Stage Embryos Research Articles

Using progesterone to follicular index ratio is better correlated with intracytoplasmic sperm injection outcome than using serum progesterone level alone.

to investigate the possibility of using the ratio of serum progesterone level to the number of follicles, according to ovarian response in the day of human chorionic gonadotrophin trigger, as a predictor of cycle outcome. A prospective intervensional study was conducted at Kamal Al-Samarai Hospital for Infertility Treatment and IVF during the period from December 2020 to September 2021. Ninety infertile women underwent intracytoplasmic sperm injection (ICSI) cycles using antagonist protocol. Moreover, once the patient reached triggering criteria, meticulous recording of follicular index together with serum estrogen level and serum progesterone level are measured. Fresh embryo transfer of cleavage stage embryo is done once serum progesterone level was less than 1.5 ng/mL. A follow-up to confirm pregnancy rate and cycle outcome was done. The study showed a positive pregnancy rate of 28.9%. The relationship between progesterone follicular index (Prog/FI) ratio and (ICSI) outcome was highly significant with a p value of 0.001. Additionally, an inverse relationship, as the ratio was lower the pregnancy rate was improved, was documented. The receiver operating characteristic (ROC) curve for progesterone follicular index ratio was 0.711 with a cut off value of 0.0354 ng/mL in addition to a sensitivity of 65.6 and a specificity of 65.4. The serum progesterone level is an independent factor for the prediction of intracytoplasmic sperm injection (ICSI) outcome, whereas the progesterone follicular index ratio can be used as a potential marker for predicting ICSI outcome in fresh embryo transfer.

ABA exerts a promotive effect on the early process of somatic embryogenesis in Quercus aliena Bl

Quercus aliena, a native Chinese tree species, is significant in industry and landscaping. However, it is traditionally propagated by seeds with many limitations, such as pest infestations, seed yield and quality. Thus, this study firstly introduces a somatic embryogenesis (SE) system for Q. aliena, enhancing its cultivation prospects. Thereinto, the development stage of zygotic embryo had a significant effect on SE, only immature embryos in 10–11 weeks after full bloom (WAF), rich in endogenous abscisic acid (ABA), could induce SE. Exogenous application ABA had positive roles in the early development process of both primary and secondary SE, while its antagonist had opposite roles. Transcriptome analysis showed that transcription regulation occupied the major position. Mfuzz cluster and WGCNA co-expression analysis showed that 24 candidate genes were involved in the SE process. The expression of the 24 genes were also affected by exogenous ABA signals, among which QaLEC2, QaCALS11 and QaSSRP1 occupied the important roles. Additionally, the callose content were also affected by exogenous ABA signals, which had significantly positive correlations with the expression of QaLEC2 and QaCALS11. This study not only established an efficient reproduction system for Q. aliena, but also revealed the difference in embryogenic ability of zygotic embryos from the aspects of transcriptome and endogenous hormone content, and lay a foundation for clarifying the molecular mechanism of SE, and provided a reference for exploring the vital roles of ABA in SE.

Evolutionary origin of the chordate nervous system revealed by amphioxus developmental trajectories.

The common ancestor of all vertebrates had a highly sophisticated nervous system, but questions remain about the evolution of vertebrate neural cell types. The amphioxus, a chordate that diverged before the origin of vertebrates, can inform vertebrate evolution. Here we develop and analyse a single-cell RNA-sequencing dataset from seven amphioxus embryo stages to understand chordate cell type evolution and to study vertebrate neural cell type origins. We identified many new amphioxus cell types, including homologues to the vertebrate hypothalamus and neurohypophysis, rooting the evolutionary origin of these structures. On the basis of ancestor-descendant reconstruction of cell trajectories of the amphioxus and other species, we inferred expression dynamics of transcription factor genes throughout embryogenesis and identified three ancient developmental routes forming chordate neurons. We characterized cell specification at the mechanistic level and generated mutant lines to examine the function of five key transcription factors involved in neural specification. Our results show three developmental origins for the vertebrate nervous system: an anterior FoxQ2-dependent mechanism that is deeply conserved in invertebrates, a less-conserved route leading to more posterior neurons in the vertebrate spinal cord and a mechanism for specifying neuromesoderm progenitors that is restricted to chordates. The evolution of neuromesoderm progenitors may have led to a dramatic shift in posterior neural and mesodermal cell fate decisions and the body elongation process in a stem chordate.

Plant Hormone Pathway Is Involved in Regulating the Embryo Development Mechanism of the Hydrangea macrophylla Hybrid.

The research is aimed to elucidate the role of plant hormones in regulating the development of hybrid embryos in Hydrangea macrophylla. Fruits from the intraspecific cross of H. macrophylla 'Otaksa' × 'Coerulea' were selected at the globular, heart, and torpedo stages of embryo development. Transcriptome sequencing and differential gene expression analysis were conducted. The results showed that fruit growth followed a single "S-shaped growth curve, with globular, heart, and torpedo embryos appearing at 30, 40, and 50 d post-pollination, respectively, and the embryo maintaining the torpedo shape from 60 to 90 d. A total of 12,933 genes was quantified across the three developmental stages, with 3359, 3803, and 3106 DEGs in the S1_vs_S2, S1_vs_S3, and S2_vs_S3 comparisons, respectively. Among these, 133 genes related to plant hormone biosynthesis and metabolism were differentially expressed, regulating the synthesis and metabolism of eight types of plant hormones, including cytokinin, auxin, gibberellin, abscisic acid, and jasmonic acid. The pathways with the most differentially expressed genes were cytokinin, auxin, and gibberellin, suggesting these hormones may play crucial roles in embryo development. In the cytokinin pathway, CKX (Hma1.2p1_0579F.1_g182670.gene, Hma1.2p1_1194F.1_g265700.gene, and NewGene_12164) genes were highly expressed during the globular embryo stage, promoting rapid cell division in the embryo. In the auxin pathway, YUC (Hma1.2p1_0271F.1_g109005.gene and Hma1.2p1_0271F.1_g109020.gene) genes were progressively up-regulated during embryo growth; the early response factor AUX/IAA (Hma1.2p1_0760F.1_g214260.gene) was down-regulated, while the later transcriptional activator ARF (NewGene_21460, NewGene_21461, and Hma1.2p1_0209F.1_g089090.gene) was up-regulated, sustaining auxin synthesis and possibly preventing the embryo from transitioning to maturity. In the gibberellin pathway, GA3ox (Hma1.2p1_0129F.1_g060100.gene) expression peaked during the heart embryo stage and then declined, while the negative regulator GA2ox (Hma1.2p1_0020F.1_g013915.gene) showed the opposite trend; and the gibberellin signaling repressor DELLA (Hma1.2p1_1054F.1_g252590.gene) increased over time, potentially inhibiting embryo development and maintaining the torpedo shape until fruit maturity. These findings preliminarily uncover the factors affecting the development of hybrid H. macrophylla embryos, laying a foundation for further research into the regulatory mechanisms of H. macrophylla hybrid embryo development.

