Euploid Embryos Research Articles

O-004 Male and female blastocysts display differences in development such that embryos assigned an identical morphological grade may have differing viability dependent on their sex

Abstract Study question Embryo selection methods based upon morphological or morphokinetic evaluation assume that male and female embryos have identical rates of preimplantation development, but is this true? Summary answer The distribution of trophectoderm grades differs for male and female blastocysts. Furthermore, male and female embryos of identical grade may have different probabilities of viability. What is known already Previous studies have suggested that male and female embryos may have subtle differences in their rates of preimplantation development. However, this possibility remains controversial. Apart from being of scientific interest, the question of whether the sex of an embryo can affect its growth trajectory is of clinical importance. Morphological grading, the primary method used by most IVF clinics when deciding which embryo to prioritise for transfer, assumes that developmental rates are independent of sex. Similarly, morphokinetic strategies for embryo evaluation, using data gathered from time-lapse incubators, are likely to be compromised if male and female embryos have differing developmental behaviour. Study design, size, duration 1,241 blastocysts underwent PGT-A and were shown to be euploid. Chromosome analysis also revealed the sex of the embryos, although this was not disclosed to patients. Standard morphological grading was carried out blindly with respect to the sex of the embryo. Information on clinical outcomes following embryo transfer was available for a subset of 336 embryos. Data was evaluated using various statistical methods to reveal any associations between embryo sex, morphology and clinical outcome. Participants/materials, setting, methods Embryos included in this study were derived from patients undergoing routine IVF with PGT-A. The only embryos excluded were those derived from patients carrying a monogenic disease mutation or a chromosome rearrangement. Embryos underwent PGT-A at the blastocyst stage on either day-5 or day-6, using a highly validated method. The euploid embryos were divided into male and female groups and blastocyst morphological grades were considered with respect to rates of implantation, miscarriage and ongoing pregnancy/birth. Main results and the role of chance The proportion of embryos biopsied on day-5 versus day-6 was identical for males and females (68% day-5 for both). No difference was observed in blastocyst expansion or inner cell mass grading on day-5. However, a highly significant difference was noted in the distribution of trophectoderm grades (A, B, C, D according to the system of Gardner and Schoolcraft, 1999) (P < 0.0001). This was characterised by a disproportionate representation of the highest grade amongst male embryos. 21.6% of male blastocysts had trophectoderm graded ‘A’, compared to 14.9% of females. Interestingly, euploid male embryos with a ‘B’ grade trophectoderm were associated with significantly higher implantation rates than females of the same morphological grade (82.1% vs. 58.7%; P = 0.0002), but the males of this grade also experienced a higher incidence of biochemical losses (16.7% vs. 4.2%; P = 0.017). As a group, male embryos do not have greater viability than female embryos, which implies that the greater proportion of high trophectoderm grades amongst these embryos is not an indicator of superior potential. In turn, this suggests that different criteria should be used for grading trophectoderm in males and females. Limitations, reasons for caution It may be desirable to introduce parallel morphological grading systems, one for male embryos and another for female. However, this would only be applicable in cycles involving PGT-A. The observation that embryos of equal grade can have different outcomes depending on their sex should be confirmed in a prospective study. Wider implications of the findings These results suggest the development of sex-specific morphological grading strategies might provide more reliable insights into embryonic potential, increasing the likelihood of selecting a viable embryo for transfer. Consideration of differences in the development of male and female embryos will also be important when developing morphokinetic algorithms for embryo selection. Trial registration number Not applicable

O-064 Genome-wide analysis of meiotic recombination reveals a subset of oocytes with reduced frequency of recombination and increased risk of aneuploidy.

Abstract Study question Are there differences in recombination rates between male and female embryos, and is the frequency of recombination associated with the risk of aneuploidy? Summary answer There is more recombination in female meiosis than in males. Aneuploidy is associated with fewer recombination events. Genome-wide reduction in recombination predisposes oocytes to aneuploidy. What is known already Aneuploidy is believed to be the primary explanation for embryo implantation failure and miscarriage and is, therefore, of great relevance to IVF. Despite the high frequency and clinical importance of aneuploidy, the mechanisms causing a gamete to become chromosomally abnormal are only partially understood. One factor known to influence the risk of aneuploidy is meiotic recombination. An absence of recombination or atypical positioning of chiasmata predisposes to chromosomal malsegregation. Most data on the subject of meiotic recombination and aneuploidy comes from miscarriages and aneuploid births, but this provides an incomplete view since most aneuploidies are lethal at earlier developmental stages. Study design, size, duration ∼300,000 polymorphisms scattered across the genome were genotyped in DNA from 125 couples and 543 blastocysts that they produced. The inheritance of individual alleles was tracked from parents to embryos, allowing the parental origin of each chromosome to be determined. This strategy also permitted the detection of abnormal numbers of chromosomes, allowed deduction of which meiotic division aneuploidy had arisen in, and revealed the sites of meiotic recombination on each chromosome in the embryos analysed. Participants/materials, setting, methods Embryo analysis involved trophectoderm biopsy, followed by multiple displacement amplification. Parental DNA and amplified embryo samples were then tested using a microarray. 22,133 individual recombination events on 14,561 chromatids were evaluated. The relationship between aneuploidy and recombination frequency was considered. The average distance between recombination events was calculated by dividing the size of each autosome (in Mega bases – Mb) by the number of detected recombination events. An unpaired t-test was conducted for statistical analysis. Main results and the role of chance A quarter of all embryos were found to carry one or more chromosome abnormalities (altogether, 221 chromosome abnormalities were detected). Of these, 82% had a maternal (oocyte) origin, while 18% were paternal. Chromosomes 16 and 22 were the most frequently affected by aneuploidy, together accounting for 33% of all maternal chromosome abnormalities. Analysis of recombination events in maternally and paternally inherited chromosomes confirmed that female meiosis is associated with a significantly higher frequency of recombination (41.4 ±7.7 recombination events compared with 24.4±4 in chromosomes of paternal origin). Chromosomes 21 and 22 showed the lowest number of recombination events, consistent with their small size (average of 0.6 and 0.7 recombination sites per chromosome, respectively). As expected, a complete failure of recombination occurred more frequently for chromosomes with lower numbers of recombination events. Interestingly, embryos with aneuploidies of maternal origin showed evidence that the oocytes that produced them had lower recombination rates in general (not restricted to the affected chromosomes) than those producing euploid embryos (P = 0.01). Across the genome, average distances between recombination events were ∼10% lower for euploid embryos compared with aneuploid embryos (72.4 +/-14 Mb versus 81+-18 Mb) (P = 0.0004). Limitations, reasons for caution It is possible that the pattern and frequency of recombination sites that exist in sperm and oocytes might show some differences from the blastocysts tested during this study, considering that more than half of all oocytes either fail to fertilise or produce embryos that arrest before the blastocyst stage. Wider implications of the findings As expected, a higher aneuploidy rate and more recombination events were seen in female meiosis compared with male meiosis. Interestingly, aneuploidy was associated with lower levels of recombination across the whole genome, not only for the affected chromosomes, suggesting that unusually low recombination activity predisposes some oocytes to chromosome malsegregation. Trial registration number not applicable

P-734 Blastocysts derived from tripronuclear zygote including small pronuclear zygote have high diploid and euploid rate with NGS and SNP analysis.

