Pools Of Embryos Research Articles

O-250 Never a lost cause: can artificial intelligence (AI)-powered ranking help decrease time to pregnancy when only poor-quality embryos are available?

Abstract Study question Can an objectively trained AI system complement embryologists in their ranking of embryos and minimize time to pregnancy, when only poor-quality embryos are available? Summary answer An objective AI algorithm could have decreased cycle to pregnancy by 18% in cohorts of embryos where only poor-quality embryos were available. What is known already Morphokinetics is a key criterion for embryologists to rank embryos as good, fair, or poor quality, with the latter frequently being discarded. Although poor-quality embryos can still lead to a pregnancy, selection of a viable embryo remains challenging to embryologists due to their depleted morphological characteristics. Unlike embryologists, an AI can be trained to see beyond embryo morphological quality, on data that includes many poor-quality embryos that resulted in a pregnancy. As such, AI has the potential to recognize viable poor-quality embryos, which becomes especially crucial in cases where good or fair embryos are unavailable from the embryo pool. Study design, size, duration Data from a French center was acquired through a MIRI® (ESCO) time-lapse system and uploaded on EMBRYOLY, between January 2022 and March 2023. For a total of 204 patients (women’s average age 35 y.o.), embryologists were faced with having to choose an embryo to transfer among 4.3 ± 1.7 poor-quality embryos either because the cohort was composed of only poor-quality embryos or because higher quality embryos had already been transferred. Participants/materials, setting, methods A previously described deep learning algorithm trained on videos of developing embryos with known pregnancy outcome was used to score embryos according to their chances of leading to a clinical pregnancy (fetal heartbeat between 6-9 weeks). A 2D ResNet model was trained, validated and tested on 8138 images of day 5 embryos annotated by senior embryologists to identify those of poor-quality (Gardner grade ICM and/or TE = C, corresponding to 44.7% of the dataset). Main results and the role of chance The association between video predictions and pregnancy was assessed with a logistic regression. A McNemar test was used to compare cycle to pregnancy (CTP) with and without EMBRYOLY’s hypothetical impact. EMBRYOLY’s AI was able to rank poor-quality embryos, as illustrated by a 2-7% relative increase in likelihood of clinical pregnancy observed with an increase in the video score (logistic regression with unit level +0.02, OR = 1.04, 95CI = [1.02-1.07], N = 261 transferred embryos, p < 0.05). EMBRYOLY’s first choice was concordant with the embryologists’ first choice in 74% of the cases that led to a pregnancy (N = 42 embryos). For patients who had two poor-quality embryos transferred with at least one pregnancy (N = 14 cycles), the results show that if the embryologist had first transferred the highest EMBRYOLY-ranked embryo, the number of cycles to pregnancy would have been reduced by 18% (from 1.7 to 1.4, McNemar, p < 0.05), corresponding to a median of 2.3 months. Limitations, reasons for caution The endpoint used was clinical pregnancy as measured by fetal heartbeat, and does not indicate the probability of live birth. The current investigation was performed with retrospective data from a single center. In the future, a multi-centre evaluation should be performed to generalize our findings. Wider implications of the findings Our findings support that an objectively trained AI can help embryologists not miss out on poor-quality embryos that can lead to a pregnancy, which is all the more important to target when good or fair embryos are unavailable from the embryo pool. Trial registration number not applicable

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

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

P-511 Euploidy potential of day 7 embryos and the relevance in Assisted Human Reproduction treatments

Abstract Study question Is it worth waiting embryos with late blastulation (Day 7) for preimplantation genetic analysis? Summary answer Embryo biopsy on Day 7 (D7) benefits cases of ovarian insufficiency and severe male factor with a euploidy rate of 15.1%. What is known already New technologies and improvements in IVF laboratories has allowed the extended cultivation of blastocysts to increase the pool of embryos per IVF cycle, resulting in greater chances, especially in cases of low blastulation rate, blastocysts with low quality or with few cells at Day 5(D5) and Day 6(D6). The variation in the time of the compaction stage depends on many factors, which may take more time, leading some embryos to reach the blastocyst stage only on D7 of development. These embryos have a lower euploidy rate, but there is embryonic competence and these embryos can be eligible for embryo transfer. Study design, size, duration Retrospective study that evaluated the chromosomal status (by Next Generation Screening - NGS) of 33 blastocysts biopsied on day 7 of development, between 2016 and 2023. Subsequently, it was compared the rate of euploidy and mosaicism with 295 blastocysts biopsied at D5 and D6, biopsied from January to October 2023. D7 embryos were originated from IVF cycles of couples with female age between 26 and 44 years, and males age between 29 and 56 years. Participants/materials, setting, methods D7 blastocysts from fresh and own oocytes fertilized by ICSI. The euploidy and mosaicism rates was compared, as well as the morphology of the ICM and trophectoderm (A-C; by Gardner Blastocyst Grade), the day which biopsy was performed and the respective infertility factors, in the groups: Group 1: D7 euploid and Group 2: D7 aneuploid. Subsequently, the rate of euploidy and mosaicism was compared between D7 embryos (Group 3) and D5/D6 embryos (Group 4). Main results and the role of chance There was no difference (p > 0.05) between maternal age for group D7 euploid (38.4 years) vs group D7 aneuploid (37.9 years) and group 3 (38.4 years) vs group 4 (38.2 years). Embryonic morphological evaluation of D7 embryos showed that ICM was classified as B (18.2%) or C (81.8%) and trophectoderm as A (6.1%), B (24.2%) or C (69.7%). Comparisons of ICM and trophectoderm classifications between euploid (group 1) and aneuploid (group 2) in D7 blastocysts showed no difference (p > 0.05). There was a predominance of 5CC embryos in both groups (Group 1: 40.0% vs Group 2: 42.4%), with no difference between them (p > 0.05). In most cases, the biopsy was performed on two days (D5+D7 – 39.3%; D6+D7 – 35.7%), while in 10.7% of cases only on Day 7, when the infertility factor was 100% ovarian insufficiency. In cases of biopsy within 3 days (D5+D6+D7), we observed that 100% of cases were related to severe male factor. When comparing the euploidy rate of D7 embryos – group 3 (15.1%) with that of D5/D6 – group 4 (34.2%), we observed a statistical difference (p < 0.05), while, for in mosaicism rates, D7 embryos represented 6.1% and D5/D6 embryos 9.1%, with no statistical difference (p > 0.05). Limitations, reasons for caution The retrospective nature of our study may lead to bias derived from clinical practices and variability in relation to patient age, embryonic quality and infertility factor. Wider implications of the findings Extended culture and biopsy on D7 benefits cases of ovarian insufficiency and severe male factor by optimizing the IVF cycle. Although the euploidy rate in D7 embryos is lower than D5/D6 embryos, 15% of D7 embryos are euploid, demonstrating that the slower development is not caused exclusively by aneuploidy Trial registration number NA

