Euploid Blastocyst Transfer Research Articles

Transcriptomic analysis of mid-secretory endometrium reveals essential immune factors associated with pregnancy after single euploid blastocyst transfer.

Implantation is a limiting factor for treatment success in assisted reproduction. Both embryonic and endometrial factors contribute to implantation. Embryonic factors have often been ignored in previous studies about the role of endometrium in implantation. In this study, we sought to identify the endometrial genes associated with negative pregnancy outcomes following the transfer of a single euploid blastocyst. Computational analyses of the transcriptomes of mid-secretory endometria from nine pregnant and seven non-pregnant patients in a cycle preceding the transfer of a single euploid blastocyst in a vitrified-warmed cycle were performed. Principal component analysis of two reported endometrial receptivity gene sets showed close clustering of the pregnant and non-pregnant samples. Differential gene expression analysis and co-expression module analysis identified 131 genes associated with the pregnancy status. The endometrial signatures identified highlight the importance of immune and metabolic regulation in pregnancy outcome. Network analysis identified 20 hub genes that could predict pregnancy outcomes with88.9% sensitivity and 85.7% specificity. Single-cell gene expression analysis highlighted the regulation of endometrial natural killer cells, T cells, and macrophages during embryo implantation. Immune cell abundance analysis supported the dysregulation of cytotoxic immune cells in the endometria of non-pregnant women. We reported the first endometrial gene signature associated with pregnancy after elimination of embryo aneuploidy and highlighted the importance of the endometrial immune microenvironment and metabolic status in pregnancy outcomes. This article is protected by copyright. All rights reserved.

The developmental competence of human metaphase I oocytes with delayed maturation in vitro

To evaluate whether metaphase I (MI) oocytes completing maturation invitro to metaphase II ("MI-MII oocytes") have similar developmental competence as the sibling metaphase II (MII) oocytes that reached maturity invivo. Retrospective cohort study. Academic medical center. A total of 1,124 intracytoplasmic sperm injection (ICSI) cycles from 800 patients at a single academic center between April 2016 and December 2020 with at least 1 MII oocyte immediately after retrieval and at least 1 sibling "MI-MII oocyte" that was retrieved as MI and matured to MII in culture before ICSI were included in the study. None. A total of 7,865 MII and 2,369 sibling MI-MII oocytes retrieved from the same individuals were compared for the fertilization and blastocyst formation rates. For patients who underwent single euploid blastocyst transfers (n = 406), the clinical pregnancy, spontaneous pregnancy loss, and live birth rates were compared between the 2 groups. The fertilization rate was significantly higher in MII oocytes than in delayed matured MI-MII oocytes (75.9% vs. 56.1%). Similarly, the blastocyst formation rate was higher in embryos derived from MII oocytes than in those from MI-MII oocytes (53.8% vs. 23.9%). The percentage of euploid embryos derived from MII oocytes was significantly higher than that of those from MI-MII oocytes (49.2% vs. 34.7%). Paired comparison of sibling oocytes within the same cycle showed higher developmental competence of the MII oocytes than that of MI-MII oocytes. However, the pregnancy, spontaneous pregnancy loss, and live birth rates after a single euploid blastocyst transfer showed no statistically significant difference between the 2 groups (MII vs. MI-MII group, 65.7% vs. 74.1%, 6.4% vs. 5.0%, and 61.5% vs. 70.0%, respectively). Compared with oocytes that matured invivo and were retrieved as MII, the oocytes that were retrieved as MI and matured to MII invitro before ICSI showed lower developmental competence, including lower fertilization, blastocyst formation, and euploidy rates. However, euploid blastocysts from either cohort resulted in similar live birth rates, indicating that the MI oocytes with delayed maturation can still be useful even though the overall developmental competence was lower than that of their invivo matured counterparts.

Reproductive genetics laboratory may impact euploid blastocyst and live birth rates: a comparison of 4 national laboratories' PGT-A results from vitrified donor oocytes

To evaluate potential variation in the euploid blastocyst rate and live birth rate (LBR) per single frozen euploid blastocyst transfer, among 4 unique United States reproductive genetics laboratories. Analyses were limited to blastocysts derived from vitrified donor oocytes, to minimize variation in oocyte quality. Retrospective cohort study from 2016 to2020. Donor Egg Bank Database. Patients undergoing invitro fertilization with donor oocytes. We excluded patients with uterine factor, male factor, or surgically extracted sperm. Only healthy women <34 years old were accepted as oocyte donors. Next generation sequencing platforms for chromosomal analysis MAIN OUTCOME MEASURE(S): Euploid blastocyst rate and LBR per euploid transfer. Secondary outcomes included the rate of aneuploidy, mosaic calls, biochemical pregnancy loss, and miscarriage rate. A total of 2,633 embryos were included. Four laboratories had >200 embryos tested. Euploid blastocyst rate was significantly higher in laboratory A (73.6%) vs. B (63.3%), C (60.9%), and D (52.3%). Mosaic rate was significantly lower for Laboratories B (2.8%) and C (5.5%) vs. Laboratories A (9.9%) and D (11.5). The LBR was significantly higher in laboratory A (58.73%) vs. laboratory D (47.3%). There were no significant differences in the implantation or miscarriage rate among laboratories. In this large study, controlling for oocyte quality, some preimplantation genetic testing for aneuploidy (PGT-A) laboratories report a significantly higher euploid blastocyst rate with concurrent higher LBR. This type of comparison is important as it provides insight into the role of the PGT-A process in outcomes. Further research is needed to evaluate the differences in laboratory techniques and bioinformatic algorithms accounting for variable outcomes across PGT-A laboratories.