Abstract Study question What is diploid and euploid frequency of blastocysts derived from tripronuclear zygote (3PN) including a small pronucleus (2.1PN) with NGS and SNP analysis? Summary answer Diploid rate of the blastocysts derived from 3PN including 2.1PN was 91.3%, in which euploid, aneuploid and mosaic embryos were 18.7%, 61.3% and 11.3%, respectively. What is known already A 3PN zygote is thought to be produced when two sperm enter into the same oocyte during in vitro fertilization (IVF) or whenthe second polar body is not extruded on both IVF and intracytoplasmic sperm injection (ICSI). It has been reported that 3PN is observed around 3-10%, and the embryos are usually discarded by the reason for genetic risks for triploid. However, some studies reported that blastocyst derived from 3PN and/or 2.1PN (defined as small PN < 20μm) contained normal diploid and euploid embryos by the preimplantation genetic testing, and that normal healthy babies were born from these embryos. Study design, size, duration We conducted a cross-sectional study between July 2019 and June 2022 in single reproduction center. We included 80 blastocysts in this study which generated from 3PN zygotes including 2.1PN zygotes. Of these, 71 blastocysts were from ICSI and 9 blastocysts were from IVF. These blastocysts were assessed for chromosomal ploidy by next generation sequencing (NGS) and single nucleotide polymorphism (SNP) analysis and embryonic development of these blastocysts were monitored by time-lapse imaging system. Participants/materials, setting, methods Average age of women was 39.2 years (22-47 years). The smallest pronuclear diameter who provided blastocysts were 12.7±2.8μm (8.5-19.7μm) in average. The blastocysts were divided between inner cell mass (ICM) and trophectoderm (TE) using laser system under inverted microscope, followed by NGS and SNP. The t-test was performed to analyze differences between triploid and diploid. Statistical significance was defined as P < 0.05. All participants provided written informed consent, and Institutional Review Board approval was obtained. Main results and the role of chance Diploid rate of the blastocysts derived from 3PN including 2.1PN was 91.3%, in which euploid, aneuploid and mosaic embryos were 18.7%, 61.3% and 11.3%, respectively. Of 80 blastocysts, 64 blastocysts (80%) were able to be divided ICM from TE. In terms of triploid and diploid, the results of ICM and TE were completely concordant, although 15.6% discordancy was observed between ICM and TE. Average ages of the patients with triploid, euploid, aneuploid, and mosaic embryos were 41.1±5.1 years, 36.0±6.3 years, 40.6±3.5 years, and 35.2±3.6 years, respectively. The patients’ age with aneuploid embryos was significantly higher than those with euploid and mosaic embryos (P = 0.015 and P < 0.001, respectively). Mean diameter of the smallest pronucleus of triploids and diploids were 15.2±4.1μm (8.5-19.7μm) and 12.7±2.8μm (8.5-18.9μm), respectively. Of diploid embryos, euploid, aneuploid, and mosaic were comparable in mean diameter of the smallest pronucleus. There were no differences in insemination methods (IVF and ICSI), frequency of abnormal blastomere division, embryonic developmental speed, and blastocyst grade, between triploid and diploid embryos. Limitations, reasons for caution Since this was a cross-sectional study by cytogenetics analysis of blastocysts, causal association between incidence of 3PN (including 2.1PN) and postnatal findings and significance of extra micro pronucleus are unknown. We need to assess longitudinal-study with large sample size of embryo transfer with PGT-A. Wider implications of the findings We revealed that blastocyst derived from 3PN including 2.1PN had high diploid and euploid rate by NGS and SNP analysis. Therefore, 3PN including 2.1PN embryos could be used for embryo transfer after chromosome ploidy analyses. Trial registration number Not applicable

O-120 Predicting Embryo Ploidy Status Using Time-lapse Images

Abstract Study question Can deep learning models using time-lapse images of embryo development be used to predict embryo ploidy and provide supplemental information to embryologists for clinical decision-making? Summary answer We developed a general MDBS-Ploidy model that uses time-lapse images and maternal age to predict embryo quality scores and ploidy status. What is known already Ploidy status, or the presence or lack of chromosomal abnormalities, is an important factor in a successful pregnancy. Embryos with abnormalities are classified as aneuploid, whereas those without are euploid. With the advent of artificial intelligence in computer vision and the collection of large IVF-related datasets combining images, videos, and clinical outcomes, a variety of methods have been developed to automatically assess embryo quality and other characteristics using images from time-lapse sequences. To our knowledge, a video classification model validated by heterogeneous data to predict both embryo quality score and ploidy is lacking. Study design, size, duration The training dataset for the model consisted of 1,998 time-lapse sequences captured by the Embryoscope®. Time-lapse image sequences consisted of around 360–420 frames captured during 5 days of development. PGT-A results were used as ground truth labels for all ploidy prediction tasks, with embryos classified as euploid or aneuploid. The dataset also included clinical information such as blastocyst score (BS) and maternal age at the time of oocyte retrieval. Participants/materials, setting, methods The MDBS-Ploidy consists of two steps. The first step is quality score prediction from day-5 time-lapse video input using a Bidirectional Long Short-Term Memory architecture with added output layers for multitasking. In the second step, the predicted score, in addition to maternal age, is used to predict the ploidy status of the embryo using a logistic regression model. We evaluated the performance of the MDBS-Ploidy using area under the receiver-operating-characteristic (AUROC) on a validation dataset. Main results and the role of chance For the MDBS-Ploidy quality score prediction module, the Pearson correlation between scores predicted by the MDBS-Ploidy and ground truth quality scores is 0.70 on the validation dataset, suggesting moderate correlation strength. As for the aneuploidy prediction module, the MDBS-Ploidy can discriminate between euploidy and aneuploidy with an AUROC of 0.76 ± 0.002. The MDBS-Ploidy performs comparably with a model trained on the embryologist-annotated blastocyst score. We replicate these comparative results in external validation datasets as well, namely the Embryoscope+® dataset with 1,000 embryos and the IVI Valencia Spain dataset with 543 embryos. Moreover, the MDBS-Ploidy is completely automated, requiring only time-lapse images from 96 to 112 hpi and maternal age to predict embryo ploidy status. This allows the MDBS-Ploidy to be adapted clinically without interrupting ongoing workflows. The MDBS-Ploidy also provides a certain level of explainability; embryologists can use the intermediate quality score (from the first module) to determine why an embryo is classified as a certain ploidy status. With a recall of 0.84 ± 0.004 for the validation dataset, the MDBS-Ploidy shows promise for successfully selecting euploid embryos. We believe that the MDBS-Ploidy can provide supplemental information for selecting the most viable embryo. Limitations, reasons for caution Our model primarily uses data from time-lapse microscopy. Clinics without access to this technology will be unable to use our model. Wider implications of the findings This model is clinically relevant for making decisions as to whether an embryo may be chromosomally normal. Automated quality score prediction is instrumental to embryologists who are currently annotating embryos manually. Future iterations of the model can theoretically be adopted into clinical practice because it is end-to-end and fully automated. Trial registration number not applicable

P-614 Endometrial preparation: effect of estrogen levels before the embryo transfer on the live birth rate from 14825 freezing-all cycles

Abstract Study question Is there a difference in live birth rate during discrepant estrogen levels before the embryo transfer following either hormone replacement therapy (HRT) or natural cycle(NC)? Summary answer The higher estrogen level on the day before embryo transfer may reduce the live birth rate in either HRT or NC. What is known already The endometrial preparation of frozen embryo transfer cycle is to synchronize of endometrium and embryo under the condition of exogenous estrogen and progesterone drugs in HRT or follicle monitoring in NC. Estrogen is crucial and different estrogen levels could imply variations on endometrial preparation. Information on the effect of discrepant estrogen levels before the embryo transfer on reproductive outcomes of women undergoing freezing-all cycles is scarce. Study design, size, duration Retrospective cohort study including 14825 first frozen embryo transfers following IVF/ICSI undergoing freezing-all cycles from January 2016 to December 2020. A total of 12305 (83.0%) patients received NC while 2520 (17.0%) received HRT. Participants/materials, setting, methods Effect of estrogen levels on live birth rates, stratifified by deciles and ROC to determine the cutoff values for estrogen level on the day before embryo transfer.Multivariable logistic regression analysis was performed to adjust for confounders. Main results and the role of chance The live birth rate was 44.7% and 52.4% in HRT and NC respectively. The live birth rate of HRT began to decline when the estrogen level was P60 in the deciles which the cut-off values of ROC was closed with the estrogen level was 891.6 pg/ml. The lower level of estrogen on the day before embryo transfer in HRT corresponds to a higher live birth rate（51.4% vs 32.9%）.The cut-off values of ROC in NC was statistically different with the estrogen level was 194.4 pg/ml. The lower level of estrogen on the day before embryo transfer in NC corresponds to a higher live rate（53.0% vs 48.6%）.After adjusting the possible confounding factors by multivariate logistic regression, it was found that the estrogen level on the day before embryo transfer in HRT and NC was the independent influencing factor of live birth rate with the OR of 0.57 (0.47,0.68) and 0.82 (0.74,0.90), respectively. Limitations, reasons for caution The greatest limitation of this study is its retrospective study. On the other hand, this study was performed using non-PGT cycle, although this is unlikely to affect the results,we cannot exclude the possibility that euploid embryo responds differently to endometrial state in comparison to aneuploid embryo. Wider implications of the findings In freezing-all cycles, the higher estrogen level on the day before embryo transfer may reduce the live birth rate in either HRT or NC. Clinicians should pay attention to the control of estrogen level in luteal phase. Trial registration number Nona

P-330 Is 17 alpha hydroxyprogesterone caproate an adequate alternative to improve reproductive outcomes in artificially prepared frozen embryo transfer cycles (FET)?

