total sperm counts are 38 million. After sperm washing, mean total sperm counts of Origio®, Irvine® and Prototype are 15 million respectively (p &amp;gt; 0.05). As total progressive motility is % 40 before washing, it is increased to 62%,56% and 64% after washing with Origio®, Irvine® and Prototype, respectively (p &amp;gt; 0.05). Annexin V staining is used for detection of apoptosis in sperm samples. On average, early apoptosis over the total sperm count(TSC) per subgroups are 34.67% for Prototype, 33.17% for Origio® and 30.96% for Irvine®, whereas late apoptosis ratios are found significantly lower in Prototype compared to Origio and Irvine; 8.7%, 28.26% and 27.74%, respectively. Ratio of necrosis over TSC per subgroups is 3.15% for Prototype, 13.49% for Origio® and 18.78% for Irvine® whereas viability ratios are as 53.48%, 25.08%, 22.52%, respectively.(p &amp;lt; 0.05). ROS levels in semen after washing are significantly lower for Prototype (5.9%, 10.29%, and 18.22%, Prototype, Origio® and Irvine®, respectively) (p &amp;lt; 0.05). Limitations, reasons for caution Semen samples from males aged 28 to 45, who are unable to conceive, do not smoke, and have not undergone any surgical interventions, are collected from patients after a sexual abstinence period of 3-4 days. Wider implications of the findings We propose that our serotonin-activated washing media for sperm preparation can serve as a suitable choice for selecting high-quality sperm in assisted reproductive technology programs. These molecules may enhance the success of assisted reproduction techniques when present in sperm preparation media. Trial registration number SerotoneX

P-044 Increased CYP1A1 expression enhances sperm apoptosis in idiopathic infertile men

Abstract Study question How are the CYP1A1 and Caspase-9 genes expression and the relationship among the expression of these genes with sperm parameters in idiopathic infertile men? Summary answer The CYP1A1 and Caspase-9 gene expression showed a significant increase in patients in comparison with the healthy group. What is known already CYP1A1 (Cytochrome P450 Family 1 Subfamily A Member 1) plays a crucial role in xenobiotic metabolism induced by aryl hydrocarbon receptor (AHR). AHR, as a ligand-activated transcription factor is involved in exogenous ligand activation. CYP1A1 is one of the most essential enzymes in phase 1 of detoxification, metabolism, and bio-activation of carcinogens and is the most well-known target gene for AHR. Monooxygenase activity of CYP1A1 may lead to oxidative stress and the formation of reactive intermediates with destructive effects such as apoptosis in testicular germ cells. CYP1A1 may lead to apoptosis via Caspase-9 activation. Study design, size, duration In this study, we aimed to determine the CYP1A1 and Caspase-9 gene expression. Participants/materials, setting, methods The infertile idiopathic and healthy fertile men were selected and evaluated for sperm analysis and gene expression. The expression levels of CYP1A1 and Caspase-9 were also determined by real time PCR. Main results and the role of chance The sperm count, morphology and motility were significantly lower in patients compared to the control group (p &amp;lt; 0.05). The CYP1A1 and Caspase-9 gene expression showed a significant increase in patients in comparison with the healthy group (p &amp;lt; 0.05). Limitations, reasons for caution Further investigations of the effect of CYP1A1 and Caspase-9 expressions on infertility by using more infertile patients and also studying the effect of other important genes such as AHR can show us more accurate results. Wider implications of the findings Our results suggest that increased CYP1A1 and Caspase-9 expression could increase the rate of male infertility. In addition, the increase in Caspase-9 expression could lead to a reduced number of sperms and an increased infertility rate by increased apoptosis in the spermatogenic lineage. Trial registration number not applicable

Ambient Ozone Exposure, Semen Plasma Metabolites, and Sperm Quality Decline among Adult Men in Wuhan China.

Ambient ozone (O3) exposure may be associated with a reduction of semen quality, yet the potential biological mechanisms remain unclear. We investigated the effects of certain seminal plasma metabolites on mediating the links between O3 exposure and the deterioration of semen quality. The untargeted metabolomics analysis was performed on semen samples of 200 Chinese adult men to determine candidate metabolites associated with characteristics of semen quality. Mediation analysis was adopted to examine whether these metabolites modulated the links between O3 exposure and semen quality. We found a significant reduction in sperm concentration by -28.1% (95% CI: -41.7%, -11.3%), and sperm count by -29.2% (95% CI: -43.7%, -11.0%) associated with each 10 μg/m3 increase in ambient O3 concentration during the period of sperm development. We delineated 7 metabolites in seminal fluid that substantially mediated the links between O3 exposure and declined semen quality, including myristoleic acid, aspartyl-isoleucine, phenylethyl primeveroside, ACar (18:2), ACar (18:1), FAHFA (22:6/22:3), and LPS (22:5). Among these, myristoleic acid exhibited the most pronounced mediation effects, with its indirect effect of which accounts for 46.4% of the overall association. Our findings suggested that exposure to ozone decreased sperm quality by disrupting fatty acid metabolism, particularly myristoleic acid.

Hirsutella sinensis intensifies testicular function and spermatogenesis in male mice with high-fat diet-induced obesity.

Hirsutella sinensis (HS) is a mycelium isolated from the fruiting body of the medicinal mushroom Cordyceps sinensis . This study explored whether HS treatment affects reproductive dysfunction in a high-fat diet (HFD)-induced mouse model and regulates various mechanisms, focusing on oxidative stress, apoptosis, inflammation, and autophagy. Twenty-four C57BL/6J (B6) mice were randomly divided into a standard chow diet (NCD)- or HFD-fed group for 24 weeks. During the final 8 weeks, half of the HFD-fed mice were orally administered HS (HFD + HS). Biochemical markers, including glucose, insulin, triglycerides, and total cholesterol, were assessed, and hormones, including testosterone, follicle-stimulating hormone (FSH), and luteinizing hormone (LH), were analyzed. Liver and testicular histology, as well as sperm quality markers such as sperm motility, sperm count, and percentage of sperm with normal morphology, were observed. The activities of the testicular antioxidants superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx) and the products of lipid peroxidation, such as malondialdehyde (MDA), were measured. The protein expression levels of apoptosis-, autophagy- and inflammation-related markers were measured. The HFD-fed mice had abnormal sex hormone levels, poor sperm quality, and a destroyed testicular structure, with increased oxidative stress and apoptosis in the testis. HS supplementation in HFD-fed mice attenuated testicular apoptosis by suppressing the Bax/Bcl-xl ratio and cleaved caspase 3 protein expression. The HS-treated mice exhibited improved reproductive function, possibly due to reduced oxidative stress and apoptosis, suggesting that HS has a protective effect against HFD-induced testicular damage. Male mice supplemented with HS exhibited attenuated poor semen quality and reduced testosterone levels brought about by HFD-induced obesity by reducing oxidative stress.