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Study question Is the duration of compaction, KID- (Known Implantation Data) and iDAScore (Intelligent Data Analysis for embryo evaluation) associated to clinical pregnancy rate? Summary answer Duration of compaction, KID- or iDAScore can be used to select the best embryo for transfer to increase clinical pregnancy rate. What is known already The development of human preimplantation embryos follows a programmed timeline in which a series of critical events occur. Compaction is a typical event at 3 to 4 days post fertilisation that is characterised by flattening of the blastomeres and the formation of tight junctions between the blastomeres. It is considered the first morphological event in the differentiation process of the embryo. Time-lapse technology introduced continuous monitoring of the embryo throughout development in the IVF laboratory. Evaluation of the developmental events combined with calculating KID- and iDAScore can optimise the selection of the most competent embryos for transfer and/or cryopreservation. Study design, size, duration This single-centre retrospective observational study included 158 IVF/ICSI cycles with fresh single embryo transfer (SET) was performed between December 2018 and November 2021. Embryos were cultured during 5 days in cleavage/blastocyst medium (Coopersurgical) in the EmbryoScope + (Vitrolife). Transferred embryos were evaluated for start of compaction, time to full compaction and duration of compaction. Embryo quality was calculated using KID- and iDAScore. These parameters were compared between the clinically pregnant and non-pregnant group (primary outcome). Participants/materials, setting, methods Only IVF/ICSI cycles with ejaculated sperm and fresh SET on day 5 were included. MNC, IVM and PGT cycles were excluded. Time zero was the start of injection or insemination. Pregnancy was confirmed by hCG and clinical pregnancy was defined by gestational sac visualisation at ultrasound. GraphPad Prism and R-studio were used for statistical analysis. For prediction of clinical pregnancy, univariate logistic regression was used. Other significant differences were determined using t-test. Main results and the role of chance Out of 158 fresh ET, 101 (63.9%) had a positive hCG, of which 88 (55.6%) achieved clinical pregnancy. All 158 transferred blastocysts were annotated to calculate KID- and iDAScore. There was no statistical difference in age between the two groups (34.7 years vs 35.0 years; p = 0.69). Start of compaction was heterogeneous (between 50.9 and 98.3 hours post injection/insemination; mean=76.5±7.7), as well as the blastomere number at its initiation (between 4 and 16 blastomeres; mean=11.8±2.1). Univariate logistic regression showed that each individual parameter, i.e. duration of compaction (p = 0.02), KID-score (p = 0.001) and iDAScore (p = 0.0006) was different between the clinically pregnant and non-pregnant group. The total duration of compaction was significantly shorter in the clinical pregnant group (mean=8.6±3.4 hours vs 10.2±4.7 hours; p = 0.01; t-test). In the pregnant group the KIDscore (mean=7.7±1.4 vs 6.7±2.3; p = 0.0007) and iDAScore (mean=8.9±0.7 vs 8.3±1.3; p = 0.0002) were significantly higher. During partial compaction, cells were rather excluded (93%) than extruded from the process; 17 embryos underwent this process, 10 of which resulted in a clinical pregnancy. Limitations, reasons for caution As this is a retrospective study, the influence of uncontrolled variables cannot be excluded. In the future, different models will be applied that can combine duration of compaction, KID- and iDAScore in a larger study. Wider implications of the findings Our analysis confirms previous findings that KID- and iDAScore are good predictors of clinical pregnancy. We also show that duration of compaction can be used as a potential predictor for pregnancy, especially in IVF clinics that have no access to KID- or iDAScore. Trial registration number not applicable

