Abstract Study question What is the underlying mechanism of sexual dimorphism in the preimplantation embryo? Summary answer Cyclin D1 (CCND1) expression is upregulated in female vs male pre-implantation embryos, potentially contributing to early sexual dimorphism What is known already Sexual dimorphism starts as early as the pre-implantation embryo. In-vitro studies have demonstrated faster development in XY vs XX embryos. Clinical studies have shown skewed male to female ratios in live births from in-vitro fertilization, likely due to bias in selecting the most developed embryo for transfer. CCND1 is a gene encoding the protein Cyclin D1, a highly conserved cell cycle regulator. Responding to mitogenic signals, CCND1 heterodimerizes with cyclin-dependent kinases and promotes progression from G1 to S phase of the cell cycle. Prior studies from our lab showed higher expression of CCND1 in XX vs XY pre-implantation mouse embryos. Study design, size, duration This was a retrospective study of 70 vitrified-thawed human embryos, from 27 patients, donated for research. Blastocysts were vitrified and thawed per our institutional protocol. Following exclusion for aneuploidy, 61high-quality blastocysts, 31 XX and 30 XY, were analyzed for differential expression of autosomal genes between the two groups. Participants/materials, setting, methods Sequencing was performed per the Harvard Bauer Sequencing Core protocol, using pre-amplified cDNA libraries from single embryos. RNA-sequencing data were processed via the Smart-seq2 Single Sample Pipeline. Aneuploidy screening was performed using the digital karyotyping method: A Z-score is calculated for each autosome per sample. Embryos were sexed similarly: summing counts of X and Y specific genes and generating a Z score for chromosome-wide expression. Differential gene expression analysis was performed using DESeq2 in R Main results and the role of chance Mean (SD) oocyte age was 34.4 (2.6) years. CCND1 gene expression was significantly higher in XX blastocysts when compared to XY by 1.55 log2fold change with an adjusted p-value p = 0.005. Furthermore, the gene expression for cyclin dependent kinases 1A and 1C (CDK1A, CDK1C) was also significantly upregulated in XX blastocysts vs XY with log2fold changes of 1.5 (p = 0.01) and 1.7 (p = 0.018) respectively. Limitations, reasons for caution This was a retrospective study, including embryos from patients with different infertility diagnoses and stimulation protocols. In addition, 8 patients contributed embryos to both groups. Furthermore, due to the nature of embryos donated for research, all embryos from an IVF cycle cohort were not included due to previous embryo transfers. Wider implications of the findings This is the first study to show that expression of CCND1 and its related kinases are upregulated in pre-implantation human female embryos vs male. Given the pathway’s impact on mitosis, this could be a crucial finding, shedding light into early sexual differentiation, especially relating to early development and blastulation. Trial registration number No

Abstract Study question Does increasing the dosage of triptorelin acetate from 0.2 mg to 0.3 mg result in higher clinical pregnancy rates in POSEIDON Groups 3 and 4? Summary answer Increasing the dosage of triptorelin acetate from 0.2 mg to 0.3 mg does not significantly enhance oocyte maturation or retrieval outcomes What is known already Emerging research suggests that different doses of GnRH agonists yield varying outcomes, particularly in subgroups with distinct reproductive profiles. However, most randomized controlled trials (RCTs) evaluating GnRH agonist doses have been conducted in predicted good ovarian responders or women with adequate ovarian reserve. There is a paucity of data on the optimal dose of GnRH agonists, particularly triptorelin acetate, in low prognosis patients classified under POSEIDON Groups 3 and 4 . This gap in the literature is significant, as these patients represent a unique challenge in ART due to their reduced ovarian reserve and advanced reproductive aging. Study design, size, duration This multicenter, randomized, open-label, controlled trial included 654 patients classified as POSEIDON Groups 3 and 4 (AMH < 1.2 ng/dL, AFC ≤5) who were randomly assigned to receive either 0.2 mg (Group A) or 0.3 mg (Group B) of triptorelin acetate for triggering final oocyte maturation. Participants/materials, setting, methods An interim analysis was performed when around 50% of the sample was included in the study. Key outcomes included the number of oocytes retrieved, the percentage of mature oocytes (M2), the duration of ovarian stimulation, and clinical pregnancy rates. Main results and the role of chance Of the 295 patients included in the interim analysis (155 in Group A and 140 in Group B), the mean age was 33.89 years, with