Early Blastocyst Research Articles

Role of EZH2-mediated H3K27me3 in placental ADAM12-S expression: implications for fetoplacental growth

BackgroundEnhancer of zeste homolog 2 (EZH2)-mediated histone 3 lysine 27 trimethylation (H3K27me3) is a transcription silencing mark, which is indispensable for cell lineage specification at the early blastocyst stage. This epigenetic repression is maintained in placental cytotrophoblasts but is lifted when cytotrophoblasts differentiate into syncytiotrophoblasts. However, the physiological impact of this lift remains elusive. Here, we investigated whether lifting EZH2-mediated H3K27me3 during syncytialization upregulates the expression of a short secretory isoform of a disintegrin and metalloprotease 12 (ADAM12-S), a well-recognized placenta-derived protease that cleaves insulin-like growth factor binding protein 3 to increase insulin-like growth factor (IGF) bioavailability for the stimulation of fetoplacental growth. The transcription factor and the upstream signal involved were also explored.MethodsHuman placenta tissue and cultured primary human placental cytotrophoblasts were utilized to investigate the role of EZH2-mediated H3K27me3 in ADAM12-S expression and the associated transcription factor and upstream signal during syncytialization. A mouse model was used to examine whether inhibition of EZH2-mediated H3K27me3 regulates placental ADAM12-S expression and fetoplacental growth.ResultsEZH2 and ADAM12 are distributed primarily in villous cytotrophoblasts and syncytiotrophoblasts, respectively. Increased ADAM12-S expression, decreased EZH2 expression, and decreased EZH2/H3K27me3 enrichment at the ADAM12 promoter were observed during syncytialization. Knock-down of EZH2 further increased ADAM12-S expression in trophoblasts. Syncytialization was also accompanied by increased STAT5B expression and phosphorylation as well as its enrichment at the ADAM12 promoter. Knock-down of STAT5B attenuated ADAM12-S expression during syncytialization. Epidermal growth factor (EGF) was capable of inducing ADAM12-S expression via stimulation of STAT5B expression and phosphorylation during syncytialization. Mouse studies revealed that administration of an EZH2 inhibitor significantly increased ADAM12-S levels in maternal blood and fetoplacental weights along with decreased H3K27me3 abundance and increased ADAM12-S expression in the placenta.ConclusionsLifting EZH2-mediated H3K27me3 increases ADAM12-S expression during syncytialization with the participation of EGF-activated STAT5B, which may lead to elevation of ADAM12-S level in maternal blood resulting in increased IGF bioavailability for the stimulation of fetoplacental growth in pregnancy. Our studies suggest that the role of EZH2-mediated H3K27me3 may switch from cell lineage specification at the early blastocyst stage to regulation of fetoplacental growth in later gestation.

Remdesivir impairs mouse preimplantation embryo development at therapeutic concentrations

Remdesivir (RDV) is the first antiviral drug to be approved by the US Food and Drug Administration for the treatment of COVID-19. While the general safety of RDV has been studied, its reproductive risk, including embryotoxicity, is largely unknown. Here, to gain insights into its embryotoxic potential, we investigated the effects of RDV on mouse preimplantation embryos cultured in vitro at the concentrations comparable to the therapeutic plasma levels. Exposure to RDV (2–8 µM) did not affect the initiation of blastocyst formation, although the maintenance of the cavity failed at 8 µM due to increased cell death. While exposure to 2–4 µM permitted the cavity maintenance, expressions of developmental regulator genes associated with the inner cell mass (ICM) lineage were significantly diminished. Adverse effects of RDV depended on the duration and timing of exposure, as treatment between the 8-cell to early blastocyst stage most sensitively affected cavity expansion, gene expressions, and cell proliferation, particularly of the ICM than the trophectoderm lineage. GS-441524, a major metabolite of RDV, did not impair blastocyst formation or cavity expansion, although it altered gene expressions in a manner differently from RDV. Additionally, RDV reduced the viability of human embryonic stem cells, which were used as a model for the human ICM lineage, more potently than GS-441524. These findings suggest that RDV is potentially embryotoxic to impair the pluripotent lineage, and will be useful for designing and interpreting further in vitro and in vivo studies on the reproductive toxicity of RDV.

Silencing XIST on the future active X: Searching human and bovine preimplantation embryos for the repressor.

X inactivation is the means of equalizing the dosage of X chromosomal genes in male and female eutherian mammals, so that only one X is active in each cell. The XIST locus (in cis) on each additional X chromosome initiates the transcriptional silence of that chromosome, making it an inactive X. How the active X in both males and females is protected from inactivation by its own XIST locus is not well understood in any mammal. Previous studies of autosomal duplications suggest that gene(s) on the short arm of human chromosome 19 repress XIST on the active X. Here, we examine the time of transcription of some candidate genes in preimplantation embryos using single-cell RNA sequencing data from human embryos and qRT-PCR from bovine embryos. The candidate genes assayed are those transcribed from 19p13.3-13.2, which are widely expressed and can remodel chromatin. Our results confirm that XIST is expressed at low levels from the future active X in embryos of both sexes; they also show that the XIST locus is repressed in both sexes when pluripotency factors are being upregulated, during the 4-8 cell and morula stages in human and bovine embryos - well before the early blastocyst (E5) when XIST on the inactive X in females starts to be upregulated. Our data suggest a role for DNMT1, UHRF1, SAFB and SAFB2 in XIST repression; they also exclude XACT and other 19p candidate genes and provide the transcriptional timing for some genes not previously assayed in human or bovine preimplantation embryos.