Study question Do human pre-implantation embryos express CD147, does it play a role during implantation, and can it be used as non-invasive marker for embryo selection? Summary answer CD147 is expressed throughout pre-implantation development. Blocking results in implantation failure in vitro, but CD147 secretion has no immediate link with implantation in vivo. What is known already Embryo selection remains a subjective process with room for improvement. Researchers focus on alternative techniques to reduce time to pregnancy. While preimplantation genetic testing and time-lapse studies increased our understanding, they failed to efficiently improve embryo selection. CD147, a cluster domain well studied in cancer, has been proven to positively influence implantation in mice. When the protein is blocked, the embryo develops to blastocyst but fails to implant. This study aims to visualize the expression of CD147 throughout human pre-implantation development and to understand its role during implantation in humans, using both an in vitro and in vivo model. Study design, size, duration CD147 expression was studied in oocytes (n = 13) and embryos (n = 30) between 3 and 7 days post-fertilisation (dpf) with immunohistochemistry. To investigate its function, 5dpf blastocysts (n = 45) were co-cultured with Ishikawa cells for two days in an in vitro attachment model with two distinct anti-CD147 antibodies (ab666 and ab119114). Attachment models containing embryos (n = 8) were stained using ActinGreen™ probes. Additionally, spent culture medium was collected in the IVF lab after fresh blastocyst transfer (n = 153, sequential medium). Participants/materials, setting, methods Fresh immature oocytes and vitrified/warmed embryos were obtained after patients’ informed consent, fixed in 4% paraformaldehyde and stained using ab666. The counterstained samples were visualized with confocal microscopy. For blocking experiments, embryos were incubated in ab666 or ab119114 (Abcam) and co-cultured in the attachment model. Attachment was measured after 24hrs by gentle pipetting of the medium. Attached embryos were stained for F-actin. CD147 concentrations in spent embryo culture medium were determined by ELISA (EH78RB, Thermo-Fisher-Scientific). Main results and the role of chance In immature oocytes, CD147 was present on the oolemma only. After fertilization, CD147 protein was restricted to the membrane of all blastomeres but fluorescence intensity decreased gradually during cleavage stages. After embryonic genome activation the expression increased again mainly on the membranes of the cells. After blastulation (5 dpf), the protein was expressed in all cells but mainly concentrated on the membranes. With the expansion of the blastocysts (6-7 dpf) CD147 became more abundant on the membranes of trophectoderm cells (TE), the signal being the strongest on the apical membranes and TE junctions. Blocking of CD147 binding in blastocysts was achieved with two anti-CD147 antibodies ab666 and ab119114 with respectively 52% and 70 % reduction of attachment on Ishikawa cells compared to non-treated control groups (n = 45, p Finally, secreted CD147 was found in culture medium of embryos 5 dpf. Most of the blastocysts (150/153) secreted CD147 with concentrations ranging from 40–229 pg/ml. No correlation with pregnancy outcome or morphology was noted, indicating that CD147 cannot be used as non-invasive marker for embryo selection. Although these results clearly highlight the importance of CD147 in human embryo implantation in vitro, more research is needed to investigate its role. Limitations, reasons for caution The experiments have been performed using an in vitro model for attachment, the impact of additional interfering factors in vivo cannot be ruled out. Wider implications of the findings Protein expression highlights the importance of CD147 in human blastocysts, especially for the attachment function of the apical TE membrane. Blocking CD147protein reduces implantation dramatically, proving its involvement in establishing a successful pregnancy. In addition, secretion of CD147 demonstrates its potential paracrine role during the interaction with the endometrium. Trial registration number not applicable

Study question Are aneuploid cells in human preimplantation embryos eliminated by apoptosis due to proteotoxic stress and autophagy-mediated apoptosis? Summary answer Proteotoxic stress, autophagy and apoptosis are differentially activated in aneuploid embryos, showing that aneuploid cells are eliminated by these mechanisms during early human embryogenesis. What is known already Aneuploidies are a common feature of human preimplantation embryos which could explain low success rates after in vitro fertilization (IVF). While most aneuploidies of meiotic origin are detrimental, transfer of euploid-aneuploid mosaic embryos can lead to healthy live-births. Moreover, the proportion of aneuploid cells are lower in blastocysts when compared to cleavage stage embryos. In the mouse, aneuploid cells are eliminated from the epiblast by autophagy-mediated apoptosis in a p53-dependent manner. We propose that in human embryos, aneuploidy causes chronic protein misfolding which leads to autophagy-induced apoptosis. Study design, size, duration Eighty-one blastocysts