Sperm Selection Research Articles

P-072 Efficacy of a microfluidic chip technology for sperm selection in intrauterine insemination cycles

Abstract Study question Is there an advantage of using a new sperm selection method by microfluidics (MFSS) over standard density gradient (DG) for couples undergoing intrauterine insemination (IUI)? Summary answer MFSS reduced sperm processing time and selected spermatozoa with greater progressive motility and superior genomic integrity, suggesting a better clinical outcome than DG. What is known already The most conservative, cost-effective reproductive treatments for infertile couples are timed intercourse and IUI. The latter is generally performed using DG to enrich and purify the most motile spermatozoa; it requires technician effort and skills as well adequate processing time. Moreover, during DG centrifugation, spermatozoa are exposed to potentially harmful silica gel particles and eventual reactive oxygen species (ROS). The recent availability of a microfluidic chamber has been proposed as a method to safely process spermatozoa in an expedited manner while reducing human intervention. Therefore, we decided to test MFSS in a laboratory that executes over 2,000 IUI cycles/year. Study design, size, duration Since September 2022, 93 couples underwent 103 IUI-MFSS. Post-processing semen parameters and clinical outcomes were compared with those of 103 age-matched couples who underwent 103 IUI-DG during the same period. To confirm findings, 45 couples undergoing their first IUI-MFSS had post-processing semen parameters and clinical outcomes compared with 45 first-time IUI-DG couples, serving as a matched control. Finally, within the same 66 couples, outcomes were compared between their initial IUI-DG cycle and a subsequent IUI-MFSS. Participants/materials, setting, methods Men had normal raw semen parameters according to the most recent WHO criteria. DG was performed according to WHO21 guidelines, while MFSS as per manufacturer protocol (ZyMōt® Multi 850µL). Stimulation protocols were similar between all patients. Before and after processing, volume, concentration, and motility were compared between the two processing methods. Sperm chromatin fragmentation (SCF) was assessed by TUNEL (In Situ Cell Death Detection Kit; normal threshold,≤15%). For all comparisons, +bHCG and clinical pregnancy (+FHB) were compared retrospectively. Main results and the role of chance Ninety-three couples (maternal age,37.3±4; paternal age,39.2±6) underwent 103 IUI-MFSS cycles and were matched for age and abstinence days (1–4d) to 103 IUI-DG patient/cycles. IUI-DG and IUI-MFSS yielded 0.5±0mL volume, 76.8±40x106/mL and 41.3±26x106/mL concentrations, and 89.4±3% and 98.3±1% motility, respectively (P<0.001). IUI-DG resulted in a 20.4% +bHCG (21/103) and 15.5% +FHB (16/103). IUI-MFSS yielded a 19.4% +bHCG (20/103) and 16.5% +FHB (17/103). Forty-five couples (maternal age,36.8±4; paternal age,37.8±6) who underwent their first IUI with MFSSwere matched to a first-time IUI-DG cohort with similar semen parameters and abstinence. IUI-DG led to a 0.5±0 mL volume, 77.3±43x106/mL concentration, and 90±2% motility. IUI-MFSS yielded a lower concentration (45.6±27x106/mL), but a remarkable increase in motility (98.3±1%) (P<0.001). IUI-DG yielded a 13.3% +bHCG (6/45) and an 8.9% +FHB (4/45). IUI-MFSS yielded a 15.6%(7/45) +bHCG and an 11.1% +FHB (5/45). Finally, we assessed 66 couples who underwent IUI-DG and subsequently IUI-MFSS. IUI-DG resulted in a 77.5±39x106/mL concentration and 89.0±3% motility, while IUI-MFSS resulted in a lower concentration (36.9±21x106/mL), but an enhanced motility (98.3±1%)(P<0.001). IUI-DG yielded a 15.2% +bHCG (10/66) and an 8.9% +FHB (5/66), while IUI-MFSS yielded a 15.2% +bHCG (10/66); all became clinically pregnant. In all comparisons, SCF was 6.5±3% in raw samples, became 4.7±2% after DG, and negligible after MFSS at 0.8±0.6%(P<0.001). Limitations, reasons for caution IUI-MFSS cycles yielded a higher number of progressively motile spermatozoa with superior genomic integrity and a comparable clinical outcome to IUI-DG. This study came at a higher cost due to the device and offered only preliminary results. Therefore, it needs to be repeated in a larger study population. Wider implications of the findings DG is time consuming, labor intensive, and its outcome is technician-dependent. While reducing exposure to chemicals and ROS, MFSS grants higher motility and superior genomic integrity. If a greater clinical outcome is confirmed in a larger cohort, MFSS may prove to be a superior sperm processing method for IUI. Trial registration number not applicable

P-076 Sperm selection by thermotaxis: a novel technique to enhance assisted reproductive technologies’ outcomes

Abstract Study question Does sperm selection by thermotaxis increase competent spermatozoa count? Summary answer When compared to capacitation alone, our findings suggest an improvement in the availability of high quality spermatozoa after thermotaxis in terms of motility and morphology. What is known already Thermotaxis is a natural mechanism that guides competent spermatozoa through the female genital tract. It has been previously shown that, in the mice model, thermotaxis may improve pregnancy rates following in vitro fertilization (IVF). However, thus far, there is little information to support these findings with human spermatozoa. Study design, size, duration A prospective single-center paired pilot analysis was performed between April 2021 and April 2022 including 21 male individuals, 12 of whom were sperm donors and 9 who were patients with asthenozoospermia (defined as < 32% progressive sperm motility). Participants/materials, setting, methods Only otherwise healthy men between 18 and 45 years old were included. All subjects performed a sperm collection at the clinic after a maximum of 3 days following the last ejaculation. Sperm concentration, motility, morphology (all using the WHO 2010 criteria) and DNA fragmentation (TUNEL) were assessed within the same sample at three time-points: fresh sample, after capacitation and following the extra thermotaxis procedure. Main results and the role of chance The average age of the participants was 34.9 ± 6.6 years old. The mean sperm concentration was 66.9 ± 39.1 million/mL (91.4 ± 26.1 in the donor subset (group A) versus 34.2 ± 28.0 in the asthenozoospermia patient (group B) subgroup). The median sperm motility was 62.0% (66.5% in group A versus 24.0% in group B). The mean sperm morphology was 7.2% (7.7% in the group A versus 6.7% in the group B). When compared to the sample in which only capacitation was performed, a statistically significant increase in spermatozoa motility (52.10% vs 82.14%, p < 0.05) and normal morphology (11% vs 16%, p < 0.05) was observed following capacitation plus thermotaxis. Conversely, DNA fragmentation did not vary significantly within the capacitated samples before and after thermotaxis (13% vs 14%). Limitations, reasons for caution The present pilot study is based on a limited number of participants and should be extended further. Wider implications of the findings The combination improvement in terms of morphology and progressive motility following thermotaxis may optimize selection of sperm cells and ultimately increase pregnancy rates after IVF. In the light of our experience, it is possible that this technique, which does not require high-end equipment, may increase the usable competent spermatozoa count. Trial registration number NCT04849156

