Good Quality Embryos Research Articles

P-420 Dysregulated microRNAs in uterine fluid from women with recurrent implantation failure are linked to endometrial receptivity and implantation

Abstract Study question Is the microRNA content different in uterine fluid (UF) from women with recurrent implantation failure (RIF) compared to healthy fertile women? Summary answer The miRNA content is altered in the UF of women with RIF compared to healthy fertile women. What is known already Previous studies indicate that microRNAs secreted from endometrial cells into the UF are involved in endometrial receptivity and embryo implantation. Moreover, endometrial miRNAs are dysregulated in women with RIF and poor endometrial receptivity has been suggested as a putative cause of the condition. Study design, size, duration This is a descriptive experimental case-control study where microRNA abundancy in UF was compared between women with RIF (n = 34) and healthy fertile women (n = 17). Study participants were recruited at two university clinics in Stockholm, Sweden, and Tartu, Estonia. UF samples were collected vaginally in the receptive phase on day LH + 7-9 by flushing the uterine cavity with sterile saline. RIF was defined as three failed in vitro fertilization (IVF) cycles with good-quality embryos. Participants/materials, setting, methods To identify miRNAs in UF we performed small RNA sequencing. Differential expression analysis (DESeq2) was used to compare the abundancy of miRNAs in UF between the two groups. Dysregulated miRNAs were externally validated using relevant published datasets and further analyzed using tools such as target gene prediction (miRTarBase) and biological KEGG pathway analysis (g:Profiler). Technical validation was performed on two miRNAs with quantitative real-time PCR (RT-PCR). Main results and the role of chance In total, we identified 61 differentially abundant UF microRNAs with a false discovery rate of < 0.05 and fold change (FC) of -2 or 2. Out of these, 34 were up-regulated and 27 were down-regulated. External validation based on published literature showed that several of the dysregulated miRNAs are expressed in endometrial epithelial cells and have been described in extracellular vesicles from the endometrium and in the context of endometrial receptivity and RIF. Using publicly available single-cell transcriptome data, we found that the predicted target genes of our microRNAs are expressed both in the trophectoderm of human blastocysts and in epithelial cells of mid-secretory endometrium. Furthermore, the predicted miRNA target genes were significantly enriched in 25 pathways critical for endometrial receptivity and embryo implantation. Technical validation of hsa-miR-486-5p (FC -20.32; p-value=0.004) and hsa-miR-92b-3p (FC -9.72; p-value=0.004) were successfully in line with the sequencing data. Limitations, reasons for caution The sample size of this descriptive study was limited. A larger study cohort should be used to validate the differentially abundant microRNAs. Moreover, further in-vitro and in-vivo studies are needed to establish the role of identified miRNAs and their predicted target genes and enriched pathways in the pathogenesis of RIF. Wider implications of the findings RIF represents a true challenge in the IVF clinic. We show that total miRNAs can be comprehensively mapped in UF and constitute a promising source of non-invasive biomarkers for RIF that could be further evaluated for its clinical utility. Our findings also give insight into the molecular mechanisms of RIF. Trial registration number Not applicable

P-778 High altitude significantly affects pregnancy, implantation, live birth, and miscarriage rates in patients subjected to in vitro fertilization procedures with donated oocytes

Abstract Study question Is high altitude an important factor that affects the clinical outcomes in patients subjected toin vitrofertilization with donated oocytes? Summary answer Pregnancy, implantation, and live birth rates are significantly impacted by paternal chronic hypoxia in IVF procedures involving egg donors. What is known already High altitude is characterized by low temperatures, high UV radiation, and a hypoxic environment that significantly affect human physiology, including endocrine, cardiovascular, metabolic, and reproductive functions. Hypoxia can affect male reproduction differently, such as at the hormonal level, during spermatogenesis and gene expression. More importantly, hypoxia can affect key events during early embryo development, with time of exposure being the determining factor in the stage of fertilization and embryonic development in mammals. Study design, size, duration The following work is a retrospective nonrandomized study based on data from 85 IVF donor egg cycles performed at high altitude (Huancayo-Peru, 3,300 meter above sea level;n = 42) and at sea level (Guayaquil-Ecuador;n = 43) between July 2019 and December 2020. The same protocols of culture and embryo classification were used in both assisted reproduction laboratories. Participants/materials, setting, methods Anonymous oocytes donors (18-29 years old) underwent physical, gynecological and psychological examinations, with no family histories of hereditary or chromosomal diseases reported. According to seminal samples, IVF or ICSI was elected as the insemination procedure. Extended embryo culture until blastocyst stage was done in all procedures. Pregnancy, implantation, live birth, and miscarriage rates were compared between groups. Main results and the role of chance Out of a total of 983 included oocytes, 529 corresponded to high level patients; and 454, to sea level patients. Fertilization rates (95.1% vs 92.1%) and good-quality embryos at day 3 (79.7% vs 83.8%) were similar between high altitude and sea level groups (P > 0.05). The number of transferred embryos in recipients at high altitude was significantly higher compared to those transferred in recipients at sea level (1.90±0.29 vs 1.62±0.49) (P < 0.05). Blastocyst formation rates (39.3% vs 44.3%), pregnancy rates (61.9% vs 71.4%), implantation rates (48.8% vs 64.7%) and live birth rates per embryo transfer (46.3% vs 65.7%) were significantly lower in the patients at high altitude compared to those from sea level (P < 0.05). Significantly more miscarriages were observed at high altitude (23.1% vs 6.5%) (P < 0.05). Seminal parameters were similar between both evaluated groups. Limitations, reasons for caution The retrospective nature of this study and the small sample size may be reason for caution. Wider implications of the findings The negative effect of high altitude on reproduction has been shown previously in men, hypoxia could cause a degeneration in the germinal epithelium that affects spermatogenesis. We show that paternal chronic hypoxia affects preimplantation embryonic development, pregnancy, implantation, live birth, and miscarriage rates in IVF procedures using donated oocytes. Trial registration number not applicable

P-065 Phenotype, genetic analysis and treatment strategy of acephalic spermatozoa

Abstract Study question Do severe acephalic spermatozoa syndrome (ASS) patients with different genes variants obtain good fertilization rate after intracytoplasmic sperm injection (ICSI)? Summary answer Despite highly abnormal sperm morphology, severe ASS patients with SUN5, TSGA10 and PMFBP1 different variations have a favorable pregnancy outcome following ICSI. What is known already ASS is a rare cause of infertility characterized by the production of two main subtypes: one type has small “pin-head” ends devoid of any nuclear material, and the other comprises spermatozoa with misaligned head-midpiece junctions. Many researchers have suggested that variants in TSGA10 without a proximal centriole might influence early embryonic development. Some case reports studies with very few cases showed that ICSI could help patients with ASS father children. Study design, size, duration This retrospective cohort study included 14 infertile men with severe ASS and 100 fertile men in Reproductive and Genetic Hospital of CITIC-Xiangya in China, from January 2020 to December 2021. Participants/materials, setting, methods Patients and fertile men were recruited respectively from Outpatient Department and Sperm bank of our hospital in China. 14 severe ASS were analyzed by whole exome sequencing. Mutational analysis in the 14 severe ASS and 100 fertile men were performed. Identified SUN5, TSGA10 and PMFBP1 variations were further investigated using bioinformatics, RT-PCR, Western Blot, indirect immunofluorescence assay, papanicolaou staining, CASA, transmission electron microscopy and ICSI treatments. Clinical data were collected from the fertility center. Main results and the role of chance In 14 patients with severe acephalic spermatozoa,16 homozygous or heterozygous variants were identified from SUN5,TSGA10 and PMFBP1. We performed deep phenotyping in acephalic spermatozoa with different variants, and found that, other than the two major subtypes, multiple variations in different genes that affect sperm heads in humans produce diverse phenotypes with subtle differences. Our findings were supported by Papanicolaou staining, SEM and TEM analyses. The rates of fertilization, good-quality embryos and pregnancy for 13 patients following ICSI were 85.71% (174/203), 55.17% (96 /174) and 69.23% (9/13), respectively. There were no differences in ICSI results between ASS patients with different genes mutations. The defects of acephalic spermatozoa caused by different genetic variations may only affect the morphology of sperm. ICSI can be used in the treatment of acephalic spermatozoa and obtain favorable fertilization rate without affecting the quality of early embryos and clinical pregnancy outcome. For individuals with ASS, ICSI using intact spermatozoa (or separated heads and tails) is suggested. If no sperm heads in their ejaculate, the spermatozoa can be collected from the testes. Very few intact spermatozoa or loose sperm heads will be available, even if normal morphology of spermatozoa is 0% and 100% spermatozoa are acephalic. Limitations, reasons for caution Only a small number ASS patients was available because of its rare incidence. Patients with ASS can be effectively treated with ICSI. However, the results in this study should be treated with caution, which involves the TSGA10, SUN5 and PMFBP1 genes and cannot be extrapolated to other genes. Wider implications of the findings The identification of novel variants in SUN5, TSGA10 and PMFBP1 broadens the spectrum of ASS genetic causes. Our study is the first to thoroughly evaluate the phenotypes and pathogenic genotypes of patients with severe ASS in China. SUN5, TSGA10 and PMFBP1 mutation-associated male infertility can be overcome by ICSI. Trial registration number kq2014032

