Sperm Fertility Research Articles

P-046 Sperm mitochondrial DNA copy number and mitochondrial function in normozoospermic men may predict fertilization rates after intracytoplasmic sperm injection

Abstract Study question Do sperm mitochondrial DNA copy number (mtDNAcn) and mitochondrial function correlate with fertilization rates after intracytoplasmic sperm injection (ICSI)? Summary answer We reveal a negative correlation between sperm mtDNAcn and fertilization rates after ICSI, while higher mitochondrial function corresponded to an increase in sperm fertilization capacity. What is known already Over 40% of male-factor infertility is of unknown origin (unexplained and idiopathic in nature). As current diagnoses remain largely descriptive, the identification of markers predicting the fertilization capacity of sperm will hold the key towards improved treatment. Mitochondria are fundamental for supporting spermatogenesis and fertilization. A recent study showed that altered levels of sperm mitochondrial proteins led to oocyte-activation deficiency, resulting in fertilization failure after ICSI. Poor semen parameters have also been linked to high mtDNAcn, however the relationship between mtDNAcn in sperm and oocyte-activation capacity remains unexplored. Further characterization may uncover prognostic markers associated with sperm-related fertilization failure. Study design, size, duration Twenty normozoospermic semen samples were included in the study. These were obtained from oocyte donation ICSI cycles, performed between March 2021 and November 2022. Both fresh and cryopreserved sperm samples were analysed. Five million sperm were collected from the fresh ejaculate for genomic DNA (gDNA) isolation and mtDNAcn quantification, while the remainder of the sample was used for mitochondrial functional analysis. These analyses were also performed on washed samples, cryopreserved after swim-up. Participants/materials, setting, methods Mean paternal age and BMI were 40 ± 5.8 years and 25 ± 2.3 kg/m2, respectively. We evaluated mtDNAcn using qPCR, by assessing the ratio of mitochondrial-to-nuclear genes (NT-ND4/B2M). For our functional studies, we quantified mitochondrial membrane potential (MMP) in individual spermatozoa by JC-1 staining and flow cytometry. Flow cytometry analysis was performed on ≥ 10 million sperm treated with a mitochondrial uncoupler for 15 minutes (1 µM trifluoromethoxy-carbonylcyanide-phenylhydrazone, FCCP) and controls. Spearman correlation was applied. Main results and the role of chance We observed variable fertilization rates in our cohort (69.0 ± 24.9%). One sperm sample resulted in total fertilization failure (TFF) after ICSI and was analyzed independently. In fresh samples, mtDNAcn (5.8 AU ± 2.61) correlated negatively with progressive motility (rs = -0.65) and fertilization rate (rs = -0.32). Notably, the sample resulting in TFF presented with the highest mtDNAcn value (13.9 AU ± 2.77). Flow cytometry analysis revealed two distinct sperm populations, corresponding to motile and immotile sperm. While immotile sperm presented with low or undetectable JC-1 aggregates (red signal), motile sperm showed different levels of MMP (ratio INTred/INTgreen) (2.3 AU ± 1.83). In motile sperm, our results revealed a positive correlation between MMP and fertilization rate (rs = 0.59). As with raw semen, we observed a negative association between MMP and mtDNAcn in motile sperm (rs = -0.53). Limitations, reasons for caution Cryopreservation is known to affect sperm motility and active mitochondria in sperm, however its effect on MMP intensity remains to be established. Our limited sample size warrants careful interpretation. Wider implications of the findings Our findings suggest a negative correlation between sperm mtDNAcn and fertilization rates after ICSI, while fertilization capacity improved with increasing MMP. Sperm mtDNAcn and MMP may have prognostic value for male fertility and ICSI outcomes. Further validation may deliver clinically useful markers, aiding infertility diagnosis, treatment and counselling. Trial registration number NA

P-098 Cryopreservation process deregulates the expression of pathways important for fertility in human spermatozoa

Abstract Study question Does cryopreservation impact on transcriptome of conventional frozen human spermatozoa? Summary answer The cryopreservation of human sperm alters pathways important for fertility, although the upregulation of some genes can compensate for the harmful effects of freezing. What is known already Sperm cryopreservation is a widely used procedure for storing gametes for later use, for preserving fertility in patients before gonadotoxic treatments or surgery, and for sperm donation programs. The cryopreservation technique can cause damage to sperm through induction of DNA alterations, fragmentation and oxidation, and altered motility, mitochondrial activity and morphology. Transcriptomic alterations have been reported in cryopreserved sperm from various animal species, but no experimental data are available on the effect of cryopreservation on the transcriptome-wide profile of human sperm. Study design, size, duration Semen samples were obtained from 20 normospermic men from April to May 2022. Each sample was divided in two aliquots. From one aliquot total RNA was immediately extracted. The second aliquot was slowly cryopreserved and after a week of storage in liquid nitrogen total RNA was extracted. A total of 13 paired RNA samples passed quality control and were analyzed after randomization into 4 pools, each of 6 patients. Participants/materials, setting, methods The men who participated in this study had a median age of 35.0 years (range: 29.0-46.0). RNA was extracted by miRNeasy Micro Kit (Qiagen), analyzed by High Sensitivity RNA on 2200 Tape Station system (Agilent), amplified by Ovation Pico WTA SystemV2 (Nugen), labeled and hybridized on GE 4x44K v2 microarrays (Agilent). The paired Significance Analysis of Microarray (SAM) was performed using the limma hgug4112a.db and samr packages in R/BioConductor. Main results and the role of chance The expression profiles of cryopreserved sperm significantly differed from those of fresh sperm in 219 down-regulated and 28 up-regulated unique transcripts. The gene ontology analysis disclosed that cryopreservation downregulates genes involved in sperm motility by the mitochondria function (CABS1, SPATA19) and the correct organization of the sperm midpiece and flagellum cytoskeleton (ACTL7A, AKAP4, C9ORF24, CAPZB, CCIN, IQCG, ODF2, SPATA6, SPATA6L, TBC1D21), in fertilization (ACTL7A, AKAP4, PLCZ1, PRSS37, TUBGCP3), early embryo development (AGT, CLMN, DCC, DHRS3, EGFLAM, FAM20A, FREM1, GPI, HEMGN, KRTDAP, IFT172, MICAL2, MLF1, NF2, PGRMC2, PHACTR1, POPDC3, RO60, ROBO1, RORA, SGCA, SPRR2D, TCF4, TNC, TNNI3), oxidant detoxification (TXNDC29) and DNA repair (BRIP1, ERCC6, TRIP12), calcium ion binding and homeostasis (AMY1C, ANO1, ANO2, ASGR1, CABS1, CPNE9, FREM1, KCNIP2, ITPR3, PKD2L1, PLCZ1, SELENOK, SYN3, TNNI3). Upregulated genes were enriched in pathways related to the negative regulation of DNA damage response (ALOX15B, CD74, RPL10, SNAI1, THBS1), cellular response to calcium ion (ALOX15B, FOSB, HLA-DQB1, S100A4, THBS1), regulation of transcription (CD74, FOSB, HLA-DRB1, MAMLD1, RASD1, RPL10, SNAI1, TCF15, TLE1), protein stabilization (CD74), protein ubiquitination (BAG1, DCAF6, ERCC6, HERPUD2, KLHL7, MARCHF8, NF2, PSMA6, RNF133, SERGEF, TRIP12, UBE2DNL, UBL3, UBQLN3, ZNRF3) and ubiquitin protein ligase binding (GPI, HSPA1L, PRKAR2A, STX8). Limitations, reasons for caution Data validation in a larger sample cohort and by quantitative PCR would be useful, although we have applied stringent criteria for gene selection (FDR = 0). Further research should be undertaken to experimentally test whether the length of cryostorage can have any effect on the gene expression profile of sperm. Wider implications of the findings Specific pathways are deregulated by cryopreservation, in accordance with altered sperm morphology, motility and molecular integrity reported in literature. Functional enrichment analyses also revealed gene expression changes to compensate for the sperm cryoinjury. This finding is noteworthy for safety issue of sperm banking i.e., fertility preservation, gamete donation. Trial registration number not applicable

