Recurrent Pregnancy Loss Research Articles

O-176 Hoping for the best, expecting the worst: pregnancy after loss

Abstract Recurrent pregnancy loss (RPL) is an extremely distressing condition and can be both physically and emotionally traumatising. The condition is compounded by the fact that even after diagnostic procedures, the majority of cases, remain unexplained. The early waiting period of a new pregnancy, when confirmation by ultrasound scan of an ongoing and viable pregnancy is awaited, represent a particularly challenging time for those affected by this condition. This waiting period is associated with high levels of distress due to the anxiety of possibly experiencing a further miscarriage. However, there is a lack of understanding amongst some health professionals of the emotional burden of this difficult waiting period, furthermore, limited support and counselling is available, and many women are left to manage their distressing emotions without coping support. While some seek frequent confirmation of viability by ultrasound scans, this approach is not feasible in most clinical settings and, in any case, appears to be of limited efficacy in reducing anxiety. An alternative approach was to provide the woman with tools that could bolster their ability to cope with this challenging period. Our programme of research has investigated the use of the Positive Reappraisal Coping Intervention (PRCI), a self- administered coping technique, based on the principles of positive reappraisal, as a method of providing much needed emotional support. Qualitative research findings have provided deep insight into the lived experience of RPL during the early waiting stages of a new pregnancy, establishing that for those with a history of RPL, this is a traumatic time of great uncertainty, emotional turmoil and immense personal significance, where those affected ‘hope for the best, but expect the worst.’ Whilst our programme of research is still ongoing, studies to date, have demonstrated that study participants were receptive to the use of the PRCI and that it appeared to convey benefits, with no apparent downside. It was a ‘go to’ resource in times of strong emotional need and one that stimulated positive coping, providing respite from the stressor of uncertainty. There is a compelling need to provide those experiencing RPL with increased emotional support to help them manage the stressful early waiting period of a new pregnancy. Findings suggest that PRCI could provide a useful adjunct to care pathways and has the potential to be made more widely available as an effective, safe, convenient, and low-cost intervention to provide emotional support in RPL clinics.

P-389 Miscarriage in recurrent pregnancy loss (RPL) patients is associated with decreased peripheral T follicular helper cells and double-positive T cells

Abstract Study question Is there a relation between certain T cell subpopulations in the peripheral blood and the reproductive outcome in patients with recurrent pregnancy loss (RPL)? Summary answer We found that miscarriage in RPL patients is associated with low quantities of peripheral T follicular helper cells (TFH) and double-positive T cells (DPT). What is known already Previous studies showed that, the quantity of certain types of immune cells, such as natural killer cells and B cells has been associated with recurrent miscarriages. It is also well known that the first trimester is characterized by inflammation profile, required for successful implantation and specific Th1/Th2-type immune microenvironment. Follicular T helper cells and rare T-cells populations such as double-positive and double-negative T cells also have an important effect on the post-implantation processes. However, the effect of these specific immune cell types and their relative quantities in relation to the reproduction outcome is not fully understood. Study design, size, duration This observational study was performed from November 2022 to September 2023. Blood samples were collected from 65 pregnant RPL women (≥2 gestational losses) aged 28-49 years during the first trimester, at 8-10 weeks of pregnancy. Patients were followed up and divided into two groups according to their reproductive outcome: live birth or miscarriage. The exclusion criteria were history of recent inflammatory, autoimmune or oncological diseases, recent antibiotic treatment, and endometrial pathologies. Participants/materials, setting, methods The percentage of eight peripheral T cell subpopulations were measured by flow cytometry: T helpers (CD3+CD4+CD8-), T killers (CD3+CD4-CD8+), double-positive T cells (DPT) (CD3+CD4+CD8+), double-negative T cells (CD3+CD4-CD8-), T follicular helper cells (TFH) (CD3+CD4+CD8-CD185+), T helper type 1 (TH1) (CD4+CD183+CD196-CD194-), type 2 (TH2) (CD4+CD183-CD194+CD196-), and type 17 (TH17) (CD4+CD183-CD194+CD196+) cells. Study groups were compared by Student’s t-test using SPSS v.21 (IBM Corp., Armonk, NY, USA). Main results and the role of chance Among the studied RPL patients, 41 women gave live birth and 24 patients had a miscarriage after the blood sample. The mean percentage and standard deviation in the of the studied T cell subpopulations was: T helpers (49.86%±14.07%), T killers (39.49%±11.50%), DPT (2.96%±1.12%), DNT (5.61%±4.76%), TFH (8.09%±3.03%), TH1 (20.86%±8.81%), TH2 (6.22%±3.68%), and TH17 (3.93%±2.37%).The performed Student’s t-test revealed significant difference only in the percentage of TFH and DPT cells between the two studied patient groups. The percentages of TFH cells and DPT cells were significantly lower in patient group with miscarriage compared to the patient group with live birth (8.07% vs. 13.35%, p = 0.04, and 1.07% vs. 2.44%, p = 0.02, respectively). Limitations, reasons for caution The study was limited in sample size. It would be beneficial future studies to include more subsets of T cells, such as TH9, TH22, and T reg. Wider implications of the findings Low percentage of peripheral TFH and DPT cells in RPL patients could be considered as an indication for potential risk of miscarriage. Routine measurement of these immune biomarkers from the peripheral blood might be beneficial for an adequate assessment and treatment of patients with previous pregnancy losses. Trial registration number not applicable

P-323 Endometriosis and adenomyosis present lower expression in uterine natural killer cells

Abstract Study question Is there an association between endometriosis and uterine natural killer cells? Summary answer Decreasing level of uterine natural killer cells had positively associated with Endometriosis and Adenomyosis. What is known already Uterine natural killer cells (uNK) represent the predominant lymphocyte population within the decidua during implantation and in the initial stages of pregnancy. Dysregulation of uNK function has been associated with reproductive challenges, including recurrent miscarriage (RM) or recurrent implantation failure (RIF). This suggests an underlying disruption in immune activity during implantation. Endometriosis is a chronic and inflammatory disorder that usually affects the pelvic cavity and can cause severe pain during menstruation. However, there are paucity studies focus on the role of uNK in endometriosis and adenomyosis. The aim of this study was to identify the association between endometriosis and uNK. Study design, size, duration In this Prospective cohort study, 60 participants were recruited from May 2023 to January 2024 at the outpatient department of Obstetrics and Gynecology in Chang Gung Memorial Hospital, Kaohsiung Medical Center in Taiwan. The inclusion criteria were as follows: adult women of childbearing age were eligible to participate. Old age (>45 years old) patients, or patient with underlying medical disease, immune diseases and malignancy, or patient using immunosuppressant were excluded. Participants/materials, setting, methods Endometrial tissues were collected during the mid-luteal phase using an endometrial curette. The level of uNK were measured by immunohistochemistry with CD56+, and was presented in absolute number per high power filed. CD138 was also measured, and the level above 5 cell/HPF was defined as chronic endometritis. Endometriosis and adenomyosis were confirmed histologically. RM was defined as ≧2 previous miscarriages. RIF was defined as ≧2 previous failures to achieve clinical pregnancies after embryo transfer. Main results and the role of chance At least one uNK/HPF was revealed in 58 cases, 0-10uNK/HPF in 15 cases (24.5%), 11-20 uNK/HPF in 19 cases (31.6%) and above 20 uNK/HPF in 26 cases. After adjusting for covariates, patient with endometriosis and adenomyosis were positively associated with lower uNK level(low vs. high uNK(cut-off value: 20 uNK/HPF), odds ratio (OR) (95% confidence interval (CI)), 9.434 (1.101–83.33), p = 0.032; 2.000 (1.515–2.639), p = 0.015). Moreover, there are 14 patients with RM (23.3%), 26 patients with RIF (43.3%) and 7 patients with CE (11.7%). However, our study did not find significantly association between RM or RIF or CE with uNK levels. Limitations, reasons for caution Because of the single-center, Asian origin of our patients, and the sample size was relatively small. Further large-scale longitudinal studies are required to verify our findings and evaluate the underlying pathophysiology between endometriosis and uNK. Wider implications of the findings Our study highlights the significantly decreased uNK level in endometrium of women with endometriosis and adenomyosis, which may indicate some alteration in immune environment under endometriosis. Further research is warranted for understanding the immuno-modulation in endometriosis. Trial registration number not applicable