no significant differences in baseline characteristics. The mean number of oocytes retrieved was similar between groups (4.73 in Group A vs. 4.62 in Group B). Although the percentage of mature oocytes was higher in Group B (65.87%) compared to Group A (61.30%), this difference was not statistically significant (p = 0.293). Notably, clinical pregnancy rates were significantly higher in Group A (61.74%) compared to Group B (45.07%) (p = 0.049). The lower dosage (0.2 mg) was associated with better clinical pregnancy rates, underscoring the importance of personalized treatment strategies in ART for low-prognosis patients Limitations, reasons for caution The population concentrates on low-prognosis patients in POSEIDON groups, which may impair the findings' generalizability to the larger population undergoing ART. Due to its retrospective nature, the capacity to demonstrate causality between triptorelin dosage and outcomes may be limited, and confounding variables may influence the results. Wider implications of the findings The significantly higher clinical pregnancy rates observed in the 0.2 mg group suggests that 0.2 mg triptorelin acetate may be more effective than 0.3 mg in achieving higher clinical pregnancy rates in low prognosis patients classified under POSEIDON Groups 3 and 4. Trial registration number Yes

Abstract Study question What is the impact of lack of awareness on menstrual cycle characteristics and fertile window among UK based women, especially those trying to conceive (TTC)? Summary answer Lower fertility awareness is reported by those TTC for more than a year, potentially impacting conception chances, highlighting a need for improved reproductive education. What is known already Despite being a fundamental biological process, the menstrual cycle remains shrouded in stigma, leading to widespread misconceptions. Limited menstrual health and fertility education contributes to misunderstandings about ovulation timing and the fertile window, resulting in mistimed intercourse and delays in conception. Poor fertility awareness also results in delays in seeking medical help and hinders diagnosis of conditions like polycystic ovary syndrome (PCOS). With rising infertility rates and later family planning, accessible, evidence-based fertility education is crucial. This study aims to identify key gaps in menstrual health literacy and fertility awareness to inform targeted educational interventions and improve reproductive health outcomes. Study design, size, duration A retrospective, cross-sectional study analysing self-reported data from 386,811 UK-based women aged 18–58 who completed an online health assessment (OHA) between September 2020 and January 2025. Participants provided self-reported demographic, menstrual, and medical history, lifestyle behaviours, fertile window (FW) knowledge, reproductive intentions and time trying to conceive. The findings were stratification by cycle characteristics, age and time spent trying to conceive. Participants/materials, setting, methods From the 386,811 participants included, a sub analysis was done on 97,414 women who were actively TTC. Responses were categorised by menstrual cycle characteristics ( regularity and length). age group and duration of time trying to conceive. Descriptive statistical analyses were conducted using R to assess trends and misconceptions in fertility awareness. The study setting was an online platform, making it accessible to a diverse population. Main results and the role of chance Of 386,811 participants, 60.8% reported regular cycles, 26.5% irregular, 12.5% had no periods and 22.2% did not know their cycle length, particularly those under 25 (33.4%). 41.5% with irregular cycles were unaware of cycle length, while 33.9% had a clinically regular length (21–35 days). 18% of those reporting regular cycles fell outside this range, with misidentification most common in under 25s (23.5%). Among 97,414 TTC, 16.7% were unaware of cycle length, and 12.8% misidentified their cycles as regular. Fertile window knowledge was low, with 13.4% unaware and 27.6% unsure of when it was. Awareness was lowest among under-25s (42.5%) and highest in those aged 36–40 (67.1%). Only 53.7% of those TTC for over a year correctly identified when their fertile window was, they were also more likely to be unaware of cycle length (20.2%) and misidentify cycles as regular (13.8%) compared to those TTC for less than a year (15.3% and 12%, respectively). A greater proportion of those who had never been pregnant were unsure (28.5%) or unaware (15%) of when their fertile window compared to those who had previously been pregnant (26.7% and 11.7%, respectively). Limitations, reasons for caution Self-reported data may introduce recall bias and inaccuracies. The study population was limited to digital health users, potentially affecting generalisability. Wider implications of the findings Findings highlight significant gaps in menstrual and fertility knowledge, particularly among younger women and those TTC for over a year, which can hinder conception efforts. Integrating accessible, evidence-based fertility education into public health initiatives could empower individuals to make informed reproductive decisions, access medical intervention earlier and improve conception outcomes. Trial registration number No