Abstract Study question Is 17 alpha-hydroxyprogesterone caproate an adequate option to improve reproductive outcomes in patients with low serum progesterone (P4) level the day of frozen embryo transfer? Summary answer Patients with serum P4<10ng/ml may benefit with the addition of intramuscular 17alpha-hydroxyprogesterone caproate injections, and have similar ongoing pregnancy rates than patients with serum P4≥10ng/ml. What is known already Indications of embryo cryopreservation have expanded in the last years. Recently published papers highlight that low serum progesterone levels before frozen embryo transfers decrease pregnancy and live birth rates and increase miscarriage rates, even in cases of euploid embryos transfers. Other studies have published a beneficial effect with the addition of daily subcutaneous progesterone injections in patients with low serum progesterone levels around the time of embryo transfers. In Argentina subcutaneous progesterone has been available since October 2022, not before. 17alpha-hydroxyprogesterone caproate is a synthetic progestin used for luteal phase support and approved for the prevention of preterm birth. Study design, size, duration This prospective cohort study was performed between May 2021 and August 2022 in 114 patients undergoing 138 FET cycles after an artificial endometrial preparation with estradiol valerate 4mg daily and micronized vaginal progesterone 200mg, three times daily. Participants/materials, setting, methods Patients <50 years old with triple-layer endometrium ≥7mm underwent frozen embryo transfer. Progesterone was measured immediately before the embryo transfer. Group 1 (N = 47): P4>10ng/ml; Group 2 (N = 46): P4<10ng/ml with addition of 17 alpha-hydroxyprogesterone caproate after embryo transfer; Group 3 (N = 46): P4<10ng/ml without addition of 17 alpha-hydroxyprogesterone. Primary endpoint was to compare ongoing pregnancy rate beyond week 12 between the three groups. Secondary endpoints were pregnancy rate and miscarriage rate. Main results and the role of chance Parameters were comparable between the groups in terms of age, body mass index, estradiol, P4 levels at the beginning of endometrial preparation and number of embryos transferred. Ongoing pregnancy rate was: Group 1 = 38.3% (18/47); Group 2 = 39.1% (18/46); Group 3 = 28.3% (13/46) (p = 0,337). Pregnancy rate was: Group 1 = 48,9% (23/47); Group 2 = 50% (23/46); Group 3 = 50% (23/46) (p>0,05). Miscarriage rate was: Group 1 = 21.73% (5/23), Group 2 = 26.1% (6/23) and Group 3 = 43.47% (10/23) (p = 0,208). Although there were no significant differences between the groups, both, Group 1 and Group 2, had higher ongoing pregnancy rates and lower miscarriage rates than group 3. This is probably due to the limited sample size. Limitations, reasons for caution The main limitation of our study is the sample size and the fact that most of the embryos were not genetically evaluated before being transferred, which would eliminate a variable that could interfere with the results obtained. Only micronized progesterone and 17alpha-hydroxyprogesterone caproate were studied. Wider implications of the findings The addition of exogenous 17alpha-hydroxyprogesterone caproate on the embryo transfer day may improve ongoing pregnancy rates in cases of low serum progesterone levels, being a simple and safe strategy to individualize luteal phase support when no other form of progesterone administration is available. Trial registration number NOT APPLICABLE

P-713 Preimplantation DNA methylation screening (PIMS) illustrates the outcome of assisted reproductive technology

Abstract Study question It is unknown whether using DNA methylation can improve the outcome of ART and can exclude the imprinted gene diseases. Summary answer Our study provides a criterion on selecting blastocyst according to DNA methylome, which can improve live birth rate and decrease birth defect rate. What is known already How to select the embryos is one key factor that can determine the successful rate of ART. As aneuploidy exists in 20-30% of blastocysts, preimplantation genetic screening (PGS) has been widely used, which increases the live birth rate. Unfortunately, the live birth rate remains around 50% even with the help of PGS. DNA methylation is known to play important function during embryogenesis. Previous study has shown that a large proportion of human embryos have abnormal DNA methylome. However, whether DNA methylation pattern can affect the clinical outcome of ART has not been investigated in clinics. Study design, size, duration To determine whether PIMS method can increase live birth rate during ART, the whole genome DNA methylation of 3-5 biopsied cells from trophectoderm were measured. The methylation level and chromosome copy number variation (CNV) were analyzed by using the methylome data. elective single embryo transfer (eSET) will be performed for embryos with euploid chromosome. The live birth rate, pregnant rate and abortion rate will be examined. 182 families including 800 blastocysts were enrolled in PIMT. Participants/materials, setting, methods Eligible couples include advanced maternal age, recurrent pregnancy loss with unknown reason, recurrent implantation failure with unknown reason, severe oligoteratozoospermia. Eligible couples must have two or more good-quality blastocysts. We call a blastocyst as good quality if the blastocyst is 4BC or better according to the Gardner morphologic criteria on day 5-6 of embryo culture. Main results and the role of chance No one has ever performed clinical trial to investigate the impact of DNA methylation of blastocyst on the outcome of artificial assisted reproductive (ART). Here, we performed PIMS using the biopsied trophectoderm of 800 blastocysts. Chromosome copy number variation and DNA methylation pattern were analyzed by using methylome data. 162 of euploid embryos were transferred. Our data show that significant variation of DNA methylation level of blastocysts can be observed in both younger women (£37 years old) and older women (>37 years). Blastocysts with global methylation level around 0.26 have the highest birth and pregnancy rate, but the lowest abortion rate. The higher the difference methylation value from 0.26 is, the lower the birth rate becomes. Furthermore, by using artificial intelligence method according to different methylated regions between the embryos succeeding in live-birth and those failing in live-birth, we can robustly predict the clinical outcome of blastocysts. PIMS method can find the abnormality of DNA methylation states in the imprinted control regions which can cause the imprinted diseases. Our study provides a criterion on selecting blastocyst according to DNA methylome during ART, which can potentially improve the live birth rate and decrease the birth defect rate. Limitations, reasons for caution Because of the low coverage of single cell DNA methylome, PIMS method cannot cover all imprinted control regions. Wider implications of the findings PIMS method can potentially find more human diseases which is caused by DNA methylation mutation. Trial registration number NCT03642574

O-260 The influence of the vaginal microbiome on clinical outcomes in patients undergoing a frozen embryo transfer: a prospective pilot study