Facioscapulohumeral Dystrophy: Molecular Basis and Therapeutic Opportunities.

Facioscapulohumeral dystrophy (FSHD) is caused by misexpression of the early embryonic transcription factor Double Homeobox Protein 4 (DUX4) in skeletal muscle. DUX4 is normally expressed at the 4-cell stage of the human embryo and initiates a portion of the first wave of embryonic gene expression that establishes the totipotent cells of the embryo. Following brief expression, the DUX4 locus is suppressed by epigenetic silencing and remains silenced in nearly all somatic cells. Mutations that cause FSHD decrease the efficiency of epigenetic silencing of the DUX4 locus and result in aberrant expression of this transcription factor in skeletal muscles. DUX4 expression in these skeletal muscles reactivates part of the early totipotent program and suppresses the muscle program-resulting in a progressive muscular dystrophy that affects some muscles earlier than others. These advances in understanding the cause of FSHD have led to multiple therapeutic strategies that are now entering clinical trials.

Live Birth after Cleavage-Stage versus Blastocyst-Stage Embryo Transfer in Assisted Reproductive Technology: A Randomised Controlled Study.

Blastocyst stage transfer appears to improve pregnancy outcomes. The aim of this study is to evaluate the pregnancy results between fresh cycle blastocyst stage embryo transfer and cleavage stage embryo transfer in patients who undergo intracytoplasmic sperm injection (ICSI). This randomised clinical trial study was conducted at the Infertility Research Centre of Milad Hospital in Mashhad, Iran from 2018 to 2020 on 240 infertile women who presented for their first ICSI procedure. These patients were assigned to receive either cleavage embryo transfer (n=112) or blastocyst stage transfer (n=107). Pregnancy outcomes were measured in both groups. There were no differences regarding age, body mass index (BMI), serum follicle-stimulating hormone (FSH), duration of infertility, and aetiology of infertility between the groups (P>0.05). There were more follicles, total oocytes, and metaphase II (M2) oocytes in the blastocyst stage group. Considerably more cleavage stage embryos were transferred compared to the number of transferred blastocysts (P=0.001). The blastocyst group had more vitrified embryos than the cleavage group (P=0.000). The rates of implantation (P=0.332), chemical pregnancy (P=0.165), clinical pregnancy (P=0.694), and live births (P=0.727) were higher in the blastocyst group, but they were not significantly different. The rate of abortion was also not significantly higher in the blastocyst group (P=0.296). Blastocysts transferred in the fresh cycle of an ICSI procedure may be more advantageous compared to cleavage stage embryo transfer (registration number: IRCT20181030041503N1).

A Simple, Rapid, and Effective Method for Tumor Xenotransplantation Analysis in Transparent Zebrafish Embryos.

In vivo studies of tumor behavior are a staple of cancer research; however, the use of mice presents significant challenges in cost and time. Here, we present larval zebrafish as a transplant model that has numerous advantages over murine models, including ease of handling, low expense, and short experimental duration. Moreover, the absence of an adaptive immune system during larval stages obviates the need to generate and use immunodeficient strains. While established protocols for xenotransplantation in zebrafish embryos exist, we present here an improved method involving embryo staging for faster transfer, survival analysis, and the use of flow cytometry to assess disease burden. Embryos are staged to facilitate rapid cell injection into the yolk of the larvae and cell marking to monitor the consistency of the injected cell bolus. After injection, embryo survival analysis is assessed up to 7 days post injection (dpi). Finally, disease burden is also assessed by marking transferred cells with a fluorescent protein and analysis by flow cytometry. Flow cytometry is enabled by a standardized method of preparing cell suspensions from zebrafish embryos, which could also be used in establishing the primary culture of zebrafish cells. In summary, the procedure described here allows a more rapid assessment of the behavior of tumor cells in vivo with larger numbers of animals per study arm and in a more cost-effective manner.

High-sensitivity whole-mount in situ Hybridization of Mouse Oocytes and Embryos Visualizes the Super-resolution Structures and Distributions of mRNA Molecules

Mammalian oocytes accumulate more than ten thousand mRNAs, of which three to four thousand mRNAs are translationally repressed. The timings and sites of translational activation of these dormant mRNAs are crucial for promoting oocyte maturation and embryonic development. How these mRNAs are accumulated and distributed in oocytes is therefore a fundamental issue to be explored. A method that enables visualization of mRNA molecules with high resolution in a simple manner would be valuable for understanding how oocytes accumulate and regulate the dormant mRNAs. We have developed a highly sensitive whole-mount in situ hybridization method using in vitro-synthesized RNA probes and the tyramide signal amplification (TSA) system optimized for mouse oocytes and embryos. By using this method, Pou5f1/Oct4, Emi2, and cyclin B1 mRNAs were detected in immature oocytes and 2-cell stage embryos. Confocal microscopy showed that these mRNAs formed granular structures in the oocyte cytoplasm. The structures of Pou5f1/Oct4 and cyclin B1 mRNAs persisted in 2-cell stage embryos. Pou5f1/Oct4 RNA granules exhibited a solid-like property in immature oocytes and became liquid-like droplets in 2-cell stage embryos. Double-staining of cyclin B1 mRNA with Emi2 or Pou5f1/Oct4 mRNA revealed that these mRNAs were distributed as different RNA granules without overlapping each other and that the size of cyclin B1 RNA granules tended to be larger than that of Emi2 RNA granules. The structures and distribution patterns of these mRNAs were further analyzed by N-SIM super-resolution microscopy. This analysis revealed that the large-sized RNA granules consist of many small-sized granules, suggesting the accumulation and regulation of dormant mRNAs as basal-sized RNA granules. The method established in this study can easily visualize the structure and distribution of mRNAs accumulated in mammalian oocytes and embryos with high sensitivity and super-resolution. This method is useful for investigating the cellular and molecular mechanisms of translational control of mRNAs by which maturation and early developmental processes are promoted.