Empirical Characterization of False Discovery Rates of Differentially Expressed Genes and Transcriptomic Benchmark Concentrations in Zebrafish Embryos.

High-throughput transcriptomics (HTTr) is increasingly applied to zebrafish embryos to survey the toxicological effects of environmental chemicals. Before the adoption of this approach in regulatory testing, it is essential to characterize background noise in order to guide experimental designs. We thus empirically quantified the HTTr false discovery rate (FDR) across different embryo pool sizes, sample sizes, and concentration groups for toxicology studies. We exposed zebrafish embryos to 0.1% dimethyl sulfoxide (DMSO) for 5 days. Pools of 1, 5, 10, and 20 embryos were created (n = 24 samples for each pool size). Samples were sequenced on the TempO-Seq platform and then randomly assigned to mock treatment groups before differentially expressed gene (DEG), pathway, and benchmark concentration (BMC) analyses. Given that all samples were treated with DMSO, any significant DEGs, pathways, or BMCs are false positives. As expected, we found decreasing FDRs for DEG and pathway analyses with increasing pool and sample sizes. Similarly, FDRs for BMC analyses decreased with increasing pool size and concentration groups, with more stringent BMC premodel filtering reducing BMC FDRs. Our study provides foundational data for determining appropriate experiment designs for regulatory toxicity testing with HTTr in zebrafish embryos.

Implicit bias in diagnosing mosaicism amongst preimplantation genetic testing providers: results from a multicenter study of 36 395 blastocysts.

Does the diagnosis of mosaicism affect ploidy rates across different providers offering preimplantation genetic testing for aneuploidies (PGT-A)? Our analysis of 36 395 blastocyst biopsies across eight genetic testing laboratories revealed that euploidy rates were significantly higher in providers reporting low rates of mosaicism. Diagnoses consistent with chromosomal mosaicism have emerged as a third category of possible embryo ploidy outcomes following PGT-A. However, in the era of mosaicism, embryo selection has become increasingly complex. Biological, technical, analytical, and clinical complexities in interpreting such results have led to substantial variability in mosaicism rates across PGT-A providers and clinics. Critically, it remains unknown whether these differences impact the number of euploid embryos available for transfer. Ultimately, this may significantly affect clinical outcomes, with important implications for PGT-A patients. In this international, multicenter cohort study, we reviewed 36 395 consecutive PGT-A results, obtained from 10 035 patients across 11 867 treatment cycles, conducted between October 2015 and October 2021. A total of 17 IVF centers, across eight PGT-A providers, five countries and three continents participated in the study. All blastocysts were tested using trophectoderm biopsy and next-generation sequencing. Both autologous and donation cycles were assessed. Cycles using preimplantation genetic testing for structural rearrangements were excluded from the analysis. The PGT-A providers were randomly categorized (A to H). Providers B, C, D, E, F, G, and H all reported mosaicism, whereas Provider A reported embryos as either euploid or aneuploid. Ploidy rates were analyzed using multilevel mixed linear regression. Analyses were adjusted for maternal age, paternal age, oocyte source, number of embryos biopsied, day of biopsy, and PGT-A provider, as appropriate. We compared associations between genetic testing providers and PGT-A outcomes, including the number of chromosomally normal (euploid) embryos determined to be suitable for transfer. The mean maternal age (±SD) across all providers was 36.2 (±5.2). Our findings reveal a strong association between PGT-A provider and the diagnosis of euploidy and mosaicism. Amongst the seven providers that reported mosaicism, the rates varied from 3.1% to 25.0%. After adjusting for confounders, we observed a significant difference in the likelihood of diagnosing mosaicism across providers (P < 0.001), ranging from 6.5% (95% CI: 5.2-7.4%) for Provider B to 35.6% (95% CI: 32.6-38.7%) for Provider E. Notably, adjusted euploidy rates were highest for providers that reported the lowest rates of mosaicism (Provider B: euploidy, 55.7% (95% CI: 54.1-57.4%), mosaicism, 6.5% (95% CI: 5.2-7.4%); Provider H: euploidy, 44.5% (95% CI: 43.6-45.4%), mosaicism, 9.9% (95% CI: 9.2-10.6%)); and Provider D: euploidy, 43.8% (95% CI: 39.2-48.4%), mosaicism, 11.0% (95% CI: 7.5-14.5%)). Moreover, the overall chance of having at least one euploid blastocyst available for transfer was significantly higher when mosaicism was not reported, when we compared Provider A to all other providers (OR = 1.30, 95% CI: 1.13-1.50). Differences in diagnosing and interpreting mosaic results across PGT-A laboratories raise further concerns regarding the accuracy and relevance of mosaicism predictions. While we confirmed equivalent clinical outcomes following the transfer of mosaic and euploid blastocysts, we found that a significant proportion of mosaic embryos are not used for IVF treatment. Due to the retrospective nature of the study, associations can be ascertained, however, causality cannot be established. Certain parameters such as blastocyst grade were not available in the dataset. Furthermore, certain platform-related and clinic-specific factors may not be readily quantifiable or explicitly captured in our dataset. As such, a full elucidation of all potential confounders accounting for variability may not be possible. Our findings highlight the strong need for standardization and quality assurance in the industry. The decision not to transfer mosaic embryos may ultimately reduce the chance of success of a PGT-A cycle by limiting the pool of available embryos. Until we can be certain that mosaic diagnoses accurately reflect biological variability, reporting mosaicism warrants utmost caution. A prudent approach is imperative, as it may determine the difference between success or failure for some patients. This work was supported by the Torres Quevedo Grant, awarded to M.P. (PTQ2019-010494) by the Spanish State Research Agency, Ministry of Science and Innovation, Spain. M.P., L.B., A.R.L., A.L.R.d.C.L., N.P.P., M.P., D.S., F.A., A.P., B.M., L.D., F.V.M., D.S., M.R., E.P.d.l.B., A.R., and R.V. have no competing interests to declare. B.L., R.M., and J.A.O. are full time employees of IB Biotech, the genetics company of the Instituto Bernabeu group, which performs preimplantation genetic testing. M.G. is a full time employee of Novagen, the genetics company of Cegyr, which performs preimplantation genetic testing. N/A.