Abstract Study question Are Lactobacillus-dominant vaginal microbial compositions advantageous for clinical outcomes and is the specific Lactobacillus species relevant? Summary answer Favourable clinical outcomes are increased in patients with a Lactobacillus-dominant vaginal microbiome, particularly in those patients with microbial communities dominated by L.crispatus and L.jensenii. What is known already The clinical relevance of the urogenital microbiome is emerging as a topic in female reproductive health. Recent evidence suggests that the composition of the vaginal microbiome may have an impact on clinical outcomes in patients undergoing assisted reproductive treatment. A Lactobacillus-dominant microbiome (LDM), typically composed of L.crispatus, L.gasseri, L.iners or L.jensenii, is considered to be associated with favourable outcomes. In contrast, a non-Lactobacillus-dominated microbiome (NLDM), consisting predominantly of anaerobe species, may be linked to poor outcomes. The evidence surrounding this relationship is controversial and the importance of relative Lactobacillus abundance in the vagina in relation to clinical outcomes remains unclear. Study design, size, duration A single-centre prospective pilot study included 81 patients (mean age: 38.2) undergoing frozen embryo transfer using their own (n = 52) or donated (n = 29) oocytes. A vaginal swab was taken on the embryo transfer day and the microbiome analysed. Implantation, ongoing pregnancy and early miscarriage rates were compared between different microbiome compositions. To minimise the confounding effect of aneuploidy on clinical outcomes, a patient sub-group (n = 54) receiving donor oocytes or a euploid embryo transfer was also considered. Participants/materials, setting, methods Quantitative PCR was utilised to determine the relative abundance of the four predominant Lactobacillus species as well as 15 species associated with vaginal dysbiosis. Microbiome compositions were grouped according to the relative Lactobacillus abundance and patients divided into an LDM (>80%) or NLDM (<80%) group. Patients were further sub-divided into community state types (CSTs) according to the dominant species present in their sample (CST-I: L.crispatus, CST-II: L.gasseri, CST-III: L.iners, CST-V: L.jensenii and CST-IV: dysbiotic species). Main results and the role of chance Relative abundance of Lactobacillus was significantly higher in samples from patients achieving an ongoing pregnancy (80.7% vs. 61.7%; p = 0.05). Implantation rates were comparable between LDM and NLDM patients (66.0% and 64.3%), but the ongoing pregnancy rate showed an apparent increase in LDM patients (58.5% vs. 39.3%; p = 0.11), concomitant with a significant decrease in the miscarriage rate (11.4% vs. 38.9%; p = 0.03). Limiting the analysis to patients at low risk of aneuploidy (young donor and PGT-A cycles), Lactobacillus abundance appeared higher in patients with ongoing pregnancies (74.8% vs. 58.0%; p = 0.34). Likewise, LDM patients showed higher ongoing pregnancy (61.3% vs. 43.5%; p = 0.27) and lower miscarriage (5.0% vs. 33.3%; p = 0.06) rates, although significance was not achieved in this restricted sample. Comparison of CST groups indicated better outcomes in CST-I and CST-V compared to other groups. The ongoing pregnancy rate was significantly higher (65.7% vs. 41.3%; p = 0.04), alongside a lower miscarriage rate (8.0% vs. 32.1%; p = 0.04). When only considering patients with low aneuploidy risk, similar results were obtained (implantation rate: 78.9% vs. 57.1%, p = 0.14; ongoing pregnancy rate: 73.7% vs. 42.9%, p = 0.04; miscarriage rate: 6.7% vs. 25.0%, p = 0.21). Of note, there were no differences in baseline characteristics (age, ethnicity & BMI) or embryo morphologies between groups. Limitations, reasons for caution All patients were from a single fertility clinic and the study population was predominantly composed of white women. Consequently, results may not be applicable to women of different ethnicities. Patients with a low risk of aneuploid embryo transfer represented a small subgroup where meaningful statistical analysis was not always possible. Wider implications of the findings This study identifies a correlation between Lactobacillus species colonisation in the vagina and successful clinical outcomes, suggesting that the vaginal microbiome modulates the chances of IVF success. The results are promising, providing motivation for further, larger studies, involving more diverse populations in order to draw definitive conclusions. Trial registration number not applicable

O-065 A multi-center randomized controlled trial of intraovarian injection of platelet rich plasma for women with poor ovarian response

Abstract Study question Does intraovarian injection of autologous platelet-rich plasma (PRP) improve oocyte yield in young patients with poor ovarian response (POR)? Summary answer Intra-ovarian PRP injection did not result in increased number of oocytes retrieved or improvement of any other relevant measured outcomes. What is known already POR is one of the major hurdles to overcome in patients undergoing controlled ovarian hyperstimulation (COH) for in vitro fertilization (IVF). Several methods have recently emerged to attempt follicular reactivation in these patients with a poor prognosis. One of them, the intra-ovarian injection of autologous PRP, demonstrated promising results in retrospective and prospective cohort studies. Study design, size, duration This was a multi-center, randomized controlled trial (RCT) to evaluate the efficacy of PRP in improving IVF outcomes in women with POR. Patients who met inclusion criteria (<38 years, two or more prior cycles with < 3 oocytes retrieved, and without single gene disorders, prior ovarian surgery, endometriomas, BMI >35, or severe male factor infertility) were randomized to either receive an autologous intra-ovarian PRP injection (PRP group) or no intervention (control group) prior to COH. Participants/materials, setting, methods A total of 83 patients were randomized to PRP group (n = 41) or control group (n = 42). After treatment, patients underwent COH, oocyte retrieval, intracytoplasmic sperm injection (ICSI), preimplantation genetic testing for aneuploidy (PGT-A), and single frozen euploid embryo transfer. Number of MII oocytes obtained was the primary outcome. Secondary outcomes included ovarian reserve tests (antral follicle count [AFC] and anti-Mullerian hormone [AMH]), blastocyst and euploid blastocyst yields, and sustained implantation. Main results and the role of chance No significant differences were observed in number of MII oocytes retrieved (3.1 ± 3.3 vs 2.8 ± 2.4 in PRP vs control, respectively; p = 0.9), blastocysts (1.3 ± 2.1 vs 1.0 ± 1.3, p = 0.8), or euploid blastocysts (0.9 ± 1.6 vs 0.8 ± 1.1; p = 0.5) per cycle. Similarly, no differences were observed in the likelihood of obtaining at least one euploid blastocyst (37 vs 45%, p = 0.4; relative risk [RR], 95% confidence interval [CI] 0.9, 0.6-1.2) or the rate of sustained implantation (29 vs 31%, p = 0.9; RR 1.0, 0.7-1.3). Pre- and post-treatment AFC in both groups (PRP: from 5.2 ± 3.2 to 7.9 ± 4.5, p < 0.0001; control: from 5.6 ± 3.3 to 6.8 ± 4.8, p = 0.02) were statistically significantly different, whereas AMH was different in the PRP group only (PRP: 0.73 ± 0.46 to 0.99 ± 0.98, p = 0.02; control: 0.70 ± 0.59 to 0.73 ± 0.57, p = 0.59). Limitations, reasons for caution Oocyte retrieval took place the cycle immediately following treatment; therefore, we could not evaluate long-term effects of PRP on ovarian reserve or response. Lack of a placebo group would limit interpretability had a difference between groups been observed. The effect of PRP on patients >38 years old was not studied. Wider implications of the findings Intra-ovarian PRP injection did not improve reproductive outcomes by any relevant measure. Our findings do not support wide utilization of PRP to improve IVF outcomes in women with POR. Trial registration number NCT04163640

O-145 From mathematical modeling to real-life clinical data: live birth rates up until the tenth blastocyst transfer without PGT-A

Abstract Study question What is the cumulative live birth rate (CLBR) from the first until the tenth consecutive blastocyst transfer, across all age-groups, if no PGT-A is applied? Summary answer Our clinical data confirm existing mathematical models: without PGT-A, a CLBR of 70.0% after three, 88.9% after seven and 95.9% after ten transfers was observed. What is known already The rate of ‘true’ repeated implantation failure (RIF) from endometrial origin is an important topic of debate in our scientific community. Its existence was challenged with the reporting of CLBRs >90% after three consecutive euploid blastocyst transfers. These findings are not directly applicable when PGT-A is not routinely performed. Mathematical models have been developed to tackle this issue by taking into account (1) the anticipated euploidy rate based on the oocyte’s age and (2) the probability of an euploid embryo to implant. So far, no actual patient data have confirmed the validity of this model. Study design, size, duration This is a single-center, retrospective, observational cohort study including 4320 unique women who underwent up to ten consecutive blastocyst transfers between 2010 and 2020. All age categories were included, as well as fresh/frozen and single/double embryo transfers. Patients using donor oocytes, PGT or cleavage stage embryo transfers were excluded or led to drop-out in the study. Participants/materials, setting, methods Patient characteristics were retrieved and cumulative outcomes were analyzed. A Kaplan-Meier curve was plotted for CLBR. For each transfer cycle the live birth rate (LBR), double embryo transfer rate (DET) and multiple pregnancy rate were calculated. Main results and the role of chance The mean age of the patients included in the study was 31.8 (±4.5) years at the time of the first ovarian stimulation. The mean body mass index was 23.7 (± 4.3) kg/m2 and mean basal FSH 6.8 (±2.3) IU/L. The mean number of stimulations per patient was 1.21 (±0.5) and a total mean number of 4.6 (±3.2) blastocysts were obtained per patient. The mean time to the pregnancy leading to live birth was less than one year (0.3±0.7). We observed the highest LBR (46.3%, 2002/4320) after the first blastocyst transfer followed by 35.4% (593/1672), 32.3% (275/851), 32.2% (147/456), 32.8% (78/238), 21.3% (26/122), 23.9% (17/71), 25.6% (10/39), 31.8% (7/22), 18.2% (2/11) for the second until the 10th transfer, respectively. The Kaplan-Meier curve showed CLBRs to mount from 70.0% after the third, up to 88.9% after the seventh and 95.9% after the tenth blastocyst transfer. We noticed a steady increase in DET rate from only 6.4% in the first up to > 27% as of the fourth and >44% as of the seventh transfer with acccompanied multiple pregnancy rates of 1.3% over 10.9% and 11.8%, respectively. Limitations, reasons for caution The main limitation is the study’s retrospective nature. As our center performs cleavage stage and blastocyst transfer, the included population is a good prognosis one as embryology allowed to perform extended culture. Nevertheless, to investigate RIF due to endometrial causes, this could also be seen as an asset. Wider implications of the findings These patient data confirm mathematical models and further question the prevalence of insurmountable, endometrial origin RIF. Preserved LBRs (even in higher cycle ranks) and a CLBR >95% provide hope and reassurance to couples with failed embryo transfers and encourage them to continue treatment if blastocysts are available. Trial registration number NA