O-265 Mapping decidualization resistance at multi-omic level

Abstract Study question What are the cellular and molecular features of decidualization resistance (DR) at single-cell resolution? Summary answer There is an absence of the stromal-epithelial transition with aberrant crosstalk among cell types in essential pathways (e.g., endoglin, WNT) confirmed at the protein level. What is known already DR refers to the inability of the endometrium to undergo decidualization changes preparing the endometrium for a conceptus, noted in disorders like endometriosis, recurrent pregnancy loss, miscarriage and preeclampsia. The molecular mechanisms causing this disorder remains unknown. It has been demonstrated that there is a transcriptomic signature of DR in the endometrium of former preeclampsia patients, during and years after suffering from the disease. Our objective is to dilucidated what is DR at single-cell resolution. Study design, size, duration This cross-sectional study compares late secretory phase endometrial biopsies from patients that previously have suffered severe Preeclampsia (sPE). The sample size consisted of 11 sPE patients and 12 control participants for single-cell RNA sequencing (scRNA-seq), 8 sPE patients and 8 controls for spatial transcriptomic mapping covering 95 tissue regions, and 10 sPE patients and 10 controls for proteomic analysis. The study spanned 2 years, from sample collection to omics analysis. Participants/materials, setting, methods Late secretory phase endometrial biopsies from sPE patients and controls underwent molecular analyses. Methods included 10x Genomics protocol for single-cell RNA sequencing, MiloR to calculate cell abundance, CellChat to infer cell-to-cell communication and ClusterProfiler for gene ontology. We solved spatial mapping at transcriptomic level using Nanostring. Proteomics data was collected by laser-capture microdissection coupled mass spectrometry. Protein-protein networks, involving PR and ESR1 receptors as master regulators of decidualization, were built using String. Main results and the role of chance Initial morphological analyses of endometrial biopsies collected during the late secretory phase revealed gross anatomical defects in glandular morphology that were histologically confirmed. scRNA-seq revealed sPE-associated alterations in cell composition involving stromal and epithelial subtypes, denoting a higher proportion of cells expressing proliferative markers in the stroma (MMP11, SFRP1) and epithelium (IHH, EMID), and a near absent epithelial-stromal transition that is normally linked to decidualization. Further, abnormal abundance and gene dysregulation were detected in the three decidualized stages identified. Mapping cell-cell communication networks confirmed aberrant crosstalk among cell types involved in essential pathways (e.g., endoglin, WNT, SPP1). Spatial transcriptomic in a replication cohort of samples revealed a landscape of dysregulated genes in stromal, luminal and glandular enriched regions, corroborating sPE-associated DR. Laser capture microdissection/mass spectrometry of samples revealed differential protein expression at a global level and confirmed many of the scRNA-seq results, such as sPE-exclusive proteins related to proliferation and the absence of a stromal to epithelial transition at a pathway level. These data lend insights into potential molecular mechanisms underlying the pathogenesis of DR. Limitations, reasons for caution DR analysed in this work is associated with sPE but we do not know if this decidualization defect would be the same in other DR associated conditions such as endometriosis, recurrent pregnancy loss, or miscarriage. Wider implications of the findings Here we present the most comprehensive snapshot of DR to date, providing a full range of markers potentially responsible for this decidualization disorder linked to sPE. We concluded that stromal and epithelial cell populations are altered revealing a near absence of the stromal-epithelial transition, cellular crosstalk and glandular constitution. Trial registration number not aplicable

P-740 Septoplasty versus expectant management in women with septate uterus and a previous history of a live birth

Abstract Study question Does hysteroscopic septoplasty improve reproductive outcome in patients who were diagnosed to have a septate uterus (SU) after a previous live birth? Summary answer This study suggests that hysteroscopic septoplasty improves the chance of a live birth in patients with both SU and a history of previous live birth. What is known already SU accounts for 50% of uterine anomalies and can be associated with adverse pregnancy outcomes. Many observational studies reported significant improvement in pregnancy outcomes following hysteroscopic septoplasty. However, some investigators have reported good reproductive outcome among women with SU without surgical interventions. A recent randomized controlled trial, albeit with a small sample size, reported no difference in reproductive outcomes between patients who underwent hysteroscopic septoplasty and those who had no surgery (Rikken 2021). Therefore, more studies are needed to be able to counsel patients with SU presenting with reproductive failure, especially those with a history of a previous live birth. Study design, size, duration This retrospective study included 114 patients who had a previous live birth and subsequently complained of reproductive failure. A SU was diagnosed during their work up between 2005-2022. Patients presented with secondary infertility (98.3%) or with recurrent pregnancy loss (RPL) [1.7%]. Forty-two patients presented with both secondary infertility and RPL (36.8%). Participants/materials, setting, methods Diagnosis of SU was suspected on transvaginal 3D ultrasound scan and was confirmed on a diagnostic hysteroscopy at an infertility clinic affiliated with an academic hospital. Patients were offered the option of hysteroscopic septoplasty or expectant management. Infertility treatment options depended on the underlying etiology and included spontaneous conception, oral fertility medications, intrauterine insemination, and in-vitro fertilization/embryo transfer. Eighty-two patients underwent hysteroscopic septoplasty (Group 1), while 32 patients declined surgery (Group 2). Main results and the role of chance There were no significant differences in mean age (years), BMI (Kg/m2), infertility duration, baseline FSH levels (mIU/mL), number of miscarriages, and gestational age at delivery before treatment between the two groups. There were no significant differences in incidence of ≤ 3 or ≥ 3 pregnancies, nor incidence of one live birth or > 1 live birth between the two groups. There were no significant differences in the underlying etiology, except for a significantly higher incidence of endometriosis (P = 0.004) and diminished ovarian reserve (P = 0.04) in Group 1 compared to Group 2. There were no significant differences in the incidence of partial septate uterus (PSU) [angle of indentation <90 degrees] and complete septate uterus between the two groups. However, there was significantly lower incidence of PSU with angle of indentation > 90 degrees (P = 0.028) and higher mean septum length in mm (18.9±1.0 vs 13.8±1.1, P = 0.024) on hysteroscopy in Group 1 compared to Group 2. After treatment, there were significantly higher pregnancy (68.3% vs 37.5%, p = 0.003) and live birth (61.7% vs 25%, P = 0.000) rates, but no significant difference in miscarriages (4.9% vs 6.3%), preterm birth (14% vs 25%) rates, or method of pregnancy in Group 1 compared to Group 2. Limitations, reasons for caution Our study has limitations being retrospective in nature with a small sample size. Therefore, more studies are required to support our findings. However, our pilot study explored management options in a particular group of patients with SU who initially proved their fertility and subsequently presented with reproductive failure. Wider implications of the findings Our data support the notion that patients with SU can have a normal reproductive outcome, although those who undergo septoplasty are expected to have a better chance of conception and live birth. During counselling of patients with SU, both surgical and expectant management can be offered. Trial registration number not applicable

P-327 A multicenter retrospective study of the investigation of detailed immunological inflammatory grading for chronic endometritis and their correlation with the endometrial microbiome