Establishment of cell lines from individual zebrafish embryos.

With the increasing use of fish as model species for research, cell cultures derived from caudal fin explants as well as pre-hatching stage embryos have provided powerful in vitro tools that can complement or serve as an ethically more acceptable alternative to live animal experiments. The widely-used protocols to establish these lines require, as a starting point, homogeneous pools of embryos or viable adult fish which are large enough for collecting sufficient fin tissue. This excludes the use of fish lines with adverse phenotypes or lines that exhibit mortality at early developmental stages and so can only be propagated as heterozygotes. Specifically, when no visually overt mutant phenotype is detectable for identifying homozygous mutants at early embryonic stages, it is then impossible to sort pools of embryos with the same genotypes to generate cell lines from the progeny of a heterozygote in-cross. Here, we describe a simple protocol to generate cell lines on a large scale starting from individual early embryos that can subsequently be genotyped by polymerase chain reaction. This protocol should help to establish fish cell culture models as a routine approach for the functional characterization of genetic changes in fish models such as the zebrafish. Furthermore, it should contribute to a reduction of experiments which are ethically discouraged to avoid pain and distress.

Transmission of Mycoplasma bovis infection in bovine in vitro embryo production

Mycoplasma bovis (M. bovis) causes several costly diseases in cattle and has a negative effect on cattle welfare. There is no effective commercial vaccine, and antimicrobial resistance is common. Maintaining a closed herd is the best method to minimize the risk of introduction of M. bovis. Assisted reproduction is crucial in a closed herd to make genetic improvements. M. bovis has been found in commercial semen, and contaminated semen has been the source of disease in naïve dairy herds. The objective of this study was to evaluate M. bovis transmission in bovine in vitro embryo production (IVP) using several possible exposure routes. We used a wild-type M. bovis strain isolated from semen at a final concentration of 106 CFU/mL to infect cumulus–oocyte complexes, spermatozoa, and 5-day-old embryos. We also used naturally contaminated semen in fertilization. Blastocysts were collected on day 7–8 and zona pellucida (ZP)-intact embryos were either washed 12 times, including trypsin washes as recommended by the International Embryo Technology Society (IETS), or left unwashed. Washed and unwashed embryos, follicular fluids, maturation medium, cumulus cells, fertilization medium, and G1 and G2 culture media, as well as all wash media were analyzed using enrichment culture followed by real-time PCR detection of M. bovis. Altogether, 76 pools containing 363 unwashed embryos and 52 pools containing 261 IETS washed embryos were analyzed after oocytes, spermatozoa, or 5-day-old embryos were infected with M. bovis or naturally contaminated semen was used in fertilization. We could not detect M. bovis in any of the embryo pools. M. bovis was not found in any of 12 wash media from different exposure experiments. M. bovis did not affect the blastocyst rate, except when using experimentally infected semen. Contrary to an earlier study, which used a cell co-culture system, we could not demonstrate M. bovis in embryo wash media or tight adherence of M. bovis to ZP-intact embryos. Naturally infected semen did not transmit M. bovis to embryos. We conclude that by using our IVP system, the risk of M. bovis transmission via IVP embryos to recipient cows is very low.

