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Mechanisms controlling trophoblast (TB) proliferation and differentiation during embryo implantation are poorly understood. Human trophoblast stem cells (TSC) and BMP4/A83-01/PD173074-treated pluripotent stem cell-derived trophoblast cells (BAP) are two widely employed, contemporary models to study TB development and function, but how faithfully they mimic early TB cells has not been fully examined. We evaluated the transcriptomes of TB cells from BAP and TSC and directly compared them with those from peri-implantation human embryos during extended embryo culture (EEC) between embryonic days 8 to 12. The BAP and TSC grouped closely with TB cells from EEC within each TB sublineage following dimensional analysis and unsupervised hierarchical clustering. However, subtle differences in transcriptional programs existed within each TB sublineage. We also validated the presence of six genes in peri-implantation human embryos by immunolocalization. Our analysis reveals that both BAP and TSC models have features of peri-implantation TB s, while maintaining minor transcriptomic differences, and thus serve as valuable tools for studying implantation in lieu of human embryos., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Study Question: What are the implications of the presence cytoplasmic strings (Cyt-S) and their quantity and dynamics for the pre-implantation development of human blastocysts?, Summary Answer: Cyt-S are common in human embryos and are associated with faster blastocyst development, larger expansion, and better morphological quality., What Is Known Already: Cyt-S are dynamic cellular projections connecting inner cell mass and trophectoderm (TE) cells, that can be observed during blastocyst expansion. Their prevalence in human embryos has been estimated to be between 44% and 93%. Data relevant to their clinical implications and role in development are lacking, limited, or controversial., Study Design, Size, Duration: Retrospective study conducted at a single IVF center between May 2013 and November 2014 and involving 124 pre-implantation genetic testing for aneuploidy cycles in a time-lapse incubator with ≥1 blastocyst biopsied and vitrified (N = 370 embryos assessed). These cycles resulted in 87 vitrified-warmed single-euploid blastocyst transfers., Participants/materials, Setting, Methods: ICSI, continuous blastocyst culture (Days 5-7), TE biopsy of fully expanded blastocysts without Day 3 zona pellucida drilling, qPCR to assess uniform full-chromosome aneuploidies, and vitrification were all performed. Only vitrified-warmed euploid single-embryo-transfers were conducted. Blastocyst morphological quality was defined according to Gardner's criteria. The AI-based software CHLOE™ (Fairtility) automatically registered timings from time of starting blastulation (tSB) to biopsy (t-biopsy, i.e. blastocyst full-expansion) as hours-post-insemination (hpi), embryo area (including zona pellucida in µm2), and spontaneous blastocyst collapses. One senior embryologist manually annotated Cyt-S presence, quantity, timings, and type (thick cell-to-cell connections and/or threads). All significant associations were confirmed through regression analyses. All couples', cycles', and embryos' main features were also tested for associations with Cyt-S presence, quantity, and dynamics., Main Results and the Role of Chance: About 94.3% of the patients (N = 117/124) had ≥1 embryo with Cyt-S. Out of a total of 370 blastocysts, 55 degenerated between blastulation and full-expansion (N = 55/370, 14.9%). The degeneration rate among embryos with ≥1 Cyt-S was 10.8% (N = 33/304), significantly lower than that of embryos without Cyt-S (33.3%, N = 22/66, P < 0.01). Of the remaining 315 viable blastocysts analyzed, 86% (N = 271/315; P < 0.01) had ≥1 Cyt-S, on average 3.5 ± 2.1 per embryo ranging 1-13. The first Cyt-S per viable embryo appeared at 115.3 ± 12.5 hpi (85.7-157.7), corresponding to 10.5 ± 5.8 h (0.5-31) after tSB. Overall, we analyzed 937 Cyt-S showing a mean duration of 3.8 ± 2.7 h (0.3-20.9). Cyt-S were mostly threads (N = 508/937, 54.2%) or thick cell-to-cell connections becoming threads (N = 382/937, 40.8%) than thick bridges (N = 47/937, 5.0%). The presence and quantity of Cyt-S were significantly associated with developmentally faster (on average 6-12 h faster) and more expanded (on average 2700 µm2-larger blastocyst's area at t-biopsy) embryos. Also, the presence and duration of Cyt-S were associated with better morphology. Lastly, while euploidy rates were comparable between blastocysts with and without Cyt-S, all euploid blastocysts transferred from the latter group failed to implant (N = 10)., Limitations, Reasons for Caution: Cyt-S presence and dynamics were assessed manually on seven focal planes from video frames recorded every 15 min. The patients included were mostly of advanced maternal age. Only associations could be reported, but no causations/consequences. Lastly, larger datasets are required to better assess Cyt-S associations with clinical outcomes., Wider Implications of the Findings: Cyt-S are common during human blastocyst expansion, suggesting their physiological implication in this process. Their presence, quantity and dynamics mirror embryo viability, and morphological quality, yet their role is still unknown. Future basic science studies are encouraged to finally describe Cyt-S molecular nature and biophysical properties, and Artificial Intelligence tools should aid these studies by incorporating Cyt-S assessment., Study Funding/competing Interest(s): None., Trial Registration Number: N/A., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Study Question: Could an artificial intelligence (AI) algorithm predict fetal heartbeat from images of vitrified-warmed embryos?, Summary Answer: Applying AI to vitrified-warmed blastocysts may help predict which ones will result in implantation failure early enough to thaw another., What Is Known Already: The application of AI in the field of embryology has already proven effective in assessing the quality of fresh embryos. Therefore, it could also be useful to predict the outcome of frozen embryo transfers, some of which do not recover their pre-vitrification volume, collapse, or degenerate after warming without prior evidence., Study Design, Size, Duration: This retrospective cohort study included 1109 embryos from 792 patients. Of these, 568 were vitrified blastocysts cultured in time-lapse systems in the period between warming and transfer, from February 2022 to July 2023. The other 541 were fresh-transferred blastocysts serving as controls., Participants/materials, Setting, Methods: Four types of time-lapse images were collected: last frame of development of 541 fresh-transferred blastocysts (FTi), last frame of 467 blastocysts to be vitrified (PVi), first frame post-warming of 568 vitrified embryos (PW1i), and last frame post-warming of 568 vitrified embryos (PW2i). After providing the images to the AI algorithm, the returned scores were compared with the conventional morphology and fetal heartbeat outcomes of the transferred embryos (n = 1098). The contribution of the AI score to fetal heartbeat was analyzed by multivariate logistic regression in different patient populations, and the predictive ability of the models was measured by calculating the area under the receiver-operating characteristic curve (ROC-AUC)., Main Results and the Role of Chance: Fetal heartbeat rate was related to AI score from FTi (P < 0.001), PW1i (P < 0.05), and PW2i (P < 0.001) images. The contribution of AI score to fetal heartbeat was significant in the oocyte donation program for PW2i (odds ratio (OR)=1.13; 95% CI [1.04-1.23]; P < 0.01), and in cycles with autologous oocytes for PW1i (OR = 1.18; 95% CI [1.01-1.38]; P < 0.05) and PW2i (OR = 1.15; 95% CI [1.02-1.30]; P < 0.05), but was not significantly associated with fetal heartbeat in genetically analyzed embryos. AI scores from the four groups of images varied according to morphological category (P < 0.001). The PW2i score differed in collapsed, non-re-expanded, or non-viable embryos compared to normal/viable embryos (P < 0.001). The predictability of the AI score was optimal at a post-warming incubation time of 3.3-4 h (AUC = 0.673)., Limitations, Reasons for Caution: The algorithm was designed to assess fresh embryos prior to vitrification, but not thawed ones, so this study should be considered an external trial., Wider Implications of the Findings: The application of predictive software in the management of frozen embryo transfers may be a useful tool for embryologists, reducing the cancellation rates of cycles in which the blastocyst does not recover from vitrification. Specifically, the algorithm tested in this research could be used to evaluate thawed embryos both in clinics with time-lapse systems and in those with conventional incubators only, as just a single photo is required., Study Funding/competing Interests: This study was supported by the Regional Ministry of Innovation, Universities, Science and Digital Society of the Valencian Community (CIACIF/2021/019) and by Instituto de Salud Carlos III (PI21/00283), and co-funded by European Union (ERDF, 'A way to make Europe'). M.M. received personal fees in the last 5 years as honoraria for lectures from Merck, Vitrolife, MSD, Ferring, AIVF, Theramex, Gedeon Richter, Genea Biomedx, and Life Whisperer. There are no other competing interests., Trial Registration Number: N/A., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Following blastocyst hatching, ungulate embryos undergo a prolonged preimplantation period termed conceptus elongation. Conceptus elongation constitutes a highly susceptible period for embryonic loss, and the embryonic requirements during this process are largely unknown, but multiple lipid compounds have been identified in the fluid nourishing the elongating conceptuses. Peroxisome proliferator-activated receptors mediate the signaling actions of prostaglandins and other lipids, and, between them, PPARG has been pointed out to play a relevant role in conceptus elongation by a functional study that depleted PPARG in both uterus and conceptus. The objective of this study has been to determine if embryonic PPARG is required for bovine embryo development. To that aim, we have generated bovine PPARG knock-out embryos in vitro using two independent gene ablation strategies and assessed their developmental ability. In vitro development to Day 8 blastocyst was unaffected by PPARG ablation, as total, inner cell mass, and trophectoderm cell numbers were similar between wild-type and knock-out D8 embryos. In vitro post-hatching development to D12 was also comparable between different genotypes, as embryo diameter, epiblast cell number, embryonic disk formation, and hypoblast migration rates were unaffected by the ablation. The development of tubular stages equivalent to E14 was assessed in vivo, following a heterologous embryo transfer experiment, observing that the development of extra-embryonic membranes and of the embryonic disk was not altered by PPARG ablation. In conclusion, PPARG ablation did not impaired bovine embryo development up to tubular stages., (© The Author(s) 2024. Published by Oxford University Press behalf of Society for the Study of Reproduction.)

