IVF Patients Research Articles

Amino Acid Profiling of Follicular Fluid in Assisted Reproduction Reveals Important Roles of Several Amino Acids in Patients with Insulin Resistance.

The global prevalence of insulin resistance (IR) is increasing continuously, influencing metabolic parameters and fertility. The metabolic changes due to IR can alter the molecular composition of plasma and other body fluids. Follicular fluid (FF) is derived mainly from plasma, and it is a critical microenvironment for the developing oocytes. It contains various metabolites and amino acids, and the quality of the oocytes is linked at least partially to amino acid metabolism. Our goal was to quantitatively determine the amino acid (AA) profile of FF in IVF patients and to compare IR and non-insulin resistance (NIR) groups to investigate the AA changes in their FF. Using UHPLC-based methods, we quantified the main 20 amino acids from human FF samples in the IR and NIR groups. Several amino acids (aspartate, glycine, glutamate, and cysteine) differed significantly (p < 0.05 or less) between the two groups. The most significant alterations between the IR and NIR groups were related to the glutathione metabolic pathway involving glycine, serine, and threonine. Since insulin resistance alters the amino acid composition of the FF, the oocytes may undergo metabolism-induced changes resulting in poor oocyte quality and less fertility in the insulin resistance groups.

Fertility specialists’ views, behavior, and attitudes towards the use of endometrial scratching in Italy

BackgroundEndometrial scratching (ES) or injury is intentional damage to the endometrium performed to improve reproductive outcomes for infertile women desiring pregnancy. Moreover, recent systematic reviews with meta-analyses and randomized controlled trials demonstrated that ES is not effective, data on the safety are limited, and it should not be recommended in clinical practice. The aim of the current study was to assess the view and behavior towards ES among fertility specialists throughout infertility centers in Italy, and the relationship between these views and the attitudes towards the use of ES as an add-on in their commercial setting.MethodsOnline survey among infertility centers, affiliated to Italian Society of Human Reproduction (SIRU), was performed using a detailed questionnaire including 45 questions with the possibility to give “closed” multi-choice answers for 41 items and “open” answers for 4 items. Online data from the websites of the infertility centers resulting in affiliation with the specialists were also recorded and analyzed. The quality of information about ES given on infertility centers websites was assessed using a scoring matrix including 10 specific questions (scored from 0 to 2 points), and the possible scores ranged from 0 to 13 points (‘excellent’ if the score was 9 points or more, ‘moderate’ if the score was between 5 and 8, and ‘poor’ if it was 4 points or less).ResultsThe response rate was of 60.6% (43 questionnaires / 71 infertility SIRU-affiliated centers). All included questionnaires were completed in their entirety. Most physicians (~ 70%) reported to offer ES to less than 10% of their patients. The procedure is mainly performed in the secretory phase (69.2%) using pipelle (61.5%), and usually in medical ambulatory (56.4%) before IVF cycles to improve implantation (71.8%) without drugs administration (e.g., pain drugs, antibiotics, anti-hemorrhagics, or others) before (76.8%) or after (64.1%) the procedure. Only a little proportion of infertility centers included in the analysis proposes formally the ES as an add-on procedure (9.3%), even if, when proposed, the full description of the indications, efficacy, safety, and costs is never addressed. However, the overall information quality of the websites was generally “poor” ranging from 3 to 8 and having a low total score (4.7 ± 1.6; mean ± standard deviation).ConclusionsIn Italy, ES is a procedure still performed among fertility specialists for improving the implantation rate in IVF patients. Moreover, they have a poor attitude in proposing ES as an add-on in the commercial setting.

Remote IVF: a clinical and laboratory guide to performing remote oocyte retrievals.

The aim of this guide is to describe different scenarios when remote IVF would be needed, considerations around how to plan for the procedure, proper equipment in the procedure room, and proper transportation of oocytes from the procedure room. There are two different scenarios for remote IVF: (1) IVF clinics designed knowing the embryology laboratory is nonadjacent and (2) IVF clinics that routinely provide care to patients in their clinic and want to provide care to those who are ineligible for a retrieval under anesthesia in an outpatient facility. This guide will focus on both scenarios. Much of the advice can be used for IVF clinics that routinely perform oocyte retrievals nonadjacent to their embryology laboratories. Special considerations are needed when patients with complex comorbidities require high-level of care and hospital-level monitoring while under anesthesia and/or post-oocyte retrieval, and are thus unable to be treated in the standard facility. For these reasons we have created a comprehensive guide to nonadjacent, or off-site, oocyte retrievals for reproductive endocrinology and infertility (REI) physicians, nurses, and embryologists to use when planning care for IVF patients. Going forward, we will refer to both these scenarios as remote IVF.

Use of autologous adipose-derived mesenchymal stem cells for ovarian rejuvenation in poor responder IVF patients: a phase 1 randomized placebo-controlled double-blind crossover study

Background: Despite the application of various methods to augment ovarian responsiveness, the management of poor ovarian responders remains challenging and pregnancy rates following in vitro fertilization are poor. Advances in adult stem cell research and their clinical application has prompted interest in their use in assisted reproduction. We report the first double-blind, randomized, placebo-controlled clinical study using autologous human stromal vascular fraction (SVF) containing adipose-derived stem cells (ADSCs) for ovarian rejuvenation. Materials and methods: Thirty patients were recruited. Twenty-one had lower-than-expected reserves for their age and 9 had premature ovarian insufficiency. Patients were randomized into a placebo group (10) and an intervention group (20). SVF was obtained from adipose tissue following abdominal liposuction; the ADSC component was characterized using flow cytometry. Three equal insertions, adjusted based on ovarian volume, were performed at monthly intervals via an ultrasound-guided transvaginal needle puncture. The SVF was not cultured before transplantation. Those in the placebo group were then crossed over to the intervention group and received a single SVF (maximally concentrated) insertion (crossover group). Results: The median viable SVF cell number inserted per patient over 3 months, and the percentage of mesenchymal stem cells (MSC) thereof, was 1.6×106 and 13.2%, respectively. Resulting anti-Mullerian hormone (AMH) changes were variable over the treatment course with a notable placebo effect. Patients with premature ovarian insufficiency showed no change in AMH, both to intervention and placebo. Despite this, a temporary return of menses was noted in a third of patients while on treatment. Patients with low reserves for age showed an increase in AMH, although not statistically significant when compared to placebo. In the crossover group, insertions were limited to one intervention comprising all cells; here a significantly higher median of 3.4×106 SVF cells were injected containing an average of 16.9% MSCs. No significant change in AMH was noted. To date 12 patients have undergone ovarian stimulation and in vitro fertilization after stem cell therapy; of these 9 have had embryo transfers with a resulting pregnancy rate of 33%. There were also 2 spontaneous pregnancies. Conclusion: Although the application of SVF-derived ADSCs for ovarian rejuvenation remains experimental, the current study provides further support for the safety of this approach and presents encouraging results as to its efficacy in assisted reproduction.