O-101 Add-ons in the ART lab

Abstract Study question Which laboratory based add-ons are effective and safe to use in ART treatment? Summary answer In total, more than thirty recommendations were formulated on the use of add-ons, with about a third being laboratory based tests or interventions What is known already Treatment add-ons are defined as not essential for the treatment, but optional additional treatments that are sometimes offered on top of routine fertility treatment. There are a wide range of treatment add-ons available, including diagnostic tests, drugs, equipment, holistic or alternative therapies, laboratory and surgical interventions, all claiming to potentiate the effect of the treatment and/or to lessen side effects, improve pregnancy or live birth rate, reduce the risk of miscarriage or shorten the time to pregnancy. Add-ons and can be at extra cost (or not), and evidence on efficacy and safety is often missing. Presumably they can be both beneficial or non-beneficial, as there may be absence of evidence to either. Study design, size, duration The ESHRE working group for good practice recommendations on add-ons in ART has produced recommendations useful for professionals, patients and policy makers. More than 30 add-ons were identified, and evaluated for efficiency and safety, and wherever possible also for cost. The literature was identified in a systematic search, reviewed and critically appraised. In the absence of any clear scientific evidence, judgement was based on the professional experience and consensus of the multidisciplinary development group. The guidelines are thus based on the best available evidence and expert agreement. Prior to publication, the guidelines were reviewed by independent international reviewers. Participants/materials, setting, methods Interventions were characterized in three sections: 1) Diagnosis and diagnostic tests, 2) Laboratory tests and intervention, and 3) Clinical management. Outcomes were efficacy (pregnancy or live birth rate), risk of miscarriage, time to pregnancy, cost, and safety/risk aspects. Some examples of add-ons in the group of laboratory tests and interventions are oocyte and sperm activation, mitochondria load and transfer, sperm selection, assisted hatching, genetic testing and addition of hyaluronic acid to media. Main results and the role of chance The guideline development group formulated more than 30 recommendations. This lecture will focus on the rationale and the recommendations of the laboratory add-ons currently used in fertility clinics. We will discuss the potential beneficial effects on the IVF treatment, its efficacy, and its safety. While most add-ons are not recommended for the general population of ART patients, where specific patient populations might benefit, indications were formulated. The guideline has been in stakeholder review and is prepared for submission in 2023. Limitations, reason for caution Of the recommendations, none are based on high quality evidence and only a few are based on moderate quality evidence. Hence 90% of the recommendations are supported only by low quality RCTs, observational data, professional experience, or consensus of the development group. Wider implications of the findings These recommendations aim to provide guidance to health care professionals and laboratory experts in the field of reproductive medicine, to help towards safe and efficient ART treatments for patients where they can participate in informed decision making with realistic expectations and be fully informed about their chances to pregnancy and live birth. Study funding and competing interest(s) All costs relating to the development process were covered from ESHRE funds. There was no external funding of the development process or manuscript production.

P-107 MICROFLUIDIC SPERM SEXING: An XLD (X-linked Disorder) model for preventing genetic disorders in newborns

Abstract Study question Can sperm sexing based on unique surface membrane markers – enable genotypic sperm sorting by allosomal content to prevent certain X-linked disorders in newborns? Summary answer In an X-Linked-Disorder prevention model, we utilized a microfluidic device functionalized with H-Y monoclonal antibodies attached to magnetic beads to enrich Y-chromosome bearing sperm sorts. What is known already About 4% of newborns are at risk of being born with an X-linked disease such as Fragile X Syndrome. The current clinically approved mitigation plan for preventing such genetic disorders is to consider Preimplantation Genetic Testing for Monogenetic diseases (PGT-M) which involves an invasive microsurgical procedure on fragile embryos. To minimize this invasiveness, we proposed a less invasive microsurgical exclusion approach to prevent such genetic disorders by genotypically sorting sperm cells that can be used for IUI/IVF/ICSI – with microfluidics – using their allosomal characteristics. Study design, size, duration Semen samples purchased from FairFax Cryobank from anonymous donors were used in immuno-magnetic microfluidic sorting protocols with the use of phycoerythrin (PE) binding Tetrameric Antibody Complexes (TAC) coupled with magnetic beads to tag H-Y monoclonal antibody (mab) binding activities on the sperm surface membrane. Participants/materials, setting, methods Immuno-magnetic sorting was conducted with specially fabricated microfluidic-sperm-sorting (MSS) chips. Sort yield and purities of positively selected sperm populations were assessed by flow-cytometry, immunofluorescence, fluorescent-in-situ-hybridization (FISH) and by quantitative-PCR (qPCR – Cq & Tm). Quantitative experiments were run in triplicates and the results were expressed as absolute means for comparison between paired variables. Statistical analyses were performed using Student’s t-test and a p-value of less than 0.05 was considered to be statistically significant. Main results and the role of chance For the assessment of H-Y sperm sorts, our preliminary data showed that flow cytometry was able to confirm the positive sperm selection (which were mostly Y-chromosome bearing sperm cells) in a sperm genotyping protocol – initially utilizing immuno-magnetic sort tubes prior to flowing through microfluidic chips. Both immuno-magnetic sort tubes and the microfluidic chips were able to optimally confirm from qPCR-Cq means – the negative selection – being the X-chromosome bearing sperm that may have been largely unselected by the H-Y-mab. Values for Cq means compared under H-Y-qPCR data sets were statistically significant with a p value of 0.000023 (p values is significant at < 0.05). Our study was able to elucidate a potential less invasive clinical application of genotypically sorting sperm cells with mab-functionalized microfluidic chips for an XLD-model. Molecular down stream applications like flow cytometry may therefore be utilized to experimentally confirm sort purities of positively selected sperm populations (with H-Y markers) – based on the utility of the SMCX and SMCY gene in the X and Y sperm chromosome bearing sperm cells respectively. Limitations, reasons for caution Physiological effect of magnetic fields of varying tesla or gauss strengths on sperm selected with immuno-magnetic beads may warrant further safety studies prior to actual clinical applications. Optimal selection results may be achieved with the use of freshly collected normospermic rather than frozen-thawed samples. Wider implications of the findings Although we had de-emphasized the clinical application of the microfluidic sperm sexing protocol for legitimate use in sex selection and gender balancing, this study design was solely focused on adapting a less invasive method of preventing X-linked genetic disorders with the potential exclusion of microsurgical procedures leading to PGT-M. Trial registration number NA

P-093 Single sperm morphokinetic variables during ICSI at the time of sperm aspiration into the microneedle

Abstract Study question Is it possible to classify sperm morphology as normal or abnormal based on their kinetic characteristics? Summary answer Using different classifiers, it was found that balanced dataset the classification scores of individual sperm morphology can be optimized during their motility just before ICSI What is known already Sperm selection plays a crucial role during ICSI. This selection by the embryologists is accompanied by two factors: motility parameters of the sperm to be injected, as well as the morphology. Motility affects morphological decision-making which may be subjective due to spermatozoa not being in a narrow vertical space in a PVP droplet, i.e. counting chamber. Machine/Deep Learning models -as classifiers- are gaining popularity in IVF as it could minimize subjectivity in gamete selection. SiD software is an algorithm that from a group of spermatozoa characteristics can support the selection of a single sperm during real time ICSI. Study design, size, duration In this prospective study 1699 individual spermatozoa were video-recorded (resolution of 200 X 200 pix.) during sperm selection in a 7%PVP solution. Motility variables (VSL, VCL, LIN, VAP, ALH, WOB, STR, MAD) were obtained from each video using the software SiD. Each sperm was classified as normal or abnormal. Based on motility variables, different classification models were used to make a morphokinetic association of the variables with the classification of each sperm. Participants/materials, setting, methods 1699 individual spermatozoa were classified and labeled by three senior embryologists into two categories: normal or anormal. To belong to any category, at least two embryologists had to agree. A normalization was applied to the motility data obtained with SiD. Machine learning classification models were applied. The three classification models with the best results were selected to optimize the hyper-parameters and improve their performance. Main results and the role of chance A set of motility variables were obtained using the software SiD1 (IVF2.0 Ltd., UK) these were used as features for each of the sperm samples and were tagged either as normal or abnormal having a total of 257 normal samples and 1442 abnormal samples. Different classification algorithms were used to perform sperm classification as normal and abnormal. Then hyperparameter tuning algorithms such as GridSearch were used to compute the optimum values, finding KNN algorithm to achieve the best results to classify normal and abnormal spermatozoa with 80% accuracy. Given the nature of the unbalanced dataset, the F1 score was calculated, achieving 0.77, other algorithms such as Decision-Tree and Random-Forest were used achieving similar results in the F1 score. We will continue growing the dataset until it is balanced and run the same algorithms expecting the performance to improve. Limitations, reasons for caution Each clinic's laboratory setup, including the camera used to record the ICSI procedure, is a clinic-specific configuration. Sperm abnormality detection is sensitive to camera resolution, showing the classifiers are resolution-dependent observers. To replicate these results, it is important to consider the quality of the camera. Wider implications of the findings Using the selected kinematics features, it is possible to classify individual spermatozoa during motility as having normal or abnormal morphology. Results may improve using standard morphological examination of each population and having more normal sperm samples. Trial registration number Not applicable