Study Question: Does the morphological quality on Day 3 influence the pregnancy outcomes of euploid blastocysts?, Summary Answer: The morphological quality on Day 3 affects the clinical pregnancy rate (CPR) and live birth rate (LBR) of low-quality euploid blastocysts., What Is Known Already: The morphological grading of Day 3 embryos affects the pregnancy outcome of cleavage-stage embryos and is an excellent indicator to predict embryo development potential. However, it is still unclear whether morphological quality on Day 3 is associated with pregnancy outcomes of the euploid blastocyst., Study Design, Size, Duration: This retrospective cohort study comprised 1275 patients who received single euploid blastocyst transfer between January 2016 and August 2021 at a tertiary teaching hospital., Participants/materials, Setting, Methods: Patients were grouped into two groups according to the morphological grading on Day 3 of transferred blastocysts: high-quality (HQ, including Grades I and II) Day 3 embryos and low-quality (LQ, Grade III) Day 3 embryos. The primary outcomes were CPR and LBR. Interactions of development days (Day 5 and Day 6) and morphological quality (high- and low-quality) of blastocysts with morphological quality of Day 3 embryos on pregnancy outcomes were tested in the stratified analysis and logistic regression models. The multivariate logistic regression analysis was conducted to investigate the independent effect of the morphological quality of Day 3 embryos on pregnancy outcomes after adjusting for potentially confounding factors., Main Results and the Role of Chance: The CPR and LBR of the HQ Day 3 embryos group were statistically higher than those of the LQ Day 3 embryos group (CPR: 59.73% versus 49.70%, respectively, P = 0.015; LBR: 49.73% versus 41.21%, respectively, P = 0.041). The development days of blastocysts did not exhibit a multiplicative interaction with the morphological quality of Day 3 embryos on the CPR (P for interaction = 0.648) and LBR (P for interaction = 0.925). The morphological quality of blastocysts exhibits a multiplicative interaction with the morphological quality of Day 3 embryos on the CPR (P for interaction = 0.020) and LBR (P for interaction = 0.012). After adjusting for potential confounders, the HQ Day 3 embryo group was positively associated with the CPR (adjusted odds ratio (aOR): 2.10, 95% CI: 1.31-3.36, P = 0.002) and LBR (aOR: 1.97, 95% CI: 1.20-3.25, P = 0.008) of LQ blastocysts. However, the morphological quality on Day 3 was not significantly associated with the CPR (aOR: 0.95, 95% CI: 0.58-1.55, P = 0.835) and LBR (aOR: 0.86, 95% CI: 0.53-1.40, P = 0.550) of HQ blastocysts., Limitations, Reasons for Caution: Selection and confounding bias introduced by the retrospective design cannot be completely eliminated in this study, although multivariable logistic analysis was conducted to adjust for potential confounders. Also, some subgroups had small sample sizes, which may reduce statistical power. Moreover, participants in our study only received single euploid blastocyst transfer, and whether the results could apply to blastocysts with unknown ploidy status is unclear., Wider Implications of the Findings: This study found that the morphological quality on Day 3 was significantly associated with the CPR and LBR of LQ blastocysts; Therefore, when only LQ euploid blastocysts are available for transfer, blastocysts derived from HQ Day 3 embryos are recommended., Study Funding/competing Interest(s): No external funding was obtained. The authors have no conflicts of interest to declare., Trial Registration Number: N/A., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Study Question: Do embryos displaying abnormal cleavage (ABNCL) up to Day 3 have compromised live birth rates and neonatal outcomes if full blastulation has been achieved prior to transfer?, Summary Answer: ABNCL is associated with reduced full blastulation rates but does not impact live birth rates and neonatal outcomes once full blastulation has been achieved., What Is Known Already?: It is widely accepted that ABNCL is associated with reduced implantation rates of embryos when transferred at the cleavage stage. However, evidence is scarce in the literature reporting birth outcomes from blastocysts arising from ABNCL embryos, likely because they are ranked low priority for transfer., Study Design, Size, Duration: This retrospective cohort study included 1562 consecutive autologous in vitro fertilization cycles (maternal age 35.1 ± 4.7 years) performed at Fertility North, Australia between January 2017 and June 2022. Fresh transfers were performed on Day 3 or 5, with remaining embryos cultured up to Day 6 before vitrification. A total of 6019 embryos were subject to blastocyst culture, and a subset of 664 resulting frozen blastocysts was included for live birth and neonatal outcome analyses following single transfers., Participants/materials, Setting, Methods: ABNCL events were annotated from the first mitotic division up to Day 3, including direct cleavage (DC), reverse cleavage (RC) and <6 intercellular contact points at the 4-cell stage (<6ICCP). For DC and RC in combination, the ratios of affected blastomeres over the total number of all blastomeres up to Day 3 were also recorded. All pregnancies were followed up until birth with gestational age, birthweight, and sex of the baby being recorded., Main Results and the Role of Chance: Full blastulation rates for embryos showing DC (19.5%), RC (41.7%), <6ICCP (58.8%), and mixed (≥2) ABNCL types (26.4%) were lower than the rates for those without ABNCL (67.2%, P < 0.01 respectively). Subgroup analysis showed declining full blastulation rates with increasing ratios of combined DC/RC affected blastomeres over all blastomeres up to the 8-cell stage (66.2% when 0 affected, 47.0% when 0.25 affected, 27.4% when 0.5 affected, 14.5% when 0.75 affected, and 7.7% when all affected, P < 0.01). However, once full blastulation had been achieved, no difference was detected between DC, RC, <6ICCP, and no ABNCL blastocysts following single frozen transfers in subsequent live birth rates (25.9%, 33.0%, 36.0% versus 30.8%, P > 0.05, respectively), gestational age (38.7 ± 1.6, 38.5 ± 1.2, 38.3 ± 3.5 versus 38.5 ± 1.8 weeks, P > 0.05, respectively) and birthweight (3343.0 ± 649.1, 3378.2 ± 538.4, 3352.6 ± 841.3 versus 3313.9 ± 509.6 g, P > 0.05, respectively). Multiple regression (logistic or linear as appropriate) confirmed no differences in all of the above measures after accounting for potential confounders., Limitations, Reasons for Caution: Our study is limited by its retrospective nature, making it impossible to control every known or unknown confounder. Embryos in our dataset, being surplus after selection for fresh transfer, may not represent the general embryo population., Wider Implications of the Findings: Our findings highlight the incremental impact of ABNCL, depending on the ratio of affected blastomeres up to Day 3, on subsequent full blastulation. The reassuring live birth and neonatal outcomes of ABNCL blastocysts imply a potential self-correction mechanism among those embryos reaching the blastocyst stage, which provides valuable guidance for clinical practice and patient counseling., Study Funding/competting Interest(s): This research is supported by an Australian Government Research Training Program (RTP) Scholarship. All authors report no conflict of interest., Trial Registration Number: N/A., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.)

Study Question: What clinical and laboratory differences emerge from parallel direct comparison of embryos reaching the blastocyst stage between Days 4, 5, 6, and 7 (Days 4-7)?, Summary Answer: Increasing times to blastocyst formation are associated with a worse clinical outcome and perturbations in developmental patterns appear as early as the fertilization stage., What Is Known Already: Previous evidence indicates that later times to blastocyst development are associated with a worse clinical outcome. However, the vast majority of these data concern Day 5 and Day 6 blastocysts, while Day 4 and Day 7 blastocysts remain less thoroughly investigated. In addition, studies comparing in parallel the developmental patterns and trajectories of Day 4-7 blastocysts are lacking. This leaves unanswered the question of when and how differences among such embryos emerge. Acquisition of such knowledge would significantly contribute to understanding the relative impact of intrinsic and extrinsic causes of embryo developmental kinetics and competence., Study Design, Size, Duration: This retrospective study involved time-lapse technology (TLT) monitoring of Day 4 (N = 70), Day 5 (N = 6147), Day 6 (N = 3243), and Day 7 (N = 149) blastocysts generated in 9450 ICSI cycles. Oocyte retrievals were carried out after clomiphene citrate-based minimal ovarian stimulation, between January 2020 and April 2021., Participants/materials, Setting, Methods: Couples included in the study presented with different diagnoses, mainly male factor and unexplained infertility. Cases involving cryopreserved gametes or surgically retrieved sperm were excluded. Microinjected oocytes were assessed by a combined TLT-culture system. Day 4-7 blastocyst groups were compared in terms of morphokinetics (pronuclear dynamics, cleavage patterns and timings, and embryo quality) and clinical outcome. Clinically usable blastocysts were cryopreserved and transferred in single vitrified-warmed blastocyst transfers (SVBT)., Main Results and the Role of Chance: From 19 846 microinjected oocytes, 17 144 zygotes (86.4%) were obtained. Overall, the blastocyst development rate was 56.0%. Rates of blastocysts formation on Days 4, 5, 6, and 7 were 0.7%, 64.0%, 33.8%, and 1.6%, respectively. The average expanded blastocyst development times were 98.4 ± 0.4, 112.4 ± 0.1, 131.6 ± 0.1, and 151.2 ± 0.5 h in the Day 4-7 groups, respectively. Female age was positively associated with longer times to blastocyst development. Rates of both inner cell mass (ICM) and trophectoderm (TE) morphological grade A blastocysts were negatively associated with the day of blastocyst development (P < 0.0001). The differences in development times and intervals increased progressively until blastocyst expansion (P < 0.0001 for all development times). Strikingly, such differences were already markedly evident as early as the time of pronuclear fading (tPNf) (20.6 ± 0.3, 22.5 ± 0.0, 24.0 ± 0.0, 25.5 ± 0.3; Days 4-7, respectively; P < 0.0001). Rates of cleavage anomalies (tri-/multi-chotomous mitosis or rapid cleavage) occurring at the first or second/third division cycles were also positively associated with longer times to blastocyst development. Implantation, ongoing pregnancy, and live birth rates were progressively reduced with increasing blastocyst development times (P < 0.0001), even after stratification for maternal age. When controlled for female age, male age, number of previous embryo transfer cycles, morphological grade of the ICM and TE, and progesterone supplementation, the probabilities of implantation, clinical, and ongoing pregnancy and live birth were significantly decreased in Day 6 blastocysts in comparison to Day 5 blastocysts. Follow-up data on birth length, weight, and malformations were comparable among the four blastocyst groups., Limitations, Reasons for Caution: The study is limited by its retrospective design. Having been obtained from a single centre, the data require independent validation., Wider Implications of the Findings: This study extends previous data on the relation between time of blastocyst formation and clinical outcome. It also indicates that differences in developmental times and patterns of Day 4-7 blastocysts occur as early as the fertilization stage, possibly dictated by intrinsic gamete-derived factors., Study Funding/competing Interest(s): This study was supported by the participating institutions. The authors have no conflict of interest to declare., Trial Registration Number: N/A., (© The Author(s) 2023. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Study Question: Does the type of incubator used to culture human preimplantation embryos affect development to the blastocyst stage and alter amino acid utilization of embryos in assisted reproduction?, Summary Answer: Culturing embryos in a time lapse system (TLS) was associated with a higher Day 5 blastocyst formation rate and altered amino acid utilization when measured from Day 3 to Day 5 compared to the standard benchtop incubator., What Is Known Already: Culture environment is known to be important for the developing preimplantation embryo. TLSs provide a stable milieu allowing embryos to be monitored in situ, whereas embryos cultured in standard benchtop incubators experience environmental fluctuations when removed for morphological assessment., Study Design, Size, Duration: A prospective clinical trial randomizing 585 sibling embryos to either the TLS (289 embryos) or the standard benchtop incubator (296 embryos) over a 23-month period in a UK University Hospital Fertility Clinic., Participants/materials, Setting, Methods: Participants were aged 42 years or under, had an antral follicle count of ≥12 and ≥6 2 pronucleate zygotes. Zygotes were cultured individually in 25 µl of medium. Randomized embryos were graded and selected for transfer or cryopreservation on Day 5. For those embryos produced by women who underwent stimulation with recombinant FSH injections and were triggered with hCG, spent medium was collected on Day 5 for amino acid analysis by high pressure liquid chromatography. Clinical pregnancy was defined as the presence of a foetal heart beat on ultrasound scan at 7 weeks., Main Results and the Role of Chance: Overall, blastocyst formation rate on Day 5 was significantly higher in embryos cultured in the TLS (55%) compared to the standard incubator (45%; P = 0.013). Similarly, there was an increase in the number of blastocysts suitable for cryopreservation in the TLS (31%) compared to the standard incubator (23%; P = 0.032). There was a significant difference in the utilization of 12 amino acids by blastocysts cultured from Day 3 to Day 5 in the TLS compared to the standard incubator. Embryos cultured in the TLS displayed an increased total amino acid utilization (P < 0.001) and reduced amino acid production (P < 0.001) compared to those in the standard incubator. Irrespective of incubator used, embryos fertilized by ICSI depleted significantly more amino acids from the medium compared to those fertilized by conventional IVF. There was no difference in the mean score of blastocysts transferred, or the clinical pregnancy rate after transfer of embryos from either of the incubators., Limitations, Reasons for Caution: The study was not powered to discern significant effects on clinical outcomes., Wider Implications of the Findings: The metabolism and development of preimplantation embryos is impacted by the type of incubator used for culture. Further research is required to investigate the long-term implications of these findings., Study Funding/competing Interest(s): NIHR Southampton Biomedical Research Centre Commercial and Enterprise Incubator Fund funded this study. The TLS was provided on loan for the study by Vitrolife. The authors declare no conflict of interests., Trial Registration Number: ISRCTN73037149., Trial Registration Date: 12 January 2012., Date of First Patient’s Enrolment: 21 January 2012., (© Crown copyright 2022.)