Abstract Study question There exists refractory chronic endometritis which does not respond to antibiotic treatment. This study aims to elucidate its pathogenesis and develop detection methods. Summary answer Through treatment intervention for chronic endometritis (CE), the inflammatory grade decreased overall and the bacteria most associated with endometritis were Lactobacillus iners. What is known already CE and endometrial dysbiosis have been reported to be associated with recurrent pregnancy loss (RPL) and recurrent implantation failure (RIF), but there is insufficient data confirming the interrelation between them. Moreover, the diagnosis of CE typically involves single immunohistochemical staining with CD138, but the diagnostic criteria vary across reports (e.g., McQueen’s method, Liu’s method, etc.) and moreover, use of this classical inflammatory assessment methods raises concerns about their sufficiency in evaluating the pathogenesis of endometritis in cases of RPL and RIF. Study design, size, duration A retrospective analysis was conducted on 206 samples of endometrial specimens collected from infertile patients at seven participating assisted reproductive healthcare facilities from 2021 to 2023. Among these, 73 samples (from 48 cases) had undergone both histological examination and simultaneous endometrial microbiome testing, and data on pregnancy outcomes (including implantation success and ongoing pregnancy rate) were collected until December 2023 for these cases. Participants/materials, setting, methods The endometrial specimens from cases with RIF or RPL were obtained through intrauterine aspiration in luteal phase. Immunohistochemical staining using CD138, CD56, and CD8 antibodies was performed. A composite grading for endometritis, based on histological findings and immunohistochemistry intensity, was established (INF grading; grade 0 to 5). Furthermore, a portion of the specimens underwent microbiome analysis using next-generation sequencing targeting the 16SrRNA, exploring correlations between bacterial taxa, abundance, histological findings, and pregnancy outcomes. Main results and the role of chance Among analyzed 48 cases, 11 cases underwent repeated endometrial microbiome analysis post-treatment again, while 7 cases had additional testing without improvement in chronic endometritis (CE) or endometrial dysbiosis. The McQueen’s method diagnosed 27 cases with CE, while the Liu method identified 4 cases. According to our INF grading, 11 cases were classified as endometritis grade 3 or above. Successful embryo implantation was achieved in 18 cases (37.5%), with ongoing pregnancy in 13 cases (27.1%). CE treatment involved broad-spectrum oral antibiotics, mainly doxycycline and metronidazole, resulting in improvement to grade 2 or below in 9 out of 11 cases with INF grade 3 or above. However, clear associations between pregnancy outcomes and INF grades were not apparent, with the most significant correlation found in CE diagnosis using Liu’s method. Analyzing biomarkers for CE through LEfSe (Linear discriminant analysis effect size), both McQueen’s method and INF grading 2 or above were associated with the intrauterine presence of Lactobacillus iners. Estimating endometrial inflammation based on intrauterine Lactobacillus iners presence yielded an AUC of 0.733 for McQueen’s CE and 0.723 for INF grade 2 or above. Limitations, reasons for caution This study is a retrospective analysis, and the treatment protocols and intervention criteria for chronic endometritis vary among different facilities. The number of cases is also limited, and it is desirable to conduct a unified prospective multicenter cohort study with consistent protocols. Wider implications of the findings The combination of the endometrial microbiome testing and detailed inflammatory grading suggests the potential for establishing appropriate treatment intervention strategies and predicting the success rate of infertile treatment. Since Lactobacillus iners tends to colonize extensively in the vaginal environment of East Asian women, it is necessary to explore region-specific measures. Trial registration number not appicable

P-436 Uterine NK cells underexpress KIR2DL1/S1 and LILRB1 receptors recognizing HLA-C2 and HLA-G on extravillous trophoblasts in reproductive failure

Abstract Study question Is there a difference in frequency, receptor expression and cytokine production of uterine NK cells (uNK) subpopulations in women with reproductive failure compared to controls? Summary answer Reproductive failure is associated with global underexpression of uNK receptors interacting with extravillous trophoblasts (EVT) that may result in defective implantation and placentation. What is known already A significant proportion of infertility, recurrent miscarriage and recurrent implantation failure are unexplained. This may be caused by immunological dysfunction at the maternal-fetal interface resulting in defective implantation and placentation. uNK is thought to play an important role in early pregnancy by cross-talking with EVT via KIR/HLA-C, LILRB1/HLA-G and CD94/HLA-E interactions to regulate EVT invasion and spiral arteriole remodeling in the decidua. Dysregulation of uNK function may cause reproductive failure; however it is unclear whether NK cell over- or under-activation is the pathology. Previous studies have been conducted on uNK obtained after miscarriage, which may be confounded by inflammatory processes. Study design, size, duration Here, we conducted a cross-sectional study to assess frequency, phenotype and function of matched pNK and uNK during the window of implantation in 16 women with reproductive failure compared to 11 controls. Women with reproductive failure cohort included unexplained subfertility (9), recurrent miscarriage (4) and recurrent implantation failure (3). The women were subsequently followed up after IVF and comparison made between women who did and did not fall pregnant. Participants/materials, setting, methods Matched peripheral blood and endometrial tissue were obtained from participants during the mid-secretory phase. Samples were accurately timed for 7-9 days after ovulation by urine LH test or histological dating. Flow cytometry was used to assess the frequency, phenotype and function. Phenotype was measured by KIR, LILRB1 and CD94 and receptor expression. Function was measured by degranulation (anti-CD107a internalization) and cytokine production (TNF-α, GM-CSF, IFN-γ and IL-8), with and without stimulation by PMA and ionomycin. Main results and the role of chance The expression of KIR2DL1/S1 and LILRB1 were significantly lower in reproductive failure group for both uNK (total uNK, uNK2 and 3) and pNK. The common finding in both uNK and pNK suggests that this is not tissue specific and may have an immunogenetic aetiology. We have previously found that degranulation peaks during secretory phase and in first trimester pregnancy. TNF-α production was also found to peak in secretory phase. Here, we found that degranulation activity of total uNK, as well as TNF-α production all uNK subsets was significantly lower in reproductive failure compared to control group. However, no significant difference was found in women in the reproductive failure cohort who fell pregnant compared to women who did not fall pregnant or miscarried after IVF. Contrary to findings from previous studies, we have not found any difference in uNK or pNK frequencies in any of the groups that were examined. Taken together, our findings suggest that reproductive failure is associated with global underexpression of receptors important for interaction with HLA-C and HLA-G on EVT, resulting in reduced uNK activation. This in turn may lead to dysregulation of implantation and placenta formation in early stage of pregnancy. Limitations, reasons for caution KIR2DL1/S1 antibody used does not distinguish between KIR2DL1 and KIR2DS1 which exerts inhibitory and activating effects respectively. Larger cohort studies on both pNK and uNK that consider KIR2DL1 and KIR2DS1 separately is needed to confirm which of these receptors are specifically reduced in reproductive failure. Wider implications of the findings Overall, our findings suggest that reproductive failure is associated with global underexpression of NK cell receptors leading to impaired implantation and placentation. This is the first study to examine uNK subsets during window of implantation and will serve as a platform to focus on particular uNK subsets in future studies. Trial registration number Not applicable

O-201 Antinuclear antibodies in women with recurrent early pregnancy loss: prevalence and impact on subsequent pregnancy outcome