P-131 Predicting Embryo Utilisation Rate on Day 5 using an Artificial Neural Network: A Multicentre Retrospective Study

Abstract Study question Can artificial intelligence (AI) identify which patient and cycle specific variables are most important for predicting blastocyst utilisation rate (BUR) on day 5? Summary answer Number of mature oocytes (MII) injected is the most significant variable for predicting BUR. Highest BUR association with MII is found when six MII injected. What is known already The day 5 usable blastocyst rate originating from the accumulation of embryos being transferred and cryopreserved is one of the most informative key performance indicators (KPI) in an IVF laboratory. As per the Vienna consensus, the blastocyst development rate competency value is set as ≥ 40% with the benchmark value set as ≥ 60%. Artificial intelligence (AI) has been integrated into clinical settings as a technological advancement aimed to improve clinical success rates. Identifying the predictors that can result in an increased BUR on day 5 can be an additional tool towards improved cycle outcomes. Study design, size, duration In this retrospective, multicentre study, we evaluated six variables for predicting BUR using an artificial neural network (ANN). Study was performed in two fertility centres, between March 2021 and August 2022. A total of 865 cycles were included. Cycle exclusion criteria: Preimplantation genetic testing (PGT), advanced maternal age (AMA) > 38, infertility aetiology of endometrial origin, severe oligospermia and epididymal/testicular sperm extraction. Primary outcome measure was the BUR on day 5 for the different variables. Participants/materials, setting, methods A total of 865 cycles were analysed using the ANN model with six variables: Number of MII oocytes injected (ICSI), use of autologous/donated gametes, maternal age at oocyte pickup (OPU), sperm concentration, progressive sperm motility rate, and fertilisation rate. Cycles were divided into training and test set through stratified random sampling: 73.2% (633) training and 26.8% (232) test. BUR on day 5 was grouped into <60% and ≥60% as per Vienna consensus benchmark values. Main results and the role of chance Number of MII oocytes was found to be the most important variable (100%) followed by the type of gametes used (54.1%), sperm concentration (32.9%), age at OPU (24.7%), progressive sperm rate (23.4%) and fertilisation rate (17.1%).The effect of MII injected on BUR was then investigated and results indicated an inverse correlation, with increasing MII injected resulting in decreased BUR (correlation coefficient of r = 0.344,p<0.001). According to the performed model, 6 injected MII produces the higher rate of utilisation (62.9%). Performance of ANN model was assessed by positive predicted value (PPV), negative predicted value (NPV), false positive rate (FPR), false negative rate (FNR), overall accuracy (OA), sensitivity and specificity. PPV for the training and test sets was 62.2% (194) for the <60% EUR on day 5 group and 79.8% (256) for ≥60% EUR on day 5 group and 64.8% (83) for the < 60% EUR on day 5 and 81.7% (85) for ≥60% EUR on day 5 group, respectively. OA obtained from training and test sets were 71.1% and 72.4% for the <60 and ≥60% EUR on day 5 groups, respectively. The area under the curve (AOC) to predict the UR on day 5 group was found to be 77.6%. Limitations, reasons for caution The results represent the experience gained from current practice and not of a prospective controlled study. Clinical outcomes using this approach were not explored. Several other patient and cycle variables can also be investigated. Wider implications of the findings The number of oocytes retrieved is associated with live birth rate with 15 being the ideal maximum. Increasing oocyte numbers however, can bear greater risks. Predicting embryo utilisation rate on Day 5 can guide the way towards personalised treatment and safety. Trial registration number Not applicable

P-401 Is cancer type associated with semen quality and infertility among men who desire fertility preservation?

Abstract Study question To investigate the association between cancer-types and semen-parameters before gonadotoxic-treatments, and to evaluate which patients have the highest risk of diminished spermatogenesis prior to therapy. Summary answer sperm quality among cancer patients before gonadotoxic treatment can differ according to the type of cancer. What is known already Many studies have reported impaired sperm quality at cancer diagnosis before any given gonadotoxic regiments. Except for testicular cancer and Hodgkin’s Lymphoma, baseline data on semen quality in case of different types of malignancies among male cancer patients are limited or based on low sample size Study design, size, duration A retrospective population-based cohort analysis was performed, 237 oncological male patients who were referred for fertility preservation between 1995-2020 within 90 days of cancer diagnosis and prior to cancer treatment Participants/materials, setting, methods The study population included 237oncological male patients who were referred to the Sperm Bank, Fertility, and IVF unit for the purpose of fertility preservation. Sperm analysis was performed within 90 days of cancer diagnosis and prior to therapy. The pre-treatment sperm analyses were based on the World Health Organization (WHO) guidelines Main results and the role of chance Histological examination of malignant diseases demonstrated 20 subtypes of cancers, the major groups included 49 patients with testicular cancer (TC), 72 patients with Hodgkin lymphoma (HD), 35 patients with other lymphoma, 29 patients with other hematological cancer, 16 with sarcoma and 36 with other solid malignant tumors. Our results show significant difference in sperm concentration between the different cancer groups (P value 0.007), the lowest median concentration of spermatozoa was found in the testicular cancer and sarcoma cancer patients 10 million per ml compared to the other cancer groups (other hematologic, Hodgkin lymphoma, other lymphoma and other solid tumors). Oligospermia was observed in 59% of testicular cancer patients and 50% of the sarcoma patients. Fertility outcomes were analyzed as a secondary objective. We found that only 37 patients' partners conceived and delivered offspring. Only 16.2% of those patients used their banked sperm and conceived via IVF-ICSI. We found that 20% of testicular cancer patients and other lymphoma patients including NHL conceived spontaneously after cancer diagnosis and treatment. Limitations, reasons for caution This is a retrospective cohort study and the oncology patients sample size is relatively small. Wider implications of the findings Our study emphasizes that sperm quality among cancer-patients before gonadotoxic-treatment can differ according to the cancer type. Significant reduction in sperm quality in testicular cancer patients was demonstrated, with a significant association was found between sperm concentration and various cancer-type. Further studies are warranted to confirm our results. Trial registration number not applicable

