Trophoblast Research Articles

P-513 Non-invasive PGT (niPGT) of vitrified blastocysts cultured for 24 hours after thawing

Abstract Study question Is niPGT on culture medium exposed for 24 hours to vitrified-thawed blastocysts useful? Summary answer niPGT using a culture medium from vitrified blastocysts cultured for 24hours after thawing showed a high concordance rate with the PGTa results. What is known already Conventional PGTa, which is widely practiced around the world, requires specialized techniques and involves embryonic trophectoderm (TE) biopsy. Recently, niPGT using cell free DNA released from embryos into culture medium has been reported as an alternative to conventional PGT, and has been found to correlate well with TE-biopsy analysis results (Carmen. Rubio, 2020). Although there have been numerous reports of niPGT accuracy based on the timing of culture medium collection, there have been few comparisons of its accuracy based on different time points of medium collection from the same embryo. Study design, size, duration From March to April 2023, 128 vitrified pronuclear stage embryos from 33 patients with consent for research use, were thawed and cultured, and from Day 4, they were individually cultured for over 40 hours. 60 embryos that reached blastocyst by Day 6 were vitrified, and the spent culture medium (SCM1) was collected. The vitrified blastocysts were later thawed and cultured individually for 24 hours, and then the embryos and spent culture medium (SCM2) were collected. Participants/materials, setting, methods Embryos were cultured in a 30 μl droplet until Day 4 and a 10 μl droplet for individual culture. Culture medium was collected and embryos were subjected to chromosomal analysis by NGS. We compared the results of niPGT taken from the same embryos on days 4- 6 (SCM1) and after thawing of vitrified blastocysts (SCM2), with PGTa analysis of the embryos. Main results and the role of chance Among all media samples analyzed, results were obtained for 80.0% (48/60) SCM1 and 96.7% (58/60) SCM2 samples. The ploidy concordance rate was 81.3% (39/48) for SCM1 and 98.3% (57/58) for SCM2. The overall concordance rate, defined as the combined rate of the total concordance for all chromosomes and the partial concordance for some chromosomes was 72.9% (35/48) for SCM1 and 94.8% (55/58) for SCM2 respectively. Significant differences were identified in the informative sample rate, the ploidy concordance rate, and the overall concordance rate(p < 0.05) between SCM1 and SCM2. Fisher’s exact test was used for statistical analysis. Limitations, reasons for caution In this study, embryo culture was continued to day 6, regardless of morphology of the embryo at day 5, which differs from the usual clinical culture process. Additionally, the timing of sample collection differed between the day 4-6 culture medium sample and that for a blastocyst. Wider implications of the findings The results demonstrated that accuracy of ploidy status was higher in the SCM2 compared to the SCM1 sample, suggesting that analysis of thawed vitrified blastocyst medium maybe a more precise predictor and therefore a non-invasive alternative to conventional PGTa. Trial registration number not applicable

O-261 Frozen gametes differ in post-insemination dynamics to their fresh sibling counterparts in cycles using testicular sperm

Abstract Study question Are there differences in post-insemination events between fresh and frozen-derived gametes when testicular sperm extraction (TESE) is used? Summary answer Frozen-TESE leads to decreased fertilization, but not blastocyst euploidy rates; 2PN-lag time is significantly longer when frozen gametes are utilized compared to their fresh counterparts What is known already Studies indicate that the origin of testicular sperm influences the development potential of cleavage-stage embryos into blastocysts. Recent morphokinetic analysis suggest that embryos from frozen-TESE exhibit prolonged pronuclear stages and more frequent uneven cleavage compared to those from ejaculated sperm. Notably, no comparison with fresh-TESE was performed. To date, no studies have explored analysis in embryo morphokinetic parameters comparing fresh and frozen-TESE, especially when derived from the same patient.Such comparisons could provide a more accurate understanding of potential differences. Given that TESE occasionally involves the use of vitrified oocytes, it is essential to thoroughly examine outcomes across all combinations. Study design, size, duration Retrospective cohort study in a tertiary referral IVF centre including 72 cycles of 30 couples between January 2017 to September 2023. Following insemination by TESE-sperm, embryos were cultured in time-lapse (TL) incubator. Blastocysts ≥BL3CC (Gardner’s) underwent trophectoderm (TE) biopsy for Preimplantation Genetic Testing for aneuploidy (PGT-A) by next generation sequency (NGS). Participants/materials, setting, methods Gamete sources were fresh (fresh-TESE) or frozen-thawed-TESE (frozen-TESE) from the same patients with non-obstructive azoospermia (NOA), and fresh or vitrified (frozen) oocytes from the same female partner. Fertilization, usable blastocysts, and euploidy rates were recorded. Morphokinetics were annotated for time (t) of PB2 (tPB2), tPNa (PN-appearance), tPNf (PN-fading), t2-t9, CP (compaction), tM (Morula), tSB (start blastulation), tB (blastocyst) and timings between each developmental event. Main results and the role of chance Among included 980 oocyte/TESE dyads, 253 (25.8%) used fresh oocyte/sperm, 141 (14.4%) frozen oocyte/fresh sperm, 558 (56.9%) fresh oocyte/frozen sperm and 28 (2.9%) frozen oocyte/sperm. In oocyte/sperm dyads using fresh-TESE, normal fertilization (2PN) (52.0% vs. 44.5%) were higher compared to dyads using frozen-TESE (P < 0.01). Mixed-effects logistic-regression accounting for dependency structure between oocytes/sperm dyads of the same couple, showed an association of frozen-TESE with lower fertilization (OR: 0.64, 95%CI: 0.45-0.89, P = 0.008) and lower blastulation (³BL3CC)/MII (OR: 0.53, 95%CI: 0.37-0.77, P = 0.001), but similar blastulation (³BL3CC)/2PN (OR: 0.69, 95%CI: 0.45-1.07, P = 0.09). After accounting for female age, euploidy per tested blastocyst was similar whether fresh or frozen sperm was used (OR: 0.91, 95% CI: 0.46-1.79, P = 0.788). Frozen oocyte use on the other hand was not significantly associated with these outcomes (P > 0.05 for all). Analysis of morphokinetic parameters revealed that use of frozen dyads was associated with longer duration of 2PN after tPNa to tPNf: in frozen-TESE, 2.10h longer (95%CI: 0.55-3.64, P = 0.008); and in frozen oocytes, 2.34h longer (95%CI: 0.31-4.36, P = 0.024). Among embryos reaching blastulation, frozen-TESE embryos reached 6-cell stage earlier (tPNf to t6, 2.4h earlier, P = 0.009) but they were somewhat delayed in reaching expansion from there on (t6 to tEB, 4.4 hours later, P = 0.059). Limitations, reasons for caution In certain oocytes, insemination was conducted using either fresh or frozen immotile sperm that showed unresponsiveness to activation methods. However, the samples size hinders a comprehensive comparison. Wider implications of the findings Use of frozen-thawed TESE from NOA-patients may decrease fertilization and consequently lower blastocyst yield, but not euploidy rates. Synchronizing fresh-TESE with oocyte retrieval appears safer to maximize fertilization. Regardless of the gamete type, frozen-sourced sperm or oocyte impact 2PN-lag time, possibly indicating nuclear repair mechanisms post-freezing, requiring further investigation. Trial registration number 2310-ABU-020-VF

A healthy live birth after mosaic blastocyst transfer in preimplantation genetic testing for GATA1-related cytopenia combined with HLA matching

