DNA Fragmentation Research Articles

P-273 Image-based oocyte model predictive of blastocyst development correlates with ploidy status across a broad spectrum of PGT-A indications, accounting for confounding variables

Abstract Study question Does MAGENTA, an AI assessment of oocyte quality, indicate ploidy potential of the developed blastocysts in the presence of confounding variables? Summary answer MAGENTA correlates to oocytes that develop into euploid embryos across diverse demographics, highlighting its robust ability to assess oocyte quality and genetic potential. What is known already MAGENTA is a non-invasive AI-image analysis tool that assesses images of mature oocytes and provides a score from 0-10, where higher scores indicate higher chances of developing into a blastocyst. MAGENTA has also displayed a correlation to blastocyst morphological quality. The ploidy status of blastocysts plays a significant role in cycle success and is often utilized for the selection of embryos to transfer. Despite the selection of embryos based on their genetic normality, many transfers are unsuccessful. With most chromosomal errors arising in the oocyte, assessment at the gamete level is critical to improving outcomes. Study design, size, duration A retrospective study using CRM Weill Cornell Medicine data included 3,342 mature oocytes that developed into blastocysts and underwent PGT-A in 2021. Blastocysts tested as mosaic (n = 753) or inconclusive (n = 13) were excluded. Images of the oocytes were obtained immediately post-ICSI using EmbryoScope time-lapse incubators (Vitrolife, Sweden). Blastocyst morphological grades were categorized by quality: highest (ICM+TE grade A), high (ICM+TE grade A/B), medium (ICM+TE grade B), or low quality (ICM or TE grade C/D). Participants/materials, setting, methods Data was obtained from 629 patients (females 26-45 years old) with an average of 5.3 blastocysts (range 1-20) with PGT-A results per patient. MAGENTA assessed each oocyte image and provided a score (0-10), with higher scores representing a greater potential to develop into a blastocyst embryo. In this dataset, 1827 (55%) blastocysts were tested as euploid and 1515 (45%) were tested as aneuploid. PGT-A was conducted for various specific and non-specific indications in these patients. Main results and the role of chance MAGENTA was positively associated with oocytes that developed into blastocysts of increasing quality, with significance between highest and high-quality (7.4 vs 7.0,p<0.05) and medium and low-quality (6.9 vs 6.1,p<0.01). Of the blastocysts, the MAGENTA score further displayed significant differences between oocytes that developed into euploid (7.0) compared to aneuploid blastocysts (6.6) (p < 0.001;Welch’s t-test). Although MAGENTA does not predict ploidy, a threshold of 8.8 MAGENTA score was determined to best distinguish between ploidy outcomes with 61% euploidy rate above this threshold compared to 51% below, a significantly different proportion (p < 0.001;Two-Sample Proportions z-test). Therefore, oocytes scored ≥8.8 display a 19.6% relative increase in chance of developing into a euploid blastocyst. To account for female age, subgroup analysis was conducted and found MAGENTA scores to be significantly different between oocytes that developed into euploid and aneuploid blastocysts for patients <35 years old (7.2 vs 6.6, p < 0.01;Welch’s t-test) and ≥35 years old (6.8 vs. 6.5, p < 0.05;Welch’s t-test). Blastocyst morphological grade was also investigated as a confounding factor. No significant differences existed between MAGENTA scores of euploid and aneuploid blastocysts among the low, high, or highest-quality. Significantly, differences were only found within medium quality blastocysts (7.0 vs 6.6,p<0.05), which had the greatest sample size (n = 1854). Limitations, reasons for caution MAGENTA was not trained to predict euploid embryo development from images of mature oocytes. Further data is required to discern the correlation of MAGENTA to chromosomal ploidy status, apart from blastocyst quality. Additionally, investigation into sperm DNA fragmentation as a potential contributor to ploidy determination is warranted. Wider implications of the findings MAGENTA can identify oocytes of better quality that not only have higher chances of blastocyst development and higher morphological grades, but also indicates oocytes that are more likely to become euploid embryos across patients of various PGT-A indications and a broad age demographic. Trial registration number not applicable

P-018 Study of sperm DNA fragmentation in the infertile population before, during and after the SARS-CoV-2 pandemic: a nationwide retrospective cohort study

Abstract Study question Did the values of single- and double-strand sperm DNA breaks (SSB and DSB) increase during the SARS-CoV-2 pandemic? Summary answer During the SARS-CoV-2 pandemic (2020), the mean values of SSB and DSB in infertile patients were statistically higher compared to the periods 2018-2019 and 2021-2022. What is known already Some studies have found lower sperm concentration and motility in patients during and after SARS-CoV-2 infection (COVID-19). These effects appear to be reversible, with significant improvements being observed after three months post-infection (complete spermatogenesis cycle). On the contrary, vaccination against this virus does not seem to affect semen parameters. The objective of the study was to compare the values of SSB and DSB sperm DNA fragmentation in the periods before the pandemic (2018-2019), during (2020, highest number of infections and social confinement), and after (2021-2022, lower infection rate, end of social confinement and vaccination period). Study design, size, duration The average SSB and DSB sperm DNA fragmentation values of 11,215 patients referred from 74 fertility centres throughout Spain from January 2018 to December 2022 were retrospectively compared. Participants/materials, setting, methods Semen samples from infertile patients were analysed using the Comet assay in alkaline or neutral pH conditions to evaluate SSB or DSB, respectively. Three periods were compared (two years before, during 2020, and two years after). Mean SSB and DSB values were statistically compared among these periods using the Kruskal-Wallis and Mann-Whitney tests. Differences were established at the 99% confidence interval. Main results and the role of chance The average SSB values obtained were: 41.67%±15.33 in 2018-2019; 43.92%±17.84 in 2020, and 41.68%±18.58 in 2021-2022. The average DSB values obtained were: 65.64%±14.27 in 2018-2019; 67.77%±13.90 in 2020, and 65.23%±15.18 in 2021-2022. The SSB and DSB values were statistically higher in 2020 compared to the periods 2018-2019 and 2021-2022 (p < 0.001 in both cases). No differences were detected between the previous period (2018-2019) and the following period (2021-2022) (p = 0.101 and p = 0.235, respectively). Limitations, reasons for caution The observed increase in SSB and DSB values during 2020 cannot be directly attributed to the SARS-CoV-2 infection (COVID-19). It is possible that it is associated with other concurrent factors. The studied samples were only from infertile patients, not from the general male population. Wider implications of the findings Given that the SARS-CoV-2 virus still exists in 2021-2022 but the social situation has normalized, the increase in fragmentation observed during 2020 could be more related to other aspects of the pandemic, such as social confinement, stress, among others. Trial registration number Not applicable

Biosynthesis, Antibacterial and Antifungal Activities of Ag Nanoparticles Prepared Using Piper nigrum Fruit Extract