The proteomic analysis of bovine embryos developed in vivo or in vitro reveals the contribution of the maternal environment to early embryo

BackgroundDespite many improvements with in vitro culture systems, the quality and developmental ability of mammalian embryos produced in vitro are still lower than their in vivo counterparts. Though previous studies have evidenced differences in gene expression between in vivo- and in vitro-derived bovine embryos, there is no comparison at the protein expression level.ResultsA total of 38 pools of grade-1 quality bovine embryos at the 4–6 cell, 8–12 cell, morula, compact morula, and blastocyst stages developed either in vivo or in vitro were analyzed by nano-liquid chromatography coupled with label-free quantitative mass spectrometry, allowing for the identification of 3,028 proteins. Multivariate analysis of quantified proteins showed a clear separation of embryo pools according to their in vivo or in vitro origin at all stages. Three clusters of differentially abundant proteins (DAPs) were evidenced according to embryo origin, including 463 proteins more abundant in vivo than in vitro across development and 314 and 222 proteins more abundant in vitro than in vivo before and after the morula stage, respectively. The functional analysis of proteins found more abundant in vivo showed an enrichment in carbohydrate metabolism and cytoplasmic cellular components. Proteins found more abundant in vitro before the morula stage were mostly localized in mitochondrial matrix and involved in ATP-dependent activity, while those overabundant after the morula stage were mostly localized in the ribonucleoprotein complex and involved in protein synthesis. Oviductin and other oviductal proteins, previously shown to interact with early embryos, were among the most overabundant proteins after in vivo development.ConclusionsThe maternal environment led to higher degradation of mitochondrial proteins at early developmental stages, lower abundance of proteins involved in protein synthesis at the time of embryonic genome activation, and a global upregulation of carbohydrate metabolic pathways compared to in vitro production. Furthermore, embryos developed in vivo internalized large amounts of oviductin and other proteins probably originated in the oviduct as soon as the 4–6 cell stage. These data provide new insight into the molecular contribution of the mother to the developmental ability of early embryos and will help design better in vitro culture systems.

REBIOPSY OF EMBRYOS WITH UNDETERMINED PGT-A RESULTS DOES NOT LEAD TO A HIGHER RATE OF UNDETERMINED REBIOPSY RESULTS

Pre-Implantation Genetic Testing for Aneuploidy (PGT-A) has become a powerful tool for improving pregnancy outcomes. When PGT-A results are “undetermined”, offering no insight on the chromosomal makeup of an embryo, many patients are skeptical about seeking rebiopsy of those embryos due to concerns that inconclusive results may be indicative of poor embryo or DNA quality that will prevent a conclusive result even after rebiopsy. Due to limited research on the relationship between undetermined embryos and their rebiopsy results, there is a prevalent exclusion of those embryos as transferable options. This limits the pool of utilizable embryos. In this study, we aim to determine whether conclusive results can still be provided via rebiopsy of those “undetermined” embryos, by assessing whether the rate of undetermined results is consistent among first-time biopsied and rebiopsied embryos.

O-255 Clinical and laboratory factors associated with pregnancy outcomes in patients undergoing frozen euploid blastocyst transfer

Abstract Study question Which variables do have an impact on the pregnancy and live birth rates (LBR) when euploid frozen embryo transfers (FET) are performed? Summary answer Day of trophectoderm biopsy, Body Mass Index (BMI) and endometrial preparation protocol have an impact on pregnancy rate (PR) and LBR in FET cycles. What is known already Preimplantation genetic testing for aneuploidy (PGT-A) and morphological grading of embryos are the two main criteria to select a blastocyst from a pool of embryos, having the highest implantation potential. However, other clinical and laboratory variables might play a crucial role for a successful outcome when top quality euploid embryos are transferred in a FET cycle. It has been described that higher BMI increases the odds for miscarriage when compared with non-obese women. Study design, size, duration This analysis includes 1660 FET cycles with data from of pregnancy rates, miscarriage rate and LBR. Embryos were obtained from 2564 IVF/ICSI cycles of couples with primary or secondary infertility at ART Fertility Clinics UAE, from November 2016 to December 2020. Participants/materials, setting, methods A total of 1660 FET cycles with 2439 euploid blastocysts were included. FET cycles with mosaic or segmental aneuploid embryos were excluded. PGT-A was performed on trophectoderm cells, using Next Generation Sequencing (NGS). Biopsy was performed on day 5, 6 or 7 blastocysts. Endometrial preparation included ovulatory natural cycles (NC) and hormone replacement therapy (HRT) cycles, chosen according to physician’s discretion. Ethical approval was obtained from the Research Ethics Committee (REFA023b). Main results and the role of chance Patients’ characteristics are described as mean±SD (min-max): age: 33.5±5.43(19-47), AMH: 3.30±3.05ng/mL(0.01-23.00), BMI: 27.1±4.87Kg/m2(13.1-43.90), mean number of blastocysts transferred: 1.4±0.49(1-3). Patients were categorized according to age (years) in 4 categories (&amp;lt;30,n=404; 30-34,n=487; 35-40,n=595; &amp;gt;40,n=174) and no association with pregnancy rate (PR, p = 0.856), biochemical miscarriage rate (BMR, p = 0.940), clinical miscarriage rate (CMR, p = 0.06) nor LBR (p = 0.154) was found. BMI (kg/m2) was divided into four groups according to World Health Organization: underweight (&amp;lt;18.5;n=32), normal weight (18.5–24.9;n=555), overweight (25–29.9;n=622), and obese (≥30;n=426). Although no differences were seen for PR or BMR between groups (p = 0.507 and p = 0.343, respectively), CMR was significantly lower for normal BMI group (p &amp;lt; 0.001) and LBR significantly higher when compared to the overweight and obese group (&amp;lt;18.5kg/m2=68.42%; 18.5–24.9kg/m2=68.35%; 25–29.9kg/m2=60.14%; ≥30kg/m2=53.29%; p &amp;lt; 0.001). No differences were observed on the outcomes when AMH was sub-divided as per Bologna Criteria (&amp;lt;1.3ng/mL,n=327; ≥1.3ng/mL,n=1090). Regarding endometrial preparation, NC protocol showed significantly lower BMR and CMR (7.93% vs 12.27%,p=0.026; 8.44% vs 17.97%,p&amp;lt;0.001), and higher LBR (70.33% vs 55.06%,p&amp;lt;0.001) compared to HRT. Day of trophectoderm biopsy had a significant higher PR for day 5 (day 5=75.58% vs day 6=61.1% and day 7=23.81%, p &amp;lt; 0.0001), yet no differences were observed for BMR, CMR nor LBR. Limitations, reasons for caution Although the large number of FET included, performed in the same centre with same methodology, the retrospective study design is a limitation. We could not discard other hypothetical variables contributing to miscarriage such as KIR-HLA discrepancies, or other obstetric factors affecting late miscarriage and live birth. Wider implications of the findings Evaluating the factors associated with pregnancy outcomes should be considered prior to euploid frozen embryo transfer for personalized treatment approach and adequate blastocyst selection. Women with higher BMI should be aware of higher risk of miscarriage and lower LBR although an euploid blastocyst is transferred. Trial registration number not applicable