BackgroundGATA1-related cytopenia (GRC) is characterized by thrombocytopaenia and/or anaemia ranging from mild to severe. Haematopoietic stem cell transplantation (HSCT) is a healing therapeutic choice for GRC patients. We identified a novel pathogenic variant (GATA1: c.1019delG) in a boy with GATA1-related cytopenia. Then we performed preimplantation genetic testing (PGT) in this GRC family. After a mosaic embryo transfered, a healthy and HLA-compatible with the proband baby was delivered.Case presentationThe proband is a 6-year-old boy who was diagnosed to have transfusion-dependent anaemia since 3 year old. Whole-exome sequencing (WES) showed that the proband has a hemizygous variant c.1019delG in GATA1, which is inherited from his mother. His parents decided to undergo PGT to have a health and HLA-compatible offspring. After whole genome amplification (WGA) of biopsied trophectoderm (TE) cells, next generation sequencing (NGS)-based PGT was preformed to analyse embryos on chromosomal aneuploidy, target mutation and HLA typing. There were 3 embryos HLA-matched to the proband. The genotypes of the 3 embryos were heterozygous variant, hemizygous variant, normal respectively. After a heterozygous, mosaic partial trisomy (chr)16, and HLA-matched embryo transfer, a healthy baby was delivered and whose HSCT is compatible with the proband.ConclusionsNGS-based PGT-HLA is a valuable procedure for the treatment of GATA1-related cytopenia caused by GATA1 variants, or other haematological disorders, oncological and immunological diseases. Furthermore, our study reconfirms that mosaic embryos transfer would bring healthy offspring.

Involvement of METTL3-mediated m6A methylation in the early development of porcine cloned embryos

METTL3-mediated N6-methyladenosine (m6A) modification is critical for gametogenesis and early embryonic development. However, the function of METTL3-mediated m6A modification in the early development of somatic nuclear transfer embryos (SCNT) remains unclear. Here, we found that METTL3 mRNA and protein levels exhibit dynamic changes during the early development of porcine SCNT embryos. The levels of METTL3 mRNA and protein in SCNT embryos at specific developmental stages differ from those in parthenogenetic activation (PA) counterparts. SiRNA injection effectively reduced the levels of METTL3 mRNA and protein in 4-cell embryos and blastocysts. METTL3 knockdown significantly reduced the cleavage and blastocyst rates of SCNT embryos. METTL3 knockdown significantly reduced the number of total cells and trophectoderm (TE) cells in the resulting blastocysts and perturbed cell lineage allocation. In addition, METTL3 knockdown reduced the levels of m6A modification in 4-cell embryos and blastocysts. Importantly, METTL3 knockdown decreased the expression levels of CDX2, GATA3, NANOG and YAP, and increased the expression levels of SOX2 and OCT4. Taken together, these results demonstrate that METTL3-mediated m6A modification regulates early development and lineage differentiation of porcine SCNT embryos.

O-198 The decline in semen quality parameters enhances mosaicism in trophectoderm biopsy of human blastocysts

Abstract Study question Do poor sperm parameters impact chromosomal analysis compared to sperm with normal parameters? Summary answer Fertilization with more than two abnormal patterns, including severe oligoasthenoteratozoospermia sperm, may increase the rate of embryo mosaicism. What is known already Sperm cells constitute the genetic half of the embryo, and the male factor plays a crucial role in embryo chromosome integrity. While PGT-A is widely used to enhance IVF outcomes, the influence of male factors on embryo genetics remains underexplored. We aimed to determine the effect of semen profile on the outcome of PGT-A cycles performed at the blastocyst stage. Study design, size, duration A single-center retrospective study conducted between January 2019 and July 2023 analyzed 609 blastocysts with Next Generation Sequencing (NGS) of trophectoderm (TE) biopsies. Four study groups were formed based on sperm parameters: normozoospermia (N), moderate male factor (MMF), severe oligoasthenoteratozoospermia (s-OAT), obstructive azoospermia (OA). Participants/materials, setting, methods Patients treated with a minimal ovarian stimulation protocol. PGT-A cycles were divided into three paternal age groups. OUTCOMES: Euploidy, aneuploidy, mosaicism rates; blast formation time; good-quality blastocyst rates. METHODS: Multivariate logistic analysis, Chi-squared tests, and STATA 12.1 statistical software. Main results and the role of chance In the 609 TE biopsied blastocysts, euploidy rate was 49.9%, aneuploidy rate was 31.5%, and mosaicism rate was 18.6%. Paternal age ≥43 increased aneuploidy risk compared with <35 (40.1% vs 26.1%, p < 0.05). The euploidy rate and aneuploidy rate were no different in MMF, s-OAT, and OA compared with N (44.8%, 40.6%, and 42.1% vs. 54.2%; 36.8%, 25.0%, and 31.6% vs. 30.5%). Mosaicism rates were significantly difference between sperm parameter groups (N 17.1%, MMF 18.4%, s-OAT 34.4% and OA 26.3%, p < 0.05). In logistic regression analysis adjusted for confounders, severe oligoasthenoteratozoospermia showed 2.5 times higher mosaicism rate (aOR 2.58; 95% CI 1.08-5.08, p = 0.027). The good-quality blastocyst rate was significantly reduced in the OA compared with N, MMF, and s-OAT (21.1% vs 43.4%, 39.2% and 37.5%, p < 0.05). No impact of semen parameters on blastocyst formation time and vitrification time outcomes was observed. Limitations, reasons for caution Retrospective, single-center study. Excludes DNA fragmentation index and TESE results, impacting sperm quality evaluation. Wider implications of the findings Severe oligoasthenoteratozoospermia is positively associated with mosaicism rate. Poor sperm parameters lead to mosaic embryos. Couples undergoing PGT-A for male factors should discuss potential outcomes. Patients with abnormal semen parameters should consider pretreatment for improved semen quality. Results useful for genetic counseling and clinical decision-making. Trial registration number None

P-533 Impact on clinical outcomes of day-6 extended culture and reduced culture media volume applied to non-invasive PGT-A