Abstract Study question What is the prevalence of antinuclear antibodies (ANAs) in women with recurrent early pregnancy loss (REPL) and do they affect subsequent pregnancy outcome? Summary answer The prevalence of ANAs in women with REPL was 13.0%, and their presence did not negatively impact subsequent pregnancy outcomes, including EPL and livebirth rates. What is known already An etiological factor cannot be identified in more than half of the couples experiencing REPL. Immunologic mechanisms could be involved, with the presence of autoimmune disease (AD) associated to lower reproductive outcomes. For the mere presence of ANAs, in the absence of AD, the evidence on the association with REPL is controversial, with most studies having a small sample size with important clinical and methodological heterogeneity. Study design, size, duration This is a single-centre, retrospective cohort study from a tertiary referral hospital including 960 women referred for REPL between January 2014 and December 2019. During the entire study period, maternal serum ANA detection was part of the routine diagnostic REPL work-up, defined as two or more pregnancy losses before the 10th week of gestation, including previous spontaneous conceptions and pregnancies following autologous IVF/ICSI treatment. Participants/materials, setting, methods Women under the age of 40 at ANA analysis were eligible for inclusion. Confirmed AD, uterine anomalies, parental chromosomal abnormalities and uncontrolled endocrine disorders were exclusion criteria. The prevalence of positive ANA titers and the impact of ANA positivity on the outcome of a subsequent pregnancy were investigated with EPL rate as the main outcome parameter and biochemical pregnancy rate, clinical miscarriage rate and live birth rate as secondary outcome measures. Main results and the role of chance Positive ANA screening at a titer of ≥ 1/80 was observed in 125/940 women (13.0%), a prevalence in line with previously published data in the general population not experiencing REPL. There were no statistically significant differences in maternal age, body mass index, ethnicity, or conception method between ANA-positive and -negative women. No higher number of previous EPLs was observed in ANA-positive compared to ANA-negative women (respectively 2.7 versus 2.8, p = 0.06). Women with ANAs had significantly more thyroid antibodies (21.6% versus 10.2%, p < 0.001). For a subsequent pregnancy, no association was observed between ANA positivity and reproductive outcome with an unadjusted EPL rate of 30/86 (34.9%, ANA-positive) versus 206/509 (40.5%, ANA-negative, p = 0.32). Logistic regression analysis did not identify ANAs as an independent predictive risk factor for additional EPL (OR 0.73, 95%CI 0.45-1.20, p = 0.22). Similarly, there was no significant difference in live birth rates between both groups (p = 0.5), with 54/86 (62.8%) in ANA-positive and 300/509 (58.9%) in ANA-negative patients. Limitations, reasons for caution The current data are limited by the absence of a formal control group without REPL, the retrospective nature of the study and potential bias due to unmeasured confounders. Wider implications of the findings These findings further questions systematic ANA measurement in the diagnostic work-up of REPL without AD. Furthermore, when ANAs are detected, patient counselling and treatment adaptations should be considered with caution, given our inability to eventually detect any significant differences in the pregnancy outcomes between ANA-positive and -negative REPL patients. Trial registration number not applicable

P-319 Chronic endometritis prevalence, associated factors and pregnancy outcome among infertile women

Abstract Study question What are the prevalence, associated factors and pregnancy outcome of chronic endometritis(CE) among infertile women? Summary answer The overal prevalence of chronic endometritis in infertile women was 24.7%. Diagnosis rate of CE was higher in primary infertility compared to other infertility groups. What is known already Chronic endometritis is defined as a chronic inflammation of uterine endometrium. The prevalence of chronic endometritis varies across different groups and diagnostic criteria. The pathophysiology of chronic endometritis remained unclear, therefore, associated factors of chronic endometritis are uncertain. Study design, size, duration We perfomed an single-center, retrospective study between May 2019 and July 2023. By doing so, we collected data on 486 infertile women. Participants/materials, setting, methods A total of 486 infertile women, who underwent hysteroscopy by single surgeon, were recruited. The diagnosis of chronic endometritis was confirmed by presence of plasma cell per section in hematoxylin-eosin stain or CD138 immunohistochemical staining. Preoperative information (including age, body mass index, smoking, alcohol consumption, clinical presentation of infertility, endometrial thickness at ovulatory phase, anti-mullerian hormone (AMH) level and infertility factors) and post-operative information (including endometrial histology and pregnancy outcome) were collected. Main results and the role of chance The prevalence of chronic endometritis in the 486 infertile women with hysteroscopy guided endometrial biopsy was 24.7%. Prevalence of chronic endometritis was 28.1%, 19%, 22.2%, 27.2% in women with primary infertility, secondary infertility, recurrent pregnancy loss (RPL) and repeated implantation failure (RIF), respectively. A multivariate analysis revealed that overweight was associated with lower risk of chronic endometritis than underweight (relative risk (RR) = 0.56, 95% confidence interval (CI): 0.32-0.98, p = 0.042). Primary infertility was associated with higher risk of chronic endometritis than non-primary infertility (RR = 1.44, 95% CI: 1.02-2.03, p = 0.039). The pregnancy rate (including spontaneous pregnancies) was comparable between women with treated chronic endometritis and without chronic endometritis; 54.7% versus 61.7% in clinical pregnancy rate and 82.8% vs 85.4% in ongoing pregnancy rate. The prevalence of chronic endometritis with coexisting endometrial lesion (endometrial polyps, submucous myoma, or both of them) was 61.7%. Limitations, reasons for caution Our retrospective study has a less powerful design compared to a prospective randomized controlled study. Wider implications of the findings In women with primary infertility, the diagnosis rate of chronic endometritis was higher compared to other infertility groups. Therefore, even in cases of primary infertility, chronic endometritis should be considered as a potential cause of infertility. Trial registration number not applicable

P-755 Effect of female age and specific cycle characteristics on success in modified natural frozen-thawed single euploid embryo transfer cycles (mNC/FET)

Abstract Study question Does the age of woman and specific cycle characteristics impact the success of modified natural frozen-thawed single euploid embryo transfer cycles (mNC/FET)? Summary answer The success rate of mNC/FET cycles is similar in all age groups. As the LH level increased ( above 23.5ng/dL) the success was significantly increased. What is known already The success of frozen embryo transfer can depend on various factors such as the patient’s age, the number of transferred embryos, the duration of embryo culture, and the method used to prepare the endometrium (Holschbach et al. 2023). However, there is currently insufficient evidence in the literature about the effect of age and cycle characteristics on the effectiveness of mNC/FET cycles in patients who have received single top and good quality euploid embryos. Study design, size, duration This study, conducted at Istanbul Memorial Hospital, ART and Reproductive Genetics Center, is a retrospective, single-centre analysis of 583 cycles of euploid mNC/FET in women aged 22-44 years. The study period was from July 2017 to January 2024. The cycles were divided into two groups based on the age of the women. Group A (n = 408) included women under the age of 38, while Group B (n = 175) included women aged 38 or above. Participants/materials, setting, methods The study investigated success predictors in mNC/FET with top and good-quality embryos. Excluded were women with recurrent pregnancy losses, recurrent implantation failure, high BMI (>35kg/m2), uterine/endometrial problems, severe male infertility, and autoimmune diseases. The mNC/FET success was a pregnancy lasting 12 weeks or more. The analysis included patient demographics and cycle outcomes. Chi-square, t-tests, and Mann-Whitney U tests assessed variables with a p-value < 0.005, indicating statistical significance. Main results and the role of chance No significant demographic differences were observed between the two groups, except for AMH levels. AMH levels were significantly higher in younger group than older group (3.02 ng/ml vs. 1.96 ng/ml, p = 0.000). The mNC/FET success rates were similar, with 76% in the younger group and 73% in the older group. Estradiol levels the day before trigger day were significantly higher in older group (278.02 pg/ml vs. 237.84pg/ml, p = 0.028). Both groups had similar mean LH levels (32.50 IU/L and 32.41 IU/L, respectively), estradiol levels and endometrial thickness on the trigger day. In the younger group, LH rise was observed later, follicular phase was longer, and embryo transfer day was later than the older group (p = 0.001). Univariate regression analysis revealed that increased LH level was an important independent variable in achieving mNC/FET success, with a 1.001-fold increase (p = 0.043). The LH levels of 23.5 IU/L or higher predicted high mNC/FET success with 60% sensitivity and 43% specificity based on the Receiver Operating Characteristic curve. (AUC: 0.606, %95 CI 0.540-0.671, p = 0.002). Prolonged infertility duration and previous ET failures reduced mNC/FET success by 0.889 and 0.544-fold, respectively (p = 0.002, p < 0.001). Higher β-hCG levels on days 14 and 16 increased mNC/FET success by 1.038-fold and 1.008-fold, respectively (p < 0.001). Limitations, reasons for caution Our study has limitations as it was retrospective. Prospective randomized studies are needed to evaluate the differences between the young (age <38) and old patient groups (age ≥38) based on mNC/FET success. The association between LH rise and mNC/FET success needs verification with a larger sample. Wider implications of the findings The mNC/FET success rates are similar in young (age <38) and old patient groups (age ≥38). However, prolonged infertility duration and previous failed ET cycles decreased the success of mNC/FET. Additionally, It is important to schedule trigger time after the optimum LH rise, which is 23.5 IU/L or higher. Trial registration number not applicable

P-318 Reproductive ageing: the seed, the soil, or both?