Fertilization underpins the vital transition from gametic meiosis to embryonic mitosis. For decades, in human IVF, microscopic observation at a single time point has limited our appreciation of the morphokinetic complexity of this process. More recently, the introduction of time lapse technology-also enhanced by combination with artificial intelligence-has revealed the finest morphokinetic details of the beginning of human development. Overall, a picture has finally emerged in which the precise timing, morphology and geometry of several fertilization events offer clues to predict the fate of the embryo-a key aspect of assisted reproduction. In this scenario, correct unfolding of intra- and interpronuclear rearrangements emerge as a crucial factor to create a platform able to preserve genetic and cellular integrity at the first mitotic cleavage., (© The Author(s) 2022. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Study Question: Is it possible to develop a comprehensive pipeline for all-in-one preimplantation genetic testing (PGT), also suitable for parents-only haplotyping and, for the first time, third-party reproduction?, Summary Answer: Optimized reduced representation sequencing (RRS) by GENType, along with a novel analysis platform (Hopla), enables cheap, accurate and comprehensive PGT of blastocysts, even without the inclusion of additional family members or both biological parents for genome-wide embryo haplotyping., What Is Known Already: Several haplotyping strategies have proven to be effective for comprehensive PGT. However, these methods often rely on microarray technology, whole-genome sequencing (WGS) or a combination of strategies, hindering sample throughput and cost-efficiency. Moreover, existing tools (including other RRS-based strategies) require both prospective biological parents for embryo haplotyping, impeding application in a third-party reproduction setting., Study Design, Size, Duration: This study included a total of 257 samples. Preliminary technical validation was performed on 81 samples handpicked from commercially available cell lines. Subsequently, a clinical validation was performed on a total of 72 trophectoderm biopsies from 24 blastocysts, tested for a monogenic disorder (PGT-M) (n = 15) and/or (sub)chromosomal aneuploidy (PGT-SR/PGT-A) (n = 9). Once validated, our pipeline was implemented in a diagnostic setting on 104 blastocysts for comprehensive PGT., Participants/materials, Setting, Methods: Samples were whole-genome amplified (WGA) and processed by GENType. Quality metrics, genome-wide haplotypes, b-allele frequencies (BAFs) and copy number profiles were generated by Hopla. PGT-M results were deduced from relative haplotypes, while PGT-SR/PGT-A results were inferred from read-count analysis and BAF profiles. Parents-only haplotyping was assessed by excluding additional family members from analysis and using an independently diagnosed embryo as phasing reference. Suitability for third-party reproduction through single-parent haplotyping was evaluated by excluding one biological parent from analysis. Results were validated against reference PGT methods., Main Results and the Role of Chance: Genome-wide haplotypes of single cells were highly accurate (mean > 99%) compared to bulk DNA. Unbalanced chromosomal abnormalities (>5 Mb) were detected by GENType. For both PGT-M as well as PGT-SR/PGT-A, our technology demonstrated 100% concordance with reference PGT methods for diverse WGA methods. Equally, for parents-only haplotyping and single-parent haplotyping (of autosomal dominant disorders and X-linked disorders), PGT-M results were fully concordant. Furthermore, the origin of trisomies in PGT-M embryos was correctly deciphered by Hopla., Limitations, Reasons for Caution: Intrinsic to linkage-analysis strategies, de novo single-nucleotide variants remain elusive. Moreover, parents-only haplotyping is not a stand-alone approach and requires prior diagnosis of at least one reference embryo by an independent technology (i.e. direct mutation analysis) for haplotype phasing. Using a haplotyping approach, the presence of a homologous recombination site across the chromosome is biologically required to distinguish meiotic II errors from mitotic errors during trisomy origin investigation., Wider Implications of the Findings: We offer a generic, fully automatable and accurate pipeline for PGT-M, PGT-A and PGT-SR as well as trisomy origin investigation without the need for personalized assays, microarray technology or WGS. The unique ability to perform single-parent assisted haplotyping of embryos paves the way for cost-effective PGT in a third-party reproduction setting., Study Funding/competing Interest(s): L.D.W. is supported by the Research Foundation Flanders (FWO; 1S74619N). L.R. and B.M. are funded by Ghent University and M.B., S.S., K.T., F.V.M. and A.D. are supported by Ghent University Hospital. Research in the N.C. lab was funded by Ghent University, VIB and Kom op Tegen Kanker. A.D.K and N.C. are co-inventors of patent WO2017162754A1. The other authors have no conflicts of interest., Trial Registration Number: N/A., (© The Author(s) 2022. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Study Question: Can vascular endothelial growth factor (VEGF)-loaded silica supraparticles (V-SPs) be used as a novel mode of delivering VEGF to the developing preimplantation embryo in vitro?, Summary Answer: Supplementation of embryo culture media with V-SPs promoted embryonic development in a manner equivalent to media supplemented with free VEGF., What Is Known Already: VEGF is a maternally derived growth factor that promotes preimplantation embryonic development in vitro. However, its use in clinical media has limitations due to its low stability in solution., Study Design, Size, Duration: This study was a laboratory-based analysis utilising a mouse model. V-SPs were prepared in vitro and supplemented to embryonic culture media. The bioactivity of V-SPs was determined by analysis of blastocyst developmental outcomes (blastocyst development rate and total cell number)., Participants/materials, Setting, Methods: SPs were loaded with fluorescently labelled VEGF and release kinetics were characterised. Bioactivity of unlabelled VEGF released from V-SPs was determined by analysis of embryo developmental outcomes (blastocyst developmental rate and total cell number) following individual mouse embryo culture in 20 µl of G1/G2 media at 5% oxygen, supplemented with 10 ng/ml recombinant mouse VEGF in solution or with V-SPs. The bioactivity of freeze-dried V-SPs was also assessed to determine the efficacy of cryostorage., Main Results and the Role of Chance: VEGF release kinetics were characterised by an initial burst of VEGF from loaded spheres followed by a consistent lower level of VEGF release over 48 h. VEGF released from V-SPs resulted in significant increases in total blastocyst cell number relative to the control (P < 0.001), replicating the effects of medium freely supplemented with fresh VEGF (P < 0.001). Similarly, freeze dried V-SPs exerted comparable effects on embryonic development (P < 0.05)., Large Scale Data: N/A., Limitations, Reasons for Caution: In this proof of principle study, the effects of V-SPs on embryonic development were only analysed in a mouse model., Wider Implications of the Findings: These findings suggest that SPs represent a novel method by which a targeted dose of therapeutic agents (e.g. bioactive VEGF) can be delivered to the developing in vitro embryo to promote embryonic development, an approach that negates the breakdown of VEGF associated with storage in solution. As such, V-SPs may be an alternative and effective method of delivering bioactive VEGF to the developing in vitro embryo; however, the potential use of V-SPs in clinical IVF requires further investigation., Study Funding/competing Interest(s): This work was funded by the University of Melbourne. The authors have no conflict of interest to declare., (© The Author(s) 2021. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Study Question: Do embryos with different developmental competence exhibit different DNA methylation profiles at the blastocyst stage?, Summary Answer: We established genome-wide DNA methylome analysis for embryo trophectoderm (TE) biopsy samples and our findings demonstrated correlation of methylation profile of trophectoderm with euploidy status and with maternal age, indicating that genome-wide methylation level might be negatively correlated with embryo quality., What Is Known Already: DNA methylation is a fundamental epigenetic regulatory mechanism that affects differentiation of cells into their future lineages during pre-implantation embryo development. Currently there is no established approach available to assess the epigenetic status of the human preimplantation embryo during routine IVF treatment., Study Design, Size, Duration: In total, we collected trophectoderm biopsy samples from 30 randomly selected human blastocysts and conducted whole-genome bisulfite sequencing (WGBS) to evaluate their DNA methylation profile. Nested linear models were used to assess association between DNA methylation level and ploidy status (aneuploidy [n = 20] vs. euploidy [n = 10]), maternal age (29.4-42.5 years old), and time of blastulation (day 5 [n = 16] vs. day 6 [n = 14]), using embryo identity as a covariate., Participants/materials, Setting, Methods: TE biopsy samples were obtained and submitted to bisulfite conversion. For WGBS, whole-genome sequencing libraries were then generated from the converted genome. An average of 75 million reads were obtained for each sample, and about 63% of the reads aligned to human reference. An average of 40 million reads used for the final analysis after the unconverted reads were filtered out., Main Results and the Role of Chance: We revealed an increase of genome-wide DNA methylation level in aneuploid embryo TE biopsies compared to euploid embryos (25.4% ± 3.2% vs. 24.7% ± 3.2%, P < 0.005). We also found genome-wide DNA methylation level to be increased with the maternal age (P < 0.005). On a chromosomal scale, we found monosomic embryos have lower methylation levels on the involved chromosome while no drastic change was observed for the involved chromosome in trisomies. Additionally, we revealed that WGBS data precisely revealed the chromosome copy number variance., Limitations, Reasons for Caution: Though our results demonstrated a negative correlation of genome-wide methylation level and embryo quality, further WGBS analysis on a greater number of embryos and specific investigation of its correlation with implantation and live birth are needed before any practical use of this approach for evaluation of embryo competence., Wider Implications of the Findings: This study revealed a change in genome-wide DNA methylation profile among embryos with different developmental potentials, reinforcing the critical role of DNA methylation in early development., Study Funding/competing Interest(s): No external funding was received for this study. Intramural funding was provided by the Foundation for Embryonic Competence (FEC). E.S. is a consultant for and receives research funding from the Foundation for Embryonic Competence; he is also co-founder and a shareholder of ACIS LLC and coholds patent US2019/055906 issued for utilizing electrical resistance measurement for assessing cell viability and cell membrane piercing., Trial Registration Number: N/A., (© The Author(s) 2021. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Study Question: What factors associated with embryo culture techniques contribute to the rate of medium osmolality change over time in an embryo culture incubator without added humidity?, Summary Answer: The surface area-to-volume ratio of culture medium (surface area of the medium exposed to an oil overlay), as well as the density and height of the overlaying oil, all interact in a quantitative way to affect the osmolality rise over time., What Is Known Already: Factors such as medium volume, different oil types, and associated properties, individually, can affect osmolality change during non-humidified incubation., Study Design, Size, Duration: Several experimental designs were used, including simple single-factor completely randomized designs, as well as a multi-factor response surface design. Randomization was performed at one or more levels for each experiment. Osmolality measurements were performed over 7 days, with up to 8 independent osmolality measurements performed per treatment group over that time. For the multi-factor study, 107 independent combinations of factor levels were assessed to develop the mathematical model., Participants/materials, Setting, Methods: This study was conducted in a research laboratory setting. Commercially available embryo culture medium and oil was used. A MINC incubator without water for humidification was used for the incubation. Osmolality was measured with a vapor pressure osmometer after calibration. Viscometry and density were conducted using a rheometer, and volumetric flasks with an analytical balance, respectively. Data analyses were conducted with several commercially available software programs., Main Results and the Role of Chance: Preliminary experiments showed that the surface area-to-volume ratio of the culture medium, oil density, and oil thickness above the medium all contributed significantly (P < 0.05) to the rise in osmolality. A multi-factor experiment showed that a combination of these variables, in the form of a truncated cubic polynomial, was able to predict the rise in osmolality, with these three variables interacting in the model (P < 0.05). Repeatability, as measured by the response of identical treatments performed independently, was high, with osmolality values being ± 2 of the average in most instances. In the final mathematical model, the terms of the equation were significant predictors of the outcome, with all P-values being significant, and only one P-value > 0.0001., Limitations, Reasons for Caution: Although the range of values for the variables were selected to encompass values that are expected to be encountered in usual embryo culture conditions, variables outside of the range used may not result in accurate model predictions. Although the use of a single incubator type and medium type is not expected to affect the conclusions, that remains an uncertainty., Wider Implications of the Findings: Using this predictive model will help to determine if one should be cautious in using a specific system and will provide guidance on how a system may be modified to provide improved stability during embryo culture., Study Funding/competing Interest(s): This study was funded by Cook Medical. The author is a Team Lead and Senior Scientist at Cook Medical. The author has no other conflicts of interest to declare., Trial Registration Number: N/A., (© The Author(s) 2021. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Study Question: Is there an association between patients' reproductive history and the mean euploidy rates per biopsied blastocysts (m-ER) or the live birth rates (LBRs) per first single vitrified-warmed euploid blastocyst transfers?, Summary Answer: Patients' reproductive history (as annotated during counselling) showed no association with the m-ER, but a lower LBR was reported after euploid blastocyst transfer in women with a history of repeated implantation failure (RIF)., What Is Known Already: Several studies have investigated the association between the m-ER and (i) patients' basal characteristics, (ii) ovarian stimulation strategy and dosage, (iii) culture media and conditions, and (iv) embryo morphology and day of full blastocyst development. Conversely, the expected m-ER due to women's reproductive history (previous live births (LBs), miscarriages, failed IVF cycles and transfers, and lack of euploid blastocysts among prior cohorts of biopsied embryos) still needs investigations. Yet, this information is critical to counsel new patients about a first cycle with preimplantation genetic testing for aneuploidy (PGT-A), but even more so after former adverse outcomes to prevent treatment drop-out., Study Design, Size, Duration: This observational study included all patients undergoing a comprehensive chromosome testing (CCT)-based PGT-A cycle with at least one biopsied blastocyst in the period April 2013-December 2019 at a private IVF clinic (n = 2676 patients undergoing 2676 treatments and producing and 8151 blastocysts). m-ER were investigated according to women's reproductive history of LBs: no/≥1, miscarriages: no/1/>1; failed IVF cycles: no/1/2/>2, and implantation failures after previous transfers: no/1/2/>2. Among the 2676 patients included in this study, 440 (16%) had already undergone PGT-A before the study period; the data from these patients were further clustered according to the presence or absence of euploid embryo(s) in their previous cohort of biopsied blastocysts. The clinical outcomes per first single vitrified-warmed euploid blastocyst transfers (n =1580) were investigated according to the number of patients' previous miscarriages and implantation failures., Participants/materials, Setting, Methods: The procedures involved in this study included ICSI, blastocyst culture, trophectoderm biopsy without hatching in Day 3, CCT-based PGT-A without reporting segmental and/or putative mitotic (or mosaic) aneuploidies and single vitrified-warmed euploid blastocyst transfer. For statistical analysis, Mann-Whitney U or Kruskal-Wallis tests, as well as linear regressions and generalised linear models among ranges of maternal age at oocyte retrieval were performed to identify significant differences for continuous variables. Fisher's exact tests and multivariate logistic regression analyses were instead used for categorical variables., Main Results and the Role of Chance: Maternal age at oocyte retrieval was the only variable significantly associated with the m-ER. We defined five clusters (<35 years: 66 ± 31%; 35-37 years: 58 ± 33%; 38-40 years: 43 ± 35%; 40-42 years: 28 ± 34%; and >42 years: 17 ± 31%) and all analyses were conducted among them. The m-ER did not show any association with the number of previous LBs, miscarriages, failed IVF cycles or implantation failures. Among patients who had already undergone PGT-A before the study period, the m-ER did not associate with the absence (or presence) of euploid blastocysts in their former cohort of biopsied embryos. Regarding clinical outcomes of the first single vitrified-warmed euploid blastocyst transfer, the implantation rate was 51%, the miscarriage rate was 14% and the LBR was 44%. This LBR was independent of the number of previous miscarriages, but showed a decreasing trend depending on the number of previous implantation failures, reaching statistical significance when comparing patients with >2 failures and patients with no prior failure (36% versus 47%, P < 0.01; multivariate-OR adjusted for embryo quality and day of full blastocyst development: 0.64, 95% CI 0.48-0.86, P < 0.01). No such differences were shown for previous miscarriage rates., Limitations, Reasons for Caution: The sample size for treatments following a former completed PGT-A cycle should be larger in future studies. The data should be confirmed from a multicentre perspective. The analysis should be performed also in non-PGT cycles and/or including patients who did not produce blastocysts, in order to investigate a putative association between women's reproductive history with outcomes other than euploidy and LBRs., Wider Implications of the Findings: These data are critical to counsel infertile couples before, during and after a PGT-A cycle, especially to prevent treatment discontinuation due to previous adverse reproductive events. Beyond the 'maternal age effect', the causes of idiopathic recurrent pregnancy loss (RPL) and RIF are likely to be endometrial receptivity and selectivity issues; transferring euploid blastocysts might reduce the risk of a further miscarriage, but more information beyond euploidy are required to improve the prognosis in case of RIF., Study Funding/competing Interest(s): No funding was received and there are no competing interests., Trial Registration Number: N/A., (© The Author(s) 2021. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Study Question: Do perturbations of embryo morphogenesis at compaction affect blastocyst development and clinical outcomes in assisted reproduction cycles?, Summary Answer: Cell exclusion and extrusion, i.e. cell disposal occurring respectively before or during morula compaction, affect blastocyst yield and quality, as well as rates of pregnancy and live birth., What Is Known Already: Despite its pivotal role in morphogenesis for blastocyst organisation and cell fate determination, compaction at the morula stage has received little attention in clinical embryology. Time lapse technology (TLT) allows detailed morphokinetic analysis of this developmental stage. However, even in the vast majority of previous TLT studies, compaction was investigated without a specific focus. Recently, we reported that compaction may be affected by two clearly-distinct patterns of cell disposal, exclusion and extrusion, occurring prior to and during compaction, respectively. However, the crucial question of the specific relevance of partial compaction for embryo development and competence in ART has remained unanswered until now., Study Design, Size, Duration: This study involved the assessment of laboratory and clinical outcomes of 2,059 morula stage embryos associated with 1,117 ICSI patients, who were treated with minimal stimulation and single vitrified-warmed blastocyst transfer (SVBT) from April 2017 to March 2018. Patterns of morula compaction were assessed and analyzed in relation to embryonic and clinical outcomes., Participants/materials, Setting, Methods: Following ICSI, time-lapse videos were analysed to annotate morphokinetic parameters relevant to both pre- and post-compaction stages. According to their morphokinetic history, morulae were classified as: (I) fully compacted morulae (FCM); (II) partially compacted morulae (PCM), showing cells (a) excluded from the compaction process from the outset (Exc-PCM), (b) extruded from an already compacted morula (Ext-PCM), or (c) showing non-compacted cells arisen from both patterns (Exc/Ext-PCM). The number of excluded/extruded cells was also annotated. Possible correlations of compaction patterns with 13 morphokinetic parameters, abnormal cleavage, blastocyst yield and morphological grade, clinical and ongoing pregnancy rates, and live birth rate were evaluated. Other factors, such as patient and cycle characteristics, possibly associated with compaction patterns and their outcomes, were investigated., Main Results and the Role of Chance: Full compaction was observed in 39.0% of all embryos. However, partially compacted morulae (PCM) showing excluded (Exc-PCM), extruded (Ext-PCM) cells, or indeed both phenotypes (Exc/Ext-PCM) were frequently detected (24.8%, 16.6%, and 19.6%, respectively) and collectively (61%) exceeded fully compacted morulae. Blastomere exclusion or extrusion affected one or several cells, in different proportions. In comparison to FCM, the developmental pace of the three PCM groups, observed at 13 developmental stages starting from pronuclear fading, was progressively slower (P < 0.0001). Developmental delay at post-compaction stages was more pronounced in the group showing both patterns of partial compaction. Blastomere exclusion and/or extrusion had a large negative impact on blastocyst development. In particular, rates of blastocyst formation and cryopreservation were very low in the Ext-PCM and Exc/Ext-PCM groups (P < 0.0001). Rates of blastocysts with ICM or TE of highest quality (Grade A) were severely affected in all PCM groups (P < 0.0001). In 1,083 SVBTs, blastocysts derived from all PCM groups produced much lower clinical pregnancy, ongoing pregnancy, and live birth rates (P < 0.0001). All three patterns of partial compaction emerged as factors independently associated with live birth rate, even after multivariate logistic regression analysis including maternal/paternal age, female BMI, and number of previous embryo transfers as possible confounding factors., Limitations, Reasons for Caution: The retrospective design of the study represents a general limitation., Wider Implications of the Findings: This large-scale study represents a further important demonstration of embryo plasticity and above all indicates new robust morphokinetic parameters for improved algorithms of embryo selection., Study Funding/competing Interest(s): This study was exclusively supported by the participating institutions. The authors have no conflicts of interest to declare., Trial Registration Number: NA., (© The Author(s) 2021. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

During the peri-implantation period of pregnancy in sheep, there is an initial period of loose apposition of the elongating conceptuses (embryos and associated placental membranes) to the endometrial luminal epithelium (LE) that is followed by adhesion of the conceptus trophectoderm to the endometrial LE for implantation. Integrins and maternal extracellular matrix (ECM) molecules are major contributors to stable adhesion at implantation, and the β3 integrin subunit (ITGB3) is implicated in the adhesion cascade for implantation in several species including the sheep. We blocked mRNA translation for trophectoderm-expressed ITGB3 by infusing morpholino antisense oligonucleotides into the uterine lumen of pregnant ewes on Day 9 to assess effects on conceptus elongation, and on Day 16 to assess effects on early placental development in sheep. Results indicate that sheep conceptuses elongate and implant to the uterine wall in the absence of ITGB3 expression by the conceptuses; however, loss of ITGB3 in conceptuses decreased the growth of embryos to Day 24 of gestation, and decreased expression of secreted phosphoprotein 1 (SPP1) and nitric oxide synthase 3 (NOS3). Abundant SPP1 was localized around the blood vessels in the placental allantoic membrane in normal sheep pregnancies. We hypothesize that NOS3 and SPP1 positively influence the development of the vasculature within the allantois, and that decreased expression of NOS3 and SPP1, in response to knockdown of ITGB3 in conceptuses, alters development of the vasculature in the allantois required to transport nutrients from the endometrium to support growth and development of the embryo., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Study Question: Which lab-related factors impact the culture system's capacity to maintain a stable osmolality during human embryo culture?, Summary Answer: Incubator humidity, the volume of mineral oil, the type of culture media and the design of time-lapse dishes have been identified as important parameters that can cause an impact on media evaporation and consequently osmolality during culture., What Is Known Already: Culture medium is a critical component in human embryo culture. Minimizing its evaporation during culture is an adequate strategy to stabilize osmolality and, as a result, improving culture conditions and clinical outcomes., Study Design, Size, Duration: The studied variables included media composition and supplementation; volume of mineral oil; incubator humidification; and the type of dish and incubator used. Additionally, six time-lapse dish models were compared in their ability to prevent evaporation., Participants/materials, Setting, Methods: Dishes were incubated in parallel to analyze osmolality during culture between groups: synthetic oviductal medium enriched with potassium versus human tubal fluid medium; protein versus no protein supplementation; dry versus humid atmosphere; high versus low volume of mineral oil. Additionally, media evaporation was compared between six models of time-lapse dishes with distinct designs, cultured in a joint incubator. Two of them were retested in their corresponding incubator to analyze the dish-incubator fit. Daily osmolality measurements were compared between groups. Linear regression was performed to analyze evaporation rates., Main Results and the Role of Chance: Protein supplementation did not significantly affect evaporation. Contrarily, humidity levels inside the incubators, the volume of mineral oil and the type of culture media, played an important role in osmolality stabilization. The design of time-lapse dishes and their recommended preparation protocol heavily influenced their evaporation rates, which were further altered by each incubator's characteristics. Media with initially high osmolalities had a bigger risk of reaching hypertonic levels during culture., Limitations, Reasons for Caution: While numerous, the studied variables are limited and therefore other factors could play a role in osmolality dynamics, as well. Incontrollable atmospheric factors could also result in some variation in the observed results between different centers and laboratories., Wider Implications of the Findings: Published literature has extensively described how hypertonic media may impair embryo development and negatively affect clinical outcomes; therefore, maintaining a stable osmolality during culture should be considered essential. This work is of interest both for embryologists when analyzing their culture system and methodologies, as well as manufacturers in charge of designing IVF consumables., Study Funding/competing Interest(s): This study was privately funded., Trial Registration Number: N/A., (© The Author(s) 2021. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Study Question: Do daily manipulations of preimplantation embryos with polycarbonate (PC)-made bisphenol A (BPA)-releasing strippers influence embryo development?, Summary Answer: Compared to glass strippers, PC strippers enhance the blastocyst development rate but this does not seem to be BPA-related., What Is Known Already: PC strippers have been shown to release tiny amounts (around 0.5 ng/ml BPA) of BPA in routine human IVF procedures. A chronic exposure to BPA either in vivo or in vitro during the preimplantation period can impact post-implantation and post-natal development. BPA can act rapidly by binding to membrane receptors and inducing rapid non-genomic effects., Study Design, Size, Duration: This experimental study using mouse embryos had a balanced design and blinded evaluations of the endpoints., Participants/materials, Setting, Methods: In vivo fertilized zygotes were obtained from outbred Swiss CD1 mice crossings after an ovarian stimulation. The zygotes were allocated to three daily handling conditions (HCs) and cultured until Day 4 in a single human commercial medium. Each day, the embryos were handled for 20 s either in a PC stripper (HC1) or in a glass stripper (HC2). In HC3, the embryos were pre-exposed to 0.5 ng/ml BPA before being handled for 20 s in a glass stripper. Handling operations were repeated on Days 1, 2 and 3. Embryo development was assessed blindly on Day 4. Expanded blastocysts were selected for a transcriptomic analysis using Agilent Sureprint G3 Mouse GE v2 microarrays and the retrotransposon LINE1-Orf2 expression was analysed using qRT-PCR, as a proxy for a global evaluation of the epigenetic status., Main Results and the Role of Chance: Compared to the embryos manipulated in HC2 (n = 243), those in HC1 (n = 228) developed significantly more often to the blastocyst stage (55 vs 46%; P < 0.05). It appears the effect of these PC strippers was not BPA-related because embryos pre-exposed to BPA (HC3, n = 230) showed no difference in the blastocyst rate when compared to HC2 (43 vs 46%). When analysing same-stage blastocysts, we noticed no difference in the embryo gene expression between the three HC groups., Large Scale Data: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE148868., Limitations, Reasons for Caution: Our results using a mouse model designed to mimic human conditions (outbred strain, human commercial IVF dishes and a unique commercial human embryonic culture media) are reassuring since no gene was found to be differentially expressed, including LINE-1 genes, as a proxy for a global evaluation of the epigenetic status. However, no global epigenetic analysis of the genome has been performed. Furthermore, we did not evaluate post-implantation events, although BPA exposure during peri-conception could affect foeto-placental and post-natal development., Wider Implications of the Findings: Based on the precautionary principle, several European countries banned the use of BPA in baby bottles and food packaging several years before European Agencies took an official position. The question of applying this principle to plastics in closed contact with human embryos is raised. Further studies are needed for a decision to be made., Study Funding/competing Interest(s): This study was supported by a grant from the Agence de Biomédecine (AOR 2016). The authors declare no competing interest., (© The Author(s) 2020. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Study Question: Are there phase-specific changes in the early secretory (ES) phase human tubal lavage proteome that can inform and potentially optimize IVF culture media?, Summary Answer: The human tubal lavage proteome during the ES phase relative to the menstrual phase reveals substantial differential protein abundance in pathways such as glycolysis, redox homeostasis and activation of 14-3-3 zeta-mediated signaling., What Is Known Already: The Fallopian tube is uniquely suited to the development of the preimplantation embryo as it transits the tube during the ES phase of the menstrual cycle. Euploid cleavage-stage embryo arrest may reflect incomplete recapitulation of in-vivo conditions by current media formulations., Study Design, Size, Duration: Proteome-wide analysis of distal tubal lavage specimens collected from 26 healthy women undergoing open microtubal anastomosis surgery from January 2013 to January 2018 was performed. Specimens were grouped by menstrual cycle phase in order to analyze phase-specific differences in protein abundance. For the murine embryo assay, single-cell embryos (N = 482) were collected from superovulated wild type C57BL/6 female mice and cultured in microdrops over 5 days for the assessment of blastocyst development., Participants/materials, Setting, Methods: Human tubal lavage specimens were processed for label-free mass spectrometry. Reported menstrual cycle day was confirmed by measuring serum hormones. Key protein targets in the ES phase were validated via immunoblot. The ES phase-specific increase in 14-3-3 zeta protein was confirmed via ELISA of conditioned media obtained from primary human Fallopian tube epithelial cell culture. A murine embryo assay was performed to investigate the impact of graduated concentrations of 14-3-3 zeta on the blastocyst development rate., Main Results and the Role of Chance: Comparison of the ES and menstrual phase human tubal lavage proteomes revealed 74 differentially expressed proteins with enrichment of pathways and biological processes involved in the regulation of carbohydrate metabolism, oxidative stress and cell survival. The adapter-regulator protein 14-3-3 zeta was among the most significantly increased in the ES phase. Supplementation of embryo culture media with 14-3-3 zeta at concentrations tested did not significantly improve the murine blastocyst development., Limitations, Reasons for Caution: Although select associations were recapitulated in the conditioned media from sex steroid exposed primary human tubal epithelial cells, cell culture represents an in-vitro approximation. Changes to embryo culture media, such as protein supplementation, must undergo rigorous preclinical safety testing prior to adoption for human use., Wider Implications of the Findings: This study represents the first description of the human Fallopian tube lavage proteome across the menstrual cycle, revealing a unique proteomic signature during the ES phase. Although supplementation of culture media with 14-3-3 zeta at appropriate concentrations showed no significant impact on the murine blastocyst development rate, other biologically plausible candidate proteins for individual or high throughput testing strategies are identified., Study Funding/competing Interest(s): This work was funded in part by an Army Medical Department Advanced Medical Technology Initiative grant from the United States Army Medical Research and Materiel Command's Telemedicine and Advanced Technology Research Center. There are no competing interests., Trial Registration Number: N/A., (© The Author(s) 2020. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Study Question: Do the long-term health outcomes following IVF differ depending upon the duration of embryo culture before transfer?, Summary Answer: Using a mouse model, we demonstrate that in male but not female offspring, adverse cardiovascular (CV) health was more likely with prolonged culture to the blastocyst stage, but metabolic dysfunction was more likely if embryo transfer (ET) occurred at the early cleavage stage., What Is Known Already: ART associate with increased risk of adverse CV and metabolic health in offspring, and these findings have been confirmed in animal models in the absence of parental infertility issues. It is unclear which specific ART treatments may cause these risks. There is increasing use of blastocyst, versus cleavage-stage, transfer in clinical ART which does not appear to impair perinatal health of children born, but the longer-term health implications are unknown., Study Design, Size, Duration: Five mouse groups were generated comprising: (i) natural mating (NM)-naturally mated, non-superovulated and undisturbed gestation; (ii) IV-ET-2Cell-in-vivo derived two-cell embryos collected from superovulated mothers, with immediate ET to recipients; (iii) IVF-ET-2Cell-IVF generated embryos, from oocytes from superovulated mothers, cultured to the two-cell stage before ET to recipients; (iv) IV-ET-BL-in-vivo derived blastocysts collected from superovulated mothers, with immediate ET to recipients; (v) IVF-ET-BL-IVF generated embryos, from oocytes from superovulated mothers, cultured to the blastocyst stage before ET to recipients. Both male and female offspring were analysed for growth, CV and metabolic markers of health. There were 8-13 litters generated for each group for analyses; postnatal data were analysed by multilevel random effects regression to take account of between-mother and within-mother variation and litter size., Participants/materials, Settings, Methods: C57/BL6 female mice (3-4 weeks old) were used for oocyte production; CBA males for sperm with human tubal fluid medium were used for IVF. Embryos were transferred (ET) to MF1 pseudo-pregnant recipients at the two-cell stage or cultured in synthetic oviductal medium enriched with potassium medium to the blastocyst stage before ET. Control in-vivo embryos from C57BL6 × CBA matings were collected and immediately transferred at the two-cell or blastocyst stage. Postnatal assays included growth rate up to 27 weeks; systolic blood pressure (SBP) at 9, 15 and 21 weeks; lung and serum angiotensin-converting enzyme (ACE) activity at time of cull (27 weeks); glucose tolerance test (GTT; 27 weeks); basal glucose and insulin levels (27 weeks); and lipid accumulation in liver cryosections using Oil Red O imaging (27 weeks)., Main Results and the Role of Chance: Blastocysts formed by IVF developed at a slower rate and comprised fewer cells that in-vivo generated blastocysts without culture (P < 0.05). Postnatal growth rate was increased in all four experimental treatments compared with NM group (P < 0.05). SBP, serum and lung ACE and heart/body weight were higher in IVF-ET-BL versus IVF-ET-2Cell males (P < 0.05) and higher than in other treatment groups, with SBP and lung ACE positively correlated (P < 0.05). Glucose handling (GTT AUC) was poorer and basal insulin levels were higher in IVF-ET-2Cell males than in IVF-ET-BL (P < 0.05) with the glucose:insulin ratio more negatively correlated with body weight in IVF-ET-2Cell males than in other groups. Liver/body weight and liver lipid droplet diameter and density in IVF-ET-2Cell males were higher than in IVF-ET-BL males (P < 0.05). IVF groups had poorer health characteristics than their in-vivo control groups, indicating that outcomes were not caused specifically by background techniques (superovulation, ET). No consistent health effects from duration of culture were identified in female offspring., Large Scale Data: N/A., Limitations, Reasons for Caution: Results from experimental animal models cannot be extrapolated to humans. Nevertheless, they are valuable to develop conceptual models, in this case, in the absence of confounding parental infertility, in assessing the safety of ART manipulations., Wider Implications of the Findings: The study indicates that longer duration of embryo culture after IVF up to blastocyst before ET leads to increased dysfunction of CV health in males compared with IVF and shorter cleavage-stage ET. However, the metabolic health of male offspring was poorer after shorter versus longer culture duration. This distinction indicates that the origin of CV and metabolic health phenotypes after ART may be different. The poorer metabolic health of males after cleavage-stage ET coincides with embryonic genome activation occurring at the time of ET., Study Funding/competing Interest(s): This work was supported through the European Union FP7-CP-FP Epihealth programme (278418) and FP7-PEOPLE-2012-ITN EpiHealthNet programme (317146) to T.P.F., the Biotechnology and Biological Sciences Research Council (BBSRC) (BB/F007450/1) to T.P.F., and the Saudi government, University of Jeddah and King Abdulaziz University to A.A. The authors have no conflicts of interest to declare., (© The Author(s) 2020. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.)