Nanomaterials are promising alternatives to antibiotics with the potential to handle the menace of emerging multidrug resistance in pathogenic microbes. We report a simple biosynthetic approach to prepare Ag nanoparticles (NPs) with strong antibacterial and antifungal actions by using Piper nigrum fruit extract as a bioreducing agent. The biosynthesized material was characterized by SEM, AFM, TEM, HRTEM, XRD and UV–Visible absorption spectroscopy. SEM, AFM and TEM analyses provided evidence that the average diameter of the Ag NPs was 18[Formula: see text]nm, while XRD and HRTEM confirmed the FCC structure of Ag NPs. The biosynthesized Ag NPs demonstrated extremely strong antibacterial activity against Bacillus oceanisediminis and Escherichia coli, and showed extremely strong antifungal action against Schizosaccharomyces pombe, inhibiting their growth at very low concentrations of 3[Formula: see text][Formula: see text]g/mL, 4[Formula: see text][Formula: see text]g/mL and 3[Formula: see text][Formula: see text]g/mL, respectively. The molecular origin of their antibacterial and antifungal activity was studied using various molecular and biochemical assays. The results of these assays revealed a major role for reactive oxygen species (ROS) in the antifungal and antibacterial actions of the biosynthesized Ag NPs. Furthermore, morphological changes in cells and cell wall damage upon Ag NP treatment were observed by scanning electron microscopy. In addition, significant enhancement in protein leakage and fragmentation of DNA was also observed upon treatment of S. pombe, B. oceanisediminis and E. coli with Ag NPs. In conclusion, the observed extremely strong antibacterial and antifungal activities of biosynthesized Ag NPs are due to the oxidative stress triggered by ROS, causing cell wall damage and subsequent protein leakage and DNA fragmentation.

P-068 Fertility education improves knowledge and semen quality: an open-label randomized controlled trial on 382 healthy Japanese men

Abstract Study question Does fertility education improve semen quality in healthy men? Summary answer Healthy men who received online video-based education had significantly improved fertility knowledge and semen quality. What is known already Modifiable lifestyle and environmental factors can affect semen quality, but men generally have limited knowledge about fertility and are not aware of the impact of these factors on their reproductive health. Prior research indicates that fertility education increases fertility awareness, but whether this effect extends to modifications in lifestyle and thus to semen quality remains unexplored. Study design, size, duration Participants (N = 382) were randomised into two groups. The intervention group (IG) watched a five-minute online video series about healthy lifestyles and fertility risks (e.g., sedentary time, smoking, high temperature) every four months, a total of three times. At the same time points, the control group (CG) received a general health web article. Assessments of fertility knowledge, health behaviour, and semen quality were assessed before the intervention (T1) and one year later (T2) for both groups. Participants/materials, setting, methods Participants were male employees recruited during annual health check-ups at six workplaces in Japan. Those aged 20-39 years who planned to have children in the future participated. The primary short-term outcome was semen quality, measured by sperm concentration, sperm count, sperm motility, and DNA fragmentation index (DFI). Secondary outcomes were Cardiff Fertility Knowledge Scale (CFKS) scores and health behaviours (e.g., reduced sedentary time). All analyses were conducted on an intention-to-treat basis. Main results and the role of chance Of the 382 participants, 208 (55%) completed the T2 survey: 53% (99/188) in the IG and 56% (109/193) in the CG. T1 characteristics, fertility knowledge, health behaviours, and semen quality were well balanced between groups. Generalised estimating equation models showed a significant gain in fertility knowledge from T1 to T2 in both groups: +13.2 points on CFKS (95% confidence interval [CI]: 9.7 to 16.7) in the IG and +4.7 (1.3 to 8.1) in the CG, with a significant interaction between group and time (P<0.001). Total motile sperm count showed a significant interaction between group and time (P=0.046), with a significant improvement over time in the IG (+58.1 [15.2 to 101.1] million/ejaculate) but no change in the CG (-2.6 [-43.8 to 38.6]). DFI showed a marginally significant interaction (P=0.064) and an improvement over time in the IG (-0.72 [-1.54 to 0.10]) compared with the CG (+0.35 [-0.43 to 1.13]). The other semen indicators and health behaviours showed a favourable trend in the IG, but the interactions between group and time did not reach statistical significance. Limitations, reasons for caution Difficulties in recruiting participants and high attrition led to a small sample size, which may limit the statistical power to detect a significant link between knowledge gain and improved sperm quality via altered health behaviours. Volunteer bias towards men more interested in fertility may limit the generalisability of the findings. Wider implications of the findings Low-cost short video interventions could improve semen quality at the population level. As a future direction, the preventive effects of fertility education on infertility and the causal pathway should be explored in larger studies. Trial registration number UMIN000045389

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 & 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 & 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 & 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 & pregnancy outcomes were collected pre and post-operatively. Variables were examined before & after varicocelectomy. X2 test and Kruskal-Wallis test were used to analyze categorical and numerical values, respectively. A p value < 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 & 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%) & 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 & progressive motility & DNA fragmentation. Pregnancy outcomes improved postoperatively for both spontaneous (32.3%) & 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 & choose the best management for this group of patients. Trial registration number NA

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-103 Sperm selection for ICSI by rheotaxis and thermotaxis in In-Situ handmade microfluidics of fluidic walls: preliminary results of a Randomized Controlled Trial