P-187 Post-thaw embryo quality is not predictive of live birth rates in frozen embryo transfer cycles: a retrospective cohort study

Abstract Study question To determine whether post-thaw change in embryo quality is associated with live birth outcomes. Summary answer Post-thaw embryo morphology is not predictive of live birth rates in single frozen embryo transfer cycles. What is known already Embryo quality is typically evaluated via morphological assessment of embryos based on the Gardner criteria. For medical or elective reasons, embryos are commonly frozen for future use through the vitrification process. Research has shown that embryo quality pre-vitrification correlates highly with freeze-thaw survival, implantation, pregnancy, and subsequent live birth rates. Good-quality embryos are therefore more likely to be selected for vitrification. However, it is not unusual to find a decline in the quality of these vitrified embryos upon thawing them for embryo transfer. Study design, size, duration We retrospectively identified a patient cohort based out of the Ottawa Fertility Centre in Ottawa, Ontario, Canada, between 2016 and 2020. Participants/materials, setting, methods Frozen single embryo transfer (FET) cycles involving good-quality expanded blastocysts deriving from autologous oocytes were selected for inclusion. We compared FET cycles involving good post-thaw embryo quality to those with worsened/poor post-thaw embryo quality. The primary outcome was live birth after FET. Secondary outcomes included rates of positive serum human chorionic gonadotropin, clinical intrauterine pregnancy, miscarriage, and ectopic pregnancy. We fit a multivariate logistic regression model, adjusting for patient and cycle characteristics. Main results and the role of chance A total of 962 single FET cycles were analyzed. There were 826 embryos that preserved their pre-vitrification quality post-thaw and 136 embryos that exhibited poorer quality on post-thaw assessment. Baseline characteristics were mostly comparable between the two groups under study. In the multivariate regression model, the adjusted odds of live birth was not significantly different in the group with good post-thaw embryo quality compared to that in the group with worse/poor post-thaw embryo quality (odds ratio [OR] 1.30, 95% confidence interval [CI] 0.86-1.96, p = 0.21). Similarly, no significant associations were found between post-thaw embryo quality and the secondary outcomes of positive BhCG (OR 1.19, 95% CI 0.81-1.75), clinical intrauterine pregnancy (OR 1.29, 95% CI 0.87-1.87), miscarriage (OR 0.97, 95% CI 0.53-1.77), and ectopic pregnancy rates (OR 0.41, 95% CI 0.14-1.24). Limitations, reasons for caution Although a multivariate regression model was used to adjust for clinically relevant confounders, there remains the possibility for residual confounding given the observational nature of our study. Wider implications of the findings The results of our study suggest that, after adjusting for patient and cycle characteristics, post-thaw embryo quality does not impact live birth outcomes. Trial registration number Not applicable

P-276 Embryo development of fresh versus vitrified metaphase II oocytes in low prognosis patients: a retrospective sibling-oocyte monocentric study

Abstract Study question Do fresh and vitrified/warmed oocytes have the same developmental potential in low prognosis patients defined by POSEIDON criteria? Summary answer Embryos derived from vitrified/warmed oocytes show lower competence for embryo development when compared to those obtained from fresh oocytes in low prognosis patients. What is known already The strategy of oocyte accumulation has been proposed for the management of low-prognosis patients with the aim to increase the number of oocytes available for fertilization and further the number of embryos. The effect of oocyte vitrification/warming on the embryo developmental potential has mainly been studied in oocyte donation programs. Investigations on sibling-oocyte failed to show difference in the morphological embryo development, but the evaluation stopped after 2-3 days of embryo culture. In addition, morphokinetic evaluation revealed delayed development of embryos derived from vitrified oocytes, from the first cleavage to the time of blastulation. Study design, size, duration Single-center sibling-oocyte retrospective monocentric study including low-prognosis patients, defined by POSEIDON criteria, who had undergone an accumulation of oocyte strategy for managing their infertility, between November 2013 and January 2021. Only patients presenting fresh and vitrified/warmed oocytes at the time of insemination were analyzed for the sibling-oocyte comparison. Main study outcomes were fertilization, cleavage, blastulation rates, day-2 and day-5 embryo quality, and the rate of embryos available for transfer/cryopreservation. Participants/materials, setting, methods Forty-five patients were included, with a mean age of 34.8 ± 3.4 years. Oocyte vitrification/warming was performed using the Cryotop method. Top-quality and good-quality embryos were defined at day-2 respectively as 4 and 3-5 adequate-sized blastomeres, without multinucleation and containing <20% of cytoplasmic fragmentation, and at day-5 according to Gardner and Schoolcraft’s classification, top ≥B4 (AB/BA/AA) and good ≥ B3BB. Statistical analyses used mixed effects logistic regression to take into account the sibling-oocyte design. Main results and the role of chance A total of 656 oocytes were inseminated, 225 fresh and 431 vitrified/warmed oocytes. The oocyte survival rate after warming was 82%, 95%CI [76-87%]. There was no difference between fresh and vitrified/warmed oocytes groups regarding the degeneration rate following ICSI (5.0% vs 4.8% respectively, p = 0.95), the fertilization rate (74.6% vs 71.4% respectively, p = 0.42) or the cleavage rate on day-2 (98.3% vs 95.4% respectively, p = 0.15). However, the embryo development was significantly different from day 2, with higher day 2 top-quality and good-quality embryos rates in the fresh oocytes group when compared to the vitrified/warmed oocytes group (top-quality: 38.3% vs 24.8% respectively, p = 0.01; good-quality 58.0% vs 45.2% respectively, p = 0.02). The same results were observed on blastocysts when extended culture was decided, with a higher blastulation rate in the fresh oocyte group (56.7% vs 31.7% respectively, p < 0.001) and higher top-quality and good-quality day 5 blastocyst rate (top-quality: 23.4% vs 5.6% respectively, p = 0.01; good-quality 23.9% vs 7.9%, p = 0.01).The rate of embryos available for transfer/cryostorage was similar in both groups (40,7 % vs 38,8 %, p = 0,71). The cumulative live-birth rate was 33,3% (4/45 from vitrified oocytes group, 5/45 from fresh oocytes group, 6/45 from mixed oocytes group). Limitations, reasons for caution The retrospective nature of the study, on a limited number of limited number of patients represents an inherent limitation. Embryo assessment was subjective, the evaluator knowing the oocyte’s source group. The impact of the morphological alterations observed in embryos derived from vitrified/warmed oocytes need to be confirmed with clinical outcomes. Wider implications of the findings The majority of studies comparing fresh and vitrified/warmed oocytes were performed in egg-donation programs. This study focuses on low-prognosis patients, who reflect perhaps more the patients who consult in ART centers. Further prospective studies are needed to confirm our findings. Trial registration number Not applicable