Study Question: Does double vitrification and warming of human blastocysts having undergone biopsy once or twice have an impact on the clinical outcome?, Summary Answer: The clinical pregnancy rate obtained with double vitrification single biopsy blastocysts was comparable to that obtained with single vitrification single biopsy blastocysts in our center in the same time period (46%; 2016-2018), whereas that obtained with double-vitrified double-biopsied blastocysts seemed lower and will need further study., What Is Known Already: Genetic testing on cryopreserved unbiopsied embryos involves two cryopreservation procedures. Retesting of failed/inconclusive-diagnosed blastocysts inevitably involves a second round of biopsy and a second round of vitrification as well. To what extent this practice impacts on the developmental potential of blastocysts has been studied to a limited extent so far and holds controversy. Additionally, the obstetrical/perinatal outcome after the transfer of double-vitrified/single or double-biopsied blastocysts is poorly documented., Study Design, Size, Duration: This retrospective observational study included 97 cycles of trophectoderm biopsy and preimplantation genetic testing (PGT) on vitrified-warmed embryos followed by a second round of vitrification between March 2015 and December 2019., Participants/materials, Setting, Methods: In 36 warming cycles, no biopsy was performed on the embryos before the first vitrification (single biopsy group). In 61 warming cycles, the embryos had been biopsied on Day 3 (n = 4) or on Day 5/6 (n = 57) before the first vitrification (double biopsy group). A second biopsy was mostly indicated in cycles of failed or inconclusive diagnosis at the first biopsy. Two cycles involved a more specific mutation test for X-linked diseases on male embryos and one cycle involved testing for a second monogenic indication supplementary to a previously tested reciprocal translocation. Post-warming suitability for biopsy, availability of genetically transferable embryos and clinical outcome of subsequent frozen-thawed embryo transfer (FET) cycles were reported. Neonatal follow-up of the children was included., Main Results and the Role of Chance: In total, 91 cleavage-stage embryos and 154 blastocysts were warmed, of which 34 (37.4%) and 126 (81.8%), respectively, were of sufficient quality to undergo trophectoderm biopsy and were subsequently vitrified for a second time. Out of these, 92 underwent biopsy for the first time (single biopsy), whereas 68 underwent a second biopsy (double biopsy). After diagnosis, 77 blastocysts (48.1%) were revealed to be genetically transferable (44 in the single biopsy group and 33 in the double biopsy group). In 46 warming cycles, 51 blastocysts were warmed and 49 survived this second warming procedure (96.0%). Subsequently, there were 45 FET cycles resulting in 27 biochemical pregnancies and 18 clinical pregnancies with fetal heartbeat (40.0% per FET cycle: 44.0% in the single biopsy group and 35.0% in the double biopsy group, P = 0.54). Thirteen singletons were born (eight in the single biopsy group and five in the double biopsy group), while three pregnancies were ongoing. A total of 26 embryos (13 in each group) remain vitrified and have the potential to increase the final clinical pregnancy rate. The neonatal follow-up of the children born so far is reassuring., Limitations, Reasons for Caution: This is a small retrospective cohort, thus, the implantation potential of double vitrification double biopsy blastocysts, as compared to double vitrification single biopsy blastocysts and standard PGT (single vitrification, single biopsy), certainly needs further investigation. Although one could speculate on birthweight being affected by the number of biopsies performed, the numbers in this study are too small to compare birthweight standard deviation scores in singletons born after single or double biopsy., Wider Implications of the Findings: PGT on vitrified-warmed embryos, including a second vitrification-warming step, results in healthy live birth deliveries, for both single- and double-biopsied embryos. The neonatal follow-up of the 13 children born so far did not indicate any adverse effect. The present study is important in order to provide proper counseling to couples on their chance of a live birth per initial warming cycle planned and concerning the safety issue of rebiopsy and double vitrification., Study Funding/competing Interest(s): None., Trial Registration Number: N/A., (© The Author(s) 2020. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Study Question: Is there a relationship between blastocyst metabolism and biomarkers of embryo viability?, Summary Answer: Blastocysts with higher developmental potential and a higher probability of resulting in a viable pregnancy consume higher levels of glucose and exhibit distinct amino acid profiles., What Is Known Already: Morphological and morphokinetic analyses utilized in embryo selection provide insight into developmental potential, but alone are unable to provide a direct measure of embryo physiology and inherent health. Glucose uptake is a physiological biomarker of viability and amino acid utilization is different between embryos of varying qualities., Study Design, Size, Duration: Two hundred and nine human preimplantation embryos from 50 patients were cultured in a time-lapse incubator system in both freeze all and fresh transfer cycles. A retrospective analysis of morphokinetics, morphology (Gardner grade), KIDScore, artificial intelligence grade (EmbryoScore), glucose and amino acid metabolism, and clinical pregnancies was conducted., Participants/materials, Setting, Methods: ICSI was conducted in all patients, who were aged ≤37 years and previously had no more than two IVF cycles. Embryos were individually cultured in a time-lapse incubator system, and those reaching the blastocyst stage had their morphokinetics annotated and were each assigned a Gardner grade, KIDScore and EmbryoScore. Glucose and amino acid metabolism were measured. Clinical pregnancies were confirmed by the presence of a fetal heartbeat at 6 weeks of gestation., Main Results and the Role of Chance: Glucose consumption was at least 40% higher in blastocysts deemed of high developmental potential using either the Gardner grade (P < 0.01, n = 209), KIDScore (P < 0.05, n = 207) or EmbryoScore (P < 0.05, n = 184), compared to less viable blastocysts and in blastocysts that resulted in a clinical pregnancy compared to those that failed to implant (P < 0.05, n = 37). Additionally, duration of cavitation was inversely related to glucose consumption (P < 0.05, n = 200). Total amino acid consumption was significantly higher in blastocysts with an EmbryoScore higher than the cohort median score (P < 0.01, n = 185). Furthermore, the production of amino acids was significantly lower in blastocysts with a high Gardner grade (P < 0.05, n = 209), KIDScore (P < 0.05, n = 207) and EmbryoScore (P < 0.01, n = 184)., Limitations, Reasons for Caution: Samples were collected from patients who had ICSI treatment and from only one clinic., Wider Implications of the Findings: These results confirm that metabolites, such as glucose and amino acids, are valid biomarkers of embryo viability and could therefore be used in conjunction with other systems to aid in the selection of a healthy embryo., Study Funding/competing Interest(s): Work was supported by Virtus Health. D.K.G is contracted with Virtus Health. The other authors have no conflict of interest to declare., Trial Registration Number: N/A., (© The Author(s) 2020. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Uterine infection is associated with infertility in women and dairy cows, even after the resolution of infection. However, the mechanisms causing this persistent infertility are unclear. Here, we hypothesized that induced endometritis in non-lactating dairy cows would reduce the developmental competence of oocytes. Non-lactating Holstein cows received an intrauterine infusion of endometrial pathogenic bacteria (Escherichia coli and Trueperella pyogenes; n = 12) or vehicle control (n = 11) on day 2 of the estrous cycle. Bacterial infusion increased expression of endometrial inflammatory mediators, and a mucopurulent discharge in the vagina confirmed the establishment of endometritis. Oocytes were collected by transvaginal ultrasound-guided ovum pickup on days 2, 24, 45, and 66 following infusion and subjected to in vitro fertilization and embryo culture. Bacterial infusion resulted in fewer cleaved oocytes developing to morulae compared to vehicle-infused controls (30.7 versus 45.0%), with the greatest effect observed in oocytes collected on day 24. Development to morula was inversely correlated with endometrial expression of IL6 on day 6. The expression of genes associated with embryo quality did not differ significantly between morulae from bacteria-infused and control cows. Artificial insemination 130 days after intrauterine infusion resulted in normal, filamentous embryos that produced interferon tau 16 days after conception in both infusion groups. This model of experimentally induced uterine infection successfully resulted in endometritis and a reduction in the proportion of oocytes that developed to morulae following in vitro fertilization. In conclusion, endometritis reduced the capacity of oocytes to develop to morulae., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

MicroRNA-21 is expressed in bovine, murine, and human cumulus cells with its expression in murine and bovine cumulus cells correlated with oocyte developmental potential. The aim of this study was to assess the relationship between cumulus cell MIR-21 and human oocyte developmental potential. These studies revealed that both the immature and mature forms of MicroRNA-21 (MIR-21-5p) were elevated in cumulus cells of oocytes that developed into blastocysts compared to cumulus cells of oocytes that arrested prior to blastocyst formation. This increase in MicroRNA-21 was observed regardless of whether the oocytes developed into euploid or aneuploid blastocysts. Moreover, MIR-21-5p levels in cumulus cells surrounding oocytes that either failed to mature or matured to metaphase II but failed to fertilize, were ≈50% less than the MIR-21-5p levels associated with oocytes that arrested prior to blastocyst formation. Why cumulus cells associated with oocytes of reduced developmental potential expressed less MIR-21-5p is unknown. It is unlikely due to reduced expression of either the receptors of growth differentiation factor 9 or rosha Ribonuclease III (DROSHA) and Dicer Ribonuclease III (DICER) which sequentially promote the conversion of immature forms of MicroRNA-21 to mature MicroRNA-21. Furthermore, cultured cumulus cells treated with a MIR-21-5p inhibitor had an increase in apoptosis and a corresponding increase in the expression of PTEN, a gene known to inhibit the AKT-dependent survival pathway in cumulus cells. These studies provide evidence for a role of MicroRNA-21 in human cumulus cells that influences the developmental potential of human oocytes., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Characterization of the molecular factors regulating early embryonic development and their functional mechanisms is critical for understanding the causes of early pregnancy loss in monotocous species (cattle, human). We previously characterized a stage specific functional role of follistatin, a TGF-beta superfamily binding protein, in promoting early embryonic development in cattle. The mechanism by which follistatin mediates this embryotropic effect is not precisely known as follistatin actions in cattle embryos are independent of its classically known activin inhibition activity. Apart from activin, follistatin is known to bind and modulate the activity of the bone morphogenetic proteins (BMPs), which signal through SMAD1/5 pathway and regulate several aspects of early embryogenesis in other mammalian species. Present study was designed to characterize the activity