Abstract Study question Do the stage of development, and the grading of embryos have an impact on live birth rate (LBR) in day 5 single fresh embryo-transfer cycles? Summary answer Grade 2-4 embryo stage of development according to ESHRE consensus is associated with a significantly higher LBR compared to grade 1 embryo stage of development. What is known already Defining the impact of the stage of development, and the grading of day 5 embryos on live birth rate is of pivotal relevance to improve the estimation of prognosis and could be helpful in deciding whether to extend embryo culture. The scant available evidence shows the absence of a correlation between the stage of development, and the grading of embryos and the LBR in day 5 single fresh embryo-transfer cycles. However, the reliability of such findings is limited by the small sample sizes and the introduction of relevant confounding factors. Study design, size, duration This is a retrospective cohort study including 2107 transfer cycles on day 5 of culture, performed between 2015 and 2023 at IRCCS Humanitas Research Hospital, Fertility Center (Milan, Italy). Participants/materials, setting, methods Women who underwent single fresh blastocyst transfer on day 5 of culture could be considered for study entry. Pre-implantation Genetic Testing (PGT) cycles and transfers of embryos obtained from cryopreserved oocytes were excluded. The stage of development, and the grading of embryos were classified according to the ESHRE and Istanbul consensus on embryo assessment. Main results and the role of chance Grade 2 embryo stage of development was associated with a significantly higher LBR compared to grade 1 embryo stage of development both at the univariate (odds ratio (OR) 1.57, 95% confidence interval (CI) 1.23, 2.00) and at the multivariate analysis (adjusted OR (aOR) 1.53, 95%CI 1.19, 1.96). Grade 3-4 embryo stage of development was associated with a significantly higher LBR compared to grade 1 embryo stage of development both at the univariate (OR 1.97, 95%CI 1.57, 2.48) and at the multivariate analysis (adjusted aOR 1.91, 95%CI 1.52, 2.40). No significant differences emerged when Grade 2 embryo stage of development was compared to Grade 3-4 embryo stage. After having the restriced the analysis to Grade 2-4 embryos, we observed that both fair and poor trophoectoderm (TE) and inner cell mass (ICM) characteristics were associated with a significantly lower LBR compared to good TE and ICM characteristics (OR 0.73, 95%CI 0.56, 0.94 and OR 0.40, 95%CI 0.26, 0.61, respectively). These findings were confirmed at the multivariate analysis. Limitations, reasons for caution The study design as well as the partially subjective evaluation of blastocyst expansion limit the reliability of results. Wider implications of the findings Women who undergo day 5 fresh embryo-transfer, should be counselled about their prognosis on the basis of both embryo stage of development and TE/ICM characteristics. Our results also provide the rational basis for designing studies comparing day 5 fresh embryo-transfer to culture extension and freeze all in day 6/7. Trial registration number No

Abstract Study question Can the cyclophosphamide equivalent dose (CED) given to young boys be used to predict future infertility? Summary answer Our data suggests that CED alone is not a good predictor of subsequent infertility. What is known already Currently young males are referred for fertility preservation based on the estimation of risk of infertility after gonadotoxic treatment utilizing the calculation of the cyclophosphamide equivalent dosage (CED). It has been generally assumed that, in young males, a CED of 3-4 g/m2 results in a risk level of 50 to 80% for future infertility and that ≥ 6 g/m2 results in a higher risk of infertility. However, data regarding the relative CED and subsequent adult recovery of spermatogenesis for these males is sparse. Study design, size, duration Testicular Tissue Cryopreservation (TTC) is offered at our centre1 to families of young boys with childhood cancer or immunologic disorders who will receive gonadotoxic treatment with >3 g/ m2 CED. A number of these boys are now adults and our aim was to assess the impact of this chemotherapy on their subsequent fertility as assessed by the presence of sperm in ejaculates. Participants/materials, setting, methods TTC has been performed for prepubertal or peripubertal boys (total =255), of whom 67 are now aged ≥ 18 years. 