Ефективність ембріологічного етапу екстракорпорального запліднення в пацієнток із прогнозованою слабкою відповіддю яєчників

Purpose - to show the efficacy of the embryological stage of in vitro fertilisation (IVF) in women with infertility and predicted poor ovarian response to stimulation with various gonadotropins. Materials and methods. This article analyzes the embryological stage of IVF in 140 women with infertility and predicted poor ovarian response. The main clinical group included 75 women who underwent controlled ovarian superstimulation with urinary gonadotropins in the protocol along with gonadotropin-releasing hormone antagonist. Group II (comparison) included 65 women who underwent stimulation of superovulation as per the similar scheme with recombinant gonadotropins. Oocyte pickups, fertilization, cultivation, and embryo transfer were carried out as per standard techniques. Confirmation or exclusion of biochemical pregnancy was carried out by determining the level of total chorionic gonadotropin in the blood serum 14 days after embryo transfer. When calculating the results, the analysis applied methods that were adopted in medical and biological statistics and described in the Glantz Manual. Data were processed by means of StatSoft Statistica 8.0 software package. Results. Among female patients of Groups I and II - where duration of ovarian stimulation did not exceed 10 days - the average duration of using gonadotropins estimated 8.4±1.3 and 9.5±1.2 days respectively (p&lt;0.05). In patients with a longer administration of drugs compared, the average duration of gonadotropin stimulation was definitely higher when using recombinant gonadotropins, i.e. 12.8±2.0 days versus 10.4±1.4 days (p&lt;0.05). When evaluating the follicle-stimulating hormone (FSH) dose used as part of gonadotropin products compared, it was found that in female patients of Groups I and II - where duration of gonadotropin stimulation did not exceed 10 days - the FSH course dose averaged 2,700±300 IU respectively. With the gonadotropin stimulation lasting for more than 10 days, the FSH dose in Group I increased on average up to 3,000±300 IU; in Group II, it increased on average up to 3,300±300 IU (p&lt;0.05). The number of viable embryos on Day 5-6 of cultivation - in the groups compared - averaged to 5.4±2.1, whereas 4.3±2.3 (p=0.014), and their percentage of the number of obtained zygotes reached 91.2±4.1% and 90.0±5.6% (p=0.041). On Day 5, the process of cultivating viable embryos in the groups decreased down to 4.2±1.5 and 3.6±1.7 (p=0.005), whereas their percentage of the number of obtained zygotes decreased down to 77.2±3.5% and 76.0±4.8% (p=0.012). When conducting the cytomorphological examination of embryos under cultivation, it was found that percentage of good quality embryos in the total pool of embryos aged 5 days was 60.4±13.6% and 54.6±14.2% (p=0.011) respectively, whereas in the total pool of embryos aged 6 days, the percentage was 72.6±14.7% and 68.7±16.4% (p=0.013). Conclusions. The conducted comparative study of the effects of human menopausal gonadotropin and the combination of recombinant FSF + recombinant luteinizing hormone at the pre-transfer stage in all examined female patients with expected poor response to gonadotropins showed that the administration of these drugs was accompanied by definitive differences within a number of parameters of stimulated cycles; in terms of statistics, significant are the conclusions as follows: reducing the stimulation period in women who had duration of gonadotropin for less than 10 days; decreasing the average dose of FSH if the gonadotropin stimulation period lasted less than and more than 10 days; improving the survival rate of embryos aged 5 and 6 days; increasing the percentage of good quality embryos among embryos aged 5 and 6 days. The research was carried out in accordance with the principles of the Helsinki Declaration. The study protocol was approved by the Local ethics committee of the participating institution. The informed consent of the patient was obtained for conducting the studies. No conflict of interests was declared by the author. Key words: infertility, assisted reproductive technologies, controlled ovarian stimulation, urinary gonadotropins, recombinant gonadotropins.

Dynamic Changes in the Proteome of Early Bovine Embryos Developed In Vivo.

Early embryo development is a dynamic process involving important molecular and structural changes leading to the embryonic genome activation (EGA) and early cell lineage differentiation. Our aim was to elucidate proteomic changes in bovine embryos developed in vivo. Eleven females were used as embryo donors and pools of embryos at the 4–6 cell, 8–12 cell, morula, compact morula and blastocyst stages were analyzed by nanoliquid chromatography coupled with label free quantitative mass spectrometry. A total of 2,757 proteins were identified, of which 1,950 were quantitatively analyzed. Principal component analysis of data showed a clear separation of embryo pools according to their developmental stage. The hierarchical clustering of differentially abundant proteins evidenced a first cluster of 626 proteins that increased in abundance during development and a second cluster of 400 proteins that decreased in abundance during development, with most significant changes at the time of EGA and blastocyst formation. The main pathways and processes overrepresented among upregulated proteins were RNA metabolism, protein translation and ribosome biogenesis, whereas Golgi vesicle transport and protein processing in endoplasmic reticulum were overrepresented among downregulated proteins. The pairwise comparison between stages allowed us to identify specific protein interaction networks and metabolic pathways at the time of EGA, morula compaction and blastocyst formation. This is the first comprehensive study of proteome dynamics in non-rodent mammalian embryos developed in vivo. These data provide a number of protein candidates that will be useful for further mechanistic and functional studies.

