Stages Of Preimplantation Development Research Articles

Determinants of thermal homeostasis in the preimplantation embryo: a role for the embryo's central heating system?

A number of factors may impinge on thermal homeostasis in the early embryo. The most obvious is the ambient temperature in which development occurs. Physiologically, the temperature in the lumen of the female tract is typically lower than the core body temperature, yet rises at ovulation in the human, while in an IVF setting, embryos are usually maintained at core body temperature. However, internal cellular developmental processes may modulate thermal control within the embryo itself, especially those occurring in the mitochondria which generate intracellular heat through proton leak and provide the embryo with its own 'central heating system'. Moreover, mitochondrial movements may serve to buffer high local intracellular temperatures. It is also notable that the preimplantation stages of development would generate proportionally little heat within their mitochondria until the blastocyst stage as mitochondrial metabolism is comparatively low during the cleavage stages. Despite these data, the specific notion of thermal control of preimplantation development has received remarkably scant consideration. This opinion paper illustrates the lack of reliable quantitative data on these markers and identifies a major research agenda which needs to be addressed with urgency in view of laboratory conditions in which embryos are maintained as well as climate change-derived heat stress which has a negative effect on numerous clinical markers of early human embryo development.

New insights into the (epi)genetics of twinning.

Spontaneous dizygotic (DZ) twins, i.e. twins conceived without the use of ARTs, run in families and their prevalence varies widely around the globe. In contrast, monozygotic (MZ) twins occur at a constant rate across time and geographical regions and, with some rare exceptions, do not cluster in families. The leading hypothesis for MZ twins, which arise when a zygote splits during preimplantation stages of development, is random occurrence. We have found the first series of genes underlying the liability of being the mother of DZ twins and have shown that being an MZ twin is strongly associated with a stable DNA methylation signature in child and adult somatic tissues. Because identical twins keep this molecular signature across the lifespan, this discovery opens up completely new possibilities for the retrospective diagnosis of whether a person is an MZ twin whose co-twin may have vanished in the early stages of pregnancy. Here, we summarize the gene finding results for mothers of DZ twins based on genetic association studies followed by meta-analysis, and further present the striking epigenetic results for MZ twins.

Characterization of the long noncoding RNA transcriptome in human preimplantation embryo development.

Infertility remains a human health burden globally. Only a fraction of embryos produced via assisted reproductive technologies (ARTs) develop to the blastocyst stage in vitro. lncRNA abundance changes significantly during human early embryonic development, indicating vital regulatory roles of lncRNAs in this process. The aim of this study is to obtain insights into the transcriptional basis of developmental events. scRNA-seq data and SUPeR-seq data were used to investigate the lncRNA profiles of human preimplantation embryos. The top 50 highly expressed unique and shared lncRNAs in each stage of preimplantation development were identified. Comparative analysis of the two datasets was used to verify the consistent expression patterns of the lncRNAs. Differentially expressed lncRNAs were identified and subjected to functional enrichment analysis. The lncRNA profiles of human preimplantation embryos in the E-MTAB-3929 dataset were similar to those in the GSE71318 dataset. The ratios of overlap among the top 50 highly expressed lncRNAs between two pairs of stages (2-cell stage vs. 4-cell stage and 8-cell stage vs. morula) were aberrantly low compared with those between other stages. Each stage of preimplantation development exhibited unique and shared lncRNAs among the top 50 highly expressed lncRNAs. Among the between-group comparisons, the 2-cell stage vs. 4-cell stage showed the highest number of differentially expressed lncRNAs. Functional enrichment analysis revealed that differentially expressed lncRNAs and their associated super enhancers and RNA binding proteins (RBPs) are closely involved in regulating embryonic development. These lncRNAs could function as important cell markers for distinguishing fetal germ cells. Our study paves the way for understanding the regulation of developmental events, which might be beneficial for improved reproductive outcomes.