P-202 Impact of multinucleation at the two-cell stage on pregnancy outcomes: a day 3 vs. day 5 transfer analysis

Abstract Study question Does multinucleation at the two-cell stage significantly affect pregnancy outcomes, and how does it differ between day 3 and day 5 embryo transfers? Summary answer Multinucleation at the two-cell stage has a negative effect on pregnancy outcomes in day 3 embryo transfers, but not in day 5 transfers. What is known already Time-lapse imaging provides a valuable tool to study multinucleation in human embryos, although the impact on development and pregnancy remains debated. Literature shows conflicting findings, with most studies associating multinucleation in the early stage embryo with altered developmental potential and decreased pregnancy outcomes. However, other studies suggest a positive correlation, indicating that multinucleated embryos can progress successfully through development and lead to positive pregnancy outcomes. These discrepancies underscore the complexity of and highlight the need for understanding the implications of multinucleation in the context of human embryo development and pregnancy. Study design, size, duration A non-interventional retrospective study on multinucleation was performed on all cycles cultured in a time-lapse incubator in a Dutch fertility clinic between November 2021 and July 2023. In total, 915 embryos (out of 406 cycles) were analyzed and 406 resulting transfers (both fresh and frozen-thawed) were included in this study. Participants/materials, setting, methods Fresh single-embryo transfer was performed on day 3 (for cycles with less than 4 2PN-embryos) or day 5 (for cycles with 4 or more 2PN-embryos). Embryos were vitrified on day 5 or 6 (at least B3BB Gardner score). Nucleation status at the two cell stage was annotated for all transferred and vitrified embryos. Ongoing pregnancies were compared for embryos with or without multinucleation (mononucleation) at the two cell stage. Main results and the role of chance Multinucleation at the two cell stage was observed in 40% (n = 366/915) of all embryos. Multinucleated embryos transferred on day 3 showed a decreased ongoing pregnancy result (17%; n = 9/54) compared to the mononucleated day 3 transfers (29%; n = 24/83). For day 5 transfers, no difference in ongoing pregnancy rates was observed between the multinucleated (37%; n = 37/101) and mononucleated (37%; n = 62/168) groups. These results indicate that development to the blastocyst stage negates any possible negative effect multinucleation can have on cleavage stage embryos. Limitations, reasons for caution A mayor limitation to this study is the small sample size of specifically day 3 transfers. Patients with more than one transfer were allowed to be included. Confounding factors were not assessed. Additionally, different types of multinucleation might result in different clinical effects. Wider implications of the findings Although additional research is needed, these results suggest that assessment of nuclear status at the two-cell stage is important for day 3 transfer strategies, but less so for day 5 blastocyst transfers. Trial registration number Not applicable

O-069 The first mitotic spindle instability impacts nucleation, fragmentation, and development in human embryos

Abstract Study question How multinucleation and fragmentation occur in the first mitosis in human embryos? Summary answer The first mitotic spindles had heterogeneity, and the shapes and movements of the spindles were strongly associated with multinucleation and fragmentation. What is known already Studies have shown that many problems occur during the first mitosis, such as fragmentation and multinucleation. However, the cell biologic mechanisms whereby these errors occur during the first mitosis in human embryos remain unknown. To clarify this aspect, it is necessary to analysis the dynamics of DNA and cytoskeleton using live human embryos. Although microinjections of DNA or mRNA into mammalian embryos were widely used for live imaging during early mitosis, the method into human poses the risk of ethical issues. Therefore, we established a method used for real-time imaging in human embryos using chemical fluorescent labeling. Study design, size, duration Frozen human two-pronuclear stage embryos were donated for research by couples who had completed fertility treatment. We obtained written informed consent from patients who donated human embryos. The mean age of patients at embryo freezing was 32.00 ± 3.03 (SD) years. We imaged thirty-one living human embryos using chemical fluorescent labeling and confocal microscopy to visualize DNA and tubulin. Participants/materials, setting, methods Thirty-one human embryos were cultured in medium containing the live-cell stain probes SPY555-DNA and SPY650-Tubulin. The Embryos were imaged using an LSM980 laser scanning confocal microscope every 10–20 min for a period of eight hours. Three-dimensional (3D) visualizations of human embryos were performed using the Imaris software. The first spindle shapes were quantified by image analysis and calculated the aspect ratio of the spindles using ImageJ. Main results and the role of chance We discovered that over 81% of two-pronuclear zygotes became multinucleated 2-cell stage embryos. We demonstrated that there was variation in the first mitotic spindles. There was a significant difference between the aspect ratio (AR) of the spindles leading to the mononuclear and multinuclear types at the 2-cell stage (p < 0.005). In addition, the low-AR spindles were unstable and often had more defocused poles than the high-AR spindles, which lead to multinucleation at the 2-cell stage (55 % vs. 0%, p < 0.05). More embryo having high-aspect ratio spindles tend to develop blastocysts than those having low aspect-ratio spindles (p < 0.05). Moreover, the movement of the central spindles varied after chromosome segregation. The embryos with more fragments at two cell stage had a significantly longer period for the central spindles to transform into midbodies compared to those with fewer fragments. The central spindles of embryos with more fragments moved a longer distance from the beginning of furrow ingression to the completion of the first division (p < 0.05). These results suggest that the first mitotic spindle instability and heterogeneity cause many errors such as multinucleation, fragmentation, and embryonic development. Limitations, reasons for caution Imaged human embryos were donated from a fertility clinic, and detailed patient information, such as underlying disease and method of ovarian stimulation, was not available. It is possible that the human zygotes used in this study had a bias in patient characteristics. Wider implications of the findings Our study showed that there were instability and heterogeneity in the first mitotic spindle, which was strongly associated with multinucleation, fragmentation, and embryonic development. Revealing the mechanism underlying spindle formation in cleavage-stage embryos will contribute to improving the success rate of assisted reproduction technologies in humans. Trial registration number not applicable

P-585 Vaginal progesterone associated with oral dydrogesterone versus vaginal progesterone for luteal support in letrozole-hcg cycles for thawed embryo transfer: a non-inferiority prospective cohort study