Natural cycle increases the live-birth rate compared with hormone replacement treatment for frozen-thawed single euploid blastocyst transfer.

A number of studies have compared the clinical outcomes between the two endometrial preparation methods: natural cycles (NCs) and hormone replacement treatment (HRT) before frozen embryo transfer, but the results were conflicting. In order to mitigate the potential effect of embryos per se, several researchers have worked on this subject for euploid blastocyst transfer, but the results were still inconsistent. Therefore, the present study was aimed to investigate the clinical outcomes between HRT and NC for autologous single vitrified-warmed euploid blastocyst transfer based on our data. A total of 598 frozen-thawed single euploid blastocyst transfer cycles in the assisted reproductive center of Northwest Women's and Children's Hospital from January 2014 to May 2021 were retrospectively analyzed. Women were stratified into the NC (n = 125) or HRT (n = 473) group according to the patient's preference and the physician's guidance. Multivariate regression models and subgroup analysis were constructed to analyze the association between endometrial preparation and live birth. Women in the NC group had a higher live birth rate (68.80% versus 58.35%, P = 0.034) and a lower risk of total pregnancy loss (8.51% versus 21.14%, P = 0.005) when compared with women in the HRT group. The biochemical pregnancy rate (75.20% versus 74.00%, P = 0.784) and clinical pregnancy rate (74.40% versus 69.98%, P = 0.334) were similar between the two groups (NC versus HRT). NC was associated with an increased odds of live birth compared with HRT by different multivariable analysis models (Model 1: adjusted odds ratio [aOR], 95% confidence interval [CI]: 0.57, 0.36 - 0.90; Model 2: aOR, 95%CI: 0.57, 0.35 - 0.92). In addition, the increased chance of live birth in the NC group was found in all subgroups. No major obstetrical complications and two malformation livebirths were reported. In women undergoing single euploid frozen blastocyst transfers, the NC group was associated with a lower pregnancy loss rate and an ultimately higher live birth rate than the HRT group. Although HRT is convenient for both clinicians and patients, the lower live birth rate should be taken into account and NC might be the first choice of endometrial preparation method.

Is there an optimal window of time for transferring single frozen-thawed euploid blastocysts? A cohort study of 1170 embryo transfers.

Is there an optimal window of time when the transfer of single frozen-thawed euploid blastocysts is associated with a maximal live birth rate (LBR)? Performing a single frozen-thawed euploid blastocyst transfer at 160 ± 4 h post-hCG trigger in modified-natural frozen-thawed embryo transfer (FET) cycles was independently associated with a higher LBR as compared to transfers outside this window; however, in natural FET cycles, LBRs were comparable across a wider range of time intervals. There is compelling evidence for maintaining embryo-endometrial synchrony to optimize clinical outcomes following FETs, which could potentially be achieved by matching the transfer time of an embryo post-ovulation to its developmental age post-oocyte retrieval. For modified-natural cycles, ovulation is widely accepted to occur ∼40 h following the hCG trigger, whilst ovulation following spontaneous LH surge onset is thought to vary from 24 to 56 h. This is a multicentered retrospective cohort study analyzing 1170 single frozen-thawed euploid blastocyst transfers following trophectoderm biopsy and preimplantation genetic testing (PGT) between May 2015 and February 2019. Limiting the analysis to single euploid embryo transfers allowed for a more accurate estimation of the endometrial synchrony factor by controlling for the developmental stage of the embryo (full blastocyst or more advanced) and its genetic composition. LBR per FET was the primary outcome measure. Patients underwent natural or gonadotrophin-induced preparation of the endometrium, with serial serum oestradiol, LH and progesterone measurements. Optimally timed transfers were predefined as those conducted 120 ± 4 h post-ovulation since biopsy and subsequent cryopreservation of full blastocysts which is usually performed at 116-124 h post-oocyte retrieval. This was considered the equivalent of 160 ± 4 h post-hCG trigger in modified-natural cycles (n = 253), as ovulation was assumed to occur ∼40 h after the hCG trigger. For natural cycles (n = 917), this was also considered to be, on average, 160 ± 4 h post the spontaneous LH surge. Thus, study groups were determined as those with optimal timing or not, and additional exploratory and subgroup analyses were performed, varying the time window in terms of onset and width, both overall and per endometrial preparation protocol. Statistical analysis was performed using the generalized estimating equations (GEE) framework to control for the clustered nature of the data while adjusting for potential confounders. Overall, LBRs were significantly higher when the transfer had been performed at 160 ± 4 h post-hCG trigger or LH surge onset compared to when it had been performed outside this window (44.7% vs 36.0%; P = 0.008). A multivariable regression GEE model including the cycle type (natural versus modified-natural), previtrification embryo quality (top versus good quality), embryo stage (fully hatched versus hatching or earlier blastocyst), vitrification day (D5 versus D6) and survival rate (>90% versus <90%) as covariates, confirmed that, overall, embryo transfers conducted 160 ± 4 h post-hCG trigger or LH surge onset (the assumed equivalent of 120 ± 4 h post-ovulation) were associated with a significantly higher LBR (relative risk (RR) 1.21, 95% CI 1.04-1.41). Subgroup exploratory analyses per endometrial preparation protocol demonstrated that these findings were primarily present in the modified-natural cycle group (RR 1.52, 95% CI 1.15-1.99), whilst the natural cycle group showed comparable LBRs across a wider range of time intervals. Moreover, the overall LBR for the natural group (36.8%; 95% CI 33.7-39.9%) was lower than that of the modified-natural group (41.3%; 95% CI 35.4-47.1%), suggesting that there likely remains a greater potential to further optimize the timing of natural cycle embryo transfers. As with all retrospective studies, the presence of residual unknown bias cannot be excluded. Additionally, patients included in this study were a selected group who underwent PGT for specific reasons and hence the results obtained might not be directly applicable to the general population or embryos that have not undergone embryo biopsy. Furthermore, the criteria utilized to interpret hormonal data from natural cycles were specifically adopted for the present study and need to be validated in further studies. The results of this study highlight the significance of embryo-endometrial synchrony for the optimization of frozen embryo transfer outcome. However, it also clearly supports that the implantation window is in most cases wide and the achievement of live birth is possible with relatively high success rates even outside the optimal window of 160 ± 4 h post-trigger for modified-natural cycles and across a range of time intervals for natural cycles. Additionally, this study suggests that implantation rates could be further optimized in natural cycles by improving methods of assessing embryo-endometrial synchrony. C. V. is supported by a National Health and Medical Research Council Early Career Fellowship (GNT1147154). No other funding was received for this study and there are no competing interests. N/A.