Establishment of a Wisent (Bison bonasus) Germplasm Bank

Simple SummaryThe wisent (European bison) is a protected species. For this reason, we undertook the use of biotechnologies—such as in vitro maturation of oocytes, in vitro fertilization of matured oocytes, in vitro culture of embryos, and embryo vitrification—to establish a wisent embryo bank. The competencies of the vitrified embryos were tested by transferring the warming embryos to cattle (interspecies embryo transfer). The pregnancy was confirmed biochemically and using USG, and although the fetuses were resorbed, the embryos’ competence for development was demonstrated. The results of these studies open the way for the cryoconservation of wisent germplasm.The wisent, or European bison (Bison bonasus), belongs to the same family (Bovidae) as the American bison and domestic cattle. The wisent is the largest mammal in Europe, and is called the “Forest Emperor”. The wisent is listed as “Vulnerable” on the IUCN Red List, and is protected by international law. Achievements in reproductive biotechnology have opened new possibilities for the cryoconservation of the wisent germplasm. Therefore, this research aimed to improve a strategy for the protection and preservation of the European bison through the creation of a wisent germplasm bank, based on the following procedures: isolation and in vitro maturation (IVM) of oocytes, in vitro fertilization (IVF) of matured oocytes, in vitro embryo culture (IVC), and embryo cryopreservation. Wisent ovaries were isolated from females outside the reproductive season, and eliminated from breeding for reasons other than infertility. Cumulus–oocyte complexes (COCs) were isolated from follicles greater than 2 mm in diameter and matured for 24 h and 30 h. After IVM, COCs were fertilized in vitro with wisent sperm. The obtained wisent zygotes, based on oocytes matured for 24 h and 30 h, were cultured for 216 h. Embryos at the morula and early blastocyst stages were vitrified and then warmed and transferred to interspecies recipients (Bos taurus). USG and biochemical tests were used to monitor pregnancies. This study obtained embryos in the morula and early blastocyst stages only after oocytes were fertilized and matured for 30 h. On average, per oocyte donor, 12.33 ± 0.5 COCs were isolated, and only 9.33 ± 0.61 COCs were qualified for in vitro maturation (75.68%), while 9.16 ± 0.48 COCs were matured (84.32%). On average, per donor, 5.5 ± 0.34 embryos were cleaved (59.96%) after 48 h post-fertilization (hpf), and 3.33 ± 0.21 achieved the eight-cell stage (36.52%) after 96 hpf, while 1 ± 0.21 morula and early blastocyst stages (10.71%) were achieved after 216 hpf. A total of six embryos (one morula and five early blastocysts) were obtained and vitrified; after warming, five of them were interspecies transferred to cattle (Bos taurus). On day 41 after fertilization, 3 out of 5 pregnancies were detected based on USG, P4, and PAG tests. However, no pregnancy was observed on day 86 after fertilization, indicating embryo resorption. This study shows that obtaining wisent embryos in vitro, and subsequent cryopreservation to create a wisent embryo bank, can be applied and implemented for the wisent protection program.

Gene Expression in Embryos From Norwegian Red Bulls With High or Low Non Return Rate: An RNA-Seq Study of in vivo-Produced Single Embryos.

During the last decade, paternal effects on embryo development have been found to have greater importance than previously believed. In domestic cattle, embryo mortality is an issue of concern, causing huge economical losses for the dairy cattle industry. In attempts to reveal the paternal influence on embryo death, recent approaches have used transcriptome profiling of the embryo to find genes and pathways affected by different phenotypes in the bull. For practical and economic reasons, most such studies have used in vitro produced embryos. The aim of the present study was to investigate the differences in the global transcriptome of in vivo produced embryos, derived from sires with either high or low field fertility measured as the non-return rate (NRR) on day 56 after first AI of the inseminated cows. Superovulated heifers (n = 14) in the age span of 12–15 months were artificially inseminated with semen from either high fertility (n = 6) or low fertility (n = 6) bulls. On day seven after insemination, embryos were retrieved through uterine flushing. Embryos with first grade quality and IETS stage 5 (early blastocyst), 6 (blastocyst) or 7 (expanded blastocyst) were selected for further processing. In total, RNA extracted from 24 embryos was sequenced using Illumina sequencing, followed by differential expression analysis and gene set enrichment analysis. We found 62 genes differentially expressed between the two groups (adj.p-value<0.05), of which several genes and their linked pathways could explain the different developmental capacity. Transcripts highly expressed in the embryos from low fertility bulls were related to sterol metabolism and terpenoid backbone synthesis, while transcripts highly expressed in the high fertility embryos were linked to anti-apoptosis and the regulation of cytokine signaling. The leukocyte transendothelial migration and insulin signaling pathways were associated with enrichments in both groups. We also found some highly expressed transcripts in both groups which can be considered as new candidates in the regulation of embryo development. The present study is an important step in defining the paternal influence in embryonic development. Our results suggest that the sire’s genetic contribution affects several important processes linked to pre-and peri implantation regulation in the developing embryo.