Abstract Study question Does sperm selection by rheotaxis and thermotaxis in In-Situ handmade microfluidics of fluidic walls optimize ICSI outcomes and workflow? Summary answer In-Situ handmade rheo/thermotaxis microfluidics of fluidic walls is as effective and more time-efficient than centrifugation density gradients to support pre-implantation embryonic development to blastocyst. What is known already Microfluidics technologies stand as the latest sperm selection methodology for ICSI. To outline their application in ART we conceived a lab-on-a-chip approach for sperm selection by In-Situ handmade microfluidics of fluidic walls (isM) in the same ICSI plate which integrates two operative zones: one to allow seminal plasma dispersion and sperm thermotaxis and another one to allow sperm rheotaxis. Our microfluidics system can be prepared with any media suitable for ICSI, disregarding centrifugation, washing, plasticware and other cells. The isM protocol proved non-inferior to centrifugation density gradients (CDG) to support embryonic development and implantation in a previous sibling-oocyte study. Study design, size, duration The present RCT was approved by our ethical committee for research with medical products (CEIm approval number 2023/394). This preliminary analysis aims to detect deviations in the pre-defined primary outcomes: Normal Fertilization Rate, Day 5/6 usable blastocyst rate per MII, and per zygote. This analysis includes data from 21 couples undergoing an ICSI treatment in our center during December 2023-January 2024. Only patients without clinical or analytical suspicion of sperm DNA fragmentation were included. Participants/materials, setting, methods Patients were randomly allocated to isM (n 9) or to CGD (n 12) groups. All participants signed an informed consent. Sperm samples were processed directly after liquefaction. For CGD, samples were prepared in 80-40% PureSperm® layers by isopycnic centrifugation followed by washing and dilution steps. The isM system was prepared by displaying three microdroplets of different volumes following a L-shape interconnection and allowing a temperature gradient of 2ºC between the first and the second microdroplet. Main results and the role of chance Sperm characteristics showed the following total mean values for the isM and CGD groups, respectively: concentration (58,2±38,0 × 106/ml and 52,7±29,1 × 106/ml) and progressive sperm concentration (16,6±17,0 × 106/ml and 16,8±13,4 × 106/ml). Women average age was 36,7±2,9 isM and 35,8±2,5 CDG. This analysis includes a total of 168 MII oocytes (n = 69 isM and n = 99 CDG), with a mean number of MII oocytes per patient of 7,4±2,1 isM and 8,3±1,9 CDG. Comparisons between the two groups showed that isM protocol optimizes ICSI outcomes by means of Normal Fertilization Rate: 88,4% vs 76,8%; p < 0,05, Day 5 A/B grade blastocyst rate: 36,1 vs 31,6%; ns, Day 5/6 usable blastocyst rate per MII: 58,0% vs 44,4%; ns and Day 5/6 usable blastocyst rate per diploid zygote: 65,6% vs 57,9%; ns. The isM protocol also optimizes the IVF workflow compared to CDG, as the time to obtain sperm for ICSI after liquefaction was 26 vs 77 minutes, respectively. Limitations, reasons for caution The study aims for a total size of 50 ICSI cycles to validate statistically our data at a power of 80%. The isM protocol depends on a free hand preparation. A ready-to-use surface would benefit the operability, albeit the use of templates and the training reduces inter and intra-operator variability. Wider implications of the findings The potential benefits of the isM protocol on fertilization could be significant for patients with a limited number of available oocytes for ICSI. According to the literature, microfluidics reduce the DNA fragmentation index in the selected sperm fraction. The isM method could be especially beneficial in patients with this pathology. Trial registration number NCT number assignment pending

P-113 Comparison of mitochondrial function between expanded and hatching human blastocysts

Abstract Study question Is there a difference in mitochondrial function between expanded and hatching fresh and vitrified blastocysts? Summary answer Freezing and thawing may lead to mitochondrial dysfunction. What is known already Mitochondria, as crucial cellular organelles, play role in energy production through the electron transport chain, cell motility and organelle movement. They also have a significant impact on embryonic development. Furthermore, during the process of embryonic hatching, the energy generated by these organelles can affect ion pump activity, blastocyst expansion, and collapse. Therefore, factors that stress embryonic mitochondria may contribute to embryogenesis and hatching. Cooling and warming, as stress factors, maybe result in organelle damage, protein denaturation, membrane disruption, and DNA fragmentation. Additionally, they can cause changes in mitochondrial function, leading to decreased ATP levels and increased ROS levels. Study design, size, duration In this study, vitrified and fresh human 8-cell embryos were collected from April 18, 2021, to July 19, 2023. The embryos were cultured until the blastocyst stage, and then four samples were assessed in four groups. These groups included vitrified expanded blastocysts, vitrified hatching blastocysts, fresh expanded blastocysts, and fresh hatching blastocysts. Participants/materials, setting, methods Vitrified embryos were sourced from the embryo bank of the Royan Institute, while fresh embryos were obtained from good-quality 3PN embryos or excess embryos from couples who did not wish to freeze them. A stock solution containing lipophilic cationic JC-1 dye in dimethyl sulfoxide was prepared. Blastocysts were then exposed to a concentration of 1 μg/ml of JC-1 diluted in HamsF10-HSA. After washing with HF-HSA, imaging was performed using a 2-photon confocal microscope. Main results and the role of chance Our results showed that mitochondrial membrane potential in expanded blastocysts of both fresh and vitrified eight-cell embryos was significantly higher than in hatching blastocysts of fresh and vitrified embryos (P < 0.05). However, there was no significant difference in mitochondrial function between the vitrified and fresh groups, neither in expanded nor hatching blastocysts. Limitations, reasons for caution Due to the human origin of the samples, the minimum necessary replicates were considered for each experimental group. Wider implications of the findings In this study, it appears that plump zona breakers can be detected in hatching blastocysts using confocal microscopy. Previous studies, such as the one conducted by Sathananthan, identified these cells using a TEM microscope. Trial registration number -

P-357 Exposure to bleomycin in vitro reduces biomarkers of human male gamete competence

Abstract Study question Does male human sperm exposure to bleomycin in vitro affect their aptitude to breed? Summary answer Bleomycin, an antitumor antibiotic, adversely impacts human male gamete morphology and functionality, thus potentially compromising male fertility. What is known already Bleomycin is a chemotherapeutic agent used to treat a number of tumors that affect males of reproductive age. Hitherto, its effect on human male reproductive capacity alone has not been previously shown. The little information available on this subject relates to in vivo animal studies and supports the feasibility of this study. Study design, size, duration Between November 2022 and June 2023, individuals of reproductive age were enrolled in this study and had their clinical semen analyzed. The remaining ejaculate was supplied by 45 participants with sperm concentration above the World Health Organization (WHO) threshold for scientific purposes. Sperm were isolated from seminal plasma in the ejaculate of each individual, diluted to a concentration of 16 X 106 sz/mL (raw). Participants/materials, setting, methods In vitro incubation was performed in sperm preparation medium (SPM; control) or bleomycin (100 µg/mL; exposed) augmented for two hours at 37oC and 5% CO2. The results of exposed group were compared to raw and control. To evaluate the competence of human sperm, markers such as acrosome and DNA integrity, mitochondrial membrane potential, sperm motility, vitality, and oxidative stress were evaluated. Ultrastructural data from transmission electron microscopy was also obtained and correlated with functional experiments. Main results and the role of chance The participants were all of reproductive age (median age of 36 years old). The raw sperm parameters fulfilled the normozoospermia threshold set by the WHO, 2021. Prior to the current trial, none of the participants had been diagnosed with cancer or had received chemotherapy, radiation therapy, or both. Bleomycin exposure for two hours in vitro significantly (P < 0.001) reduced sperm motility, vitality, and chromatin condensation as compared to raw and control sperm. Moreover, bleomycin significantly (P < 0.001) increased the percentage of sperm with DNA fragmentation (sDNAfrag) and low mitochondrial membrane potential (MMP). Broad strands were significantly (P < 0.001) attained by bleomycin-induced breaks in the sperm DNA (Head staining pattern), which were succeeded by histone-rich areas (PAR-post-acrossomic region staining pattern). Additionally, bleomycin significantly (P < 0.05) delayed the acrossomic response, but had no significantly influence on the generation of extracellular and intracellular reactive oxygen species or in its reconstitution. Modifications in ultrastructural morphology caused by bleomycin corroborate the results obtained by functional tests. Limitations, reasons for caution Bleomycin impact on male fertility competence in vivo remains crucial. Increased sample sizes, including prepubertal subjects, are needed to pinpoint the precise bleomycin’s mechanism to cause oxidative stress, apoptosis, and sDNAfrag. Long-term implications on fertility and reproductive health, besides methods to lessen or offset bleomycin’s effects, should also be pursued. Wider implications of the findings Bleomycin adversely affects male gamete competence in humans. Along with its accompanying lung toxicity, healthcare practitioners should actively monitor and further investigate bleomycin’s gonadotoxicity in males. It is advised that all cancer patients undergoing chemotherapy treatments that contain bleomycin be provided with a fertility preservation plan in the meantime. Trial registration number not applicable