23 adult males have returned to produce a semen sample. Semen was provided a minimum of 5 years after cessation of treatment in all cases. Data is available for 11 boys who had testicular tissue frozen at age ≤13 years and 12 who had sperm observed during dissection of tissue (age 13-18 years). Main results and the role of chance In those who were pre /peri pubertal at the time of TTC, 9/11 adult males had some viable sperm and 5/11 had a normal density (>20 million/ml). The lowest CED at which no sperm were detected was 6.5 g/m2. However, 2 adult males had recovery to normal density following high CED (14 and 16 g/m2). In adult males who had some sperm frozen during TTC as post pubertal males, 10/12 had some viable sperm and 4/12 had normal densities. The lowest CED at which there were no sperm detected was 12 g/m2 but recovery to normal range also took place following 12 g/m2 in 1 male. The results showed wide variability in sperm density with CED, with a trend to lower sperm density at higher CED, and a rapid decline in sperm density at around 4 g/m2 CED in both groups. Limitations, reasons for caution The number of males who have returned to produce a semen sample for evaluation is low and needs to be increased to draw more reliable conclusions. Wider implications of the findings Discussion regarding chemotherapy related future infertility should be had with all parents / boys having a CED > 4 g/m2 and options provided to preserve testicular tissue and /or sperm. Reference: 1. Ho et al., (2017) Clinical Endocrinology. 87(3): p. 279-285 Trial registration number No

Abstract Study question Do ionizing radiations affect human sperm integrity? If so, at what dose? Summary answer In vitro exposure of human ejaculated spermatozoa significantly reduces vitality, motility, and chromatin integrity without causing DNA fragmentation, telomeric damage, or altering capacitation ability. What is known already Exposure to ionizing radiation (IR) is a public health concern. However, its impact on human spermatozoa remains poorly described, particularly regarding nuclear damage. Our objective is to assess the impact of IR on functional parameters, as well as on nuclear quality. To achieve this, we expose human spermatozoa in vitro to increase doses of IR, reflecting various diagnostic and therapeutic medical scenarios. This study will help elucidate the mechanisms by which IR affects male fertility and, consequently, optimize fertility preservation strategies. Study design, size, duration We exposed human spermatozoa in vitro to increase IR doses mimicking epididymis irradiation during medical procedure: diagnostic imaging (scanner CT: 14–70 mGy) and radioiodine therapy (140 mGy). Sperm vitality, motility, DNA fragmentation, chromatin decondensation, telomere length (STL), nuclear morphometry, oxidative stress and capacitation markers post-IR were compared to their non-irradiated controls for each patient to address inter-individual variability. Participants/materials, setting, methods We used surplus semen from patient having consented to research (Germethèque Biobank). Gamma radiation conditions mimicking CT doses of 14 (n = 35), 70 (n = 39), and 140 mGy (n = 73) were experimentally calibrated. Sperm parameters (WHO 2021 guidelines), DNA fragmentation (TUNEL/SCD assays), chromatin decondensation (Chromomycin A3), STL (qPCR), nuclear morphometry (3D bioimage analysis), oxidation (Oxiselect/MDA kits) and capacitation markers -velocity (SCA© CASA), [AMPc] (ELISA), phosphotyrosine profile (Western blot) and acrosome integrity (PSA-FITC)- were evaluated. Main results and the role of chance The three dose groups showed no significant differences in initial sperm parameters or bio-clinical criteria, confirming the absence of biases. We observed a significant decrease in sperm vitality across all three radiation doses compared to the non-irradiated control condition (-3.4% for 14 mGy, -4.2% for 70 mGy, and -5.6% for 140 mGy; p < 0.001). Progressive motility was reduced at 140 mGy (37.0 ± 2.3% versus (vs.) 33.1 ± 2.0% after IR exposure; -3.9%, p < 0.001). IR induced chromatin decondensation at 70 mGy (23.6 ± 1.9% vs. 26.0 ± 1.8%, +2.3%, p < 0.05) and 140 mGy (23.5 ± 1.3% vs. 26.1 ± 1.5%, +2.5%, p < 0.01), with greater decondensation observed in samples with pre-existing sperm parameter alterations (+3.5%, p < 0.05). No significant changes were detected in DNA fragmentation levels (TUNEL or SCD assays) or sperm telomere length (STL). Exposure to 140 mGy did not significantly alter oxidative levels in seminal fluid or sperm cells, nor did it affect capacitation markers, although motility alterations were identified using SCA®. Finally, no significant global impact on 3D nuclear morphometric parameters has been