O-050 The impact of embryo biopsy on obstetric and neonatal outcomes

Abstract Background Preimplantation genetic testing of embryos (PGT) was performed first in 1990 to prevent the transmission of X-linked disease. Since then, the technique has improved to include couples who are both carriers of the same autosomal recessive disorder, to those with balanced translocations having recurrent pregnancy loss, and to women with advanced maternal age who are at higher risk for having aneuploid embryos. Originally, PGT involved biopsy of the polar body or of one blastomere of a cleavage stage embryo for diagnosis. The contemporary practice of PGT involves the culture of embryos to the blastocyst stage (5-7 days after fertilization), with biopsy of several trophectoderm cells that would become the placenta, transport of biopsy tissue off-site for genetic analysis, and cryopreservation of blastocysts until biopsy results are available. A single euploid embryo is then transferred into the uterus as a frozen embryo transfer. Objective We sought to determine if removal of these cells for preimplantation genetic testing was associated with adverse obstetrical or neonatal outcomes after frozen-thawed single embryo transfer compared to frozen-thawed single embryo transfer without biopsy. Further, we review other studies of contemporary practice of PGT to draw conclusions about the safety of the practice. Study design We linked assisted reproductive technology surveillance data from the Society for Assisted Reproductive Technology Clinic Outcome Reporting System to birth certificates and maternal and neonatal hospitalization discharge diagnoses in Massachusetts, USA, from 2014-2017, considering singleton births after frozen-thawed single embryo transfer. We compared outcomes of cycles having embryo biopsy (n = 585) to those having no biopsy (n = 2,191), adjusting for mother’s age, race, education, parity, body mass index, birth year, insurance, and all infertility diagnoses (Sites CK et al, Am J Obstet Gynecol 2021;225:285.e1-7). Results With no biopsy used as the reference, we found no differences between no biopsy and biopsy groups with respect to preeclampsia, pregnancy-induced hypertension, placental disorders (placental abruption, placenta previa, placenta accreta, placenta increta, and placenta percreta), preterm birth, low birthweight, cesarean delivery, gestational diabetes mellitus, or maternal or infant length of stay after delivery. Comparing our outcomes to 4 other studies of contemporary PGT practice of frozen-thawed embryo transfers (Zhang WY Fertil Steril 2019, Li M Am J Obstet Gynecol 2020, Makhijani R Hum Reprod 2021, He H Fertil Steril 2019), our data are consistent with no effect of biopsy on low birth weight, pregnancy-induced hypertension, gestational diabetes, placenta previa, and placenta accreta. A slight decrease in preterm delivery (RR 1.10, CI 1.02-1.18), slight reduction in cesarean section (RR 0.90, CI 0.82-0.99), and increase in intrauterine growth restriction (RR 1.21, CI 1.06-1.38) are reported with biopsy in a systematic review (Hou W, Fertil Steril 2021). The increase in risk for intrauterine growth restriction with biopsy was influenced by one study (Li M Am J Obstet Gynecol 2020). Conclusion Embryo biopsy of trophectoderm cells for PGT in contemporary practice appears to be safe generally with respect to maternal and neonatal outcomes. There is no clear increase in diagnoses related to placentation (preeclampsia, pregnancy-induced hypertension, placental disorders, preterm birth, low birthweight), cesarean delivery, gestational diabetes mellitus, or maternal or infant length of stay after delivery. Further study of a possible increase in intrauterine growth restriction with biopsy is indicated.

O-299 Sperm DNA fragmentation by TUNEL influences live birth in egg recipients after euploid embryo transfer

Abstract Study question Does sperm DNA fragmentation (SDF) by TUNEL affect reproductive success measured as live birth rate (LBR) in egg recipients after euploid embryo transfer? Summary answer SDF increases miscarriage rates in egg recipients with PGT-A. Therefore, live birth rates depends on sperm DNA damage in egg recipients after euploid embryo transfer. What is known already Sperm DNA integrity is important for optimal fertilisation, implantation and pregnancy. Although controversial, several studies have shown that sperm DNA fragmentation correlates with adverse pregnancy outcomes in infertile men. Ooplasmic sperm DNA fragmentation repair mechanisms may occur in female gametes, mainly in young women. However, research on the impact of DNA fragmentation on LBR are limited. Also, chromosomally normal embryos analysed by PGT-A offers opportunity to isolate female factor and study male factor. The aim of this study is to assess whether high level of semen DNA damage has an impact on reproductive outcomes in terms of live birth rate. Study design, size, duration This is a retrospective study of 467 PGT-A cycles in egg recipients. The study population consists of infertile couples for euploid embryo transfer (February 2017-November 2022). Semen samples were obtained to measure DNA damage from 447 men. PGT-A was performed in 1831 blastocysts. Trophectoderm biopsies (d5/d6 blastocysts) were analysed by NGS (Veriseq Illumina). Embryos were frozen and 568 blastocysts transferred in the subsequent cycle. Conventional hormone replacement was used for endometrial preparation in recipient patients. Participants/materials, setting, methods Cohort study with 447 infertile couples attending to private fertility clinic undergoing ART with their autologous semen, donated oocytes and PGT-A. SDF was measured by Terminal-deoxyribonucleotidyl-ARTSferase-mediated dUTP Nodule End Labelling (TUNEL) assay using the FITC-labelled-dUTP in situ cell death detection kit (Roche). The cohort was divided into two groups according the DNA fragmentation index (DFI): high SDF group (DFI>20%) and normal SDF group (DFI≤20%). To evaluate reproductive outcomes Chi-square and Linear regression tests were used. Main results and the role of chance Of the 447 couples included (average paternal age 41.29 + 7.32 years), 66.5% of male partners had normal semen parameters. We reported high sperm DNA fragmentation in 8% of patients. Overall, the embryo aneuploidy rate was 31.7%. For the further statistical analysis confounding factors such as, female and male age, embryo quality, day of embryo biopsy were included. PGT-A analysis showed an aneuploidy rate of 32.1% in the high DNA fragmentation group and 30.3% in the normal DNA fragmentation group (p = 0.453). Regarding clinical data, the overall implantation rate was 34.9%. There was no significant difference between elevate DNA fragmentation and normal DNA fragmentation with respect to biochemical miscarriage rate (4.3% vs 7.6%, p = 0.799). Although not significant differences between groups, a lower implantation rate near to reach significance was found in the high SDF group (20.0% vs 34.4%, p = 0.050). Pregnancy and clinical pregnancy rates were significantly lower in the high DNA fragmentation group (26.1% vs 44.9%, p = 0.042; 21.7% vs 31.3%, p = 0.044;), respectively. Also, miscarriage rate was significantly higher in the high SDF group (40% vs 12.5%, p = 0.014). Therefore, the live birth rate was significantly lower in the high SDF group (13.0% vs 32.6%, p = 0.024). Limitations, reasons for caution Larger studies including a higher number of samples are needed to confirm the correlation observed between LBR in eggs recipients with semen DNA fragmentation. Those patients with a DFI higher than control values should be treated with antioxidants. The pregnancy outcomes should be analyzed after treatment of DNA fragmentation. Wider implications of the findings Our findings suggest that autologous semen DNA fragmentation influence in live birth rate in egg recipients. Our data show that live birth rates decreased in patients with a high DFI after PGT-A. This study confirms published studies in the literature showing that ART outcomes are affected by sperm DNA fragmentation. Trial registration number Not Applicable