P-178 The interaction between spermatozoa and cumulus cells: a more physiological approach to the selection of good quality spermatozoa for assisted reproduction

Abstract Study question Does selection by cumulus cells (CCs) improve sperm quality in terms of acrosome integrity and mitochondrial functionality compared to standard techniques? Summary answer The sperm-CCs interaction method appears to be effective in selecting sperm with an intact acrosome and functioning mitochondria What is known already In nature, the selection of the best quality sperm is very demanding and involves high morphological qualities and dynamic features. Semen preparation in ART has long been carried out using either the swim-up or density gradient method; although both methods provide motile sperm, they cannot really replicate the physiological selection processes. As a result, several methods have been proposed to mimic the natural selection that occurs in the female reproductive tract, allowing the most competent sperm to provide the paternal contribution to the zygote. However, the literature on these techniques is limited and does not provide consistent conclusions Study design, size, duration Proof-of-principle study conducted at the New Fertility Group (NFG) in Rome and the Department of Molecular and Developmental Medicine at the University of Siena (Italy) from July 2022 to December 2022, including 9 semen samples. Comparison of the CCs selection model with the standard swim-up by assessing mitochondrial and acrosome integrity. In addition, mitochondrial activity was assessed by oxygraphic analysis in spermatozoa exposed to immortalised human granulosa cells (hGL5) compared to standard swim-up sorted spermatozoa Participants/materials, setting, methods The CCs-model was prepared by running two channels of medium, one with CCs (study group) and one with medium alone (control). Spermatozoa were added to each channel and incubated at 37 °C for 1 hour. Moreover, spermatozoa were also added on hGL5 and incubated for 1.5h. The collected spermatozoa were compared with controls for acrosomal integrity, assessed by FITC-labelled PSA, and mitochondrial membrane potential, assessed by Mitotracker. Finally, sperm oxygen uptake was measured by oxygraphic analysis Main results and the role of chance Assessment of sperm acrosome integrity showed a higher percentage of responding acrosomes in the CCs model compared to swim-up and controls (61% ± 0.3; 39% ± 0.4; 19% ± 0.4 p < 0.05). The CCs model also showed 83% ± 0.3 of spermatozoa with active mitochondria compared to 65% ± 0.3 in the control group (p < 0.005). A similar pattern was observed in the swim-up group. Quantitative analysis of mitochondrial membrane potential (MMP) confirmed that CCs and swim-up groups had higher MMP compared to the control group (p < 0.005). Evaluation of mitochondria in the hGL5 treated group showed active mitochondria in 78% ± 0.49 of the sperm compared to 59% ± 0.65 in the control group (p = 0.1). The same profile was detected by fluorescence quantification of MMP in the hGL5 group compared to the control group (p < 0.01). Oxygraphic analysis, as an index of oxidative phosphorylation and consequently of ATP production, was performed on hGL5 exposed spermatozoa, swim-up selected spermatozoa and controls. hGL5 exposed and swim-up selected spermatozoa showed a similar O2 consumption, about 4 times higher than unselected spermatozoa (p < 0.05) Limitations, reasons for caution The limited sample size and the heterogeneity of the samples were the main limitations of this study. Although CC and hGL5 sperm selection models appear to be more physiological, further studies on other parameters such as sperm DNA integrity are needed. Wider implications of the findings Both the CCs model and the hGL5 model appear to be effective in selecting good quality sperm. As these models avoid sperm centrifugation, further research is needed to understand their efficacy and reliability and whether they are a valid alternative to conventional sperm selection methods in terms of IVF outcomes. Trial registration number none

P-782 The influence of microfluidic preparation of spermatozoa for ICSI (intracytoplasmic sperm injection) in an unselected IVF population on fertilization rate, embryo-quality and pregnancy rate

Abstract Study question Does the microfluidic technique as a new preparation option for sperm in ICSI during IVF have a better outcome in fertilization, blastocysts and pregnancy rate? Summary answer The patients that had the sperm preparation for ICSI with the microfluidic sperm selection had similar results as with the gradient centrifugation selection. What is known already Semen analysis is a poor predictor of the fertilization potential of spermatozoa and a male factor may contribute to poor outcomes of IVF procedure despite a normal semen analysis. Traditionally, sperm processing is accomplished by density gradient centrifugation or swim-up methods but this have been shown in some studies to increase reactive oxygen species and induce sperm DNA fragmentation. The microfluidic sperm selection (ZyMot- ICSI) is based on the selection of the spermatozoa with the lowest DNA fragmentation rate but studies do not prove better clinical outcomes after this method. Study design, size, duration This is a prospective randomized study carried out in a university level fertility clinic in Oradea, Romania from January 2022 to December 2022, including 239 patients in total. The patients that underwent IVF-ICSI procedures for various indications were randomized in two groups: the control group swim-up technique was used for sperm selection for ICSI during IVF and the study group with the ZyMot-ICSI (microfluidic sperm selection technique) was used. Participants/materials, setting, methods We conducted a prospective trial comparing 119 couples that were allocated to the classic swim-up technique (control group) and 120 couples that were allocated with the microfluidic technique was used (study group) in our university level clinic to go through IVF. Inclusion criteria were couples with known infertility requiring IVF and willing to participate in the study. Exclusion criteria were: severe oligoasthenospermia (concentration < 5x10^6 spermatozoa/ml or motility <10%), oocyte or sperm donation. Main results and the role of chance The statistical analysis showed that there is no significant difference between the fertilization rate (study vs control p = 0.37 with absolute difference -1.182, 95% CI (-7.2, 5.4)) or blastocyst rate (study vs control p = 0.88 with absolute difference -0.5 95% (-6.5, 6)) nor is there a difference in clinical pregnancy. Even if microfluidic preparation of spermatozoa did not seem to improve the results it may be utilized more broadly for standard IVF, intrauterine insemination and could also improve workflow, decrease intervention by laboratory personal and provide more consistent incubation conditions. Limitations, reasons for caution The population studied was inclusive with unselected IVF indications and did not attempt to isolate male factor infertility cases or patients with a history of elevated sperm DNA fragmentation. Wider implications of the findings Microfluidic sperm preparation performs similarly to density gradient centrifugation in sperm preparation for IVF in an unselected population. Trial registration number Not applicable

O-166 Distinct molecular signatures of successful sperm in situ

Abstract Out of millions of ejaculated sperm, a few reach the fertilization site in mammals. Flagellar Ca2+ signaling nanodomains, organized by multi-subunit CatSper calcium channel complexes, are pivotal for sperm migration in the female tract, implicating CatSper-dependent mechanisms in sperm selection. Here using biochemical and pharmacological studies, we demonstrate that CatSper1 is an O-linked glycosylated protein, undergoing capacitation-induced processing dependent on Ca2+ and phosphorylation cascades. CatSper1 processing correlates with protein tyrosine phosphorylation (pY) development in sperm cells capacitated in vitro and in vivo. Using 3D in situ molecular imaging and ANN-based automatic detection of sperm distributed along the cleared female tract, we demonstrate that spermatozoa past the utero-tubal junction possess the intact CatSper1 signals. Together, we reveal that fertilizing mouse spermatozoa in situ are characterized by intact CatSper channel, lack of pY, and reacted acrosomes. These findings provide molecular insight into sperm selection for successful fertilization in the female reproductive tract.