P-066 Testis expressed 44 is essential for spermatogenesis and male fertility

Abstract Study question Dose Testis expressed 44 (TEX44) gene associated with spermatogenesis and TEX44 mutations required for male infertility ? Summary answer TEX44 is expressed in testis and participated in spermatogenesis. And mutations of TEX44 may be involved in risk of asthenozoospermia and lead to subfertile. What is known already The World Health Organization estimates that 7-9 % of male population worldwide struggle with infertility. Asthenoteratozoospermia is one of the major factors for male infertility, whereas the causes of large numbers of cases are still unknown.TEX44 is a protein coding gene, which is specifically expressed in male germ cells. However, the expression, localization pattern and precise underlying mechanisms of TEX44 in mouse and human have not been clearly during spermatogenesis and risk of asthenozoospermia. Study design, size, duration This study was an analysis of 120 Han Chinese men with asthenoteratozoospermia by whole-exome sequencing（WES）, at the Reproductive Medical Center of Peking University Third Hospital between January 2018 and January 2022.All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and national research committees of Peking University Third Hospital and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Participants/materials, setting, methods Real-time polymerase chain reaction (Q-PCR) and western blot assays (WT) were used to investigate the mRNA and protein levels of TEX44 in testes. Immunofluorescent analysis was performed on testis sections by TEX44 antibodies during the spermatogenesis. TEX44 knockout mice were generated by CRISPR/Cas9 gene-editing. Computer-assisted sperm analysis (CASA) and fertility testing were also carried out to evaluate the effect of TEX44 on spermatogenesis and male reproduction.TEX44 mutation was an analysis of asthenozoospermia patients using WES. Main results and the role of chance TEX44 mRNA and protein were found to be expressed at the highest level in mouse testis by Q-PCR and WT. Immunohistochemical results revealed TEX44 was located in the cytoplasm of elongating spermatids and exhibited specific localization to the flagellum and manchette during spermiogenesis. Tex44 knockout mice presented no detectable difference in testis-to-body weight ratios when compared with wild-type mice. However, gross testis morphology shows reduction of testis size in TEX44 knockout mice compared wild-type after 12 weeks old. Tex44(-/-) males are subfertile because of abnormal sperm movement and reduced motility.Proportions of motile and progressive movement showing spermatozoa of TEX44 knockout mice were always 30–50% lower than that in controls using CASA.Tex44-/- mice are fertile despite a significant reduction in sperm motility. But the average total number of mouses born of Tex44-/- mice were decreased (P < 0.01). In addition, WES analysis revealed two novel frameshite point mutation patients in TEX44 gene during asthenozoospermia patients. Of these missense mutations, patients with asthenozoospermia have abnormal sperm movement and reduced motility. Fertility could be rescued by the use of intra-cytoplasmic sperm injections (ICSIs). Limitations, reasons for caution Additional cases are in need of study, especially with TEX44 mutation patients from only a small number of asthenozoospermia patients. Protein analysis and function were limited. In future investigations, a larger sample size should be used and the role of the interaction proteins with TEX44 should be analyzed. Wider implications of the findings TEX44 was validated as spermatids-essential gene could play important roles in spermatogenesis. The identification and characterization of TEX44 enhances our understanding of the molecular mechanisms of spermatids differentiation and provides insight into its potential role in the asthenozoospermia.These found will provide important guidance for genetic counseling and assisted reproduction treatments. Trial registration number This study was supported by the National Natural Science Foundation of China (NO.81671513) and Beijing Natural Science Foundation (NO.7172236).

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

P-027 Absence of perinuclear theca ACTRT1 protein induces sperm head deformation and primary male infertility in humans

Abstract Study question Given that Actrt1 knockout induces teratospermia, fertilization failure and severe male infertility in mice, will ACTRT1 deficiency affect sperm morphology and male fertility in humans? Summary answer The ∼110 kb microdeletion of X chromosome only including ACTRT1 gene was identified inducing sperm head deformation and fertilization failure in an infertile Chinese man. What is known already The actin-related proteins, ACTRT1, ACTRT2, ACTL7A, and ACTL9, interact to form a multimeric complex in the sperm subacrosomal region and is vital for the attachment of acrosome to nucleus. The disruption of ACTL7A or ACTL9 have been identified to cause acrosomal detachment, total fertilization failure and male infertility in humans and mice. Actrt1-knockout mice are severely subfertile also because of malformed sperm heads with detached acrosomes and a partial deficiency in fertilization. Loss/reduced expression and/or abnormal localization of PLCζ, a well-known sperm-borne activating factor, are identified in ACTL7A/ACTL9 mutant patients as well as Actrt1-, Actl7a-, and Actl9-deficient mice. Study design, size, duration We recruited a cohort of 85 infertile men with teratospermia which is characterized by deformed sperm heads in the center for reproductive medicine from August 2019 to June 2022. Genomic DNA (gDNA) of patients was extracted from peripheral blood, then whole-exome sequencing and in silico analyses were performed to identify gene mutations. Morphological analysis, functional assays, and assisted reproductive therapy were performed in 2022. Participants/materials, setting, methods The ACTRT1 deficiency was identified by whole-exome sequencing and confirmed by whole-genome sequencing, PCR and qPCR. Family members’ gDNA was collected to define the hereditary mode. Papanicolaou-staining, scanning and transmission electronic microscopy were performed to reveal sperm morphologies. Western blot and immunostaining of spermatozoa were conducted to explore the pathological mechanism of the ACTRT1 deficiency. Intracytoplasmic sperm injection (ICSI) combined with artificial oocyte activation (AOA) was applied for the assisted reproductive therapy of ACTRT1-deletion patient. Main results and the role of chance We identified a whole-gene deletion mutation of ACTRT1 on Chromosome X in an infertile male with teratospermia, which was inherited from his mother. Papanicolaou-staining, scanning electronic microscopy, and transmission electronic microscopy showed the sperm head deformation owing to acrosome detachment, which mimicked the previously reported phenotype of Actrt1-knockout mice. The results of western blot and immunostaining suggested that ACTRT1 deficiency induced a down-regulated expression of ACTL7A and PLCζ proteins, but not ACTRT2, in human sperm samples. The detached acrosomes induced fertilization failure in assisted reproductive therapy of both Actrt1-knockout mice and the ACTRT1-deletion patient, which could be effectively rescued by ICSI combined with AOA. Limitations, reasons for caution Additional cases are needed to confirm the genetic contribution of ACTRT1 mutations to male infertility with teratospermia and fertilization failure. In addition, the proband with ACTRT1 deletion was with severe oligozoospermia and mild asthenozoospermia. The effect of ACTRT1 mutations on sperm count and motility still need more investigation. Wider implications of the findings Our results reveal a gene–disease relationship between ACTRT1 deficiency and human male infertility owing to teratospermia and fertilization failure. This report also describe a good fertility outcome of assisted reproductive therapy with ICSI and AOA for the ACTRT1-deficient patient. Trial registration number not applicable

P-154 Is there an impact of oocyte vitrification on embryological and clinical outcomes in PGT-A cycles? A propensity score matching-based study