P-194 Initial experience of using iDAScore as a tool to predict euploid blastocysts

Abstract Study question Can the annotationfree embryo scoring system (iDAScore) predict the likelihood of euploidy in embryos? Summary answer Our results have shown that there is a significant positive correlation between iDAScore and blastocyst euploidy. What is known already iDAScore (Vitrolife, Sweden) is an embryo ranking model that was developed by using artificial intelligence (AI) and deep learning. It does not require any user-dependent annotation which largely eliminates the subjectivity of manual annotation by embryologists. It has also been reported to be a robust predictor in likelihood of embryo implantation. However, the correlation between iDAScore and euploidy has not been extensively studied. Therefore, this study is to determine if iDAScore can be utilized as a predictor for embryo euploidy. Study design, size, duration A total of 860 blastocysts (mean age: 30.0; age range: 19.0-35.0) were assessed with iDAScore on day-5 and/or day-6 of embryo development from January 2022 to October 2022 at Alpha IVF & Women’s Specialists. The euploidy rates were analyzed based on the stratified iDAScores (Group A:0.0-4.9; Group B: 5.0-7.4; Group C:7.5-8.9; Group D:9.0-10.0). Participants/materials, setting, methods Scores were computed by the iDAScore software at the Vitrolife Technology Hub (Vitrolife, Gothenburg, Sweden) prior to trophectoderm biopsy for Preimplantation Genetic Testing for Aneuploidies (PGT-A) (Ion Torrent, USA). The ploidy status (euploid or aneuploid) based on PGT-A results were recorded and assessed with the corresponding iDAScore. Main results and the role of chance The euploidy rates of Group A, B, C and D were 56.7% (17/30), 60.0% (105/175), 65.9% (178/270) and 74.8% (288/385) respectively. Group D shows significantly higher euploidy rate compared to Group B and Group C (p = 0.0005, p = 0.0143). A weak significance was observed between Group D and Group A (p = 0.0505). This is probably due to smaller sample size in Group A. No significant differences were displayed among Group A, Group B and Group C (p = > 0.005). Our results demonstrated an increasing trend between iDAScore and the proportion of euploid embryos. Limitations, reasons for caution Few of the blastocysts in Group A could be selected for biopsy so the sample size was only a fraction of the other groups. Further studies and larger sample sizes would allow the findings to be stated more confidently. Wider implications of the findings Although the iDAScore algorithm scores the chance of embryo implantation, we have demonstrated its potential to assist in predicting the embryo ploidy status in a non-invasive manner. This may be used to inform the decision of which and how many blastocysts to biopsy and transfer, thus reducing treatment costs. Trial registration number not applicable

P-160 Trophectoderm biopsy techniques with and without laser use and their contribution to PGT-A results in sibling embryos

Abstract Study question Do the methods used in trophectoderm biopsy and the use of laser affect the genetic results of the harvested cells and the embryos thus examined? Summary answer The different methods of trophectoderm biopsy showed no differences in terms of euploidy rate and the specific type of chromosomal abnormalities. What is known already Next generation sequencing (NGS) is a comprehensive method for the genetic screening of preimplantation embryos and is now favored by many clinics. The method can detect structural abnormalities on all chromosomes and offers better reproductive success compared to earlier tests. However, as the test is very sensitive, biopsy of the embryo and treatment of the biopsied embryos and harvested cells are important to improve diagnostic success. Contamination of the biopsied sample can affect the results of the genetic test and lead to misdiagnosis. Study design, size, duration A prospective, randomized controlled trial of 44 couples undergoing infertility treatment with pre-implantation genetic screening (PGT-A, NGS) at the British Cyprus IVF Hospital between October 2021 and December 2022. Participants/materials, setting, methods PGT-A cases with more than two blastocysts were included in the study. Randomization was performed on days 5 and 6 of embryo development and embryos were divided into two groups before the biopsy. In group 1, embryos were biopsied using the flicking method without laser assistance. In group 2 embryos, cells were removed by pulling the desired cells with 3 to 5 laser shots at the edge of the selected cell group. Main results and the role of chance PGT-A results of 288 blastocyst-stage embryos from 44 cases were analyzed. 136 embryos were in group 1 (flicking) and 152 to group 2 (laser). The embryos in the groups were siblings, so the basic patient characteristics, number of eggs retrieved, sperm characteristics, and early embryonic details were identical between the groups. The mean number of retrieved cells was 6.19 ± 0.915 and 6.14 ± 0.849 (p = 0.764), the number of euploid embryos was 49/122 (40.2%) and 57/137 (41.6%) (p = 0.814), the number of embryos with whole chromosome aneuploidy was 46/73 (63%) and 58/80 (72.5%) (p = 0.209) and embryos with complex aneuploidy (more than two abnormal chromosomes) were 30/73 (41.1%) and 40/80 (50%) (p = 0.268) in group 1 and group 2, respectively. Limitations, reasons for caution As this is a preliminary research result, the small sample size is the major limitation. Mosaicism, which is common in preimplantation embryos and has a significant impact on the results, was not investigated. Wider implications of the findings Although the present data showed no differences between the two biopsy procedures in terms of PGT-A test results, invasive embryo biopsy procedures can damage embryos or misdirect genetic test results. Therefore, validation and universalization of the method for embryo biopsy are needed, which is emerging in recent publications. Trial registration number not applicable

P-222 Early blastulation on day 4 predicts ongoing pregnancy in fresh transfer cycles

Abstract Study question Do embryos with early blastulation (EB) on D4 show higher implantation potential compared to top D5 blastocysts in fresh elective single embryo transfer (eSET) cycles? Summary answer Fresh EB transfer with time start to blastulation (tSB)<96.6h and time of expanded blastocyst (tEB)<100h post-insemination is predictive of ongoing pregnancy for patients <35. What is known already Expansion and hatching of blastocysts are correlated to implantation potential in fresh single transfers. Thanks to time-lapse morphokinetics, several time-points can be analyzed during embryonic growth. Embryos with faster growing speed are correlated to euploid status. The tSB of < 96.6h, day 4 post-insemination, is reported to have significant predictive value of euploid embryos, which are found to be associated not only with higher expansion scores, but also with shorter tSB, full expansion, and hatching. A study reported that significantly more embryos that reached the tSB by 100h implanted compared to those that did not. Study design, size, duration Prospective observational study in a single private hospital during 2022. In 160 cycles (n = 81 cIVF and n = 79 ICSI) an EB on D4 was observed. 48 patients (n = 28 ICSI and n = 20 cIVF) underwent a fresh eSET with an embryo showing EB on day 4. Participants/materials, setting, methods Patients with EB on D4 (tSB<96.6h, tEB<100h post-insemination) were enrolled regardless of indication, female age, attempt’s rank or technique. Those with a fresh eSET on D4 were included in this study. Gardner’s classification was used to rate the blastocysts in Embryoscope + (Vitrolife). The outcome data of transfer for D4 blastocysts (B3/B4 AA-AB-BA) were compared to eSET with top D5 (B4/B5 AA) of reference group (female age ≤35). The statistical analyses were performed by SPSS software. Main results and the role of chance In the study group, all the transferred embryos were graded according to Gardner’s classification as B3AA, or B3AB, or B3BA, or B4AA, or B4AB, or B4BA. In the reference group, we chose only the fresh transfer cycles with ultra-top grade D5 embryos classified as B4AA or B5AA. The results obtained in the overall population (n = 48) were compared to those obtained in the reference group (n = 92): age 34.0 ± 3.8 vs 31.8 ± 2.3, oocyte maturity (OM) 79.8% vs 80.3%, fertilization rate (FR) 80.0% vs 77.0%, overall blastulation rate (OBR) 78.1% vs 74.3%, top blastulation rate (TBR) 59.1% vs 54.4%, total pregnancy rate (PR) 75.5% vs 61.4%, implantation rate (IR) 58.3 vs 51.2% and ongoing pregnancy rate (OPR) 58.6% vs 47.1%, and showed a statistical difference only in PR (p<0.05). The results obtained in younger patients with age 32.1 ± 3.2 (n = 34) were also compared to reference group: OM 75.1% vs 80.3%, FR 85.0% vs 77.0%, OBR 78.1% vs 74.3%; TBR 58.7% vs 54.4% were comparable between the groups. A statistical difference was observed for PR (88.0% vs 61.4%, p<0.01) IR (70.6% vs. 51.2%, p<0.05) and OPR (70.6% vs 47.1%, p<0.05). Limitations, reasons for caution The small size of the sample needs to be expanded in order to confirm the encouraging findings in a larger population, especially in older patients yielding more aneuploid embryos. The study will continue during 2023 and will compel the live birth rate. Wider implications of the findings When a D4 embryo with EB is available, the best option is the fresh transfer on D4. The increased implantation with EB seems to correlate with embryo euploidy and the advanced endometrial receptivity in stimulated cycles. Higher IR and OPR are reported even in patients with lower rate of aneuploidy. Trial registration number not applicable