P-050 Microfluidic chip-sorted spermatozoa selection yields embryo euploidy rate and reproductive outcomes similar to density-gradient centrifugation

Abstract Study question Does microfluidic chip-sorted spermatozoa selection (MSS) improve good quality blastocyst (GQB) and embryo euploidy rates compared to density gradient centrifugation (DGC)? Summary answer The use of MSS before intracytoplasmatic sperm injection (ICSI) does not improve good quality blastocist and euploidy rates comparing to DGC. What is known already The integrity of sperm DNA plays a crucial role in embryonic development. Conventional sperm selection methods, such as DGC, may potentially damage sperm DNA due to oxidative stress generated during centrifugation. One of the recent advances in sperm selection techniques is the microfluidic chamber, which, requiring less processing time and eliminating the need for centrifugation, allows for the selection of sperm with better mobility, morphology, and reduced DNA damage. Consequently, improved GQB and euploid embryo rates, as well as enhanced reproductive outcomes, are expected. Study design, size, duration Retrospective study of 1739 blastocysts from 404 couples undergoing 544 autologous cycles of ICSI/PGT-A (preimplantation genetic testing for aneuploidies) from January 2020 to December 2022. Those patients with sperm concentration <5 × 106/mL or motility <10%, altered karyotype, women aged > 43 years, uterine anomalies and thrombophilia were excluded. Besides comparing euploidy rates and blastocyst formation, reproductive outcomes were analyzed and compared. Participants/materials, setting, methods According to the sperm selection technique, two groups were established. One group underwent MSS (ZyMōt ICSI® or ZyMōt Multi®), resulting in 295 blastocysts from 96 cycles. It was compared with a control group (DGC) including 1444 blastocysts from 448 ICSI/PGT-A cycles. PGT-A was performed in day 5/6 using Next Generation Sequencing (NGS) technology. For a 95% confidence interval, statistical significance was considered at p < 0.05. Chi-square analysis was used for the statistical evaluation. Main results and the role of chance Both groups were comparable in terms of demographic data, including age, sperm analysis parameters, and cycle characteristics. There were no significant differences between the DGC and MSS groups in the women’s age (mean ± SD) (37.9 ± 3.9 vs. 38 ± 3.9 years, p = 0.99) or men’s age (38.4 ± 7.1 vs. 39.8 ± 5.9 years, p = 0.11). No significant differences were found between the DGC and MSS groups in terms of GQB rate (49.3% vs. 50.7%, p = 0.55), euploidy rate (35.8% vs. 32.2%, p = 0.23), fertilization rate (69.5% vs. 69.5%, p = 0.99), clinical pregnancy rate per transfer (63% vs. 76%, p = 0.13), clinical miscarriage (9% vs. 21%, p = 0.33), and live birth rate per transfer (44% vs. 37%, p = 0.42). The results also showed a higher number of euploid embryos per initiated cycle in the DGC group (1.154) compared to the MSS group (0.989). Despite this difference, the results were not statistically significant (p > 0.05). Limitations, reasons for caution The limitations of this study include the potential impact of selection bias, as it is a retrospective study in which groups were not randomly assigned. Additionally, the low number of blastocysts in the MSS group and the potential bias of DNA fragmentation, which was not evaluated in the study. Wider implications of the findings Both methods demonstrated similar effectiveness. While MSS could potentially be more effective in samples with high DNA fragmentation and they feature a user-friendly application, its major limitation lies in its challenging application with samples having a low concentration of motile sperm. Specific semen samples may need different sperm selection methods. Trial registration number Not Applicable

P-059 Euploidy and mosaics rates using microfluid sperm selection techniques (Zymot-ICSI®)

Abstract Study question Can Zymot-ICSI® influence the mosaic embryos rate? Is the degree of doble strands DNA fragmentation (dsSDF) affecting euploidy and mosaicism outcomes? Summary answer The use of Zymot-ICSI® in seminal samples induces a decrease in aneuploid embryos, and an increase in euploid and/or mosaic embryos independently of the dsSDF. What is known already It has been demonstrated that sperm with higher motility exhibit superior genomic integrity, establishing a direct relationship between dsSDF and the capacity for embryonic development and implantation. In a previous study focused on investigating differences in a various biopsy techniques and their impact on the genetic outcome of the embryos, an increase in the number of mosaic embryos was identified. The main objective of this study is to ascertain whether sperm selection using these techniques, specifically the use of Zymot-ICSI®, increase the euploidy rate in patients with altered fragmentation indices, and its relations with mosaics distribution. Study design, size, duration A retrospective study analyzed a total of 684 biopsied embryos in 2021, of which 339 came from seminal samples without COMET test, and 345 embryos came from seminal samples that underwent the COMET test, analysing the sperm DNA fragmentation with one DNA strand (ssSDF) and double DNA strands (dsSDF). Genetics analysis of these 345 embryos were divided into 3 groups according to the techniques used in the sperm selection before ICSI (dsSDF: <60; 60-80; >80). Participants/materials, setting, methods Differences between Swim-up/gradients(Sw/G)(n = 513) and Zymot-ICSI®/Sperm-Slow(n = 171) were assessed. 309 embryos without COMET prior to ICSI, underwent Sw/G, 5 sperm-slow and 20 Zymot-ICSI® (both prophylactically). In cases where fragmentation was high and Zymot-ICSI®(n = 98) could not be performed before ICSI for low sample quality, Sperm-slow(n = 10) or Sw/G(n = 199) techniques (with normal or altered fragmentation) were used. The existence of variations in the type of mosaicism (Simple/Complex) was also analysed with respect to the sperm selection technique used and dsSDF. Main results and the role of chance No significant differences were observed between the different sperm selection techniques used (regardless of the degree of dsSDF), in terms of euploidy, aneuploidy and mosaic values (p:0.861). According to the dsSDF, the usefulness of Zymot-ICSI® versus Sw/G is shown, decreasing the percentage of aneuploid embryos IF < 60 (20% vs 44.1%); IF > 80 (28.6% vs 43.8%), increasing the percentage of euploid embryos in IF < 60% (60% vs 46.5%) and maintaining them in IF > 80 (52.4% vs 50%). However, an increase in mosaics was observed in all the groups studied (depending on the sperm selection technique Zymot-ICSI® vs Sw/G) but independently of the dsSDF: <60: 20% vs 9.30%; 60-80: 13.4% vs 9.62%; >80: 18.2% vs 5.88%. No differences were found between the different types of mosaic embryos (simple or complex) between the different groups according to the dsSDF (p > 0.1), but there was a trend towards a decrease in complex mosaic embryos and an increase in simple mosaic embryos when using Zymot-ICSI® versus Sw/G (Complex Mosaic: 13% vs 23.2%; Simple Mosaic 87% vs 76.8%). Limitations, reasons for caution Although there is a trend towards an increase in mosaic embryos, the difference is not significant, probably due to the low number of samples in some cases. Wider implications of the findings Use of Zymot-ICSI® in seminal samples induces a decrease in aneuploid embryos, and an increase in euploid and/or mosaic embryos. The increase in mosaic embryos after the use of Zymot-ICSI® occurs independently of the dsSDF, observing a tendency towards a decrease in mosaic-complex embryos and an increase in simple embryos. Trial registration number Not applicable