Human Trophectoderm Spheroid Derived from Human Embryonic Stem Cells.

The use of human embryos for studying the early implantation processes and trophoblast is restricted by ethical concerns. The development of models mimicking the peri-implantation embryos is critical for understanding the physiology of human embryos and many pathophysiological disorders including recurrent implantation failure and miscarriage. Three-dimensional (3D) models of trophoblastic spheroids have been successfully derived from human embryonic stem cells (hESC). Simultaneous activation of the BMP pathway and blockage of the Activin/Nodal pathway favor the differentiation of hESC into trophoblast. Here we describe a 3D trophectoderm differentiation protocol with the use of BAP (BMP4, A83-01, and PD173074) to generate hESC-derived trophectoderm spheroids (BAP-EB). BAP-EB is highly reproducible and exhibits morphological and transcriptomic similarities to human early blastocysts.

Chronic low BPS exposure through diet impairs in vitro embryo production parameters according to metabolic status in the ewe

Bisphenol A (BPA), an endocrine disruptor, has been replaced by structural analogues including bisphenol S (BPS). BPA and BPS exhibited similar effects regarding reproductive functions. Moreover, metabolic status and lipid metabolism are related to female fertility and could worsen BPS effects. The objective was to determine BPS in vivo effects on folliculogenesis and embryo production after chronic exposure through diet, and the influence of metabolic status in adult ewes. Sixty primiparous 2.5 year-old ewes, undergoing a restricted or well fed diet, were exposed to BPS (0, 4 or 50 µg/kg/day) for at least three months. After hormonal oestrus synchronisation and ovarian stimulation, ewes were subjected to ovum pick-up (OPU) procedures to collect immature oocytes, that underwent in vitro maturation, fertilisation and embryo production. Body weight, body condition score and plasma glucose were higher in well-fed compared to restricted ewes, while plasma NEFA was lower during the 4–5 months after the beginning of the diets. Plasma progesterone levels increased on day 5 before OPU session in well-fed compared to restricted ewes. No effect of BPS dose was observed on follicle population, plasma AMH levels and embryo production numbers and rates. However, a significant diet x BPS dose interaction was reported for cleaved embryos, > 4-cell embryos, blastocyst and early blastocyst numbers, and plasma triiodothyronine levels. Our study showed that a contrasted diet did not affect follicle population nor embryo production in adult ewes but could affect the quality and progesterone secretion of the corpus luteum. Chronic low BPS exposure had no effect on follicular population and oocyte competence. Nevertheless, the significant diet x dose interactions observed on embryo production suggest that BPS effect is modulated by metabolic status. Further studies are required to assess the risk of BPS exposure for public reproductive health.

Generation of Sheep Induced Pluripotent Stem Cells With Defined DOX-Inducible Transcription Factors via piggyBac Transposition.

Pluripotent stem cells (PSCs) have the potential to differentiate to all cell types of an adult individual and are useful for studying mammalian development. Establishing induced pluripotent stem cells (iPSCs) capable of expressing pluripotent genes and differentiating to three germ layers will not only help to explain the mechanisms underlying somatic reprogramming but also lay the foundation for the establishment of sheep embryonic stem cells (ESCs) in vitro. In this study, sheep somatic cells were reprogrammed in vitro into sheep iPSCs with stable morphology, pluripotent marker expression, and differentiation ability, delivered by piggyBac transposon system with eight doxycycline (DOX)-inducible exogenous reprogramming factors: bovine OCT4, SOX2, KLF4, cMYC, porcine NANOG, human LIN28, SV40 large T antigen, and human TERT. Sheep iPSCs exhibited a chimeric contribution to the early blastocysts of sheep and mice and E6.5 mouse embryos in vitro. A transcriptome analysis revealed the pluripotent characteristics of somatic reprogramming and insights into sheep iPSCs. This study provides an ideal experimental material for further study of the construction of totipotent ESCs in sheep.

Occurrence and greater intensity of estrus in recipient lactating dairy cows improve pregnancy per embryo transfer