P-018 Structural and biocompatibility analysis of gold nanoparticle integrated polymeric scaffolds for infertility diagnosis

Abstract Study question Can infertility diagnosis and sperm selection be improved by using bio- nanotechnologies? Summary answer Gold nanoparticle integrated biopolymeric scaffolds show good biocompatibility when interacting with human sperm cells, being suitable to be used as cell sorting and biosensing platforms. What is known already Microfluidic platforms are used in sperm selection for increasing the in-vitro fertilization success but they are limited by the exclusive analysis of sperm cell motility. The integration of microfluidic platforms with nanomaterials such as nanopatterned substrates can greatly enhance the selectivity and the sensitivity of the detection. The accuracy can be further improved by employing the intrinsic properties of gold nanoparticles (optical properties, high biocompatibility, surface chemistry favoring the conjugation of specific biomolecules), which makes them suitable for the successful development of precise and reliable detection assays, so far used to a limited extent in infertility research and clinical approaches. Study design, size, duration Concentration, motility and morphology of spermatozoa were analyzed in semen samples collected for spermogram. The normozoospermic and teratozoospermic samples were selected and prepared by density gradient centrifugation. The sediment obtained contained 100% motile spermatozoa, which were incubated on the thin films for 24h. After 24h, the probe from each incubation dish was collected and the survival rate was analyzed using the Makler chamber. Participants/materials, setting, methods Thin films of atactic polystyrene, chitosan and mixtures of each of the two polymers with gold nanoparticles were used to produce scaffolds of microchannels. The substrates were morphologically characterized by optical microscopy, scanning transmission electron microscopy and atomic force microscopy. Normozoospermic and teratozoospermic sperm samples were collected and incubated on the scaffolds. After 24h incubation, the viability was assessed by counting the motile spermatozoa under microscopic observation. Statistical analysis was performed using two-way ANOVA test. Main results and the role of chance Analysis of the sperm cell viability incubated on the polymeric scaffolds revealed good biocompatibility with higher survival rate in the case of chitosan-gold nanoparticle integrated samples. In multiple comparisons of normozoospermic samples, the survival rates of the sample incubated on the chitosan thin films to the control sample were 18% to 80% (-62%) (p = 0.0143), 16.66% to 45% (-28.34%) (p = 0.0143), 0% to 15.38% (-15.38%) (p = 0.0239), 4.76% to 55.35% (-50.59%) (p = 0.0239). In multiple comparisons of teratozoospermic samples, the survival rates of the sample incubated on the chitosan thin films to the control sample were 14.67% to 41% (-26.33%) (p = 0.0143), 0% to 44.82% (-33.82%) (p = 0.0239). For polystyrene, the viability was 0% which excludes its further use in infertility-related assays due to its high cytotoxicity. Overall, the colloidal gold nanoparticles integrated into the polymeric matrix improved the biocompatibility of the films. For example, citrate gold nanoparticles of spherical shape and sizes of about 20 nm increased the survival rate of normozoospermic samples, while PEGylation of nanoparticles showed further improvement of the biocompatibility. The influence of the particle type, composition and coating material onto the spermatozoa viability is currently under investigation for optimizing the effectiveness of the polymeric scaffolds. Limitations, reasons for caution NA Wider implications of the findings Due to the unique properties of gold nanoparticles, the nanoparticle-polymer scaffold systems have the potential to become the foundation of a precise biosensor that can enhance the accuracy of the current approaches in infertility assessment. Trial registration number Not applicable

P-091 Deep learning-based selection of fertilization-competent human spermatozoa

Abstract Abstract withdrawn by the authors

P-029 Centrifugation-free sperm separation device offers an efficient and standardized protocol to select high quality spermatozoa

Abstract Study question Can centrifugation-free sperm separation device be used to simplify and standardize the selection of high-quality spermatozoa? Summary answer Centrifugation-free CA0 sperm separation device minimizes inter- and intra-operator variability and yields spermatozoa with comparable fertilizing properties in a variety of semen conditions. What is known already Centrifugation-based sperm separation methods have been used in assisted reproductive technology (ART) for many decades. However, the conventional methods are criticized for harmful effects due to centrifugation. To overcome the disadvantage, more noninvasive technologies have been developed and attempted to improve the sperm separation process, e.g., the formation of capillary bridge to select motile spermatozoa, migration-sedimentation technique to sort out functional spermatozoa, microfluidic sorting chip to isolate healthy sperm. While these methods provide alternatives for noninvasive sperm separation, the limitations such as inconsistency of semen quality improvement and lack of standardized procedure remain to be resolved. Study design, size, duration A randomized controlled trial of 76 men who sought ART treatment (Lee Women’s Hospital, Taichung, Taiwan) from June to October 2022 was carried out. Seventy-six neat semen specimens were categorized into 27 normozoospermic specimens, and 49 non-normozoospermic samples (semen quality below any of the WHO 5th Edition lower reference values: concentration <15 million/mL, total motility <40%, or normal morphology <4%). Participants/materials, setting, methods Neat semen samples were separated for three replicates (replicate 1-3) using LensHooke® CA0 sperm separation device (Bonraybio, Taichung, Taiwan). Three operators (operators 1–3) performed CA0 procedures on each sample to test inter-technician variability. Interclass-correlation coefficient (ICC) between replicates as well as operators were evaluated. Pre-selection and post-selection semen quality were evaluated. The parameters included total motility, progressive motility, rapid progressive motility, morphology, DNA fragmentation index (DFI), and acrosome reaction rate (AR). Main results and the role of chance CA0 selects self-propelling spermatozoa within a microenvironment created by a microporous filter membrane. The procedure involves three pipetting steps: loading semen sample, adding sperm washing medium, and recovering the processed sample. Following the standard procedure, CA0 resulted in a low intra- and inter-operator variability and ICC values between replicates and different operators were all greater than 0.9, indicating an excellent reproducibility of CA0. In addition, significant higher levels of motility and normal morphology were observed in post-selection specimens either of normozoospermic or non-normozoospermic samples (pre-selection vs. post-selection, all p < 0.0001). In paired analysis of the advanced semen parameter, our study showed noteworthy results that CA0 significantly improved DFI from 18.2% to 2.6% for normozoospermic samples; such reduction was also found in non-normozoospermic sample processing, from 13.4% to 4.2% (both, p < 0.0001). The levels of AR were significantly reduced in normozoospermic samples (from 14.4% to 5.4%) and non-normozoospermic samples as well (from 12.8% to 5.0%) (both groups p < 0.0001). In conclusion, CA0 provides an efficient, noninvasive, standardized, and reproducible sperm separation model that CA0 diminishes the variations and ensures sperm quality. Limitations, reasons for caution The presented study was a pilot trial examining the sperm quality improvement. Follow-up analysis on IUI/IVF outcomes associated with the improvement in semen quality utilizing CA0 will be assessed in future studies. Wider implications of the findings CA0 provides multifaceted benefits covering consistent clinical outcomes, simplified and standardized procedure, user-friendliness, and cost reduction. We believe CA0 not only allows noninvasive sperm separation of clinically usable but also gives the possibility of standardization on sperm separation procedure. Trial registration number CS2-22039

O-148 Cumulative embryotoxic effect of IVF plastic devices used sequentially in routine clinical practice could slow the embryo development and delay blastulation