observed to date. Limitations, reasons for caution This model provides an opportunity to analyze IR-induced alterations without patients/pathology’s interference. Differences in dose rate, exposure duration, and the spermatozoa environment (seminal instead of epididymal fluid) represented limitations. In vivo studies are essential to fully understand the impacts of IR on the regulation pathways of spermatogenesis and sperm maturation. Wider implications of the findings We show for the first time the impact of IR on human sperm quality using three radiations doses used for clinical diagnosis or treatment. Significant but minor effects on conventional parameters and chromatin decondensation were observed. Ongoing work includes epigenomic alterations from sperm cells and seminal fluid to further investigate. Trial registration number Yes

Abstract Study question Can real-world data (RWD) offer valuable insights into the management of assisted reproductive technology (ART) and enhance patient care? Summary answer OPERA project provided a descriptive analysis of RWD from ART cycles, facilitating an in-depth evaluation to enhance the efficiency and effectiveness of ART procedures. What is known already ART is widely used for infertility treatment, with outcomes influenced by patient characteristics and stimulation protocols. Randomized controlled trials (RCTs) are widely regarded as the gold standard for evaluating the effectiveness of interventions. However, a key limitation of RCTs is that they may not fully reflect the benefits and risks of treatments in the broader patient populations encountered in clinical practice. In contrast, understanding RWD is crucial. RWE offers a complementary approach to RCTs, providing valuable scientific knowledge that can inform clinical decision-making. Study design, size, duration The OPERA project, sponsored by FROM Foundation ETS, is an European observational retrospective academic multicentre study analyzing 95,108 ART cycles performed between 2016 and 2020 in 18 centers across Italy and Germany, all using the same database, MediTex.These 95,108 ART cycles include 64,081 ovarian stimulation cycles and 31,027 frozen embryo transfer (FET) cycles derived from them. Participants/materials, setting, methods Data from the MedITEX database, collected across 18 ART centers, were securely transmitted to the Electronic Data Capture system ICE and managed/anonymized in accordance with GDPR and GxP regulations. The study included patient demographics, hormonal profiles, and ART protocols. Outcomes such as oocyte retrieval, fertilization rates, embryo transfers, and pregnancy results (fresh and frozen) were analyzed. Main results and the role of chance Among the 64,081 ovarian stimulation cycles, recombinant FSH was the most commonly used gonadotropin, accounting for 33.4% of cycles; GnRH antagonists were preferred, accounting for 78.4% of cycles. The median peak estradiol level was 1,028 pg/mL. Ovarian pick-up (OPU) was achieved in 93% of cycles, while the remaining 7% of cycles were cancelled before OPU, mainly due to poor ovarian response. The median number of oocytes retrieved per cycle was 8. ICSI was the predominant insemination technique, used in 72.1% of cycles. In 41,3% of stimulation cycles it was possible to cryopreserve oocytes or embryos. For fresh embryo transfer (ET) cycles, the clinical pregnancy rate (CPR) was 33.8%, and the live birth rate (LBR) was 25.8%. For frozen embryo transfer (FET), CPR and LBR were similar, at 34.2% and 24.3%, respectively, with a slightly higher miscarriage rate in FET cycles (28.9% vs 23.8% in fresh ET). The cumulative pregnancy rate and cumulative live birth rate were 39.3% and 30.2%. Overall, OPERA data indicate that the protocols used are very safe, with the risk of Ovarian hyperstimulation syndrome remaining below 5% of initiated cycles. Limitations, reasons for caution The retrospective design may introduce bias, and data are limited to specific centers and populations. However, by integrating diverse healthcare systems, OPERA provides valuable information into treatment differences and outcomes. Such a broader dataset enhances the ability to analyze and optimize ART procedures, also through the application of artificial intelligence algorithms. Wider implications of the findings OPERA enables a range of comparisons across treatments, laboratory results, and outcomes. It offers a comprehensive descriptive analysis of ART procedures, from couple selection and treatment choices to final outcomes. With the integration of AI, the database has the potential to enhance the efficiency and effectiveness of various interventions. Trial registration number No