The aim of this study was to determine the association between occurrence and intensity of estrous expression with pregnancy success in recipient lactating dairy cows subjected to embryo transfer (ET). Two observational studies were conducted. Holstein cows were synchronized using the same timed ET protocol, based on estradiol and progesterone in both experiments. At 9 d after the end of the timed ET protocol only animals that had ovulated were implanted with a 7-d embryo [experiment 1 (Exp. 1); n = 1,401 ET events from 1,045 cows, and experiment 2 (Exp. 2); n = 1,147 ET events from 657 cows]. Embryos were produced in vivo (Exp. 1 and Exp. 2) and in vitro (only Exp. 2), then transferred to recipient cows as fresh or frozen-thawed. Pregnancy was confirmed at 29 and 58 d after the end of timed ET protocol. In Exp. 1, animals had their estrous expression monitored through a tail chalk applied on the tail head of the cows and evaluated daily for chalk removal (no estrus: 100% of chalk remaining; estrus: <50% of chalk remaining). In Exp. 2, cows were continuously monitored by a leg-mounted automated activity monitor. Estrous expression was quantified using the relative increase in physical activity at estrus in relation to the days before estrus. Estrous expression was classified as no estrus [<100% relative increase in activity (RI)], weak intensity (100-299% RI), and strong intensity (≥300% RI). Data were analyzed by analysis of variance using mixed linear regression models (GLIMMIX) in SAS (SAS Institute Inc.). A total of 65.2% (914/1,401) and 89.2% (1,019/1,142) of cows from Exp. 1 and Exp. 2, respectively, displayed estrus at the end of the ovulation synchronization protocol. In Exp. 1, cows expressing estrus before to ET had greater pregnancy per ET than those that did not [41.0 ± 2.3% (381/914) vs. 31.5 ± 2.9% (151/487), respectively]. Similarly, in Exp. 2, cows classified in the strong intensity group had greater pregnancy per ET compared with cows in the weak intensity and no estrus groups [41.3 ± 2.2% (213/571) vs. 32.7 ± 2.7% (115/353) vs. 11.3 ± 3.5% (26/218), respectively]. There was no effect of ET type on pregnancy per ET in Exp. 1. However, in Exp. 2, cows that received an in vivo-produced embryo, either fresh or frozen, had greater pregnancy per ET compared with cows that received in vitro-produced embryo. Cows receiving embryos in the early blastocyst and blastocyst stage had greater fertility compared with cows receiving embryos in the morula stage. There was an interaction between the occurrence of estrus and the stage of embryo development on pregnancy per ET, cows which displayed estrus and received a morula or early blastocyst had greater pregnancy per ET than cows that did not display estrus. In conclusion, the occurrence and the intensity of estrous expression improved pregnancy per ET in recipient lactating dairy cows and thus could be used as a tool to assist in the decision making of reproduction strategies in dairy farms.

O-213 Slow day 5 development affects implantation potential of fresh transferred embryos but not birthweight once pregnancy occurs: A multi-center retrospective cohort study

Abstract Study question Does slow development of fresh transferred day 5 embryos lead to decreased implantation potential and birthweight? Summary answer Slow day 5 development was associated with reduced implantation potential when transferred fresh but the subsequent birthweight of the resulting baby was not impacted. What is known already Slow development of in vitro cultured cleavage stage embryos is associated with reduced blastocyst development and implantation rates. There is no current consensus regarding whether to transfer fresh slow developing day 5 embryos or to extend culture for a subsequent day with potential for cryopreservation. It is therefore important to understand the true prognosis of fresh transferred day 5 embryos at less advanced developmental stages. This would provide evidence based guidelines for the decision making process in regard to embryo transfer. Study design, size, duration This is a retrospective multi-center cohort study, including 1213 consecutive patients undergoing autologous oocyte in vitro fertilization (IVF) treatment during 2016-2019,with fresh transfer of a single day 5 embryo (selection based on developmental stage and inner cell mass and trophectoderm morphology if blastocyst was at the ≥expanding stage). Cycle data were collected from 4 associated private clinics, with repeat cycles of same patients excluded to avoid clustering effect at statistical analysis. Participants/materials, setting, methods Live birth and birthweight were followed up in all 1213 fresh day 5 SETs. Multiple regression (logistic or linear) was performed to investigate association between slow day 5 development (defined as ≤ early blastocyst) and (a)live birth, (b) birthweight, and (c) gestation-adjusted birthweight (Z score) to account for gestational age, gender and compared to embryos at ≥ expanded stage. Results were expressed as adjusted odds ratio (aOR) with 95% confidence interval (CI)or coefficients (β). Main results and the role of chance No implantation was achieved following single fresh transfer of day 5 embryos that failed to reach early blastocyst stage (n = 76) and were transferred as ≤ morula stage. Live birth rate was significantly lower following single day 5 fresh transfer of an early blastocyst (n = 237, 16%), in comparison to expanding (n = 329, 27%, P = 0.001), expanded(n = 392, 41%, P = 0.000), and hatching/hatched blastocysts (n = 169, 44%, P = 0.000). After adjusting for potential confounding factors including; maternal age, hours post insemination at day 5 assessment, number of oocytes collected, number of 2PN embryos, and number of embryos frozen; multiple logistic regression showed significantly reduced likelihood of live birth resulting from early blastocysts in reference to those at the expanding (aOR=0.584, 0.371-0.917, P = 0.020), expanded (aOR=0.322, 0.208-0.501, P = 0.000), or hatching/hatched stages (aOR=0.255, 0.147-0.443, P = 0.000). However, multivariate linear regression indicated that early blastocysts resulting in a live birth (n = 39) did not lead to altered birthweight (β=-9.091, P = 0.904; β=-34.960, P = 0.343; β=-26.074, P = 0.414; respectively) or Z score (β = 0.045, P = 0.706; β=-0.051, P = 0.426; β=-0.028, P = 0.506; respectively) in reference to the expanding (n = 90), expanded (n = 160), or hatching/hatched stages (n = 75). Limitations, reasons for caution The retrospective nature of this study does not allow controlling of unknown confounders. The 4 participating clinics are associated within the same network with shared protocols, therefore, results may not be generalized to other clinics with different settings. Wider implications of the findings The findings suggest no clinical value of fresh day 5 transfer of embryos ≤morula stage. Although early blastocysts implant at reduced rate, assuring birthweight outcomes suggest clinical value. Future studies intend to investigate slow growing day 5 fresh transfers versus embryos that were slow growing but transferred after day 6. Trial registration number NA