Abstract Study question Is there an embryotoxic cumulative effect resulting from IVF plastic consumables (PC) when used sequentially under clinical routine practice? Summary answer Reduced blastulation rates and slow development were detected in some associations of PC evaluated by the Mouse Embryo Assay (MEA) suggesting possible cumulative embryotoxicity. What is known already Recommendations from European (ESHRE) and American (ASRM) societies regarding the use of IVF PC suggest that they should undergo appropriate quality control tests to detect the possible presence of embryotoxins. The main test used by manufacturers to exclude any embryotoxicity is the MEA. This assay is currently performed individually on each PC so that a certificate of conformity is delivered for each product/lot. However, several PC are used during a single IVF cycle, potentially creating an embryotoxic cumulative effect. To our knowledge, the cumulative toxicity of several PC used sequentially on gametes and embryos has not been studied so far. Study design, size, duration The objective of this prospective study was to determine if there is cumulative embryotoxicity when several PC are used together under clinical routine conditions. Ten associations, each containing 13 to 31 PC, were designed, and assessed for embryotoxicity using a MEA methodology. The tested combinations replicated the different steps used in an IVF cycle, including sperm collection and selection, oocyte pick-up, fertilization, embryo culture, embryo transfer, vitrification and thawing and sperm freezing. Participants/materials, setting, methods A defined volume of culture media was used to extract the toxicity of several PC used in every association in triplicate. The MEA tests were performed on each medium extraction with 21 one-cell stage fresh mouse embryos. Blastocyst formation rates after 96 and 120h of culture (Day 5/6), blastocyst good quality rates and hatched blastocyst rates were compared between the tested and control groups. The total cell number per blastocyst was also evaluated. Main results and the role of chance No toxicity was detected in the first two associations comprised of 22 and 23 PC, which mimicked the collection (either in sperm cups or in spermicide-free condoms) and processing of sperm samples. However, the third association comprised of 32 plastic devices replicating the sperm collection in sperm cups, processing, and freezing in high security straws, showed toxicity as the mean blastulation rate at day 5 was reduced to 20.6% compared to 95.6% in the control group (p < 0.05). Surprisingly, the mean blastulation rates observed at day 6 was 55.6% versus 100.0% in the control group (p < 0.05). The blastocysts obtained at day 6 showed a mean number of cells (n = 112.5) significantly reduced in comparison with the control group (n = 167.9) (p < 0.05), suggesting a sublethal presence of toxins which slowed embryo development down and delayed blastulation. More experiments involving toxicity assessment in other associations of PC are currently undergoing and will be presented at the conference. Limitations, reasons for caution Sensitivity could vary depending on the methodologies used for toxicity extraction and MEA. The main cause, if any, of the detected toxicity will need to be pinpointed by analyzing each element of the affected associations individually. Wider implications of the findings Also, professionals should rationalize and minimize the number of plastic consumables used during the IVF procedures to reduce the potential accumulation of toxicity in the embryo culture system. Trial registration number Not applicable

P-042 Sperm Selection Based on Speed Using Rheotaxis in a Parallel Array of Microfluidic Apertures

Abstract Study question Does selecting human spermatozoa using a dynamic microfluidic system impact semen parameters and genomic characteristics? Summary answer Flow rate adjustment in a dynamic microfluidic device selects spermatozoa with superior DNA integrity. It also appears to select a higher proportion of Y-chromosome-bearing spermatozoa. What is known already Mammalian spermatozoa demonstrate rheotactic motion against the flow. This is important in order to overcome the fluid drag force in the female genital tract to reach the oocyte. Sperm processing methods have been developed based on these principles; more recently, static and dynamic microfluidic devices have been tested due to the association between high progressive motility and greater genomic integrity. These devices also appear to have an impact on sperm sex selection. Study design, size, duration Human ejaculates (n = 7) were evaluated and simultaneously processed by density gradient centrifugation (DGC) or a novel microfluidic sperm sorter (MFSS) with an adjustable flow rate inspired by the constriction of the uterotubal junction. Concentration, motility, morphology, and SCF were assessed and compared between raw, DGC-, and MFSS-processed specimens. To identify differences in the SCF and the proportion of X- and Y-spermatozoa, spermatozoa isolated at different flow rates (150, 250, 350, and 450 μl/h) were evaluated. Participants/materials, setting, methods Ejaculates were evaluated by standard semen analysis according to WHO 2021 criteria. Following complete liquefaction, 1 mL of sample was used for DGC, and 0.8 mL of semen was used for the prototype MFSS at 0.2 mL for each flow rate. SCF was assessed by TUNEL assay with a normal threshold of ≤ 15%. Fluorescent in situ hybridization (FISH) was performed using X- and Y-chromosome probes. Main results and the role of chance A total of 7 men (35.4±5 years) had the following semen parameters: volume of 3.3±2 mL, concentration of 76.7±33 x106/mL, 45.5±1% motility, and a normal morphology of 3.2±0%. When comparing the post-processing parameters between the two methods, DGC yielded a concentration of 49.9±25 x106/mL and MFSS yielded a reduced concentration of 4.5±5x106/mL (P<0.0001). DGC yielded 91.1±1% motility and normal morphology of 3.2±0.4%. Albeit at a cost of concentration, MFSS yielded spermatozoa with significantly higher motility (96.3±3%; P<0.0001) and a higher proportion with normal morphology (4.0±0.6%; P<0.0001). Moreover, the SCF of the raw sample was 9.4±2.0%; although the SCF was 8.1±3.6% with DGC, it was 4.5±2% (P<0.001) with MFSS. An analysis was performed within the different flow rates of the MFSS; a 250 μl/h flow rate yielded the highest recovery rate (42.0±6%; P<0.001), highest concentration (5.6±4 x106/mL; P<0.01), highest motility (97.0±2.2%; P<0.05), and a normal morphology rate (4.3±1%). The SCF for this flow rate was <3%. Surprisingly, we noticed that the proportion of Y-chromosome–bearing spermatozoa positively correlated to the flow rate, suggesting that there may be a subtle difference in the kinetics of male and female spermatozoa. Limitations, reasons for caution Although our novel MFSS platform is a promising technique, it is costly and requires specific expertise and training. This is a limited observation and requires integration of clinical outcomes to prove its benefits. Wider implications of the findings A microfluidic device with a controlled flow rate allowed the fine-tuned selection of spermatozoa with superior hydrodynamic and kinetic characteristics and a higher genomic integrity. Once automated, this device may help to select spermatozoa with greater embryo developmental competence and be useful to isolate spermatozoa of the desired sex. Trial registration number N/A

P-143 Will Use of Testicular sperm in ICSI cycles increase Embryo Aneuploidy?

Abstract Study question Will use of surgically retrieved Testicular Sperms (TESA) in Intra-Cytoplasmic Sperm Injection (ICSI) cycles increase embryo aneuploidy? Summary answer Use of TESA sperms for ICSI seems safe and doesn’t increase embryo aneuploidy. TESA performed for various indications also showed no variation with embryo aneuploidy. What is known already Invention of ICSI and improved surgical sperm retrieval techniques have helped men with severe male factor infertility to father their own genetic child. Sperms retrieved from seminiferous tubules of testes are considered immature as they haven’t completed the process of spermiogensis. It is hypothesized that use of TESA sperm for ICSI can increase embryo aneuploidy, which is still uncertain. Study design, size, duration This retrospective study conducted at a private fertility clinic from 2014–2022 (n = 265 patients; total of 860 embryos evaluated). Study populationTESA sperm used and PGT-A done (n = 66). TESA group was further sub-divided into 2 groups based on the indication for TESA,Non-Obstructive Azoospermia(n = 26) and Raised SDF(n = 40). Control Population Ejaculated sperm used and PGT-A done (n = 199) Only younger women(<37yrs) with self-gametes and RIFconsidered in this study. RIF was defined as women with 2 failed IVFcycles in thepast with at least 4 blastocystsbeing transferred. Participants/materials, setting, methods All fertilized oocytes inseminated by ICSI and subjected to extended blastocyst culture. TESA done as per our clinic’s SOP and for control group sperm preparation done by density gradient method. Trophectoderm biopsy was performed and sent for PGT-A to the genetic lab. Next-Generation sequencing (NGS) was the Comprehensive Chromosomal technique (CCS) used for assessing embryo ploidy status. Aneuploidy across all groups compared. Main results and the role of chance Following were the Aneuploidy rates of TESA sperm, Ejaculate sperm group and TESA done for NOA and SDF respectively. Ejaculate Sperm – Aneuploidy%- 42% TESA Sperm (irrespective of the indication)- Aneuploidy%- 44% (p = 0.6260) TESA Sperm (done for NOA)- Aneuploidy%- 44% (p = 0.7504) TESA Sperm (done for raised SDF) – Aneuploidy%- 32% (p = 0.05) Embryo aneuploidy was comparable between Ejaculate and TESA sperm group. Though TESA sperm is considered immature, it didn’t seem to increase embryo aneuploidy. Embryo Aneuploidy from TESA sperm ICSI cycles done for NOA or raised SDF also showed no altering trend. Data from this study is suggestive that TESA sperm doesn’t alter embryo ploidy status. Limitations, reasons for caution Data is retrospective with small sample size and unequal distribution. A well designed Randomized Trial might help test the inference of this study further. Wider implications of the findings Incidence of male infertility and use of ICSI is on a rise globally. Interventions to optimize embryo implantation and reproductive outcomes will help with better success rates and shorten duration to conception. Further research is warranted with severe male factor infertility to improve sperm selection techniques and reproductive outcomes. Trial registration number not applicable