Abstract Study question Is it all about the embryo? Can a single euploid blastocyst transfer sustain reproductive outcomes in women with advanced reproductive age? Summary answer Despite euploid embryo transfer, endometrial senescence appears to play a crucial role in determining reproductive success, particularly in older patients undergoing in vitro fertilization (IVF). What is known already Advanced reproductive age significantly impairs oocyte and embryo quality. Effective crosstalk and synchronization between embryo and endometrium are pivotal for successful pregnancies, both in vivo and in vitro. Implantation failure remains a persistent challenge in IVF, exacerbated by delayed family planning trends. The transfer of euploid blastocysts through preimplantation genetic testing for aneuploidy (PGT-A) is a potential strategy to address implantation issues, decrease miscarriages, and shorten time-to-pregnancy. However, a significant proportion of IVF treatments remain unsuccessful, suggesting critical factors beyond the embryo. Study design, size, duration This retrospective study analyzed 457 single euploid blastocyst transfers performed by two doctors at a private IVF center from January to November 2023. Inclusion criteria encompassed cases with PGT-A indications (advanced maternal age, repeated implantation failure, recurrent pregnancy loss, etc.) or cases where embryo biopsy was patient-requested. The primary outcome was ongoing pregnancy rate (OPR), with secondary outcomes including pregnancy rate (PR) and clinical pregnancy rate (CPR). Participants/materials, setting, methods Participants were categorized into four groups: Group 1 (< 35 years old; N = 100), Group 2 (35-37 years old; N = 102), Group 3 (38-40 years old; N = 118), and Group 4 (> 41 years old; N = 87). A control group (oocyte donation; N = 50) with single euploid embryo transfers showed a mean recipient age of 43 years. Donors were always less than 32 years old. Endometrial preparation protocols were consistent across all groups. Chi-square test was used to compare groups. Main results and the role of chance Among the 457 transfers analyzed, with a mean female age of 37 years, the overall PR was 71.3%, CPR was 64.8%, and OPR was 60.2%. Groups showed no differences in demographic variables and cycle parameters. Group 1 demonstrated higher PR (83.3%), CPR (78.4%), and OPR (73.5%) compared to Group 2 (PR 70.9%, p = 0.034; CPR 66%, p = 0.047; OPR 62.1%, p = 0.081), Group 3 (PR 68.2%, p = 0.009; CPR 61.2%, p = 0.005; OPR 56.6%, p = 0.008), and Group 4 (PR 65%, p = 0.002; CPR 56.1%, p < 0.001; OPR 51.2%, p = 0.001). Furthermore, the control group (oocyte recipients) showed significantly lower PR (68%, p = 0.031), CPR (54%, p = 0.002), and OPR (46%, p < 0.001) compared to Group 1, but not with the other groups. These findings demonstrate that even with euploid embryos originating from young donors, older women exhibited lower pregnancy rates than young women transferring single euploid blastocysts from their own oocytes and similar rates compared to those with advanced reproductive age transferring their own euploid blastocysts, implying that endometrial ageing may disproportionately influence IVF treatment outcomes. Limitations, reasons for caution The retrospective design introduces inherent bias, notably selection bias and unmeasured confounders. This preliminary data did not account for male infertility factors and their impact on embryo quality. Wider implications of the findings Reproductive aging appears to be influenced by factors beyond the embryo itself, especially in women above 35 years. Further research is warranted to elucidate the causes and impact of reproductive aging on implantation potential, aiming to refine clinical practices and enhance the success of IVF treatments. Trial registration number not applicable

KAT8-mediated H4K16ac is essential for sustaining trophoblast self-renewal and proliferation via regulating CDX2

Abnormal trophoblast self-renewal and differentiation during early gestation is the major cause of miscarriage, yet the underlying regulatory mechanisms remain elusive. Here, we show that trophoblast specific deletion of Kat8, a MYST family histone acetyltransferase, leads to extraembryonic ectoderm abnormalities and embryonic lethality. Employing RNA-seq and CUT&Tag analyses on trophoblast stem cells (TSCs), we further discover that KAT8 regulates the transcriptional activation of the trophoblast stemness marker, CDX2, via acetylating H4K16. Remarkably, CDX2 overexpression partially rescues the defects arising from Kat8 knockout. Moreover, increasing H4K16ac via using deacetylase SIRT1 inhibitor, EX527, restores CDX2 levels and promoted placental development. Clinical analysis shows reduced KAT8, CDX2 and H4K16ac expression are associated with recurrent pregnancy loss (RPL). Trophoblast organoids derived from these patients exhibit impaired TSC self-renewal and growth, which are significantly ameliorated with EX527 treatment. These findings suggest the therapeutic potential of targeting the KAT8-H4K16ac-CDX2 axis for mitigating RPL, shedding light on early gestational abnormalities.

P-322 Development of a CD138-based histopathological scoring system to diagnose chronic endometritis

Abstract Study question Does a standardized histopathological classification system lead to a robust interobserver agreement to diagnose chronic endometritis (CE) and can an accurate computation algorithm be developed? Summary answer The proposed scoring system has a particularly good to excellent interobserver agreement among pathologists. A machine-learning algorithm outperformed pathologists in the clinically most relevant 2-tier-system. What is known already Plasma cell (PC) presence in the stromal endometrium has been proposed as a marker for CE, yet no standardized classification system that considers the amount or distribution of PCs exists. Also, interobserver agreement has been insufficiently reported in the previous publications on the topic. Without a robust histopathological score, the potential clinical impact of CE on ART treatment outcomes cannot be investigated. This issue is strongly reflected in the heterogeneity of the current diagnostic and therapeutic approaches, specifically for patients experiencing repeated implantation failure (RIF) or recurrent pregnancy loss (RPL). Study design, size, duration A 5-tier classification system based on CD138-positive PCs (Roche B-A38/automated Ventana system) was established: class0 (no PCs), classIPC (isolated PCs), class1 (³1 cluster of 5-19 PCs/0.25mm2), class2 (³1 cluster of 20-49 PCs/0.25mm2), class3 (³1 cluster of 50 or more PCs/0.25mm2). A test set of 78 endometrial biopsies (collected over two years) was selected by a certified pathologist who sorted >1000 biopsies to ensure the full spectrum of PC counts/distribution was accounted for. Participants/materials, setting, methods The 3DHistech-platform digitized the slides (pixel size 0.2738x0.2738mm) and a scoring session was set up in the Pathomation software suite. Six pathologists scored independently after training. Overall agreement was expressed by Fleiss’ Kappa values. Pairwise interobserver agreement was calculated using Cohen’s Kappa statistics. A supervised machine-learning algorithm was developed in QuPath(V0.4.2) and considered a seventh observer in the interobserver agreement calculations. Main results and the role of chance The CD138-based semiquantitative scoring system showed particularly good to excellent interobserver agreement among all observers. Overall agreement values were 0.722 for the 5-tier system and 0.858 for the 2-tier system (0+IPC versus 1 + 2+3, which was considered the clinically most relevant classification, i.e. CE absent versus diagnosed). Pairwise interobserver agreement was 0.871-0.951. Intra-class correlations varied from 0.727 to 0.839 in the 5-tier system and 0.893 to 0.965 in the 2-tier system. All findings were statistically significant (p < 0.001). The supervised machine-learning algorithm was able to compete with human pathologist observers and outperformed them in the 2-tier system. The proposed classification system provides a framework for assessing the presence and severity of endometrial PC infiltration and can guide further research. Limitations, reasons for caution Inconsistent staining intensity, non-specific epithelial staining and background interference are common problems that might limit interlaboratory use of the algorithm. Furthermore, the algorithm has not yet been associated to patient/clinical parameters. Accurate analysis of an endometrial biopsy might still be insufficient to provide a formal diagnosis of CE. Wider implications of the findings To unravel the association between PCs in endometrial biopsies (as a proxy for the diagnosis of CE) and the reproductive outcomes following ART, a standardized methodology is of crucial importance for future studies. Higher accuracy, efficieny and automation are advantages when using computer-aided diagnostics for CE. Trial registration number BUN 143202042810