O-263 Effect of clindamycin and a live biotherapeutic containing lactobacilli on the reproductive outcomes of IVF patients with abnormal vaginal microbiota: a double-blind, placebo-controlled multicentre trial

Abstract Study question Does treatment of abnormal vaginal microbiota improve the reproductive outcomes of IVF patients? Summary answer Data is being analysed and will be presented at ESHRE 2023. What is known already An increasing number of studies reported an association between abnormal genital tract microbiota and adverse reproductive outcomes in IVF patients. One hypothesis could be that vaginal microbiota ascend to the endometrium where the microbiota hampers implantation. To the best of our knowledge, this is the first study powered to investigate causality between abnormal genital tract microbiota - in this study defined by a bacterial vaginosis like vaginal microbiota - and clinical pregnancy rate in IVF patients. Study design, size, duration Double-blind, placebo-controlled multicentre trial in IVF patients diagnosed with abnormal vaginal microbiota and subsequently randomised into three parallel groups 1:1:1. The first group received clindamycin 300 mg ×2 daily for 7 days followed by vaginal Lactobacillus crispatus until the clinical pregnancy scan. The second group received clindamycin and placebo, whereas the third group received placebo/placebo. A total of 1518 patients were screened, and 338 patients were randomised. The study duration was from 2017 to 2023. Participants/materials, setting, methods IVF patients with any cause of infertility embarking on their first, second or third IVF stimulation cycle or embryo transfer were approached for inclusion. At a minimum 12 days prior to embryo transfer, patients were screened for abnormal vaginal microbiota defined by a qPCR assay, targeting high quantitative loads of Fannyhessea vaginae and Gardnerella spp. Patients were excluded if they had intrauterine malformations like polyps, septum, and fibroma. Main results and the role of chance Data and safety monitoring board has decided not to open the randomization code yet. The final dataset is being completed. Data is being analysed and will be presented at ESHRE 2023. Limitations, reasons for caution Data is being analysed and will be presented at ESHRE 2023. Wider implications of the findings The present drug intervention study used clindamycin and a live biotherapeutic containing Lactobacillus crispatus. The results of the study could have a worldwide important clinical impact on daily IVF practice as currently there is no evidence that screening and treating IVF patients with abnormal vaginal microbiota improves the reproductive outcome. Trial registration number EudraCT 2016-002385-31

P-555 Do IVF patients prefer the rectal route to the vaginal for luteal phase progesterone administration? - a cohort study

Abstract Study question How do IVF patients perceive rectal administration of progesterone for luteal phase support compared to vaginally administered progesterone? Summary answer Rectal administration of progesterone causes less discomfort in IVF patients, undergoing Hormone Replacement Therapy Frozen Embryo Transfer (HRT-FET) and is preferred compared to vaginal administration. What is known already Progesterone is essential for implantation and normal development of pregnancy in spontaneous pregnancies as well as in ART pregnancies. In IVF, progesterone is most often administered vaginally in Europe for luteal phase support, although, this route may cause cumbersome discharge, vaginal bleeding, and irritation of the vulva. For many years the vaginal route has been the gold standard, although, progesterone is well absorbed rectally and recent studies have shown that rectally administered micronized progesterone was well accepted among IVF patients. Until now, in IVF very little focus has been on patient convenience as regards luteal phase support. Study design, size, duration An interventional cohort study, conducted from January 2020 to November 2022 in a public fertility clinic. A total of 479 patients received a questionnaire (Q1) by e-mail during the period between the embryo transfer and the pregnancy test; A second questionnaire (Q2) was answered at the time of the first ultrasound scan in gestational week 7. Participants/materials, setting, methods Patients underwent an HRT-FET protocol, including vaginal progesterone (VP) (400mg/12hours). In patients with serum progesterone levels lower than 11 ng/ml on the 6th day of progesterone, additional progesterone was administered rectally (RP) (400mg/12hours) from that day until the day of pregnancy testing, and in pregnant patients until the first scan in week 7. Side effects and patient convenience of both routes were reported in a questionnaire, including 27 questions, using a visual analog scale (0-100). Main results and the role of chance A total of 73% of HRT-FET patients (349/479) answered Q1 before the pregnancy test and a total of 27 % (93/349) of the cohort received progesterone both vaginally and rectally. The response rate of Q2 in gestational week 7 was 100 % (221/221) and a total of 28 % of patients (62/221) administered progesterone both vaginally and rectally. In Q1, a total of 60% of patients (54/90) preferred RP over VP in the cohort of patients treated with both administration regimens. In Q2, a total of 62% of patients (37/60) preferred RP over VP in the cohort of patients treated with both administration regimens. The overall discomfort was lower in relation to RP in both questionnaires and, especially discharge was lower in RP compared to VP; median interquartile range (IQR) in Q1: 11 (3-23) vs. 50 (25-66) and Q2: 19 (9-28) vs. 47 (22-62), respectively. Changes in defecation pattern after RP were reported in Q1 as well as Q2, 48% (45/92) and 55% (34/61), however, the discomfort was reported as mild. An increased flatulence was reported by 50% (46/92) in Q1 and 63% (39/92) in Q2, however, again the discomfort was reported as mild. Limitations, reasons for caution Currently, it is unknown whether the present results apply to progesterone products other than the one used in the present study (Cyclogest®). The questionnaire used, although having been used in another published study with another progesterone product, was not specifically validated for this product. Wider implications of the findings The rectal route should be remembered for luteal phase support in IVF, and some patients might choose the rectal route over the vaginal for their luteal phase support due to less discharge. Trial registration number EudraCT no.: 2019-001539-29

P-571 Endometrial cortisol and estradiol levels as indicators of pregnancy prognosis and their association with State-Trait Anxiety Inventory (STAI) psychological questionnaire punctuations in IVF patients

Abstract Study question How anxiety and stress questionnaires punctuations and endometrial steroid biosynthesis metabolites levels are associated with embryo implantation and pregnancy outcomes in IVF patients? Summary answer Those patients with high cortisol and low estradiol endometrial levels, were stressed according to STAI State punctuations and showed lower rates of implantation and pregnancy. What is known already Although IVF treatments are known to be a source of stress, its relevance in reproductive success remains controversial, mainly due to the lack of accurate biomarkers. Steroid hormones, such as progesterone and estrogens, are essential for reproductive physiology, and directly related to common stress biomarkers such as cortisol. Nowadays, clinical stress evaluation is exclusively performed via highly subjective psychological questionnaires, not being standardized in the clinical setting. In this study, we measured endometrial levels of main steroid hormones -including cortisol-, stress psychological questionnaires, and reproductive outcomes for determining the relationship among potential stress biomarkers and their influence on fertility. Study design, size, duration In this prospective cohort study, a total of 55 IVF patients were included (&amp;lt;45 years old, no uterine or systemic pathologies and good quality embryos). All endometrial biopsies were collected in mid-secretory phase for metabolites measurement between 2019 and 2021. Also, these patients were psychologically evaluated by stress related questionnaires the same day of biopsy collection or days later in the same cycle. Reproductive outcomes were compared between groups established according to stress biomarkers thresholds. Participants/materials, setting, methods Concentration of eleven selected steroid metabolites was measured by ultra-performance liquid chromatography-tandem mass spectrometry. A psychological questionnaire was also assessed to measure patients’ stress and anxiety (STAI). According to metabolites’ concentration distribution, Barnard’s test was applied to compare the proportion of patients with successful and unsuccessful treatments at different thresholds. A Wilcoxon test was performed for mean metabolites’ levels comparison between stressed and unstressed patients. A p-value of 0.05 was stablished as significant. Main results and the role of chance All patients with high endometrial cortisol levels (&amp;gt;12.5 ng/g; n = 3) failed to implant (100% vs 45.8% non-implanted in &amp;gt; 12.5 ng/g and &amp;lt;12.5 ng/g patients respectively, p-value=0.047), while the totality of patients with high endometrial estradiol levels (&amp;gt;0.9 ng/g; n = 5) achieved embryo implantation and on-going pregnancy (100% vs 50% implanted in &amp;gt; 0.9 ng/g and &amp;lt;0.9 ng/g patients respectively, p-value=0.038). Those patients with high cortisol levels, all not pregnant, had also low estradiol levels and high stress state punctuation (STAI State punctuations ≥ 60). Conversely, patients with high estradiol levels, all pregnant, had also low cortisol levels and were not stressed (STAI State punctuations &amp;lt; 60). Stratification of patients into stressed (STAI State&amp;gt;60) and unstressed (STAI State&amp;lt;60) independently of their implantation results, revealed that 27% of stressed women presented high cortisol levels, while 100% of unstressed patients had low cortisol levels. In the same way, 42% of unstressed patients had high estradiol levels, while 100% of stressed patients presented low estradiol values. Hence, mean values differences of cortisol (10.54 vs 3.05 ng/g) and estradiol (0.55 vs 4.10 ng/g) between stressed and unstressed patients were not significant (p-value=0.08862 and p-value=0.2327, respectively). No relevant associations were observed with stress as a trait. Limitations, reasons for caution Due to the heterogeneous and non-normal metabolites’ concentration distribution among patients, this study would need a largest sample size for having a representative population in the different subgroups. Although we have used molecular and psychological validated stress biomarkers, a causal relationship between stress and reproductive outcomes should be further explored. Wider implications of the findings This study highlighted cortisol and estradiol as potential endometrial biomarkers that better reflect pregnancy prognosis. Further investigation is needed to find if other psychological questionnaires molecularly recognize stressed patients as a noninvasive diagnosis associated to reproductive outcomes. These patients would benefit from psychological counseling to improve their reproductive success. Trial registration number Not applicable

P-722 Impact of blastocyst quality and post-thaw embryo culture factors in non-invasive PGT-A informativity on vitrified blastocysts.