Abstract Study question Impact on clinical outcomes of day-6 extended culture and reduced culture media volume applied to non-invasive PGT-A Summary answer The implementation of changes in culture conditions to accommodate niPGT-A has no impact on blastocysts viability or pregnancy outcomes. What is known already The recent identification of embryo cell-free DNA (cfDNA) in the culture media has created the possibility of a non-invasive approach for aneuploidy testing in blastocysts. Concerns have however arisen at two levels. Firstly, the concordance rates that cfDNA results have with the full blastocyst or a trophectoderm (TE) biopsy and secondly, the impact on clinical outcomes of the changes in the embryo culture protocol that are required to implement niPGT-A in the IVF laboratory. Concordance rates of niPGT-A to invasive PGT-A have gradually improved, however the impact of culture protocol changes is not as well understood. Study design, size, duration Six IVF clinics participating in a trial examining concordance rates for invasive and non-invasive PGT-A on day-6/7 blastocysts were involved (Rubio et al., 2020). Patients were recruited from April 2018 to December 2020. In the study group, 428 PGT-A cases following the niPGT-A protocol for embryo culture were included. The control group consisted of 1,392 regular PGT-A cases that did not participate in the study and underwent the standard PGT-A culture protocol. Participants/materials, setting, methods In the control group, embryos were biopsied and vitrified on day 5, 6 or 7. In the study group, niPGT-A culture conditions started on day 4, when embryos were washed and cultured individually in 10μl of fresh media. On day 6/7 blastocysts were biopsied, vitrified and media collected for niPGT-A analysis. Clinic A cultured all embryos up to day 6/7, whereas in clinics B-F a minority of day-5 blastocysts were biopsied and transferred. Main results and the role of chance Statistical comparisons were performed between the study and the control groups. Mean female age, number of oocytes, fertilization rates and number of blastocysts biopsied were not significantly different for the study and the control group. Overall, the niPGT-A protocol did not have any detrimental effect on blastocyst quality, euploidy rates or informativity rates. Deferred single embryo transfers (SET) were performed in both groups. Regarding the overall pregnancy outcomes, no significant effect was observed on clinical pregnancy rate, miscarriage rate or ongoing pregnancy rate (OPR, ≥12 weeks) in the study group compared to the control group when stratified by day of biopsy. Clinic A showed no difference in OPR per transfer regardless of culture protocol (62.3% in control group day 5/6/7; 60.8% in control group day 6/7; and 65.6% in study group day 6/7). For the remaining clinics (B-F), a comparison was made separately between day-5 and day-6 blastocysts that were transferred and cultured according to the standard and to the niPGT-A protocol. No significant differences in OPR were observed, neither between control group day 5 (60.6%) and study group day 5 (58.7%), nor between control group day 6 (44.7%) and study group day 6 (41.9%). Limitations, reasons for caution The limitations are intrinsic to the retrospective nature of the study, and also to the fact that the study was conducted in invasive PGT-A patients and not in niPGT-A cases alone. Wider implications of the findings This study shows that changing current IVF laboratory practice to adopt niPGT-A protocols has no impact on the number of blastocysts available for transfer and overall clinical outcomes. Whether removal of the actual invasive biopsy step leads to further improvements in pregnancy rates awaits further studies. Trial registration number ClinicalTrials.gov (NCT03520933)

P-551 Application of simultaneous PGT-M/PGT-A using whole genome sequencing and haplotyping for monogenetic disorders, multiple variants, low penetrance, and de novo disorders

Abstract Study question Is the concurrent use of high-resolution PGT-A and genome-wide haplotyping with disease-specific assays for PGT-M effective in an unselected patient population at-risk for monogenetic disorders? Summary answer PGT-A/PGT-M is an effective treatment for couples/patients burdened with genetic diseases, that provides optimal family planning, prevents vertical transmission, and reduces personal and societal encumbrance What is known already Societal awareness of genetic testing expanded the use of carrier screening, diagnostic genetic tests, and higher in vitro fertilization (IVF) utilization has increased demand for preimplantation genetic testing for monogenetic disorders (PGT-M), as an alternative to invasive prenatal tests. The scope of PGT-M testing expands beyond common severe childhood disorders, to include serious and mild late onset disorders, cancer predisposition, variants of unknown significance, de novo mutations, and variants in multiple genes. This highlights novel ethical dilemmas, necessitating adjustment of generic genome wide approaches to complement, or eventually replace, targeted disease-specific PGT-M assays. Study design, size, duration This is a retrospective study of 359 couples at-risk for monogenetic disorders referred to the CReATe Fertility Centre, Toronto, Canada. Our objective was to evaluate the efficiency of concurrent use of genome wide haplotyping and disease specific assays for PGT-M and high resolution PGT-A in an unselected patient population at risk for monogenetic disorders, multiple and de novo variants, low penetrance and variants of unknown significance. Participants/materials, setting, methods All patients received comprehensive genetic counselling. For each couple/patient, DNA from gamete providers, related family member/s and whole-genome amplified (WGA)DNA from a single trophectoderm(TE) biopsy were analyzed. Whole-genome SNP-array (Karyomapping, Illumina, CA) and STR-analysis were used to obtain parental haplotypes and Sanger-sequencing was used for direct variant analysis. PGT-A was performed using Illumina platform(NextSeq 550). NxClinical (BioDiscovery) and BlueFuse Multi(Illumina) software were used for data analysis. Main results and the role of chance 1826 embryos, from 235 IVF cycles were analyzed for 88 unique genes (14 cancer predisposing, 42 autosomal recessive and 32 dominant genes) and a total of 282 unique variants. The most frequent genes tested were BRCA1/2 -21% of all cases, followed by HBB-8.7%, CFTR-8.7% and CYP21A2-8.7%. PGT-M for 11 cases was done for a de novo variant detected in a sibling and in 2 cases for a parental mosaic variant. Multiple genes and VUS were analyzed in 12 and 6 cases, respectively. PGT-A analysis showed 31.6% aneuploidy, 9.6% mosaicism and 58.8% euploidy. Genome wide haplotyping was used for euploid and mosaic embryos with an average case call rate of 95%, and average case Allele Drop Out (ADO) rate of 2.7%. Recombination events in 3% of cases prevented assignment of haplotype. Direct mutation testing was obtained for all samples and was 100% concordant with the predicted haplotypes. In cases with no predicted haplotype, direct testing established the PGT-M diagnosis. The distribution for both recessive and dominant disorders was in Mendelian order. To date 66 live births confirmed the results from PGT-M diagnosis. Limitations, reasons for caution Ethical considerations and extensive genetic counselling are prerequisites for PGT-A/M testing implementation. Simultaneous screening for embryo aneuploidy and monogenetic disorders optimizes patient treatment and family planning, but may not be available in all jurisdictions. Although the data was collected prospectively, retrospective design is a limitation. Wider implications of the findings Simultaneous PGT-A and PGT-M with direct variant testing provides optimal embryo prioritization for transfer and significantly reduces time to a healthy pregnancy in couples at risk for inherited disorders. Karyomapping is effective for generic haplotyping, with a low risk of undetected recombination events in the genomic region of interest. Trial registration number not applicable

P-586 Mild ovarian stimulation using the aromatase inhibitor letrozole (LTZ) is an effective stimulation protocol to obtain euploid blastocysts for women of an advanced maternal age