P-371 The role of pharmacogenetics for individualized adjuvant therapy in patients with recurrent implantation failures

Abstract Study question In women with recurrent implantation failure (RIF), could we improve IVF/ICSI outcomes after administration of appropriate adjuvant therapies depending on patient's genetic profile?. Summary answer In RIF patients carrying polymorphisms detected in p53, IL-11 and VEGF genes, the addition of aspirin and intralipids administration appears to be beneficial. What is known already RIF is a distressing phenomenon of obscure etiology. Among the putative factors associated with this condition, we have immunological factors (the presence of antiphospholipid antibodies, certain cytokines or NK cells in maternal serum), genetic alterations in the parents or embryos, uterine abnormalities, or the presence of endometritis. Recently, certain genetic polymorphisms in genes involved in embryo implantation mechanisms at the endometrial (p53 and VEGF), immunological (IL-11 and Apo-E) and thrombotic levels (F5, F2 and MTHFR) have been identified. Study design, size, duration 104 IVF cycles (2017-2021), corresponding to ninety-eight patients, were included in this retrospective study. RIF was defined as the failure of achieving an on-going pregnancy after the transfer of at least three good-quality embryos. Exclusion criteria were abnormal karyotype or sperm FISH, uterine malformations, or positive antiphospholipid antibodies. During the index cycle, these patients received different adjuvant therapies; the following were analyzed: hydroxychloroquine, intrauterine hCG instillation, scratching, intralipids, corticosteroids, aspirin, and low molecular weight heparin. Participants/materials, setting, methods To carry out the genetic profile, different SNPs were analyzed in p53 (rs1042522), VEGF (rs1570360), IL-11 (rs11668344), Apo-E (rs429358 and rs7412), F5 (rs6025), F2 (rs199963) and MTHFR (rs1801133 and rs1801131) genes.Taqman® Life Technologies allelic discrimination real-time-PCR and TRP-PLUS kit were used for the detection of the different alleles. The following IVF outcome variables were considered: positive b-hCG, clinical (gestational sac) and on-going (10 week) pregnancy rate. SPSSv20.0 was used for statistical analysis. Main results and the role of chance Polymorphisms associated with a higher risk for RIF were PR and PP genotype for p53 gene, GG for VEGF, GG for IL-11, E4 isoform for Apo-E, GA and AA for F5, GA and AA F2, TT and CC for MTHFR gene. Accordingly, 64% of patients included in our study showed a high-risk genetic profile for RIF. Genotype variants for p53, IL-11 and VEGF were the most prevalent among this population. A multivariate binary logistic regression for possible confounding factors (maternal age, embryo quality, oocyte origin, number of embryos transferred, PGT-A and other adjuvants treatments) showed that those patients with PR/PP genotype for p53, GG for VEGF and GG for IL-11, obtained significantly higher positive b-hCG, clinical and on-going pregnancy rates when aspirin was administered compared to other genotypes (p < 0.05). Furthermore, patients with GG genotype for VEGF, also obtained a significant improvement in positive b-hCG and clinical pregnancy rates after intralipids administration (p < 0.05). None of the other adjuvant treatments employed showed significant differences regardless of the genetic profile. Limitations, reasons for caution The inherent limitations of a retrospective analysis of a limited sample size. Wider implications of the findings IVF/ICSI outcome of RIF patients was analyzed according to the adjuvant treatment received and considering their genetic profile. Pharmacogenetics seems promising in identifying patients at high risk for RIF and may add in the optimization of specific pharmacotherapy depending on the genetic background of each infertile woman. Trial registration number not applicable

P-421 Increased myeloid-derived suppressor cells in women with recurrent miscarriage and recurrent implantation failure

Abstract Study question We sought to explore the levels of myeloid-derived suppressor cells (MDSCs) in women experiencing recurrent miscarriage (RM) or recurrent implantation failure (RIF) Summary answer MDSCs were significantly expanded in RM patients with respect to fertile healthy women (p = 0.03, 8.25 vs 7.00). What is known already RM and RIF are two distinct disorders that can be associated with a baseline pro-inflammatory state. Different surrogate inflammatory biomarkers have been associated with these disorders in a subgroup of patients, including expanded cytotoxic NK cells, increased pro-inflammatory cytokines or reduced regulatory T cells (TReg). MDSCs are a heterogeneous population of leukocytes, known for their capacity to modulate immune responses. The immunosuppressive function of MDSC in cancer has been well established, while a controversial role in autoimmune diseases has been reported. Typically, there are two major groups of MDSCs in humans: granulocytic/polymorphonuclear MDSCs (PMN-MDSCs) and monocytic MDSCs (M-MDSCs). Study design, size, duration A cross-sectional study to evaluate the levels of MDSCs in women with RM and RIF consecutively referred to our Reproductive Immunology Unit. 55 patients and 23 healthy women were evaluated from 2021 to 2022. RM was defined as the loss of two or more pregnancies and not necessarily consecutive. RIF was defined as the failure to achieve a clinical pregnancy after transferring at least four good-quality embryos. Participants/materials, setting, methods A full fertility screening was performed for all women and their partners as part of routine care. A group of healthy non-pregnant women up to 45 years old with two or more children (proven fertility) and no spontaneous miscarriage was used as control group. Freshly collected blood samples were analyzed by multiparametric flow cytometry using a BD FACS CANTO II. Peripheral blood M-MDSCs were characterized based on HLA-DR, CD14, CD33 and CD45 expression. Main results and the role of chance In this study, we evaluated 55 women, 35 that fulfilled RM criteria and 20 with RIF. In addition, a group of 23 non-pregnant fertile women was included as a control group. We characterized MDSCs as CD14+CD33+CD45+HLA-DR-/LOW, according to previous reports. We observed a significant expansion of peripheral blood MDSCs in RM group compared to the control group (8.25% [6.62-11.9] vs 7.05% [6-7.8], p = 0.03) and RIF group showed a similar increasing trend compared to controls (8.6% [5.65-12.35] vs 7.05% [6-7.8], p = 0.09). We performed ROC curves analysis to evaluate the predictive power. We established an optimal cut-off level of 8.3% in RM group, with a sensitivity of 50% and a specificity of 86.96%. Moreover, women with more than 8.3% had a higher probability of presenting RM compared to the control group (OR: 6.66; CI: 1.68-26.46). MDSCs are characterized by their ability to exert a potent immunosuppressive function. However, recent investigations have shown that MDSCs could act as pro-inflammatory cells under certain inflammatory conditions, such as autoimmune diseases. In this context, MDSCs could worsen the pro-inflammatory state in women, leading to miscarriage or implantation failure. Limitations, reasons for caution In order to validate these results, we should extend our cohort and control group. Moreover, we need to perform a functional evaluation of this subset so we could elucidate their role in these conditions. Wider implications of the findings MDSCs might be a good predictor for RM and RIF patients and with others (as expanded NK cells) may classify a subgroup of patients with a pro-inflammatory profile that can benefit from personalized therapies. Trial registration number Not aplicable

O-124 A new artificial intelligence (AI) system in the block: impact of clinical data on embryo selection using four different time-lapse incubators