Abstract Study question What are the most important factor/s involved in the informativity of non-invasive PGT-A (niPGT-A) of vitrified blastocysts? Summary answer The day of vitrification and post-thaw time in culture of vitrified blastocysts are the most important factors for niPGT-A informativity, without correlation with blastocyst quality. What is known already niPGT-A is a technique that allows the non-invasive study of the chromosomal content of blastocysts. The informativity and concordance rates for fresh (Rubio, 2020) and frozen-thawed embryos (Garcia-Pascual, 2022) have been related with the time in culture and the day of media collection. However, there is a need to understand the optimal conditions needed to obtain optimal informativity for frozen-thawed vitrified blastocyst undergoing niPGT-A. Study design, size, duration Prospective study including 135 spent blastocyst media (SBM) and the corresponding blastocyst samples from thawed day-5 and day-6 blastocysts from PGT-A (73) and niPGT-A (62) cycles collected from January 2021 until March 2022. Blastocysts were discarded under IRB approval. The embryos were divided into 3 groups depending on the day of development and the time in culture after thawing: group-1, day-5 blastocysts cultured 8h; group-2, day-5 blastocysts cultured 24h and group-3, day-6 blastocysts cultured 8h. Participants/materials, setting, methods Media and blastocysts were obtained from IVF patients (29-42 years-old). After thawing, each blastocyst was washed to remove remaining cumulus cells and individually transferred to a 10 μL droplet of fresh media or to an EmbryoScope dish with 20 μL per well. SBM and blastocysts were collected, frozen and analysed by Next Generation Sequencing. Correlation between concordance rates (overall agreement between SBM and blastocyst samples), informativity, embryo quality and time in culture were evaluated. Main results and the role of chance All the embryos had a good embryo quality (BB or greater using Gardner Criteria) but different expansion degree. Informativity was significantly lower when day-5 embryos were cultured for only 8 hours (group 1), regardless of the degree of expansion (expansion degree 3, 28.57%; degree 4, 64.29% and degree 5-6, 57.14%, p &amp;lt; 0.0005). The concordance rates between SBM and whole blastocysts were very high and no significant different in relation to the expansion degree when comparing day-5 embryos cultured for 8 hours (group 1, 90.5%) with 24 hours (group 2, 93.6%) or with day-6 embryos cultured for 8 hours (group 3, 92.3%). The data suggest that the important factor impacting the informativity rate is the day of vitrification, and the time in culture after thawing, but not the expansion degree of the embryos. Regarding the concordance rates, there is no impact of the time in culture nor the expansion degree. Limitations, reasons for caution The main limitation of this study is that all the embryos analysed had fair or good quality but different expansion degrees. Wider implications of the findings The high concordance rates observed in frozen blastocysts opens the possibility of expanding the use of niPGT-A to frozen blastocyst without an impact of the expansion degree in the results. Trial registration number Not applicable

P-772 Three-dimensional folliculometry (SonoAVC) provides a better estimation of total and mature oocyte yields compared to conventional two-dimensional assessment in oocyte donors: a prospective, cohort study

Abstract Study question Does three-dimensional folliculometry (using SonoAVC) provide a better estimate of the number of retrieved and mature oocytes in egg donors compared to conventional two-dimensional assessment? Summary answer Three-dimensional folliculometry was superior to the two-dimensional ultrasound assessment and provided an excellent prediction (+/-20%) of total oocyte yield in almost two-thirds of participants. What is known already When used for monitoring ovarian stimulation, 3-D folliculometry has been shown to reduce inter-, and intra-observer variability and was significantly faster than conventional, two-dimensional ultrasound assessment (Vandekerchove F, 2014, Rei 2019). Although so far, no study has demonstrated improved clinical outcomes in IVF patients, the impact of 3D-folliculometry was not yet thoroughly evaluated in oocyte donation programmes, where expected total and mature oocyte yield is a crucial variable. Study design, size, duration Ninety-one consecutive oocyte donors were prospectively evaluated using three-dimensional folliculometry with SonoAVC software (E8, General Electric). A single operator (DB) performed all 3D ultrasound measurements immediately before starting oocyte retrieval and the oocyte retrieval procedure too. Two-dimensional measurement of follicles (average of two perpendicular diameters) was performed two days before egg retrieval by a single operator (NR) (Mindray DC7). Oocyte donors’ age (26.2±3.7 years), BMI (23.8±3.9), and baseline AMH (4.6±2.3 ng/mL) were also registered. Participants/materials, setting, methods Voluntary, anonymous oocyte donors were stimulated with a GnRH antagonist protocol and triggered with a GnRH agonist. Good, moderate, and poor prediction by ultrasound were defined as an oocyte/follicle count ratio of 80-120% (+/-0-20%), 60-79% or 121-140% (+/- 21-40%), and less than 60% or more than 140% (+/- more than 40%), respectively. For total oocyte yield, a threshold of all follicles 13 mm or above was used, both for the two- and three-dimensional assessment. Main results and the role of chance The number of total and mature oocytes retrieved was 22.2±9.4 and 19.0±8.3, respectively with an excellent average maturity rate of 86% (range: 44-100%). No donors were excluded from the study due to suboptimal visualization of ovaries with SonoAVC. For total oocyte yield; good, moderate, and poor prediction was achieved in 37, 44, 19% of donors with two-dimensional and in 63, 29, 8% of participants with three-dimensional folliculometry, respectively (p = 0.002). Significant underestimation of total oocyte yield (defined as more than 6 „missing” total oocytes) was less frequent with 3D assessment (12 versus 25% p = 0.022). For total oocyte yield, linear regression analysis showed a significantly higher r-square value with three-dimensional assessment (0.676 vs 0.278). For mature oocytes, the prediction was less precise than for total oocyte yield. Good, moderate, and poor prediction was achieved in 40, 30, 30% of donors with two-dimensional and in 55, 30, 15% of participants with three-dimensional folliculometry, respectively (p = 0.031). For mature oocytes, linear regression analysis showed a significantly higher r-square value with three-dimensional assessment (0.664 vs 0.282). The best SonoAVC threshold to predict total and mature oocyte yields was the sum of all follicles equal or above 13 mm and total follicle count between 24-13 mm, respectively. Limitations, reasons for caution Due to practical reasons, three- and two-dimensional measurements were not performed on the same day or with the same ultrasound equipment. In future studies, 3D-folliculometry could be used at the egg collection scheduling (2-3 days before the procedure) so that predictive information on oocyte yield would be available earlier. Wider implications of the findings Apart from reducing inter-observer variability and diminishing discomfort (faster examination time), 3D-folliculometry might have a significant role in the management of OD programmes by improving the prediction of total/mature oocyte yields. 3D-measurements performed before the procedure could also play a role in the quality control of the egg collection procedure. Trial registration number Not applicable

O-212 Screening IVF embryos with polygenic risk scores: risk reduction estimates, population impact, and attitudes of patients and clinicians