Abstract Study question Does MOS with LTZ produce higher numbers of good-quality blastocysts compared with the use of clomiphene citrate (CC) for women of an advanced maternal age? Summary answer MOS using LTZ is a good ovarian stimulation protocol for a higher number of good-quality blastocysts for women of an advanced maternal age (AMA) What is known already The clinical pregnancy rate with a good blastocyst should be 40% or more among younger women, but about 35% for AMA women (≥40 years). Therefore, high-dose gonadotropins are usually used for AMA women. However, the best efforts for both patients and clinicians are sometimes in vain. Many AMA women show a lower ovarian reserve where ovarian stimulation with a lessened dose of gonadotropins is preferred. Taking CC or LTZ with MOS can reduce the amount of gonadotropins used, but it remains uncertain whether it is better to administer LTZ or CC together for MOS. Study design, size, duration This retrospective study was conducted between January 2020 and April 2021. A total of 288 women received MOS. Of course, 153 used CC (CC group) and 133 used LTZ (LTZ group). The ART outcomes were compared between these two groups. Among them, the women who were ≥40 years old were divided into two groups: CC-O40 (n = 61) and LTZ-O40 (n = 54). The ART outcomes were compared between the two groups. Participants/materials, setting, methods All women took either 100 mg of CC or 5 mg of LTZ daily for 7 days between menstrual cycle 3 (MC3) and MC9, and 225 IU of recombinant-FSH were administered on MC3, MC5, MC7 and MC9. On MC 10, when 3 or more well-developed follicles (≥ 20 mm in diameter) were confirmed, a dual-maturation trigger was performed using rec-hCG and GnRH agonist nasal spray, and then oocyte-pick up (OPU) was performed 35-36 hours afterward. Main results and the role of chance The number of retrieved oocytes in the CC group averaged 7.5 ± 5.3 (mean ± S.D.), which was significantly higher than that in the LTZ group (6.3 ± 4.7; p < 0.05). The average number of blastocysts and morphologically good blastocysts was comparable in the CC and LTZ groups (CC group; 2.8 ± 3.0 and 1.8 ± 2.4; LTZ group; 2.4 ± 2.7 and 1.4 ± 2.0, respectively, [P=NS]). For AMA women, the average number of blastocysts in the CC-O40 group was 1.7 ± 1.8, which was similar to that in the LTZ-O40 group (1.7 ± 1.8, P=NS). The blastocyst formation rate in the LTZ-O40 group was 58.5%, which was significantly higher than that in the CC-O40 group (46.6%; P < 0.05), but the rate for good blastocysts in the LTZ-O40 group (30.8%) was comparable to that in the CC-O40 group (23.3%, P=NS). The euploid rates in the LTZ group was 53.4%, which was significantly higher than that in the CC group (38.0%; P < 0.05), and the difference was significant even for AMA women (LTZ-O40; 40.5%, CC-O40; 16.7%, respectively, p < 0.05). Limitations, reasons for caution The study was a controlled trial with a limited number in the study population. Additionally, all cases had not received a trophectoderm (TE) biopsy. Further study is needed to determine the effect that an aromatase inhibitor exerts on obtaining either morphologically good blastocysts or euploid blastocysts. Wider implications of the findings AMA women showed blastocyst formation rates from LTZ with MOS significantly higher than that of the CC group, and euploid rates in the LTZ group were also significantly higher. The characteristics of an LTZ such as aromatase inhibitor enhanced the development of embryos. Trial registration number not applicable

P-563 Improved diagnosis in PGT-A samples with intermediate sex chromosomes copy number changes by adding ploidy and contamination analysis

Abstract Study question Does triple analysis of aneuploidy, ploidy and contamination improve the diagnosis in samples with intermediate copy number changes for the X and the Y chromosomes? Summary answer PGT-A with additional ploidy and contamination analysis improves the diagnosis of samples with intermediate sex chromosomes copy number changes, reducing non-informativity and the re-biopsy need. What is known already Next Generation Sequencing (NGS) in Preimplantation Genetic Testing for Aneuploidies (PGT-A) identifies chromosomal aneuploidies in trophectoderm (TE) biopsies. However, diagnosis limitations exist when the TE biopsy displays intermediate copy number changes for the X and the Y chromosomes, observed in ∼0.3% of biopsies. Few explanations are plausible, including mosaicism for the sex chromosomes, presence of exogenous DNA contamination or triploidy. A second TE biopsy (re-biopsy) could be recommended to obtain a conclusive result. However, additional Single Nucleotide Polymorphism (SNP) analysis to determine the ploidy status of the embryo and the presence of contamination in the biopsy can be an alternative. Study design, size, duration In this retrospective study, the impact on the diagnosis of SNPs analysis with a target panel was evaluated in PGT-A samples showing intermediate copy number changes for the X and the Y chromosomes. A total of 21 TE biopsies from January to December 2023 were included in the study. Participants/materials, setting, methods The TE biopsies were analysed using NGS with a proprietary protocol for library preparation, Ion Chef and Ion S5 System instruments for sequencing and a proprietary bioinformatics pipeline (v2.0) for 24-chromosomes aneuploidy testing. A re-biopsy was analysed in 8 embryos using the same protocol. Eighteen original biopsies were retrospectively evaluated for ploidy and contamination using an Ion AmpliSeq Custom Panel including SNPs (Thermo Fisher Scientific, USA) and a proprietary algorithm (v1.0) for data analysis. Main results and the role of chance The 21 TE biopsies displaying intermediate copy number changes for the X and the Y chromosomes were initially diagnosed as non-informative and candidates for re-biopsy to rescue diagnosis. A total of 8 embryos were re-biopsied (38.1%): 6 re-biopsies (75.0%) showed the same intermediate sex chromosomes copy number changes suggesting a triploid status of the embryo, 1 re-biopsy (12.5%) showed trisomy 22 suggesting DNA contamination in the original biopsy and, 1 re-biopsy (12.5%) displayed a mosaic monosomy X/XY suggesting a diploid-mosaic status of the embryo. In 18 original TE biopsies (85.7%), an additional ploidy and contamination analysis was done: 10 samples were triploid (55.6%), 6 samples showed DNA contamination (33.3%) and 2 samples were diploid (11.1%). In 5 embryos, the re-biopsy with the aneuploidy testing had been done: (i) in 4 triploid embryos the re-biopsies showed the same intermediate copy number changes for the sex chromosomes than the original biopsy, confirming the triploid status of the embryo; (ii) in 1 diploid embryo the re-biopsy showed a mosaic monosomy X/XY, confirming the diploid status and the sex chromosome mosaicism of the embryo. From the 21 embryos initially non-informative, only the 6 contaminated ones remained non-informative and candidates for re-biopsy (33.3%). Limitations, reasons for caution The limited number of samples with both analysis, ploidy/contamination by SNPs in the original sample and aneuploidy by NGS in the re-biopsy precluded to drawn stronger conclusions. This double-check would reinforce the benefit of doing additional ploidy/contamination analysis in these specific cases. Wider implications of the findings A high incidence of samples with intermediate sex chromosomes copy number are triploid, some have exogenous DNA contamination, and few are real mosaics for the sex chromosomes. Additional ploidy and contamination analysis could improve the PGT-A diagnosis and decrease mosaicism and non-informativity rates, reducing the embryos that would need re-biopsy. Trial registration number Not applicable

P-188 Growth variance score (GVS): a morphokinetic-based model with capability to distinguish euploid from aneuploid embryos