Autologous Platelet-Rich Plasma Improves Pregnancy Outcomes of Patients with Thin Endometrium Regardless Endometrial Thickening: Multicenter Retrospective Study with Elimination of Embryonic Confounders

Objective: To determine whether endometrial thickening is an important factor for pregnancy outcomes in platelet-rich plasma (PRP) treatment for patients with thin endometrium. Methods: Data from the registry database of the Japan Gynecologic PRP Study Group from April 2019 to October 2021 were analyzed retrospectively. A total of 208 patients who underwent single blastocyst transfer (SBT) after PRP due to thin endometrium (¡8 mm) in their previous cycle were included in the study. Their pregnancy outcomes were compared with their own historical data before PRP infusion. The same patient group was then divided into 136 patients whose endometrium thickened after PRP and 72 patients whose endometrium did not, and the pregnancy outcomes of the two groups were compared. Furthermore, to eliminate embryonic confounding, 28 patients who had single euploid blastocyst transfer (SEBT) with preimplantation genetic testing for aneuploidy (PGT-A) were selected from the same patient group and divided into two groups of 22 patients whose endometrium thickened and 6 patients whose endometrium did not, and the pregnancy outcomes were compared in the same method. Results: After PRP administration, the clinical pregnancy rate, live birth rate, and miscarriage rate were all significantly improved compared with the historical controls (34.1 vs 20.0, [Formula: see text]; 22.6 vs 3.9, [Formula: see text]; 33.8 vs 73.4, [Formula: see text]). There were no statistically significant differences in the respective rates between the “thickened endometrium” group and the “unthickened endometrium” group in SBT group (36.0 vs [Formula: see text], [Formula: see text]; 25.7 vs [Formula: see text], [Formula: see text]; 28.5 vs [Formula: see text], [Formula: see text], respectively). Likewise, in the SEBT with PGT-A group, there were no statistically significant differences in results between the two groups (36.4 vs [Formula: see text], [Formula: see text]; 36.4 vs [Formula: see text], [Formula: see text]; 0 vs [Formula: see text], respectively). Conclusions: PRP administration to patients with thin endometrium improves pregnancy outcomes even when endometrial thickening is not achieved.

IMPACT OF FRESH-EMBRYO PARAMETERS ON ONGOING PREGNANCY IN VITRIFIED/WARMED EUPLOID BLASTOCYST CYCLES

To evaluate the independent effect of the morphological parameters on predicting ongoing pregnancy after vitrified/warmed euploid blastocyst transfer.

ENDOMETRIAL PREPARATION BY HORMONE REPLACEMENT AND DAY 6 BIOPSIED EUPLOID BLASTOCYST TRANSFER (BT) INCREASES THE RISK OF MISCARRIAGE COMPARED WITH AN OVULATORY CYCLE AND DAY 5 BIOPSIED EUPLOID BT

Accepted theory posits that more than half of the causes of pregnancy loss involve aneuploidy of the transferred embryos or blastocysts. This explanation has recently come into question, however, due to the clinical application of preimplantation genetic testing for aneuploidy (PGT-A). In an effort to further elucidate the risk factors for miscarriage, we retrospectively evaluated the clinical data between ongoing pregnancies and cases of miscarriage following euploid blastocyst transfer.

Inflammatory Bowel Disease and Pregnancy

Inflammatory Bowel Disease and Pregnancy

HOW MANY EUPLOID BLASTOCYSTS NEED TO BE TRANSFERRED IN OBESE PATIENTS TO ACHIEVE A LIVE BIRTH? AN ENDLESS PATIENT-DOCTOR NEGOTIATION STORY

To evaluate live birth rates of single and double euploid blastocyst transfer in relation to patients' Body Mass Index (BMI).

Day of trophectoderm biopsy and embryo quality are associated with outcomes following euploid embryo transfer.