Abstract Study question IVF embryos can now be screened for complex diseases. What is the expected risk reduction? What are the attitudes of patients and clinicians? Summary answer Substantial risk reductions can be achieved, but only in the best-case scenario. The views of patients and clinicians regarding the technology are not fully aligned. What is known already It recently became possible to screen IVF embryos for a variety of complex diseases using polygenic risk scores. Prioritization of embryos for transfer based on these scores may lead to reduced disease risk in offspring. Preliminary risk reduction modeling suggested that substantial risk reductions are possible, particularly when screening for a single disease. However, multiple factors remain unexplored, such as the effect of implantation failure and assortative mating on risk estimates and the impact of embryo prioritization on population allele frequencies. The attitudes of IVF patients and clinicians towards polygenic embryo screening are also unknown. Study design, size, duration We estimated the expected risk reduction under various settings using statistical modeling, based on the liability threshold model and other quantitative and population genetic models. To learn about attitudes of patients and clinicians, we performed in-depth, semi-structured interviews with 26 USA patients and 27 USA clinicians over a period of about one year. Participants/materials, setting, methods We used R to numerically estimate risk reductions and implemented a web app in Shiny. To recruit patients and clinicians for the interviews, we used flyers (for patients), emails on publicly available lists (for clinicians), snowball sampling, and convenience sampling. Interviews were conducted over Zoom for one hour each by a single interviewer (D.B.). The study was approved by Baylor College of Medicine's IRB committee. We applied deductive and inductive thematic analysis using a team-based approach. Main results and the role of chance Statistical genetic modeling suggested that with five viable embryos, risk reductions of up to 50% can be achieved when screening for a single common disease. We developed a risk reduction estimator web app, allowing users to examine the effect of the number of embryos, the accuracy of the score, the selection strategy, and the score percentile or disease status of the parents. Assortative mating and modeling of implantation failures are expected to only slightly decrease risk reductions. Risk allele frequencies are expected to change very little across generations even if embryo selection becomes widespread. In interviews, patients accepted screening for health conditions, particularly severe diseases. Many were concerned about costs, and some were interested in screening for informative purposes. Most patients did not oppose screening for traits. In contrast, about half of clinicians were unwilling to offer screening due to concerns over utility, implementation, harm to patients, and lack of scientific validity, although all were willing to reconsider it in the future for specific conditions. Many clinicians considered screening for traits a non-medical procedure. About half agreed that screening should be regulated. Limitations, reasons for caution The risk reduction estimates may depend on the assumptions of the statistical models. As with any qualitative research, interview findings may not be generalizable. Furthermore, the design of the semi-structured interview guide may have influenced participants’ expressed attitudes. Wider implications of the findings In the best-case scenario, prioritizing IVF embryos based on polygenic scores may achieve substantial risk reductions. Several initial concerns seem unfounded. IVF patients are broadly interested in the technology but may be unaware of its many limitations. Clinicians have major concerns regarding clinical implementation and harm to patients. Trial registration number not applicable

O-103 A method for mapping sex-specific meiotic crossovers from PGT-A data elucidates the role of aberrant recombination in the origins of aneuploidy

Abstract Study question How do the number and location of meiotic crossovers contribute to the formation of aneuploidies observed in preimplantation human embryos? Summary answer Normalized across chromosomes, trisomies possess 35% fewer crossovers on average compared to disomies, while the genomic distribution of crossovers is also substantially altered. What is known already Meiotic recombination is a crucial source of genetic diversity and is also critical for ensuring the accuracy of chromosome segregation. Understanding the landscape of meiotic recombination, its variation across individuals, and the processes by which it goes awry are long-standing goals in human genetics. Current approaches for inferring the landscape of recombination either rely on population genetic patterns of linkage disequilibrium—capturing a time-averaged view—or direct detection of crossovers in gametes or multi-generation pedigrees, limiting the scale and availability of relevant datasets. Moreover, most of these methods are designed for discovering recombination using data from normal, disomic chromosomes. Study design, size, duration We present a method for mapping sex-specific recombination landscapes from low-coverage (&amp;lt;0.1×) data from preimplantation genetic testing for aneuploidy (PGT-A) of embryos with arbitrary ploidy configurations. To overcome the sparsity of these data, our method exploits its inherent relatedness structure, knowledge of haplotypes from external population reference panels, as well as the frequent occurrence of chromosome loss in embryos, whereby the remaining chromosome is phased by default. Participants/materials, setting, methods We benchmarked our method by simulating crossovers between known haplotypes. Encouraged by the performance on simulated data we extended our study to retrospective analysis utilizing de-identified PGT-A data obtained between April 2021 and August 2022 at the CReATe Fertility Centre (Toronto, Canada). The data include 20,160 embryos (2,559 IVF patients) with an average depth of coverage of ∼0.05×, facilitating the mapping of crossovers at an average resolution of ∼150 kbp. Main results and the role of chance Our benchmarking results demonstrate high sensitivity and specificity across all ancestries at a coverage of 0.05x per homolog (AUC = 0.989), with AUC declining by 0.014 and 0.053 for when coverage is reduced to 0.025x and 0.013x, respectively. Extending our analysis to real PGT-A data, we observed that our inferred sex-specific landscapes of meiotic crossovers on disomic chromosomes were strongly correlated with published genetic maps from studies based on high-coverage sequencing of parent-offspring trios (r = 0.86 for female map; r = 0.53 for male map), broadly supporting the accuracy of our method. Notably, the total length of the female genetic map was reduced by 35% for trisomies compared to disomies, consistent with the hypothesized role of reduced crossovers and exchangeless chromosomes in the origins of female meiotic aneuploidy. In addition, the genomic distribution of crossovers is also altered in a chromosome-specific manner. Examples include a reduction in crossovers near the centromere of trisomies versus disomies of chromosome 16, as well as an enrichment of crossovers on the q-arm of trisomies versus disomies of chromosome 22. Together, our results provide a detailed corroboration of the hypothesis that aberrant meiotic recombination contributes to the origins of aneuploidies. Limitations, reasons for caution The accuracy of our method is influenced by genomic heterogeneity in depth of coverage, rates of heterozygosity, and mismatches between the ancestry of the reference panel and the tested sequence. Moreover, technical errors such as spurious alignment and genotyping could hinder analysis in repetitive genomic regions. Wider implications of the findings Together, our study helps clarify the dual function of meiotic recombination in generating genetic diversity while ensuring meiotic fidelity. Our method for patient-specific mapping of meiotic recombination phenotypes may offer clues about how dysregulation of this process contributes to infertility. Trial registration number NIH r R35GM13374

P-696 Identification of novel candidate genes associated with meitotic aneuploidy in human embryos by whole-exome sequencing (WES)

Abstract Study question Can certain genetic variants in women cause a genetic predisposition to aneuploidy? Summary answer Our results suggest that the maternal variants identified in genes regulating meiotic processes could be useful genetic biomarkers for predicting a predisposition to embryonic aneuploidies. What is known already Errors in chromosome segregation during meiosis as chromosome synapsis, crossing-over and spindle building, occur frequently in human oocytes and cause aneuploidy in embryos. These errors increase dramatically in the oocytes of older women. However, the rate of producing aneuploidy oocytes varies among IVF patients for a given age. Recent publications have identified genetic variants that are crucial for producing healthy oocytes in female IVF patients. The association between maternal genetic variants and embryonic aneuploidy risk suggests the potential of using genomic data to predict embryonic aneuploidy risk. Our aim was to identify novel variants and candidate genes for embryonic aneuploidy. Study design, size, duration A prospective observational cohort study was done including 127 trophoectoderm biopsies from 29 couples who performed whole-exome sequencing (WES) and PGT-A between November 2019 and March 2022. Women were 35 years old or younger and normal karyotype for men and women. Patients were divided in two groups according to the embryo aneuploidy rate expected by their maternal age: ≤ 50% of aneuploid blastocysts as control group(n = 14), and ≥ 50% of aneuploid blastocysts as study group(n = 15). Participants/materials, setting, methods WES was performed using Trusight One Expanded Sequencing Panel (Illumina®). The following criteria were used for filtering and annotation of candidate variants: (1) minor allele frequency (MAF) &amp;lt;0.05 in the gnomAD and 1000 genomes, (2) variants in genes previously associated with chromosome segregation, chromatin cohesion, meiosis and cell division processes, (3) exonic/splicing boundaries variants, (4) variants having potentially strong/moderate functional effects on the protein evaluated using three in silico prediction algorithms (SIFT, PolyPhen-2 and MutationTaster). Main results and the role of chance Overall, the mean female and male age was 26.21 + 4.11y and 40.69 + 9.07y respectively. No differences were shown between groups for patient characteristics. Regarding PGT-A cycle no differences were seen between the number of oocytes and MII retrieved. However, the mean number of biopsied embryos per patient (5.36 + 2.50 vs 3.47 + 1.36; p &amp;lt; 0.05) as well as the percentage of embryos biopsied on day 5 (85.33 vs 32.69; p &amp;lt; 0.05) were higher in the control group. Finally, as expected, the percentage of aneuploidies was higher in the study group (18.64 vs 71.89; p &amp;lt; 0.05). 45 variants were identified in genes potentially associated to the mechanism related to the phenotype. After the variant filtering six probably pathogenic variants were identified. The variants found in the genes were: c.1397T&amp;gt;C (TLE6), c.169G&amp;gt;A (IKBKG), c.1227A&amp;gt;C (BUB1B), c.277G&amp;gt;A (TP73), c.190C&amp;gt;T (PLXNA3) and c.744C&amp;gt;G (AURKC). All variants were heterozygous and all of them were of maternal origin. The females carrying the candidate variants belonged to the study group, and two variants in different genes were identified in one patient. No potentially pathogenic variants were detected in patients from the control group. Therefore, the average detection rate of genetic variants with potential impact in embryo aneuploidy was 33.33% (5/15). Limitations, reasons for caution A notable strength of our study is the inclusion of both (male and female) genomic data. On the other hand, our study has certain limitations including the inherent challenges of any observational study, also, the pathogenicity of the identified variants should be validated in further functional studies. Wider implications of the findings We described new candidate genes that have never been associated to meiotic embryo aneuploidy and are involved in important biological processes (cell division and chromosome segregation). WES may be considered as an efficient tool to identify patients with higher risk of embryo aneuploidy allowing an individualized genetic counselling in advance. Trial registration number Not applicable