Localization of embryonic stem cells lacking E-cadherin in a mouse blastocyst

Abstract Here we present our results regarding the role of E-cadherin (CDH1, cadherin 1) in the specification of embryonic stem cells (ESCs) in the embryo environment. It has been previously shown that, when forming embryoid bodies with wild-type ESCs, ESCs lacking a functional copy of the gene encoding the adhesive protein E-cadherin (Ecad−/−) preferably sort out to the outside compartment, thus forming the primitive endoderm (PrE) lineage. However, little or no information is available regarding the dynamics of Ecad−/− cells in the actual blastocyst, and so the aim of this work was to determine in which of the three blastocyst cell lines - trophoblast (TE), epiblast (Epi), or PrE - Ecad−/− cells would be located in the embryo environment. For this purpose we injected ESCs into embryos at various stages of pre-implantation development. We used a H2B-GFP cell line expressing histone H2B conjugated with green fluorescent protein (H2BEGFP), as well as a Ecad−/− cell line, in which cells exhibit weaker adhesive properties than wild-type Esc because of their allelic deficiency in the locus encoding cadherin CDH1. We have demonstrated that these cells exhibit a trend to locate in the TE and much less frequently in the Epi, but never in the PrE. We propose that this may be due to differences in the expression of genes characteristic of these cell lines within the Ecad−/− cell colony.

P-728 Impact of calling mosaicism for a less stringent threshold on clinical pregnancies in more than 6000 PGT-A cycles

Abstract Study question How does calling mosaicism for a higher threshold affect euploidy, the chance of finding at least one euploid embryo and clinical pregnancy? Summary answer Increasing the threshold significantly increased euploidy percentage and the probability of finding at least one euploid embryo, as expected, but also the pregnancy outcomes. What is known already NGS technology has shed light on the occurrence of chromosomal mosaicism at the preimplantation stage of development. Evidence regarding clinical outcomes after transfer of embryos with putative mosaic results are accumulating (ESHRE Working Group on Chromosomal Mosaicism, 2022). A series of studies reporting the birth of healthy babies after the transfer of embryos with a chromosomal mosaic result on PGT-A (Greco et al., 2015; Kahraman et al., 2020; Viotti et al., 2019 and 2021), have been published. Data suggested lower implantation rates and higher miscarriage rates when mosaics were compared with euploid embryo transfers. Study design, size, duration This retrospective study covers a period from January 2017 to October 2022 and includes 6063 PGT-A cycles. A total of 18425 blastocysts were biopsied and analyzed with NGS. From January 2017 to December 2020, the cut-off value for reporting mosaicism and euploidy was %20; the rate of which was increased to 30% after January 2021. Chromosomes with PGT-A profiles that deviate from 2 copies by less than 30% were reported as normal. Participants/materials, setting, methods A total number of 12751 and 5674 blastocysts were biopsied between January 2017-December 2020 and January 2021-October 2022, respectively. PGT-A was performed on Ion Torrent S5 (Thermo Fisher Scientific). The mean female age for 20% and 30% cut-off groups were 36.7+-5.1, 35.9+-5.2, respectively. As this age difference was statistically significant, further analysis was performed in age subgroups < =38 and >38. Main results and the role of chance For PGT-A cycles with female age < =38, cycles with at least one euploid embryo in the 30% cut-off group increased from 74.0% to 77.5% whereas cycles with at least one mosaic embryo decreased from 40.8% to 37.0% (p = 0.0212; p = 0.0274). Cycles with at least one mosaic embryo without any euploid embryos decreased by 2% (9.2% to 7.2%) (p = 0.0522). The euploidy percentage out of all tested biopsies was significantly increased from 40.4% to 45.8% (p = 0.0001). For cycles with female age >38, ratio of PGT-A cycles with at least one euploid embryo increased from 28.1% to 30.3% in the 30% cut-off group but this was not statistically significant (p = 0.28). Cycles with at least one mosaic embryo were decreased from 15.1% to 10.8% and cycles with at least one mosaic embryo without any euploid embryo decreased from 9.5% to 5.6% in the 30% cut-off group (p = 0.006; p = 0.0018). The euploidy percentage was increased by 2.8% from 15.1% to 18.1% (p = 0.0115). The pregnancy outcomes were significantly higher for euploid embryos with the 30% cut-off compared to 20%, as clinical pregnancy was 64.5% vs. 59.1% and clinical pregnancy loss was 7.9% vs. 12.4% (p = 0.0022; p = 0.0011). Similarly, ongoing pregnancy was 5.2% higher, but non-significant. Limitations, reasons for caution These are the results of a single ART center which is using the same protocols for ovarian stimulation and endometrial preparation in the two periods above mentioned. Although not expected, minor changes may have occurred; similar practices have to be reported. Wider implications of the findings Mosaicism is undeniably a true biological phenomenon. However, trophectoderm biopsy and NGS are prone to create an artefactual false mosaicism. Relaxing the mosaicism calling rate by 10% may have added some better-quality blastocysts to the euploid pool giving the opportunity to embryologists of transferring blastocysts with higher implantation potential. Trial registration number Not applicable

