Sperm Selection Research Articles

Glass wool column filtration for stallion sperm selection: A comparative analysis with the single-layer colloid centrifugation.

Glass wool column filtration (GWCF) selects human, bull, boar, dog and buffalo spermatozoa, but reports in the horse are scarce. Single-layer colloid centrifugation with Androcoll-E™ is currently the standard procedure to select good-quality equine sperm. This study was designed to assess GWCF (50 and 75 mg columns; GWCF-50 and GWCF-75, respectively) efficacy to select good-quality sperm from fresh and frozen-thawed equine semen, and to compare its performance with Androcoll-E™ colloid centrifugation. Percentage total motile (TM), progressively motile (PM), morphologically normal (MN), osmotically competent (HOS+) and acrosome-intact/osmotically competent (AI/HOS+) sperm were determined. In studies done with fresh semen samples (n = 17), suspensions subjected to GWCF-50 showed an improvement (p < .05) in PM and HOS+ sperm after selection. With GWCF-75, an increase (p < .05) in PM, MN and HOS+ sperm was observed. Results with GWCF were comparable or better than with Androcoll-E™ selection. Sperm recovery was similar between procedures for all semen parameters. Total sperm count recovery was lower after GWCF-75 (GWCF-50 = 60.0; GWCF-75 = 51.0; Androcoll-E™ = 76.0 million sperm; median; p = .013), but results on total progressive sperm count were similar (GWCF-50 = 23.0; GWCF-75 = 27.0; Androcoll-E™ = 24.0 million sperm; median; p = .3850). Using frozen-thawed semen samples (n = 16), an improvement (p < .05) in TM, PM, NM, HOS+ and AI/HOS+ sperm was observed in GWCF-75 filtrates. Results were comparable to Androcoll-E™ centrifugation, except HOS+ that increased (p < .05) only after GWCF-75. Recovery was comparable for all parameters in frozen samples. GWCF is a simple and low-cost procedure that selects equine sperm with a quality comparable to colloid centrifugation with Androcoll-E™.

General and comparative aspects of endometritis in domestic species: a review.

Endometritis is a leading cause of sub- and infertility in domestic animal species. The healthy uterus is colonized by commensal bacteria, viruses, and yeast/fungi that represent the non-pathogenic microbiota. A shift in the number or type of organisms accompanied by immune dysfunction, however, may trigger uterine infection and inflammation. Metritis is associated with inflammation of all uterine layers (endometrium, myometrium, and perimetrium) whereas endometritis is a more superficial inflammation involving solely the endometrium. Endometritis generally occurs at two time points in domestic animal species, post-partum and post-mating. Post-partum endometritis may chronically persist, either as a low-grade disease that often manifests as a vaginal discharge but not a systemic illness (in some species termed clinical endometritis) or sometimes subclinical where features are only detected by endometrial sampling. Contamination of the uterus at the time of mating occurs by direct deposition of semen (ejaculated or artificially inseminated) into the uterus. Improper drainage of the ejaculatory fluid or an inadequate immune response may result in persistent mating-induced endometritis. Both post-partum and post-mating endometritis interferes with fertility by creating a suboptimal environment for embryo development and placentation and chronic endometritis may have an impact on sperm survival and fertilisation ability. In the post-partum animal, there may also be changes in milk production and maternal behaviour which can affect offspring health and survival. Preventive strategies for endometritis largely depend on monitoring their known risk factors, which are sometimes specific with regard to the species. Effective, non-antibiotic therapy for endometritis is not available to date. Overall, extensive research has been performed in cattle and horses to unravel key aspects of endometritis, but in sows and bitches, the available literature is scant. Thus, the need and opportunity to investigate the condition vary considerably among domestic species and necessitate their comparative assessment. This article reviews general and comparative aspects of the diagnosis and classification, pathogenesis, preventive strategies, and therapeutics of endometritis in domestic species with a specific focus on cows, mares, sows, and bitches.

Design of a gradient-rheotaxis microfluidic chip for sorting of high-quality Sperm with progressive motility

Assisted reproductive technology (ART) is an important invention for the treatment of human infertility, and the isolation of high-quality sperm with progressive motility is one of the most critical steps that eventually affect the fertilization rate. Conventional sperm separation approaches include the swim-up method and density gradient centrifugation. However, the quality of isolated sperm obtained from both approaches can still be improved by improving sorted sperm motility, minimizing the DNA fragmentation rate, and removing abnormal phenotypes. Here, we report a Progressive Sperm Sorting Chip (PSSC) for high-quality sperm isolation. Based on the rheotaxis behavior of sperm, a gradient flow field is created in the chip for progressive sperm sorting. Clinical experiment results for 10 volunteers showed that greater than 90% of isolated sperm exhibit high motility (> 25μm/s), high linearity (0.8), and a very low DNA fragmentation rate (< 5%). In addition, the whole process is label and chemical free. These features aid in gentle sperm sorting to obtain healthy sperm. This device uniquely enables the selection of high-quality sperm with progressive motility and might be clinically applied for infertility treatment in the near future.

Zinc alleviates cadmium-induced reproductive toxicity via regulating ion homeostasis, metallothionein expression, and inhibiting mitochondria-mediated apoptosis in the freshwater crab Sinopotamon henanense

Cadmium (Cd) is a carcinogenic environmental pollutant that harms male reproductive systems by lowering sperm quality, impairing spermatogenesis, and causing apoptosis. Although zinc (Zn) has been reported to alleviate Cd toxicity, the underlying mechanisms have not been fully elucidated. The aim of this work was to investigate the mitigating effects of Zn on Cd-induced male reproductive toxicity in the freshwater crab Sinopotamon henanense. Cd exposure not only resulted in its accumulation but also in Zn deficiency, decreased sperm survival rate, poor sperm quality, altered ultrastructure, and increased apoptosis in the testis of the crabs. Morever, Cd exposure increased the expression and distribution of metallothionein (MT) in the testis. However, Zn supplementation effectively mitigated the aforementioned effects of Cd, as demonstrated by preventing Cd accumulation, increasing Zn bioavailability, alleviating apoptosis, increasing mitochondrial membrane potential, decreasing reactive oxygen species (ROS) levels, and restoring MT distribution. Moreover, Zn also significantly reduced the expression of apoptosis-related (p53, Bax, CytC, Apaf-1, Caspase-9, Caspase-3), metal transporter-related ZnT1, metal-responsive transcription factor 1 (MTF1), and the gene and protein expression of MT, while increasing the expression of ZIP1 and Bcl-2 in the testis of Cd-treated crabs. In conclusion, Zn alleviates Cd-induced reproductive toxicity via regulating ion homeostasis, MT expression, and inhibiting mitochondria-mediated apoptosis in the testis of S. henanense. The information obtained in this study may serve as the foundation for further investigation into the development of mitigation strategies for adverse ecological and human health outcomes associated with Cd contamination or poisoning.