Abstract Study question Can AI algorithms assist embryologists in evaluating embryos from any time-lapse system (TLS) along with clinical data to better predict pregnancy outcomes and reduce time-to-pregnancy? Summary answer Our algorithm (Embryoly) significantly increases accuracy in predicting clinical pregnancy by 26.9% amongst embryos deemed of fair and good quality when clinical data is included. What is known already Embryologists routinely use defined morpho-kinetic criteria to decide which embryo to transfer, and yet, many embryos deemed of good quality fail to lead to a pregnancy. Thus, AI algorithms to assist embryologists in objectively selecting the most promising embryos are in demand. To date, several reports indicate that AI algorithms are capable of predicting pregnancy clinical outcomes but to the best of our knowledge they only consider visual data (or together with a small set of clinical features) from individual TLI systems to generate their predictions. Study design, size, duration A dataset of 6790 embryos (97.82% known clinical pregnancy outcome, 31.47% frozen transfers) from 2519 patients from 11 European fertility centers recorded with 4 different TLS (GERI-Merck, Embryoscope & EmbryoscopePlus-Vitrolife and MIRI-Esco) was used to train and validate Embryoly. Nine out of 93 clinical factors were identified as being the most predictive, including woman age, woman and man BMI and AMH levels. Performances were evaluated on a separate test dataset (393 videos). Participants/materials, setting, methods Clinical pregnancy outcome was predicted using a 3D convolutional neural network that analyzed up to 5 days of embryo development. The output score was further analyzed considering the clinical features to generate a second clinical score. Both predictions were compared to those of 10 senior embryologists made on the same test dataset (with and without clinical features). Embryo quality was assessed as: poor, fair, good. Unless specified otherwise, McNemar test was used for statistical tests. Main results and the role of chance Overall accuracy of embryologists in predicting clinical pregnancy based on videos alone was 57.25% (CI 95% : 52.34% - 62.16%) compared to 60.56% (CI 95% : 55.71% - 65.41%) for Embryoly (p = 0.35). When videos were analyzed together with the clinical factors, overall accuracy of embryologists was significantly lower than Embryoly (60.05% [CI 95% : 55.19% - 64.91%] vs 68.19% [CI 95% : 63.57% - 72.82%], p-value=0.015, respectively). Clinical factors significantly increased our accuracy by 7.63% (p-value=0.030). More specifically, Embryoly algorithms fared better in terms of detecting false positives (31.30% vs 19.34%) compared to embryologists, with a specificity of 74.4% vs. 58.6%, respectively. If we consider only embryos of fair and good quality (71.50% of our test dataset) Embryoly’s accuracy was 13.52% higher than that of embryologists. This translates into AI having an even better ability to detect false positives for embryos that could be seen as good candidates for transfer (20.28% false positives against 42.70% for the embryologists). Embryoly performs differently across selected TLS when analyzing videos alone, but not when clinical data was also considered (chi2 test, p < 0.001 and 0.5, respectively). Further work will investigate these discrepancies across TLS. Limitations, reasons for caution As of today, Embryoly’s accuracy in predicting the outcome of poor-quality embryos is not different to that of embryologists (79.46% vs 84.96%; p-value=0.19). We are improving this by exposing Embryoly to more “poor quality” embryos, so as to also identify poor quality embryos with unexpected potential for implantation. Wider implications of the findings Our pioneering findings support the use of AI for a standardized and couple-centered care in clinical embryology, integrating male and female factors with embryo development analyses from multiple TLS. Our approach has the potential to cost-effectively reduce time to pregnancy and is another step toward a personalized embryo transfer strategy. Trial registration number Not applicable

P-162 GDF9 concentration in embryo culture medium is linked to human embryo quality and viability

Abstract Study question Is the GDF9 concentration in Day 3 human embryo culture medium linked to embryo quality and clinical outcome? Summary answer The embryo quality and clinical outcome appears to be linked to the GDF9 concentration in Day 3 culture medium. What is known already Previous studies have demonstrated that the GDF9 level in follicular fluid and cumulus granulosa cells can be used as biomarker for predicting embryo quality. Study design, size, duration In study 1, a total of 280 embryos from 70 patients who obtained at least 4 embryos with 6-10 blastomeres (2 transferable and 2 non-transferable embryos) at Day 3 were enrolled. The corresponding GDF9 concentration in culture media were compared between transferable and un-transferable embryos and also compared among good, fair and poor quality embryos. In study 2, a total of 119 embryos from 61 patients (29 implanted and 32 non-implanted patients) were enrolled. Participants/materials, setting, methods The expression pattern of GDF9 in human embryos was investigated using Q-PCR and immunofuorescence. GDF9 concentration in embryo culture media was quantified by ELISA assay. Statistical analyses defined correlations between GDF9 concentration and embryo quality, in study 1, and implantation and live birth, in study 2. Main results and the role of chance GDF9 mRNA and protein were detected from human oocytes to eight-cell embryos and displayed a slow decreasing trend. In study 1, GDF9 concentration in culture medium is lower for transferable embryos compared with non-transferable embryos (331 pg/mL (quartiles: 442, 664 pg/mL) vs. 518 pg/mL (quartiles: 328, 1086 pg/mL), P < 0.001), and increased commensurate with the diminution of the embryo quality (P < 0.001). In study 2, signifcantly lower GDF9 concentration was detected for implanted embryos than non-implanted embryos (331 pg/mL (quartiles: 156, 665 pg/mL) vs. 518 pg/mL (quartiles: 328, 1086 pg/mL), P < 0.001). The same trend was found between the embryos that led to live birth and those that failed. Limitations, reasons for caution Further randomized controlled trials are needed for exploring whether GDF9 assay of spent embryo culture medium can improve the clinical outcome in IVF. Wider implications of the findings GDF9 in culture medium has the potential to be a non-invasive biomarker for embryo selection. Trial registration number not applicable

P-093 A comparison of the effects of IMSI and HOST for sperm selection on morphokinetic parameters and ploidy status in sibling embryos

Abstract Study question Are hypo-osmotic swelling test (HOST) and intracytoplasmic morphologically selected sperm injection (IMSI) both effective alternative methods for sperm selection? Summary answer Similar results were obtained in embryo morphokinetics between HOST and IMSI. What is known already HOST is a simple, low-cost and rapid test based on the swelling of the tail of the sperm in a hypoosmotic environment. Less DNA fragmentation, membrane integrity and aneuploidy rates have been found in sperm selected by the HOST method. It also allows identification of viable sperm in a totally immotile sperm sample. IMSI, on the other hand, is a method for identifying the morphologically best sperm using high-magnification microscope. In studies, IMSI has been associated with high fertilization rates. However, it is a time-consuming method that requires expensive equipment. Study design, size, duration Time-Lapse Monitoring (TLM)-incubated sibling embryos of 25 male factor patients who applied for ART treatment to Istanbul, Sisli Memorial ART and Genetics Center in 2021 were included into the study. Sibling embryos were grouped according to their sperm selection method. In the IMSI group 170 embryos, in the HOST group 173 embryos were evaluated retrospectively in terms of the morphokinetic parameters, the rates of blastulation, Top- Quality (TQ) and Good Quality (GQ) embryos and euploidy. Participants/materials, setting, methods Blastocyst grading was performed according to Gardner’s classification. Blastocysts 3AA,4AA,4AB,4BA,4BB,5AA,5AB,5BA,5BB were accepted as TQ+GQ. Blastulation rates was determined including the embryos at least 2 expansion. PGT-A results were obtained by Next Generation Sequencing(NGS) method. The comparison of the TLM parameters and clinical features of the patients were performed using Student’s t-test. Chi-square test was used for comparing the rates of fertilization, blastulation, TQ+GQ embryo and euploidy in each group. Main results and the role of chance No statistical difference was observed between IMSI and HOST groups in terms of morphokinetic parameters (tPN, tPNf, t2, t3, t4, t5, t6, t7, t8, t9, tSC, tM, tSB, tB, tEB) (p > 0.05). There were higher rates of normal fertilization in the HOST group (90.8%) than in the IMSI group (82.4%) and of TQ+GQ embryo (HOST: 50%, IMSI: 47.3%). However, these diffrences were not statistically significant (p > 0.05). There was statistically lower abnormal fertilization rate was found in HOST group (2.3% vs 7.1%, p = 0.038). When PGT-A results were compared (HOST:19 embryos, IMSI: 19), slightly higher results were observed in IMSI group than HOST group ( 63%, 58% respectively) (p > 0.05). Limitations, reasons for caution This was a retrospective of a small number of cases. Wider implications of the findings The similarity of the outcome in these initial findings studying a small number of cases, suggest that this study should be extended. Trial registration number None