Abstract Study question Does micronized vaginal progesterone (MVP) associated with oral dydrogesterone (DYG) an alternative for luteal support in Letrozole-hcg cycles for thawed embryo transfer? Summary answer The association of MVP and DYG is a non-inferior alternative to only MVP for luteal support in thawed embryo transfer in Letrozole-hcg cycles. What is known already Endometrial preparation is essential for thawed embryo transfer. Several protocols involving different luteal support with progesterones are available. Moreover, the presence of corpus luteum decreases the obstetrical morbidity mainly linked to hypertensive disorders. Letrozole-hcg administration is a good alternative for frozen embryo transfer (FET). However, the literature lacks information about the comparison among the different protocols for luteal support in Letrozole-hcg cycles. Dydrogesterone is an oral progesterone with some unique characteristics which could be utilized with some advantage for luteal support, including oral administration and some immunological activity. Study design, size, duration This is a prospective non-inferiority cohort study performed at an infertility clinic including patients performing the first FET cycle during 2023. Reproductive outcomes were compared between patients receiving 600 mg (3x200 mg) of MPV or 40 mg of DYG plus 400 mg of MVP after Letrozole-hcg administration. Participants/materials, setting, methods All patients (aged 23-39 years) undergoing their first FET cycle during 2023 were included in this study (n = 169), divided into two groups: MVP+DYG (n = 54) or MVP (n = 115) for luteal support after Letrozole-hcg. Letrozole was administered for 5 days (2,5 mg/bid) and hcg was done when a dominant follicle reached 18 mm with a follicular endometrium measuring at least 7 mm. Main results and the role of chance Both groups were equivalent in terms of age, infertility aetiology, BMI and AMH (P > 0.05). Moreover, the proportion of cleavage or blastocyst stage was also similar between both groups (57% blastocyst transfer for MVP+DYG and 50% blastocyst transfer for MVP group, P > 0.05). The number of transferred embryos was (mean ±SD) 1.6±0.5 for both groups, P > 0.05. Furthermore, the number of top-quality embryos (cleavage stage or blastocyst) was also similar, P > 0.05. Moreover, clinical pregnancy (> 12 weeks) was 42% in MVP+DYG group versus 31% in the MVP group (-0.22-1.27, P = 0.156) and the early pregnancy loss per positive hcg test did not differ (18% for MVP+DGY and 22% for MVP, P = 0.687). In addition, we performed a multivariable analysis controlling possible confounders for clinical pregnancy: BMI, age, AMH, embryo quality, infertility aetiology and embryo stage during the transfer, and only age (0.86; 95%CI -0.229 to -0.064, P = 0.001) was related to clinical pregnancy. Limitations, reasons for caution The main limitation is the cohort design of our study, with an inherent risk of bias (selection bias). Moreover, we calculated this study to achieve the power for a non-inferiority comparison, not superiority comparison. Wider implications of the findings This is the first study comparing MVP+DYG to only MVP in patients submitted to FET using the Letrozole-hcg protocol. We demonstrated in this non-inferiority study the usefulness of adding DYG. Moreover, our results were promising showing a trend towards better pregnancy rates after MVP+DYG. Trial registration number not applicable

P-770 Health outcomes of taiwanese infants after different embryo stages in both fresh and frozen transfer procedures

Abstract Study question Does freezing embryos have any effects on neonatal and postnatal health? Does extended embryo culture have any impact on these health outcomes? Summary answer frozen embryo transfer (FET) infants were more likely be large gestational age, but with lower odds of recurrent upper respiratory infection and dermatitis. What is known already Infants born from frozen embryo transfers tend to have higher birth weights and are less likely to be small for gestational age. There are no notable variations in congenital malformations or child development. However, there’s a lack of long-term follow-up data. Study design, size, duration In this retrospective cohort study conducted in Taiwan, data from the national assisted reproductive technology (ART) database spanning from January 1st, 2013, to December 31st, 2017, was utilized. The study categorizes cases into fresh and frozen embryo transfer groups, with additional subdivisions based on cleavage and blastocyst stage transfers. Singletons born in each group were then linked to the National Health Insurance Database for follow-up on health conditions until December 13th, 2018. Participants/materials, setting, methods We collected data on singleton births resulting from assisted reproductive technology procedures performed in Taiwan from January 1st, 2013, to December 31st, 2017. Our analysis included 10,669 eligible infants. We investigated factors such as gestational age, birth weight, and various systemic diseases, encompassing neurological disorders, facial and eye abnormalities, cardiovascular diseases, respiratory disease, gastrointestinal disorders, renal system disorders, musculoskeletal disorders, skin and submucous disorders, chromosomal abnormalities, infections, and birth injuries. Main results and the role of chance Compared to infants conceived from fresh embryos, those born to mothers who underwent frozen embryo transfer (FET) were more likely to be large for gestational age (OR 1.63, 95% CI 1.30-2.04 for blastocyst and OR 1.69, 95% CI 1.36-2.10 for cleavage stage), and to have a neonatal birth weight of ≥ 4000g (OR 1.88, 95% CI 1.15-3.07 for blastocyst and OR 2.64, 95% CI 1.63-4.28 for cleavage stage). However, they were less likely to be small for gestational age (OR 0.68, 95% CI 0.56-0.82 for blastocyst and OR 0.68, 95% CI 0.56-0.82 for cleavage stage). Infants born from frozen embryo transfers also experienced lower odds of recurrent upper respiratory infection (OR 0.72, 95% CI 0.60-0.86 for blastocyst and OR 0.60, 95% CI 0.52-0.69 for cleavage stage), as well as eczema and dermatitis (OR 0.75, 95% CI 0.62-0.91 for blastocyst and OR 0.75, 95% CI 0.62-0.91 for cleavage stage). There was no observed effect on infant health between blastocyst and cleavage stage embryo transfers. Limitations, reasons for caution Our retrospective study design may not possess the same level of rigor as a randomized controlled trial (RCT). The follow-up period in our study ranged from 1 to 5 years, and some diseases may not manifest within this short-term timeframe, necessitating longer-term tracking for more comprehensive observation. Wider implications of the findings The result highlights the potential benefits of FET in reducing the risk of low birth weight. The observed decrease respiratory infections and skin disorders among FET infants suggests a possible advantage in immune system development or environmental adaptability. Further research with longer-term tracking is warranted. Trial registration number KMUHIRB-E(I)-20210222

O-293 Mastering consistency: a similar training allows to standardize embryo transfer performance, but experience does not involve further improvements