Cryopreservation Induces Acetylation of Metabolism-Related Proteins in Boar Sperm

Cryodamage affects the normal physiological functions and survivability of boar sperm during cryopreservation. Lysine acetylation is thought to be an important regulatory mechanism in sperm functions. However, little is known about protein acetylation and its effects on cryotolerance or cryodamage in boar sperm. In this study, the characterization and protein acetylation dynamics of boar sperm during cryopreservation were determined using liquid chromatography–mass spectrometry (LC-MS). A total of 1440 proteins were identified out of 4705 modified proteins, and 2764 quantifiable sites were elucidated. Among the differentially modified sites, 1252 were found to be upregulated compared to 172 downregulated sites in fresh and frozen sperms. Gene ontology indicated that these differentially modified proteins are involved in metabolic processes and catalytic and antioxidant activities, which are involved in pyruvate metabolism, phosphorylation and lysine degradation. In addition, the present study demonstrated that the mRNA and protein expressions of SIRT5, IDH2, MDH2 and LDHC, associated with sperm quality parameters, are downregulated after cryopreservation. In conclusion, cryopreservation induces the acetylation and deacetylation of energy metabolism-related proteins, which may contribute to the post-thawed boar sperm quality parameters.

Comparison of Cryoresistance of Testicular and Urinal Spermatozoa of the Toad &lt;i&gt;Bufo bufo&lt;/i&gt; (Amphibia, Anura, Bufonidae) during Slow Freezing

A comparative study of the morphofunctional parameters of testicular (non-activated) and urinal (activated) spermatozoa of the common toad Bufo bufo (L., 1758) before and after cryopreservation was carried out. Cryopreservation was performed by a slow method. Sucrose (10%) and dimethylformamide (12%) were used as cryoprotectants. It was shown that the pool of testicular spermatozoa contains significantly more cells with abnormal morphology both before and after cryopreservation. The osmoticity of the base medium for the cryoprotective solution (Ringer’s solution for amphibians – 230 mOsm or urinal plasma – 45 mOsm) had little effect on the survival of testicular and urinal spermatozoa during cryopreservation. Comparison of the morphological and functional characteristics of spermatozoa before and after freezing-thawing indicates that the cryoresistances of testicular and urinal spermatozoa differ slightly.

Comparison of the effects of sperm selection methods on assisted reproduction outcomes in male infertility

Objective: To compare physiological intracytoplasmic sperm injection (PICSI) and the hypoosmotic swelling test (HOST) sperm selection techniques in terms of embryo development, implantation rate and live birth rates in cases of severe or moderate oligoastheneospermia. Materials and Methods: The electronic material and files of cases admitted to the Medipol University IVF Centre between 2013 and 2022 were analyzed retrospectively. This research included a total of 143 cases with moderate or severe oligoasthenospermia, 80 cases with PICSI as sperm selection technique and 63 cases with HOST as sperm selection technique. Cases of severe or moderate oligoasthenospermia as defined by the World Health Organisation (WHO) are included in this study. The demographic parameters of both groups, including female age, paternal age, duration of infertility, AMH (anti-Mullerian hormone), body mass index (BMI), endometrial thickness, and the number of prior attempts, were analyzed. Implantation, pregnancy loss, and rates of live births were compared between two groups. Results: Female age, paternal age, AMH, endometrial thickness, BMI, and the number of previous trials were similar between the groups included in the study. There were not any significant differences between the groups in terms of fertilization (.18±6.92 vs 9.11±6.35, p=0.345), TQ-GQ blastocyst development (2.36±2.40 vs 1.64±2.35, p=0.097), and the number of embryos transferred (1.59±0.50 -1.76±0.44 p= 0.141). The live birth rates between the PICSI and HOST groups did not vary statistically (p=0.790), however, the rate of implantation was significantly higher in the HOST group (p=0.043). Sperm selection by PICSI or HOST method did not affect fertilization, TQ-GQ blastocyst development, and the rates of pregnancy loss and live birth. Conclusion: Although there were not any differences in embryo development, pregnancy loss, and live birth rates comparing the two methods, the HOST group had a higher implantation rate. Keywords: PICSI, HOST, Male infertlity, live birth

Expression of aldose reductase in mouse endometrial epithelial cells and its role in sperm capacitation

The survival, motility and capacitation of sperm in the female reproductive tract are important prerequisites for fertilization. The uterus is the main location for sperm capacitation. One of the most important physiological functions of the endometrial epithelium is to create a suitable uterine environment under the regulation of ovarian hormones, to ensure sperm capacitation. The composition of uterine fluid directly affects sperm capacitation. Fructose is an important component of semen that supports sperm viability and motility. Aldose reductase, a rate-limiting enzyme in the polyol pathway, metabolizes sorbitol and fructose, thereby supplying cells with necessary energy for functional activities. Existing studies have reported the presence aldose reductase in the endometrium, leading us to hypothesize that its expression in endometrial epithelium might promote sperm capacitation by maintaining the uterine environment. Yet, the mechanism of regulation has not been clarified. In this study, we investigated the expression of aldose reductase in mouse endometrial epithelium and its potential role in sperm capacitation. We initially investigated the periodic characteristics of glucose, fructose and sorbitol in uterine fluid. We then studied the temporal and spatial characteristics of aldose reductase in the endometrial epithelium. Next, we examined the effect of aldose reductase on glucose, fructose and sorbitol in uterine fluid. Finally, we explored the effect of aldose reductase on sperm capacitation and fertilization. The results showed that glucose and fructose content in uterine fluid and the expression of aldose reductase fluctuated periodically during physiological periods. Inhibition of aldose reductase in the endometrial epithelium interfered with sperm capacitation and fertilization by reducing the fructose levels in the uterine fluid. To conclude, the aldose reductase-mediated polyol pathway in endometrial epithelial cells is essential to maintain an appropriate fructose environment in the uterine fluid for sperm capacitation and fertilization.