P-529 High Live Birth Rates After Low Dose Immunization Therapy (LIT) Pre and Post Pregnancy With Partner Lymphocytes In Patients With Recurrent Miscarriage (RM)

Abstract Study question Could Lymphocyte Immunization Therapy effectively improve pregnancy and live birth rate in Unexplained Recurrent Miscarriage patients? Summary answer The use of paternal lymphocytes pre and post pregnancy significantly increases the pregnancy success and Live Birth Rate In Patients With Recurrent Miscarriage (RM) What is known already RM affects 1–2% of couples who attempt to conceive and has been defined as three consecutive pregnancy losses within 20 weeks of pregnancy confirmation. Although many potential causes have been established for RM, about 50% of these remain idiopathic and unexplained owing to immunology factors. Meta-analyses have observed better effectiveness and safety of LIT in treating couples with RM shown improvement in pregnancy outcomes. Still, results are conflicting due to different screening criteria and therapeutic protocols. Objective of present study is to evaluate effectiveness of low dose LIT in patients with uRM and Th1/Th2/Treg paradigm disorders Study design, size, duration In self-controlled, prospective study, after IEC clearance and obtaining Informed consent, 19 RM patients underwent immunization with partner lymphocytes from August 2021 to October 2022. Inclusion criteria were at least three clinical pregnancies that culminated in abortion before 20 weeks of gestation or at least three cycles of IVF/ICSI with at least two PGT+ embryos and &amp;gt;8mm endometrium during ET and no pregnancy, normal coagulation tests, autoimmune antibodies negative, normal couple karyotyping. Participants/materials, setting, methods Maternal BMI of 30 and age of 45 was the upper limit with peripheral blood Th1/Th2/Treg cells proportions and concentrations of TNF-a, IFN-g, TGF-β1, IL-2, IL-4, IL-6, IL-10, and blocking antibodies were detected using flowcytometry/ELISA as per Seragen’s Immuno profiling platform. 30-50ml paternal blood was used to purify lymphocytes which were intradermally administered to the wife before Embryo Transfer and post-pregnancy confirmation. Main results and the role of chance Proportion of Th1 cells was significantly decreased while the proportions of Th2 cells and Treg cells were significantly increased in immunotherapy patients after treatment. In addition, the concentration of TGF-β1 in serum was significantly higher after immunotherapy than before. The concentration of TNF-a, IFN-g was significantly high before therapy were significantly decreased after therapy. LIT effectively induced the production of blocking antibodies in all the patients. 16/19 (84%) uRM patients underwent ET post LIT became pregnant and 4 of the pregnant patient had delivered healthy babies (21%). 42% (8/19) of patients have reported uneventful ongoing pregnancies crossed &amp;gt;14weeks. 4 patients who were pregnant for more than 12-14 weeks had miscarriages (21%). 1 patient (5%) did not conceive. 1 patient (5%) conceived spontaneously and crossed 20 weeks of uneventful gestation. 1 patient (5%) opted to try to conceive naturally hence ET is not planned. The cumulative positive uneventful pregnancy outcome was 68%, approximately the same as the figures reported earlier. LIT is associated with high live birth rates, especially in women with RM. It not only ameliorates patient’s cellular immune function but also further increases patient’s pregnancy success rate with high safety, which is worthy of clinical application and promotion. Limitations, reasons for caution This prospective self-control study with small sample size lacks randomized control group. Beneficial effects observed in this study are in sync with published data. Larger study with patients recruited based on inclusion criteria of unexplained recurrent miscarriage due to immunological factors is proposed for recommending LIT as routine therapy. Wider implications of the findings Our study findings supports LIT as a beneficial treatment for RM/RIF in IVF patients. LIT may be considered safe and effective therapy in individual cases and based on the immunoprofiling (Th1/Th2/Treg paradigm disorders), after all other potential causes of RM or RIF have been ruled out. Trial registration number Not applicable

O-134 Pretreatment with luteal estradiol for programming antagonist cycles compared to no pretreatment in advanced age women treated with corifollitropin alfa: a randomized controlled trial

Abstract Study question Does luteal estradiol pretreatment improve the number of oocytes retrieved in women aged 38 to 43 years treated in antagonist protocol with corifollitropin alfa? Summary answer Programming antagonist cycles with luteal estradiol did not increase the number of retrieved oocytes in advanced age women, however it was effective for programming. What is known already Previous studies showed that programming IVF cycles with estradiol pretreatment is a valuable tool for the organization of IVF centers and patients. However, variable effects of estradiol pretreatment were observed on the number of retrieved oocytes: no difference in normal responders, improved yield in oocyte donors and conflicting results in poor responders but no improvement in those fulfilling the bologna criteria. In advanced age women, recruitable follicles tends to decrease in number and to be more heterogeneous in size but it remains unclear if estradiol pretreatment could yield more oocytes through its negative feed-back effect on FSH intercycle rise. Study design, size, duration This non-blinded randomized controlled trial was conducted between 2016 and 2022. Participants were 324 women aged 38 to 43 years who requested IVF treatment. The primary endpoint was the comparison of the total number of retrieved oocytes between pretreated (P) and no pretreated (NP) patients. Secondary endpoints were duration and total dosage of recombinant FSH, cancellation rate, total number of metaphase II oocytes and obtained embryos, fresh transfer live birth rate (LBR) and cumulative LBR. Participants/materials, setting, methods This multicentric study (6 centers) enrolled women with regular cycles, weight &amp;gt;50 kg and BMI &amp;lt;32, IVF rank 1-2. According to computerized randomization, micronized estradiol 2mg twice a day was started on day (D) 20-24 continued until Wednesday beyond the onset of menses followed by administration of corifollitropin alfa on Friday i.e. stimulation (S)1 or from D1-3 of a natural cycle in unpretreated patients. GnRH antagonist was started at S6 and additional rFSH at S8. Main results and the role of chance Basal characteristics were similar in estradiol P (n = 147; number (mean [SD]) of treatment days 9.8[2.6]) and NP patients (n = 144), both displaying however a wide range of AMH levels (0.3 to 11.6 ng/ml) for advanced age patients. The cancellation rate showed a trend to be lower in P group (11 vs 19 %, p = 0.11). The mean number of retrieved oocytes was lower in P compared to NP patients (8.4[6.1] vs 9.1[6], respectively). The treatment difference was -0.7[-2.1 ;0.8] p = 0.03 one tailed test . No significant differences were observed in the number of MII oocytes (7[5.5] vs 7.3[5.2], p = 0.72) and the number of obtained embryos (5[4.6] vs 5.2[4.2], p = 0.60) between the two groups. Oocytes retrieval was more often on working days in P patients (91.9 vs 74.2 %, p &amp;lt; 0.001). However, the duration of stimulation was significantly longer (11.7[1.7] vs 10.8[1.8] days, p &amp;lt; 0.001) and the extra rFSH dosage in addition to corifollitropin alfa was significantly higher (1040[548] vs 778[504] IU, p &amp;lt; 0.001) in P than NP patients. The LBR after fresh transfer in P patients (16.2%[16/99]) was not significantly different from that in NP patients (18.5%[17/92]), p = 0.82, nor the cumulative LBR per patients 19.1 % vs 26.4 %, p = 0.16 respectively. Limitations, reasons for caution Our stimulated women older than 38 years obtained a wide range of collected oocytes (0 to 29) suggesting very different stages of ovarian aging in both groups. Estradiol pretreatment is more susceptible to increase oocyte yield at the stage of ovarian aging characterized by asynchrony of a reduced follicular cohort Wider implications of the findings Programming antagonist cycles with luteal estradiol pretreatment is effective in advanced age women to better schedule oocyte retrievals on working days. However, the potential benefit on the number of collected oocytes remains to be demonstrated in a more selected population displaying the characteristics of Poseïdon groups 3 and 4. Trial registration number ClinicalTrials.gov NCT02884245