P-176 A novel electrophoretic sperm isolation system achieves equivalent ICSI outcomes to the combined density gradient centrifugation and swim-up method in a significantly shorter processing time

Abstract Study question Does an electrophoretically sperm isolation method, the Felix™ system, have clinical or operational advantages over the combined density gradient centrifugation and swim-up (DGCS) method? Summary answer The Felix™ system produces results that are comparable to the DGCS method, however, Felix™ has the ability to isolate spermatozoa more efficiently. What is known already The isolation of healthy spermatozoa to improve ART outcomes has received enormous attention in the literature recently. Although swim-up and density gradient centrifugation are the most common sperm separation methods, the optimal technique is still unclear. Recently, good sperm selection approaches using various non-centrifugal devices have emerged. Previous reports have shown that a quick and simple sperm separation device, the Felix™ system, can isolate high-quality spermatozoa and improve the results of ART. The Felix™ system combines electrophoresis and separation membranes to collect negatively charged mature normal sperm. Study design, size, duration A prospective oocyte sibling study was conducted between March 2023 and December 2023. The study involved infertile couples (n = 45) with a semen volume of 2.3 mL or more, a motile sperm concentration of 2 million/mL or more, maternal age under 43 years, and at least four mature oocytes at the time of oocyte retrieval. Ejaculated semen was prepared separately using DGCS and Felix™ on the day of oocyte retrieval and subsequent ICSI results were analyzed. Participants/materials, setting, methods In the DGCS group, semen was layered on a 45/90% of sperm grad and centrifuged for 15 minutes, followed by a 30 min swim-up. Subsequently, 0.3 mL of supernatant was collected. In the Felix™ group, 0.3 mL of sperm was collected following six minutes of membrane hydration and electrophoresis. Sperm concentration, total motility, progressive motility and DNA fragmentation (SDFR) were measured. Fertilization, blastocyst development, good-quality blastocyst development, and embryo utilisation rates were also assessed. Main results and the role of chance The average female age at the time of oocyte retrieval was 36.5±4.0 years, and the average male age was 38.1±5.9 years. Of 401 oocytes, 200 and 201 oocytes underwent ICSI using sperm prepared by DGCS and Felix™, respectively. The average processing time of Felix™ was significantly shorter than that of DGCS (54.0±4.3 vs. 7.4±0.2 minutes, p < 0.001, respectively). No significant difference was observed between the DGCS and Felix™ groups for sperm concentration (p = 0.49), motility (p = 0.05), progressive motility (p = 0.34), and SDFR (p = 0.09). Furthermore, there were no significant differences between the two groups with regards to fertilization rate (81.0% vs. 80.6%, p = 0.92), blastocyst development rate (52.9% vs. 58.4%, p = 0.60), good-quality blastocyst development rate (26.1% vs. 35.7%, p = 0.06), and embryo utilisation rate (54.3% vs 58.0%, p = 0.50). Limitations, reasons for caution Although the DGCS method was used as a comparison group, it is necessary to compare the Felix™ system with DGC alone, swim-up alone, or other sperm isolation methods. Larger, more powered studies are required to determine the impact on pregnancy and live birth outcomes. Wider implications of the findings The Felix™ system is a viable option for sperm isolation. While DGCS requires complicated procedures and time-consuming process including centrifugation and incubation; Felix™ requires six minutes only. The Felix™ system reduces processing times significantly and therefore can improve laboratory operations and workflow. Trial registration number Not Applicable

P-717 Add-ons availability and quality of online information provided by Brazilian IVF clinics

Abstract Study question How do Brazilian fertility clinic websites advertise four IVF add-ons: assistedhatching (AH), preimplantation genetic testing for aneuploidies (PGT-A), ERA and sperm DNA fragmentation test. Summary answer Available information, about ivf add-ons in Brazil, on researched websites is heterogeneous and is not evidence-based according to the last published recommendations. What is known already Over 1 billion people worldwide use online resources to obtain health-related information. People struggling with infertility are increasingly turning to online resources for infertility-related content and social support. “Add-on” procedures are actively promoted on some fertility clinic websites as means to improve IVF success rates. However, the actual contribution of these interventions to IVF success rates remains debatable. ESHRE has recently issued 42 recommendations were formulated on the use of add-ons in IVF. At present, no study has evaluated the type and quality of the information provided on the Brazilian IVF clinics’ websites regarding the merits of “add-ons. Study design, size, duration Prospective evaluation of the availability and quality of information on four common add-ons provided by IVF clinics in the southeast of Brazil between October-November 2023. Clinics were identified using the national registry (SisEmbrio) as well as through manual online search. As only public online available information was used no ethical approval was necessary. Statistical analyses were restricted to descriptive of the frequency of each add-on and frequency of claims made in relation to their use. Participants/materials, setting, methods One reviewer took screenshots of the included clinic pages and claims and a second reviewer double-checked everything. The folowing information was colletced: availability of of the four add-ons; information on the effectiveness on: implanation rate(IR), pregnancy rate (PR) or live-birth rate (LBR); information on the scientifc uncertainty of the use of add-on and possible negative impact; for PGT-A if the clinic informed that the data obtained should be confirmed by prenatal diagnostic testing. Main results and the role of chance The websites of 122 IVF clinics were included. Nine clinics had no website or were offline. PGT-A was the most frequent available add-on (n = 77; 68%) but only two of them (2,5%) informed on PR. There was no information on IR nor LBR. Thirteen clinics (16,8%) acknowledged it could have adeverse effects but only one mentioned the scientifc uncertainty related to its use. Forty (35%) websites displayed AH with 7 reporting on possible negative effects but none disclosed success rates (PR, IR or LBR). Eight clinics admitted AH had uncertain benefits and seven noted possible negative impact. The sperm DNA fragmentation test was presented in 37 (33%) websites but none reported on potential adverse effects nor success rates in terms of PR, IR nor LBR. No website provided information on the uncertainty of the benefit of the test. The ERA-test was advertised in 28 (25%) websites with no information on its benefits on sucess rates. Three clinics advised it could have a negative impact and six agreed its effectiveness was unclear. The cost was never pesented for any of the add-ons researched in accordance to national regulations. Of note, 36 websites advertised other add-ond such as MSOME, ZYMOT and endometrial scrathcing. Limitations, reasons for caution The study was restricted to 122 clinics websites in the southeast of Brazil. Evalaution was restricted to four commons add-ons and the quality of the information provided could not be assessed as the clinics follow no specific regulation on how to advertise scientifc information to their patiens and general public. Wider implications of the findings The information provided by Brazilian IVF clinics could be misleading. The use of add-ons is surrounded by scientific uncertainly related to their benefits and cost-effectiveness therfeore it is of utmost importance that clinics reviewt heir websites and offer sound scientific data to patients and the general public as well. Trial registration number Not applicable