P-205 Blastocyst derived from oocytes with smooth endoplasmic reticulum aggregates (SERa) has similar clinical and perinatal outcomes with those of oocytes without SER

Abstract Study question This study was to investigate effect of SERa on the fertilization rate, embryonic development after ICSI, and clinical and perinatal outcomes after single blastocyst transfer. Summary answer SERa (+) derived embryo can be selected as embryos for transfer when no available SERa (-) derived embryos. What is known already Based on findings that the risk of congenital abnormalities in the newborn is higher in ovum with SERa in the cytoplasm, the Istanbul consensus workshop at the 2011 meeting of the ESHRE recommended against fertilizing ovum with SERa due to these risks. However, there have been several reports of healthy infants born from embryos derived from SERa, suggesting that, while more long-term follow-up is necessary, healthy births are possible from such embryos. In 2017, the 2011 recommendations were reviewed in the Alpha/ESHRE consensus (Vienna), which said the approach should be determined on a case-by-case basis. Study design, size, duration We retrospectively investigated 23,007 oocytes which was retrieved between January 2016 and March 2020. Of these, 1,038 oocytes (4.5%) with visible SERa comprised SERa (+), while 21,969 oocytes (95.5%) without SERa comprised SERa (-). Participants/materials, setting, methods SERa were observed under the microscopy after denudation. The rate of fertilization, good-quality day-3 embryos, good-quality day-5 blastocysts, and day-5, 6 or 7 blastocysts were evaluated for both groups. We also compared the rate of clinical pregnancy, live birth, miscarriage, and birth defects in single blastocyst transfer between SERa (+) derived 114 blastocysts and SERa (-) derived 6,290 blastocysts from January 2016 and December 2018. Main results and the role of chance The results are shown. 2PN fertilization rate outcomes after ICSI (SERa(-) eggs vs. SERa(+)eggs),81.4%(17,873/21,969) vs.79.4% (823/1,038),and good-quality day3 rate was 61.1%(10,927/17,873)vs.60.9% (501/823) which was not significantly different. Good-quality day5 blastocyst rate was 46.5% (7,876/16,955) vs. 39.8%(304/763), and day 5 blastocyst success rate was 60.8% (10,317/16,955) vs.54.3% (414/763), which were both significantly lower with SERa(+). (P < 0.001) The day 6 blastocyst success rate was 69.9% (11,849/16,955) vs. 65.5% (500/763) (P = 0.01), and the day 7 blastocyst success rate was 70.9% (12,024/16,955) vs. 67.5% (515/763) (P = 0.04), which were all significantly lower with SERa(+).The clinical pregnancy rate was 39.4% (2,481/6,290) vs. 35.1% (40/114), the live birth rate was 27.7% (1,745/6,290) vs. 26.3% (30/114), and the miscarriage rate was 27.5% (683/2,481) vs. 20.0% (8/40) and the congenital abnormality rate was 1.6% (29/1,757) vs. 0% (0/30) for SERa(-) embryos and SERa(+) embryos, respectively, which were not significantly different. Blastocyst derived from oocytes with SERa has similar clinical and perinatal outcomes with those of oocytes without SERa. Significant differences were examined using the chi-squared test, with p < 0.05, indicating a significant difference. Limitations, reasons for caution Embryos derived SERa (+) were transferred when the patient did not want any more oocytes retrievals, no embryos derived SERa (-) were available, and only if the couple desired embryo transfer after the problems associated with SERa (+) embryos were fully explained. Wider implications of the findings To the best of our knowledge, this study is the largest number of live births investigating the outcome of SERa (+) derived embryos. SERa (+) derived embryo can be selected as embryos for transfer when no available SERa (-) derived embryos. Trial registration number Not Applicable

P-048 Fertilization rate and embryonic development after intracytoplasmic sperm injection (ICSI) using a microfluidic sperm selection device without centrifugation

Abstract Study question Can the use of a microfluidic sperm selection device without centrifugation simplify the procedure without affecting fertilization rates and embryonic development after ICSI? Summary answer The microfluidic device can be used to select sperm in a simple procedure without reducing the fertilization or embryo development rate. What is known already In human ART, it is essential to process the semen and adjust the sperm sample according to the intended purpose, rather than using raw semen. Current sperm preparation methods at most IVF clinics include the density gradient centrifugation method, which utilizes the difference in density between the maturation stages of sperm, followed by washing using centrifugation. However, these methods require a lot of steps and the procedure is complicated. Recently, several non-centrifugal sperm processing devices have become available. One of them is a simple sperm conditioning method using a microfluidic device. Study design, size, duration This was a prospective study using sibling oocytes including 20 ART patients treated with 24 cycles and where there were 320 zygotes after ICSI. The duration of the study was 8 months (May 2020 to December 2020). Participants/materials, setting, methods For sperm preparation without centrifugation, a microfluidic device “ZyMōtⓇ Multi 850μL” was used. According to the labelled use, 850μL of semen was required with “ZyMōtⓇ” device (ZyMōt group), and the rest of the semen was processed according to routine laboratory procedure by monolayer density gradient centrifugation and washing by centrifugation (DGC group). Oocytes from the same patient were randomly divided into 2 batches and ICSI was performed using sperm treated with each method. Main results and the role of chance The 2PN formation rate in the ZyMōt group was 84.5% (142/168), which was not significantly different from 82.9% (126/152) in the DGC group. There was also no significant difference in 1PN formation rate (3.0% vs. 3.3%), multi PN formation rate (3.6% vs. 3.3%) and the non-fertilization rate (8.3% vs. 10.5%). The good quality embryo rate at Day 3 was 25.0% (31/124) in the ZyMōt group and 24.5% (27/110) in the DGC group, with no significant difference. The Day 5 blastocyst rate was 37.9% (47/124) in the ZyMōt group and 36.7% (40/109) in the DGC group, and the cumulative blastocyst rates by Day 7 were 54.0% (67/124) and 57.8% (63/109), respectively, with no significant difference. Limitations, reasons for caution This study was limited to samples with a motile sperm concentration of more over 1.0 × 106 in raw semen. Wider implications of the findings These results demonstrated that sperm processing using the microfluidic device without centrifugation does not affect the fertilization or blastocyst development rate after ICSI and that the sperm processing procedure can be simplified by using this device. Trial registration number not applicable

P-652 Contribution of the D19S884 allele 8 of the FBN3 gene and the Hippo signaling to the reproductive and metabolic phenotype of PCOS patients