P-312 Automated sperm selection software (SiD) is as good as human selection for ICSI

Abstract Study question Automated sperm selection or human eye-based sperm selection, does it make a difference on IVF lab outcomes? Summary answer Single sperm selection by the AI SiD was as proficient as highly experienced embryologists and embryo development may have improved for inexperienced embryologists. What is known already The implementation of artificial intelligence in reproductive medicine has proven effective for a variety of applications. One application for this technology is single sperm selection during ICSI. The computer assisted program SiD, was designed to grade sperm, in real-time, based on motility parameters transforming qualitative sperm assessments into a quantitative score. SiD’s assistance can be used to select the most optimal sperm for ICSI. The application of sperm selection by AI is expected to bypass user bias, fatigue, limited training, while also optimising time. However, there are limited studies correlating the usage of AI for sperm selection with ICSI outcomes. Study design, size, duration Between August and November 2022, sibling MII oocytes were randomly divided in two groups; 1-ICSIgroup: ICSI performed with sperm selected by the embryologist based on motility and 2- SID-ICSIgroup: ICSI performed with sperm selected using SiD software. Priority was given to sperm classified as “Best” by SID software for ICSI. “good” sperm were considered as second choice. Morphology selection was performed before ICSI in both groups. Embryos were cultured during 6 days in same conditions. Participants/materials, setting, methods Sperm preparation was performed using a microfluidic chamber (Zymot™). Sperm selection was performed as previously described, and ICSI was performed following routine procedures. Biological outcomes were analysed in both groups and compared including fertilization rate, cleavage rate, embryo and blastocyst development rate, top quality embryo rate and blastocyst development rate. Biological outcomes in both groups were also compared based on embryologist seniority. Finally, embryo morphokinetics parameters were analysed in both groups through manual TLT annotations. Main results and the role of chance A total of 226 MII oocytes were included in “ICSI group” and 227 in “SID-ICSI group. A non significant trend towards better outcomes was observed in the SID-ICSI group for all biological outcomes including fertilization rate (81,5% vs 80,5%), cleavage rate (98,4% vs 97,3%), day 3 embryo development rate (77,8% vs 73,1%), top quality development rate at day 3 (50,8% vs 50,0%), blastocyst development rate at day 5 (49,7% vs 45,1%), good quality blastocyst development rate at day 5 (48,1% vs 46,2%), top quality blastocyst development rate at day 5 (25,4% vs 23,1%). Similarly, a non-significant increase was observed in all these outcomes when comparing both groups when sperm selection is performed by a junior embryologist. Conversely, no difference was seen in both groups when sperm selection is performed by a senior embryologist. Finally, no significant difference was observed in the SID-ICSI group for all fertilization events (tPB2, cytoplasmic wave, tPN1, tPN2, presence of cytoplasmic halo, tPNf, and disappearance of cytoplasmic halo) other cleavage timings (t2-&amp;gt;t10, tM, tSC, tEC, tSB) and embryonic cell cycles (ECC1, ECC2, ECC3 s2, s3). Limitations, reasons for caution The ZymotTM microfluidic chamber was used for sperm preparation. This device optimises sperm selection, giving rise to an already optimised population of spermatozoa. Future investigations evaluating the effect of SiD automated sperm selection software on biological outcomes in samples prepared with a less effective technique would potentially yield conversing results. Wider implications of the findings This pilot study demonstrated that the usage of the automated sperm selection software SiD gives rise to similar biological outcomes and embryo morphokinetics, indicating its effectiveness in sperm selection. We expect this program would be useful in the presence of less experienced laboratory members helping by standardizing sperm selection. Trial registration number NA

P-059 Sperm DNA fragmentation (SDF) after cryopreservation and sperm selection: implications for clinical pregnancies and live births after intrauterine insemination with donor sperm

Abstract Study question Can SDF assessment pre-cryopreservation, post-thaw and after post-thaw sperm selection predict clinical outcome in a heterologous intra-uterine donor insemination program (IUI)? Summary answer SDF increased post-cryopreservation in donor sperm and after post-thaw density gradient without affecting clinical pregnancy, live birth and miscarriage rates. What is known already Sperm cryopreservation is effective for preservation of male fertility and facilitates the storage of donor semen, while infectious disease screening can be completed and confirmed negative. Sperm viability and motility are the most vulnerable parameters during the freeze-thaw process. Inherent reactive oxygen species may induce SDF via oxidative stress. Controversial results are reported regarding induction of SDF after cryopreservation due to differences in cryo tolerance, methods of cryopreservation and methods used to evaluate SDF. Sperm selection post-thaw is indispensable to select a normal motile fraction for insemination. Again, results are controversial regarding influence on SDF levels after sperm selection post-thaw. Study design, size, duration This was a prospective, observational study. Between March 2015 and March 2019, eighteen potential sperm donors were screened via a three step plan – semen assessment, medical assessment and serological blood tests. In accordance with the Belgian legislation (2007), a sperm donor was matched to obtain a maximum of 6 pregnancies in acceptor women/couples, allowing for more than one offspring/woman or couple. A total of 106 acceptors were matched for heterologous intra-uterine insemination. Participants/materials, setting, methods Semen samples were collected after 2-7 days abstinence and standard semen parameters analyzed within 60 mins after ejaculation. Within the same time frame SDF test was performed using TUNEL assay both before and after cryopreservation and after a two-step discontinuous density gradient centrifugation post-thaw. Samples were cryopreserved by the conventional slow freezing method using sperm freeze solution. A yield of ≥ 2 M progressive spermatozoa post-thaw after selection was sufficient to contemplate IUI. Main results and the role of chance Mean female age at first cycle was 33.9 ± 4.1 years and donor age 28.5 ± 5.6 years. Out of 429 cycles, 100 (23.3%) resulted in clinical pregnancy. Excluding three ongoing pregnancies, we counted 75 live births (17.6% of cycles or 77.3% of pregnancies), while 21 pregnancies ended in miscarriage (4.9% of cycles or 21.6% of pregnancies) and 1 resulted in stillbirth. Progressive motility (59.3 ± 12.5%) decreased significantly after cryopreservation (30.0 ± 13.2%; p &amp;lt; 0.001), but increased post-thaw after density gradient centrifugation (61.6 ± 16.0%; p &amp;lt; 0.001). On the contrary, SDF (12.0 ± 5.9%) increased post-cryopreservation (26.3 ± 14.5%; p &amp;lt; 0.001) and further increased after sperm selection (34.9 ± 22.1%; p = 0.04). Using multilevel mixed-effects logistic regression (Odds ratio [95%CI]), we found that female age significantly influenced clinical pregnancy (0.911 [0.847-0.981]; p = 0.01), live birth (0.894 [0.834-0.959]; p = 0.002) and miscarriage rates (1.180 [1.033-1.347]; p = 0.015). There was no significant age-adjusted effect of SDF post-thaw after density gradient selection on clinical pregnancy (1.007 [0.994-1.021]; p = 0.30), live birth (1.001 [0.988-1.014]; p = 0.84) or miscarriage (1.021 [0.997-1.046]; p = 0.08). Median time-to-live birth was 4 cycles. Analyses of time-to-live birth with mixed-effects Cox models revealed no significant association with progressive motility; SDF before or after cryopreservation and after sperm selection post-thaw. Limitations, reasons for caution Results concern a small donor population, with high normal semen parameters and should therefore be extrapolated cautiously to subfertile men with normal and subnormal semen parameters. Wider implications of the findings In a donor IUI program with strict selection on conventional sperm parameters, differences in SDF before and after cryopreservation and gradient centrifugation do not change IUI outcomes, although cryopreservation and centrifugation increase SDF. Findings could be useful for the optimization of sperm freezing and selection in different assisted reproductive procedures. Trial registration number not applicable

P-006 In men with Raised Sperm DNA Fragmentation Index, would advance Sperm Preparation techniques alter Embryo Euploidy?