P-537 Intentional endometrial injury significantly improves ongoing pregnancy rates of an oocyte donation program in patients without recurrent implantation failure (RIF): a randomized controlled trial.

Abstract Study question Is endometrial scratching a useful add-on to be applied in assisted reproduction patients without recurrent implantation failure (RIF)? Summary answer Endometrial scratching results in significantly higher clinical and ongoing pregnancy rates per intention to treat (ITT) among women undergoing oocyte donation without RIF. What is known already The effect of endometrial injury or endometrial scratch (ES) on reproductive outcomes has been widely studied by randomized controlled trials but from the information obtained regarding its benefit on improving endometrial receptivity remains controversial. Oocyte donation programs (OD) provides with the ideal setting for this research, as the recipient's endometrial priming guarantees the homogeneity of the endometrium avoiding a deleterious differential effect of the stimulation on endometrial receptivity and makes comparable the quality of the oocytes and transferred embryos, then limiting the confounding factors involved in studies with autologous oocytes and their frozen surplus embryos, better addressing the research question. Study design, size, duration A multicentric, open-label, randomized controlled trial has been conducted in a private setting from Oct 2013-Nov 2022. Eligible recipients were randomly assigned in a 1:1 ratio to either ES (by pipelle biopsy in the luteal phase of the menstrual cycle prior to the embryo transfer, n = 303) or no intervention (NES, n = 310), through a computer-generated randomization list, and embryo transfers (ET) performed in the cycle following the intervention. Participants/materials, setting, methods 18-44 years aged ovum recipients with preserved ovarian function, 19-29.9 kg/m2, first/ second OD fresh embryo transfer, endometrial thickness &amp;gt; 6 mms, and 1-2 optimal quality blastocysts transferred. Exclusion: any adverse condition, RIF with OD, ET not performed in the cycle following the intervention. Outcomes included ongoing implantation rate (ongoing sacs/ embryo transferred), biochemical, clinical and ongoing pregnancy rates analyzed per ITT basis and per protocol strictly completed (PP, treatment correctly received, embryo transfer achieved). Main results and the role of chance A total number of 458 recipients underwent OD-embryo transfer, 226 in scratching arm and 232 in the NES group. No differences existed in mean age and BMI of recipients and oocyte donors, oocytes retrieved and microinjected, fertilization rates, blastocysts transferred, endometrial thickness and estradiol /progesterone levels after their endometrial priming, nor other clinically relevant parameters. The biochemical pregnancy rate was 62.6%95%CI(56.6-68.3) and 55.6%95%CI(49.7-61.3) in the ES arm and in NES arm on the ITT analysis (OR 1.073 95%CI(0.990-1.163), p = 0.088); and 75.3%95%CI(69.0-80.7) vs. 70.7%95%CI(64.3-76.4), respectively PP (OR1.048 95%CI(0.965-1.137), p = 0.266. The clinical pregnancy rate was 57.1%95%CI(51.0-63.1) and 47.5% 95%CI(41.7-53.3) in the ES arm and in NES arm on the ITT analysis (OR = 1.102 95%CI(1.015-1.196); p = 0.021), and in the per protocol analysis, 68.6% 95%CI(62.0-74.6) vs 60.3%95%CI(53.7-66.6) respectively, OR = 1.086 95%CI(0.995-1.186), p = 0.066. The ongoing pregnancy rate was 45.1%95%CI(39.1-51.2) and 36.3% 95%CI(30.8-42.1) in the ES arm and in NES arm on the ITT analysis (OR = 1.092 95%CI(1.007-1.183); p = 0.033), and in the per protocol analysis, 53.8%95%CI(47.0-60.5) vs 46.1%95%CI(39.6-52.8) respectively, OR = 1.080 95%CI(0.985-1.184), p = 0.101. The ongoing implantation rate was in the PP analysis, 61.4%95%CI(55.1-67.8) in ES, and 56.5%95%CI(50.1-68.2) in NES respectively, p = 0.276, while in the ITT analysis, 51.3% 95%CI(45.4-57.2) vs 44.4% 95%CI(38.7-50.1), p = 0.098 in the ES arm and in NES arm. Limitations, reasons for caution The number of patients not fulfilling the complete procedure probably avoided to find significant differences on the PP analysis, and during the study, there may be some clinical and/or laboratory changes through the years, that should have been affecting outcomes on both groups. Wider implications of the findings This RCT shows that ES in the luteal phase of the cycle preceding the OD embryo transfer in recipients without RIF would pose a significant benefit thus its consideration for all IVF patients can be advised at this point. Trial registration number NCT01955356

P-479 Prognosis for ongoing pregnancy after recurrent implantation failure (RIF) following IVF/ICSI treatment