Abstract Study question Does longitudinal growth of euploid embryos differ from aneuploids? Summary answer Euploid embryos had significantly lower growth variance score (GVS), indicating steadier growth when observed with time-lapse monitoring, compared to aneuploid embryos. What is known already A major quest for optimizing embryo selection is the identification of morphokinetic parameters to distinguish euploid from aneuploid embryos. Although some studies have identified altered morphokinetic parameters related to embryo ploidy, outcomes were not reproducible, and could not provide sufficient evidence to apply it. Moreover, longitudinal changes during this temporal process of embryo development, their reference standards, and the impact of such changes on embryonic fate remains unknown. Aiming to develop a growth variance model, using the reference ranges of embryonic morphokinetic growth as a single growth trajectory might provide a better additive value to distinguish euploid from aneuploid embryos. Study design, size, duration This observational, retrospective cohort study was performed in a tertiary referral IVF centre between December 2020 and March 2023. The study included a total of 677 blastocysts from 147 cycles tested for Preimplantation Genetic Testing for aneuploidy (PGT-A) by next generation sequency (NGS). All embryos were monitored by time-lapse (TL) system during culture. Participants/materials, setting, methods Growth variance score (GVS) is a measure of embryonic growth pattern calculated using the TL morphokinetic reference ranges (from tPB2 until tEB), developed for euploid embryos. Higher GVS scores indicate more erratic growth while lower scores indicate steadier growth. Trophectoderm (TE), inner cell mass (ICM), and expansion grade were assessed immediately before biopsy using Gardner’s criteria. Euploid and aneuploid blastocysts were compared in terms of ICM, TE grade, day of biopsy and GVS score. Main results and the role of chance Among tested blastocysts, 383 were euploid (56.6%) and 294 of them were abnormal (43.4%). Euploid embryos had significantly lower GVS compared to aneuploid embryos (mean difference: 0.30 lower, 95% CI: 0.16 to 0.45; P < 0.001). Among day 5 biopsies, euploids had significantly lower GVS compared to aneuploids (mean difference: 0.27 lower, 95% CI: 0.04-0.51; P = 0.019). Among Day 6+ biopsies, euploids also had lower GVS compared to aneuploids, but the difference did not reach statistical significance (mean difference: 0.09 lower, 95% CI: -0.08-0.28, P = 0.30). Regression analysis showed GVS was significantly and independently associated with euploidy after adjusting for day of biopsy and morphological grade (aOR: 0.83, 95% CI: 0.70 – 0.99; P = 0.048). We compared two embryo selection modalities, the first one using morphology and biopsy day, the second one incorporating GVS to morphology and biopsy day. In 90% (95% CI: 87.2 – 92.8%) of the cycles, both modalities made a concordant selection by choosing the same embryo as the best. In the remaining 10% (95% CI: 6.4% - 14.7%) of the cycles, they made a discordant choice and the euploidy rate of embryos chosen by the combination of GVS and morphology were higher by 20.4% (95% CI: -0.06 to 44.0%, P = 0.060). Limitations, reasons for caution To confirm its reproducibility in other clinical and laboratory settings, GVS should be replicated with larger sample sizes and in external cohorts. Wider implications of the findings The GVS model might emerge as a valuable prognostic tool to select euploid blastocysts within patient’s own pool of embryos. GVS could be easily incorporated into laboratory work-flow when a time-lapse monitor is available to prioritize embryos for transfer, given external validations are successful. Trial registration number Not applicable

P-528 Establishing sequencing coverage depth for preimplantation genetic testing for monogenic disorders (PGT-M) using a specific targeted next generation sequencing (tNGS)

Abstract Study question What is the sufficient sequencing coverage read depth (RD) for direct variant analysis for PGT-M using a specific tNGS assay? Summary answer Downsampling modelling experiments identified a minimum sequencing depth of 50X that is recommended for PGT-M with this specific tNGS assay. What is known already Properly developed and validated multiplex PCR amplification and NGS-based analysis reliably produces PGT-M diagnoses with low allele and amplification failure at RD of hundreds of reads when analyzing a 3-10 cell trophectoderm (TE) biopsy. However, the RD can be affected by amplification efficiency in a specific genomic region and therefore affect the reproducibility of variant detection: the higher the number of aligned reads, the higher the confidence of base calling at a particular position. Downsampling is a powerful bioinformatics tool for modelling comparisons between samples or between replicates, identifying analytical sequencing depth limits for accurate clinical diagnosis. Study design, size, duration This is a retrospective comparative diagnostic test accuracy (DTA) and verification study. Twenty-three embryos from 8 patients that had undergone PGT-M with clinically established Taqman qPCR genotyping were used as positive controls. The primary outcome was to establish the lowest RD point of down sampling where allelic frequency is reliably detected at heterozygosity between 20-80% in known positive controls. A further 30 PGT-M clinical cases were used to verify analytical performances under clinical conditions. Participants/materials, setting, methods Embryo biopsies were pre-amplified with a multiplex primer pool for PGT-M and PGT-A, using a two-step PCR strategy. Sequencing was performed on Illumina NextSeq550. Reads were aligned to a human reference genome (GRCh37/hg19) and processed with an in house bioinformatic pipeline. The RD and alternative allele frequencies (AAF) for PGT-M was calculated. The reads for the heterozygous variants were downsampled to 10X, 20X, 30X, 50X, 100X, 200X, 300X, 500X, and 1000X to establish the cutoff. Main results and the role of chance One or two TE biopsies (N = 44) of 23 embryos that were 100% concordant with previous PGT-M results were tested for 1 to 3 of the 12 variants (MLH1 c.199G>A, RB1 c.1589A>G, SLC16A2 c.604G>A, GNPTAB c.1123C>T, GNPTAB c.3314A>G, WFS1 c.1391C>G, USH2A c.10073G>A, USH2A del exons 5-11, F5 c.1601G>A, GAA c.693-1G>C, GAA c.875A>G, and HEXA c.1274_1277dupTATC). PGT-A analysis of multiple biopsies from the same embryo demonstrated consistent karyotypes (N = 44). The 19 heterozygous variants from 17 biopsies exhibited average RD of 763X (238X -1980X) with average AAF of 50% (44%- 56%). The reads for the heterozygous variants were downsampled to different levels (100,000 replicates). The distribution of the AAF were compared among the different depth levels. At a RD of 50X, the AAF displayed an interquartile range (IQR) of 8% (46%-54%) between the maximum and minimum values from 20% to 78%. Compared to 30X (IQR: 43%-57%, with minimum 13% and maximum 90%), and 100X (IQR: 47%-54%, with minimum 30% and maximum 71%), the RD at 50X generated reliable heterozygous calls with minimum depth requirement. 245 biopsies from 30 clinic cases were analyzed by tNGS-based PGT-M. 90 samples with heterozygous variant calls showed average RD of 979X (57X-5803X) with average AAF of 47% (29%-585). Limitations, reasons for caution The 50X cutoff is based on the heterozygous variants with theoretical 50% AFF and is specific for this PGT-A assay. This evaluation should be corroborated for complicated variants with homologous genes, which might require different thresholds. The aneuploidy at specific chromosome can cause the deviation of AFF from 50%. Wider implications of the findings This specific tNGS-based PGT-M can provide direct variant analysis at a 50X read depth with a high level of consistency compared to established methodologies while allowing for simultaneous aneuploidy screening using the same biopsy and procedural optimization. Trial registration number not applicable

P-516 Minimizing mosaicism: impact of trophectoderm (TE) biopsy specimen size on mosaic blastocysts in preimplantation genetic testing for aneuploidy (PGT-A)

Abstract Study question Is the size of TE biopsy specimens related to the chromosomal status of the blastocysts in PGT-A? Summary answer Smaller biopsy specimens are associated with higher proportion and altered composition of mosaic blastocysts in PGT-A. What is known already Concordance between TE biopsy and inner cell mass or whole embryos is influenced by mosaicism type, degree, biopsy location, number of cells biopsied, and cell loading. Particularly, mosaic embryos and those with structural rearrangements have a higher level of inconsistency. Recent studies have demonstrated the potential reclassification of chaotic embryos as euploid, leading to successful live births. This study aimed to investigate the potential influence of TE biopsy specimen size on the chromosomal status of blastocysts undergoing PGT-A. Study design, size, duration This retrospective study, conducted from October, 2023, to January, 2024, included 246 blastocysts from patients undergoing TE biopsy and PGT-A at the blastocyst stage on days 5, 6, or 7. Next-generation sequencing (NGS) was used to obtain PGT-A results, categorizing blastocysts into euploid, aneuploid, and mosaic based on their genetic composition. Participants/materials, setting, methods The biopsy specimen area was measured using the Image J program. Mosaic blastocysts were categorized into either a chaotic mosaic group or another group, which included blastocysts with fewer than three chromosomal aneuploidies and segmental mosaicism. Further analysis was conducted by classifying blastocysts into high-level mosaic (>30% aneuploidy) and low-level mosaic (≤30% aneuploidy) groups. Group differences were analyzed using t-tests and chi-square, with a significance threshold set at p<0.05. Main results and the role of chance In the analysis of expanded mosaic blastocysts, the chaotic mosaic group had smaller biopsy specimens than those in the comparison group (p<0.05). The high-level mosaic group also had significantly smaller biopsy specimens than the low-level mosaic group (p<0.05). When categorizing the biopsied sample size into two groups—below 1350 µM2 (n = 98) and above 1500 µM2 (n = 97)—the rates of mosaic embryos were 37% (36/98) and 30% (29/97), respectively, showing a numerical difference without statistical significance. These findings suggest a correlation between the size of biopsy specimens and the chromosomal status of mosaic blastocysts, particularly indicating that a smaller sample size is associated with an elevated chance of mosaic embryos showing chaotic or high-level mosaic characteristics. Limitations, reasons for caution The biopsy specimens were photographed, and their areas were measured using the Image J program, lacking a comprehensive representation of the three-dimensional sample morphology. This constraint could potentially affect the accuracy and in-depth understanding of the spatial characteristics of the biopsy specimens. Wider implications of the findings The study emphasizes the crucial role of optimizing biopsy specimen size in PGT-A. The identified correlation with the chromosomal status in mosaic embryos, especially the association with chaotic or high-level mosaic embryo outcomes in smaller samples, highlights the need for careful size considerations. Trial registration number not applicable