To compare clinical outcomes following transfer of euploid blastocysts of varying quality biopsied on day 5 versus day 6. Retrospective cohort study to evaluate embryo transfer outcomes for women undergoing autologous cryopreserved next generation sequencing euploid single embryo transfer from 10/2015 to 2/2022 at an academic IVF program. The primary outcome was live birth rate (LBR). Secondary outcomes included ongoing pregnancy rate (OPR), implantation rate (IR), and miscarriage rate (SAB rate). Five hundred and fifty-five transfers from 418 patients were analyzed. Euploid embryos biopsied on day 5 resulted in higher LBR compared to those biopsied on day 6 (62.3% vs. 49.6%; aRR 0.81 95% CI 0.65-0.996). When stratified by biopsy day and blastocyst quality, there was no difference in IR, OPR, and SAB rate for good, fair, and poor quality blastocysts biopsied on day 5 versus day 6. However, day 5 good quality embryos were associated with a higher LBR compared to day 6 good quality embryos (74.3% vs. 51.3%; aRR 0.69; 95% CI 0.48-0.999). There were no significant differences in LBR for fair and poor quality embryos biopsied on day 5 versus day 6. Overall LBR are higher for euploid embryos biopsied on day 5 versus day 6. When stratified by embryo quality and day of biopsy, LBR are significantly higher for good quality day 5 versus day 6 embryos. When choosing between multiple euploid embryos, day 5 biopsied good quality embryos should be preferentially selected for transfer over day 6 embryos of the same quality.

Is Cleavage Stage Multinucleation Indicative of Lower Pregnancy Potential in Euploid Blastocysts?

Background: Cleavage stage multinucleation is a morphological assessment IVF labs may use to rank embryos for transfer, as blastocysts that were multinucleated at the 2-cell or 4-cell stage have a lower live birth rate (LBR) compared to blastocysts that were mononucleated 1 . However, when these blastocysts are PGT-A tested, the euploidy rate is similar 2 , indicating that the poorer pregnancy outcomes may not be due to aneuploidy. It is unknown if multinucleation is indicative of a lower likelihood of pregnancy for euploid blastocysts. Aim: To determine if LBR following transfer of euploid blastocysts is lower when blastocysts were multinucleated at the 2-cell or 4-cell stages. Method: Retrospective study on 360 euploid blastocysts cultured in the EmbryoScope or EmbryoScope+ between March 2018 and October 2019, and individually transferred. Time-lapse images were retrospectively assessed for multinucleation at the 2-cell and 4-cell stage. A multinucleated embryo was defined as having [Formula: see text] 1 cell with [Formula: see text] 2 nuclei. Results: Blastocysts that were multinucleated at the 2-cell stage (n=139) had a similar LBR to mononucleated embryos (40.3% vs 41.6%). Multinucleation at the 4-cell stage (n=43) also did not significantly affect LBR (37.2% vs 43.6%). Multivariate logistic regression analysis found no significant effect of multinucleation on LBR. Biochemical pregnancy, clinical pregnancy (fetal heart), and miscarriage rates were also similar between multinucleated and mononucleated embryos. Conclusion: This study found no evidence that multinucleation at the 2-cell or 4-cell stage influences pregnancy rate in euploid blastocysts. As such, the assessment of cleavage stage multinucleation for PGT-A tested embryos may not be useful in ranking PGT-A tested embryos for transfer. Further work is required with larger numbers of embryos to confirm how the degree of multinucleation may influence LBR.

Why Euploid Blastocysts Fail

Preimplantation genetic testing for aneuploidy improves implantation and reduces miscarriage rates, but still about 30 to 35% of euploid blastocysts fail to implant and 10 to 13% of pregnancies following euploid blastocyst transfer miscarry. Both endometrial and embryo factors may contribute to failed euploid embryo transfers. Endometrial factors include uterine structural abnormalities, altered timing of endometrial receptivity, glandular-stromal disynchrony, and inflammatory states. Recent studies question the clinical benefit of some recent assays for endometrial receptivity. Other recent studies question the value of surgical correction of uterine septae and fibroids to prevent further pregnancy loss. Embryo factors include aneuploidy, either undetected by PGT-A performed on the blastocyst trophectoderm (i.e. false negative test) or de novo non-disjunction. Sub chromosomal genetic variation, e.g. copy number variants and de novo retrotransposon insertions, may impair embryo viability. We employ a “genetics first” approach, which relies on genetic analysis of products of conception, obtained by uterine evacuation, to evaluate miscarriage after euploid blastocyst transfer. Non-invasive genetic analysis of first trimester pregnancies also may help characterize the cause of embryo loss after euploid embryo transfer. Early results suggest that analysis of cfDNA and trophoblast retrieval and isolation from the cervix (TRIC) may allow women experiencing early pregnancy loss to learn about the genetic state of their miscarriage, without an invasive surgical procedure to obtain products of conception.