Abstract Study question Does oocyte vitrification affect euploid blastocyst rate (EBR) per cohort of inseminated oocytes and/or clinical outcomes per euploid blastocyst transfer? Summary answer Vitrified-warmed oocytes are subject to ≈90% cryo-survival and lower blastulation rates than fresh cohorts, but similar euploidy and clinical outcomes per euploid blastocyst transfer. What is known already Cryopreservation was a game-changer in IVF. It is an essential step of the IVF journey with a plethora of clinical (e.g., reduction of OHSS while fully-exploiting the ovarian reserve, implementation of chromosomal/genetic testing, cumulative perspective in defining IVF success), logistic (e.g., higher flexibility), and social (e.g., fertility preservation) advantages. Vitrification represents the gold-standard protocol. Studies in sibling oocytes showed that vitrification may slightly reduce blastocyst development without impact on embryo chromosomal constitution. Here we aimed at measuring the impact (if any) of oocyte vitrification on EBR per cohort of inseminated oocytes and clinical outcomes per euploid blastocyst transfer. Study design, size, duration We gathered a dataset of first PGT-A cycles with vitrified-warmed (N = 54) or fresh (N = 3916) inseminated MII-oocytes (April-2013 to July-2022). Patients were matched 1:1 for maternal age at retrieval (37.2 ± 3.4 versus 37.1 ± 3.4 years) and MII-oocytes used (8.7 ± 4.1 versus 9.0 ± 5.1) through propensity-score-matching resulting in 49 cycles per arm. The primary outcome was EBR per cohort of inseminated MII-oocytes. The secondary outcomes were all embryological and clinical outcomes, including cumulative-live-birth-rate (CLBR) per concluded warming/fresh cycle. Participants/materials, setting, methods ICSI of all (cryo-survived) MII-oocytes, trophectoderm biopsy without day3 zona-drilling, and comprehensive-chromosome-testing to assess full-chromosome non-mosaic aneuploidies were performed. Cryopreservation was performed via vitrification. Only euploid blastocyst transfers were conducted. The groups were comparable also for BMI and sperm factor. The reasons for oocyte vitrification were supernumerary oocytes (N = 22, 45%; days between vitrification and warming: 573.7 ± 503.9), postponement of sperm collection (N = 7, 14%; 69.3 ± 113.4 days) and fertility preservation (N = 20, 41%; 1160 ± 779.7 days). Main results and the role of chance 8.7 ± 4.1 oocytes were warmed and 7.9 ± 3.7 survived (survival rate: 90.3 ± 14.7%). The zygotes were 5.4 ± 2.9 and 6.5 ± 4.0 in the vitrified-warmed and fresh groups, respectively (p = 0.2; fertilization-rates: 69.7 ± 22.3% versus 73.6 ± 18.4%, p = 0.5). The blastocysts were 2.1 ± 1.8 and 3.0 ± 1.9 (p = 0.01; blastulation-rates per zygotes: 34.9 ± 26.2% versus 49.4 ± 26.7%, p = 0.02). The euploid blastocysts were 1 ± 1.2 and 1.4 ± 1.6 (p = 0.2; euploidy-rates per biopsied blastocysts: 49.7 ± 34.3% versus 45.7 ± 36.3%, p = 0.5). The EBR per inseminated oocytes were 12.7 ± 13.3% and 17.5 ± 18.1% (p = 0.3). AA-blastocyst rates were also similar (N = 48/101, 47.5% versus N = 77/149, 51.7%, p = 0.6), while the day5-blastocysts rate was lower among vitrified-warmed cohorts (N = 12/101, 11.9% versus N = 51/149, 34.2%, P < 0.01). Nonetheless, the clinical outcomes per euploid transfer (LBR: N = 15/34, 44.1% versus N = 16/38, 42.1%, p = 0.99; miscarriage-rate per clinical pregnancy: N = 1/16, 6.3% versus N = 4/20, 20%, p = 0.4), gestational age (37.8 ± 1.5 versus 38.4 ± 1.4 weeks, p = 0.32) and birthweight (3471.7 ± 575 versus 3153.8 ± 449.3 g, p = 0.1) were comparable. 85.7% (N = 42/49) and 77.6% (N = 38/49) of the warming/fresh cycles were concluded with similar CLBR: 33.3% (N = 14/42) versus 39.5% (N = 15/38; p = 0.6). Among the 22 cohorts of vitrified-warmed oocytes because supernumerary, the EBR per inseminated oocytes was similar to their sibling fresh cohorts (11.7 ± 13.4% versus 13.8 ± 11.1%; p = 0.6). Also, 68.1% warming cycles were concluded (N = 15/22) eliciting 20% CLBR (N = 3/15), comparable to their sibling fresh cohorts (N = 4/22, 18.2%). Limitations, reasons for caution Retrospective design with limited sample size. About half of the patients in the vitrified-warmed group cryopreserved oocytes because supernumerary. These women were characterized by better ovarian reserve markers and response to ovarian stimulation than controls. Nonetheless, these parameters do not affect our primary outcome. Wider implications of the findings Oocyte vitrification is crucial in IVF. It involves higher flexibility with clinical (full ovarian reserve exploitation, oocyte accumulation), social (fertility preservation), and ethical (cryo-storage of supernumerary oocytes) benefits. Beyond a moderate impact (degeneration after warming, lower blastulation-rates), euploidy-rates and clinical outcomes are not affected by this procedure. Trial registration number not applicable

O-070 Paternal age is associated with mitochondrial vulnerability to sperm cryopreservation

Abstract Study question Is paternal age associated with lower mitochondrial activity in fresh and cryopreserved semen? Summary answer Paternal age does not appear to severely compromise sperm mitochondrial activity in fresh semen but determines higher mitochondrial vulnerability to sperm cryopreservation. What is known already The impact of paternal age on male fertility has not been fully clarified although growing evidence suggests age-related loss of sperm quality. Mitochondrial activity has been recognized as a reliable marker of sperm functionality, reflecting motility, vitality and fertilization competence. Sperm cryopreservation has been largely utilized in fertility preservation and ART schemes, despite its potential harm on cell membranes including those of the mitochondria. The influence of advanced paternal age (APA) on sperm vulnerability to cryopreservation-induced cell damage is still not known. Study design, size, duration Twenty three normospermic patients, 11 of which ≤ 35 (non-APA) and 12 ≥ 42 years old (APA), provided semen samples by masturbation after 1-5 days of abstinence, between April and August of 2022. A 250µl semen aliquot was frozen. Sperm concentration, motility, vitality, morphology and mitochondrial functionality were compared in fresh semen from non-APA vs. APA patients, while motility and mitochondrial functionality were compared in thawed semen from both groups with the two-tailed Student t-test. Participants/materials, setting, methods Fresh and thawed semen samples were provided by patients under evaluation for couple infertility treatment in our fertility center. Semen was cryopreserved with CryoSperm (Origio) and analyzed after rapid thawing and washing/dilution in Sperm Preparation Medium (Origio). Sperm vitality was assessed with VitalScreen (FertiPro) and mitochondrial functionality was evaluated with MitoTracker Red CMXRos (Cell Signalling Technology), through the percentage of stained cells and fluorescence intensity measured in 75 spermatozoa per patient with the ImageJ software. Main results and the role of chance Mean ages for non-APA and APA patients were 32.6 and 45.3 years, respectively. Age groups did not differ for any of the parameters assessed in fresh semen (volume: 2.3±1.1 vs. 2.6±1.2mL; concentration: 58±32 vs. 47±20 x106/mL; rapid progressive motility 5±5 vs. 5±8%; slow progressive motility 40±10 vs. 36±10%; non-progressive motility 10±5 vs. 10±3%; immotile: 44±9 vs. 49±13%; normal morphology: 6±3 vs. 5±1%; vitality: 71±8 vs. 66±15%, for non-APA and APA, respectively). However, non-APA patients presented a higher percentage of spermatozoa with rapid progressive motility (5±7 vs. 0%; p = 0.04) and a lower percentage of immotile spermatozoa (76±12 vs. 88±9%; p = 0.02) after thawing. Regarding mitochondrial functionality, no differences were observed in fresh semen from different age groups [93±11 vs. 92±7% stained cells; 3.33±1.57 vs. 3.35±1.49 (fluorescence arbitrary units), for non-APA and APA, respectively). In cryopreserved semen, however, although age groups did not differ for the percentage of stained spermatozoa (92±10 vs. 90±5%), fluorescence intensity was higher in spermatozoa from non-APA patients (2.53±1.63 vs. 1.76±0.54; p < 0.01). Limitations, reasons for caution Our study is limited by the potential interference of confounding factors not equally distributed in age groups. The conclusions from this study must be confirmed in other patient populations with different race/genetics and habits. Wider implications of the findings Our study sheds light on the impact of paternal age on sperm quality, a topic highly relevant to reproductive medicine still not fully understood. We provide evidence that APA is associated with higher vulnerability of mitochondria to the stress induced by cryopreservation and its metabolic consequences. Trial registration number not applicable