P-443 An association between olfaction and unexplained recurrent pregnancy loss

Abstract Study question Is there a male body-odor associated with unexplained recurrent pregnancy loss (uRPL)? Summary answer The body-odor of men from uRPL couples may have different chemical composition than control men. What is known already Recurrent pregnancy loss is defined as 2-3 consecutive pregnancy losses, affecting about 1% of all couples, and in about half of them, the reason for the pregnancy losses in unexplained (uRPL). In our previously published study, we found that women who experienced uRPL have altered perceptual and brain responses to men’s body-odors in comparison to control women. This suggests a possible link between uRPL and the olfactory system, resonating with the Bruce effect in mice. In the Bruce effect, the particularities of male body-odor are also a factor in the effect. Study design, size, duration We set out to ask whether the body-odor (BO) of men in uRPL is different from the BO of control men. To test this, we used two main approaches: have women smell BOs from uRPL-men and control-men and rate the perceived odor; and sample the BOs using a machine (electronic nose) and test if the composition of the BOs is objectively different. Participants/materials, setting, methods We collected BOs from spouses of uRPL and control women. We recruited women with uRPL, control women (with no history of pregnancy losses) and nulliparous women to sniff the BOs and rate them for perceived intensity, pleasantness, sexual attraction, and fertility. We then used an electronic nose (eNose, Airsense) to sample the BOs and used a Linear SVM classifier on this data, to try and classify the BOs to the two groups. Main results and the role of chance Twenty-one women with uRPL and 24 control women sniffed and rated 34 BO jars (17 uRPL-men). We found that uRPL-men BO was rated as more pleasant, sexually attractive and fertile than control men (rmANOVAs for each parameter but intensity revealed a significant effect of male group (all F(1,43)>14.24, all p < 0.001), no women-group effect and no interactions). We then recruited 51 nulliparous women, not related to the original study, to do the same task, and the results replicated: nulliparous women rated uRPL-men’s BO significantly more pleasant, sexually attractive and fertile than control men’s BO (all t(50)>2.88, all p < =0.006), while the intensity was not different (t(50)=0.87, p = 0.38). We then used a PEN3 eNose to sample 37 male BOs (18 uRPL). The PEN3 eNose contains 10 different metal oxide sensors, and each sensor is coated in a unique material that makes it sensitive to different sets of chemical compounds. Based on the data from the eNose, a Linear SVM classifier successfully classified the odors to uRPL-men or control-men at 69.9% accuracy in a five-fold cross-validation test (p < 0.001 estimated by repeating the process 1000 times and shuffling the labels). Limitations, reasons for caution This study shows an association between the body-odor of men and uRPL, however it shows a correlative connection (not causative). Therefore, the interpretation for these current results are limited. Wider implications of the findings These initial results suggest that uRPL-men’s and control-men’s BO have different chemical composition. This implies a possible contribution of men to the effect we observed in our previously published study, and combined, further strengthens a link between uRPL and the olfactory system that we continue to investigate. Trial registration number not applicable

P-388 The science of frozen embryo transfer, is modified natural cycle better?

Abstract Study question Do live birth rate, obstetric outcomes and number of clinic visits differ between frozen embryo transfer in modified natural and artificial cycle? Summary answer Modified natural cycle yielded a higher live birth rate for the similar number of visits, less hypertension, but higher rate of gestational diabetes. What is known already A frozen embryo transfer in a modified natural cycle entails procedure timing such that it coincides with the woman’s ovulatory cycle. Conversely, a frozen embryo transfer in an artificial cycle involves controlled hormonal stimulation, with the subsequent scheduling of embryo transfer. Based on the current evidence, there is no difference in pregnancy rates between modified natural cycle and artificial cycle. Frozen embryo transfer during an artificial cycle allows for greater flexibility for the patient and physicians. However, it seems to be associated with higher risk of adverse obstetric and neonatal outcomes. Study design, size, duration 1206 frozen embryo transfer cycles between January 1st, 2022, and December 31st, 2022 were retrospectively studied. Patients older than 40, with recurrent implantation failure, and recurrent pregnancy loss were excluded. Patients were divided according to their age, BMI, AMH, and the type of embryo transfer protocol. Compared outcomes were endometrial thickness, biochemical pregnancy rate, clinical pregnancy rate, miscarriage rate, number of clinic visits prior to transfer, obstetric complications, livebirth rate, and livebirth weight. Participants/materials, setting, methods Patients in the modified natural cycle group were followed by ultrasound and when the endometrium reached a thickness ≥ 7mm, and a dominant follicle ≥ 15 mm, HCG trigger was given, and the embryo (blastocyst) transferred 7 days later. In the artificial cycle group, patients received estrogen supplementation either orally or by patches and when the endometrium reached a thickness ≥ 7 mm, embryo transfer was scheduled following intramuscular progesterone administration for 6 days. Main results and the role of chance Patients with modified natural cycle (n = 250) embryo transfers have better clinical pregnancy rate compared to the embryo transfers (n = 250) in artificial cycles (63,6% vs 55,2% p = 0,05), and significantly better live birth rate (57,2% vs 43,9% p = 0,003) despite similar biochemical pregnancy rate (64% vs 61% p = 0,48). The miscarriage rate was significantly lower in modified natural compared to artificial cycles (10,2 % vs 20,99%, p = 0,008). Similar results were found with the transfer of PGTA tested embryos. There was no difference in the endometrial thickness between the 2 groups. The number of visits was higher in the modified natural cycle group, but the difference was not clinically significant (1,57 days vs 1,2 days). When comparing the obstetric outcomes, patients with a modified natural cycle transfer had a lower risk of hypertension (6,6% vs 13,5 % p = 0,10), but a significantly higher risk of gestational diabetes (19% vs 8,3% p = 0,02). No difference was observed regarding the birth weight, percentage of preterm birth or mode of delivery. Regression analysis was performed to identify any cofounder that might have affected the clinical pregnancy rate, and the results showed that the type of transfer was the only factor affecting the outcomes (OR: 1,23 CI: 1,03- 1,46) Limitations, reasons for caution The main limitation of this study is its retrospective nature. A randomized controlled study should be performed to confirm these findings. Wider implications of the findings Modified natural embryo transfer could be used to improve pregnancy outcomes. With proper scheduling, embryo transfer could be done with a similar number of visits for modified natural cycles and artificial cycles. More studies are needed to assess the increased risk of gestational diabetes with natural cycle embryo transfer. Trial registration number NCT06053827

O-322 What is the number of IVF cycles needed to produce at least one euploid blastocyst in patients of all ages with varied AMH levels