Impact of inactivated SARS-CoV-2 vaccination on embryo ploidy: a retrospective cohort study of 133 PGT-A cycles in China

BackgroundUnsubstantiated concerns have been raised on the potential correlation between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination and infertility, leading to vaccine hesitancy in reproductive-aged population. Herein, we aim to evaluate the impact of inactivated SARS-CoV-2 vaccination on embryo ploidy, which is a critical indicator for embryo quality and pregnancy chance.MethodsThis was a retrospective cohort study of 133 patients who underwent preimplantation genetic testing for aneuploidy (PGT-A) cycles with next-generation sequencing technology from June 1st 2021 to March 17th 2022 at a tertiary-care medical center in China. Women fully vaccinated with two doses of Sinopharm or Sinovac inactivated vaccines (n = 66) were compared with unvaccinated women (n = 67). The primary outcome was the euploidy rate per cycle. Multivariate linear and logistic regression analyses were performed to adjust for potential confounders.ResultsThe euploidy rate was similar between vaccinated and unvaccinated groups (23.2 ± 24.6% vs. 22.6 ± 25.9%, P = 0.768), with an adjusted β of 0.01 (95% confidence interval [CI]: -0.08–0.10). After frozen-thawed single euploid blastocyst transfer, the two groups were also comparable in clinical pregnancy rate (75.0% vs. 60.0%, P = 0.289), with an adjusted odds ratio of 6.21 (95% CI: 0.76–50.88). No significant associations were observed between vaccination and cycle characteristics or other laboratory and pregnancy outcomes.ConclusionsInactivated SARS-CoV-2 vaccination had no detrimental impact on embryo ploidy during in vitro fertilization treatment. Our finding provides further reassurance for vaccinated women who are planning to conceive. Future prospective cohort studies with larger datasets and longer follow-up are needed to confirm the conclusion.

P-786 Perinatal outcomes of assisted reproduction technology (ART) pregnancies after transfer of mosaic blastocysts: a single centre matched cohort study

Abstract Study question Do viable pregnancies after transfer of mosaic blastocysts present different perinatal outcomes as compared to those deriving from transfer of euploid blastocysts? Summary answer Pregnancies from transfer of mosaic vs. euploid blastocysts showed an increased rate of fetal structural malformations and postpartum haemorrhages (PPH). What is known already The incidence of mosaicisms in the trophoectoderm is a relatively frequent event and most true embryo mosaicisms result in spontaneous intrauterine demise. The confirmation of the mosaic aneuploidy in the fetus is related to the type and grade of the aneuploidy with higher risk for chromosomes 13, 14, 15, 16, 18 and 21 and X monosomy. On the other hand, selected mosaic blastocysts have been transferred with subsequent birth of healthy neonates. However, the risk of abnormal obstetric outcomes after transfer of mosaic embryos remains to be completely elucidated. Study design, size, duration Explorative cohort study between 2016 and 2021 including consecutive viable ART pregnancies developing from transfer of mosaic blastocysts (n = 39 cases). Transfer of available mosaic embryos was considered for patients with a poor response to ovarian stimulation or recurrent implantation failures and no available euploid blastocysts, after genetic counselling. Cases were matched for maternal age, parity and body mass index to women with viable pregnancies after transfer of euploid blastocysts (n = 39, controls) and followed-up until delivery. Participants/materials, setting, methods All patients recruited at San Raffaele, Scientific Institute of Milan, underwent a prenatal genetic testing by trophoectoderm biopsy and next-generation sequencing analysis. Patients with a viable fetus at 12 weeks gestation were included and underwent detailed ultrasound assessment at 12, 20 and 32 weeks including fetal biometry and anatomical survey. Frequency of adverse pregnancy and perinatal outcomes including maternal, fetal and delivery complications were compared between patients who received a mosaic versus a euploid blastocyst. Main results and the role of chance No difference in indications to preimplantation diagnosis was found between cases and controls. In the group who transferred mosaic embryos, median rate of mosaicisms was 35% (IQR 30%-40%) and they were characterized by heterogeneous anomalies including complex aneuploidies (n = 12), autosomal monosomies (n = 13), trisomies (n = 8) and duplications/deletions (n = 6). Invasive testing was performed in 62.0% of mosaics (5 chorion villous samplings; 19 amniocenteses; 2 anomalies: confined placental mosaicism trisomy 15 after transfer of mosaic monosomy 6 and fetal microduplication 6p after transfer of mosaic deletion 10p) vs 2.5% of controls (p &amp;lt; 0.001). Structural malformations were observed in 6 neonates in the study group (tanatophoric skeletal dysplasia, complex central nervous system anomaly, congenital diaphragmatic hernia, cerebral arteriovenous fistulae, pyeloureteral junction stenosis, diffuse haemangiomatosis) compared to 1 in controls (pulmonary stenosis) (p = 0.04). One neonate per group was followed-up for future surgery. PPH rate was significantly higher in cases vs controls (15 vs 7; p = 0.04). Neonatal survival at hospital discharge was not different in the two groups (p = 0.30). No significant differences were recorded in rates of preterm birth, small or large for gestational age, fetal growth restriction, miscarriage, preeclampsia and median birthweight centile or gestational age at birth. Limitations, reasons for caution Data from our exploratory study should be interpreted cautiously given the limited sample size which makes it challenging to infer a causal relationship between the association found and transfer of mosaic blastocysts. Wider implications of the findings The higher rates of structural malformations and PPH in ART pregnancies after transfer of mosaic embryos strongly suggest the need for strict monitoring of these patients. First and second trimester ultrasound screening for structural defects, and preventive measures to minimize bleeding at delivery should be implemented. Trial registration number not applicable

P-387 Autologous Platelet-Rich Plasma (PRP) intrauterine infusion before embryo transfer could improve the treatment outcomes, especially for transferring euploid blastocysts, in recurrent implantation failure patients