Abstract Study question Is there any variation in the live birth rate (LBR) per vitrified-warmed single euploid blastocyst transfer (SEBT) among clinicians trained at the same IVF center? Summary answer The LBR per SEBT (overall: 53%) remained consistent among equally trained transfer operators (range: 48%-60%) regardless of the number of prior procedures performed. What is known already The number of ART treatments and practitioners has increased over the years. To ensure high standards, an efficient quality control system should monitor and standardize operators’ performance by leveraging statistically-sound key-performance-indicators (KPIs). The embryo transfer procedure is a clinically relevant step, particularly susceptible to variations among clinicians, irrespective of their experience. However, the outcome of “LBR per transfer” is influenced by various confounders, such as embryo stage, morphology, reproductive history, uterine conditions, and others. Our objective was to assess potential differences among transfer practitioners who underwent the same training at a single IVF center in the least biased scenario possible. Study design, size, duration Retrospective single center study. Out of 8663 transfers performed by 4 operators (all trained by operator-1) between 2013 and 2021, we selected 421 first vitrified-warmed transfers with day5 euploid blastocysts whose morphology was >Gardner’s BB-grade in non-obese women, thereby excluding all main confounders on our primary outcome (= LBR per SEBT across different operators). The study was powered to exclude a 18%-decrease (β = 0.8, α = 0.05) in the LBR versus operator-1 (ratio comparator:control=1:2). Participants/materials, setting, methods We conducted ICSI, blastocyst biopsy without day3 zona-drilling, and aneuploidy testing to exclude full-chromosome uniform aneuploidies. All blastocysts were transferred ∼2 hours after warming. Uterine assessment was conducted to exclude putative conditions associated with lower LBR. Endometrial preparation protocol (modified-natural cycle versus hormone-replacement-therapy), maternal age at transfer, experience of prior implantation(s) (yes/no), the embryologists involved in the transfer procedure, were assessed as putative further confounders. All significant differences were confirmed through regression analyses. Main results and the role of chance The overall LBR per vitrified-warmed SEBT was 53.4% (N = 225/421). Operator-1 conducted 3960 transfer procedures during the study period, with 167 eligible for this study, achieving a 55.7% LBR (N = 93/167). Operator-2,-3, and -4 respectively conducted 2533, 1087, and 1083 procedures during the study period, with 134, 62, and 58 eligible for this study. Their LBRs were 50% (N = 67/134, p-value versus operator-1=0.2), 48.4% (N = 30/62, p-value=0.2), and 60.3% (N = 35/58, p-value=0.3). Multivariate logistic regression analysis, adjusted for endometrial preparation protocol and embryologists involved in the transfer procedure, confirmed the absence of an association (Operator-2 vs. 1 multivariate-OR: 0.83, 95%CI 0.5-1.39, adjusted-p=0.49; Operator-3 vs. 1 multivariate-OR: 0.75, 95%CI 0.40-1.40, adjusted-p=0.37; Operator-4 vs. 1 multivariate-OR: 1.40, 95%CI 0.72-2.70, adjusted-p=0.32). Importantly, the number of prior transfer procedures conducted by each clinician during the study period also showed no association with the primary outcome (OR = 1.0, p-value=0.82). From an IVF center perspective, the LBR per SEBT remained stable across the 9-year study period, showing minimal non-significant fluctuations. These outcomes describe an IVF center where equally-trained clinicians adopt the same standard-operating-procedures to manage vitrified-warmed embryo transfers. In this context, the LBR per SEBT represents an ideal KPI to assess the learning curve of operators in training. Limitations, reasons for caution Over 2500 SEBTs are required to rule out a 5%-decrease in the LBR. Yet, such a substantial number is better suited for comparing centers’ rather than practitioners’ performance. It would be intriguing to investigate potential differences among operators who underwent different training regimens but operate within the same clinic. Wider implications of the findings No correlation exists between the number of prior embryo transfer procedures conducted and further improvements, therefore initial operators’ training is crucial for standardizing their performance. LBR per SEBT is a strong KPI, though poorly-actionable in the short-term. It’s better suited for center comparisons, prompting internal procedure reviews when differences arise. Trial registration number not applicable

P-127 The RNA m6A landscape of mouse oocytes and preimplantation embryos

Abstract Study question Despite the significance of N6-methyladenosine (m6A) in gene regulation, the requirement for large amounts of RNA has hindered m6A profiling in mammalian early embryos. Summary answer We apply picoMeRIP-seq to map m6A in mouse oocytes and preimplantation embryos. We define the landscape of m6A during the maternal-to-zygotic transition. What is known already N6-methyladenosine (m6A), the most prevalent internal modification in eukaryotic messenger RNA (mRNA), plays key regulatory roles in many biological processes (for example, RNA stability, splicing, transport and translation) and is involved in a variety of physiological processes (for example, cell differentiation and reprogramming, embryonic development and stress responses). Study design, size, duration We recently developed an m6A mapping assay for low RNA input, low-input methyl RNA immunoprecipitation and sequencing (picoMeRIP-seq), which enables mapping of the m6A methylome using a low number of cells. In this article, we applied picoMeRIP-seq to multiple developmental stages of mouse oocytes and early embryos to profile their m6A landscapes. Participants/materials, setting, methods We use mouse as study materials.Using picoMeRIP-seq, we generated transcriptome-wide m6A maps for six stages (GV, MII, zygote, two-cell, eight-cell and blastocyst), with two biological replicates per stage and 49–110 oocytes/embryos per replicate. Main results and the role of chance We define the landscape of m6A during the maternal-to-zygotic transition, including stage-specifically expressed transcription factors essential for cell fate determination. Both the maternally inherited transcripts to be degraded post fertilization and the zygotically activated genes during zygotic genome activation are widely marked by m6A. In contrast to m6A-marked zygotic ally-activated genes, m6A-marked maternally inherited transcripts have a higher tendency to be targeted by microRNAs. Moreover, RNAs derived from retrotransposons, such as MTA that is maternally expressed and MERVL that is transcriptionally activated at the two-cell stage, are largely marked by m6A. Our results provide a foundation for future studies exploring the regulatory roles of m6A in mammalian early embryonic development. Limitations, reasons for caution The effect of m6A on the turnover of retrotransposon-derived transcripts abundant in oocytes and early embryos needs to be further addressed in future studies. Wider implications of the findings Our results provide a foundation for future studies exploring the regulatory roles of m6A in mammalian early embryonic development. Trial registration number not applicable

P-548 An investigation into the mechanisms of active enhancers regulating the initial lineage differentiation in mammals