P-007 A Four Arm Prospective Randomized Control Trial to Investigate the Role of Advance Sperm Selection Techniques for Raised Sperm DNA Fragmentation Index

Abstract Study question Role of Testicular Sperm Aspiration (TESA), Magnetic Activated Cell Sorting (MACS), Microfluidics & Daily ejaculation, to reduce raised Sperm DNA Fragmentation Index (SDF) & evaluate impact on outcomes? Summary answer Amongst the advance sperm selection methods, Microfluidic sperm sorting seems to significantly reduce the SDF and offer the best reproductive outcomes What is known already Raised SDF is known to affect the reproductive outcomes of a couple. There are various sperm processing methods/interventions shown to reduce the sperm DNA fragmentation, but their effectiveness to optimize reproductive outcomes is still debatabl. Study design, size, duration RCT approved by the Institutional Ethical Committee. IEC No.:1312/Inst/TG/2019. Couples undergoing IVF with raised SDF were randomized into four arms by computer generated randomized numbers from October 2019 to May 2022 (n = 466). Of those 373 patients recruited to, TESA (n = 129), MACS (n = 92), Microfluidics (n = 116) Daily ejaculation (n = 36) . Males with SDF > 25% & Age 25-40 yrs were included. Women>37 yrs, BMI >30 and men with Smoking, Binge alcohol, varicocele excluded. Couples with one failed IVF were offered SDF. SDF>25%was considered raised and were included in study. Participants/materials, setting, methods After Oocyte Pick Up (OPU), sample processed for ICSI and was sent for post intervention SDF. TESA done as per SOP of clinic. MACS & Microfluidics done as per instruction from manufacturer. Daily ejaculate group, patient ejaculated daily for 1week prior to OPU and at OPU, fresh ejaculate processed through density gradient. Men with high SDF, after randomization were subjected to OPU, ICSI, Blastocyst Culture, Freeze all policy and Frozen Embryo Transfer with 2blastocysts Main results and the role of chance Primary outcome-magnitude of reduction in SDF post intervention Secondary outcomes – Blastocyst Rate (BR), Implantation rates (IR), Live birth (LBR)Mean of SDF on Raw Semen Sample Vs Post Intervention Sample: TESA – 44% Vs 25.75% Microfluidics - 34.4% Vs 9.8% (p < 0.05; Significant) MACS – 32.2% Vs 27.95% Daily Ejaculation 39.37% Vs 19.66% Microfluidics seem to be beneficial intervention in significantly reducing the SDF. MACS seemed to have offered the least reduction of SDF. BR: This was comparable between all groups with no statistical significance Reproductive Outcomes for TESA, Microfluidics, MACS & Daily ejaculation: IR – 39.3% Vs 57.27% Vs 46.4% Vs 61% (p = 0.1) LBR – 60% VS 65% Vs 64% Vs 61% (p = 0.41) Miscarriage Rates and Multiple Pregnancy rates were comparable between the groups and statistically not significant. We observe that microfluidics was the best sperm processing method as it significantly reduced the DFI and had the best reproductive outcomes. It is noninvasive and can be done easily. Even though there was no statistical significance, Daily ejaculation yielded equivocal outcomes than the other 3 groups. Considering the ease, cost effectiveness and optimal reproductive outcomes Daily Ejaculation can also be offered to patients with raised SDF. Though TESA had acceptable outcomes, considering its invasiveness adequate counselling is warranted. Outcomes for MACS were guarded and be suggested with caution. Limitations, reasons for caution The sample size is small in the daily ejaculate arm as there were many dropouts. The unequal size of the arms would affect the results. So, we are continuing the randomization and intend to look into the results with equal size of the arms in the future Wider implications of the findings Published literature has shown raised SDF to be detrimental to reproductive outcomes. There is an urgent need for an efficient intervention which is safe, economical, easy to use and offers the best reproductive outcomes. Daily ejaculation, a traditional concept which is cost effective needs due consideration over other techniques. Trial registration number CTRI. Trial No.: REF/2021/05/043439

P-020 Association between male age and the frequency of sperm abnormalities

Abstract Study question Does advancing male age influence the frequency of occurrence of sperm abnormalities? Summary answer Advancing male age was associated with increased incidence of certain head and tail defects – elongated head, nuclear vacuoles, and coiled tail. What is known already Evaluation of male reproductive potential is based mainly on sperm motility and sperm morphology. Morphological sperm features serve as a reliable indicator in predicting the fertilizing capacity of sperm. Nowadays, there are various sperm selection techniques based on sperm morphology. However, advancing male age has been associated with decrease in semen quality. Therefore, the aim of this study was to examine the effect of aging on the frequency of occurrence of sperm abnormalities. Study design, size, duration This retrospective study includes 3980 men aged between 15 and 72 years, with a mean of 36 years. Semen samples were collected between October 2015 and December 2022 in a private in-vitro hospital. Men were divided into three groups according to their age: 1) from 15 to 25 years (n = 144), 2) from 25 to 45 years (n = 3447), and 3) over 45 years (n = 393). Participants/materials, setting, methods Sperm morphology was evaluated according to the Kruger’s strict criteria. Totally, 23 types of abnormalities were determined. Head defects included small, large, amorphous, elongated, round, pear-shaped, double, detached head, acephalic cells, small or large acrosomal areas, and spermatozoa without acrosome. Midpiece defects included thick, thin, bent, asymmetric midpiece and cytoplasmic droplets. Tail defects included short, coiled, and double tail. Acrosomal vacuoles and nuclear vacuoles were also evaluated. Statistics: ANOVA, followed by LSD post-hoc test. Main results and the role of chance The results are presented as mean±SD. Among the studied population the percentage of normozoospermic men was 44%, while the percentage of teratozoospermic semen samples was 56%. The total mean frequencies of occurrence of head, midpiece and tail abnormalities were 85.9±7.9%, 34.1±12.8% and 4.7±5.1%, respectively. Only 3 (13%) of the analysed 23 types of sperm abnormalities were significantly more common with advanced male age – elongated head, nuclear vacuoles, and coiled tail.The frequency of occurrence of elongated head differed significantly between the age groups 1-2, and 1-3: 11.38±10.1% vs. 13.79±11.58%, p = 0.015, and 11.38±10.1% vs. 14.4±11. 8%, p = 0.008, respectively. No difference in the incidence of this defect was observed between groups 2 and 3 (p > 0.05).Furthermore, the frequency of nuclear vacuoles significantly increased with age as observed in the following age group comparisons 1-2, 1-3, and 2-3: 4.28±4.93% vs. 5.4±6.1%, p = 0.04, 4.28±4.93% vs. 6.48±7.89%, p < 0.001, and 5.4±6.1% vs. 6.48±7.89%, р=0.001, respectively.Finally, coiled tail was observed more frequently in age groups 2 and 3 compared to age group 1: 13.79±11.58% vs. 11.38±10.1%, p < 0.001, and 14.4±11. 8% vs. 11.38±10.1%, p < 0.001, respectively. Limitations, reasons for caution This is a single-institution retrospective study. Wider implications of the findings The findings of this study showed that aging is associated with an increased percentage of several sperm abnormalities: spermatozoa with elongated head, nuclear vacuoles, and coiled tail. Further studies are required to clarify the negative impact of aging on spermatogenesis leading to morphological defects. Trial registration number NA