O-171 Apicobasal RNA asymmetries regulate cell fate in the early mouse embryo

Abstract Assessing the morphological appearance of an embryo as it progresses from zygote to blastocyst is the primary method used by embryologists to evaluate embryo viability and developmental potential. Standard embryo grading techniques, using time-lapse microscopy, analyse morphological characteristics such as size, shape, number, and appearance of blastomeres. The aim of this study was to determine how individual cells of the embryo function on a subcellular level to build a healthy embryo. RNA localisation plays indispensable roles in establishing asymmetries and coordinating cell fate decisions during early embryogenesis across various non-mammalian species. To direct the spatiotemporal distribution of RNA within the cells of an embryo, the microtubule cytoskeleton provides highly sophisticated trafficking pathways. Yet, it remains unknown whether subcellular heterogeneities exist during mammalian preimplantation development and how they contribute to cell fate determination. Using advanced live imaging, we visualised global RNA transcripts at high spatiotemporal resolution from fertilisation to the blastocyst stage in living preimplantation mouse embryos. We discovered apicobasal RNA asymmetries specific to outer cells of the 16-cell stage mouse embryo, coinciding with cell fate decisions and the emergence of the pluripotent inner cell mass. Highly clustered RNAs accumulated proximal to the basal membrane, while more dispersed RNA foci were identified apically as the embryo reaches the late 16-cell stage. The targeted distribution of membrane-less RNA molecules was facilitated by the microtubule cytoskeleton, associated with lysosomes which serve as RNA transport vehicles. We discovered that apically located RNA foci were more dynamic and accompanied an enrichment of translation components. Furthermore, we uncovered that the established RNA asymmetries enhanced translation capacity in apical regions compared to basal regions. These spatiotemporal RNA heterogeneities were unevenly inherited by outer and inner daughter cells during subsequent cell divisions. Notably, embryos that were unable to establish RNA asymmetries failed to allocate cells to the trophectoderm and inner cell mass. Our study provides insights into a subcellular mechanism driving asymmetric RNA localisation and compartmentalised translational regulation in outer cells of the 16-cell stage embryo, which may contribute to cell fate decisions during mammalian preimplantation embryogenesis. We envision that when paired with classical morphological assessment criteria of the embryo, these findings may assist with selecting the embryo with the highest developmental potential.

P-531 Embryo grading in PGT-A testing: does it really matter?

Abstract Study question Can embryo morphology grading be a good parameter to predict the embryo transfer’s outcome when euploid embryos are used? Summary answer Gardner’s grading is a useful predictor of LBR per euploid embryo transferred. Morphologic grading should be used for the embryo selection among euploid blastocysts. What is known already When preimplantation genetic testing for aneuploidies (PGT-A) is not performed, the selection of the embryo(s) to be transferred is based on embryo morphology. In PGT-A cycle, some studies have minimised the importance of embryo morphology, when euploid embryos are present. However, others still consider it as good predictor. Understanding the role of morphology in biopsied euploid embryos may help in optimizing frozen embryo transfer cycles. Study design, size, duration We retrospectively analysed PGT-A frozen embryo transfers with reported outcome, performed in our clinics between 2018 and 2022. The cycles included were divided into two groups based on Gardner’s grading of euploid embryos transferred: “good” embryos (AA, AB, BA and BB; n = 263) versus “poor” embryos (including AC, CA, BC, CB, and CC; n =112). Participants/materials, setting, methods We included in the study only single euploid embryo transfers, double embryos transfers and mosaic embryo transfers excluded. Body mass index (BMI), oocyte age and outcomes (pregnancy and live birth rates) were compared between the two groups. Data was statistically analysed using MedCalc Software, where P < 0.05 was considered as significant. Main results and the role of chance A total of 375 embryo transfer cycles were included in the study. Maternal ages were not significantly different between groups (good vs poor embryos) (37.5 vs 38.2). Similarly, oocyte age (37.7 vs 38.1, respectively), and BMI (22 vs 22.7, respectively) did not differ between good and poor embryos. On the contrary, clinical pregnancy (56.6% vs 40.1%) and live birth rates (27.7% vs 13.4%) were significantly higher when morphologically good grade embryos were transferred (p < 0.05). In fact, both rates were significantly higher after the transfer of blastocysts that exhibited A-grade or B-grade in inner cell mass and/or trophectoderm, as opposed to the transfers of blastocysts that exhibited a C-grade trophectoderm and/or inner cell mass. Limitations, reasons for caution The findings apply to the participating centres and may not represent worldwide trends. Data was analysed retrospectively, but details of the follow-up on PGT pregnancies and babies born were limited. The degree of blastocyst expansion and the day of biopsy were not taken into consideration in this study. Wider implications of the findings The Gardner embryo quality assessment improves the clinics’ ability to prioritize embryos after PGT. Preferential transfer based on morphologic criteria increases the chances of a positive outcome in terms of live birth rates. Trial registration number not applicable