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-210 Utilization of coconut water as an additive to improve the human sperm quality following freezing-thawing Process

Abstract Study question Is the supplementation of the cryosolution with coconut water (CW) improve the motility and DNA integrity of spermatozoa after cryopreservation? Summary answer Coconut water as an additive to the cryosolution found to decrease the sperm DNA fragmentation and improve the sperm motility post-thawing. What is known already Despite the benefits of cryopreservation, it has adverse effects on sperm cell in different scenarios. Decrease in motility, viability and DNA fragmentation are the most common effects of cryopreservation on sperm function. The freezing/thawing shock produces physical and chemical stresses on the spermatozoa that could change the composition of the lipids in their plasma membrane resulting in increased production of reactive oxygen species (ROS) and increases the oxidative stress. Thus, supplementing the cryosolution with antioxidants may improve the recovery rate of cryopreserved spermatozoa by reducing the oxidative damage. Coconut water is characterized by its high contents of proven antioxidant properties. Study design, size, duration The study was conducted on 70 semen samples from patients attended the fertility center in Al-Sadder medical city, Najaf, Iraq, for routine semen analysis over a period of ten months from March to December 2022. All the patients gave their consent for participation in this study. The samples had normal semen parameters according to World Health Organization (WHO) 2010 standard criteria. Participants/materials, setting, methods Sperm motility and DNA fragmentation index (SDF) test were done for each sample, then, each semen sample divided to three parts: P1: cryopreserved with glycerol-containing cryosolution only (SpermFreezTM), P2: cryopreserved with glycerol-containing cryosolution and 5%coconut water (CW), and P3: cryopreserved with glycerol-containing cryosolution and 10% CW. After 1-month vapor-dependent cryopreservation, all samples were thawed and the sperm motility assed using computer-assisted sperm analyzer (CASA) and DFI was evaluated using Acridine orang stain method. Main results and the role of chance The percentage of total sperm motility and progressive motility in fresh samples were (39% and 35% respectively) and the level of sperm DNA fragmentation was (21%). The total and progressive sperm motility decreased significantly (P < 0.05) post-thawing, while the SDF increased. After cryopreservation the percentage of total and progressive sperm motility exhibited a significantly elevated levels in P2 (10.1% and 7.9% respectively) and P3 (14.6% and 11.3% respectively) than in P1 (6.8% and 4.2% respectively), however, the level of DFI significantly (P < 0.05) decreased in both P2 (27.4%) and in P3 (24.5%) than in P1 (30.1%). In comparison between the two concentrations of CW, P3 recorded a significant increase in the recovery of sperm total and progressive motility (14.6% and 11.3% respectively) than P2 (10.1% and 7.9% respectively). Furthermore, the level of DFI in P3 (24.5%) decreased significantly than its level in P2 (27.4%). Limitations, reasons for caution This study was to evaluate the efficiency of CW in the improvement of human sperm motility and DNA integrity post-thawing. Further studies required to evaluate the effect of using CW in sperm cryopreservation on the success rate of the conventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Wider implications of the findings In this study, we found that the additive of CW to the glycerol-containing cryosolution (SpermFreezTM) can improve the human sperm motility and DNA integrity post-cryopreservation. The best recovery rate of sperm motility and DNA integrity was with the supplementation of 10% of CW to the cryosolution. Trial registration number Not applicable

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 <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-074 In-vitro vitamin D treatment on human sperm improves sperm motility, capacitation, and oocyte activation

Abstract Study question Does in-vitro vitamin D treatment on fertile and infertile human sperm improves sperm motility and alter protein markers involved in capacitation and oocyte-activation. Summary answer In-vitro vitamin D treatment enhance functionality of sperm by increase intracellular calcium ion, thus increasing sperm motility and capacitation status. What is known already Many studies have been done to investigate the relationship between vitamin D and male infertility. It is known that low serum vitamin D in human contribute to male infertility. However, the effect of in-vitro vitamin D treatment on sperm remains unclear, particularly on the effect of vitamin D following ejaculation into female reproductive tract. This includes processes involved in fertilisation such as capacitation, hyperpolarization, oocyte activation and sperm-oocyte fusion. Therefore, we examined whether in-vitro vitamin D treatment on sperm can improve the sperm parameters, hence will then improve fertilisation and pregnancy. Study design, size, duration Randomised controlled trial with prospective interventions (in-vitro vitamin D treatment). Based on power analysis, 40 subjects both fertile and infertile men who came for semen analysis were recruited at University Malaya Medical Centre from January 2022 to January 2023. All men were consented to participate in this study. Each washed semen sample were aliquoted to serve as control and receive in-vitro vitamin D treatment. Participants/materials, setting, methods Total of 40 samples were included for final analysis. All semen samples were washed and treated with 1nM vitamin D in-vitro for 45 minutes at 37ºC. Motility, intracellular calcium released, capacitation status, mitochondria membrane potential and other basic semen parameters were measured after in-vitro vitamin D treatment for fertile and infertile samples. Furthermore, immunofluorescence of sperm origin oocyte activation factor, Phospholipase C Zeta (PLCζ), was done to investigate fluorescence intensity per cell using Image J. Main results and the role of chance In both fertile and infertile sample group, total motility is increased after in-vitro vitamin D treatment (MD = 8.727%, P < 0.001). Fluo-4 AM stain shows there are increase of intracellular calcium release after in-vitro vitamin D, with mean difference of Corrected Total Cell Fluorescent (CTCF) for Fluo-4 AM (MD = 25043, P = 0.0228. Chlortetracyclin (CTC) test for capacitation status and immunofluorescence of phosphotyrosine shows there are increase CTCF (MD = 0.02595, P = 0.0142) after in-vitro vitamin D treatment. Staining of JC-1 shows there are more hyperpolarised mitochondria membrane in in-vitro vitamin D treatment group compare to the control group. Ratio of red to green fluorescent is 0.639 in control group and 2.22 in in-vitro vitamin D treatment group. Moreover, immunofluorescence of sperm origin oocyte activation factor, Phospholipase C Zeta (PLCζ) show in-vitro vitamin D improve the expression of these protein. Mean difference of CTCF between control and vitamin D treatment group for PLCζ is 8.013x 10-3, P = 0.0226. Thus, in-vitro vitamin D enhance functionality of sperm by increase intracellular calcium ion, thus increasing sperm capacitation status and sperm membrane hyperpolarization. Limitations, reasons for caution Although power analysis is used to calculate sample size, however bigger sample size always needed to increase the accuracy. Moreover, patients’ sample are examined and treated after 3 hours due to limited collection room in the facility. Wider implications of the findings Vitamin D can be used as a tool in in-vitro fertilisation (IVF) procedure and can be added to sperm preparation medium due to its implication in increasing capacitation and hyperpolarised sperm membrane to increase chance of successful IVF. Trial registration number not applicable