Abstract Study question How many cycles are needed to get at least one blastocyst in patients of different AMH levels and varied ages? Summary answer Age and AMH determine the number of cycles, which ranges from 1 cycle in young patients up to 10 cycles in over-44-year-old patients. What is known already AMH secretion by ovarian granulosa cells initiates during the 36th week of gestation in pre-antral and antral follicles. This persists throughout a woman’s reproductive lifespan. AMH serves as a biomarker for assessing ovarian reserve and forecasting the ovarian response to stimulation. AMH is strongly associated with the quantity of antral follicles and the production of oocytes. AMH can be utilised to determine the initial dosage of FSH in an IVF cycle, as well as serving as a quantitative indicator of the number of oocytes produced. Study design, size, duration In this retrospective cohort study, data was collected from 16,417 ICSI cycles with Preimplantation Testing for Aneuploidy (PGT-A) conducted at three referral fertility centers from Jan 2019 to Dec 2023. We excluded cycles without serum AMH, endocrinopathies or recurrent pregnancy loss, males with total sperm count of less than 5 million and total motility less than 30%, and those with parental chromosomal abnormalities. Participants/materials, setting, methods Patients were stratified into three different categories of AMH (ng/ml), low (< 1.1 ng/mL), intermediate (>1.1 to < 3.0 ng/mL) and high (> 3.0 ng/mL). Each category was subdivided into 6 age sub-categories as follow (< 35), (35 to < 38), (38 to < 40), (40 to < 42), (42 to < 44), (> 44). All of the cycles underwent blastocyst stage biopsy and PGT-A utilising next generation sequencing (NGS). Main results and the role of chance Euploidy rate was statistically insignificant for all AMH levels in the same age group. However, euploid embryo numbers vary significantly. Euploid embryos numbers are shown in low, medium, and high AMH by age group. In the (<35) years was 1.9±1.5, 2.9±2.3, and 4.7±3.5, p < 0.0001. In the (35 to > 38) years was 1.3±0.8, 2.1±1.8, and 3.2±2.6, p < 0.0001. In the (38 to > 40) years was 0.9±0.8, 1.7±1.4, and 2.6±1.7, p < 0.0001. In the (40 to > 42) years was 0.6±0.5, 0.9±0.7, and 1.5±1.4, p < 0.0001. In the (42 to > 44) year was 0.26±0.2, 0.49±0.38, and 0.6±53, p < 0.0001. In the (≥44) years was 0.10±0.10, 0.23±0.21, and 0.40±0.37, p = 0.0170. We calculated the number of cycles needed to produce at least one grade BB or higher euploid blastocyst from the mean number of euploid embryos per cycle: The number of cycles is presented in low, medium, and high AMH. In the (<35) and (35 to > 38), 1 cycle is needed. In (38 to > 40) years, 2 cycles, 1 cycle and 1 cycle. In the (40-42) group 2 cycles, 2 cycles and 1 cycle. In the (42 to < 44) group 4 cycles, 3 cycles and 2 cycles. In the (≥44) group 10 cycles, 5 cycles and 3 cycles. Limitations, reasons for caution The retrospective nature of the study is the main limitation. Wider implications of the findings The study offers valuable insights for patient counselling regarding the required number of cycles to achieve euploid embryos, as well as for financial budgeting for both patients and insurance companies. Trial registration number Not applicable

O-236 Why one is not enough when studying pregnancy loss - a European perspective

Abstract Pregnancy loss is a distressing but still poorly understood condition, ending one in four pregnancies. With more than 30 million losses worldwide yearly, it is posing significant challenges to individuals, families, and healthcare systems. This presentation delves into the imperative for a multifaceted approach to studying pregnancy loss. By examining pregnancy loss through multiple lenses including fetal and parental genetics, male and female physiology, and psychosocial, lifestyle and political dimensions, we can achieve an improved understanding of the causes, impact, prevention, and treatment of pregnancy loss. Historically, pregnancy loss has been considered a part of the reproductive journey – a course of nature. And until a couple has suffered from recurrent pregnancy loss, the physiological and mental impact has been neglected. This is an outdated approach that prevents a full understanding of the consequences and underlying etiologies hindering the potential of prevention and treatment, and thereby contributes to maintaining the women’s health gap. Moreover, current practice ignores the association of euploid pregnancy loss with an increased risk of future losses and later maternal health consequences such as diabetes and cardiovascular disease. In Copenhagen, we are currently running a large-scale pregnancy loss study (Copenhagen Pregnancy Loss, COPL) involving 3000 pregnancy losses. By including the full trio (fetus, female, and male) we are gaining new insight to the mechanisms causing pregnancy loss and the consequences hereof, both in men and women. We know from previous cytogenetic investigation of lost pregnancies, that about 60% are caused by fetal aneuploidy. But the remaining part is still not well understood and leaves the opportunity of treatments and preventions if we knew the underlying mechanism. That we aim to cover by building the largest database and biobank in the field. Moreover, collected data from the COPL study will be linked to nationwide Danish registries including comprehensive information on medical history, social economic status, educational status, medication, and ancestry. We have validated the use of cell-free fetal DNA in maternal blood to separate the losses caused by fetal genetics or not. This differentiation is essential to address the heterogeneous nature of pregnancy loss, but also to provide an individual answer to the couples. In 1000 consecutive pregnancy losses between gestational age 5 and 22 weeks, we were able to provide a cffDNA based result of the fetal ploidy status in 89% of cases. When comparing the method to direct sequencing of the pregnancy tissue, the cffDNA-based testing had a higher success-rate and identified 85% of the aneuplodies (sensitivity) with a 93% specificity. These results show the potential and feasibility of the method to distinguish euploid and aneuploid pregnancy loss for improved clinical management and benefit of future research. From our close collaboration with couples that are experiencing a devastating event as pregnancy loss, we have seen the huge unmet need for better handling, but also a deep commitment and motivation from the men and women involved. We have investigated the conflicting feelings emerging in a next pregnancy and a need for closer support and care. We belief, that it takes a trio to improve the understanding and handling of pregnancy loss.

P-245 Evaluating the impact of embryos undergoing multiple freeze-thaw cycles in preimplantation genetic testing for aneuploidy (PGT-A) on treatment outcomes in IVF