P-251 Differences in fertilization, blastocyst, and ploidy rates in intracytoplasmic sperm injection (ICSI) versus conventional insemination for patients undergoing in vitro fertilization (IVF)

Abstract Study question Are there differences in fertilization, blastocyst development, or euploidy rates when comparing ICSI versus conventional insemination among patients with male and non-male factor infertility? Summary answer Fertilization rate was lower for male factor ICSI. There were no differences in blastocyst development. Euploidy rate was lower for non-male factor ICSI. What is known already ICSI was developed for male factor infertility due to its requirement for a very small number of viable sperm. Its use has expanded beyond male factor due to some studies suggesting enhanced fertilization, and lower risks of failed fertilization or inadvertent contamination of embryo biopsy specimens. There are potential risks associated with ICSI, including imprinting disorders, birth defects, and significantly increased cost. It is also unclear whether ICSI contributes to genetic abnormalities by bypassing the egg’s natural sperm selection and potentially disrupting the meiosis apparatus. Study design, size, duration This retrospective cohort study of 576 patients was conducted at a single clinic from January 2021 to December 2021. Patients were grouped into (1) ICSI with male factor (n = 201), (2) ICSI with non-male factor (n = 160), and (3) conventional insemination with non-male factor (n = 215). Primary outcome was ploidy status, determined by calculating the percentage of euploid, aneuploid, and mosaic (both low and high mosaic) among the three groups. Secondary outcomes included fertilization and blastocyst development rates. Participants/materials, setting, methods Patients undergoing autologous IVF at a single clinic, with PGT-A performed at a single lab were included. Fertilization rate was defined as the percentage of 2PNs per number of mature eggs injected/inseminated. Blastocyst rate was defined as the percentage of blastocysts per 2PNs. Ploidy rate was defined as the percentage of euploid/aneuploid/mosaic blastocysts per total biopsied blastocysts for which a result was obtained. Percentage data was compared using N-1 Chi-squared test. Main results and the role of chance There were no significant differences in patient age, number of oocytes retrieved, or number of mature oocytes among the three groups. Fertilization rate for male factor ICSI was significantly lower compared to both non-male factor ICSI (74.2% vs 77.8%, p = 0.005) and conventional insemination (74.2% vs 76.8%, p = 0.018). There was no significant difference in fertilization rate between non-male factor ICSI and conventional insemination. There were no significant differences between blastocyst rates on Day 5, 6, or 7, or total blastocyst rate among the three groups. The euploidy rate for conventional insemination was significantly higher than that for non-male factor ICSI (53.4% vs 46.8%; p = 0.008). Non-male factor ICSI yielded lower euploidy rate than male factor ICSI (46.8% vs 52.4%, p = 0.034). Aneuploidy rate for non-male factor ICSI was significantly higher than both male factor ICSI (36.1% vs 31.0%, p = 0.041) and conventional insemination (36.1% vs 31.0%, p = 0.030). Mosaicism levels were similar between the three groups. There were no significant differences in number of biopsied embryos with no results: 5 for ICSI male factor group (0.59%), 6 for ICSI non-male factor (0.96%), and 10 for conventional insemination (0.89%). Limitations, reasons for caution This was a retrospective analysis of patients from a single clinic, with a relatively small sample size so results may not be generalizable to all populations. Extensive demographic information and baseline characteristics (including reason for ICSI performed among non-male factor patients) were not analyzed so there may be confounding factors. Wider implications of the findings This is the first study that we are aware of to investigate the ploidy status of embryos created among non-male factor patients undergoing ICSI. Conservative use of ICSI can significantly decrease the cost of IVF. Larger studies are needed to further elucidate the role for ICSI among these patients. Trial registration number not applicable

P-041 Enhancing Embryos of the Desired Sex in Couples Undergoing PGT-A while Minimizing Embryo Wastage

Abstract Study question Can we enhance the proportion of euploid embryos of the desired sex for couples undergoing ICSI and PGT-A with sex selection while reducing embryo wastage? Summary answer Using a novel sperm selection technique, we generated higher proportions of embryos and offspring of the desired sex without impairing clinical outcomes or offspring health. What is known already Although controversial, the wish for offspring of a specific sex is common in the U.S. Many couples, particularly those requiring IVF with PGT-A, request a euploid embryo of a specific sex. This is often associated with the generation of more conceptuses of the unwanted sex, resulting in embryo wastage. We propose utilizing a simple and safe sex selection technique (SST) to skew spermatozoa toward the desired sex so that concurrent embryos can be generated in higher proportions. Study design, size, duration Over a 7-year period, ejaculates from male partners of couples (n = 144) undergoing ICSI with PGT-A were processed using SST to enrich spermatozoa for their preferred sex. Standard sperm processing was carried out for couples undergoing ICSI exclusively to assess conceptus aneuploidy, comprising the control group (n = 1,440). The proportion of male and female spermatozoa in the initial and selected specimens, PGT-A results, and ICSI outcomes following frozen embryo transfer (FET) were compared between the two groups. Participants/materials, setting, methods A total of 1,584 couples were treated in 2,723 ICSI cycles. Standard sperm processing was performed for 1,440 couples who did not have offspring sex preferences, comprising the control. For 144 consenting couples, SST was used to enrich spermatozoa for either sex (IRB 1306014043). To confirm sex enrichment and ploidy, ≥1,000 sperm cells were screened by fluorescent in-situ hybridization (FISH) for nine chromosomes. Embryology and PGT-A outcomes were compared between the control and SST cohorts. Main results and the role of chance Couples (n = 1,440) from the control cohort (maternal age, 37.1±4yrs; paternal age, 39.1±6yrs) underwent 2,541 ICSI cycles, yielding an 80.1% fertilization rate (15,901/19,859). PGT-A confirmed that 46.6%(n = 4,479) of conceptuses were female and 53.4%(n = 5,132) were male. These couples achieved 76.2%(866/1136) implantation and 64.9%(737/1136) clinical pregnancy rates, resulting in 624 healthy deliveries (48% female, 52% male). From the study cohort (n = 144), 79 desiring female offspring (maternal age, 37.9±4yrs; paternal age, 40.8±6yrs) obtained an 81.6% sperm sex enrichment. They underwent 98 ICSI cycles, achieving a 76.7%(792/1032) fertilization rate, resulting in a greater proportion of female embryos (79.1%, 351/444) than the control (P<0.05), of which 78.6% (276/351) were euploid. Following FET, 39 couples obtained a 79.5% (31/39) implantation rate, yielding 27 clinical pregnancies with deliveries, thus far, of 16 female singletons, all developing normally. The remaining 65 couples (maternal age, 37.6±3yrs; paternal age, 40.8±5yrs) preferring male offspring obtained 80.8% male sperm enrichment and underwent 84 ICSI cycles, achieving a 74.9%(723/965) fertilization rate with a greater proportion of 79.3%(249/314) male embryos compared with the control (P<0.05), of which 66.3%(165/249) were euploid. Following FET, their implantation rate was 90.0%(36/40), yielding 25 clinical pregnancies that resulted in the deliveries, thus far, of 20 male singletons, all developing normally. Limitations, reasons for caution Although SST does not absolutely grant offspring of a specific sex, it allowed couples to obtain a greater proportion of conceptuses of their preferred genotype. This method does not replace PGT-A for sex selection, but rather enhances the generation of embryos of the desired sex, thereby reducing embryo wastage. Wider implications of the findings We confirmed a significantly higher proportion of conceptuses of the desired sex. Thus far, embryo developmental competence, aneuploidy rates, delivery rates, and offspring health were not impaired by SST-processed spermatozoa. This supports the safety of SST, rendering it effective and ethically palatable. Trial registration number n/a