O-276 Fertility preservation and sperm quality in adolescent transgender patients prior to hormonal treatment

Abstract Study question This study investigated the take-up, hormonal profile and sperm quality in adolescents undergoing fertility preservation via different methods, prior to medical intervention for gender dysphoria. Summary answer Fertility preservation via surgical sperm retrieval (SSR) or masturbation, is possible in this group, however long-term data is required to check gamete health. What is known already As increasing numbers of adolescents with gender dysphoria (GD) start gonadotrophin-releasing hormone agonists (GnRHa) to delay puberty to minimise psychological distress. However, the uncertainty of the long-term effects of these medications highlights the importance of undergoing fertility preservation (FP) prior to starting any hormone treatment. Study design, size, duration Data for adolescents wanting to FP was prospectively maintained. A total of 122 patients were referred for FP, of which 23 declined (19%). Participants/materials, setting, methods Young people were counselled for FP and serum testosterone, FSH, and LH were recorded prior to providing semen samples. Semen samples were classified by concentration; normal >15mil/ml, oligospermia <15mil/ml, poor <1sperm/slide, azoospermia. Insufficient samples, or unwillingness to masturbate, meant SSR was offered in a stepwise manner using electroejaculation (EE), TESE and mTESE. Main results and the role of chance Most patients (n = 78, 64% - median age 16.7) were able to produce semen by masturbation, cryopreserving an average of 6.6 straws. Sperm concentration was normal in 44% of samples produced, 36% were oligozoospermic, 9% were poor sperm concentration, and 12% were azoospermic. Mean blood results showed: testosterone 12.32nmol/l, FSH 3.83 IU/L, LH 4.39 IU/L. 21 patients required SSR - median age of 14.9. EE was successful in 4 patients, 9 underwent TESE, and 8 underwent mTESE. 4 mTESE patients were azoospermic. Success rate was 77%, with an average of 5.5 vials stored. Semen parameters in this cohort were poor - however possibly adequate for ICSI. Hormone levels were similar to the masturbation cohort: mean testosterone 11.7nmol/l, FSH 3.4 IU/L, LH 3.5 IU/L. 3 patients used GnRHa pre-SSR. Following a washout period, 1 patient remained azoospermic despite a testosterone of 21 nmol/l. The other 2 patients had sperm found with abnormal morphology and motility. This is the largest UK cohort of transgender girls referred for FP. The results showed that semen parameters were abnormal in 67% of the masturbation samples produced, and within the SSR group, all patient’s testosterone levels were >8nmol/l, with an average of 5.5 vials saved. Limitations, reasons for caution The quantity of long-term data on whether adolescents end up using their gametes post FP is a factor to consider with this study, as more evidence is needed in order to display the actual long-term usage of gamete cryopreservation. Wider implications of the findings Masturbation is often possible in this group, but alternatively, SSR provides patients with a safe opportunity to potentially have their own biological children in the future. Trial registration number not applicable

O-013 Metabolic characteristics of offspring born to women with polycystic ovary syndrome in childhood

Abstract Study question Does polycystic ovary syndrome (PCOS) affect the metabolic characteristics of offspring in childhood? Summary answer Girls born to PCOS mothers had increased cholesterol (CHOL) and low-density lipoprotein cholesterol (LDL) levels, whereas boys had comparable CHOL and LDL levels to controls. What is known already Maternal PCOS status may negatively influence childhood growth, endocrine and metabolic function. The long-term impact of PCOS on children health is poorly understood. Current findings reported by previous studies are divergent. Propensity score matching can reduce bias where strong confounding by indication is expected. Study design, size, duration This was a cohort study including 223 singletons born to women with PCOS and 2326 singletons born to non-PCOS women. All offspring were conceived by in-vitro fertilization (IVF) or Intracytoplasmic sperm injection (ICSI) from January 2001 to January 2018 in reproductive center of Women’s hospital, Zhejiang University, School of medicine. Growth and development parameters and metabolic parameters were collected at age 3-6 years in Follow-up center of Women’s hospital, Zhejiang University, School of medicine. Participants/materials, setting, methods We used the propensity score (PS) to match PCOS women and non-PCOS women in a 1:4 ratio. The variables in the PSM included maternal age at conception, paternal age at conception, maternal height, maternal preconception BMI, IVF or ICSI, fresh-embryo transfer (ET) or frozen-embryo transfer (FET), gender of offspring, age at follow-up, year of follow-up. The PCOS group consisted of 217 patients and the non-PCOS group consisted of 787 patients after PS matching. Main results and the role of chance Maternal preconception weight (58.3±0.6 vs. 56.0±0.2, P<0.001) and body mass index (BMI) (23.0±0.2 vs. 21.9±0.1, P<0.001) were significantly higher in mothers with PCOS before PS matching. After PS matching, parental demographic characteristics were similar between groups including maternal preconception weight and BMI. In PS matching cohort, the height, weight, BMI of children at age 3-6 years were comparable between PCOS group and non-PCOS group. Singletons born to PCOS women had significantly higher CHOL levels (4.43±0.05 vs. 4.29±0.01, P=0.013) and LDL levels (2.45±0.04 vs. 2.37±0.01, P=0.049) compared with non-PCOS group. Even though the girls had similar BMI in childhood between the two groups(15.26±0.15 vs.15.13±0.80, P=0.449), the girls born to PCOS women still exhibited elevated CHOL levels (4.52±0.08 vs. 4.33±0.04, P=0.024) and LDL levels (2.55±0.06 vs. 2.39±0.03, P=0.007). These differences in CHOL (4.34±0.07 vs. 4.26±0.04, P=0.196) and LDL levels (2.36±0.05 vs. 2.36±0.03, P=0.866) were not observed in male offspring between the two groups. Limitations, reasons for caution Mothers’ weight gain during pregnancy, offspring lifestyle were not available for this study. This was a study carried out in a single center and was therefore susceptible to bias. Further studies remain necessary to fully confirm these results. Wider implications of the findings Our results support CHOL and LDL lipid metabolism as two of the earliest metabolic abnormalities features of the female offspring born to PCOS women. These findings suggest that further studies are needed to investigate the potential mechanisms and long-term metabolic changes associated with these differences. Trial registration number not applicable

P-056 Oxidative stress and human semen quality: new results for diagnostic process of male infertility