Abstract Study question In men with Raised Sperm DNA Fragmentation Index (DFI), Would advance sperm preparation techniques at Intra-Cytoplasmic Sperm Injection (ICSI) alter embryo euploidy status? Summary answer In men with raised Sperm DFI, use of advance sperm selection techniques are safe and do not appear to alter ploidy status of the embryos What is known already MACS (Magnetic Activated Cell Sorting), Microfluidic Sperm Sorting (MF), surgically retrieved Testicular Sperm (TESA) and Double Density Gradient sperm processing (DDG) have been shown to optimize sperm selection at ICSI for men with raised sperm DFI. Would any of these sperm selection methods alter embryo ploidy status is not proven Study design, size, duration This was a retrospective observational study conducted at a private fertility centre in-between years 2015 &amp; 2022. Couples where women (&amp;lt;37yrs) had idiopathic Recurrent Implantation Failures (RIF) and men had raised sperm DFI (&amp;gt;25% DFI) and underwent Pre-Implantation Genetic Testing (PGT) were evaluated (n = 473). Based on sperm selection method the study population was divided into four groups. TESA (n = 84), MACS (n = 105), MF(n = 135), DDG (n = 148).A total of 1802 embryos were evaluated in this study. Participants/materials, setting, methods All women underwent Controlled Ovarian Stimulation (COS) and oocyte retrieval and male partners underwent sperm processing as per the clinics SOP. Appropriate counselling and written consents were obtained from all couples, where applicable. All oocytes subjected to ICSI, extended blastocyst culture and trophoectoderm (TE) biopsy. Post biopsy embryos were vitrified and TE tissue subjected to Next-Generation Sequencing (NGS) to assess the ploidy status. Ploidy status between various sperm selection methods was compared. Main results and the role of chance Euploidy rates between the study groups were as follows: TESA (n = 311 embryos screened)- 48% MACS (n = 445 embryos screened) – 51% MF (n = 489 embryos screened) – 61% DDG (n = 557 embryos screened)- 54% Though numerically the ploidy status was highest in MF group for euploid embryos, there seems to be no statistical significance between the groups (p-value = 0.973). Different sperm selection techniques do not seem to alter embryo ploidy status. Ejaculated sperm or Testicular sperms had similar euploidy status. Use of Testicular sperm, which is considered immature doesn’t seem to increase embryo aneuploidy. MACS, MF and DDG all seemed to have similar euploid embryos. This further warrants research to validate the role of MACS and MF as a routine intervention to optimize sperm selection and reproductive outcomes. Limitations, reasons for caution Retrospective design, un-equal group size and small sample size, a well-designed study is needed to further validate the findings of this study. Wider implications of the findings Role of advance sperm selection methods to optimize embryo implantation is yet to be proven. Data from this study showed no alarming increase in embryo aneuploidy in younger women and was comparable with published literature. Use of advance sperm selection techniques seems safe and doesn’t alter embryo ploidy status. Trial registration number not applicable

P-073 Comparison of human preimplantation embryo development after sperm selection for ICSI using the microfluidic ZyMōt device versus density gradient centrifugation

Abstract Study question Does microfluidic sperm selection using ZyMōt device has a significant effect on blastocyst development, ploidy, and improves pregnancy outcomes when compared with density-gradient centrifugation. Summary answer The study demonstrated that sperm selection using a microfluidic device had no impact on euploid blastocyst rates or clinical pregnancy outcomes. What is known already Selecting competent spermatozoa with the highest genomic integrity for ICSI is a key to achieving normal embryo development and a healthy live birth. Several sperm preparation techniques have long been scrutinized with varied success. The novel microfluidic ZyMōt device appears promising for improving the quality of sperm used in Assisted Reproductive Technologies. Preliminary tests have shown that ZyMōt may increase sperm motility and considerably decrease DNA fragmentation, which could potentially enhance euploid blastocyst development rates. However, the impact of this sperm selection method on clinical outcomes has not been fully investigated. Study design, size, duration This is a retrospective cohort study performed in a single academic IVF centre (IRB # 16367). Data were collected from 1286 ICSI cycles in 2020-2021 (730 with ZyMōt and 556 with DGC), including 867 cycles with PGT-A outcomes. Morphological grading of 6640 blastocysts cultured in a time lapse imaging incubator (Embryoscope™) and ploidy of 4783 blastocysts using high resolution NGS (Illumina platform and Blue Gnome) were compared between the two study groups. Participants/materials, setting, methods All male partners were normo-spermic according to standard semen analysis. ICSI results for a total of 7277 mature oocytes injected with spermatozoa processed with ZyMōt device and 5977 oocytes injected with spermatozoa processed by gradient-density centrifugation were compared. For the analysis of 1478 euploid embryo transfer outcomes, all cases included a single blastocyst transferred in a frozen-thaw cycle. Associated patient characteristics, including age and fertility diagnosis were collected. Main results and the role of chance A significantly higher fertilization rate was observed with ZyMōt compared to the gradient prepared sperm (83.4% vs 81.0%, P = 0.0004, respectively). The blastocyst rates (61.3%, vs 60.4%, P = 0.34) as well as the proportion of high-quality blastocysts were similar in both groups (P = 0.20). Euploidy rates were not different between all female age groups, including young egg donors (≤ 30y of age) to patients with advanced maternal age (&amp;gt;40 y of age). Microfluidic sperm selection did not improve clinical pregnancy rates after euploid embryo transfer (42.9% vs 41.4%; P = 0.58). Limitations, reasons for caution Although this is the largest study to date evaluating application of the ZyMōt device, the retrospective nature and cohort design limited the interpretation of our results. The patient population did not include male factor cases and sperm DFI data was not available. Embryo transfer outcomes were limited to clinical pregnancies. Wider implications of the findings The ZyMōt device offers a convenient alternative to standard sperm processing by density gradient method. Further studies are needed to fully determined the impact of ZyMōt on embryo quality and clinical outcomes in different age and diagnosis groups of patients; particularly for those with high DFI and poor IVF/ICSI outcomes. Trial registration number not applicable

P-193 Comparison of ICSI outcomes and euploidy rates between AI and non-AI sperm selection