Abstract Study question How do patients perceive IVF counseling, and how does it impact conversion from second consultation to treatment, and likelihood of discontinuation after a failed attempt? Summary answer Being aware of psychological-support availability, diminished-ovarian-reserve, and stronger belief in a multicycle-approach associated with higher conversion-rate. Older women with no/fewer embryo-transfers failed showed lower treatment-discontinuation. What is known already ART has enabled millions of infertile women and men worldwide to seek treatment, yet it remains underutilized. Despite their strong motivation to achieve a live birth, treatment discontinuation after failure is a significant challenge. For ART patients, understanding realistic success probabilities and adopting a multicycle perspective are essential for empowering decision-making and preparing them to cope with potential setbacks. In this context, the role of reproductive counselling becomes pivotal. Effective counselling not only supports patients in navigating their journey but also addresses the reasons behind treatment discontinuation, ultimately improving the overall quality of infertility care and patient outcomes. Study design, size, duration Control arm of a prospective study (March-December 2024) comparing standard versus AI-based personalized prognostic counseling strategies. Out of 97 patients recruited after their second IVF counseling, 65 anonymously filled-in a survey (67%) to outline their opinion on factors impacting on IVF success, personal versus gynecologists’ expectations, and satisfaction with the information received. The outcomes were: conversion from second counseling to IVF treatment and treatment-discontinuation ≥3 months after a first failed IVF attempt. Participants/materials, setting, methods On average, the patients were 36 years-old, had 2.6ng/ml AMH and 24.3Kg/m2 BMI, 77% with an academic education, >70% with >1year of infertility, 34% with diminished-ovarian-reserve. Of them, 45% were already counseled and 5% treated elsewhere. The conversion-rate second counseling to treatment was 88% (N = 57/65). The treatment-discontinuation after a first failed IVF attempt was 46% (N = 11/24). We adopted Fisher’s exact-/t-tests to investigate the association between survey’s answers and these outcomes, accounting for confounders. Main results and the role of chance Among the patients, 89% prefer highly detailed IVF-counseling. 85% prioritized achieving a live-birth within short timeframe over less-invasive (12%) or less-expensive treatments (3%). When asked about factors impacting success, 69% acknowledged lifestyle-habits, 82% ovarian-reserve, 65% semen-quality, 55% infertility-diagnoses, 39% reproductive-history, and 52% BMI as significant contributors. All patients thoroughly read consent-forms and 66% declared graphical-material was used by gynecologists to estimate their prognosis; 27% had higher expectations than those provided. The possibility of requiring multiple-attempts was anticipated for 42 patients (65%), all acknowledging the importance of multicycle counseling; 83% to enhance their chances of success, 52% to shorten time between attempts, 88% to obtain realistic estimates of expenses, and 95% of time spent in treatment. Psychological-support was mentioned to 21 patients (32%). Patients were highly satisfied with the explanations regarding medications (4.3/5), techniques (4.8/5), costs (4.6/5), prognosis (4.5/5), consent-form (4.6/5), informative-materials (4.3/5), and gynecologists’ empathy (4.5/5). Features associated with higher conversion rates included awareness of psychological-support availability (N = 21/21,100% versus N = 36/44,82%;p=0.035), diminished-ovarian-reserve (N = 22/22,100% versus N = 35/43,81%,p=0.029), and stronger belief in a multicycle-approach (4.5/5 among converted versus 3.9/5 among non-converted patients). Features associated with higher treatment-discontinuation included younger maternal-age (36.2 versus 38.5years,p=0.011) and number of transferred blastocysts failing to implant/miscarrying (1.12 versus 0.2,p=0.008). Limitations, reasons for caution The small sample size precluded additional sub-analyses with sufficient statistical power (e.g., among different gynecologists). The continuation of the study will involve a larger sample size and will examine the influence of AI-based personalized prognostic counseling. The treatment-discontinuation rate is still incomplete as 18 first cycles are still ongoing. Wider implications of the findings Evidence-based counseling with a multicycle perspective could improve patient conversion rates and reduce treatment discontinuation, particularly among better-prognosis women experiencing implantation failures. Additionally, mentioning psychological support as an available option may provide further benefits. Trial registration number Yes