P–252 The correlation of first cleavage and blastulation timing to the euploid status of embryos after PGT-A

Abstract Study question To examine the correlation of first cleavage and blastulation timing on euploidy rates in IVF cycles after PGT-A. Summary answer The timing of blastulation is observed earlier in the euploid embryos. What is known already Embryo evaluation is one of the most critical processes that affect the clinical outcome in IVF cycles. Conventional morphologic assessment and morphokinetic assessment using time lapse technology are performed in order to select the embryo with the higher implantation potential to be transferred. It is stated that embryos with faster developmental potential, especially early forming blastocysts, show increased euploidy rate and higher implantation potential. Study design, size, duration This study includes ICSI/PGT-A treatments completed between May 2018 and December 2019. 117 blastocysts were biopsied and their euploidy status was analyzed by NGS. These embryos resulted from 32 different ICSI treatments. PGT-A was performed due to: a) repeated IVF failure, b) advanced maternal age, c) recurrent pregnancy loss.ICSI was implemented in all cases and blastocysts were vitrified awaiting the genetic results. Single euploid blastocyst transfer followed and clinical pregnancy rate was monitored. Participants/materials, setting, methods Based on the genetic results, the biopsied embryos were divided into two categories; group A representing the euploid embryos and group B the aneuploid embryos. The timing of 1st cleavage and the timing of blastulation, by means of forming a blastocoel, were investigated and compared between the two groups. The rate of early blastocysts in the two groups was also analysed. Early blastocysts are considered those formed at 96h ±2 of embryo culture post ICSI. Main results and the role of chance After the genetic analysis of the biopsied embryos, 37 blastocysts were included in group A-Euploid embryos and 80 blastocysts in group B-Aneuploid embryos. The mean time of the 1st cleavage division was similar between the two groups, with marginally no statistical significance (group A-euploid:25.9h, group B-aneuploid: 26.9h ,p&amp;gt;0.05). Regarding the blastulation time, it was achieved earlier in group A-Euploid, at a mean time of 102.6h, compared to the mean time of 106h in group B-Aneuploid (p &amp;lt; 0.05). Between the cohort of the Euploid embryos (group A), there was a higher rate of early blastulating embryos, compared to the cohort of aneuploid embryos (Group B) (24% VS 17.5%), although it was not statistically significant (p &amp;gt; 0.05). After transferring 1 euploid blastocyst, the ongoing pregnancy rate was monitored in 76.5%, independently of the 1stcleavage and blastulation time of the transferred embryo. Limitations, reasons for caution Further investigation in larger randomized studies is required, as only a limited number of cases were included in this study. Further analysis of the ongoing pregnancy rate between the euploid blastocysts, depending on other morphokinetic parameters would be of paramount significance, as well. Wider implications of the findings: High clinical pregnancy rates observed independently of the analyzed time points, indicate high success rates obtained after PGT-A/NGS. Additionally, success rates show that trophectoderm biopsy is not hazardous for the embryo viability, if performed properly. Concluding, genetic testing combined with time-lapse microscopy may provide further information to improve IVF outcomes. Trial registration number N/A

O-148 Sperm Aminopeptidase N as a predictive biomarker of blastocyst development and embryo viability

Abstract Study question To evaluate human sperm APN as a prognostic factor for determining high-quality embryos. Summary answer The human sperm APN has the potential to become new molecular prognostic biomarker for having high-quality and viable embryos. What is known already Prognosis and diagnosis of male fertility is one of the major concerns in reproductive medicine. Approximately 30%-40% of men with otherwise normal fertility parameters are still unable to achieve pregnancy. The predictive clinical value of a semen analysis to identify fertile or infertile males is limited; therefore, new sperm diagnostic or prognostic methodologies are urgently required. Sperm Aminopeptidase N (APN) may be a relevant molecular marker due to its high concentration in sperm cells and its role in sperm physiology, such as motility, acrosome reaction, and embryo development. Study design, size, duration A prospective study that involves a total of 81 couples and 611 embryos who underwent oocyte-donation cycles at the Clínica IVI Bilbao (Spain) between September 2014 and July 2015. Participants/materials, setting, methods This study was set in an assisted reproduction unit and in an academic research laboratory. All semen samples were examined and classified following WHO guidelines. Spermatozoa were isolated from semen on discontinuous colloidal silica gradient (45%-90%) technique. Embryo quality and development were determined according to the Spanish Association of Reproduction Biology Studies (ASEBIR) criteria. Flow cytometry analyses of quantitative and semi-quantitative sperm human APN levels. Main results and the role of chance The obtained results proved that the most evolved and viable blastocysts were associated with low sperm APN levels. Expanding, expanded, hatching/hatched and viable blastocysts come from semen samples which showed lower APN levels than early blastocysts, blocked and non viable blastocyst. The cumulative probability of having more evolved blastyocysts increased 1.38-fold at day 5 and 1.98-fold at day 6 of embryo development as well as the likelihood of having viable embryo increased 1.48-fold when semen samples with low APN levels are used during the ICSI technique. Limitations, reasons for caution Data obtained from a single Fertility Clinic. A multi-centrum study will be required. Wider implications of the findings The human sperm APN has the potential to become new molecular prognostic biomarker for having high-quality embryos that could help to diagnose male infertility, especially when seminal parameters are close to the threshold values. Trial registration number Not applicable

P–138 When is low quality really low? Should we transfer low-grade blastocysts?