P-015 Impaired spermatogenesis in colitis: the role of interferon-γ mediated ferroptosis in sertoli cells by colitic CD4+ T cells

Abstract Study question Does colitis have an impact on spermatogenesis and what could be the potential mechanism? Summary answer Colitis causes sertoli cell ferroptosis and disrupts spermatogenesis in mice due to the migration of colitic CD4+ T cells that produce interferon-γ. What is known already In recent years, there has been a significant increase in rheumatic and inflammatory diseases. Epidemiological data reveals that dysregulated immune conditions can directly or indirectly impact male fertility and sexual function in patients of reproductive age. Inflammatory bowel disease (IBD), which encompasses Crohn’s disease and ulcerative colitis, is a chronic, recurring gastrointestinal inflammatory disorder characterized by bloody stools and weight loss. A study from a Swedish national cohort showed that IBD patients had diminished sperm fertility and increased sperm DNA fragmentation compared to healthy individuals. However, the precise mechanisms underlying male fertility problems in IBD remain unclear. Study design, size, duration In this study, C57BL/6 mice were kept under specific pathogen-free conditions. A murine colitis model that simulates human colitis histological features and symptoms was created using Dextran Sulfate Sodium (DSS). To establish the model, 2-2.5% DSS was added to the mice’s drinking water from days 0 to 7, followed by a three-day recovery period. Participants/materials, setting, methods Testis, epididymis and colon sections were stained with hematoxylin and eosin for histological analysis. The localization and phenotype of infiltrated lymphocytes in epididymis and testis were studied by immunostaining and flow cytometry. Sperm functions including motility, viability, and DNA integrity were assessed by standard assays. Iron, malondialdehyde, and reactive oxygen species (ROS) levels were analysed by commercial kit. Main results and the role of chance Colitis was found to impair male fertility in DSS-induced colitic mice, causing testis and epididymis tissue damage along with inflammatory cell infiltration. Colitis altered the immune cell phenotypes in the testis and epididymis. The proportion and absolute cell count of CD4+ T cells and CD19+ B cells increased after colon inflammation. Testicular sertoli cells in colitic mice exhibited ferroptosis, characterized by increased levels of iron deposition, malondialdehyde, and ROS. We assessed sperm parameters from the cauda epididymis of mice. Compared to the control group, there were significant reduction in sperm concentration, motility, viability and DNA integrity in the group with murine colitis induction. Consistently, we observed lower numbers of active pups from the fathers of the colitis group compared to control mice. CD4+ T cells with interferon-γ producing capabilities accumulated in the testes of colitis mice. Leukocyte flow cytometric analysis and parabiotic mouse model further demonstrated that colitis promoted pathogenic CD4+ T cells migration to the testis through circulation. The treatment of anti-CD4 antibody, anti-interferon-γ antibody or ferroptosis inhibitor can attenuate the colitis-associated testis pathologies. Limitations, reasons for caution Our study was conducted exclusively in mice. The pathogenic effect and the corresponding mechanism of colitis on male fertility in humans still needs to be confirmed. Wider implications of the findings Our team identified the mechanism linking dysregulated gut immune cell activity to testicular dysfunction. By identifying specific molecules on colitic immune cells, which allow their infiltration into the testis, may pave the way for a new therapeutic approach potentially improving the prognosis of colitis and related fertility impairments. Trial registration number not applicable

P-271 Deep sequencing of spermatozoal accessible chromatin reveals complex arrangements underlying spermatozoal chromatin packaging and fertilization

Abstract Study question Could structural aspects of sperm DNA packaging contribute to fertilization and early embryo development? Summary answer Structural aspects of sperm DNA include complex patterns of repetitive-DNA and developmental gene packaging that could delineate a paternal impact on fertilization and early development. What is known already Genetics, epigenetics and transcriptomics of ejaculate human spermatozoa could have important effects on the establishment of embryonic chromatin architecture that may impact early fetal development. Spermatozoa are transcriptionally silent; however, a small fraction (∼5%-15%) of their chromatin escapes repackaging by protamines and remains in a more open nucleosomal conformation that may be poised for transcription. The sensitivity of this fraction to progressive salt/detergent disruption is likely assayed in sperm chromatin dispersion (SCD) or halo tests that are commonly used in assessments of sperm DNA fragmentation. Study design, size, duration Eleven healthy male donors and 9 women undergoing IVF treatment agreed to participate in this study. Eleven normozoospermic semen samples were donated by male donors, aged 25-39 years, who abstained from sexual activity for 3 days. Seven MII oocytes and 5 pronuclear zygotes were donated from 5 and 4 women, respectively, undergoing IVF treatment. The samples were collected within a time frame of 6 months. Female donors provided either MII oocytes or pronuclear zygotes. Participants/materials, setting, methods Sperm chromatin was processed by differential salt extraction tailored with endonuclease or micrococcal nuclease treatment and RNA isolated using established protocols. Oocytes and pronuclear zygotes were analyzed at the single cell level. DNA library construction employed the NEBNext Ultra approach (NEB) and RNA libraries the Ovation Single Cell (NuGEN) for Illumina. Bioinformatics analysis included FastQC, HISAT2, StringTie, GREAT, edgeR and other source-software. Public datasets were employed to further increase the statistical power. Main results and the role of chance The aim of our approach was to dissect the accessible sperm chromatin fraction, identifying regions of great importance that could contribute towards the formation of the zygote and/or early embryonic development. Differing extraction methods revealed complementary accessible and resistant fractions in sperm, with accessible regions being significantly enriched for chromatin important in very early embryonic development. These chromatin fractions contained various genomic features including promoter sequences, CpG-islands and CTCF-binding sites that could attract transcription binding factors in the paternally-derived chromatin. Accessible sperm chromatin also contains high levels of specific DNA repeat classes of retroviral origin, known to impact early gene expression and gene expression programs. Accessible sperm chromatin was strongly associated with developmental and regulatory signatures with Bonferroni P values ranging between 2.07x10-4 and 1.03x10-20, potentially revealing a role for paternal chromatin in the initial start-up of early zygotic development. We further investigated paternally- and maternally- derived transcripts and identified mono-allelically transcribed genes present in the zygotes, that were entirely absent from all seven oocyte transcriptomes, suggesting a paternal origin, and potentially important roles following oocyte fertilization. Limitations, reasons for caution Due to the restraints of obtaining human material, the study relied on a relatively small sample size. However, sequencing results showed quite homogeneous clustering in both DNA/RNA samples, with at least 5 biological replicates from at least 4 donors per group, potentially reducing the impact of low sample size. Wider implications of the findings A better understanding of differentially accessible sperm chromatin and residual RNA, could help support the discovery of biomarkers that can be used to select more competent spermatozoa for IVF cycles, resulting in higher success rates in couples undergoing ART. Trial registration number not applicable