O-225 May embryonic cfDNA in the culture media play a role as a non-invasive assessment of aneuploidy in PGT

Abstract Non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) in spent blastocyst media (SBM) is a promising technique that has the potential to revolutionize current preimplantation genetic testing procedures. This technique is based in the analysis of the cell-free DNA (cfDNA) released by the embryo during the latest stages of preimplantation development. It allows for the detection of chromosomal abnormalities in embryos without the need for invasive procedures that can harm the embryo (reviewed in Navarro-Sánchez et al., 2022). Our group among others has conducted different studies in this field, and the early clinical application of the cfDNA analysis in the culture medium has been proposed as a screening tool, giving a priority score for embryo transfer based on the media results (Rubio et al., 2019, 2020). In the final analysis of our multicenter study in day-6 and day-7 blastocysts, we have been able to compare the chromosomal results of the SBM with trophectoderm (TE) biopsies in 2,187 samples, and with the inner cell mass ICM in 230 blastocysts. In this study, the analysis of the cfDNA showed a concordance rate of 87% with the ICM biopsy, which is currently considered the gold standard for PGT-A. However, it is important to continue evaluating the factors that may affect the informativity and concordance rates of cfDNA-based assays, such as the culture day when medium is collected, contamination with external and/or cumulus cell DNA, and previous manipulation of the embryos. Multivariate analysis has shown that the longer the time in culture, the higher the informativity rate, and female age had the greatest impact on concordance rates with TE biopsies. Regarding technical aspects, the SBMs with higher number of reads after sequencing resulted in higher concordance rates, reflecting the importance of analysing samples with sufficient DNA quantity. Finally, the predictive value in terms of concordance with TE biopsies was higher for aneuploid blastocyst. No significant independent correlation was observed for embryo quality. The improvements in IVF lab protocols and the use of time-lapse and short incubation protocols for vitrified-thawed blastocysts are also noteworthy, as they may further increase the concordance rates of cfDNA-based assays with blastocysts. In fact, concordance in previously vitrified-thawed blastocysts as result as high as 90%. Regarding clinical outcomes, several groups have evaluated non-invasive PGT-A with transfer of euploid embryos showing good results. In a pilot study with only 7 SETs in couples with PGT-A or PGT-SR indication, 5 pregnancies were achieved resulting in 5 livebirths (Xu et al., 2016). In a second study, in couples with recurrent spontaneous abortion or repeated implantation failure, 50 transfers of euploid media were performed resulting in a 58% clinical pregnancy rate, with 27 healthy babies born (Fang et al., 2019). Similar results were described in good prognosis patients <38 years of age with ongoing pregnancy rates comparable to PGT-A (61.5%), and higher than conventional IVF or ICSI (48.5%) (Franco et al., 2021). Overall, the high concordance rates with trophectoderm biopsies and inner cell mass biopsies are encouraging, and the clinical outcomes with transfer of euploid embryos are promising. It is exciting to see the progress in non-invasive preimplantation genetic testing using cfDNA in culture medium, and it will be interesting to see how this technology evolves and its potential role as a screening or diagnostic tool in the future.