Abstract Study question Is the intrauterine infusion of autologous PRP prior to embryo transfer, especially euploid blastocyst transfer, effective in solving recurrent implantation failure? Summary answer Autologous PRP intrauterine infusion before embryo transfer (ET), especially before transferring euploid blastocysts, could improve the treatment outcomes in RIF patients. What is known already Firstly, autologous PRP intrauterine infusion has been proposed to improve the outcomes of ET in patients having thin endometrium or a history of RIF. Hypothetically, growth factors and cytokines in PRP assist in preparing endometrium surface as well as conducting critical early molecular signaling pathways between the embryos and the uterus. We have developed and optimized a specific PRP infusion protocol for RIF (presented at ESHRE 2021, P-428). Secondly, various reports have suggested that transferring euploid blastocysts have better results than unscreened ones. Here we evaluate the effects of PRP infusion, together with transferring euploid blastocysts in RIF treatment. Study design, size, duration From 11/2020 - 07/2021, all patients who had one PRP infusion before ET were recorded (n = 130). RIF patients were classified as follows: group A (unscreened blastocysts, n = 51 patients) included having at least two ET failures (beta-hCG &amp;lt; 5 IU/L). The embryos in this group did not undergo preimplantation genetic testing for aneuploidy (PGT-A). Group B (euploid blastocysts, n = 50 patients) were cases having at least one ET failure using euploid blastocysts previously screened by PGT-A. Participants/materials, setting, methods Endometrium preparation was followed hormone replacement protocol using E2 and P4 with autologous PRP infusion 48 hours before ET. All ETs in this study were frozen blastocyst ones. Apart from groups A and B, a small group of patients (n = 29) might not have previous unsuccessful ET but had relatively thin endometrium (&amp;lt; 9 mm), hence requiring PRP treatment. Beta-hCG, yolk sac and clinical pregnancy outcomes were recorded. A Chi-square test was applied. Main results and the role of chance 76/130 patients in this study had beta-hCG positive (58.5%). Here, we focused on RIF, therefore only data of groups A and B were analyzed (n = 29 cases without previous ET failures excluded). In group A (n = 51), 26 cases had beta-hCG positive (51%); 21 cases (41.2%) had yolk sac and then clinical pregnancy; five cases had only biochemical pregnancy. In group B (n = 50), 26 cases had beta-hCG positive (52%), 23 cases had gestational sac and then clinical pregnancy (46%); three cases had biochemical pregnancy. PRP infusion had positive impacts on implantation (all groups had &amp;gt;50% beta-hCG positive). The rates of beta-hCG positive in groups A and B were both lower than this rate in total patients suggesting that relatively thin endometrium (&amp;lt; 9 mm) may not critically require PRP infusion. The beta-hCG rates of groups A and B were statistically indifferent (p &amp;gt; 0.05). However, group B had higher average age than group A (37.7 ± 6.5 vs 33.06 ± 4.92), and had several poorer prognosis patients such as patients having low AMH, endometriosis or hydrometrocolpos. This result implied that PGT-A could help older and poorer prognosis RIF patients equal treatment outcomes with the younger and better prognosis RIF patients. Limitations, reasons for caution There were fewer biochemical pregnancy events in group B than group A (three vs. five), however, the study had a small sample size requiring ongoing enrolment to confirm this conclusion. This is impossible for this type of intervention study to have control (a group without PRP infusion). Wider implications of the findings On the side of the endometrium, PRP intrauterine infusion showed positive effects in assisting RIF patients to have implantation. On the embryo's side, embryo selection, especially PGT-A for euploid ones, could critically contribute to successful implantation and pregnancy. Trial registration number Not applicable

P-408 IL-10 and IL-17 pathway enrichment in uterine fluid-derived extracellular vesicles (UF-EVs) is associated with failed implantation in women with recurrent implantation failure undergoing PGT-A