Abstract Study question Does exist a correlation between the presence of D19S884 allele 8 (A8) and Hippo pathway dysregulation with the metabolic and ovarian PCOS profiles? Summary answer The presence of the A8 allele affects insulin resistance and hyperandrogenism. Moreover, PCOS ovarian phenotype could be related to a dysregulation of Hippo pathway genes. What is known already The specific D19S884 A8 allele of the FBN3 gene has been associated with an increased probability of polycystic ovarian syndrome (PCOS). D19S884 participates in FBN3 alternative splicing and produces Asprosin-3 and Fibrilin-3 proteins that may be related to PCOS clinical manifestations. Asprosin-3 has been recently identified as a glucose modulator and could be related to metabolic profiles of PCOS. Fibrilin-3 is an extracellular matrix protein, and together with a dysregulation of the Hippo pathway, could be responsible for constraining follicular growth in the PCOS ovaries. Study design, size, duration Descriptive and cross-sectional study with 93 women (25-37 years old) undergoing an IVF cycle between 2019 and 2021 at Hospital la Fe (Valencia, Spain). Thirty patients were considered PCOS while the remaining sixty-three were non-PCOS controls. After recruitment women were screened for A8 allele, metabolic status, hormone profile and Hippo pathway genes. The IVF cycle parameters were recorded to determine their relationship with study variables. Participants/materials, setting, methods Patients with two or more Rotterdam-criteria or with polycystic ovarian morphology (PCOM) were considered as PCOS. After signed informed consent, blood samples and cumulus cells were obtained at egg retrieval. Hormone and metabolic parameters were analyzed and the homeostasis model assessment of insulin resistance (HOMA-IR) established. Genomic DNA was isolated to assess the presence of A8 allele by capillary electrophoresis. The expression of Hippo pathway genes, BIRC1 and CCN2, was analyzed by Taqman qPCR assay. Main results and the role of chance PCOS patients showed increased body mass index (Control:23.2±3.7; PCOS:25.3±4.5, p = 0.04) antimüllerian hormone levels (Control:17.7±7.7pmol/L; PCOS:39.7±22.9pmol/L, p = 0.00), antral follicle count (Control:15±7; PCOS:27±13, p = 0.001) and a reduced number of good quality embryos (Control:1.3±0.9; PCOS:0.8±1.0, p = 0.02). The A8 allele was detected in 15% of the recruited patients, with a 13% incidence in controls and 19% in PCOS. Interestingly, PCOS A8+ patients presented the PCOM phenotype. Women with the A8 allele (A8+) showed higher DHEAS levels (A8-:1899.7±1256.9; A8+:2642.5±555.2, p = 0.046) and free androgen index (A8-:1.1±0.8; A8+:2.0±1.2, p = 0.046) when compared to A8- ones. Similar results were obtained when the comparison was made according to clinical diagnose (Control A8+ vs. Control A8- and PCOS A8+ vs PCOS A8-). Moreover, the overall A8+ patients associated an increase in glucose levels (A8-:85.7±10.9mg/dL; A8+:93.0±6.01mg/dL, p = 0.02) and HOMA-IR (A8-:1.7±1.1; A8+:2.5±1.2). These metabolic findings were observed in control women with the allele but not in PCOS. The PCOS group showed a downregulation of CCN2 (Fold-change (FC): -3.8±9.1, p = 0.02) in cumulus cells. Interestingly, those women with the A8 allele showed downregulated BIRC1 levels (FC A8+: -2.7±4.8 and A8-: 0.2±2.7; p = 0.04). These gene dysregulations could be related with a deficient oocyte-cumulus communication, abnormal follicular growth and an ovarian stroma stiffness alteration. Limitations, reasons for caution These promising results need to be confirmed in a larger population of PCOS to allow validation of the observed effects according to PCOS sub-phenotypes. Moreover, transcriptomic analysis and functional enrichment assays should be carried out to clarify the contribution of Hippo pathway and A8 allele to PCOS pathogenesis. Wider implications of the findings The obtained results suggest that the presence of the A8 allele influences the metabolic profile related to insulin resistance and androgen levels, as well as the activation status of Hippo pathway. Moreover, this signaling pathway seems to be dysregulated in PCOS patients, suggesting its possible role in the ovarian phenotype. Trial registration number Not applicable

O-165 Impact of endometriosis on the oocyte

Abstract Available evidence indicates that women with endometriosis tend to have lower implantation rates following IVF than those without endometriosis. Whether this reduction in implantation rate is due to compromised endometrial receptivity or reduced embryo quality remains controversial. However, studies with the oocyte donor model in which reciprocal transfers are performed involving oocytes/embryos from women with or without endometriosis support the conclusion that oocyte quality is the main factor compromising implantation rate. This conclusion is consistent with documented elevations of inflammatory cytokines, reactive oxygen species and growth- and angiogenic factors in follicular fluid and peritoneal fluid of women diagnosed with endometriosis. In this lecture, we will review evidence in support of negative effects of endometriosis on oocyte quality. We will discuss the results of studies directly assessing oocyte quality through gross morphological analyses and transmission electron microscopy assessments on human oocytes. Given the paucity of human oocytes available for research, we will then consider studies on animal oocytes matured in vitro in medium supplemented with either follicular fluid or peritoneal fluid from women with endometriosis. We will then proceed to reviewing the clinical IVF literature assessing the relationship between endometriosis and embryo quality, as the latter is predominantly driven by oocyte quality. We will specifically appraise studies investigating any association between endometriosis and 1) the proportion of good quality embryos and blastocyst formation rate, as categorized by conventional morphology assessment; and 2) risks of aneuploidy, and aberrations in embryo morphokinetics identified by time-lapse imaging. We will close by a brief discussion of the challenges that prevail in untangling negative effects of endometriosis on oocyte quality.

P-697 The association between serum prolactin levels and outcomes of in vitro fertilization

Abstract Study question Is there an association between serum prolactin levels and outcomes of in vitro fertilization (IVF) in infertile women? Summary answer Serum prolactin levels are negatively associated with the proportion of good quality embryos from the total number of embryos obtained at IVF What is known already It is widely known that hyperprolactinemia is a powerful inhibitor of the hypothalamic-pituitary-ovarian axis leading to anovulation. However, few studies have tried to establish the impact of slightly higher PRL levels on the outcomes of in-vitro fertilization. Although some researchers have stipulated that PRL may play a positive role in oogenesis, embryo development and luteal function, due to the scarcity of data, this matter is considered an ongoing debate. Study design, size, duration We performed a retrospective study in the Department of Reproductive Medicine of a private hospital. The medical records of all consecutive patients who underwent IVF between January 2014 and December 2021 with all causes of infertility were reviewed. Participants/materials, setting, methods Patients with various causes of infertility undergoing IVF-ICSI were included in the study. Outcomes such as numbers of oocytes obtained, numbers of embryos were assessed and the proportion of good quality embryos from the total number of embryos (GQE/TNE) obtained was calculated. The study group included 544 patients with a mean age of 34.4±4.46 years, mean AMH of 3.99 1.34 ng/mL and mean serum prolactin levels of 25.07±23.7 ng/ml. Main results and the role of chance we found no association between serum prolactin levels and oocytes or embryos number. Serum level of prolactin was negatively correlated with the proportion of GQE/TNE (r = 0.101, p = 0.042). When patients were divided in subgroups according to ovarian reserve, the patients in the fourth quartile of prolactin had a lower proportion of GQE/TNE (p = 0.041) in comparison with those from the inferior quartiles only in the reduced ovarian reserve group (serum AMH below 1.1 ng/ml). Limitations, reasons for caution Patients included in this study are infertile patients with indications for IVF treatment. Therefore, the results of this study should be used with caution in other populations. Wider implications of the findings Our study suggest that serum prolactin levels might be negatively associated with in-vitro fertilization outcomes. If confirmed by further studies, treatment could be given to lower prolactin levels, even if mildly elevated, thus assuring more successful outcomes. Trial registration number NA

O-065 The effect of double vitrification on embryo reproductive potential and clinical outcome