Abstract Study question Can artificial intelligence (AI) assisted sperm selection improve intracytoplasmic sperm injection (ICSI) outcomes and euploidy rates? Summary answer AI selected spermatozoa showed no statistical significance in various measures of ICSI outcomes but an increasing trend in euploidy rate can be observed. What is known already All ICSI practitioners would like to have an objective tool for the selection of sperm to be injected as it is one of the factors in producing successful ICSI. Sperm selection assistant (SiD: (IVF 2.0 Limited, UK) is a real-time AI spermatozoa identifier that assesses all spermatozoa in a visual field based on their motility patterns individually. Mendizabal-Ruiz et. al. (2022) have reported promising data showing SiD may reduce subjectivity in sperm selection, which can potentially be an alternative method to our current practice. Study design, size, duration From June 2022 to December 2022, 89 ICSI cycles (mean age: 35.3; age range: 26.0–47.0) were done in Alpha IVF &amp; Women’s Specialists. A sibling AI (Group A) and non-AI (Group B) assisted sperm selection study with ICSI was performed on the oocytes retrieved from these cycles. Of which, 76 cycles were planned for Preimplantation Genetic Testing for Aneuploidies (PGT-A) on utilizable blastocysts. Participants/materials, setting, methods All matured oocytes were injected using Piezo-ICSI method (Prime Tech Ltd, Japan). Half of the oocytes of each cycle were injected with SiD selected sperm whereas the other half by manual selection. All injected oocytes were cultured up to day-7 and trophectoderm biopsy for PGT-A screening (IonTorrent, USA) was done on utilizable blastocysts prior to vitrification (Cryotec, Japan). The fertilization, blastulation, utilization and euploidy rates were assessed in both groups. Main results and the role of chance In Group A, the fertilization rate (2PN), abnormal fertilization rate ( &amp;gt; 2PN), blastulation rate from 2PN, blastocyst utilization rate from 2PN and euploidy rate from utilizable blastocysts were 81.5% (419/514), 2.1% (11/514) 77.1 (323/419), 57.5% (241/419) and 45.2% (71/157) respectively. In Group B, the fertilization rate (2PN), abnormal fertilization ( &amp;gt; 2PN), blastulation rate from 2PN, blastocyst utilization rate from 2PN and euploidy rate from utilizable blastocysts were 79.7% (427/536), 2.8% (15/536), 75.9% (324/427), 59.0% (252/427) and 35.5% (60/169) respectively. No significant differences were observed in the fertilization rate (p = 0.4828), abnormal fertilization rate (p = 0.5545), blastulation rate from 2PN (p = 0.6861), blastocyst utilization rate (p = 0.7278) and euploidy rate (p = 0.0898). Although the ICSI outcomes are comparable between the two groups, AI assisted sperm selection showed an increasing trend in euploidy rates. SiD may be considered as a tool to assist embryologist in selecting better spermatozoa during ICSI procedures. Limitations, reasons for caution The patient sample size was small but further studies with a larger sample size certainly seem to be justified, particularly in association with PGTA on utilizable blastocysts. The value of SiD shall be greatly enhanced when spermatozoon morphology can also be measured. Wider implications of the findings This is more evidence suggesting that SiD may improve ICSI outcomes and euploidy rate when compared to manual selection by an embryologist. In addition, it could be used to train and assist embryologists learning ICSI procedures. Trial registration number not applicable

O-300 Human sperm selection using cumulus oophorus complexes compared with conventional sperm preparation method on sperm quality and ICSI outcomes: A pilot study

Abstract Study question We compared a sperm selection protocol using cumulus oophorus complexes with a swim up preparation, evaluating the effects on sperm parameters and ICSI outcomes Summary answer Spermatozoa selected using cumulus oophorus complexes showed higher rates of normal morphology, good motility compared with basal semen and the traditional pellet swim up protocol. What is known already It is known that the head of a mature spermatozoa has a hyaluronan receptor that allows them to bind with hyaluronan acid. Cumulus oophorus complexs (COCs) extracellular matrix is made mainly by hyaluronic acid (HA). Various studies have confirmed the importance of COCs in fertilization, demonstrating that spermatozoa that are able penetrate through a layer of COCs are more likely to have a normal morphology and be more effective in producing acrosome reactions than spermatozoa that are not able to do. Moreover, these spermatozoa have been shown to have higher chromatin integrity and zona-binding capacity, resulting in increased fertilization competence. Study design, size, duration A prospective study was conducted between July and December 2022. A total of 50 participants were recruited for the study, randomly selected between patients attending ICSI. The patients were classified as group A when the sperms were selected using pellet swim up and COC selection and as group B when it was used pellet swim up treatment alone. Patients older than 39 years, with endometriosis or repeated IVF failure or severe male factor were excluded. Participants/materials, setting, methods 3 drops of 30 mlt aligned and connected each other were prepared, covered with mineral oil. In group A, in the middle drop, was transferred COC fragments, while the central drop was empty in group B. In the left drop were added 20 mlt of sperm suspension after swim up, and from the right drops were recovered the sperms after passing the middle drop with or without COC, that were used for ICSI treatment. Main results and the role of chance The aim of the study was to verify the effectiveness of COCs, collected after ovarian stimulation, in selecting the best sperms to be used in ICSI procedure. No statistical difference was found between group A and B in the median age of female patients, FSH/LH units administred, E2 and endometrium thickness at triggering using hCG, in the sperm concentration, motility and normal morphology in the baseline seminal samples. We found a statistical difference in the traditional seminal parameters comparing the two different treatments. In particular, we found an higher rates of sperms with rapid and progressive motility and normal morphology in group A between basal semen sample and after sperm selection using COC filter (86.7 vs 31.3%; 8.2 vs 4.4 %). We found a lower sperm concentration in group A vs group B after sperm treatment (2.3 vs 4.7 M/ml), and an higher rates of rapid progressive motility and normal morphology in group A vs group B (86.7 vs 38.6 %; 8.2 vs 4.6%). We found an higher but not statistically significative pregnancy and implantation rates in group A compared with group B probably because of the limited number of patients analyzed (25 vs 19.2 %; 25 vs 20.6%) Limitations, reasons for caution The study was conducted on a small participant cohort. We analyzed the traditional sperm parameters, but it is necessary to evaluate the chromatine integrity of the sperms after COC selection to confirm that the higher sperm competence in fertilization and embryo development could be based on a better genetic pattern Wider implications of the findings Data seem to demonstrate that the use of COC allows to select higher competent sperms if compared with traditional pellet swim up treatment. The clinical and biological ICSI outcomes seem to be improved by COC treatment but it is necessary to increase the sample size in future experimental trial Trial registration number not applicable

P-061 In situ microfluidics of fluidic walls: a novel deviceless and cost-effective approach for sperm selection in the same ICSI-dish