Abstract Study question At What Age and Embryo Grade Does iDAScore Effectively Predict Euploid Embryos? Summary answer Higher mean iDA scores associate with lower genetic abnormalities across all ages, notably in ≥ 41 patients and lower-grade embryos (BC, CB), where predictability is stronger. What is known already A higher iDA score is known to correlate with increased euploidy rates, indicating better embryo viability. Previous studies have shown that euploid embryos have higher implantation and live birth rates, making genetic assessment crucial in ART. However, it remains unclear whether the predictive value of the iDA score is consistent across different maternal ages and embryo grades. Advanced maternal age is associated with increased aneuploidy, while lower-grade embryos tend to have poorer outcomes. This study aims to explore the relationship between PGT-A results and iDA scores, considering maternal age and embryo grading, to enhance embryo selection strategies Study design, size, duration This retrospective data review study analyzed PGT-A fresh IVF cycles conducted at a single center between May 2022 and December 2023. Participants/materials, setting, methods This retrospective study analyzed 1,527 embryos from 308 fresh PGT-A IVF cycles of 254 patients at a single center (May 2022–December 2023). Participants were categorized into <35, 35–37, 38–40, ≥41 years. Embryos were classified as euploid, aneuploid, or mosaic (high-level ≥50%, low-level <50%). Embryo quality was good (AA, AB, BA, BB) or poor (BC, CB). Mean iDA scores were compared across genetic status, quality, and age groups. Main results and the role of chance This study examined the relationship between iDA scores and PGT-A results across maternal ages and embryo grades. In all age groups (<35, 35–37, 38–40, ≥41 years), euploid embryos had significantly higher iDA scores than aneuploid embryos (p < 0.05). No significant difference was found between euploid and mosaic embryos in participants under 41, but in those ≥41, iDA scores followed a significant trend: euploid > mosaicism > aneuploid (p < 0.05). When mosaics were subdivided into high-level (≥50%) and low-level (<50%), younger groups showed no significant differences. However, in participants ≥41, low-level mosaics had significantly higher scores than high-level mosaics (p < 0.05), while high-level mosaics and aneuploids were similar. For embryo grading, good-quality embryos (AA, AB, BA, BB) showed no significant differences across groups. In lower-quality embryos (BC, CB), euploid and mosaic embryos had significantly higher scores than aneuploids (p < 0.05), particularly in participants <40. However, in 35–37 years, iDA scores did not predict PGT-A outcomes in lower-quality embryos. Notably, BB-grade embryos showed no correlation between high iDA scores and PGT results, reinforcing the stronger predictive value of iDA scores in lower-quality embryos. These findings suggest that IDA scores may be more effective for embryo selection in older patients and lower-quality embryos. Limitations, reasons for caution This study is limited by its retrospective, single-center design and uneven sample distribution (<35: 290, 35–37: 282, 38–40: 364, ≥41: 591 embryos), as PGT-A is more commonly used in older patients. This may impact statistical power in younger groups. IDA scores need further validation and should complement other assessment tools. Wider implications of the findings iDA scores may help embryo selection in ≥ 38-year-old patients and lower-quality embryos (BC, CB) when PGT-A is not feasible due to cost. However, BB-grade embryos and younger patients (35–37 years) showed limited predictability. Further studies should validate its clinical utility and integration with AI-based embryo assessment. Trial registration number No

Abstract Recurrent pregnancy loss (RPL), also known as recurrent miscarriage (RM), is a devastating condition affecting approximately 2.5% of women trying to conceive. This condition is generally defined as the occurrence of two or more spontaneous miscarriages, before 24weeks gestation. It is well known that the leading cause of miscarriages is embryonic aneuploidy, a condition closely linked to maternal age. Chromosomal abnormalities are detected in around 40% of gestational losses in patients experiencing recurrent miscarriages. However, despite significant advances in the field, many pregnancies end in miscarriage with no explanation that can be provided to patients; it is estimated that 50% of couples diagnosed with RPL are of unknown etiology. Beyond genetics, several factors have been proposed as responsible for RPL: uterine abnormalities, endocrinological disorders, immunological disorders, antiphospholipid syndrome, male factors and lifestyle habits. Research into the causes of RPL should focus on enriched populations and understanding the mechanisms of action of harm, it is only then we can discover therapeutic interventions that are likely to show benefit.