that were diagnosed by PGT as euploid (n = 49) or uniformly combined abnormal (CA, n = 32), i.e. 2 or more chromosomes were abnormal in every cell, were warmed. Sixty-seven were suitable for trophectoderm (TE) biopsy, 54 biopsies were successfully tubed and sent for RNA-sequencing while the remainder of the embryos was fixed for immunostaining. Thirty-three day-3 embryos were overnight incubated in 0.5µM reversine allowed to develop into blastocysts and treated as the PGT embryos. Participants/materials, setting, methods After TE biopsy, we live-stained the embryos with either Caspase-3/7 or 8 and subsequently fixed them. The biopsies underwent RNA-sequencing using the SMART-seqv4 and the fixed embryos were immunostained for LC3B, p62 (autophagy) and HSP70 (proteotoxic stress). Confocal imaging was performed using a Zeiss LSM800 confocal microscope and the presence of signal was quantified using the Zen Blue 2.0 and Arivis software. Main results and the role of chance Forty-two percent of the embryos in which we induced aneuploidies using reversine developed into blastocysts, which is comparable to untreated embryos. After immunostaining, we observed that CA and reversine-treated (RT) embryos contained less cells than euploid embryos (median number of nuclei: 43.5, 47, 90, respectively). This correlates with a higher expression of apoptotic markers Caspase-3/7 in CA embryos (p = 0.0199) and Caspase-8 in both aneuploid groups (CA: p = 0.0085 and RT: p = 0.0394). Aneuploid embryos showed significantly increased HSP70 levels (median intensity per cell: euploid=165, CA = 313, RT = 400), LC3B (median puncta per cell: euploid=3.07, CA = 10.10, RT = 19.62) and p62 (median puncta per cell: euploid=17.60, CA = 30.53), suggesting increased proteotoxic stress and autophagy. Preliminary analysis of the RNA-sequencing data reveals enrichment for pathways such as the p53-pathway, protein secretion, TNFA signaling via NFkB and apoptosis, supporting the hypothesis of a link between aneuploidy and apoptosis. Limitations, reasons for caution No functional tests e.g. with inhibitors of autophagy were carried out. RNA-sequencing was carried out on a small sample; we will expand this sample in the near future. Wider implications of the findings This study shows for the first time the mechanism by which aneuploid cells are eliminated from the human preimplantation embryo, explaining how mosaic embryos can still lead to a healthy and genetically normal live birth. Trial registration number not applicable

Study question Can mitochondrial DNA (mtDNA) variants explain the differences in birthweight between ART and spontaneously conceived (SC) individuals and how do they originate? Summary answer Children born after ART carry more frequently a different mtDNA variant composition, both maternally inherited and de novo, which are predictive of their birthweight percentile. What is known already Children born after ART show an increased risk of lower birthweight and of developing a mild abnormal cardio-metabolic profile later in life. Variation in the mtDNA associates with overall health in the general population, including cardio-metabolic fitness, and can result in changes in mitochondrial function. We hypothesized that mitochondrial DNA variants could explain the differences in birthweight between ART and SC individuals and that these differences may result from maternal transmission and/or from the ovarian stimulation (OS) used in ART. Study design, size, duration We deep-sequenced the mtDNA of 472 individuals of who 283 ART and 189 SC, 182 mother-child pairs and 113 single oocytes from both natural menstrual cycles and OS cycles. The mtDNA was compared between groups and Fisher linear discriminant analysis was used as predictive model for the birthweight percentile. Participants/materials, setting, methods Mitochondrial DNA was enriched by long-range PCR and subsequently sequenced on an Illumina platform. mtDNA server and MuTect were used for variant calling for variants with a load higher than 1.5%, versus the reference NC_012920.1. An orthogonally rotated factor analysis was used to reduce the dimensionality of the studied dependent variables in the complex data of the heteroplasmic variants. Main results and the role of chance ART individuals carried more frequently haplogroup U4 (p = 0.004) and component analysis indicated that they carry a different mtDNA heteroplasmic variant composition than SC individuals (p = 0.01), driven by non-synonymous protein-coding and rRNA-coding variants. These differences were also predictive of the risk of a lower birthweight percentile, especially for the SC children, together