Abstract Study question Is oxidative stress (OS) evaluated in human spermatozoa a predictive marker of better semen quality? Summary answer CellROX®Orange is a new fluorescent probe able to detect Reactive Oxygen Species (ROS) identifying a viable oxidized sperm fraction related to a better sperm performance. What is known already OS, defined as an unbalance between ROS production and antioxidant defences, is considered one of the causes of male infertility. OS evaluation in spermatozoa represents an important goal of research in this field because this parameter could be helpful to predict sperm fertilization ability to improve Assisted Reproductive Technology outcomes. By using different probes and methods for OS evaluation in spermatozoa or in semen, several studies were performed demonstrating a negative role of ROS on sperm functions. Such studies were not conclusive for the small number of included subjects, the high variability in the cohorts and the lack of validation. Study design, size, duration An observational study was conducted on 121 semen samples from patients undergoing routine semen analysis for couple infertility in the Andrology Laboratory of Careggi University Hospital of Florence from September 2021 to March 2022. Washed and Swim-up selected spermatozoa were incubated with two fluorescent probes, CellROX® Orange and Dihydroethidium (DHE), and revealed by flow cytometry. Participants/materials, setting, methods After routine semen analysis, the percentage of oxidized spermatozoa was evaluated with CellROX® Orange and DHE, and then correlated with standard semen parameters and sperm DNA fragmentation (sDF, evaluated by TUNEL/PI). Sperm kinematic parameters and hyperactivated motility were also assessed by C.A.S.A. system. Furthermore, CellROX® Orange positivity and phosphatidylserine membrane exposure (determined by Annexin V staining) or Caspase 3 and 7 activities (measured by FLICA™) were concomitantly evaluated. Main results and the role of chance We demonstrated that CellROX® Orange is able to detect hydrogen peroxide only in viable spermatozoa and the percentage of CellROX® Orange positive spermatozoa were positively associated with semen quality and negatively with sDF. To confirm these results, we performed the same experiments by using DHE, another probe which reveals hydrogen peroxide and superoxide anion in both viable and unviable cells. Similarly to CellROX® Orange, a positive correlation with semen quality and a negative one with sDF were found in viable spermatozoa. Conversely, in unviable spermatozoa, opposite associations were observed. These results indicate that oxidative status revealed by the two probes is related to a better sperm quality. To further investigate this possibility, we double labelled spermatozoa with CellROX® Orange and Annexin V (a marker of early signs of apoptosis) as well as with CellROX® Orange and FLICA™ (that detects caspase activity, a late sign of apoptosis). We found that CellROX® Orange mostly identifies spermatozoa without apoptotic features. Furthermore, we evaluated CellROX® Orange positivity in Swim-up selected spermatozoa, finding significantly higher levels in these samples respect to unselected samples, demonstrating once again that this probe identifies spermatozoa with better quality. Limitations, reasons for caution Our results indicate that within the viable oxidized spermatozoa there are cells with physiological and non-physiological intracellular ROS levels, however, they do not explain at what levels ROS cease to be functional and become deleterious for spermatozoa. Wider implications of the findings ROS evaluation in viable spermatozoa with the commercially available probes CellROX® Orange and DHE, allows the identification of the oxidized sperm fraction related to a sperm better performance. Therefore, the two probes may be useful to determine such fraction, likely improving the diagnostic process of male infertility. Trial registration number not applicable

P-106 The inhibition of sperm SIRT1 enhances fertilization and modifies subsequent embryonic epigenetic quality

Abstract Study question Is SIRT1 present in sperm and thus involved in fertilization and/or sperm epigenetic code? Do the SIRT1-driven epigenetic marks be transmitted to the progeny? Summary answer SIRT1 localized at the sperm head and influences fertilization and sperm epigenetic code via its deacetylation activity. SIRT1-modulated epigenome is transmitted to zygotes and blastocysts. What is known already The addition or removal of acetyl group from lysine residua (i.e. acetylation) post-translationally modulates proteins through the action of acetylases and deacetylases, respectively. SIRT1 (also Sirtuin 1) is capable to deacetylate a number of proteins, including histones, with roles in male reproduction. Testicular SIRT1 deficit leads to subfertility and increases number of abnormal and immotile spermatozoa that fail to fertilize. However, neither sperm SIRT1 presence nor its function has been investigated despite the significance of acetylation in the events around fertilization. Moreover, the epigenetic mode of action of sperm SIRT1 highlights its relevance for the success of the pre-implantation embryo. Study design, size, duration Mouse sperm of 14-18-weeks-old males of CD-1 x C57Bl6 hybrid strain were isolated from the epididymis. Sperm were incubated under capacitating conditions in presence of sirtinol (5 µM), a selective SIRT1 inhibitor, or the vehicle (0.1% v/v DMSO) for 1 h in 37 °C and 5% CO2. Participants/materials, setting, methods Motility and acrosome reaction was evaluated as previously stated (Ritagliati et al., 2018). Moreover, sperm proteins were extracted into RIPA-buffer and acetylated proteins were depicted by western blot. Finally, in vitro fertilization and embryo culture were performed. Above parameters defining IVF outcomes (i.e., fertilization, cleavage, and blastocyst rate), immunocytochemistry of acetylated histone 4 in the 16th residua of lysine (H4K16) in zygotes and blastocyst was quantified. Main results and the role of chance SIRT1 was localized in the sperm head, apparently at the perinuclear theca and perforatorium. Inhibition of SIRT1 leads to the increase of lysine acetylation (2.28 vs 1, fold change values) and hyperactivation (16.55 ± 0.79 vs 10.84 ± 0.79 %sperm), the enhanced motility that facilitates sperm to penetrate the oocyte, as well as the acrosome reaction (28.69 ± 0.41 vs 17.98 ± 0.41 %sperm). Subsequently, these spermatozoa were able to better fertilize oocytes in vitro (90.5 ± 7.21 vs 66.25 ± 7.21, %). Interestingly, zygotes derived from sirtinol-pre-treated spermatozoa developed to 2-cell embryos at a higher rate than those fertilized by untreated spermatozoa (79.33 ± 7.21 vs 67.00 ± 7.21, %). Moreover, we observed significant hyperacetylation of H4K16 in the male pronuclei of zygotes and embryos derived from sirtinol-treated sperm (8.19 ± 1.02 vs 6.10 ± 2.09 and 45.55 ± 13.96 vs 27.62 ± 13.96, integrated density of the signals, respectively). Limitations, reasons for caution Although acetylation of H4K16 was accumulated in male pronuclei, we do not know which subset of genes was affected. Therefore, we only can assure that SIRT1-driven epigenetic marks are transmitted to the offspring, but their consequences should be further elucidated. For knowledge transfer to humans, a verification study is required. Wider implications of the findings the reduction in testicular SIRT1 expression is an age-related phenomenon. Therefore, our results support SIRT1 as the basis for new interventions against age-related male infertility. Moreover, due to the high reactivity of SIRT1 to its inactivation, SIRT1 becomes a target to pharmacologically enhance sperm fertilization ability in assisted reproductive techniques. Trial registration number Animal procedures were conducted in accordance with Act No. 246/1992 Coll. on the Protection of Animals Against Cruelty under the supervision of the Animal Welfare Committee of the Ministry of Education, Youth and Sports of the Czech Republic, approval ID MSMT-11925/2016-3.