Simultaneous cellular and molecular phenotyping of embryonic mutants using single-cell regulatory trajectories.

SummaryDevelopmental progression and cellular diversity are largely driven by transcription factors (TFs); yet, characterizing their loss-of-function phenotypes remains challenging and often disconnected from their underlying molecular mechanisms. Here, we combine single-cell regulatory genomics with loss-of-function mutants to jointly assess both cellular and molecular phenotypes. Performing sci-ATAC-seq at eight overlapping time points during Drosophila mesoderm development could reconstruct the developmental trajectories of all major muscle types and reveal the TFs and enhancers involved. To systematically assess mutant phenotypes, we developed a single-nucleus genotyping strategy to process embryo pools of mixed genotypes. Applying this to four TF mutants could identify and quantify their characterized phenotypes de novo and discover new ones, while simultaneously revealing their regulatory input and mode of action. Our approach is a general framework to dissect the functional input of TFs in a systematic, unbiased manner, identifying both cellular and molecular phenotypes at a scale and resolution that has not been feasible before.

Loss of iqgap1 (IQ Motif Containing GTPase Activating Protein 1) in Zebrafish Leads to Intracerebellar Hemorrhage, Morphological Abnormalities and Activates Hematopoietic Response

Defects in multiple cell signaling molecules lead to disruptions of vascular integrity given the need for fine-tuned regulation of the cell adhesion complexes. These genetic defects have been linked to the development of intracerebral hemorrhage (ICH). There is genetic evidence in humans for ICH due to some genetic variants, while other variants have been identified in preclinical animal models. Signaling adaptor proteins play a crucial role in cell signaling by promoting interactions between effector proteins and even enabling integration of different pathways. IQGAP1 is a conserved signaling adaptor known for its roles in cell adhesion, cancer and for other cell biological effects. We engineered a zebrafish null mutant in the zebrafish iqgap1 gene by introducing an 11-bp deletion using a CRISPR/Cas9 genome editing method and characterized its phenotype. Homozygous mutants exhibit severe brain hemorrhage and morphological abnormalities, which are ultimately lethal, in about 30-40% of cases, whereas the other embryos survive to adulthood. We visualized the expression pattern of iqgap1 relative to the established fli1a vascular marker and found that iqgap1 strongly overlapped with fli1a expression, but was expressed much more broadly in tissues, such as muscle, branchial arches, and the caudal hematopoietic tissue (equivalent to the mammalian fetal liver).Critically, iqgap1 exhibited co-localization with fli1a in the blood vessels of the central nervous system, whose disruption is likely responsible for the brain hemorrhage. Whole embryo RNA sequencing-based comparison of hemorrhage-positive iqgap1-/- embryo pools with wild-type embryos at 52 hours post-fertilization (hpf) shortly after the onset of hemorrhage identified approximately 800 differentially regulated genes. The most striking feature of this dataset was up-regulation of hematopoietic markers especially those of erythrocytes, neutrophils, mast cells and HSPCs (hematopoietic stem and progenitor cells), but not macrophages. We have confirmed by in situ hybridization with marker gene probes that erythrocyte and neutrophil production is up-regulated most strongly in iqgap1-/- embryos undergoing some level of hemorrhage. By contrast, fli1a endothelial and stem cell marker was downregulated. This animal model provides a compelling genotype-phenotype correlation, implicating IQGAP1 as a new player in vascular disorders such as ICH and identifying a previously unrecognized relationship between IQGAP and regulation of hematopoiesis. Furthermore, this model is now poised to identify ameliorating and exacerbating modifier lesions and potential therapeutic agents that restore normal vascular integrity and prevent ICH. DisclosuresRobitaille: Novartis: Consultancy. Berman: Oxford Immune Algorithmics: Membership on an entity's Board of Directors or advisory committees.

Early, nonlethal ploidy and genome size quantification using confocal microscopy in zebrafish embryos.

Ploidy transitions through whole genome duplication have shaped evolution by allowing the sub- and neo-functionalization of redundant copies of highly conserved genes to express novel traits. The nuclear:cytoplasmic (n:c) ratio is maintained in polyploid vertebrates resulting in larger cells, but body size is maintained by a concomitant reduction in cell number. Ploidy can be manipulated easily in most teleosts, and the zebrafish, already well established as a model system for biomedical research, is therefore an excellent system in which to study the effects of increased cell size and reduced cell numbers in polyploids on development and physiology. Here we describe a novel technique using confocal microscopy to measure genome size and determine ploidy non-lethally at 48 h post-fertilization (hpf) in transgenic zebrafish expressing fluorescent histones. Volumetric analysis of myofiber nuclei using open-source software can reliably distinguish diploids and triploids from a mixed-ploidy pool of embryos for subsequent experimentation. We present an example of this by comparing heart rate between confirmed diploid and triploid embryos at 54 hpf.

Preimplantation genetic testing for aneuploidy: are we examining the correct outcomes?