P-121 Post-warming morphokinetics as a predictive tool for embryo implantation

Abstract Study question Does frozen-thawed blastocyst morphokinetics observed using time-lapse imaging provide an indicator for implantation in intracytoplasmic sperm injection (ICSI) cycles with deferred embryo transfers? Summary answer The blastocyst morphokinetic behavior after warming may provide an indicator for implantation in ICSI cycles. What is known already Embryo cryopreservation has become a pivotal component in assisted reproduction, applicable both to surplus embryos and in freeze-all cycles. Throughout vitrification and warming processes, embryos face considerable challenges. Historically, the evaluation of fresh blastocysts has relied on the examination of morphological features and the assessment of blastocoel expansion, trophectoderm, and inner cell mass. However, for vitrified and warmed blastocysts, traditional evaluation may not suffice to predict implantation. The emergence of a TLI culture system has facilitated a comprehensive examination of embryo development. Hence, post-warming culture in TLI incubators may provide a more precise assessment of vitrified and warmed blastocyst behavior. Study design, size, duration From January to October 2022, an observational cohort study was conducted at a private university-affiliated IVF centre. This included 411 vitrified/warmed blastocysts, from freeze-all cycles, with known implantation data (no implantation and full implantation per transfer), which were evaluated by TLI. Blastocyst morphological dynamics variables after warming were recorded, and their potential association with clinical pregnancy was investigated using Generalized linear models (GzLM) adjusted for oocyte age, followed by Bonferroni post hoc test. Participants/materials, setting, methods After warming, the recorded factors were: (i) initial blastocyst area (IBA); (ii) maximum blastocyst area (MBA); (iii) Blastocyst area delta (DBA); (iv) initial ZP thickness (IZP); (v) minimum ZP thickness (MZP); (v) ZP thickness delta (delta ZP area); and (iv) blastocyst expansion (BE). Embryos received a final score from 0 to 9, according to the sum of the scores from each parameter and were grouped as: Low total-score, medium total-score), and high total-score. Main results and the role of chance No significant difference was observed when maternal age (37.8 ± 5.3, 37.2 ± 5.2, and 37.4 ± 5.7, for low, medium and high total-score groups respectively, p = 0.743) and oocyte age (34.9 ± 4.9, 34.1 ± 4.7, and 33.7± 5.2, for low, medium and high total-score groups respectively, p = 0.229) were compared between the score groups. A significant increase in the implantation rate was noted among embryos presenting higher scores, followed by those with median scores, while embryos with lower scores presented the lowest implantation rate (34.8 ± 5.3, 35.9 ± 5.2, and 39.7 ± 5.2, for low, medium and high total-score groups respectively, p < 0.001). No significant differences in the miscarriage rate were observed between the three score groups (10.0 ± 4.2, 13.2 ± 4.1, and 5.1 ± 2.9, for low, medium and high total-score groups respectively, p = 0.239.) The logistic regression analysis showed that embryos in the low total-score group presented a 12.3% decreased chance of implantation when compared with embryos in the high total-score group (OR: 0.877, CI: 0.843-0.912, p < 0.001), while embryos in the medium total-score group presented a 9.6% decreased implantation chance when compared with embryos in the high total-score group (OR: 0.904, CI: 0.870-0.939, p < 0.001). Limitations, reasons for caution The use of historical cohort groups is a drawback. Wider implications of the findings The behavior of blastocysts after warming, as indicated by blastocyst area, ZP thickness, re-expansion, along with the pre-vitrification embryo classification, may provide crucial information for selecting the optimal embryo for transfer and managing patient expectations. Trial registration number N/A

P-134 Fatty acid supplementation during warming improves pregnancy outcomes after frozen blastocyst transfers: a propensity score-matched study

Abstract Study question Does warming with fatty acids (FAs) affect the viability of human blastocysts and pregnancy outcomes after a single vitrified-warmed blastocyst transfer (SVBT)? Summary answer FA-supplemented warming solutions promoted the blastocyst outgrowth by decreasing the degeneration of trophoblast cells and improved pregnancy outcomes after SVBTs. What is known already Vitrification is considered a well-established technique and has been used worldwide. However, previous studies demonstrated the detrimental effects of vitrification on pre-implantation development and term pregnancy in oocytes and embryos. Vitrification reduces the intracellular lipid content and decreases the competence for pre-implantation development. The FA supplementation into the warming solution restores the lipid content in the ooplasm and improves developmental competence by promoting the β-oxidation pathway in mice. Although, in humans, the FA-supplemented warming solutions reportedly improved pregnancy outcomes after vitrified-warmed cleavage-stage embryo transfers, the effectiveness of these solutions on blastocyst warming remain unclear. Study design, size, duration The experimental study included 401 discarded blastocysts. The blastocysts were warmed using the FA-supplemented (FA group) or non-supplemented (control group) solutions. The blastocyst adhesion, outgrowth area, and incidence of outgrowth degeneration were assessed. The retrospective cohort study included 4,426 treatment cycles from 4,426 women who underwent SVBTs between January 2023 and August 2023 (January–May 2023, control group; June–August 2023, FA group). Pregnancy outcomes were compared statistically between the groups. Participants/materials, setting, methods The implantation competence was experimentally examined using an outgrowth model. The blastocysts were placed on fibronectin-coated dishes and cultured for 96 hours. The embryos were designated as degenerated when the outgrowth area decreased by > 10%, compared with the maximum area. For the clinical study, multiple covariates that affected the outcomes were used for propensity score matching. The patients in the FA group were matched with controls using a propensity score in a 1:1 ratio. Main results and the role of chance The adhesion rate after the outgrowth culture was comparable between the control and FA groups. However, the outgrowth was larger in the FA group than in the control group. The multivariate linear regression analysis also demonstrated similar results. Furthermore, the incidence of outgrowth degeneration was significantly decreased in the FA group. Furthermore, the subgroup analyses revealed that FA-supplemented warming solutions were effective in blastocysts donated from young patients, day 5 blastocysts, and morphologically good blastocysts. For the retrospective cohort study, the implantation, clinical pregnancy, and ongoing pregnancy rates were significantly higher in the FA group than in the control group. The multivariate logistic regression analysis also demonstrated that warming with FA-supplemented solutions significantly increased the probability of ongoing pregnancies. However, the rates of early pregnancy loss and miscarriage in the first trimester were comparable between the groups. The subgroup analyses demonstrated a similar trend to in vitro experiments; the FA-supplemented solutions were more effective in good prognosis cases (young patients, day 4/5 blastocysts, and morphologically good blastocysts) than in poor prognosis cases (advanced age patients, day 6/7 blastocysts, and morphologically poor blastocysts). Limitations, reasons for caution This was a single-centre, retrospective study, necessitating further multicentre studies to ascertain the generalizability of these findings to other clinics with different protocols and/or patient demographics. Despite using propensity score matching and multivariate models to adjust for the confounders, we cannot adjust for all possible confounders. Wider implications of the findings FA-supplemented warming solutions improved pregnancy outcomes after SVBTs, particularly in patients with a good prognosis. These warming solutions could contribute to maximizing clinical outcomes, shortening treatment periods, and reducing patient burden. Trial registration number not applicable

O-097 Implantation-on-chip: precise quantification for functional implantation failure studies