Abstract Study question Do double-vitrified embryos have impaired implantation potential compared to embryos vitrified once? Summary answer Double-vitrified embryos demonstrated significantly lower percentages of positive β-HCG, clinical pregnancy (CPR) and live birth (LBR) compared to embryos vitrified once. What is known already Since vitrification became a core player in ART, many clinics adopted a freeze-all strategy, including vitrification of more than one embryo per vitrification device. Nowadays, the guidelines’ suggestion for single embryo transfers often results in a surplus of good quality embryos post warming which are re-vitrified to avoid embryo wastage. Thus, transfers of double-vitrified embryos have been performed worldwide, leading to successful pregnancies. It remains yet to be answered whether embryos after double vitrification exhibit the same reproductive performance as embryos vitrified once. By now, limited studies including various cryopreservation methods show relatively conflicting results. Study design, size, duration This retrospective cohort study was conducted in Embryolab Fertility Clinic, Greece. Assisted reproductive cycles included in the study were performed from January 2015 to December 2020. Both PGT cycles and cryopreserved oocyte cycles were excluded. The mean women age was 32.8 years old (±5.9) in the study group and 33 (±5.9) in the control group. There was no variation in the cryopreservation methods used, since all embryos were cryopreserved by the same vitrification protocol. Participants/materials, setting, methods For the present study, 233 embryo transfers of 334 double-vitrified embryos (309 blastocysts/25 cleavage embryos) and 172 transfers of 260 embryos vitrified once (246 blastocysts/14 cleavage) were analyzed to explore the impact of double vitrification on positive β-HCG, CPR, LBR and miscarriage rate. Moreover, the performance of embryos that were cultured for at least 24 hours before re-vitrification was compared to embryos warmed and re-vitrified on the same day, for the same parameters mentioned above. Main results and the role of chance All embryos survived vitrification (100% survival rate) in both study and control group. An alpha level of .05 was used for all statistical tests. Results showed that positive β-HCG percentage was significantly lower in the double vitrification group (45.1% for double -vitrified embryos -study group-, 57.0% for embryos vitrified once -control group-, p=.02), as well as CPR (28.3% in study group - 42.4% in control group, p=.003) and LBR (26.6% in study group - 40.4% in control group, p=.004). The miscarriage rate did not significantly differ between two groups (35% in study group - 25% in control group, p=.121). Moreover, positive β-HCG percentage was significantly higher in transfers with embryos that were cultured at least 24 hours before re-vitrification (group A: 48.9%), compared to embryos that were warmed and re-vitrified on the same day (group B: 30.6%, p=.02). Interestingly, although CPR (31% in group A, 18.4% in group B, p=.08), LBR (29.4% in group A, 16.3% in group B, p=.06) and miscarriage rate (35.6% in group A, 33% in group B, p=.87) were not significantly different between these groups, there was a tendency for rates to be higher in group A compared to group B. Limitations, reasons for caution This is a retrospective study, including transfers of embryos of different stage (day 2 – day 5). However, the control group was matched to the study group, in terms of embryo stage. The number of previous treatments per couple was not included in the study. Wider implications of the findings Although double-vitrified embryos can lead to successful pregnancies, our results showed an adverse effect of double vitrification on embryo reproductive potential. To avoid lower pregnancy rates, culture of embryos until day 5 and cryopreservation of one embryo per vitrification device should be considered. Trial registration number not applicabe

O-069 Does dual stimulation with continuous FSH administration increase the total number of metaphase II oocytes in poor responders?

Abstract Study question Is there an increase in the total number of metaphase II (MII) oocytes between a conventional ovarian stimulation and a dual uninterrupted stimulation? Summary answer Dual stimulation with continuous follicle stimulating hormone (FSH) administration is not superior to conventional stimulation in terms of number of MII oocytes. What is known already In the last decade, the concept of multiple follicular waves during one menstrual cycle has gained a lot of interest. Translated into clinical practice, combining two stimulations in the same ovarian cycle has appeared beneficial with regard to the number of retrieved oocytes, without affecting the embryo quality or ploidy status. Usually, this so-called dual stimulation approach is characterized by a discontinuation of FSH administration for approximately 5 days between two consecutive stimulations. The role of dual uninterrupted ovarian stimulation has not yet been studied. Study design, size, duration This was an open-label randomized controlled trial (RCT) with superiority design, performed in a single tertiary center. Power analysis indicated a required sample of 46 patients to detect superiority (defined as two surplus MII oocytes) of the dual stimulation group. Between October 2020 and September 2021, 70 patients were screened, 48 were randomized and 46 completed the study. Participants/materials, setting, methods Women aged 25-40 with a serum anti-Müllerian hormone (AMH) level of ≤ 1.5 ng/mL, antral follicle count (AFC) of ≤ 6, or ≤ 5 oocytes after a previous stimulation, were eligible for inclusion. Randomization occurred only in case of ≤ 9 follicles of ≥ 11mm on the trigger day. In the control group, patients underwent one round of ovarian stimulation and oocyte retrieval only, while the study group had two uninterrupted rounds of ovarian stimulation and two retrievals. Main results and the role of chance Baseline characteristics were similar between both groups. The cumulative number of COC and MII oocytes after completion of the second oocyte retrieval was similar in the control and study group [5.3 ± 2.7 versus 5.3 ± 3.1 [difference 95%CI (-1.7 to 1.7), p = 0.92] and 4.1 ± 2.5 versus 4.3 ± 2.7 [difference 95%CI (-1.7 to 1.3), p = 0.82]. Likewise, a comparable number of good quality embryos at day 3 was available (3.0 ± 2.0 versus 2.7 ± 2., p = 0.63). In the study group, the cancellation rate due to insufficient response to the second round of stimulation was 39.1%. When focusing on the first stimulation in both groups, there were no significant differences regarding basal FSH, FSH consumption and the number of preovulatory follicles. After the first oocyte retrieval, the mean number of COC and MII oocytes was significantly higher in the control group (who had human chorionic gonadotropin (hCG) triggering), compared to the study group (who had Gonadotropin Releasing Hormone (GnRH) agonist triggering) (5.3 ± 2.7 versus 3.3 ± 2.2, p = 0.004 and 4.2 ± 2.4 versus 2.9 ± 2.2, p = 0.05). Likewise, the number of good quality embryos on day 3 was significantly higher (3.0 ± 2.0 versus 1.9 ± 1.7, p = 0.04). Limitations, reasons for caution This study was powered to demonstrate superiority for number of MII oocytes. Investigating the impact of dual stimulation on pregnancy rates would have required a larger sample size. Wider implications of the findings The observed suboptimal oocyte yield after agonist triggering in poor responders is a reason for concern and further scrutiny, given that previous RCTs have shown similar outcomes in normal and high responders after hCG and GnRH agonist trigger. Trial registration number NCT03846544