P-237 In vitro time to maturation of Metaphase I oocytes: a time-lapse study correlating blastocyst formation and ploidy status

Abstract Study question What is the impact of metaphase I (MI) time to mature period (TTM) regarding blastocyst development and ploidy status? Summary answer A short period of TTM (0.1 to 1.0) is related to an increase number of blastocyst formation when compared to longer TTM (1.1 to 3.0). What is known already To achieve meiotic competence, coordinated nuclear and cytoplasmic changes must occur in the oocyte to support fertilization and DNA replication and to ensure correct ploidy of the zygote and embryonic genome activation. Concomitant with chromosome condensation and migration, oocyte maturation goes by cytoplasm reorganization throughout the transitions of MI, polar body extrusion and MII. Despite the optimization of COH protocols, nearly 20% of the retrieved oocytes remain immature at the GV or MI stages. In vitro maturation of MI could increase the number of available embryos; however, longer culture time may affect oocyte quality, resulting in abnormal fertilization and cleavage. Study design, size, duration Prospective cohort study from 244 patients (n = 277 cycles) that had at least 1 MI oocyte after oocyte retrieval (OPU) following controlled ovarian hyperstimulation (COH) for in vitro fertilization (IVF) treatment according to medical referral in an single private ART center from January/2019 to November/2022. All MI oocytes were placed in a time-lapse incubator (TL, EmbryoScope, Vitrolife, Sweden) to check the exact time of first polar body extrusion (TTM to MII) before intracytoplasmic sperm injection (ICSI). Participants/materials, setting, methods Three hours after OPU, oocytes were denuded and assessed for maturation. Six-hundred and seven MI oocytes were followed in TL and those that reached the MII stage in a maximum of 6 hours post-OPU, were injected by ICSI (at 6h post-OPU) and stratified according to the TTM (0.1-1.0 hours and 1.1-3.0 hours). Fertilization rate, blastocyst formation, and ploidy were assessed. Kruskal-Wallis, chi-squared and Fisher tests were applied for statistical analysis. p < 0,05 was considered significant. Main results and the role of chance Patient mean age (n = 244) was 38,50 ± 3,40 years. From those, 25 patients (10.3%) had 2 cycles and 4 patients (1.6%) had 3 cycles with at least 1 MI oocyte. Age, anti-Müllerian hormone (AMH), #OPU, #MII, %MII (MII/OPU), #MI and MI% (MI/OPU) mean were not statistically different in patients with 1, 2 or 3 cycles in our cohort. One hundred forty-nine patients (61%) in 165 cycles (59.6%) had at least 1 oocyte that reached MII stage. In total, 248 oocytes were injected after 0.1-1.0h (group 1, n = 104, 41.9%) and 1.1-3.0h (group 2, n = 144 oocytes, 58.1%) of in vitro maturation. Age (38,99 ± 3,01 vs 38,85 ± 3,31), AMH (1,27 ± 1,29 vs 1,63 ± 1,87), #OPU (8,98 ± 4,32 vs 8,90 ± 4,34), #MII (4,41 ± 2,74 vs 4,44 ± 2,178), %MII (47.6% ± 18.8% vs 48.4% ± 17.1%) and %MI that followed to ICSI (59.2% ± 28.5% vs 54.8% ± 29.2%) were not different between groups 1 and 2 respectively. The mean number of MI that followed to ICSI were higher in group 1 (1,28 ± 0,48 vs 1,10 ± 0,32, p = 0.0008). Normal fertilization (2PN) and embryo cleavage were similar between groups (54.8% vs 45.1%, p = 0.157, 96.6% vs 93.3%, p = 0.467, respectively). However, blastocyst formation rate (#blastocyst/#fertilized) was higher in group 1 (n = 33, 55.9% vs n = 26, 34.7%, p = 0.015). Euploidy rates were similar between groups (9/28, 32% vs 4/24, 16%, p = 0.336). Limitations, reasons for caution Besides the retrospective nature of this study, male factor and infertility reason were not considered in this analysis. Due to the low number of blastocyst formation and consequentially ploidy assessment and number of embryo transfer, the results regarding euploidy rate may be underestimated and pregnancy rates were not included. Wider implications of the findings Oocytes that reached maturity up to 1 hour after denudation showed higher blastocyst formation rate, thus increasing embryo availability to transfer. In vitro-matured oocytes exhibit increased spindle and chromosomal abnormalities compared with oocytes matured in vivo, which may explain the lower rates of euploid embryos, especially in group 2. Trial registration number Not applicable.

P-662 Embryo ploidy rates following PPOS or GnRH antagonist protocol. A prospective study with repeated ovarian stimulation

Abstract Study question Is there any difference in embryo ploidy rates following progesterone primed ovarian stimulation (PPOS) using micronized progesterone or GnRH antagonist protocol? Summary answer Pituitary downregulation with a PPOS protocol with micronized progesterone results in a comparable number of euploid blastocysts with a GnRH antagonist protocol. What is known already Although the GnRH antagonist is consider by most the gold standard protocol for controlling the LH surge during ovarian stimulation for IVF/ICSI, progesterone-primed ovarian stimulation protocols (PPOS) are being increasing used in freeze-all protocols. Still, despite the promising results of PPOS protocols, an early randomized trial reported potentially lower live births in recipients of oocytes resulting following downregulation with medroxyprogesterone acetate as compared with a GnRH antagonist protocol. The scope of the current prospective study was to investigate whether PPOS with micronized progesterone results in equivalent blastocyst euploidy rates as compared with a GnRH antagonist protocol. Study design, size, duration In this prospective study, performed between September 2019 to January 2022, 44 women underwent two consecutive ovarian stimulation (OS) protocols within a period of 6 months in a GnRH antagonist protocol or in a PPOS protocol with oral micronized progesterone. Participants/materials, setting, methods Overall, 44 women underwent two OS cycles with identical fixed dose of rFSH (225 or 300 IU) in both cycles. Downregulation in the 1st cycles was performed with the use of a flexible GnRH antagonist protocol and consecutively, after a washout period of one month, control of LH surge was performed with oral micronized progesterone from stimulation day 1. After the completion of both cycles, all generated blastocysts underwent genetic analysis for aneuploidy screening (PGT-A). Main results and the role of chance Comparisons between cycles did not reveal differences between the duration of OS neither in the gonadotrophins dose. Hormonal profile on the day of trigger revealed statistically significant differences between cycles in all the tested hormones except for FSH: with significantly higher serum E2 levels, more elevated LH levels and higher Progesterone levels in PPOS cycles as compared with antagonist cycles, respectively. PPOS protocol resulted in a significantly higher number of oocytes (10.27± 5.84 versus 12.68± 8.09), (DBM -2.4 [95% CI -4.1; -0.73]), MIIs (7.34±4.15 versus 9.09± 6.12), (DBM -1.8 [95% CI -3.1; -0.43]), and 2PNs (5.66±3.35 versus 7.14± 4.99), (DBM -1.5 [95% CI -2.6.1; -0.32]) as compared with the GnRH antagonist protocol. Nevertheless, no differences were observed regarding the mean number of blastocysts (2.84±2.12 versus 2.91±2.11), (DBM -0.07 [95% CI -0.67; 0.53]) and the mean number of biopsied blastocysts (2.86±2.15 versus 2.93±2.16), (DBM -0.07 [95% CI -0.70; 0.56]). Finally, no difference was observed for the primary outcome, with a mean number of euploid embryos of 0.86 ± 0.90 vs. 1.00 ± 1.12 for the comparison of PPOS vs. GnRh antagonist. Limitations, reasons for caution The study was powered to detect differences in the mean number of euploid embryos and not in terms of pregnancy outcomes. Additionally, per protocol there was no randomisation, the first cycle was always a GnRH antagonist cycle and the 2nd a PPOS with one month of washout period in between. Wider implications of the findings The current study provides evidence that the PPOS may result in equivalent blastocyst euploidy rates compared with antagonist protocol. This may imply that in case of a freeze-all protocol, clinicians may safely consider PPOS to control the LH surge and patients could benefit from the advantages (cost and oral administration). Trial registration number NCT04108039

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

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

P-501 Live birth rates (LBR) are not influenced by a change in embryo quality imposed by embryo cryopreservation and thawing in single euploid frozen embryo transfers.