Abstract Study question What is the cumulative ongoing pregnancy rate within one year after visiting the RIF outpatient clinic in patients with RIF after IVF/ICSI treatment? Summary answer The cumulative ongoing pregnancy rate was 42.9% with a mean time to pregnancy of 8.8 months (95% CI 7.5-10.1 months). What is known already An estimated portion of 10-15% of IVF patients experience RIF. Multiple add-on treatments have been proposed, however, evidence for effective clinical therapeutic options still remain scarce. Although it is described that RIF patients might eventually benefit most from a ‘keep calm and carry on’ approach, in practice patients often seek clearer answers on their pregnancy chance and request further investigations/interventions. It would be helpful to give patients insight in their chances of achieving pregnancy after RIF, but at this moment little is known about their prognosis. This is further complicated by the lack of consistency in the definition of RIF. Study design, size, duration A prospective cohort study on 42 RIF patients after one-year follow-up was performed, as part of the MURIM (Multidisciplinary Research on Repeated Implantation Failure and Recurrent Miscarriages) study. Baseline characteristics including an endometrial assessment and ReceptIVFity (vaginal microbiome) test were collected at the RIF outpatient clinic. After a follow-up duration of minimal one year participants were asked to complete a questionnaire regarding fertility treatment and pregnancy outcome. Missing information was received via medical files. Participants/materials, setting, methods RIF patients aged 18 to 38 years old visiting the RIF outpatient clinic at Maastricht University Medical Centre+ between April 2019 until September 2021 were included. RIF was defined as consecutive implantation failure of three high quality embryos or ten embryos without a quality criterion. Clinical characteristics, pregnancy outcome and time to pregnancy was analyzed by survival analysis and cox hazard regression analysis via SPSS version 20.0. A p-value of &amp;lt; 0.05 was considered statistically significant. Main results and the role of chance Forty-two out of 44 contacted patients responded to the questionnaire (response rate = 95.5%). The ongoing pregnancy rate was 42.9% (Standard Deviation (SD): 0.50%) during the first year after visiting the RIF outpatient clinic. Mean time to ongoing pregnancy was 8.8 months (95%-CI: 7.5-10.1 months). The mean amount of embryo transfers (ETs) during this year was 2 (SD: 1.78). The ongoing pregnancy rate per ET (by survival analysis) for the first, third and sixth ET was 33.1%, 49.3% and 63.8%, respectively. To correct for a possible overestimation of the cumulative ongoing pregnancy rate per ET by normal survival analysis, pregnancy rates were calculated again using the number at risk of the total group during the first ET (pessimistic cumulative pregnancy rate). The pessimistic cumulative ongoing pregnancy rates were 33.1%, 42.9% and 48.6%, respectively. When comparing baseline characteristics by univariable cox hazard regression between women with and without ongoing pregnancy, no significant differences were found. Nineteen pregnancies were reported during the one-year follow-up, one conceived spontaneously (5.3%); four via fresh ET (IVF=15.8% and ICSI=5.3%); and 14 by frozen ET (IVF=26.4% and ICSI=47.4%). The pregnancies resulted in 14 livebirths (73.7%); one miscarriage (5.3%); one stillbirth (5.3%); and three ongoing pregnancies (15.8%). Limitations, reasons for caution Although this is the first study that prospectively describes pregnancy prognosis after RIF, it is limited by the short follow-up period and relatively small sample size. The RIF outpatient clinic consultation might have contributed to the obtained pregnancy results but the proportionality is unclear as there is no control group. Wider implications of the findings The obtained ongoing pregnancy prognosis after RIF is encouraging and justifies a conservative approach after three failed ETs. Furthermore, the high pregnancy rate indicates the need of well-defined, individualized diagnostic criteria to define RIF and to be able to deviate between couples with 'bad luck' and an underlying (treatable) cause. Trial registration number NL66835.068.18/METC18-040

O-005 The ToF-study - comparing the cumulative live birth rate of blastocyst-stage versus cleavage-stage embryo transfers in good prognosis IVF patients: a multicenter randomized controlled trial.

Abstract Study question Does blastocyst-stage embryo transfer in fresh and frozen cycles improve the cumulative live birth rate (cLBR) compared with cleavage-stage embryo transfer in IVF/ICSI treatments? Summary answer In good prognosis IVF patients (≥4 available embryos), a blastocyst-stage transfer policy did not result in a significant higher cLBR compared to cleavage-stage transfer policy. What is known already A recent Cochrane systematic review and meta-analysis concluded that fresh blastocyst-stage transfer in IVF/ICSI treatments is associated with higher rates of pregnancy in comparison to fresh transfer of cleavage-stage embryos. However, it is unknown whether a blastocyst-transfer policy also improves the cumulative live birth rate, i.e. the live birth rate derived from fresh and frozen-thawed embryo transfers following a single oocyte retrieval, in comparison to a cleavage-stage transfer policy in IVF/ICSI. Study design, size, duration In this multicenter randomized controlled trial women were randomly allocated to blastocyst-stage transfers (blastocyst group - fresh embryo transfer on day 5 after oocyte retrieval followed by vitrification of remnant blastocysts on day 5 and 6 following local criteria) or cleavage-stage transfers (cleavage-stage group - fresh embryo transfer on day 3 after oocyte retrieval followed by embryo cryopreservation on day 3 or 4). Randomization was stratified for age (≥36 or &amp;lt; 36 years). Participants/materials, setting, methods Women with a good prognosis after IVF/ICSI (defined as presenting ≥4 available embryos on day 2 of embryo culture), during their first, second, or third treatment cycle, were included. The primary outcome was the cLBR per oocyte retrieval, including associated frozen-thawed embryo transfers within 12 months after randomization (or 17 months during the COVID pandemic). Risk ratios (RR) with 95% CI adjusted for age group were calculated using log-linear binominal regression. Main results and the role of chance A total of 1202 women from 21 Dutch centers were randomly assigned to blastocyst-stage transfers (N = 599) or cleavage stage transfers (N = 603) between 2018 and 2021. At submission of this abstract, data on the primary outcome was available for 1153 (95.9%) women, 577 women in the blastocyst-stage group and 576 women in the cleavage-stage group. The cumulative live birth rate was 58.2% (336/577 women) in the blastocyst-stage group and 57.3% (330/576 women) in the cleavage stage group (RR 1.022, 95% CI 0.844-1.237; p = 0.825). The live birth rate after fresh embryo transfer was 38.0% (219/577 women) versus 29.9% (172/576 women) in the blastocyst-stage group and cleavage-stage group respectively (RR 1.282, 95% CI 1.017-1.615 p = 0.035). Interaction was found between age and day of transfer with a higher cumulative live birth rate and a higher live birth rate after fresh transfer in women of 36 years or older in the blastocyst group. Analyses on other IVF treatment outcomes, obstetrical or neonatal outcomes, patient burden, and cost effectiveness are ongoing. Limitations, reasons for caution Outcomes are only applicable for treatments of women with at least four embryos available on day two of embryo culture. Wider implications of the findings A blastocyst-stage embryo transfer policy did not result in a significant higher cumulative live birth rate in comparison to a cleavage-stage embryo transfer policy in IVF/ICSI treatments. Further research into the interaction of age with outcomes is warranted. Trial registration number NTR7034

O-210 Detection of small copy number variations as incidental findings in PGT-A: clinical utility from a multisite experience including 12,157 patients

Abstract Study question What is the technical accuracy and clinical utility of reporting small copy number variants (CNVs below 3Mb) detected by a targeted next-generation sequencing-based PGT-A platform? Summary answer Some pathogenic or likely pathogenic CNVs &amp;lt;3Mb can be accurately detected by this assay, increasing clinical utility of PGT-A for a subset of IVF patients. What is known already CNVs are linked to a wide range of phenotypes, spanning from syndromes that include reduced penetrance and variable expressivity to more severe phenotypes. Prenatally, the prevalence of pathogenic CNVs is approximately 1.5%. Most PGT-A platforms that rely on whole genome amplification and shallow sequencing have a resolution limit of 5-10 Mb, preventing the detection of smaller CNVs. Here, we report an innovative PGT-A assay that interrogates thousands of targeted sites in the genome to provide robust copy number analysis allowing for the identification of some small CNVs (incidental findings, IF), outside the primary scope of detecting whole-chromosome aneuploidies in PGT-A. Study design, size, duration Retrospective observational study performed between 2020-2022, involving 12,157 patients who underwent PGT-A performed by targeted-NGS (PGTseq-A) for whole chromosome and large segmental aneuploidies. If an IF &amp;lt; 3 Mb was detected in multiple embryos, the couple was advised to undergo follow-up analysis by chromosomal microarray (CMA) to confirm the parental origin of the CNV, define its breakpoints and determine whether it is classified as benign-likely benign (B/LB), variant of uncertain significance (VUS) or pathogenic-likely pathogenic (P/LP). Participants/materials, setting, methods The PGTseq-A assay employed amplifies 5,000 amplicons across the genome to evaluate copy number. Validation was performed using 5-cell samples from cell lines with known CNVs, and trophectoderm biopsies from embryos with known parental structural rearrangements. An IF was reported when a gain/loss of at least three consecutive amplicons appeared in at least two embryos from the same cycle. Transfer data was reviewed to determine which embryos were transferred. This study received IRB approval. Main results and the role of chance In 77 out of 12,157 PGT-A patients (0.63%;95%CI:0.5-0.8%), an IF that met reporting criteria was identified. To determine the size and pathogenicity, a CMA follow up was requested and performed on 67 couples. In all cases, one of the partners was confirmed to have the CNV identified in the embryos (100.0%: N 67/67 95%CI:94.6-100). The identified CNV was of maternal origin in 36 cases (53.7%) and of paternal origin in 31 cases (46.2%). A strong correlation was identified between PGT-A-predicted CNVs and the genomic coordinates defined by the CMA on parental DNA (r = 0.86). All CNVs intervals predicted by the PGT-A included the CMA genomic region. Twenty-seven (40.2%) were classified as B/LB, 31 (46.2%) as a VUS, and 9 (13.4%) as P/LP. Six of the nine P/LP cases (66.6%) involved imbalances of 16p. The remaining P/LP cases involved deletions of X, 15q and 18p11.32. From a review of transfer data, patients typically transferred embryos negative for IFs as first option regardless of the morphology and pathogenicity classification of CNV-positive sibling embryos. Specifically, in 8 cycles where a detected CNV was classified as B, LB or as VUS, negative embryos were prioritized for transfer despite their poorer morphology. Limitations, reasons for caution De novo CNVs were not considered by design because the reported CNVs had to be present in at least two embryos from the same cohort. Furthermore, the detection of small CNVs is not uniform in the genome and the negative predictive value of the assay for non-targeted regions is null. Wider implications of the findings This PGT-A assay accurately detects small pathogenic CNVs without prior knowledge of parental inheritance, enhancing clinical utility in a subset of patients. Additionally, reporting of VUS and B/LB findings may impact couples’ decision-making concerning embryo selection, reinforcing that scientific rigor is a necessity when evaluating the capabilities of PGT-A. Trial registration number not applicable