O-053 Link between cannabis use and fertility: effects on gamete competence and early embryonic development

Worldwide human fertility has been facing a significant decline over recent years. Therefore, attention has been focused on identifying environmental and lifestyle risk factors that may be contributing to impact human reproductive function. The use of alcohol, tobacco and certain medications have undergone substantial assessment into their effects on fertility. As physicians aim to offer the most appropriate recommendations to their patients, it is of utmost importance to understand the side effects of readily available substances and their mechanisms of action. Cannabis falls into this group of substances used regularly by women and men of reproductive age, with its consumption steadily rising with the onset of legalization in Canada in 2018 also amongst pregnant women. At present, the Society of Obstetricians and Gynecologists of Canada recommends that women of reproductive age, especially if considering pregnancy, discontinue cannabis use. There is, however, inconclusive evidence that links cannabis and fertility. This presentation will outline the most recent data from our laboratory on the effects of Delta-9 tetrahydrocannabinol (THC), the main psychoactive component of cannabis, on oocytes competency and sperm fertilization capability. In fact chronic exposure to cannabis also adversely affects the male reproductive system, inducing oligospermia, morphologically abnormal sperm, and impaired sperm motility in rodents. Using a bovine model as translational model for humans, we identified effects of THC on oocyte developmental parameters, gene expression as well on sperm parameters, such as motility, morphology, capacitation and mitochondrial potential. The general hypothesis at the basis of this research program aims to elucidate the effects of THC at clinically relevant doses on gene and epigenetic modifications in sperm, oocytes and embryos, ultimately affecting fertility and pregnancy outcome. We showed a significantly reduced ability of THC-treated oocytes to undergo nuclear maturation, ultimately leading to decreased oocyte fertilization capability and poor embryonic development. We showed that THC acts as a partial agonist at the Cannabinoid receptors 1 and 2 (CB1 and CB2), as THC effects were reverted by the addition of antagonists. The significant decrease in maturation and cleavage rate of oocytes exposed to the higher doses of THC, representative of high recreational use of Cannabis, allows us to speculate that THC might act as a selection factor for only the highest quality oocytes, that overcome the THC effects. The more immature oocytes may be negatively affected by THC by being pushed towards meiotic resumption, leading to early maturation, prior to gaining developmental competence. Levels of connexin gap-junction proteins were used as indicator of oocyte competency, as they highly correlate to oocyte fertilization potential and embryo quality. Our results demonstrated a significant decrease in connexin 37 (CX37) and connexin 43 (CX43) mRNA levels measured by digital droplet PCR (ddPCR), in oocytes only in the low dose THC group, representative of the therapeutic use of Cannabis. More recently we have also shown how THC affects methylation in granulosa cells, via disrupting methylation and de-methylation enzymes, ultimately determining oocyte competency and, therefore, fertility. THC exerts parallel effects on the male gametes as well, increasing capacitation of in vitro treated sperm, affecting their mitochondrial potential and disrupting the expression of key microRNAs in sperm that are linked to early embryonic development, therefore affecting pre-implantation development and, ultimately, pregnancy outcome. Our results suggest an overall detrimental effect of THC on the competency and fertilization potential of both female and male gametes, negatively influencing fertility and the likelihood of a viable pregnancy, especially of importance when undergoing artificial reproductive technologies procedures. Trial registration number XXXX

Micrococcal nuclease sequencing of porcine sperm suggests enriched co-location between retained histones and genomic regions related to semen quality and early embryo development.

The mammalian spermatozoon has a unique chromatin structure in which the majority of histones are replaced by protamines during spermatogenesis and a small fraction of nucleosomes are retained at specific locations of the genome. The sperm's chromatin structure remains unresolved in most animal species, including the pig. However, mapping the genomic locations of retained nucleosomes in sperm could help understanding the molecular basis of both sperm development and function as well as embryo development. This information could then be useful to identify molecular markers for sperm quality and fertility traits. Here, micrococcal nuclease digestion coupled with high throughput sequencing was performed on pig sperm to map the genomic location of mono- and sub-nucleosomal chromatin fractions in relation to a set of diverse functional elements of the genome, some of which were related to semen quality and early embryogenesis. In particular, the investigated elements were promoters, the different sections of the gene body, coding and non-coding RNAs present in the pig sperm, potential transcription factor binding sites, genomic regions associated to semen quality traits and repeat elements. The analysis yielded 25,293 and 4,239 peaks in the mono- and sub-nucleosomal fractions, covering 0.3% and 0.02% of the porcine genome, respectively. A cross-species comparison revealed positional conservation of the nucleosome retention in sperm between the pig data and a human dataset that found nucleosome enrichment in genomic regions of importance in development. Both gene ontology analysis of the genes mapping nearby the mono-nucleosomal peaks and the identification of putative transcription factor binding motifs within the mono- and the sub- nucleosomal peaks showed enrichment for processes related to sperm function and embryo development. There was significant motif enrichment for Znf263, which in humans was suggested to be a key regulator of genes with paternal preferential expression during early embryogenesis. Moreover, enriched positional intersection was found in the genome between the mono-nucleosomal peaks and both the RNAs present in pig sperm and the RNAs related to sperm quality. There was no co-location between GWAS hits for semen quality in swine and the nucleosomal sites. Finally, the data evidenced depletion of mono-nucleosomes in long interspersed nuclear elements and enrichment of sub-nucleosomes in short interspersed repeat elements.These results suggest that retained nucleosomes in sperm could both mark regulatory elements or genes expressed during spermatogenesis linked to semen quality and fertility and act as transcriptional guides during early embryogenesis. The results of this study support the undertaking of ambitious research using a larger number of samples to robustly assess the positional relationship between histone retention in sperm and the reproductive ability of boars.

Evaluation of a novel microfluidic chip-like device for purifying bovine frozen-thawed semen for in vitro fertilization

The aim of this study was to validate a novel sperm purification device, the VetCount™ Harvester, for use in bovine in vitro fertilization (IVF). The device's performance was compared to BoviPure™ gradient centrifugation, a commercially available and accepted routine technique. Semen quality parameters were assessed for frozen-thawed semen from six different bulls (n = 6) following sperm purification. For each bull two semen subsamples were purified utilizing BoviPure™ gradient centrifugation and the VetCount™ Harvester, including a third subsample as untreated control. Both treatments significantly increased the proportion of progressively motile sperm cells (84.4 ± 14.1% and 85.1 ± 7.8%, respectively) compared to the untreated semen (41.9 ± 18.8%). BoviPure™ gradient and VetCount™ Harvester selected predominantly viable acrosome intact (VAI) sperm cells with low membrane fluidity and low free intracellular calcium concentration [Ca2+]i (76.5 ± 4.4% and 78.6 ± 6.0%). Normalizing [Ca2+]i of VAI sperm cells (non-treated semen: [Ca2+]i = 1) VetCount™ Harvester purified spermatozoa (0.67 ± 0.10) showed significantly lower [Ca2+]i than BoviPure™ treated sperm (0.84 ± 0.14; P < 0.05). Subsequently, the fertilizing ability of the spermatozoa was evaluated performing a competitive fertilization assay. Sperm cells from both treatment groups were fluorescently labelled using different dyes and added in equal amounts to in vitro matured oocytes. After 18 h co-incubation, the origin of the fertilizing sperm cell was evaluated via fluorescence microscopy. In two bulls, VetCount™ Harvester selected sperm that fertilized significantly more oocytes then BoviPure™ treated sperm, in another bull it was the opposite. For three bulls no difference was observed.We conclude that the VetCount™ Harvester selects a high-quality, fertile sperm fraction from frozen-thawed bull semen. However, some considerations have to be kept in mind for the direct use of the isolated sperm fraction in IVF.