Abstract Study question Is a change of embryo quality through vitrification and thawing associated with a reduced LBR when a single euploid frozen embryo transfer is performed? Summary answer Live birth rates are not influenced by a change in embryo quality through embryo cryopreservation and thawing in single euploid frozen embryo transfer. What is known already Embryo aneuploidy is the most significant single factor and accounts for about 50% of implantation failure and miscarriage in human reproduction. Preimplantation genetic test for aneuploidy (PGT-A) presents a mean to identify euploid embryos, thereby improving pregnancy rates and reducing early pregnancy losses. However, after PGT-A on trophectoderm cells at blastocyst stage, embryos have to be cryopreserved and warmed later for the embryo transfer procedure. Embryo quality is known to impact implantation and ongoing pregnancy rates, therefore, the aim of this analysis was to evaluate the impact of a change in embryo quality between pre-vitrification and post-thaw on the outcome. Study design, size, duration This retrospective observational study included a total of 611 pregnant patients after frozen-thawed euploid single embryo transfer (FET) at a tertiary IVF centre between April 2017 and December 2020. All embryos transferred were cryopreserved after trophectoderm biopsy for PGT-A, performed either on day 5 or day 6. Each patient was only included once into the analysis. Endometrial preparation for FET was performed either in a natural cycle (NC) or as a hormonal replacement cycle (HRT). Participants/materials, setting, methods Patients with infertility, undergoing ovarian stimulation and FET in either a HRT or a NC as endometrial preparation approach, and who achieved a pregnancy, were included. Embryos were morphologically graded pre-vitrification and post-thaw based on inner cell mass and trophectoderm morphology on the day of cryopreservation. Four groups were initially established (top, good, fair, poor), hence for the analysis, fair and poor qualities were combined. Main results and the role of chance A total of 591 patient were analysed after exclusion of 20 patients due to the occurrence of late miscarriage and ectopic pregnancy. Patients’ characteristics are as follows (mean±SD (min-max)): age 33.87±5.55(20-47) years; AMH 3.20±3.28ng/mL(0.01-35.72); BMI(body mass index) 26.61±4.86kg/m2(14.84-40.31) and AFC 14.00±7.33(1-35). The changes in the embryo quality pre-vitrification and post-thaw were categorized in “no change”, “improved” or “degraded”. All groups were compared to top-quality-no change group. A binary logistic analysis evaluated the following groups regarding their combined impact on LB: top quality-degraded (odds ratio (OR)0.702; 95% confidence interval (CI)0.302-1.635, p = 0.413); good quality-no change (OR 1.022; CI:0.457-2.287; p = 0.958); good quality-degraded (OR 0.787; CI:0.330-1.880; P = 0.590); good quality-improved (OR 0.301; 0.056-1.628; P = 0.163); fair+poor quality-no change(OR 0.521; 0.203-1.340; P = 0.176); fair+poor quality-degraded (OR 0.283; 0.056-1.438; P = 0.128) and fair+poor quality-improved (OR 1.917; 0.431-8.534; P = 0.393). No impact on the probability of LB was seen in any combined group of change in embryo quality with its pre-grades. Furthermore, no association was found for age, BMI, AMH, pre-vitrification and post-thaw quality. Embryos biopsied on day 6 instead day 5 had a decreased chance of a LB (OR 0.516; 0.305-0.875, p = 0.014). Further on, NC was associated with significantly higher chance of LB compared to HRT cycle (OR 2.629; 1.606-4.305, p = <0.001). Limitations, reasons for caution Limitation is the retrospective design of the study and the grading of the embryos, which might be subjective and bearing interobserver variability. Wider implications of the findings Whereas a change in the embryo quality does not impact the chance of LB after single euploid FET, a NC approach increases the odds for LB. This fact should be considered for a personalized treatment approach and in the choice of the endometrial preparation protocol. Trial registration number not applicable

O-297 Contribution of Frozen Embryo Transfer Cycles to ICSI Clinical Outcomes in Male Factor Infertility

Abstract Study question What is the benefit of adding frozen embryo transfer (FET) cycles to ICSI clinical outcomes in male factor infertility? Summary answer FET carried out on spare embryos that reached blastocyst stage remarkably contributed to additional clinical pregnancies in ICSI cycles for male factor infertility. What is known already Current trends in reproductive medicine lean toward full-preimplantation development and eventually to PGT-A to select a single euploid embryo for transfer. The utilization of this approach, while beneficial in most couples, is not ideal for male factor infertility due to the tendency of being characterized by impaired embryo development. So, we wonder if the utilization in FET cycles of leftover embryos that reached to blastocyst stage or those that eventually reached day 5 for aneuploidy testing contributed to the clinical outcomes. Study design, size, duration In the past 7 years, we included 22,289 couples who underwent ICSI while the large majority (84.8%) received a fresh embryo transfer, of which 70.9% were transferred at day 3. Leftover embryos, together with those euploid after PGT-A at blastocyst stage, were replaced in subsequent FET cycles. The clinical outcomes including clinical pregnancy rate (CPR) and deliveries were compared between the fresh embryo transfer and those after FET in total and after PGT-A. Participants/materials, setting, methods Couples with male factor underwent ICSI in standard fashion using exclusively ejaculated sample. For fresh embryo transfer cycles, embryos were transferred either at day 3 or at day 5. For FET cycles, embryos were cultured up to exclusively blastocyst stage and cryopreserved by vitrification. For aneuploidy, NGS was carried out for PGT-A. FET was carried out in natural or programmed cycles. Main results and the role of chance In the cohort underwent fresh embryo transfer, 18,896 couples underwent 37,751 ICSI cycles, where 322,916 oocytes were injected and 243,768 (75.5%) fertilized. Additionally, 3,393 patients received 4,712 FET in total with a fertilization of 69.8% (46,163/66,171) that did not differ from the fresh transfer group. The number of average embryos transferred in fresh cycles was 2.4±2 while FET was carried out exclusively on single embryo. Fresh transfer yielded 37.3% (14,087/37,751) CPR, while overall FET cycles achieved a higher CPR at 52.2% (2,462/4,712, P<0.0001). Similarly, the delivery rate in fresh cycles was 31.8% (12004/37751) and became 45.5% (2145/4712) in the FET (P<0.0001). To identify the advantage of selecting a single euploidy embryo, we compared FET on leftover unscreened blastocyst to those that were planned for PGT-A. Spare embryo transfers involved 1774 patients in 2282 cycles with a fertilization rate of 67.7% (18,905/27,937). For the PGT-A cycles, 1619 couples in 2430 cycles achieved a comparable fertilization rate of 71.3% (27,258/38,234), In this comparison, the FET on spare unscreened embryos achieved a CPR at 47.0% (1,072/2,282) while in the PGT-A group reached CPR at 57.2% (1390/2430, P<0.0001). Similarly, the delivery rate was 37.6% (857/2,282) in the FET and PGT-A was 53.0% (1,288/2,430) (P<0.0001). Limitations, reasons for caution The comparison is retrospective and is carried out on male factor infertility where day 3 embryo transfer were performed almost exclusively on fresh cycle to overcome poor embryo development. Nonetheless, those spare embryos that reached blastocyst stage and those that electively underwent aneuploidy testing significantly contributed to enhance clinical outcomes. Wider implications of the findings ICSI can overcome most of male infertility; however, the risk of impaired embryo development proposes a transfer at cleavage stage. The advanced embryo culture condition together with time-lapse allowed us to monitor embryos up to the blastocyst stage that once transferred, improving clinical outcomes, especially for embryos with confirmed euploidy. Trial registration number N/A