Expression of Toll-Like Receptors in the Lung Tissue of Mouse Fetuses Generated by in vitro Embryo Culture and Embryo Transfer

Mouse fetuses generated by in vitro embryo culture and embryo transfer exhibit impaired lung development, altered composition of pulmonary epithelial cells associated with downregulation of several genes involved in lung development and toll-like receptor (TLR) signaling pathway. The aims of the present study were to determine the expression of all TLRs and to examine if the expression of TLRs, along with genes involved in TLR signaling pathway, is altered in the lung tissue of mouse fetuses generated through embryo culture and embryo transfer. Two experimental (EGs) and one control (CG) group were included in the study. Embryos cultured at 5% CO₂-95% air for 95 h or less than 24 h were transferred to pseudo-pregnant females to obtain fetuses comprising EG_{in vitro} (n = 18) and EG_{in vivo} (n = 18), respectively. Fetuses obtained from naturally ovulating females on day 18 of pregnancy served as the CG (n = 18). Western blot and immunohistochemistry were used to determine the expression of TLR proteins. The expression of transcripts encoding TLRs, and the genes involved in TLR signaling pathway (Lbp, Pik3r1, Pik3cb, Nfkbia, and Fos), was determined using qRT-PCR. While all TLRs were expressed by cells lining the bronchial/bronchiolar epithelium of lung tissues in all groups, some of the TLRs were expressed in a specific pattern. When compared to CG, the expression of transcripts encoding TLR-2, -3, -4, -5, -7, -8, -9, -12, -13, Lbp, Pik3r1, Pik3cb, Nfkbia, and Fos was significantly downregulated in both EGs. It appears that stress imposed on embryos at preimplantation stages of development is associated with downregulation of TLRs, along with some of the genes involved in TLR signaling pathway, in the lung tissue during the perinatal period. It remains to be determined if downregulation of TLRs, along with the genes involved in TLR signaling pathway, has any functional consequences in the adult lung tissue.

Leukocyte Telomere Length in Children Born Following Blastocyst-Stage Embryo Transfer