Questions continue to be raised regarding the benefit of genetic assessment of embryos prior to transfer in IVF, specifically with regards to preimplantation genetic testing for aneuploidy (PGT-A). To evaluate and quantify these concerns, we appraised the most recent (2012-2019) randomized controlled trials on the topic. Only two of these six studies listed cumulative live birth rates per started cycle, with both eliciting a statistically non-significant result. This article describes the concern that a focus on results from the first embryo transfer compared to cumulative outcomes falsely construes PGT-A as having superior outcomes, whilst its true benefit is not confirmed, and it cannot actually improve the true pregnancy outcome of an embryo pool.

DNA methylation status of bovine blastocysts obtained from peripubertal oocyte donors.

In the dairy industry, the high selection pressure combined with the increased efficiency of assisted reproduction technologies (ART) are leading toward the use of younger females for reproduction purposes, with the aim to reduce the interval between generations. This situation could impair embryo quality, decreasing the success rate of the ART procedures and the values of resulting offspring. Young Holstein heifers (n = 10) were subjected to ovarian stimulation and oocyte collection at 8, 11, and 14 months of age. All the oocytes were fertilized in vitro with semen from one adult bull, generating three pools of embryos per animal. Each animal was its own control for the evaluation of the effects of age. The EmbryoGENE platform was used to compare the DNA methylation status of blastocysts obtained from oocytes collected at 8 versus 14 and 11 versus 14 months of age. Age-related contrast analysis identified 5,787 and 3,658 differentially methylated regions (DMRs) in blastocysts from heifers at 8 versus 14 and 11 versus 14 months of age, respectively. For both contrasts, the DMRs were distributed nonrandomly in the different DNA regions. The DNA from embryos from 8-month-old donors was more hypermethylated, while the DNA from embryos from 11-month-old donors displayed an intermediate phenotype. According to Ingenuity Pathway Analysis, the upstream regulator genes cellular tumor antigen p53, transforming growth factor β1, tumor necrosis factor, and hepatocyte nuclear factor 4α are particularly associated with methylation sensitive targets, which were more hypermethylated in embryos from younger donors.

Erratum. Worth the wait? Day 7 blastocysts have lower euploidy rates but similar sustained implantation rates as Day 5 and Day 6 blastocysts.

STUDY QUESTION Does the reproductive potential of embryos change when blastocyst development takes longer than the traditionally accepted 5 days when accounting for aneuploidy and endometrial-embryo asynchrony? SUMMARY ANSWER Aneuploidy increases with increasing duration of blastulation, but if blastocyst morphologic quality and endometrial-embryo asynchrony are controlled for, euploid Day 7 embryos have similar sustained implantation as compared to Days 5 and 6 euploid blastocysts. WHAT IS KNOWN ALREADY The relative contributions of diminished embryo quality versus endometrial and embryo asynchrony to poor outcomes associated with embryos cultured past Day 6 are not clear. Asynchrony can be eliminated by embryo vitrification with transfer in a subsequent month after retrieval. STUDY DESIGN, SIZE, DURATION Retrospective cohort study of patients from a single center attempting conception through ICSI and utilizing preimplantation genetic testing for aneuploidy screening (PGT-A) from January 2017 to September 2018. Cycles were excluded if they utilized surgical sperm or preimplantation genetic testing for monogenetic/single gene defects. ICSI cycle outcomes from 2586 patients were evaluated for ploidy status of embryos. PARTICIPANTS/MATERIALS, SETTING, METHODS Only patients undergoing single, euploid frozen embryo transfer were included when analyzing cycle outcomes by day of blastocyst expansion of the transferred embryo (n = 2130). Ploidy rates by the day upon which an embryo was considered to be usable (denoted, 'usable blastulation day') were determined so as to assess the contribution of aneuploidy to slow embryo development. Outcomes of euploid frozen single embryo transfers (SET) of Day 7 embryos were evaluated to assess the reproductive potential associated with embryos that were slowly developing for reasons other than aneuploidy. Analyses were adjusted by maternal age and blastocyst morphology. MAIN RESULTS AND THE ROLE OF CHANCE Overall, 67.7% (n = 3508) of usable Day 5 blastocysts were euploid, 52.1% (n = 5560) of usable Day 6 blastocysts were euploid and 43.1% (n = 229) of usable Day 7 embryos were euploid (Day 5 versus Day 6: odds ratio (OR) 0.7 (95% CI, 0.64-0.76), P < 0.001; Day 5 versus Day 7: OR 0.56 (95% CI, 0.46-0.69), P < 0.001; Day 6 versus Day 7: OR 0.81 (95% CI, 0.67-0.99), P = 0.036). Stratified by Society for Assisted Reproductive Technology maternal age groups, a reduction in the prevalence of euploidy by increasing time to embryo blastulation was still seen. The sustained implantation rate (SIR) was similar after euploid SET of Days 5 and 6 embryos (overall, 68.9% (95% CI, 66.0-71.6) and 66.8% (95% CI, 63.8-69.7), respectively; P = 0.81). SIR after euploid Day 7 SET appeared slightly lower than that of Days 5 and 6 embryos (52.6% (95% CI, 35.8-69.0); (Day 5 versus Day 7: OR, 0.67 (95% CI, 0.32-1.41), P = 0.29; Day 6 versus Day 7: OR 0.58 (95% CI, 0.28-1.2), P = 0.14)) but did not achieve statistical significance. LIMITATIONS, REASONS FOR CAUTION The primary limitation is the low number of Day 7 blastocyst transfers that limits statistical power. Additionally, the retrospective nature of this study may prevent full elucidation of potential biases with respect to culture, morphologic assessment and selection of Day 7 embryos for transfer. WIDER IMPLICATIONS OF THE FINDINGS Routine culture through Day 7 may successfully increase the pool of transferrable embryos for patients who would otherwise have no usable embryos if culture terminated on Day 6. This is particularly true for older patients (i.e. greater than 35 years of age), whose embryos take longer to blastulate and, therefore, are more susceptible to cycle cancelation. Additionally, as evidenced by an adequate overall SIR of 52.6% after euploid SET of Day 7 blastocysts, embryos developing to a usable blastocyst on Day 7 are likely within the 'window of blastulation.' STUDY FUNDING/COMPETING INTEREST(S) None.