Abstract Study question Do cryopreserved embryos subjected to thaw, biopsy, re-freeze (TBR) exhibit inferior pregnancy outcomes compared to embryos undergoing standard PGT-A with a single vitrification-warming cycle? Summary answer Transfer of twice-thawed euploid embryos demonstrated comparable clinical pregnancy outcomes, yet elevated miscarriage rates following a repeated vitrification-warming cycle warrants further scientific inquiry. What is known already There is often hesitancy to test cryopreserved embryos that were not initially PGT-A tested due to concerns about embryo survival during the additional freeze-thaw cycle those embryos will endure. However, the TBR technique has shown efficacy, particularly for IVF patients with recurrent failed attempts using fresh, untested embryos. Previous studies on TBR consistently report comparable embryo survival rates to fresh cycles. Nevertheless, there is no consensus on whether transferring euploid TBR embryos adversely affects pregnancy outcomes compared to euploid fresh biopsy embryos, potentially due to the relatively low number of transferred TBR embryos in such studies. Study design, size, duration This study retrospectively analysed euploid freeze-thaw single embryo transfer (SET) data from embryos that underwent PGT-A at a large fertility clinic in London, between January 2019 and June 2022. Pregnancy outcomes were compared between freshly biopsied embryos (control group, n = 349) and cryopreserved embryos that underwent thaw and biopsy before freeze-thaw SET (twice-thawed group, n = 35). Embryos previously biopsied with PGT-A, subsequently cryopreserved, and then subjected to thaw and re-biopsy were classified in the twice-thawed group. Participants/materials, setting, methods Blastocysts cultured from fresh embryos (control group) or derived from frozen-thawed blastocysts (twice-thawed group) underwent PGT-A using standard protocols before freeze-thaw SET. The study assessed biopsy rates, post first thaw and survival rates post second thaw in the twice-thawed group, comparing to the control group. The clinical outcomes evaluated were Implantation rate (IR), Live Birth Rate (LBR), and Miscarriage Rate (MR). Statistical analysis included one-way ANOVA for continuous and Pearson’s chi-squared test for categorical variables Main results and the role of chance Blastocyst characteristics, including morphological quality (characterised into ‘GOOD’, ‘FAIR’ and ‘POOR’) and day of biopsy (5 or 6), did not significantly differ between blastocyst transfers of the twice-thawed and control group. Furthermore, maternal factors including age, number of previous PGT-A attempts and endometrial thickness did not significantly differ between groups. However, compared with the control group, the number of eggs collected was significantly higher in the twice-thawed group (15.8±8.20 vs. 25.8±18.7, p = <0.001). The biopsy rate of previously cryopreserved embryos that underwent TBR was 79%. However, there was no significant difference between the survival rate after the second thaw and before SET between the twice-thawed and control groups. There was no difference in IRs (71.1% vs. 74.3%, p = 0.687) and LBRs (56.4% vs. 40.0%, p = 0.062) between groups. Although, compared to the control, MRs were significantly higher in the twice-thawed group (20.6% vs. 46.2% p = 0.003). Limitations, reasons for caution The twice-thawed group includes patients with diminished ovarian reserve, previously undergoing single freeze-thaw SET resulting in recurrent miscarriages. Thus, further research is essential to clarify whether elevated miscarriage rates result from repeated freeze-thaw cycles. Additionally, as single freeze-thaw SET is preferred, small sample size in the twice-thawed group limits conclusions. Wider implications of the findings This study suggests employing TBR on previously frozen blastocysts yields non-inferior IRs and LBRs, with an additional freeze-thaw cycle not adversely affecting embryo survival before SET. Therefore, such techniques should be considered in previously frozen IVF embryos to reduce wastage, mitigate unforeseen fertility complications, and concurrently achieve favourable pregnancy outcomes. Trial registration number Not applicable

P-559 Pregnancy rates in conventional and non-invasive pre-implantation genetic testing – a multicentric retrospective cohort study

Abstract Study question To determine the pregnancy rates with embryo diagnosed as euploid by non-invasive preimplantation genetic testing (niPGT-A) and trophectoderm based pre-implantation genetic testing (cPGT-A) Summary answer Biochemical pregnancy rates are comparable in couples undergoing transfer with embryos diagnosed as euploid as judged by niPGT-A or cPGT-A What is known already NiPGT-A utilizes cell-free DNA in culture media to assess the chromosomal status of embryos. Several studies have shown that the niPGT-A and cPGT-A have a reasonable concordance rate and niPGT-A can be considered as an alternative in predicting the ploidy status of the embryos. However, limited information exist on the clinical outcomes of embryos deemed euploid by niPGT-A. Study design, size, duration Data of 1209 participants between June 2022 and Dec 2022 was collected from 46 centers of Indira IVF Hospital. These were couples who had experienced recurrent implantation failures (>3 failed ET), recurrent miscarriages (>2 miscarriages) or had advanced maternal age (>35 years) and opting either for niPGT-A or cPGT-A at our centres for euploid embryo transfers. Women with anatomical uterine defects and Asherman syndrome were excluded. Participants/materials, setting, methods Trophectoderm biopsy was done in 1383 Day 5 grade A embryos and PGT-A was done using NGS. Culture media of 3083 Grade A embryos were collected on D5 or D6 and frozen. NGS was done at an external facility. Embryos deemed euploid in either of the groups were transferred. Biochemical pregnancy was determined by measurements of beta hCG. Main results and the role of chance There were 906 patients in the niPGT-A group and 303 in the cPGT-A group. The clinical characteristics of the participants were comparable in both groups. Biochemical pregnancy rates were 60% and 69%, respectively. This difference was not statistically significant (p < 0.6). The euploidy rates were comparable in the niPGT-A and cPGT-A groups (40% and 46%) respectively. The aneuploidy rates were lower in the niPGT-A group as compared to the cPGT-A group (15% vs 26%). The mosaicism rates were higher in the niPGT-A group as compared to the cPGT-A group. Limitations, reasons for caution This is a preliminary data analysis of only the biochemical pregnancy rates. Clinical pregnancy, miscarriage, and live birth outcomes are subject to further investigation. Wider implications of the findings The study will aid IVF specialist to consider niPGT-A as a possible alternative to cPGT-A in clinical practice. Trial registration number ’not applicable’

P-124 Machine learning scores reveal mosaic embryo quality as the key predictor for live births, outperforming mosaicism characteristics in ART success

Abstract Study question Is it possible to score the mosaic embryos in order to prioritise them according to their pregnancy potencial? Summary answer The machine-learning score underscores the crucial role of embryo quality over mosaicism, prioritizing high-quality, day-5 biopsied embryos for enhanced live birth outcomes in ART. What is known already After embryo biopsy and PGT-A analysis, embryos can be classified as euploid, aneuploid, or mosaic, wherein a combination of chromosomally normal and altered cells is detected. The transfer of mosaic embryos has been controversial, but currently, the recommendations of international scientific societies, including ESHRE, advocate for their transfer. In case of pregnancy, a prenatal test is advised to rule out any chromosomal aberration. The non-transfer of these embryos reduces ART success chances, as chromosomally normal children may be born from them. Study design, size, duration The study design is observational and retrospective. A total of 8346 embryos from 2583 PGT-A cycles were considered (January-2017 to January-2023). Only transferred mosaic embryos (n = 263) were included in the study. The trophectoderm biopsies of D5, D6, or D7 blastocysts were analyzed by NGS using the Illumina platform (VeriSeq Illumina®, San Diego, CA, USA). The biopsied embryos were vitrified and transferred in a subsequent cycle. Participants/materials, setting, methods The primary indications for PGT-A included advanced maternal age, abnormal sperm FISH, and recurrent miscarriage or implantation failure. Clinical outcomes, along with variables related to progenitors, embryos, biopsy, ovarian stimulation, and adjuvant treatments, were recorded in a database. Machine learning models were conducted using R statistical software (v.4.3.1), and the ‘Caret’ library was utilized to implement different algorithms. ‘Autoscore,’ as an interpretable scoring tool, combined machine learning and regression modeling. Main results and the role of chance The mean maternal and paternal ages were 34.95±6.38 and 39.13±6.74 years. Biopsies were predominantly performed on day 5 (61%). Additionally, 44% of embryos were of A quality, and 49% were of B quality. Regarding clinical outcomes for mosaic embryos, biochemical, clinical pregnancy, and live birth rates were 51%, 41%, and 36%, respectively. The features used to generate the score included those related to embryo quality, biopsy, and mosaicism. Through 10-fold cross-validation, the study identified top-ranking predictors maximizing the AUC of the predictive model. The highest weight in the score was assigned to embryo quality (42%) and day of biopsy (34%), with lower weights for factors related to mosaicism: monosomy and/or trisomy (18%) and mosaicism level (6%). The machine-learning score indicates that, in transferring embryos with mosaicism, priority should be given to high-quality embryos biopsied on day 5. The type and level of mosaicism have a minor impact on the embryo’s gestational capacity, becoming relevant only when prioritizing embryos of identical quality. In such cases, embryos with lower mosaicism degrees and no monosomy demonstrate higher implantation potential. Limitations, reasons for caution The inherent limitations of a retrospective analysis highlight the need for prospective randomized studies to confirm the validity and usefulness of the mosaic embryo transfer score. Wider implications of the findings A scoring system has been developed to prioritize the transfer of mosaic embryos. The crucial variables are associated with embryo quality and the day of biopsy, reflecting the progression of embryonic development. Conversely, variables related to mosaicism play a secondary role. Trial registration number Not applicable