Abstract Study question How can we obtain precise quantitative information about what factors causally affect the first steps of embryo implantation and Repeated Implantation Failure (RIF)? Summary answer We have developed a personalized implantation-on-chip model, based on novel in vitro models of the endometrium and the embryo (i.e., blastoids), to quantify functional attachment. What is known already RIF is a multifactorial condition characterized by the persistent inability to achieve clinical pregnancy, despite the transfer of high-quality embryos. Typically diagnosed within the context of assisted reproductive technology (ART) treatment, RIF arises due to disruptions in the endometrium receptivity, the interaction between the endometrium and early embryo, or both. The endometrial factors of RIF can be linked to disrupted hormone signaling and timing, impacting endometrial receptivity and therefore the chance of conceiving. Synchronization between the blastocyst and receptive endometrium is critical for successful implantation. Ethical and technical limitations of in vivo patient research restricts our understanding of implantation failure. Study design, size, duration Organoids were transformed into endometrial monolayers within custom-made microfluidic chips. These monolayers on chip were either treated with EPCX; β-estradiol (E2), progesterone (P4), 8-Br-cAMP (C) and XAV939 (X) (referred as induced), or not treated (referred as control) for 6 days. Large number of blastoids (>100) were then infused per chip followed by 48 hours of culture. Then, the rate of adhered blastoids was measured under exposure to controlled flow rates (50, 100 and 400 µl/min). Participants/materials, setting, methods Human naïve induced pluripotent stem cells were used to form blastoids, stem cell-based models of the blastocyst, to alleviate the ethical concerns and limited access of blastocysts. Blastoids present the three blastocyst lineages required for development and offer limitless production in number. Endometrium organoids, derived from parous fertile patients (N = 3), was used to recapitulate molecular and functional receptivity by RT-qPCR-based gene expression measurements and microfluidic flow-dependent adhesion rates of blastoids, respectively. Main results and the role of chance 2-D monolayers that maintained their epithelial identity and responsiveness to hormones as measured by expression of receptivity-related genes (PAEP, SPP1 and GPX3), which were upregulated in EPCX condition. Further characterization of the epithelial monolayers showed reduced MUC1 expression in the EPCX condition, suggesting increased receptivity. Additionally, reduced acetylated α-tubulin, indicating a reduction in the number of ciliated cells, aligning with our observations. Through controlled exposure to increasing flow, functional receptivity through blastoid adhesion was precisely quantified, which showed 35% of the blastoid remained adhered in the EPCX condition, whereas only 15% remained adhered in the control. A 48 hours culture period presented a blastoid-induced endometrial gap in 45.54% of the remaining blastoids, with an average of 110.287,3 µm2 in surface area, which was associated with trophoblast cells infiltration, measuring an average migration distance of 174.8 µm from the pluripotent cell core. In addition, the substantial difference in receptivity profiles established a baseline for subsequent experiments, including the study of temporal alterations in hormone signaling, a potential problem associated with RIF. Hormone administration variations, timing and a progesterone-deficient condition are currently being tested. These results will underscore the sensitivity of our system and demonstrate its potential for applications in drug screening. Limitations, reasons for caution Currently our endometrium model consists of epithelial cells, which are only representative for implantation to a limited extent. A more complex endometrium including stromal, endothelial and immune cells could highly increase the physiological relevance of the model. Wider implications of the findings This platform has the potential to provide a diagnostic platform to identify patients with endometrium epithelium-associated RIF and factors that may improve embryo receptivity and implantation. Moreover, this approach has the potential to be pivotal for toxicological studies, and furthering our understanding of human reproductive biology. Trial registration number not applicable

Alpha 1,3 N-Acetylgalactosaminyl Transferase (GTA) Impairs Invasion Potential of Trophoblast Cells in Preeclampsia.

Preeclampsia (PE) is a pregnancy-specific disorder associated with shallow invasion of the trophoblast cells and insufficient remodeling of the uterine spiral artery. Protein glycosylation plays an important role in trophoblast cell invasion. However, the glycobiological mechanism of PE has not been fully elucidated. In the current study, employing the Lectin array, we found that soybean agglutinin (SBA), which recognizes the terminal N-acetylgalactosamine α1,3-galactose (GalNAc α1,3 Gal) glycotype, was significantly increased in placental trophoblast cells from PE patients compared with third-trimester pregnant controls. Upregulating the expression of the key enzyme α1,3 N-acetylgalactosaminyl transferase (GTA) promoted the biosynthesis of terminal GalNAc α1,3 Gal and inhibited the migration/invasion of HTR8/SVneo trophoblast cells. Moreover, the methylation status of GTA promoter in placental tissues from PE patients was lower than that in the third trimester by methylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP) analysis. Elevated GTA expression in combination with the DNA methylation inhibitor 5-azacytidine (5-AzaC) treatment increased the glycotype biosynthesis and impaired the invasion potential of trophoblast cells, leading to preeclampsia. This study suggests that elevated terminal GalNAc α1,3 Gal biosynthesis and GTA expression may be applied as the new markers for evaluating placental function and the auxiliary diagnosis of preeclampsia.

CircCUL1 inhibits trophoblast cell migration and invasion and promotes cell autophagy by sponging hsa-miR-30e-3p in fetal growth restriction via the ANXA1/PI3K/AKT axis.

Fetal growth restriction (FGR) severely affects the health outcome of newborns and represents a major cause of perinatal morbidity. The precise involvement of circCULT1 in the progression of FGR remains unclear. We performed next-generation sequencing and RT-qPCR to identify differentially expressed circRNAs in placental tissues affected by FGR by comparing them with unaffected counterparts. Edu, flow cytometry, and transwell assay were conducted to detect HTR8/SVneo cell's function in regard to cell proliferation, migration, and invasion. The interaction between circCUL1 and hsa-miR-30e-3p was assessed through dual-luciferase reporter assays, validation of the interaction between circCUL1 and ANXA1 was performed using RNA pulldown and immunoprecipitation assays. Western blot analysis was performed to evaluate protein levels of autophagy markers and components of the PI3K/AKT signaling pathway. A knockout (KO) mouse model was established for homologous mmu-circ-0001469 to assess fetal mouse growth and development indicators. Our findings revealed an upregulation of circCUL1 expression in placental tissues from patients with FGR. We found that suppression of circCUL1 increased the trophoblast cell proliferation, migration, and invasion, circCUL1 could interact with hsa-miR-30e-3p. Further, circCUL1 stimulated autophagy, modulating trophoblast cell autophagy via the ANXA1/PI3K/AKT pathway, and a notable disparity was observed, with KO mice displaying accelerated embryo development and exhibiting heavier placentas in comparison to wild-type C57BL/6 mice. By modulating the ANXA1/PI3K/AKT signaling pathway through the interaction with hsa-miR-30e-3p, circCUL1 promotes autophagy while concurrently suppressing trophoblast cell proliferation, migration, and invasion. These findings offer novel insights into potential diagnostic markers and therapeutic targets for FGR research.

Placental Methylglyoxal in Preeclampsia: Vascular and Biomarker Implications.

Preeclampsia is a multifaceted syndrome that includes maternal vascular dysfunction. We hypothesize that increased placental glycolysis and hypoxia in preeclampsia lead to increased levels of methylglyoxal (MGO), consequently causing vascular dysfunction. Plasma samples and placentas were collected from uncomplicated and preeclampsia pregnancies. Uncomplicated placentas and trophoblast cells (BeWo) were exposed to hypoxia. The reactive dicarbonyl MGO and advanced glycation end products (Nε-(carboxymethyl)lysine [CML], Nε-(carboxyethyl)lysine [CEL], and MGO-derived hydroimidazolone [MG-H]) were quantified using liquid chromatography-tandem mass spectrometry. The activity of GLO1 (glyoxalase-1), that is, the enzyme detoxifying MGO, was measured. The impact of MGO on vascular function was evaluated using wire/pressure myography. The therapeutic potential of the MGO-quencher quercetin and mitochondrial-specific antioxidant mitoquinone mesylate (MitoQ) was explored. MGO, CML, CEL, and MG-H2 levels were elevated in preeclampsia-placentas (+36%, +36%, +25%, and +22%, respectively). Reduced GLO1 activity was observed in preeclampsia-placentas (-12%) and hypoxia-exposed placentas (-16%). Hypoxia-induced MGO accumulation in placentas was mitigated by the MGO-quencher quercetin. Trophoblast cells were identified as the primary source of MGO. Reduced GLO1 activity was also observed in hypoxia-exposed BeWo cells (-26%). Maternal plasma concentrations of CML and the MGO-derived MG-H1 increased as early as 12 weeks of gestation (+16% and +17%, respectively). MGO impaired endothelial barrier function, an effect mitigated by MitoQ, and heightened vascular responsiveness to thromboxane A2. This study reveals the accumulation of placental MGO in preeclampsia and upon exposure to hypoxia, demonstrates how MGO can contribute to vascular impairment, and highlights plasma CML and MG-H1 levels as promising early biomarkers for preeclampsia.