Abstract Study question Is our novel deviceless method based on in-situ microfluidics a valuable strategy to select suitable sperm for ICSI? Summary answer The novel protocol allows selecting sperm for ICSI within 15 minutes, in the same ICSI dish and disregarding fungible supplies, centrifugation and washing steps. What is known already Microfluidics is an innovative operation in ART which integrates sperm guidance biomimicry during the in vitro sperm selection process. In contrast to other sperm preparation methods such as Wash-Swim-Up and Density- Gradients-Centrifugation, microfluidics disregards washing and centrifugation steps along the procedure. In addition, microfluidics are time-efficient methods which reduce risks associated with handling, gamete mix-up and ROS production. However, microfluidics devices are costly and likely to fail to select motile sperm in dispermic samples. With the aim of outlining the application of microfluidics in ART, we conceived a lab-on-a-chip approach for sperm selection by in-situ handmade microfluidics of fluidic walls. Study design, size, duration The microfluidics circuit conforms to Laplace and hydrostatic pressure laws and integrates the sperm guidance mechanisms of rheotaxis and boundary-following behavior. System’s efficacy is based on the volume differences, the distances between microdoplets, and their arrangement. It divides into Section 1 (dispersion and seminal plasma removal) and Section 2 (sperm sorting in response to positive rheotaxis). The system was adjusted to recover at least 20 selected progressive sperm for ICSI in less than 15 minutes. Participants/materials, setting, methods We prepared the system on a conventional IVF polystyrene round dish (15 mm diameter) using MOPS buffer, PVP and mineral oil only. We verified the flow-driven dynamics by registering the horizontal displacement (direction) and flow velocity (µm/s) using eosin-Y and methylene blue dyes. We confirmed the removal of the seminal plasma in a PSA assay. The proof-of-concept analysis was performed using fresh semen samples from 150 patients (Caucasian; mean age: 33,53±8,19). Main results and the role of chance Our design includes three main points to allow sperm separation: 1 (initial), 2 (rebalancing), 3 (final) with a total distance of 1,5 cm between 1 and 3. Our system assures a continuous microfluidic flow, with two opposed directions (from point 1 to point 2 and from point 3 to point 2). PSA assay showed a significant decrease in PSA levels &amp;gt;200000 ng/ml in the fresh sample to undeterminable levels (&amp;lt;0,0091 ng/ml) at point 3. To assess the utility to select a suitable number of sperm for ICSI we used sperm samples with the following characteristics: volume 3,6 (1,0-7,6) ml, concentration 68 (0,29-150) ×106/ml, progressive motility 24 (2-41) % and morphology 6 (1-15) % normal. The minimum and maximum times for sperm recovery at point 3 were recorded at 5 and 35 minutes, respectively. Normozoospermic samples required lower recovery times compared to dispermic (p &amp;lt; 0,001), although according to the median, samples with at least 1 × 106/ml of motile sperm need less than ten minutes recovery time. Sperm parameters: concentration, motility and morphology were inversely correlated with the recovery time (p &amp;lt; 0,0001), being morphology the parameter with the lesser influence on the outcome. Limitations, reasons for caution The present protocol depends on a free-hand preparation. Although the use of templates and the training reduces inter and intra-operator variability, a ready-to-use surface would benefit the operability. Non-inferiority studies are required to compare our method with the currently used ones (Wash-Swim-Up or Density-Gradients-Centrifugation) before clinical implementation. Wider implications of the findings Our deviceless methodology streamlines sperm selection for ICSI and reduces risks along the procedure. This novel approach represents a paradigm shift in sperm preparation, since it dispenses with centrifugation, washing and plasticware. The present protocol can be implemented to the clinic in the near-term, particularly due to its simplicity. Trial registration number na

P-063 Microfluidic technology is highly effective in selecting a sperm population with high progressive motility and low DNA fragmentation index

Abstract Study question Which sperm preparation procedure is able to select the best population of sperm? Summary answer Microfluidic sperm sorting using Fertile Plus device isolates sperm with the highest motility and lowest levels of DNA fragmentation. What is known already The sperm selection in Medically Assisted Reproduction should lead to fully functional spermatozoa able to reach the fertilization site and allow optimal fertilization and embryo development. These abilities are dependent on specific sperm parameters such as, but not limited to, motility, morphology, Acrosome Index (AI), and DNA fragmentation Index (DFI). At present, the routine sperm selection is suboptimal, does not replicates the physiological processes observed in nature and consequently may induce sperm damage. The new Microfluidic Sperm Sorting technology allows a physiological selection of sperm. However, no comparison with other selection procedures regarding all the above-mentioned parameters was reported. Study design, size, duration Prospective-observational study performed at an University Hospital-IVF Centre between April-November 2022 comparing the effect of four different sperm selection procedures on various semen parameters. Fifty-two samples with minimum 20x106 sperm/ml and progressive motility ≥30% were included. Descriptive statistics reported the mean±SD [minimum-maximum] values for each variable. Interclass correlation coefficient (ICC) was calculated for the absolute agreement in between the four different procedures, where DGC was considered the standard-of-care. An ICC-value &amp;lt;0.4 indicates a low agreement. Participants/materials, setting, methods Unused raw semen after routine diagnostic analysis were individually split in 4 fractions and processed immediately by different sperm preparation methods: 1) sperm wash (SW), 2) Density Gradient Centrifugation (DGC), 3) Magnetic Activated Cell Sorting (MACS, (Miltenyi Biotec)), and 4) Microfluidic Sperm Sorting (MSS) using Fertile Plus Device (Koek Biotechnology). Each fraction was analyzed for progressive motility, morphology, Acrosome Index (AI) and DNA fragmentation Index (DFI; TUNEL assay protocol). Main results and the role of chance The mean age of the patients was 35.9 years (SD ± 6.8). The mean value of days of abstinence was 4.1 (SD ± 6.8). Mean progressive motility rates were: 54.3±10.6% [23-86%], 74.3±11.8% [38-90%], 77.2±12.5% [37-92%] and 88.6±4.2% [73-96%] for SW, DGC, MACS, and MSS, respectively. Mean percentages of normal morphology were: 3.3±2.9% [0-13%], 4.1±3.1% [0-13%], 4.2±3.7% [0-18%], and 5.1±3.9% [0-16%] for SW, DGC, MACS, and MSS, respectively. Mean AI were: 8.5±4.9% [1-20%], 9.7±6.0% [1-30%], 8.7±4.9% [0-19%], and 10.8±6.8% [1-30%] for SW, DGC, MACS, and MSS, respectively. Mean DFI were: 6.2±4.6% [0.8-26.1%], 2.7±3.2% [ 0.2-14%], 2.1±4.3% [0.9-20.8%], and 0.2±0.4% [0.0-2.3%] for SW, DGC, MACS, and MSS, respectively. Values referring to ICC absolute agreement between the DGC and the other methods indicate MSS as the only procedure in strong disagreement regarding progressive motility [0.29, 95%CI (-0.2-0.58)] and DFI [0.17; 95%CI (-0.19-0.45)]. No disagreement regarding morphology and AI was observed between all techniques. A further paired comparison confirmed the strong disagreement between MSS and MACS [0.2; 95%CI (-0.17-0.49) and 0.21; 95%CI (-0.25-0.52)] and between MSS and SW [0.06; 95%CI (-0.04-0.25) and 0.06; 95%CI (-0.15-0.28)] regarding progressive motility and DFI, respectively. Limitations, reasons for caution Differences in DNA-damage may have been smaller between techniques if abstinence period was shorter. Due to the design of the experiment, data on clinical outcomes are not available. Wider implications of the findings The selection of a population of highly motile spermatozoa with undamaged DNA from unprocessed semen is ideally performed with MSS using Fertile Plus. Question remains how these results compare with a shorter abstinence period and whether this improves the embryological outcomes in the IVF laboratory. Trial registration number NCT05383599