P-741 Standard IV Cannula Aspiration (SIVCA): A step-by-step video demonstration of the minimally invasive testicular sperm aspiration technique with technical improvements after 4 years of experience

Abstract Study question Is there room for further technical improvements in the Standard IV Cannula Aspiration (SIVCA), for a more efficient testicular issue aspiration and sperm extraction procedure? Summary answer A more efficient SIVCA procedure can be achieved by using a higher flow-rate cannula, less negative pressure aspiration and a Luer Lock syringe. What is known already Standard IV Cannula Aspiration (SIVCA) was introduced in 2022. It showed comparable sperm retrieval rates (SRR) to mTESE (56.2 vs 57.3%; McNemar P = 1.000). But given the novelty of the technique, some technical difficulties were encountered: The 14G cannula frequently kinked during the back and forth motions of aspiration, thus having to be replaced several times during the procedure; and the aspirating syringe often detached from the catheter mid-procedure, due to the absence of a lock mechanism securing it to the flashback chamber of the cannula. Study design, size, duration 224 men were enrolled in this prospective 24 months cohort study conducted at a specialized IVF center. The men had testicular tissue aspiration performed via SIVCA, either for azoospermia or increased seminal sperm DNA fragmentation, in anticipation of their partners’ ovum pick-up for an ICSI cycle. If enough testicular tissue was aspirated, it was examined for sperm. If not more than 0.2 gm of tissue could be aspirated from either testis, the patient underwent mTESE. Participants/materials, setting, methods After general anesthesia, 5cc of Xylocaine (lidocaine HCl) 2% was instilled into each spermatic cord. A standard 14-G 310 ml/min flow-rate IV cannula was introduced into the testis. After withdrawing the needle, a 3cc Luer Lock syringe was secured to the cannula. The plunger was maximally retracted to generate −250 Torr of negative pressure and clamped. Back and forth motions were performed, mapping and aspirating tissues from different regions of each testis at a time. Main results and the role of chance Out of a total of 224 cases, an adequate testicular tissue sample of more than 0.2 gm was successfully aspirated from 218 cases, with a 0.97 probability of success. Mild hematoma and pain were encountered in 5.8% of cases (n = 13/224) and subsided within two to three weeks (Grade I on the Clavien Dindo Scale). In a previous series, 130 cases of SIVCA were performed by fully retracting the plunger of a regular 20cc syringe, generating about −520 Torr of negative pressure, while the syringe was connected - but not secured - to the flashback chamber of a 270ml/min flow-rate cannula. In that series, adequate tissue samples could successfully be aspirated in 122 cases, showing a 0.93 probability of success. By using a higher flow-rate 14G IV cannula (310 ml/min vs 270 ml/min), less negative pressure (−250 Torr vs − 520 Torr) and a Luer Lock syringe; fewer number of cannulas were lost to kinking per procedure and no inconvenient disconnections occurred mid-procedure between the cannula and the aspirating syringe owing to the Luer Lock mechanism; all while maintaining a high success rate of aspiration of sufficient testicular tissue (X2 (1, N = 354) = 2.6156, p = .10582). Limitations, reasons for caution Whenever sclerosis and/or fibrosis of testicular tissue rendered aspiration of adequate amounts of testicular tissue difficult or challenging, we reverted to surgical m-TESE. Ice packs should be applied for at least 48 to 72 hours post-procedure to forestall hematomas. Further blinded randomized control studies with larger datasets are recommended. Wider implications of the findings SIVCA is a safe, efficient and easily reproducible method of testicular sperm aspiration. Its allowance for mapping different regions of the testes in order to extract sufficient amounts of tissues, makes it an ideal initial alternative to surgical m-TESE for testicular tissue aspiration and sperm searching. Trial registration number NCT05247723

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

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

P-106 A novel sperm selection technique to select the most motile spermatozoa with superior genomic integrity for intrauterine insemination

Abstract Study question Does the utilization of a novel sperm selection method via microfluidics(MFSS) confer advantages over the conventional density gradient(DG) technique for couples undergoing intrauterine insemination(IUI)? Summary answer MFSS efficiently selected spermatozoa with increased motility and genomic integrity in an expedited manner, leading to an enhanced IUI clinical outcome. What is known already The first-line reproductive treatments for infertile couples are timed intercourse and IUI. In IUI, DG is commonly utilized to isolate motile spermatozoa. However, this method requires technical skills and processing time. Furthermore, DG exposes spermatozoa to silica gel particles and reactive oxygen species during centrifugation. MFSS represents a safer and more efficient sperm processing method to minimize human intervention. It has been reported by several investigators that MFSS selects spermatozoa with higher progressive motility and lower DNA fragmentation in ART. We have decided to investigate the efficacy of MFSS as an alternative method to select spermatozoa for IUI. Study design, size, duration Since 2017, post-processing semen parameters and Sperm Chromatin Fragmentation (SCF) was measured in the raw, DG-, and MFSS-processed specimens. Clinical outcomes were compared between IUI cycles using DG versus MFSS processing. Paired analyses were performed on patients who underwent history IUI-DG and subsequent IUI-MFSS, and cycles were split into natural (NC) or clomid or letrozole superovulation (CLO) cohorts. Participants/materials, setting, methods A total of 855 couples underwent 922 IUI-MFSS. Post-processing semen parameters and clinical outcomes were compared with those of 2325 age-matched couples who underwent 8322 IUI-DG during the same period. All male partners included in this study had normal semen parameters. DG was performed according to WHO, while MFSS as per manufacturer protocol(ZyMōt® Multi 850µL). SCF was assessed by TUNEL(normal threshold,≤15%). For all comparisons, +bHCG and clinical pregnancy(CPR,+FHB) were compared. Main results and the role of chance IUI-DG(maternal age:32.7±2;paternal age:37.9±6) resulted in a final concentration of 48.4±33x106/mL and motility of 88.1±4%. IUI-MFSS resulted in a decrease in a final concentration at 36.4±17x106/mL with a remarkable increase in motility at 98.6±1%(P<0.01). SCF was 6.0±4% in raw specimen, became 4.7±2% after DG, and 2.5±2% after MFSS(P<0.01). Overall, IUI-DG(n = 2325) resulted in a +bHCG of 12.7%(1065/8322) and a CPR(+FHB) of 11.0%(919/8322). IUI-MFSS(n = 855) resulted in a higher +bHCG of 16.1%(149/922,P<0.01) and a CPR of 12.5%(116/922). Next, we divided cycles into NC or CLO cohorts. In couples with NC, IUI-DG(n = 420) resulted in a +bHCG of 9.3%(132/1416) and a CPR of 8.8%(124/1416). IUI-MFSS(n = 107) resulted in a +bHCG of 11.4%(15/131) and a CPR of 11.4%(15/131). In CLO cycles, IUI-DG(n = 1990) resulted in a +bHCG of 13.0%(788/6048) and CPR of 10.9% (659/6048), while IUI-MFSS(n = 496) resulted in a significantly higher +bHCG at 17.4%(122/698) and CPR at 14.3%(100/698,P<0.01). In the paired sub-analysis, NC couples (n = 30) underwent IUI-DG and subsequently IUI-MFSS. IUI-DG resulted in a 9.4% +bHCG(7/74) and 8.1% CPR(6/74). IUI-MFSS yielded a 10.2% +bHCG(4/39) and 10.2% CPR(4/39). CLO couples (n = 132) underwent IUI-DG and resulted in a 13.6% +bHCG(85/623) and 11.2% CPR(70/623), followed by IUI-MFSS yielding a 15.2% +bHCG(27/178) and 12.4% CPR(22/178). Limitations, reasons for caution IUI-MFSS cycles resulted in an increased proportion of progressively motile spermatozoa, with a superior genomic integrity and higher clinical outcome. The benefit of MFSS may be somewhat limited because of the low SCF of this particular cohort and only a larger case series would be able to confirm this finding. Wider implications of the findings DG is labor intensive, technician-dependent, and unable to meaningfully ameliorate SCF. MFSS, while reducing exposure to reactive oxygen species, grants the highest sperm progressive motility with superior genomic integrity that may be responsible for an improved pregnancy outcome. Trial registration number not applicable