Embryo culture in step single and sequential media: influence on morphology and implantation potential of blastocysts

Purpose. To assess the impact of step single and sequential culture media on the morphology and frequency of blastocyst implantation. Materials and methods. In total, 514 embryos obtained from patients aged 30 to 35 years and fertilized by ICSI were studied (2017 to 2018). The pool of embryos from each woman was randomly divided into two groups: the first group was cultured in the sequential media and the second - in the single ones. Evaluation of the effect was carried out by comparing the obtained blastocysts quality cultured in the single and sequential media. Blastocyst morphology was assessed using the modified Gardner D. K. scale. Statistical analysis was performed by Mann–Whitney U-test and Pearson's chi-squared test with Yates' correction. Statistically significant differences between the compared groups were considered at P < 0.05. Results. The studies found that the use of single Origio ® 1-Step medium at embryo culture phase improves the morphological quality of the resulting blastocyst compared to embryos cultured in sequential Origio ® Sequential Cleav™ / Sequential Blast™ media. No statistically significant difference in the pregnancy and implantation rate of blastocysts obtained after embryo cultivation in the step single or sequential media was detected. Conclusions. The priority in terms of obtaining a large number of high quality blastocysts with high implantation potential is to use a step single medium for the embryo cultivation.

Worth the wait? Day 7 blastocysts have lower euploidy rates but similar sustained implantation rates as Day 5 and Day 6 blastocysts

Does the reproductive potential of embryos change when blastocyst development takes longer than the traditionally accepted 5 days when accounting for aneuploidy and endometrial-embryo asynchrony? Aneuploidy increases with increasing duration of blastulation, but if blastocyst morphologic quality and endometrial-embryo asynchrony are controlled for, euploid Day 7 embryos have similar sustained implantation as compared to Days 5 and 6 euploid blastocysts. The relative contributions of diminished embryo quality versus endometrial and embryo asynchrony to poor outcomes associated with embryos cultured past Day 6 are not clear. Asynchrony can be eliminated by embryo vitrification with transfer in a subsequent month after retrieval. Retrospective cohort study of patients from a single center attempting conception through ICSI and utilizing preimplantation genetic testing for aneuploidy screening (PGT-A) from January 2017 to September 2018. Cycles were excluded if they utilized surgical sperm or preimplantation genetic testing for monogenetic/single gene defects. ICSI cycle outcomes from 2586 patients were evaluated for ploidy status of embryos. Only patients undergoing single, euploid frozen embryo transfer were included when analyzing cycle outcomes by day of blastocyst expansion of the transferred embryo (n = 2130). Ploidy rates by the day upon which an embryo was considered to be usable (denoted, 'usable blastulation day') were determined so as to assess the contribution of aneuploidy to slow embryo development. Outcomes of euploid frozen single embryo transfers (SET) of Day 7 embryos were evaluated to assess the reproductive potential associated with embryos that were slowly developing for reasons other than aneuploidy. Analyses were adjusted by maternal age and blastocyst morphology. Overall, 67.7% (n = 3508) of usable Day 5 blastocysts were euploid, 52.1% (n =5560) of usable Day 6 blastocysts were euploid and 43.1% (n =229) of usable Day 7 embryos were euploid (Day 5 versus Day 6: odds ratio (OR) 0.7 (95% CI, 0.64-0.76), P < 0.001; Day 5 versus Day 7: OR 0.56 (95% CI, 0.46-0.69), P < 0.001; Day 6 versus Day 7: OR 0.81 (95% CI, 0.67-0.99), P = 0.036). Stratified by Society for Assisted Reproductive Technology maternal age groups, a reduction in the prevalence of euploidy by increasing time to embryo blastulation was still seen. The sustained implantation rate (SIR) was similar after euploid SET of Days 5 and 6 embryos (overall, 68.9% (95% CI, 66.0-71.6) and 66.8% (95% CI, 63.8-69.7), respectively; P = 0.81). SIR after euploid Day 7 SET appeared slightly lower than that of Days 5 and 6 embryos (52.6% (95% CI, 35.8-69.0); (Day 5 versus Day 7: OR, 0.67 (95% CI, 0.32-1.41), P = 0.29; Day 6 versus Day 7: OR 0.58 (95% CI, 0.28-1.2), P = 0.14)) but did not achieve statistical significance. The primary limitation is the low number of Day 7 blastocyst transfers that limits statistical power. Additionally, the retrospective nature of this study may prevent full elucidation of potential biases with respect to culture, morphologic assessment and selection of Day 7 embryos for transfer. Routine culture through Day 7 may successfully increase the pool of transferrable embryos for patients who would otherwise have no usable embryos if culture terminated on Day 6. This is particularly true for older patients (i.e. greater than 35years of age), whose embryos take longer to blastulate and, therefore, are more susceptible to cycle cancelation. Additionally, as evidenced by an adequate overall SIR of 52.6% after euploid SET of Day 7 blastocysts, embryos developing to a usable blastocyst on Day 7 are likely within the 'window of blastulation.' None.