P-227 ICSI outcomes after using in-situ microfluidics of fluidic walls versus DGC: a prospective non-inferiority comparative pilot study in sibling oocytes

Abstract Study question Does the novel strategy in-situ microfluidics (isM) yield comparable ICSI outcomes to the control sperm selection methodology density gradient centrifugation (DGC)? Summary answer ICSI outcomes show that handmade in-situ microfluidics of fluidic walls are as effective as DGC to achieve fertilization and usable day 5 blastocyst formation rate. What is known already Microfluidics technologies are proving their capability to select suitable sperm for ICSI. Increasingly, publications show novel and ingenious microfluidics strategies aimed to integrate sperm biomimicry during in vitro sperm selection while simplifying the IVF-workflow. We recently designed a microfluidics system which allows selecting sperm for ICSI in the same ICSI-dish, only using microfluidics and disregarding centrifugation, washing and plasticware. A previous proof-of-concept study showed that this methodology was efficient to select suitable sperm for ICSI as it could separate at least 20 progressive spermatozoa in less than 15 minutes in a clean microdroplet free of any remaining seminal plasma. Study design, size, duration The present pilot study included a total of 280 fresh MII-oocytes allocated in a 1:1 ratio to the study (isM-ICSI) and control (DGC-ICSI) groups. The statistical power was established at 80% at a CI 95%. For comparison we relied in the KPIs: ICSI normal fertilization rate (INFR) and Day 5 usable blastocyst rate (D5UBR). The non-inferiority margin (Δ) was established using 2-sigma warning limit and our historical control mean values (ΔINFR = 7% and ΔD5UBR = 10%). Participants/materials, setting, methods An informed consent was signed by all participants. The comparative study included 29 consecutive ICSI cases performed in fresh oocytes from women under 40 years old and with an oocyte yield ≥6 MII. All semen samples had ≥1 × 106/ml progressive spermatozoa and were split into three aliquots: 1-seminogram (100 µl), 2-isM (10 µl), 3-DGC (surplus). Embryos were cultured in bench-top Time-lapse incubators, using GTL-media under 6,4% CO2 and 5% O2 conditions up to blastocyst stage. Main results and the role of chance Sperm characteristics showed the following total mean values for total concentration (42,83 ± 31,1 × 106/ml), total motility (51,28 ± 14,16 %) and progressive sperm concentration (17 ± 12 × 106/ml). The mean number of MII per patient was 9,66. From the total number of oocytes (n = 280), 139 were allocated to isM (study group) and 141 to DGC (control group) groups. INFR was 72,0 % vs 76,6 %, respectively. Non-significant differences were observed (p = 0,54). We observed that the mean total number of usable blastocysts (day 5/6) per patient was 1,83 (53/29) in isM vs 1,66 (48/29) in DGC. Moreover, D5UBR was 53,0% vs 44,4 % (p = 0,26) with a difference between the study and the control of within the non-inferiority margin. We observed a lower rate of arrested embryos (up to day 6) in the study group (19,0% vs 30,0%), although results were not statistically significant (p = 0,08). Limitations, reasons for caution This pilot study includes a limited number of oocytes. The present isM protocol depends on a free-hand preparation. Although the use of templates and the training reduces inter and intra-operator variability, a ready-to-use surface would benefit the operability. Further investigation is required to evaluate clinical pregnancy and live birth outcomes. Wider implications of the findings Sperm DNA fragmentation is cited among the causes of embryo arrest. As the use of isM resulted in a lower proportion of arrested embryos, exploring its capacity to select non-fragmented sperm warrants further interest. The isM protocol streamlines sperm selection for ICSI, reduces costs and risks along the procedure. Trial registration number na

O-256 Evaluation of the fertility potential of semen using Proton NMR

Abstract Study question Is Proton NMR seminal plasma assessment a valid method for identifying biomarkers allowing to predict top-quality (TQ) embryo development? Summary answer Biomarkers important in the context of male fertility can be assessed using proton NMR What is known already Currently, the quality of sperm is assessed predominantly by examining its concentration, motility, and morphology. These parameters are insufficient in diagnosing male infertility and predicting treatment outcomes with assisted reproductive technology (ART). Metabolomics techniques have been shown to be potentially useful in identifying biomarkers related to infertility. It has been proven, using proton NMR, that there are a number of substances in the semen plasma that correlate with various physiological states of patients and may potentially indicate impaired functioning of various metabolic pathways. However, the relationship between biomarkers and embryo development has not yet been investigated Study design, size, duration This was a prospective case-control study and consisted of 30 semen samples from 13 patients who underwent IVF treatment between January – March 2022 at INVICTA Fertility Centre, Poland. Sperm motility, concentration, morphology, and DNA fragmentation of the samples were assessed. Before sperm preparation for fertilization, semen was frozen to perform 1H NMR analyses, which were preceded by centrifugation in order to separate the plasma. NMR analysis results were compared to the embryological results. Participants/materials, setting, methods It was possible to identify pre-selected biomarkers in semen plasma through the use of reference spectra and to quantify their concentrations using the internal standard addition method. Their relative concentrations (separately and in pairs) were collated with semen parameters, embryo development data and pregnancy outcomes. A Machine Learning approach was employed to identify the most important variables, which were then used for further modelling with the use of LGBM models. Main results and the role of chance NMR allows for the identification and measurement of biomarkers important in the assessment of the fertility potential of semen. The presence of amino acids, organic acids, sugar residues and fatty acid derivatives was confirmed by applying this technique. It was found that the concentration of these biomarkers is an individual feature - probably dependent on the physiological state of the organism. The results indicate that the ratios of biomarker concentrations to each other are more important than their individual concentrations. Machine learning modelling showed a strong correlation of several compounds with the chance of obtaining a TQ blastocyst. The amount of taurine, glutamic acid and choline together with its derivatives seem to be the most important factors in blastocyst development. The results indicate that if the amount of glutamic acid significantly surpasses glycerophosphocholine, choline exceeds taurine, the amount of taurine is higher than that of citrate, or the amount of choline is significantly higher than its phosphorus analogue the likelihood of obtaining a TQ blastocyst is higher. This would suggest that several metabolic pathways as the Krebs cycle, the one-carbon cycle or choline metabolism were modified signalling pathological conditions which the standard assessment of semen parameters cannot detect. Limitations, reasons for caution The NMR technique allows a fast determination of the content of many biomarkers, but results are in the mmol/l range. The results should be confirmed in a larger group of patients with different semen disorders Wider implications of the findings NMR allows for the rapid creation of unique metabolic profiles of patients, which can ensure the creation of personalized treatments for male infertility and the selection of sperm with the highest chance of obtaining a TQ blastocyst Trial registration number The studies were approved by the Ethics Committee at the Gdansk Regional Medical Board (No KB-3/22).