O-268 Re-considering the endometrial microbiome: a comparison of DNA- and RNA-based sequencing techniques within the same cohort

Abstract Study question Does the analysis of the endometrial microbiome provide the same information when using DNA or RNA sequencing-based techniques? Summary answer DNA vs. RNA-based microbiome analysis techniques demonstrate significant microbial compositional differences, meaning that the previous endometrial 16S rRNA gene-based microbiome analysis results might be overestimated. What is known already In recent years, high-throughput sequencing technologies have revolutionised the field of reproductive microbiome research. Our understanding of the composition of endometrial microbiome predominantly relies on DNA-based 16S rRNA gene profiling method. Regardless of its effectiveness and low cost, its underestimation of microbial diversity, abundance, or functionality and lack of detection precision on species level have been highlighted. The meta-transcriptome analysis, RNA-based method, overcomes these limitations and identifies functional genes that are actively expressed by the alive microbes. This study aims to elucidate for the first time the endometrial microbiome using a dual approach of 16S rRNA gene-method and meta-transcriptomic analyses. Study design, size, duration In total forty-seven women with infertility at ages 27-42 years old were enrolled in the present study. Paired endometrial samples, endometrial brushing and Pipelle biopsy were collected from each participant at the mid-secretory menstrual phase (LH + 7/9). Participants/materials, setting, methods Endometrial samples were collected using Tao Brush for 16S rRNA analysis (DNA analysis) and Pipelle endometrial curette for meta-transcriptomic analysis (total RNA analysis). QIAamp UCP Pathogen Mini Kit was used for DNA extraction and 16S rRNA gene V3-V4 regions were sequenced on MiSeq. RNA was extracted using miRNeasy Micro kit, with rRNA removal by RiboZero kit and Libraries were generated using Stranded Total RNA Prep. Taxonomy was assigned by using Kraken2 (v2.2.1), Bracken (v2.7). Main results and the role of chance To the best of our knowledge, this is the first study to compare endometrial microbial identification using both metagenomics (16S rRNA sequencing) and meta-transcriptomics (meta-RNA sequencing) within the same cohort. Analysis of the composition of the microorganisms by bacterial 16S rRNA gene sequencing revealed that the most abundant bacterial genus in the endometrium was Lactobacillus. In the meta-transcriptome analysis, no microorganisms belonging to the Lactobacillus genus were detected in high abundance. This indicates that the relative abundance of this genus at the DNA level does not necessarily imply microbial activity or the presence of viable Lactobacillus in the endometrium. The meta-transcriptomics analysis detected microbial taxa such as Staphylococcus, Bacillus, Streptococcus, and Burkholderia as the most dominant. The results suggest that DNA-based detection of microorganisms in the endometrium may be overestimated due to the presence of naked DNA sequences or microorganisms from the vagina and cervix. Furthermore, RNA-based analysis demonstrates a population of active microorganisms different from the image shown by DNA analysis. Altogether, our study results indicate that the uterine microenvironment, as previously identified through DNA sequencing, may partly reflect the vaginocervical microenvironment, with the uterine tissue being a low biomass site not dominated by lactobacilli. Limitations, reasons for caution The endometrial samples were paired; however, DNA was analysed in the endometrial brushing sample while RNA in the tissue biopsy, which might result in some differences in microbial composition. Wider implications of the findings Contrary to the general belief of the Lactobacillus dominance in the human endometrium, our study suggests that the endometrial microenvironment may be harbouring DNA fragments and/or cells of lactobacilli originating from the lower reproductive tract. Our study results call out to re-consider/re-analyse the endometrial microbiome in health and disease. Trial registration number not applicable

Comparative Analysis of the Effects of Magnesium Oxide Nanoparticles on Sperm Parameters in Fresh and Frozen Samples

Background: Freezing is a crucial technique in reproductive science utilized for the preservation of sperm samples. However, the process of freezing and thawing sperm can result in detrimental effects on sperm quality. One of the major mechanisms underlying this decline in sperm quality is the generation of reactive oxygen species during the freeze process. The purpose of the current study was to investigate the effects of magnesium oxide nanoparticles on frozen sperm parameters. Methods: Semen samples were collected from 8 fertile men, aged 30 to 42 years, with normozoospermia, following 3 to 5 days of abstinence. The samples were divided into fresh (n=3), freeze (n=3), and control (n=2) groups. Three fresh experimental groups were only exposed to MgO NPs with concentrations of 5, 25, and 50 μg/ml and three freezing experimental groups were frozen after being treated with MgO NPs, thawed, and analyzed after 30 min. Results: Our findings revealed that the progressive movement and vitality of sperm experienced a significant decline, while non-progressive and immotile sperm showed a notable increase in both fresh and frozen experimental groups exposed to MgO NPs. However, the application of MgO NPs during fresh and freezing processes demonstrated an effective preservation of pH, morphology, and DNA fragmentation in sperm cells. Conclusion: The analysis revealed that MgO NPs negatively impact sperm motility and viability in both fresh and freeze analysis. Also, the use of MgO NPs in fresh and frozen processes effectively maintains the pH, morphology, and fragmentation of DNA in sperm cells.