P-175 Human cumulus cell telomere length and its association with assisted reproduction outcomes

Abstract Study question Is there any relationship between the relative telomere length (RTL) within cumulus cells (CCs) and the outcome of assisted reproductive treatment using the corresponding oocyte? Summary answer Lower RTLs in CCs were significantly associated with embryos chosen for transfer or cryopreservation. In contrast, embryos considered non-viable (discarded) tended to have higher RTLs. What is known already Cumulus cells fulfil vital roles in support of oocyte development, including the transduction of external signals and the provision of resources via transzonal projections. Given their essential role in the acquisition of oocyte developmental competence, the biology of CCs is of clinical relevance. Telomeres are specialised structures protecting the ends of chromosomes, composed of repetitive DNA sequences and associated proteins. Telomeres shorten with each mitotic division, as well as due to oxidative damage, eventually reaching a critical threshold at which point cellular senescence occurs. Currently, published data on CCs telomere length and relationship with oocyte potential are conflicting. Study design, size, duration The study involved 182 human CC samples collected from 52 IVF patients. Quantitative PCR (qPCR) was used to measure the relative telomere length in each of the CC samples. Telomere lengths were assessed for associations with various patient characteristics (e.g. age, body mass index, infertility diagnosis). Additionally, potential relationships with clinically relevant oocyte/embryo features were investigated (fertilisation; development/morphology), as well as the eventual fate of the associated embryo (transferred; cryopreserved for potential future use; discarded). Participants/materials, setting, methods Real-time quantitative PCR was carried out using PCR primers specific for the telomere repeat. A single-copy gene was also amplified from each CC sample. Quantification of this gene was used for normalisation of the telomere data, allowing control for variation in the number of cumulus cells in each sample. Associations between RTL in CCs and patient, embryonic, and clinical factors were assessed using various statistical methods, with P-values &amp;lt;0.05 considered significant. Main results and the role of chance No associations were identified between RTL and any patient characteristics, except for BMI. The amount of CC telomeric DNA tended to be greater for patients with higher BMI (P = 0.002). When considering links between RTL and oocyte or embryonic factors, a significant relationship was detected between the quantity of telomeric DNA in CCs and whether the corresponding embryo was considered non-viable (discarded) or whether it was transferred or cryopreserved (P = 0.019). This finding raises the possibility that measurement of RTL in CCs could provide a pre-conception, non-invasive assessment of oocyte quality. In the context of fertility preservation, RTL measurement could assist in evaluating a cohort of oocytes, indicating whether the cryopreserved eggs are likely to be sufficient or whether additional cycles to generate more would be advisable. If future studies confirm that CC RTL has a strong predictive value, the possibility of limiting fertilisation to oocytes considered to have high likelihood of viability could also be considered for routine IVF cycles. It is unclear why shorter CC telomeres might be associated with oocytes of superior potential, but one possibility is that the cells may have undergone a greater proliferation (more mitoses), resulting in a more extensive cumulus mass supporting the enclosed oocyte. Limitations, reasons for caution Before drawing definitive conclusions, confirmation of the findings within a larger, independent data set is necessary. Even if confirmed, determination of the true clinical value of telomere assessment will require further, appropriately designed studies. The current study was not powered to evaluate relationships between CC telomere lengths and IVF outcomes. Wider implications of the findings Currently, simplistic morphological evaluation is the only method for assessing oocyte competence prior to fertilisation. If CCs telomere measurement is confirmed to have predictive value, a preconception test of oocyte potential could be offered. This would be extremely valuable for patients cryopreserving oocytes for fertility preservation and for donor banks. Trial registration number NA

P-792 The effect of subendometrial injection of autologous platelet rich plasma (PRP) guided by hysteroscopy in recurrent implantation failure patients - A pilot study

Abstract Study question Does the subendometrial injection of autologous platelet-rich plasma (PRP) guided by hysteroscopy achieve successful pregnancy outcome in recurrent implantation failure patients? Summary answer In this study we observed that subendometrial injection of autologous PRP is associated with higher implantation rates in recurrent implantation failure patients. What is known already The key success factors in IVF are to have a good endometrium lining and vascularity for successful implantation. These two factors directly affect the pregnancy outcomes of IVF. Although several approaches have been attempted to address these factors including estrogen treatment, aspirin, sildenafil, vitamin E, Granulocyte-Colony Stimulating Factor (G-CSF), there is no efficient method described. Additionally, some cases do not show a response to the medical treatment. In those cases, specifically, in previously failed IVF patients autologous Platelet Rich Plasma (PRP) injection into the sub-endometrial cavity with an ovum pick up needle has been preferred, as confirmed by literature. Study design, size, duration An open label, single center study. Total 130 women were selected for hysteroscopic instillation of PRP, all patients have previous unsuccessful IVF attempts in our clinic or were referred to our clinic. Study participants are of age 23 to 49 years. Study began in July 2020 and was completed in December 2022. Participants/materials, setting, methods 2 ml of autologous PRP was instilled in the junctional zone using a wallace needle through the hysteroscope's operating channel to inject the PRP beneath the superficial endometrium from day 6-14 of menstrual cycle. FET preparation was started from Day 2/3 of next menstrual cycle using 6 mg/daily estradiol hemihydrate (EV Active Tab CORONA Remedies). Main results and the role of chance Women who received hysteroscopic instillation of PRP in the sub-endometrial junction had higher pregnancy rates (82/130 [63.07%] (P-value = &amp;lt;0.05). In the subsequent cycle monitored to day 15, Endometrial thickness (ET) was 7 mm or thicker in 90 of 130 patients [69.23%] (P-value = &amp;lt;0.05) and 6-7 mm in 8 of 130 patients [6.15%]. Endometrial thickness did not improve in 32 of 130 patients [24.61%] and remained below 6 mm. Subendometrial blood flow increased significantly in 92 of 130 patients [70.76%] (P-value = &amp;lt;0.05). The mean increase in ET was 1.5 to 2 mm. The instillation of PRP caused no side effects and was well tolerated by all patients. In 47 patients (36.15%) we did not achieve clinical pregnancy while 1 had a biochemical pregnancy Based on paired ‘t’ test, it is concluded that there is statistically significant improvement in ET, vascularity and successful pregnancy outcome after PRP instillation. Limitations, reasons for caution Little is known about the best dosage and time for the instillation of PRP in endometrium. The importance of standard yet simple techniques and a highly repeatable protocol for clinical use is deciding factor which yet to finalize. It involves invasive procedure so may cause infections and anesthesia related complications. Wider implications of the findings Although PRP has still not gained universal acceptance in previously failed IVF patients for a variety of reasons, emerging evidences suggests that PRP could be an effective option compared to conventional approach especially, in patients with thin endometrium, advanced maternal age and primary ovarian insufficiency. Trial registration number Not applicable