Abstract Study question Can the UF-EVs transcriptome provide markers of implantation in women diagnosed with recurrent implantation failure (RIF)? Summary answer Enrichment in IL-10 and IL-17 pathway transcripts in UF-EVs of women with RIF might identify those at risk for negative outcome after euploid blastocyst transfer. What is known already Our group has recently found that transcriptomic changes occur in UF-EVs during the window of implantation and closely resemble those occurring in the endometrial tissue. Patients affected by RIF represent a debated and heterogeneous population of patients who currently undergo several investigations, empirical therapies and embryo transfer attempts, with inconclusive results. We investigated whether UF-EVs can provide markers to define subgroups of patients within the RIF population, to predict outcomes and tailor interventions. Study design, size, duration We herein present the first sub-analysis of our previous larger observational study on the human UF-EVs transcriptome (Giacomini et al., 2021). RIF was defined by &amp;gt; 3 failed embryo transfers (ETs) with high quality embryos and patients affected by RIF and undergoing their first PGT-A cycle were enrolled (n = 19). Fertile women undergoing PGT-A+PGT-M for monogenic diseases and achieving successful implantation were used as controls (n = 29). PGT-A was performed by Next Generation Sequencing. Participants/materials, setting, methods RNA-Seq was performed on UF-EVs collected on day LH + 7 of the cycle preceding that of the blastocyst transfer. Differential Gene Expression (DGE) analysis was performed between RIF patients failing implantation (Group A) versus RIF patients achieving implantation (Group B) and between Group A and fertile controls achieving implantation (Group C). Pre-ranked gene set enrichment (GSEA) with WebGestalt was used for pathway enrichment analysis, with Normalized Enrichment Score (NES) indicating the strength of the enrichment. Main results and the role of chance RIF patients had a mean (± SD) age of 36.6 (± 3.4) and 5.1 ± 2.1 (mean ± SD) previous failed ETs. DGE comparisons between UF-EVs of RIF women who failed implantation (n = 10) showed 135 up- and 32 down-regulated genes compared to Group B, and 258 up- and 44 down-regulated genes compared to Group C, with 63 genes showing consistent significantly different ‘expression’ values in both comparisons. UF-EVs of Group A were enriched in transcripts belonging to the IL-17 pathway compared to both groups (Group A vs B, NES=2.2044, FDR &amp;lt;0.0001; Group A vs C, NES=2.3584, FDR &amp;lt;0.0001).Also transcripts belonging to the IL-10 pathway were enriched compared to both groups (Group A vs B NES=2.2741, FDR = 0.002; Group A vs C NES=2.2047, FDR &amp;lt; 0.0001). Among genes enriched in both DGE comparisons, C-X-C motif chemokine ligand (CXCL)1, CXCL2, CXCL8, and prostaglandin-endoperoxide synthase 2 (PTGS2) belonged to both the IL-10 and IL-17 pathway. On multivariate analysis controlling for relevant confounders (i.e. number of previous failed ETs), expression levels of CXCL8 emerged as a significant negative predictor of implantation (OR 0.675, 95%CI 0.493-0.925, p = 0.01). Limitations, reasons for caution As the main limitation of our study, we collected UFs in the cycle immediately preceding (and not corresponding to) the euploid ET. This approach was considered the most appropriate in relation to the current paucity of data regarding the safety of UF aspiration in the same cycle of an ET. Wider implications of the findings UF-EVs represent a source of transcriptomic markers related to the endometrial immune profile in RIF patients. Enrichment in the IL-17 and IL-10 pathways in this population could define the subgroup who is at risk for implantation failure even after euploid ET and who could potentially benefit from tailored immunotherapies. Trial registration number not applicable

P-128 Role of PGT-A and ERA to optimise reproductive outcomes of couples with recurrent implantation failure

Abstract Study question In couples with Recurrent Implantation Failure (RIF), can Pre-implantation Genetic Testing for Aneuploidy (PGT-A) or Endometrial Receptive Array (ERA) help to optimise the reproductive outcomes? Summary answer In RIF patients PGT-A or ERA individually do not seem to optimize outcomes. PGT-A in-combination with ERA seems to be a beneficial intervention. What is known already Role of PGT-A and ERA to optimize reproductive outcomes has been ambigious. In this current study we wanted to evaluate their efficacy in RIF patients. Study design, size, duration This is a retrospective data of couples undergone treatment for RIF at our private fertility centre from January 2014 to July 2019. The study population was divided to four groups based on the treatments offered: Group A: ERA (n = 47) Group B: PGT-A (n = 54) Group C: ERA+PGT-A (n = 49) Group D: No PGT-A/ No ERA (n = 189) Participants/materials, setting, methods Couples with minimum two failed transfers were recruited. ICSI was the method of insemination. Blastocysts were subjected to genetic testing through Next-Generation Sequencing(NGS). All embryo transfers performed in a Frozen Embryo Replacement Cycle(FET). ERA was done as per our institutes Standard Operating Procedures(SOP) and for ERA patients ET was done as per the receptivity status. Elective Single Euploid Blastocyst transfer (eSET) was done in grpB&amp;C un-screened double Blastocyst Transfer (DET) was done in grpA&amp;D. Main results and the role of chance Clinical Pregnancy Rate (CPR), Miscarriage Rate (MR) Implantation Rate (IR), Multiple Pregnanacy rates (MPR) and Live Birth Rate (LBR) was evaluated in all groups. Outcomes of groups A, B, C &amp; D were as follows: CPR – 68.09% Vs 62% Vs 68.42% Vs 70% IR – 48.89% Vs 47.50% Vs 65.22% Vs 45% MR – 12.77% Vs 9.25% Vs 5.26% Vs 17.89% MPR – 16% Vs 0% Vs 0% Vs 17% LBR – 51.06% Vs 46.8% Vs 63.16% Vs 45% CPR was comaparble between all groups ERA &amp; PGT-A group seems to have the best IR, LBR with least miscarriage rates. For RIF patients role of PGT or ERA when offered individually, doesn’t seem to optimize outcomes. PGT-A in-combination with ERA seems to be beneficial to optimize outcomes. Limitations, reasons for caution This data is retrospective and there is a need for a well designed RCT to further validate the conclusion of this study. Caution needs to be exercised while offering PGT and ERA as they incurr additional financial burden to the patients. Wider implications of the findings Embryo Transfer cycles with a personalised window of implantation and transfer of a single Euploid embryo can help RIF patinets to have the best reproductive outcomes and can also help shorten the time to pregnancy. Trial registration number Not applicable

O-255 Clinical and laboratory factors associated with pregnancy outcomes in patients undergoing frozen euploid blastocyst transfer