P-359 Effect of estradiol pretreatment on antagonist ICSI cycles outcome

Abstract Study question Does estrogen pretreatment in antagonist ICSI cycles improves the outcome? Summary answer Pretreatment with estrogen in antagonist cycles reduced the days of stimulation, the number of hMG ampoules and enhanced the implantation rate. What is known already It has been previously reported that, programming of antagonist cycles with E2 only, started in the luteal phase and continued beyond the menses till the first day of stimulation, yielded similar results as the long agonist protocol. E2 pre-treatment in the luteal phase has an inhibiting role on follicular growth through its negative feedback on the FSH increase during the luteal follicular transition phase. This effect stops as soon as E2 administration is terminated. Study design, size, duration This was a prospective randomized controlled trial performed in a private IVF centers from June 2020 to February 2021. One hundred and fourteen infertile couples undergoing intracytoplasmic sperm injection embryo transfer cycles (ICSI-ET) were included. Participants/materials, setting, methods Group A includes 57 patients were pretreated with a dose of 4 mg estradiol valerate (progynova 2 mg; Bayer) once daily starting 10days before expected menses. Ovarian stimulation was started one day after the last estradiol dose, in a dose ranging from 225 to 375 IU of hMG. While in group B , 57 patients received stimulation in a dose of 225 to 375 IU of hMG starting on cycle day 2 without estradiol pretreatment. Main results and the role of chance The daily dose of hMG was significantly lower in The study group (P = 0.048), the stimulation days were significantly higher in the control group (P = 0.049), the total number of hMG ampoules was significantly higher in the control group (P = 0.041), and the implantation rate was higher in the study group (P = 0.045) , also the pregnancy rate (positive pregnancy test) was higher in the study group (p = 0.044) but there was no significant statistical difference detected as regards the clinical pregnancy rate between both groups (P = 0.180) , the serum estradiol on day of hCG ( p = 0.055), serum progesterone level on hCG day (P = 0.71) and the endometrial thickness (p = 0.780) . Also there is no significant statistical difference between the two groups as regarding number of follicles on day of hCG. (P = 0.803), the number of mature oocytes (P = 0.703) and no significant difference in the number of good quality embryos (p = 0.532) and the ratio of mature oocytes to number of follicles on day of hCG (P = 0.835), with no significant statistical difference between both groups. Limitations, reasons for caution The limitation of this study is the small number of patients as we included only 114 patients. Wider implications of the findings Luteal estradiol in antagonist cycles improved the outcomes of ICSI procedure by reducing the number of gonadotropin ampoules used for COH and reduced duration of stimulation, also the implantation rate is improved by luteal estradiol administration. Trial registration number NCT05197374

P-419 Does adjuvant usage in the luteal phase improve live birth rates of ICSI - ET patients with previous implantation failure?

Abstract Study question Does adjuvant usage in the luteal phase improve live birth rates of ICSI-ET (intracytoplasmic sperm injection - embryo transfer) patients with previous implantation failure? Summary answer Combination of low molecular weight heparin, aspirin, and prednisolone in the luteal phase doesn't improve live birth rates of ICSI-ET patients with previous implantation failure. What is known already RIF affects around 10% of patients undergoing IVF worldwide. The exact pathology of implantation failure, although transferring a good quality embryo, is still unknown. There are numerous treatment options, However, there is no conclusive evidence that prophylactic treatment improves the pregnancy rate in infertile women during IVF. Study design, size, duration This retrospective study included 1095 patients with > 2 failed either fresh or frozen embryo transfer cycles between 2014 Jan – 2021 Jan. Participants/materials, setting, methods Patients were divided into two groups. Group-A consisted of patients with only vaginal progesterone for luteal phase support. Group-B consisted of patients with triple (daily subcutaneous low molecular weight heparin, daily p.o. 150 mg aspirin, and daily p.o. 16 mg prednisolone) luteal phase supplementation to vaginal progesterone. Demographic parameters, cycle characteristics, embryology, and pregnancy outcomes were compared, and the live birth rate was the study's primary outcome. Main results and the role of chance There were 5910 ICSI&ET cycles between the years Jan 2014 and Jan 2021. A total of 3853 Patients with single day5-6 good quality blastocyst transfer was selected from the database, and appropriate patients for inclusion criteria with at least two previous failure implantation history were included. As a result, a total of 1095 fresh or frozen-thawed ET cycles were included in the study. There were 617 patients without (Group A) and 478 patients with adjuvant use (Group B). Demographic parameters were similar between groups. Although higher embryology outcomes (Group A vs. B, No.of oocyte; 11 (7-17) vs. 13 (8-19), No. of MII; 9(5-14) vs. 10 (6-15), No. of 2PN; 5 (3-9) vs. 6 (4-10), p < 0.01), positive b-hCG rates, miscarriage rates, live birth rates were similar between groups (Group A vs. B, positive b-hCG; 30.8% (190/617) vs. 35.4% (169/478), Miscarriage rates; 4.4% (27/617) vs. 6.7% (32/478), Live Birth Rates; 20.4% (126/617) vs. 23.8% (114/478)). When the live birth rates were stratified depending on the number of previous failed transferred cycles, the live birth rates were still comparable between the groups. Limitations, reasons for caution The study's retrospective design limits the power of the results. The study's main limitation is the lack of the euploidy information of transferred embryos. Wider implications of the findings Our findings suggest that empirical usage of adjuvants such as low molecular weight heparin, aspirin, and prednisolone does not improve live birth rates in patients with previous implantation failure. These results support the present literature. Trial registration number Not applicable

O-279 The type of gonadotrophin: Does it improve ART outcome?

Abstract Ovarian stimulation is a vital clinical component of an IVF cycle, with gonadotrophins being the main stimulation drugs. Follicle stimulating hormone (FSH), the key driver of follicle recruitment during an ovarian cycle is a complex glycosylated protein comprised of an α and β subunit with four glycosylation sites. Differences in glycosylation lead to heterogeneity with the FSH isoforms varying in their receptor binding and biological activity. The cyclic physiological variation of FSH isoforms and the FSH threshold/ window concept lead to the selection of a single dominant follicle during a menstrual cycle. Surpassing the FSH threshold and extension of the FSH window with ovarian stimulation using gonadotrophins salvages follicles with higher FSH sensitivity leading to multi-follicular development and retrieval of multiple oocytes. The first IVF birth in 1978 was from a natural cycle and was preceded by several unsuccessful attempts at oocyte retrieval. Ovarian stimulation was therefore incorporated into IVF treatment to overcome the inefficiencies encountered with a natural cycle. The objective of ovarian stimulation during IVF treatment is to obtain several mature oocytes, leading to good quality embryos for immediate replacement and cryopreservation for future use. An ideal stimulation wherein gonadotrophins are the mainstay is one that achieves maximal efficacy, safety and cost-effectiveness. Several gonadotrophin preparations have been available for clinical use over the years since the first use of pregnant mare serum gonadotrophin (PMSG) in 1940 and the first successful birth using human pituitary gonadotrophin (hPG) in 1961. These initial preparations were soon abandoned due to concerns with antigenicity and safety. Subsequent important milestones in the development gonadotrophins for clinical use include the urinary derived products, recombinant FSH preparations, long acting FSH and the biosimilars. Given the selection of gonadotrophins currently available, the apparent question is if they differ? Circulating FSH isoforms change during different stages of the menstrual cycle and through the reproductive stages. More acidic isoforms are predominant during the early follicular phase, with less acidic or basic isoforms predominant in the late follicular phase of the menstrual cycle, and more acidic isoforms during the menopausal stage. Similarly, the different FSH products vary in their properties owing to differences in their production and purification. Urinary derived products are more acidic compared to recombinant preparations and the different recombinant products vary in degree of acidity due to differences in the extent and pattern of glycosylation. The acidic and basic properties of gonadotrophin types influence metabolic stability, half-life and their biological activity. The question then remains if these differences in bioactivity affect clinical outcomes? Clinical effectiveness of the various gonadotrophins types should be addressed in the context of the different pituitary suppression regimens, ovarian response categories and patient variables given the implementation of individualised stimulation protocols. It is also imperative to clearly define successful clinical outcomes incorporating both efficacy and safety. Cumulative live birth rate (CLBR) following fresh and frozen embryo transfers resulting from one episode of ovarian stimulation should be adopted as the measure of the overall efficiency along with OHSS as a measure of immediate safety. There are several studies and systematic reviews comparing the different gonadotrophin types and products for various outcomes including surrogate endpoints, with inconsistent results attributed to differences in the inclusion/ exclusion criteria, heterogeneity across studies and varied quality of the primary studies. Furthermore, there have been sparse studies reporting on CLBR and there is a continuing debate on what would be considered as a relevant clinical difference in efficacy. In addition to efficacy and safety, the ultimate choice of gonadotrophin should be based on regional availability, patient acceptability and cost-effectiveness for a live birth.