P-103 Three-dimensional oviductal organoid model to study spermatozoa-oviduct interaction in humans

Abstract Study question Is it possible to establish an in vitro model that recapitulating human oviduct to investigate sperm-oviduct interaction Summary answer Three-dimensional (3D) oviductal organoid model was established from either primary human oviductal tissues or immortalized human OE-E6/67 cell line, based on our protocol. What is known already The sperm reservoir in oviduct is formed to maintain the fertilizing capacity of spermatozoa until ovulation by the binding of sperm to the epithelial lining of the oviduct, which is also significant to select sperm with normal chromatin structure and superior fertilization ability. The regulation and mechanism of sperm reservoir formation in human is still unknown, mainly due to the ethical issue and technical difficulty in obtaining enough human oviduct. We previously demonstrated that sperm fucosyltransferase-5 (sFUT5), a carbohydrate-binding protein on spermatozoa, is responsible for spermatozoa-oviduct interaction in humans. Study design, size, duration Human oviductal epithelial cell line immortalized by HPV 16 E6/E7 open reading frame by retroviral expression system was employed to establish the co-culture model. Fresh human samples (human spermatozoa,N = 30; 1-2 cm primary oviductal tissue,N = 15) were collected in hospital with signed consent. Fluorescent pre-labelled sperm were co-cultured with organoids for 2 hours. At least three replicates were conducted for each assay. Participants/materials, setting, methods Morphological characterization and markers expressions of the organoid were determined by immunohistochemistry and confocal microscopy analysis. The sperm binding capacity of the oviductal organoid was studied by co-culture system. Sperm chromatin structure of the organoid-bound sperm was assessed by flow cytometry-based TUNEL assay. Affinity chromatography followed by nano-liquid chromatography–mass spectrometry was used to identify the sFUT5-binding proteins on ovidutal epithelial cells (OECs) and their expressions on OECs were validated by immunostaining/flow cytometry. Main results and the role of chance The organoid exhibited a hollow structure surrounded by a single layer of OECs. Immunohistochemistry analysis revealed the high expressions of epithelial marker E-cadherin (basolateral membrane), secretory marker pair-box 8 (PAX8) and proliferative marker Ki67. Expression of ciliated cell marker, acetylated tubulin (AcTUB) can also be detected. The modification of culture environment successfully reversed the OEC organoids polarity as demonstrated by immunostaining using antibodies against ZO-1 (apical marker) and E-cadherin (basolateral marker), making the organoid accessible during the co-culture. These apical-out OEC organoids have significantly higher sperm binding capacities than their basal-out counterparts. Furthermore, OEC organoid-bound spermatozoa possessed more intact DNA than the unbound spermatozoa. The results indicate the relevance of using OEC organoid-spermatozoa co-culture model as a tool to assess spermatozoa-oviduct interaction in humans. By using immuno-affinity chromatography and mass spectrometry analysis, cell adhesion molecule 4 (CADM4) was then identified as a potential sFUT5-interacting protein on OECs. This result was further supported by flow cytometry and immunofluorescent staining. 3-D oviductal organoid model demonstrates profound potential for in vitro investigation of human oviduct physiology. Limitations, reasons for caution Our 3D model may not fully represent the entire cellular complexity and tubular architecture of the oviductal epithelium. Further investigation on the sperm morphology and DNA integrity and analysis on organoid transcriptome will enhance our understanding on oviduct in human reproduction. Wider implications of the findings The results from co-culture may give a clue to future study on whether oviductal organoid can be applied clinically for functional sperm selection in in vitro fertilization. Our apical-out organoid model may also be applied to study tubal ectopic pregnency. Trial registration number not applicable

P-108 Does DNA Fragmentation.level significantly impact clinical outcomes in patients undertaking Assisted Reproduction with using donor oocytes?

Abstract Study question Does an abnormal Sperm DNA Fragmentation Index significantly impact clinical outcomes in patients undergoing Assisted Reproduction with ICSI using donor oocytes? Summary answer No statistical difference was seen in pregnancy or miscarriage rates between high or low DNA fragmentation indexes with oocyte donation cycles using intracytoplasmic sperm injection. What is known already High levels of sperm DNA fragmentation are proposed to have an adverse impact on reproduction. While subject to debate, generally accepted ranges of > 30% considered high and associated with poorer outcomes, with levels <15% considered normal. Impact in the intermediate range if 15-30% is less understood. Potential interventions for elevated DFI include ICSI, lifestyle changes, antioxidant therapy or more controversially TESE. It is thought that high quality oocytes have the potential to repair sperm DNA damage- thus the use of younger donors with higher fertility in oocyte donation cycles may improve outcome and overcome a high DFI. Study design, size, duration A retrospective cohort analysis was performed on our database of donor oocyte FET treatment between January 2016 and December 2020, with 1370 cycles identified Screening to identify those couples where pre- treatment sperm DFI was performed as part of the treatment workup, and DFI was analysed in 9% of these cycle (n = 124). Clinical outcomes were recorded including positive HCG, clinical pregnancy rate, miscarriage rate and ongoing pregnancy rate at 12/40 gestation, and compared between groups. Participants/materials, setting, methods Oocyte donation cycles with baseline Sperm DFI measurements in a tertiary academic centre were identified. Donor treatments were chosen to reduce the impact of female factor variation on results. 49 cases had normal DFI(<15%), and were compared to 21 patients in the high group ( > 30). 54 patients were on the intermediate range (15-30%) and were excluded from the analysis. Pregnancy and miscarriage rates were compared between high and low groups to assess impact of DFI. Main results and the role of chance Elevated DFI has been proposed to have an abnormal impact on assisted reproduction outcomes. Other studies have suggested that ICSI may compensate for this, or that healthy oocytes may be able to negate any detrimental impact from the sperm. With autologous gamete treatments, large variations in the expected prognosis are due to female characteristics such as age, ovarian reserve or gynaecological disorders such as endometriosis or PCOS. This use of younger egg donors with normal AMH and the exclusion of any identifiable pelvic abnormality allows for some standardisation of the female cohort, therefore allowing a more direct assessment of the impact of any male factor abnormality on the outcome. Positive HCG rates per embryo transfer were the same between DFI groups. Normal 53.1% (26/49) vs High 47.65% (10/21) p = 0.68 Similarly there was no difference in Clinical Pregnancy Rates based on DFI level Normal 44.9% (22/49) vs High 47.7% (10/21) p = 0.83 Miscarriage rates per positive HCG test were again not impacted by male sperm DFI: Normal 30.7% (8/26) vs High 33.3 (3/10) p = 0.72 The study data demonstrates that in treatment cycles using a combination of ICSI and good quality oocytes, the clinical outcomes do not appear to be impacted by raised DFI Limitations, reasons for caution We previously demonstrated that fertilisation and blastulation rates were unaffected by DFI. The study was limited by a relatively small sample size, as DFI analysis was not routinely measured before treatment. Larger multi-centre studies to increase the study population and statistical power would be useful for confirmation of the findings. Wider implications of the findings Our data suggests that biochemical, clinical and ongoing pregnancy rates, and also miscarriage rate, were not adversely impacted by high DFI in donor oocyte cycles.This could be attributed to the ability of a high quality oocyte to repair sperm DNA damage when combined with ICSI for sperm selection and fertilisation. Trial registration number Not applicable