Abstract Study question What is the live birth rate after single, low-grade blastocyst (LGB) transfer? Summary answer The live birth rate for LGBs is 28%, ranging between 15–31% for the different inner cell mass (ICM) and trophectoderm (TE) subgroups of LGBs. What is known already Live birth rates following LGB transfer are varied and have been reported to be in the range of 5–39%. However, these estimates are inaccurate as studies investigating live birth rates following LGB transfer are inherently limited by sample size (n = 10–440 for LGB transfers) due to LGBs being ranked last for transfer. Further, these studies are heterogenous with varied LGB definitions and design. Collating LGB live birth data from multiple clinics is warranted to obtain sufficient numbers of LGB transfers to establish reliable live birth rates, and to allow for delineation of different LGB subgroups, including blastocyst age and female age. Study design, size, duration We performed a multicentre, multinational retrospective cohort study in 9 IVF centres in China and New Zealand from 2012 to 2019. We studied the outcome of 6966 single blastocyst transfer cycles on days 5–7 (fresh and frozen) according to blastocyst grade, including 875 transfers from LGBs (&amp;lt;3bb, this being the threshold typically applied to LGB studies). Blastocysts with expansion stage 1 or 2 (early blastocysts) were excluded. Participants/materials, setting, methods The main outcome measured was live birth rate. Blastocysts were grouped according to quality grade: good-grade blastocysts (GGBs; n = 3849, aa, ab and ba), moderate-grade blastocysts (MGBs; n = 2242, bb) and LGBs (n = 875, ac, ca, bc, cb and cc) and live birth rates compared using the Pearson Chi-squared test. A logistic regression analysis explored the relationship between blastocyst grade and live birth after adjustment for the confounders: clinic, female age, expansion stage, and blastocyst age. Main results and the role of chance The live birth rates for GGBs, MGBs and LGBs were 45%, 36% and 28% respectively (p &amp;lt; 0.0001). Within the LGB group, the highest live birth rates were for grade c TE (30%) and the lowest were for grade c ICM (19%). The lowest combined grade (cc) maintained a 15% live birth rate (n = 7/48). After accounting for confounding factors, including female age and blastocyst characteristics, the odds of live birth were 2.33 (95% CI = 1.88–2.89) for GGBs compared to LGBs and 1.56 (95% CI = 1.28–1.92) for MGBs compared to LGBs following fresh and frozen blastocyst transfers (p &amp;lt; 0.0001, odds ratios confirmed in exclusively frozen blastocyst transfer cycles). When stratified by individual ICM and TE grade, the odds of live birth according to ICM grade were 1.31 (a versus b; 95% CI = 1.15–1.48), 2.82 (a versus c; 95% CI = 1.91–4.18) and 2.16 (b versus c; 95% CI = 1.48–3.16; all p &amp;lt; 0.0001). The odds of live birth according to TE grade were 1.33 (a versus b; 95% CI = 1.17–1.50, p &amp;lt; 0.0001), 1.85 (a versus c; 95% CI = 1.45–2.34, p &amp;lt; 0.0001) and 1.39 (b versus c; 95% CI = 1.12–1.73, p = 0.0024). Limitations, reasons for caution Despite the large multicentre design of the study, analyses of transfers occurring within the smallest subsets of the LGB group were limited by sample size. The study was not randomised and had a retrospective character. Wider implications of the findings: LGBs maintain satisfactory live birth rates (averaging 28%) in the general IVF population. Even those in the lowest grading tier maintain modest live birth rates (15%; cc). It is recommended that LGBs not be universally discarded, and instead considered for subsequent frozen embryo transfer to maximize cumulative live birth rates. Trial registration number Not applicable

Gliomagenesis is orchestrated by the Oct3/4 regulatory network.