and functional requirement of BMP signaling during bovine early embryonic development and to investigate if follistatin involves BMP signaling for its stage specific embryotropic actions. Immunostaining and western blot analysis demonstrated that SMAD1/5 signaling is activated after embryonic genome activation in bovine embryos. However, days 1-3 follistatin treatment reduced the abundance of phosphorylated SMAD1/5 in cultured embryos. Inhibition of active SMAD1/5 signaling (8-16 cell to blastocyst) using pharmacological inhibitors and/or lentiviral-mediated inhibitory SMAD6 overexpression showed that SMAD1/5 signaling is required for blastocyst production, first cell lineage determination as well as mRNA and protein regulation of TE (CDX2) cell markers. SMAD1/5 signaling was also found to be essential for embryotropic actions of follistatin during days 4-7 but not days 1-3 of embryo development suggesting a role for follistatin in regulation of SMAD1/5 signaling in bovine embryos., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Study Question: Is it possible to develop an automated annotation tool for human embryo development in time-lapse devices based on image analysis?, Summary Answer: We developed and validated an automated software for the annotation of human embryo morphokinetic parameters, having a good concordance with expert manual annotation on 701 time-lapse videos., What Is Known Already: Morphokinetic parameters obtained with time-lapse devices are increasingly used for the assessment of human embryo quality. However, their annotation is time-consuming and can be slightly operator-dependent, highlighting the need to develop fully automated approaches., Study Design, Size, Duration: This monocentric study was conducted on 701 videos originating from 584 couples undergoing IVF with embryo culture in a time-lapse device. The only selection criterion was that the duration of the video must be over 60 h., Participants/materials, Setting, Methods: An automated morphokinetic annotation tool was developed based on gray level coefficient of variation and detection of the thickness of the zona pellucida. The detection of cellular events obtained with the automated tool was compared with those obtained manually by trained experts in clinical settings., Main Results and the Role of Chance: Although some differences were found when embryos were considered individually, we found an overall concordance between automated and manual annotation of human embryo morphokinetics from fertilization to expanded blastocyst stage (r2 = 0.92)., Limitations, Reasons for Caution: These results should undergo multicentric external evaluation in order to test the overall performance of the annotation tool. Getting access to the export of 3D videos would enhance the quality of the correlation with the same algorithm and its extension to the 3D regions of interest. A technical limitation of our work lies within the duration of the video. The more embryo stages the video contains, the more information the script has to identify them correctly., Wider Implications of the Findings: Our system paves the way for high-throughput analysis of multicentric morphokinetic databases, providing new insights into the clinical value of morphokinetics as a predictor of embryo quality and implantation., Study Funding/competing Interest(s): This study was partly funded by Finox-Gedeon Richter Forward Grant 2016 and NeXT (ANR-16-IDEX-0007). We have no conflict of interests to declare., Trial Registration Number: N/A., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Full-grown oocytes are transcriptionally quiescent. Following maturation and fertilization, the early stages of embryonic development occur in the absence (or low levels) of transcription that results in a period of development relying on maternally derived products (e.g., mRNAs and proteins). Two critical steps occur during the transition from maternal to embryo control of development: maternal mRNA clearance and embryonic genome activation with an associated dramatic reprogramming of gene expression required for further development. By combining an RNA polymerase II inhibitor with RNA sequencing, we were able not only to distinguish maternally derived from embryonic transcripts in bovine preimplantation embryos but also to establish that embryonic gene activation is required for clearance of maternal mRNAs as well as to identify putative transcription factors that are likely critical for early bovine development., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

Study Question: Does the quality of a single transferred blastocyst affect singleton birthweight in frozen-embryo transfer (FET) cycles?, Summary Answer: The transfer of a poor-quality blastocyst was associated with lower mean birthweight and gestation-adjusted birthweight (Z-scores) when compared with the transfer of an excellent-quality blastocyst during FET cycles., What Is Known Already: Embryo quality is a strong predictor of IVF success rates. However, very few studies have examined the effect of embryo quality on singleton birthweight., Study Design, Size, Duration: This retrospective study involved singleton live births born to women undergoing frozen-thawed single blastocyst transfers during the period from January 2010 to December 2017 at a tertiary care centre., Participants/materials, Setting, Methods: A total of 1207 women who fulfilled the inclusion criteria were included and were grouped into four groups depending on the blastocyst quality: excellent, good, average and poor. The primary outcome measure was singleton birthweight. The Z-score was employed to calculate the birthweight adjusted for gestational age and newborn gender. Multiple linear regression analysis was performed to investigate the relationship between embryo quality and neonatal birthweight after adjustment for some potential confounders., Main Results and the Role of Chance: In the primary multivariable model, singletons from the poor-quality blastocyst group weighed 183.5 g less than those from the excellent-quality blastocyst group (95% CI: -295.1 to -71.9 g, P = 0.001) in terms of mean birthweight after accounting for patient characteristics, IVF treatment parameters, the year of treatment and newborn gender. Likewise, poor-quality blastocyst transfer was associated with lower gestation-adjusted Z-scores than the transfer of excellent-quality blastocysts (β = -0.35, 95% CI: -0.59 to -0.12, P = 0.003)., Limitations and Reasons for Caution: The current study was limited by its retrospective design and the fact that our analysis was restricted to women with singleton births from single blastocyst transfers. Future prospective studies are required to confirm our findings., Wider Implications of the Findings: Our findings provide new insight into the relationship between embryo quality and neonatal outcomes by showing that poor-quality blastocyst transfer was associated with a decrease in singleton birthweight., Study Funding/competing Interest(s): This study was supported by the National Key Research and Development Program of China (grant no. 2018YFC1003000), the National Natural Science Foundation of China (grant nos. 81771533, 81571397 and 31770989), and the China Postdoctoral Science Foundation (Grant no. 2018M630456). The authors have no conflicts of interest to declare., Trial Registration Number: Not applicable., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Study Question: Do different oxygen levels during human IVF embryo culture affect embryo utilization, cumulative IVF success rates per cycle and neonatal birthweight?, Summary Answer: After 2 days of culture, a lower oxygen level (5%) leads to more good-quality embryos and more embryos that can be cryopreserved, and thereby to a higher cumulative live birth rate per cycle when compared to embryo culture in 20% oxygen, while birthweights are similar., What Is Known Already: Several studies have compared IVF outcome parameters after embryo culture in a more physiological level of 5% oxygen and the atmospheric level of 20%. Although there is consensus that embryo development improves in 5% oxygen, effects on pregnancy and live birth rates are mainly seen in blastocyst, but not cleavage-stage transfers. A major drawback of these studies is that only fresh embryo transfers were included, not taking additional frozen-thawed transfers from these cycles into account. This might have underestimated the effects of oxygen level, especially in cleavage-stage embryo transfers. Furthermore, little is known about the effect of oxygen level during culture on birthweight., Study Design Size Duration: This is a cohort study in 871 consecutive patients who had an IVF cycle between January 2012 and December 2013, and 5-7 years follow-up to allow transfer of frozen-thawed embryos. Based on daily availability of positions in the incubators, all oocytes and embryos of one cycle were allocated to one of the three incubators with traditional ambient oxygen levels (6% CO 2 and 20% O 2 in air), or to a fourth incubator that was adjusted to have low oxygen levels of 5% (6% CO 2 , 5% O 2 and 89% N 2 ). Embryos were cultured under 5 or 20% oxygen until Day 2 or 3, when embryos were transferred or cryopreserved, respectively. Clinical and other laboratory procedures were similar in both groups., Participants/materials Setting Methods: To compare embryo characteristics and (cumulative) pregnancy outcomes between the two oxygen groups, for each patient only the first cycle in the study period was included in the analysis, resulting in 195 cycles in the 5% group (1627 oocytes) and 676 in the 20% oxygen group (5448 oocytes). Embryo characteristics were analysed per cycle and per embryo and were corrected for maternal age, cycle rank order, fertilization method (IVF or ICSI) and cause of subfertility. Perinatal data from the resulting singletons ( n  = 124 after fresh and 45 after frozen-thawed embryo transfer) were collected from delivery reports from the hospitals or midwife practices., Main Results and the Role of Chance: In the 5% oxygen group, there were significantly more embryos of good quality (45.8 versus 30.9% in the 20% group, adjusted odds ratio (OR) [95% CI] = 1.9 [1.6-2.4]). This did not result in higher live birth rates per cycle, but after fresh transfers more good-quality spare embryos could be cryopreserved (46.1 versus 29.7%, adjusted OR [95% CI] = 2.0 [1.7-2.5]).After a follow-up period of 5-7 years, in which 82.4% of the cryopreserved embryos from the 5% oxygen group and 85.4% from the 20% oxygen group were thawed, the percentage of patients with at least one live birth resulting from the study cycle was significantly higher in the low oxygen group (adjusted OR [95% CI] = 1.5 [1.01-2.2]). In 124 live born singletons from fresh embryo transfers and in 45 from transfers of cryopreserved embryos, birthweight was similar in both oxygen groups after correction for confounding factors., Limitations Reasons for Caution: This is a retrospective study, and treatment allocation was not randomised. The study was not powered for a predefined birthweight difference. With the number of live births in our study, small differences in birthweight might not have been detected. The selection of embryos to be cryopreserved was based on embryo morphology criteria that might be different in other clinics., Wider Implications of the Findings: Improved embryo utilization by more cryopreservation leading to higher cumulative live birth rates per cycle favours the use of 5% instead of 20% oxygen during human IVF embryo culture. This study also demonstrates that for comparison of different IVF treatment regimens, the cumulative outcome, including transfers of fresh and frozen-thawed embryos, is to be preferred instead of analysis of fresh embryo transfers only., Study Funding/competing Interests: No external funding was received for this study. None of the authors has a conflict of interest to declare., Trial Registration Number: NA., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.)