with the absence of haplogroup T, the presence of homoplasmic tRNA-variants, pregnancy-induced hypertension and the embryo culture medium used. The differences in heteroplasmic variation observed in the ART children resulted from both maternal transmission (p = 0.03) and de novo mutagenesis (p = 0.02). Mothers of ART children showed a similar mtDNA genotype as their children and differed in the same variant composition when compared to the mothers of SC children (p = 0.03). Furthermore, the comparison of oocytes from the same donors retrieved in natural menstrual cycles and after one OS cycle showed that OS does not increase de novo mutagenesis. Additionally, clinical parameters such as the total dosage of FSH units, the number of oocytes retrieved, and maternal age did not show any correlation with the differences observed in ART individuals. Limitations, reasons for caution This study is observational with no functional tests being performed. Wider implications of the findings We demonstrate an association between a lower birthweight percentile and a mtDNA variant composition which is more frequently carried by ART children. These non-disease associated mtDNA variants could cause a suboptimal mitochondrial function affecting the birthweight. Long-term health consequences of these differences remain to be further elucidated. Trial registration number Not applicable

Study question Is there any indication for presence of viral RNA in FF, CC, immature oocytes or endometrial biopsy (EB) of SARS-CoV–2 patients undergoing ovarian stimulation? Summary answer Viral RNA is undetectable in FF, CC and EB with RT-PCR. However, S-protein expression on corona radiata cells suggests susceptibility to SARS-CoV–2 infection. What is known already The effects of a SARS-CoV–2 infection on the female reproductive system are still poorly understood. Theoretically, co-localisation of the angiotensin converting enzyme (ACE2) and transmembrane serine protease 2 (TMPRSS2) on human blastocysts implies susceptibility to viral infection, mediated by the coronavirus spike (S) protein. To date, SARS-CoV–2 RNA was undetectable in mature oocytes from COVID–19 patients, despite the expression of ACE2 and TMPRSS2. The presence of viral RNA in endometrial tissue, immature oocytes, CC or FF has not yet been investigated in samples from patients with positive nasopharyngeal SARS-CoV–2 test. Study design, size, duration This is a prospective, single-centre, observational study including ten patients with a positive nasopharyngeal swab for SARS-CoV–2, performed 48 hours before oocyte retrieval (OR), from September 2020 to January 2021. A patient was eligible if she preferred to continue treatment following adequate counselling of the unknown but presumably low risk for vertical transmission. Since a freeze-all strategy was applied, an EB was performed. Participants/materials, setting, methods During OR, all protective measures were taken. Pooled FF, CC and EB from each patient were tested for viral RNA presence with RealStar® SARS-CoV–2 RT-PCR-Kit1.0 (Altona-Diagnostics). Ct values Main results and the role of chance SARS-CoV–2 RNA was undetectable in the pooled FF, CC and EB from all patients included in the study. Histological analysis of the EB showed no pathological modifications, including inflammatory reaction, as compared to biopsies collected from swab negative patients. After staining with anti-S antibody, cultured EnSC and immature oocytes tested negative for the S-protein. However, the binding of anti-S antibody was demonstrated on the corona radiata cells remaining on the zona pellucida after oocyte denudation for intra-cytoplasmatic sperm injection, indicating presence of SARS-CoV–2. In that case, the explanation for the undetectable viral RNA in CC could be that the viral RNA concentration remained under the detection limit of the currently used RT-PCR test. Limitations, reasons for caution This study was conducted in a small population (ten patients included) with different viral load, with mild or without symptoms of COVID–19. Another important limitation is the absence of validation of the RT-PCR protocol for the investigation of other types of samples than nasopharyngeal swabs. Wider implications of the findings: The absence of SARS-CoV–2 RNA in all samples analysed represents a step further in reassuring a safe ART program for COVID–19 patients. However, the presence of S-protein on corona radiata cells warrants further investigation, since the theoretical possibility to infect human oocytes and/or embryos cannot be ruled out. Trial registration number NCT04425317