Glioblastoma multiforme (GBM) is a lethal brain tumor characterized by developmental hierarchical phenotypic heterogeneity, therapy resistance and recurrent growth. Neural stem cells (NSCs) from human central nervous system (CNS), and glioblastoma stem cells from patient-derived GBM (pdGSC) samples were cultured in both 2D well-plate and 3D monoclonal neurosphere culture system (pdMNCS). The pdMNCS model shows promise to establish a relevant 3D-tumor environment that maintains GBM cells in the stem cell phase within suspended neurospheres. Utilizing the pdMNCS, we examined GBM cell-lines for a wide spectrum of developmental cancer stem cell markers, including the early blastocyst inner-cell mass (ICM)-specific Nanog, Oct3/4,B, and CD133. We observed that MNCS epigenotype is recapitulated using gliomasphere-derived cells. CD133, the marker of GSC is robustly expressed in 3D-gliomaspheres and localized within the plasma membrane compartment. Conversely, gliomasphere cultures grown in conventional 2D culture quickly lost CD133 expression, indicating its variable expression is dependent on cell-culture conditions. Incomplete differentiation of cytoskeleton microtubules and intermediate filaments (IFs) of patient derived cells, similar to commercially available GBM cell lines, was seen. Subsequently, in order to determine whether Oct3/4 it was necessary for CD133 expression and cancer stemness, we transfected 2D and 3D culture with siRNA against Oct3/4 and found a significant reduction in gliomasphere formation. These results suggest that expression of Oct3/4,A- and CD133 suppress differentiation of GSCs.

The Metabolic Signature of In Vitro Produced Bovine Embryos Helps Predict Pregnancy and Birth after Embryo Transfer.

In vitro produced (IVP) embryos show large metabolic variability induced by breed, culture conditions, embryonic stage and sex and gamete donors. We hypothesized that the birth potential could be accurately predicted by UHPLC-MS/MS in culture medium (CM) with the discrimination of factors inducing metabolic variation. Day-6 embryos were developed in single CM (modified synthetic oviduct fluid) for 24 h and transferred to recipients as fresh (28 ETs) or frozen/thawed (58 ETs) Day-7 blastocysts. Variability was induced with seven bulls, slaughterhouse oocyte donors, culture conditions (serum + Bovine Serum Albumin [BSA] or BSA alone) prior to single culture embryonic stage records (Day-6: morula, early blastocyst, blastocyst; Day-7: expanding blastocyst; fully expanded blastocysts) and cryopreservation. Retained metabolite signals (6111) were analyzed as a function of pregnancy at Day-40, Day-62 and birth in a combinatorial block study with all fixed factors. We identified 34 accumulated metabolites through 511 blocks, 198 for birth, 166 for Day-62 and 147 for Day-40. The relative abundance of metabolites was higher within blocks from non-pregnant (460) than from pregnant (51) embryos. Taxonomy classified lipids (12 fatty acids and derivatives; 224 blocks), amino acids (12) and derivatives (3) (186 blocks), benzenoids (4; 58 blocks), tri-carboxylic acids (2; 41 blocks) and 5-Hydroxy-l-tryptophan (2 blocks). Some metabolites were effective as single biomarkers in 95 blocks (Receiver Operating Characteristic – Area Under the Curve [ROC-AUC]: 0.700–1.000). In contrast, more accurate predictions within the largest data sets were obtained with combinations of 2, 3 and 4 single metabolites in 206 blocks (ROC-AUC = 0.800–1.000). Pregnancy-prone embryos consumed more amino acids and citric acid, and depleted less lipids and cis-aconitic acid. Big metabolic differences between embryos support efficient pregnancy and birth prediction when analyzed in discriminant conditions.

Maternal Neutrophil Depletion Fails to Avert Systemic Lipopolysaccharide-Induced Early Pregnancy Defects in Mice.

Maternal infection-induced early pregnancy complications arise from perturbation of the immune environment at the uterine early blastocyst implantation site (EBIS), yet the underlying mechanisms remain unclear. Here, we demonstrated in a mouse model that the progression of normal pregnancy from days 4 to 6 induced steady migration of leukocytes away from the uterine decidual stromal zone (DSZ) that surrounds the implanted blastocyst. Uterine macrophages were found to be CD206+ M2-polarized. While monocytes were nearly absent in the DSZ, DSZ cells were found to express monocyte marker protein Ly6C. Systemic endotoxic lipopolysaccharide (LPS) exposure on day 5 of pregnancy led to: (1) rapid (at 2 h) induction of neutrophil chemoattractants that promoted huge neutrophil infiltrations at the EBISs by 24 h; (2) rapid (at 2 h) elevation of mRNA levels of MyD88, but not Trif, modulated cytokines at the EBISs; and (3) dose-dependent EBIS defects by day 7 of pregnancy. Yet, elimination of maternal neutrophils using anti-Ly6G antibody prior to LPS exposure failed to avert LPS-induced EBIS defects allowing us to suggest that activation of Tlr4-MyD88 dependent inflammatory pathway is involved in LPS-induced defects at EBISs. Thus, blocking the activation of the Tlr4-MyD88 signaling pathway may be an interesting approach to prevent infection-induced pathology at EBISs.

Bioinspired 3D Culture in Nanoliter Hyaluronic Acid-Rich Core-Shell Hydrogel Microcapsules Isolates Highly Pluripotent Human iPSCs.