Abstract Study question Which variables do have an impact on the pregnancy and live birth rates (LBR) when euploid frozen embryo transfers (FET) are performed? Summary answer Day of trophectoderm biopsy, Body Mass Index (BMI) and endometrial preparation protocol have an impact on pregnancy rate (PR) and LBR in FET cycles. What is known already Preimplantation genetic testing for aneuploidy (PGT-A) and morphological grading of embryos are the two main criteria to select a blastocyst from a pool of embryos, having the highest implantation potential. However, other clinical and laboratory variables might play a crucial role for a successful outcome when top quality euploid embryos are transferred in a FET cycle. It has been described that higher BMI increases the odds for miscarriage when compared with non-obese women. Study design, size, duration This analysis includes 1660 FET cycles with data from of pregnancy rates, miscarriage rate and LBR. Embryos were obtained from 2564 IVF/ICSI cycles of couples with primary or secondary infertility at ART Fertility Clinics UAE, from November 2016 to December 2020. Participants/materials, setting, methods A total of 1660 FET cycles with 2439 euploid blastocysts were included. FET cycles with mosaic or segmental aneuploid embryos were excluded. PGT-A was performed on trophectoderm cells, using Next Generation Sequencing (NGS). Biopsy was performed on day 5, 6 or 7 blastocysts. Endometrial preparation included ovulatory natural cycles (NC) and hormone replacement therapy (HRT) cycles, chosen according to physician’s discretion. Ethical approval was obtained from the Research Ethics Committee (REFA023b). Main results and the role of chance Patients’ characteristics are described as mean±SD (min-max): age: 33.5±5.43(19-47), AMH: 3.30±3.05ng/mL(0.01-23.00), BMI: 27.1±4.87Kg/m2(13.1-43.90), mean number of blastocysts transferred: 1.4±0.49(1-3). Patients were categorized according to age (years) in 4 categories (&amp;lt;30,n=404; 30-34,n=487; 35-40,n=595; &amp;gt;40,n=174) and no association with pregnancy rate (PR, p = 0.856), biochemical miscarriage rate (BMR, p = 0.940), clinical miscarriage rate (CMR, p = 0.06) nor LBR (p = 0.154) was found. BMI (kg/m2) was divided into four groups according to World Health Organization: underweight (&amp;lt;18.5;n=32), normal weight (18.5–24.9;n=555), overweight (25–29.9;n=622), and obese (≥30;n=426). Although no differences were seen for PR or BMR between groups (p = 0.507 and p = 0.343, respectively), CMR was significantly lower for normal BMI group (p &amp;lt; 0.001) and LBR significantly higher when compared to the overweight and obese group (&amp;lt;18.5kg/m2=68.42%; 18.5–24.9kg/m2=68.35%; 25–29.9kg/m2=60.14%; ≥30kg/m2=53.29%; p &amp;lt; 0.001). No differences were observed on the outcomes when AMH was sub-divided as per Bologna Criteria (&amp;lt;1.3ng/mL,n=327; ≥1.3ng/mL,n=1090). Regarding endometrial preparation, NC protocol showed significantly lower BMR and CMR (7.93% vs 12.27%,p=0.026; 8.44% vs 17.97%,p&amp;lt;0.001), and higher LBR (70.33% vs 55.06%,p&amp;lt;0.001) compared to HRT. Day of trophectoderm biopsy had a significant higher PR for day 5 (day 5=75.58% vs day 6=61.1% and day 7=23.81%, p &amp;lt; 0.0001), yet no differences were observed for BMR, CMR nor LBR. Limitations, reasons for caution Although the large number of FET included, performed in the same centre with same methodology, the retrospective study design is a limitation. We could not discard other hypothetical variables contributing to miscarriage such as KIR-HLA discrepancies, or other obstetric factors affecting late miscarriage and live birth. Wider implications of the findings Evaluating the factors associated with pregnancy outcomes should be considered prior to euploid frozen embryo transfer for personalized treatment approach and adequate blastocyst selection. Women with higher BMI should be aware of higher risk of miscarriage and lower LBR although an euploid blastocyst is transferred. Trial registration number not applicable

The effect of trophectoderm biopsy technique and sample handling on artefactual mosaicism.

To determine whether embryo mosaicism prevalence in preimplantation genetic testing for aneuploidy (PGT-A) cycles is associated with the trophectoderm biopsy technique used (a. number of laser pulses or b. the use of flicking or pulling) or the time to tubing. Prospective observational study performed in a single IVF-PGT-A setting from May 2019 to May 2021. Trophectoderm biopsies were analysed by next-generation sequencing. Mosaicism was analysed in relation to the biopsy methodology (number of laser pulses and pulling vs flicking), time elapsed from biopsy to tubing (min), and time of sample cryostorage from tubing to amplification (days). As a secondary objective, the number of laser pulses and biopsy methodology were studied in relation to clinical outcomes of transferred euploid blastocysts. None of the analysed variables were associated to mosaicism prevalence. Multivariable regression analysis demonstrated that mosaicism prevalence was comparable either when > 3 laser pulses were used as compared to ≤ 3 (13.9% vs 13.8%, aOR = 0.8726 [0.60-1.28]) and pulling compared to flicking (13.1% vs 14.0%, aOR = 0.86 [0.60-1.23]). Moreover, neither the number of laser pulses during biopsy (> 3 vs ≤ 3) nor the technique used (pulling vs flicking) were associated with clinical pregnancy after the transfer of frozen-thawed euploid blastocysts (54.9% vs 55.2%, aOR = 1.05 [0.53-2.09]; 61.1% vs 52.9%, aOR = 1.11 [0.55-2.25], respectively). Our results suggest that, as long as the biopsy and tubing procedures are performed following standardized high quality procedures, no specific approach would increase the generation of artefactual mosaicism as a result of trophectoderm biopsy. Trophectoderm biopsies should be performed regardless of the methodology but always aiming on minimising blastocyst manipulation.