STUDY QUESTION: Is a reduction in the oxygen tension from 5 to 2% during extended culture from Day 3 onwards beneficial for human blastocyst development in vitro? SUMMARY ANSWER: A reduction in oxygen concentration from 5 to 2% O2 after Day 3 did not improve embryo development, quality and utilization rate. WHAT IS KNOWN ALREADY: The human embryo leaves the fallopian tube to reach the uterine cavity around Day 3-4 post-ovulation. As the oxygen concentration ranges from 5 to 7% in the fallopian tube and decreases to 2% in the uterus, reducing the oxygen tension during extended culture from Day 3 onwards seems more physiological. We aim to mimic the in-vivo environment during in-vitro embryo culture. Therefore, we compared the effect of extended culture performed at 5% (control arm) or 2% oxygen (O2; study arm) tension on blastocyst formation and quality. STUDY DESIGN, SIZE, DURATION: Between December 2016 and September 2017, in two prospective studies, sibling embryos were randomized on Day 3 to either 5% O2 (control) or 2% O2 (study) for extended culture. In the control arms of both studies 1 and 2, the dishes with blastocyst medium were pre-equilibrated overnight in 5% O2, 6% CO2 and 89% N2 at 37°C. In the 2% study groups, the overnight pre-equilibration of blastocyst media was performed in either 2% O2 (study 1, 99 cycles) or 5% O2 (study 2, 126 cycles). The latter provides a gradual transition from 5 to 2% O2 environment for the study arm. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Embryo culture until Day 3 was always performed in 5% O2; if at least four embryos of moderate to excellent quality were obtained on Day 3, the sibling embryos were randomized to either 5% O2 or 2% O2 for extended culture. The endpoints were embryo development and quality on Day 5/6 and the utilization rate (embryos transferred and cryopreserved). Statistical analysis was performed using the chi-square test, a P-value of

Study question Does extended culture to the blastocyst stage affect singleton birthweight after either fresh or vitrified-warmed embryo transfer? Summary answer Singleton birthweight z-scores did not vary significantly after a fresh blastocyst transfer, whereas the additional effect of vitrification remains inconclusive. What is known already Observational studies have associated extended culture with an increased risk of preterm birth and low birthweight. On the contrary, in terms of birthweight and gestational age, singletons born after vitrification have been associated with a better perinatal outcome when compared to those born following a fresh transfer. Study design, size, duration Our post-hoc cohort analysis on neonatal outcomes included 447 liveborn singletons was derived from a recent retrospective analysis on cumulative live birth rates after cleavage-stage and blastocyst transfers. These babies were born following a fresh single cleavage-stage transfer (FCT Day 3, n = 113), fresh single blastocyst transfer (FBT Day 5, n = 218), vitrified-warmed cleavage-stage transfer (VCT Day 3, n = 58) or vitrified-warmed blastocyst transfer (VBT Day 5, n = 58). Participants/materials, setting, methods Singleton birthweight was the primary outcome measure. Gestational age and gender of the newborn were accounted for by using birthweight z-scores in a multivariable linear regression analysis, adjusting for other confounders (maternal age, BMI, parity and smoking behaviour). Vanishing twins were excluded from the analysis. Main results and the role of chance A significantly lower z-score was observed after blastocyst transfer compared to cleavage-stage transfer in the vitrified-warmed Day 5 group (P = 0.013), a difference not observed in the fresh transfer groups (P = 0.32). Following multivariable regression analysis [adjusted regression coefficient (95% confidence interval)], the FCT and FBT groups showed no significant influence on the birthweight z-scores after fresh transfer [-0.19 (-0.44; 0.05)], but the transfer of vitrified blastocysts (VBT) was associated with a lower birthweight [-0.52 (-0.90; -0.15)] compared with the transfer of vitrified cleavage-stage embryos (VCT). Limitations, reasons for caution The present cohort was relatively small, especially in the vitrified-warmed subgroups. Pregnancy-associated factors possibly influencing birthweight (such as diabetes, hypertension, pre-eclampsia) were also not accounted for in the analysis. Wider implications of the findings Different ART procedures, including extended culture and vitrification, may hold potential safety issues. These results require further confirmation in future larger studies. Study funding/competing interest(s) None. Trial registration number N/A.