Abstract Study question What role do active enhancers play in determining the fate of early embryonic cells? Are there differences in active enhancers between mouse and human blastocysts? Summary answer Tissue-specific active enhancers in inner cell mass (ICM) and trophoblast (TE) are essential for blastocyst formation, regulating gene expression through interactive loops with promoters. What is known already At the two-cell stage of nuclear transfer embryos, the absence of interaction between Zscan4d promoter and distal active enhancer leads to reduced Zscan4d expression, resulting in lower developmental rates. Overexpressing the demethylase Kdm4d alleviates reprogramming-resistant H3K9me3 modification, restoring the structural loop between the Zscan4d promoter and active enhancer and enhancing developmental efficiency. These findings strongly suggest a close association between the formation of regulatory chromatin interactions in early embryonic development and histone modifications. Indicating active enhancers marked with H3K27ac may regulate differentiation gene expression through distal interactions in blastocyst. Study design, size, duration Analyze variations in histone H3K27ac modification, chromatin accessibility, higher-order structures, and transcriptomes during mouse embryo differentiation from 8-cell stage to ICM and TE formation. Identify tissue-specific active enhancers in ICM and TE, validate their gene expression regulation, explore their role in early differentiation-associated genes in mice. Compare and clarify similarities and differences in enhancer regulation mechanisms during early embryo development in humans and mice. Participants/materials, setting, methods The 8-cell, morula and blastocyst stage embryos were collected from the oviducts and uterus of super-ovulated female ICR mice mated with male mice. The clinical human blastocysts were collected and separated the ICM and TE. Low-input multi-omics sequencing, including Cut-Tag, ATAC-seq, Hi-C, and RNA-seq, was performed on the embryonic samples. After analyzing and identifying active enhancers, tissue-specific ones were selected and validated for their impact on gene expression via CRISPR-Cas9 knockout in embryos. Main results and the role of chance Inhibiting ep300 expression in mouse embryos through siRNA interference reduces blastocyst formation from 90% to approximately 40%. Introducing the small molecule inhibitor A485 at the 8-cell stage, blocking the active center of CBP/p300 enzymes, completely arrests embryos at the morula stage, preventing their development into blastocysts. Emphasizing the crucial role of correctly establishing active enhancers marked by H3K27ac modification in embryonic differentiation. Multi-omics data demonstrates high repeatability and quality. PCA clustering analysis reveals a clear differentiation path in the distribution of H3K27ac-modified histones, chromatin accessibility sites, and the transcriptome from the 8-cell stage to the blastocyst. Differences in H3K27ac histone modification distribution between the ICM and TE indicate the presence of tissue-specific active enhancers in blastocyst. The disparities in H3K27ac modification distribution between blastocyst ICM and TE primarily occur in distal gene regions rather than proximal ones. Moreover, these distal enhancers exhibit a positive correlation with the expression of lineage-specific genes, suggesting a probable regulatory role of distal enhancers in gene modulation during embryonic differentiation. Tissue-specific enhancer-promoter (EP) interaction loops emerge during embryonic differentiation, allowing direct interaction between tissue-specific active enhancers and promoters, thereby activating the expression of tissue-specific genes. Limitations, reasons for caution While DNA sequences with H3K27ac modification are currently considered markers for active enhancers, H3K4me modification is another histone marker for enhancers. However, enhancers associated with H3K4me modification may not necessarily be in an activated state. Future research can simultaneously incorporate appropriate detection methods for H3K4me modification. Wider implications of the findings After elucidating the mechanisms through which active enhancers regulate mouse differentiation, comparing the differences in active enhancers during the first lineage differentiation process between mice and humans can help identify factors that enhance human blastocyst quality. This can potentially improve clinical outcomes in assisted reproduction. Trial registration number not applicable

P-138 Effect of biofeedback from the blastocysts on the cumulative pregnancy rate

Abstract Study question The study is to compare cumulative pregnancy rates of embryos transferred with and without spent culture media to confront biofeedback’s role in clinical outcomes Summary answer Transferring the blastocysts with spent culture media improved the pregnancy rates. Biofeedback was observed from the blastocyst stage. The blastocyst freezing and transfer approach improved. What is known already Biofeedback is often encountered in regulating menstrual cycles and other events in continuing gestations with estrogen, progesterone, beta hCG..etc. A similar biofeedback is possible with growing blastocyst for implantation. Since the in-vitro culture of embryos is devoid of its occurrence, it may lead to asynchrony of subsequent events and implantation. There is no scientific evidence from metabolomics on the characterization of these substances or secretions from spent culture medium that includes cell-free DNA giving feedback and its role in improving clinical outcomes. Study design, size, duration This retrospective multi-centric study included the 3591 frozen embryo transfers from 1st Apr 2022 to 31st Nov 2023 out of 4623 women who underwent oocyte retrieval from 1st Jan 2022. Patients were divided into study (group A) and control (group B). In group A, 1035 women received the transfers from spent culture media; in group B, 2556 women received transfers after shifting the blastocysts to fresh culture media before loading into the ET catheter. Participants/materials, setting, methods All the transfers considered in this study were day-5 blastocysts, irrespective of their frozen state. Day-5 frozen-thawed were incubated short-time (∼1 hour) before the transfer. Whereas, the frozen embryos and morulas were cultured till day 5. In the study group, all cultures were conducted in 100ul of culture media under oil per embryo to maintain consistency amongst the group. The rest of the laboratory and clinical procedures were consistent between the two groups. Main results and the role of chance The cumulative pregnancy rate was significantly higher in group A than in group B (60.9% Vs 55.6%; OR = 1.23; 95% CI, 1.07 -1.44, Z-score 2.901, P < 0.003). The effect of spent culture media was further observed by regrouping the day-3 or 4 frozen (group A1) and day-5 frozen patients (group A2). Day-5 thawed blastocysts transferred after short incubation to allow the secretions to directly deposit on the endometrium. Group A1 might have a high concentration of secretions due to its accumulation from the embryo or morula stages. Pregnancy rates in group A1 were higher than A2 but not statistically significant (61.7% Vs 59.7%; OR = 1.08, 95% CI, 0.84-1.4, Z-score 0.654, P = 0.51). The less impact indicates that the feedback came from blastocysts rather than embryos. Limitations, reasons for caution It is not evident how these secretions contribute to biofeedback. Quantification, identifying their time of release and recovery from the qualitative tests for their therapeutic use are the limitations. Nevertheless, spent media can be employed if found beneficial. Wider implications of the findings We have taken a large possible data set for control against the test group and took cumulative pregnancy rate as the primary outcome indicator to prove biofeedback from blastocysts can improve the clinical outcomes irrespective of the age and other factors of infertility. Trial registration number Not applicable