Study Question: Is there an effect of male factor infertility (MFI) on either early or late morphokinetic parameters obtained during embryonic culture to blastocyst stage in a time-lapse imaging (TLI) incubator?, Summary Answer: Neither mild nor severe MFI had an impact on overall time to blastocyst or duration of individual cleavage stages in the total embryo population., What Is Known Already: Prior studies have suggested that paternal DNA and sperm quality affect embryo morphokinetic parameters, but the impact of MFI is not fully understood., Study Design, Size, Duration: This retrospective cohort study, at a major academic fertility centre, included 536 couples (women, ≤44 years of age) undergoing IVF between September 2013 and September 2016., Participants/materials, Setting, Methods: Data from 4126 embryos cultured to the blastocyst stage in a TLI-monitored incubator were retrospectively reviewed. Embryos derived from the sperm of men with MFI were compared with those derived from patients with other infertility diagnoses. Generalized fixed and random effects models, t-test and χ2 were used as appropriate., Main Results and the Role of Chance: Couples with MFI had a higher rate of ICSI utilization and fewer usable embryos on average, and the men were older compared with couples with other diagnoses. Additionally, the women in MFI couples were younger and had higher antral follicle counts (AFCs) and higher anti-Müllerian hormone (AMH) levels compared with the other women undergoing IVF. When controlling for maternal and paternal ages, AMH and fertilization method (conventional IVF versus ICSI), neither mild nor severe MFI affected duration of individual cleavage stages or overall time to the blastocyst stage, when all or only usable embryos were examined (coefficient 0.44 hours in all embryos, P = 0.57; coefficient 0.39 hours in usable embryos, P = 0.60). Whether the sperm was surgically extracted similarly had no significant effect on embryo morphokinetic parameters. When the fertilization method was assessed independently, ICSI lengthened the overall time to blastocyst stage by 1.66 hours (P = 0.03) on average, primarily due to an increase in duration of the time from 5-cell embryo stage to early blastulation (P5SB)., Limitations, Reasons for Caution: This large cohort study avoided embryo selection bias due to random assignment of embryos to the TLI incubators. However, our findings may not be generalizable to groups under-represented in our clinic population. Future studies should also evaluate the impact of male hormonal status and detailed sperm morphology, such as head versus flagellum defects, on embryo morphokinetic development., Wider Implications of the Findings: Our findings suggest that the fertilization method rather than MFI per se impacts time to early blastulation. The clinical implications of this effect on embryo development warrant further investigation., Study Funding/competing Interest(s): There were no sources of funding for this study. There are no competing interests., Trial Registration Number: N/A., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Study Question: What is the effect of antioxidants acetyl-L-carnitine, N-acetyl-L-cysteine and α-lipoic acid (A3) in vitrification and warming solutions on mouse blastocyst development and viability?, Summary Answer: The combination of three antioxidants in vitrification solutions resulted in mouse blastocysts with higher developmental potential in vitro and increased viability as assessed by both an outgrowth model in vitro and fetal development following uterine transfer., What Is Known Already: The antioxidant combination of acetyl-L-carnitine, N-acetyl-L-cysteine and α-lipoic acid present in IVF handling and embryo culture media has significant beneficial effects on mouse embryo and fetal development, especially under oxidative stress., Study Design, Size, Duration: The study was a laboratory-based analysis of an animal model. Rapid cooling through vitrification was conducted on F1 mouse blastocysts, with antioxidants (A3) supplemented in vitrification and/or warming solutions, followed by culture and embryo transfer., Participants/materials, Settings, Methods: Pronucleate oocytes were collected and cultured in groups to Day 4 blastocysts. Expanded blastocysts were vitrified and warmed in solutions with and without the A3 antioxidants and cultured for a further 24 h. Blastocyst cell number and allocation, apoptosis and histone acetylation levels were all quantified, and viability through outgrowths and transfers assessed., Main Results and the Role of Chance: Mouse blastocysts vitrified with no antioxidants had significantly lower cell numbers (P  < 0.001) and higher apoptotic cells (P  < 0.05) compared to non-vitrified embryos. Addition of combined A3 antioxidants to the vitrification and warming solutions resulted in a significant increase in inner cell mass cell (ICM) number (P  < 0.001) and total cell number (P  < 0.01), and an increase in outgrowth area (P < 0.05), which correlated with the increased fetal weight (P < 0.05), crown rump length (P < 0.05) and limb development (P < 0.05) determined following transfer compared to embryos with no antioxidants. Furthermore, while blastocyst vitrification significantly reduced acetylation levels (P < 0.05) compared to non-vitrified embryos, the inclusion of A3 antioxidants helped to ameliorate this., Limitations, Reasons for Caution: Embryo development was only examined in the mouse., Wider Implications of the Findings: Results in this study demonstrate that vitrification and warming of blastocysts have significant detrimental effects on embryo histone acetylation and subsequent viability. The presence of antioxidants in the vitrification solutions helps to alleviate the negative effects of cryopreservation. Our data indicate that antioxidants need to be present in the medium at the time of exposure to increased oxidative stress associated with vitrification and that prior exposure (i.e. during culture or IVF alone) is insufficient to protect cells against cryo-induced injury. Hence, A3 antioxidants may assist in maintaining the viability of vitrified human embryos in ART through the reduction of oxidative stress., Study Funding/competing Interest(s): This work was funded by a research grant from Vitrolife AB (Sweden). The authors have no conflict of interest to declare., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

The introduction of time-lapse imaging to clinical in vitro fertilization practice enabled the undisturbed monitoring of embryos throughout the entire culture period. Initially, the main objective was to achieve a better embryo development. However, this technology also provided an insight into the novel concept of morphokinetics, parameters regarding embryo cell dynamics. The vast amount of data obtained defined the optimal ranges in the cell-cycle lengths at different stages of embryo development. This added valuable information to embryo assessment prior to transfer. Kinetic markers became part of embryo evaluation strategies with the potential to increase the chances of clinical success. However, none of them has been established as an international standard. The present work aims at describing new approaches into time-lapse: progress to date, challenges, and possible future directions., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Study Question: Is transfer of vitrified blastocysts associated with higher perinatal and maternal risks compared with slow-frozen cleavage stage embryos and fresh blastocysts?, Summary Answer: Transfer of vitrified blastocysts is associated with a higher risk of preterm birth (PTB) when compared with slow-frozen cleavage stage embryos and with a higher risk of a large baby, hypertensive disorders in pregnancy (HDPs) and postpartum hemorrhage (PPH) but a lower risk of placenta previa when compared with fresh blastocysts., What Is Known Already: Transfer of frozen-thawed embryos (FETs) plays a central role in modern fertility treatment, limiting the risk of ovarian hyperstimulation syndrome and multiple pregnancies. Following FET, several studies report a lower risk of PTB, low birth weight (LBW) and small for gestational age (SGA) yet a higher risk of fetal macrosomia and large for gestational age (LGA) compared with fresh embryos. In recent years, the introduction of new freezing techniques has increased treatment success. The slow-freeze technique combined with cleavage stage transfer has been replaced by vitrification and blastocyst transfer. Only few studies have compared perinatal and maternal outcomes after vitrification and slow-freeze and mainly in cleavage stage embryos, with most studies indicating similar outcomes in the two groups. Studies on perinatal and maternal outcomes following vitrified blastocysts are limited., Study Design, Size, Duration: This registry-based cohort study includes singletons born after frozen-thawed and fresh transfers following the introduction of vitrification in Sweden and Denmark, in 2002 and 2009, respectively. The study includes 3650 children born after transfer of vitrified blastocysts, 8123 children born after transfer of slow-frozen cleavage stage embryos and 4469 children born after transfer of fresh blastocysts during 2002-2015. Perinatal and maternal outcomes in singletons born after vitrified blastocyst transfer were compared with singletons born after slow-frozen cleavage stage transfer and singletons born after fresh blastocyst transfer. Main outcomes included PTB, LBW, macrosomia, HDP and placenta previa., Participants/materials, Setting, Methods: Data were obtained from the CoNARTaS (Committee of Nordic ART and Safety) group. Based on national registries in Sweden, Finland, Denmark and Norway, the CoNARTaS cohort includes all children born after ART treatment in public and private clinics 1984-2015. Outcomes were assessed with logistic multivariable regression analysis, adjusting for the country and year of birth, maternal age, body mass index, parity, smoking, parental educational level, fertilisation method (IVF/ICSI), single embryo transfer, number of gestational sacs and the child's sex., Main Results and the Role of Chance: A higher risk of PTB (<37 weeks) was noted in the vitrified blastocyst group compared with the slow-frozen cleavage stage group (adjusted odds ratio, aOR [95% CI], 1.33 [1.09-1.62]). No significant differences were observed for LBW (<2500 g), SGA, macrosomia (≥4500 g) and LGA when comparing the vitrified blastocyst with the slow-frozen cleavage stage group. For maternal outcomes, no significant difference was seen in the risk of HDP, placenta previa, placental abruption and PPH in the vitrified blastocyst versus the slow frozen cleavage stage group, although the precision was limited.When comparing vitrified and fresh blastocysts, we found higher risks of macrosomia (≥4500 g) aOR 1.77 [1.35-2.31] and LGA aOR 1.48 [1.18-1.84]. Further, the risks of HDP aOR 1.47 [1.19-1.81] and PPH aOR 1.68 [1.39-2.03] were higher in singletons born after vitrified compared with fresh blastocyst transfer while the risks of SGA aOR 0.58 [0.44-0.78] and placenta previa aOR 0.35 [0.25-0.48] were lower., Limitations, Reasons for Caution: Since vitrification was introduced simultaneously with blastocyst transfer in Sweden and Denmark, it was not possible to explore the effect of vitrification per se in this study., Wider Implications of the Findings: The results from the change of strategy to vitrification of blastocysts are reassuring, indicating that the freezing technique per se has no major influence on the perinatal and maternal outcomes. The higher risk of PTB may be related to the extended embryo culture rather than vitrification., Study Funding/competing Interest(s): The study is part of the ReproUnion Collaborative study, co-financed by the European Union, Interreg V ÖKS. The study was also financed by grants from the Swedish state under the agreement between the Swedish government and the county councils, the ALF agreement (LUA/ALF 70940), Hjalmar Svensson Research Foundation and NordForsk (project 71 450). There are no conflicts of interest to declare., Trial Registration Number: ISRCTN11780826., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Study Question: Can time-lapse imaging systems make it possible to identify novel early non-invasive biomarkers to predict live birth?, Summary Answer: From mostly high-grade embryos, out of 35 morphometric, morphologic and morphokinetic variables, only pronuclei (PN) position at time of PN juxtaposition and the absence of multinucleated blastomeres at the 2-cell stage (MNB2cell), were potentially associated with live birth., What Is Known Already: Previous studies indicate that some kinetic markers may be predictive of blastocyst development and embryonic implantation. Certain teams have suggested including some of them in decisional algorithms for embryo transfers., Study Design, Size, Duration: Using a time-lapse incubator (EmbryoScope, Unisense FertiliTech), we retrospectively explored the associations between the morphometric, morphologic and morphokinetic parameters of oocytes, zygotes and embryos, and their associations with live birth. This study assessed 232 embryos from single embryo transfers after ICSI cycles performed between January 2014 and December 2017., Participants/materials, Setting, Methods: The morphometric, morphologic and morphokinetic parameters (18, 4 and 13, respectively) of oocytes, zygotes and early embryos were studied retrospectively. The associations between these parameters were examined using a Spearman's correlation, Mann-Whitney or chi-squared test as appropriate. We examined whether these parameters were associated with outcomes in univariate and multivariate logistic regression analyses., Main Results and the Role of Chance: Central PN juxtaposition was associated with a 2-fold increase in the odds of live birth (OR = 2.20; 95% CI, [1.26-3.89]; P = 0.006), while the presence of MNB2cell was associated with half the odds of live birth (OR = 0.51; 95% CI, [0.27-0.95]; P = 0.035). These two parameters were independent of embryo kinetics. The 33 remaining parameters had no significant association with the capacity of transferred embryos to develop to term., Limitations, Reasons for Caution: Even though the population size was relatively small, our analyses were based on homogeneous cycles, i.e. young women whose transferred embryos were found to be high-grade according to conventional morphology evaluation. In addition, our conclusions were established from a specific, highly selected population, so other study populations, such as women in an older age bracket, may yield different results. Finally, because we assessed day 2/3 transfers, our findings cannot be generalized to embryos cultured up to the blastocyst stage., Wider Implications of the Findings: It would be interesting to explore, prospectively, whether PN localisation is a relevant measure to predict embryo development when added into further algorithms and whether this parameter could be suitable for use in other IVF clinics. Further studies are needed, notably to explore the added value of timing evaluation in cohorts of embryos with low or intermediate morphology grade, as well as in other maternal populations (i.e. older women)., Study Funding/competing Interest(s): No external funding was used for this study. P. Sagot received funding from the following commercial companies: Merck Serono, Finox Biotech, Ferring, MSD France SAS, Teva Sante ́ SAS, Allergan France, Gedeon Richter France, Effik S.A., Karl Storz Endoscopie France, GE Medical Systems SCS, Laboratoires Genevrier, H.A.C. Pharma and Ipsen.All the authors confirm that none of this funding was used to support the research in this study. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the journal policies on sharing data and materials., (© The Author(s) 2019. Published by Oxford University Press.)