Human induced pluripotent stem cells (iPSCs) are ideal for developing personalized medicine. However, the spontaneous differentiation of human iPSCs under conventional 2D and 3D cultures results in significant heterogeneity and compromised quality. Therefore, a method for effectively isolating and expanding high-quality human iPSCs is critically needed. Here, a biomimetic microencapsulation approach for isolating and culturing high-quality human iPSCs is reported. This is inspired by the natural proliferation and development of blastomeres into early blastocyst where the early embryonic stem cells-containing core is enclosed in a semipermeable hydrogel shell known as the zona pellucida (Zona). Blastomere cluster-like human iPSC clusters are encapsulated in a miniaturized (≈10 nanoliter) hyaluronic acid (HA)-rich core of microcapsules with a semipermeable Zona-like hydrogel shell and subsequently cultured to form pluripotent human iPSC spheroids with significantly improved quality. This is indicated by their high expression of pluripotency markers and highly efficient 3D cardiac differentiation. In particular, HA is found to be crucial for isolating the high-quality human iPSCs with the biomimetic core-shell microencapsulation culture. Interestingly, the isolated human iPSCs can maintain high pluripotency even after being cultured again in 2D. These discoveries and the bioinspired culture method may be valuable to facilitate the human iPSC-based personalized medicine.

Effect of the Re-Vitrification of Embryos at Different Stages on Embryonic Developmental Potential.

BackgroundUsing re-vitrified human embryos for frozen-warmed embryo transfer (FET) is a valuable option when there are no other cryopreserved embryos to use, however, except for the PGT cases, no published data are available for FET with human embryos that were re-vitrified at different developmental stages.ObjectiveTo evaluate the effect of re-vitrification of embryos at different stages on embryonic developmental potential.MethodThis study included clinical retrospective and mouse experimental studies. For the retrospective study, a total of 25 FET cycles with re-vitrified day 3 embryos (re-vitrification group 1) and 54 FET cycles with re-vitrified day 5 blastocysts (re-vitrification group 2) between January 2015 and December 2019 were included in this study. The corresponding FET cycles with once-vitrified embryos were identified using propensity score (PS) matching according to the time of embryo transfer. For the mouse experimental study, we divided embryos into 5 groups: fresh (group 1), vitrified at the 8-cell stage (group 2), vitrified at the early blastocyst stage (group 3), vitrified at the 8-cell stage, and re-vitrified at the 8-cell (group 4) or early blastocyst stage (group 5). The fresh embryos was selected as control group. The primary outcome in this study was delivery outcomes.ResultsNo significant difference in delivery rate was detected between re-vitrification group 1 (24.00%) and the corresponding control group (28.00%). However, re-vitrification group 2 (46.3%) showed a significant decrease in delivery rate compared with the two corresponding control groups (63.89% and 64.12%) (P < 0.05). Our experiment using mouse embryos also confirmed the clinical data, and showed that re-vitrification at the blastocyst stage following the first round of vitrification at the 8-cell stage reduced the delivery rate. In addition, both re-vitrified groups showed a significantly higher expression level of BAX. However, only re-vitrification at the blastocyst stage increased the expression level of CASPASE3. ConclusionsRe-vitrification at the 8-cell and blastocyst stages has different effects on embryonic developmental potential, as re-vitrification at blastocyst stage following a previous vitrification at 8-cell stage reduced the delivery rate, while vitrification at the 8-cell stage twice achieved comparable pregnancy outcomes to the once-vitrified group.

P38-MAPK-mediated translation regulation during early blastocyst development is required for primitive endoderm differentiation in mice

Successful specification of the two mouse blastocyst inner cell mass (ICM) lineages (the primitive endoderm (PrE) and epiblast) is a prerequisite for continued development and requires active fibroblast growth factor 4 (FGF4) signaling. Previously, we identified a role for p38 mitogen-activated protein kinases (p38-MAPKs) during PrE differentiation, but the underlying mechanisms have remained unresolved. Here, we report an early blastocyst window of p38-MAPK activity that is required to regulate ribosome-related gene expression, rRNA precursor processing, polysome formation and protein translation. We show that p38-MAPK inhibition-induced PrE phenotypes can be partially rescued by activating the translational regulator mTOR. However, similar PrE phenotypes associated with extracellular signal-regulated kinase (ERK) pathway inhibition targeting active FGF4 signaling are not affected by mTOR activation. These data indicate a specific role for p38-MAPKs in providing a permissive translational environment during mouse blastocyst PrE differentiation that is distinct from classically reported FGF4-based mechanisms.

Oxidative stress induced by methomyl exposure reduces the quality of early embryo development in mice.

Methomyl is a widely used carbamate insecticide and environmental oestrogen that has adverse effects on the reproductive system. However, there have been no reports on the effect of methomyl on early embryos in mammals. In this study, we explored the effect of methomyl exposure on the quality of early embryonic development in mice and the possible mechanisms. During in vitro culture, different concentrations of methomyl (10, 20, 30 and 35 μM) were added to mouse zygote medium. The results showed that methomyl had an adverse effect on early embryonic development. Compared with the control group, the addition of 30 μM methomyl significantly reduced the rate of early embryo blastocyst formation. Methomyl exposure can increase oxidative stress and impair mitochondrial function, which may be the cause of blastocyst formation. In addition, we found that methomyl exposure promoted apoptosis and autophagy in mouse blastocysts. The toxic effect of methomyl on early embryos may be the result of oxidative stress induction. Taken together, our results indicate that methomyl can cause embryonic development defects in mice, thereby reducing the quality of early embryo development.