P-110 Live birth of blastocyst culture in women with previous poor blastocyst formation: a cohort study

Abstract Study question Do cumulative live birth rates(CLBRs) in the second cycle differ between blastocyst-stage and cleavage-stage transfer in women with low blastocyst progression during initial IVF ? Summary answer The CLBRs following subsequent oocyte retrievals were comparable for extended culture with blastocyst-stage embryo versus cleavage-stage transfers. What is known already Fresh blastocyst-stage embryo transfer increases live birth rates when compared to cleavage- stage embryo transfer in women with good prognosis. However, it remains unclear whether a blastocyst-culture approach can still be considered for women with a history of failed or reduced blastocyst formation. Study design, size, duration A retrospective cohort study evaluating outcomes of the second IVF/ICSI in women who had a history of poor blastocyst development in their first-time retrievals at a single fertility center from September 2019 to December 2021. Women fulfilled embryo quality criteria of having at least two good-quality cleavage-stage embryos (graded 1-2) on day 3 were included for analysis. Participants/materials, setting, methods A total of 396 patients were analyzed and categorized into two groups. Group D5/6: Extending culture of all embryos to blastocyst-stage in vitro followed by blastocyst transfer; Group D3: transfer or cryopreservation of one or two of the highest quality cleavage-stage embryo(s) on Day3.The main outcome measure was the CLBR per oocyte retrieval.Multivariable logistic regression analyses were used to assess the association between two culture approaches and live birth outcomes after adjusting for possible confounders. Main results and the role of chance The CLBRs (57.01% versus 49.48%) were numerically higher in women in D5/6 group (N = 107) than those in D3 group (N = 289) although the difference was attenuated and did not reach statistical significance after controlling for confounders (aOR: 1.27, 95%CI 0.78, 2.07,P=0.345). All subgroup analyses showed similar conclusions as the main model, even restricting on women with a limited number of quality embryos 3 days post-fertilization. Among cycles with only 2 or 3 good-quality cleavage embryos, Day3 transfers were still associated with lower CLBR compared to all blastocyst culture and transfer (aOR 0.66, 95% CI 0.51-0.87). Limitations, reasons for caution The retrospective nature of the present study, alongside the selection bias towards blastocyst culture, may predispose to the risk of residual confounding. The cohort of women characterized by previous failed or reduced blastocyst formation was comparatively small. Larger prospective studies focusing on the unique group are still needed. Wider implications of the findings Even in poor prognosis IVF patients with a history of poor blastocyst formation and/ or blastulation failure, all blastocyst culture and transfer can still be considered when at least two good-quality embryos are available at day 3. These findings are clinically relevant and require additional investigation. Trial registration number Not Applicable

O-297 Prediction of pregnancy outcomes of single vitrified-warmed blastocyst transfer using combination of an automatic classification algorithm applied on cleavage stage embryos and blastocyst morphology

Abstract Study question Does combination of embryo assessment algorithm on cleavage-stage based on the automatic annotation of morphokinetic and blastocyst morphology in embryo selection improve implantation outcomes? Summary answer Combination of embryo assessment algorithm on cleavage-stage based on the automatic morphokinetic annotation and blastocyst morphological evaluation was showed significantly predictive for implantation outcomes. What is known already Conventional morphological assessment is still the gold standard for embryo selection. Timelapse monitoring of embryo development may represent a superior way to culture and select embryos in vitro. Nowadays, time-lapse culture systems are becoming more popular, many algorithms have been developed for embryo selection based on embryo morphokinetics, providing additional information for morphological assessment. However, manual annotations of developmental events and application of algorithms can be time-consuming and subjective processes. Automation of morphokinetic annotation can be a potential approach that reduces subjectivity in embryo selection. Study design, size, duration The observational, retrospective study was conducted in a single centre between 2020-2023 and included 511 single vitrified-warmed blastocyst transfer cycles. Blastocyst selected for transfer on Day 5,6 based on conventional morphological evaluation. Embryos were evaluated on Day 3 with a score from 1 (best) to 5 (worst) (from highest to lowest developmental potential) by the automatic embryo assessment algorithm. The correlation of the algorithms scoring and pregnancy outcomes was qualified by generalized estimating equations (GEEs). Participants/materials, setting, methods Embryos were cultured in timelapse GERI incubator and classified on day 3 by automatic embryo assessment algorithm depending on four parameters: P2 (t3-t2), P3 (t4-t3), oocyte age, and number of cells. The correlation of the algorithms scoring and pregnancy outcomes was qualified by generalized estimating equations (GEEs). Three GEE model using embryo assessment algorithm, conventional morphological assessment, and a combination of both classification system as the predictor were compared. Main results and the role of chance The implantation rate was higher with lower the scores generated by the embryo assessment algorithm. A GEE model showed the negative association between higher embryo score and lower odds of implantation. The OR of Score 3; 4; 5 vs 1 were 0.335; 0.259; 0.245 (95% CI 0.194-0.580; 0.138-0.485; 0.111-0.544, P = 0.000), respectively, for implantation. The AUC of the model using embryo assessment algorithm for implantation potential = 0.641. The AUC of the model combining both embryo assessment algorithm and conventional morphological assessment for implantation potential = 0.733. The differences were statistically significant (z-statistic = 3.896, p = 0.0001). Limitations, reasons for caution The sample size was enough to confirm the ability of the model for embryo selection. However, the retrospective nature of this study may be a limitation. Wider implications of the findings Using combination of timelapse technology with automated embryo assessment and conventional morphological assessment can predict the success rates of assisted reproduction cycles. To our knowledge, this is the first study using embryo dataset from single vitrified-warmed blastocyst transfer cycles with this embryo assessment algorithm. Trial registration number not applicable

P-237 Fertilization prediction of mature oocyte based on microscopic image of first polar body morphology using YOLOv8