P-039 Pregnancy outcomes and genetic analysis of globozoospermic patients: A case series and review of the literature

Abstract Study question How should men with globozoospermia be managed in Assisted Reproductive Technologies (ART)? Summary answer IMSI (intracytoplasmic morphologically selected sperm injection), without oocyte activators (OA) allows to obtain a live birth, even when there is a mutation of DPY19L2 gene. What is known already Globozoospermia is a rare syndrome found in less than 0.1% of the infertile population. It is characterized by the presence of spermatozoa with round heads devoid of acrosomes. Two main genes are involved in the transmission of the disease: SPATA 16 and DPY19L2. Only Assisted Reproductive Technologies (ART) such as ICSI (intra-cytoplasmic sperm injection) and IMSI, with or without OA allow these couples to obtain pregnancies. There are no live births described after IMSI without OA in patients with a DPY19L2 mutation. Study design, size, duration Four infertile couples followed in the Department of ART at the Poissy Hospital Center, were studied between 2011 and 2022.All patients had total globozoospermia. Three patients were tested for the DPY19L2 gene mutation. Patients were managed in ART with the IMSI technique without the use of OA. Participants/materials, setting, methods The first patient had a homozygous mutation of the DPY19L2 gene. Two live births were obtained after IMSI. The second patient was not DPY19L2 mutated. 2 IMSI were performed, resulting in a biochemical pregnancy. The third patient had a homozygous mutation of the DPY19L2 gene. An IMSI was performed without obtaining a pregnancy, a second attempt is scheduled soon. The fourth patient refused genetic testing and IMSI management. Sperm donation was performed without achieving pregnancy. Main results and the role of chance In this case series, we present the management of four globozoosperm couples in the Reproductive Biology and Andrology Department at the Poissy Hospital Center. We also present the first two live births after performing an IMSI, without OA, in a globozoospermic patient with a homozygous mutation of DPY19L2 gene. In fact, according to our review of the literature, 53 case reports or articles of patients having benefited from ART with or without OA have been reported in globozoospermic patients. In these couples, 60 children were born. However, no live births had been reported until now in couples managed in IMSI without OA, where men had globozoospermia with DPY19L2 mutation. Limitations, reasons for caution The use of OA to increase the membrane permeability of oocyte to calcium and increase fertilization rates. The highest number of live births in the globozoosperm population occurs after ICSI or IMSI with OA. There is no described live birth without the use of OA in a DPY19L2 mutated patient. Wider implications of the findings The use of OA remains debated and is not authorized in some countries. Although our study found low fertilization rates after IMSI, it’s possible to obtain live births. The selection of spermatozoa with an acrosome outline at high magnification is thus an interesting alternative to the use of oocyte activators. Trial registration number not applicable

P-048 Vitrification of human semen impairs the mobility of spermatozoa when compared to the conventional slow-freezing

Abstract Study question Does vitrification provide a better method for human sperm cryopreservation than slow freezing in terms of survival, mobility, maturity, and DNA fragmentation? Summary answer Vitrification of human semen yields similar outcome as slow-freezing considering survival, mobility, DNA fragmentation, and maturity but the mobility after thawing is lower. What is known already Slow freezing is the most common technique for cryopreservation of human sperm. Unfortunately, many sperm cells die during the process or lose their mobility and are thereafter unable of fertilization. Meanwhile, vitrification of animal sperm showed good outcomes in terms of sperm survival and motility while being easier to perform than slow freezing. So far, many studies focused on survival and mobility of human sperm after vitrification compared to slow freezing, with results still not unified. They also neglected the importance of sperm maturity and DNA fragmentation caused by the process. DNA damage can affect embryo development or cause abortion. Study design, size, duration The study was performed from March January to July 2022. It included 69 male participants who underwent a diagnostic spermiogram at the Division of gynaecology and obstetrics of University Medical Centre Ljubljana. All participants gave written consent of using the remaining of sample, after performing the spermiogram, for our study. Sperm samples were divided in three aliquots, one saved as native sample, one intended for vitrification, and one intended for slow freezing. Participants/materials, setting, methods Participants in the study had normal sperm quality parameters according to 5th edition of WHO manual. Slow freezing was performed using Sperm Freezing medium (Origio). For vitrification sperm was diluted in solution consisting of Sperm preparation medium (Origio), sucrose, and HSA in 1:1 (v/v) ratio, incubated for five minutes and then vitrified by adding 30 microliter droplets directly into liquid nitrogen. After thawing sperm survival, mobility, maturity, and DNA fragmentation were assessed. Main results and the role of chance The data are presented as mean values (± standard deviation) when normally distributed and as medians (IQR) when not normally distributed. The patient’s age was 35.0±5.1 years. In native sperm sample, sperm concentration was 69.6±59.0x106 cells per millilitre and total motility was assessed to be 60.0% (50.0%-70.0%). After thawing we noticed significantly lower mobility (p &amp;lt; 0.001), while also the mobility was significantly lower after vitrification when compared to slow-freezing (17.6% (12.4%-25.3%) and 14.5% (9.0%-22.6%), p = 0.007). Interestingly, there was no difference in survival after thawing the samples (20.5±10.7 % after slow freezing and 23.7±13.7 % after vitrification). Also there was no difference in maturity of sperm when checked with protamine staining (native vs. slow-freezing vs. vitrification; 53.6±15.2 % vs. 53.9±16.0 % vs. 52.4±15.2 %, p = 0.905) and also when checked with hyaluronan binding assay (78.0% (67.5%-82.4%) vs. 79.4% (71.7%-82.95%) vs. 75.3% (67.5%-81.1%), p = 0.607) . While sperm maturity was not affected with cryopreservation, DNA fragmentation was significantly higher after cryopreservation (native vs. slow-freezing vs. vitrification; 19.7±9.5%, vs. 24.7±11.6 vs. 27.1±15. %, p = 0.049). Limitations, reasons for caution We believe our study would be better by obtaining higher number of participants and by being performed over longer time period. We would also like to further optimise vitrification protocol. More knowledge is also needed in poor sperm samples, since we only included sperm samples of good quality. Wider implications of the findings We believe our finding are of great importance for the field of andrology. Our results show vitrification of human sperm can be comparable to slow freezing in most of parameters analysed, but lower mobility after vitrification is reason for caution. Trial registration number 20210024