Abstract Study question Can a machine learning-based model predict successful fertilization of oocyte using first polar body morphology? Summary answer With small imbalance oocyte dataset, the most successful model to predict fertilization of mature oocyte is YOLOv8lcls with 97.47% accuracy, 100.00% specificity, and 94.87% sensitivity. What is known already The development of oocytes is crucial in the development and fertility of embryos in In Vitro Fertilization (IVF). There are several internal and external factors that could affect the fertilization competency of oocytes, one of which is the first polar body (PBI). The importance of the quality of PBI in ICSI has long been studied and has been evaluated by scoring systems regarding Istanbul Consensus. However, there is still a debate regarding the influence of PBI on the developmental competency of oocytes. Study design, size, duration This study involved the analysis of retrospective data including the microscopic light image of meiosis II (MII) oocytes at the time of Intracytoplasmic sperm injection (ICSI), along with polar body scoring by embryologists and the fertilization outcome of 1006 MII oocytes from a single public IVF clinic, between 2020-2022. After ICSI, fertilized oocytes were classified into 2 groups: 2PN and non-2PN (e.g., 0PN, 1PN, > =3PN). Participants/materials, setting, methods Light microscopic images of oocytes in the metaphase of meiosis II (MII) stage are captured, followed by the prediction of the likelihood of 2PN development after fertilization through intra-cytoplasmic sperm injection (ICSI). The image has its PBI segmented by the CNN and then cropped out of the original image. The PBI image is then fed into the prediction CNN to obtain the final result. The model performance was determined by evaluating accuracy, sensitivity, and specificity. Main results and the role of chance There were 1006 images of MII oocytes included in this study. After ICSI, 860 oocytes were fertilized resulting in the 2PN stage embryos and were classified as 2PN, whereas 146 oocytes were not fertilized or abnormally fertilized and were classified as non-2PN. Among these images, 782 oocytes received a PBI score of A, 677 of which are 2PN and 105 are non-2PN. The B grade consists of 224 images, of which are 183 2PN, and 41 non-2PN. The dataset was split into 3 subsets for training (70%), validation (10%), and testing (20%). The images are augmented by horizontal flipping to increase the amount of data and allow the model to observe several positions of the PBI. The YOLOv8 segmentation model is applied to segment the PBI and locate its position. The image is then rotated to move the PBI to the top and the PBI is cropped based on its position. Lastly, the cropped PBI image is used to predict the development by using the YOLOv8 Classify model trained on an augmented dataset. The YOLOv8l-cls model achieves the highest accuracy at 97.47%, specificity at 100.00%, and sensitivity at 94.87%. Limitations, reasons for caution The model does not include information on sperm quality, which also essentially contributes to the fertilization process. 2PN stage prediction is just the initial and surrogate outcome in IVF treatment. The model’s interpretation of the PBI score remains inconclusive. Therefore, additional experiments using dataset with balanced PBI grading are recommended. Wider implications of the findings The developed model is the first step in paving the way to establish and implement tools for assisting embryologists in selecting and prioritizing the high-potential oocyte for fertilization. Additional factors such as patient characteristics, other features of oocytes, and male factors should be further integrated to improve model performance. Trial registration number NA

O-148 Does presence of endometriosis adversely affect oocyte morphology? Evaluation of a large number of oocytes obtained from endometriosis patients, 27204 oocytes

Abstract Study question Does presence of endometriosis adversely affect oocyte morphology? Evaluation of a large number of oocytes obtained from endometriosis patients, 27204 oocytes Summary answer In the presence of endometriosis, oocyte morphology is not impaired. The rate of reaching blastocyst and fertiilization rate, also pregnancy outcomes are not affected negatively. What is known already Although there are hypothesis, theories that altered steroidogenesis and folliculogenesis, higher oxidative stress, reactive oxygen species, altered cell cycle progression, inflammation and angiogenesis in the follicular environment exposes oocytes to a hostile inflammatory environment and alters oocyte quality. There has been much debate and conflicting evidence as to whether the poorer IVF outcomes in women with endometriosis is related to altered oocyte quality. There is some evidence to suggest that impaired oocyte morphology in women with endometriosis may have an adverse impact on fertilization rate, however, most studies have shown that there is no difference in pregnancy outcomes following IVF. Study design, size, duration This retrospective, single center study evaluated 29130 ART cycles from August 2011 to March 2023,based on data obtained from Istanbul Memorial Hospital,ART and Reproductive Genetics Center.Study group included endometriosis patients(n = 4602 cycles, 27204 oocytes) and control group included non-endometriosis patients(n = 24528 cycles, 178774 oocytes).We analyzed demographic and cycle characteristics, oocyte morphology in ART cycles between the two groups.Futhermore, we compared pregnancy outcomes in frozen-thawed embryo transfer(FET) cycles (total number:11116 FET cycles; endometriosis group:2255 cycles,non-endometriosis group:8861 cycles). Participants/materials, setting, methods Patients diagnosed with endometrioma by ultrasound, diagnosed with endometriosis by laparoscopy or patients who underwent endometrioma surgery or adenomyosis detected on ultrasound were included in the study (endometriosis) group. In the control group, the patients without endometriosis were included. Mann Whitney U test and Pearson Chi-square test used. Cliff's Delta effect size (????) for non-parametric tests and Phi effect size (p) for categorical data were reported. Main results and the role of chance The sample was very large, so the statistical results were given in terms of effect size, not only p value calculated. Female age was similar. Female body mass index, number of previous cycles, duration of infertility, AMH, total gonadotropin dosage used, duration of ovarian stimulation, estradiol level on trigger day, number of aspirated oocytes, mature and fertilized oocytes, maturation and fertilization rate, rate of blastulation, rate of usable blastocyst (top and good quality), number of embryos transferred, blastocyst stage embryo transfer cycles were statistically different between the two groups (p < 0.001). However when the effect size examined, all variables were found to have a negligible association by Cliff's Delta or Phi effect size calculations. Oocytes obtained from endometriosis patients had statistically significantly higher severe central granulation, large perivitellin space, thick zona, polar body defect abnormalities compared to non-endometriosis patients (p < 0.001), Phi effect size showed negligible association for all variables. In endometriosis group compared to non endometriosis group; biochemical pregnancy (68.9% vs 72.2%, p:0.002, Phi:0.030), clinical pregnancy (61.5% vs 64.5%, p:0.007, Phi:0.025), total pregnancy loss (22% vs 24.4%, p:0.05, Phi:0.022) were statistically higher, but Phi effect sizes were negligible. Live birth were similar (52.5% vs 53.2 P:0.56) between the two groups. Limitations, reasons for caution The principal limitation of the study is retrospective design of the analysis. But the strength of the study is that included 27204 oocytes from endometriosis patients. Wider implications of the findings To our knowledge, this study includes the largest case group that investigates the endometriosis and oocyte morphology. The results show that endometriosis does not have a negative impact on oocyte morphology. Additionally, it has been shown that the presence of endometriosis does not have a negative effect on pregnancy outcomes. Trial registration number *