ABSTRACT Several recent studies have suggested an association between children conceived by assisted reproductive technology (ART) and a distinct growth pattern in early life, which has been linked to diseases of older age. Telomere shortening, specifically a shorter leukocyte telomere length (LTL), has also been linked to age-related diseases including cardiometabolic diseases and cancer. Initial telomere length is shaped by a telomere reset process during gamete fertilization and the early stages of preimplantation development. Given that ART involves the in vitro manipulation of oocytes and embryos, this study aimed to evaluate the association between ART-related factors and LTL in children. Whole genome-sequencing (WGS) data from the Nanjing and Suzhou centers of the China National Birth Cohort (CNBC) were obtained from 1137 individuals from 365 parent-children families, including 202 children conceived using ART and 205 conceived spontaneously. The association was determined between blastocyst-stage transfer and shorter telomere length in 180 children conceived by ART in the same centers. In addition, qPCR was used to perform validation on fingerstick blood samples from 406 children conceived using ART at 3 different centers in China. Data from qPCR were then compared with data obtained from WGS in 70 children in the discovery cohort with sufficient DNA samples. Associations between parental factors and demographics with LTL were examined in the discovery cohort. LTL attribution with aging was calculated using data from the discovery cohort, 1185 individuals aged 40–69 years from healthy controls in the Nanjing Lung Cancer Cohort with LTL measured using WGS, and 1452 East Asian adults aged 40–69 years in the UK biobank with LTL measured using qPCR. Results of association analyses found paternal LTL (β = 0.27, P = 3.26 × 10−8), maternal LTL (β = 0.26, P = 2.41 × 10−7), plurality (twins vs singletons; β = −0.34, P = 0.010), sex of children (male vs female; β = −0.22, P = 0.032), gestational age (β = 0.05, P = 0.026), and conception type (ART-conceived pregnancy vs spontaneously conceived, β = −0.35, P = 5.98 × 10−4) were all significantly associated with LTL in children. Multivariate regression analysis revealed that children conceived by ART had a significantly shorter LTL than those conceived spontaneously, even when adjusting for parental age at conception (adjusted β = −0.41, P = 3.33 × 10−4). Transfer of blastocyst-stage embryos was found to be significantly associated with shorter LTL (β = −0.54, P = 2.69 × 10−3). In addition, when LTL in children from spontaneous pregnancies was compared with LTL in children from ART pregnancies through cleavage-stage versus blastocyst-stage transfer, the LTL of the cleavage-stage group was comparable to the spontaneous pregnancy group, whereas the LTL of the blastocyst-stage group was shorter (β = −0.67, P = 7.81 × 10−8). This result suggests that the difference in LTL between transfer stages might explain the difference between the ART-conceived and spontaneous pregnancy groups. No associations between COS protocols, fertilization methods, or embryo transfer cycles on shorter LTL in children were observed. The multicenter validation cohort had similar associations between blastocyst-stage transfer and shorter LTL. Shortened LTL associated with blastocyst transfer was equivalent to 13.93 years (95% CI, 4.64–23.22 years), 13.42 years (95% CI, 4.52–22.21 years), and 10.85 years (95% CI, 1.03–20.67 years) of aging between 40–69 years in the discovery and 2 validation cohorts, respectively. The results of this study demonstrate that children conceived by ART are associated with a shorter LTL compared with those conceived spontaneously, and transfer of blastocyst-stage embryos was associated with shorter LTL in children than is the transfer of cleavage-stage embryos.

Cell-free deoxyribonucleic acid analysis in preimplantation genetic testing

Detection of chromosomal aneuploidies and monogenic disorders in preimplantation embryos is essential for selecting the best embryo for transfer during invitro fertilization to achieve a healthy pregnancy. Preimplantation genetic testing (PGT) is typically performed on preimplantation embryos to select a genetically normal embryo for transfer. A trophectoderm biopsy is necessary for PGT; this is an invasive procedure to the embryo that requires specialized equipment and highly trained embryologists, resulting in high costs associated with invitro fertilization treatment. Moreover, the biopsy procedure may increase the likelihood of developing pregnancy complications, such as preeclampsia and hypertensive disorders. Therefore, there is a need for noninvasive embryo screening strategies. The presence of cell-free deoxyribonucleic acid in the embryo culture medium presents an opportunity to screen for genetic abnormalities. Cell-free deoxyribonucleic acid is released by embryos in the latter stages of preimplantation development, and its analysis has been proposed as a noninvasive approach for PGT. Here, we review studies reporting the concordance rates between cell-free deoxyribonucleic acid and trophectoderm biopsies, or whole blastocysts, in couples undergoing PGT. Noninvasive PGT results are promising for aneuploidy detection, with some early evidence of successful clinical application. Further research is required to explore its application for the detection of structural rearrangements and monogenic disorders.

Insulin-like Growth Factor Binding Protein 3 Increases Mouse Preimplantation Embryo Cleavage Rate by Activation of IGF1R and EGFR Independent of IGF1 Signalling

The viability of embryos cultured in vitro is poor compared to those that develop in vivo. The lack of maternally derived growth factors in vitro may contribute to this problem. Insulin-like growth factor binding protein 3 (IGFBP3) is one such growth factor that has been identified in the maternal reproductive system. This study examined the role of autocrine and exogenous IGFBP3 in mouse preimplantation embryos. Embryos expressed IGFBP3 across all stages of preimplantation development, and addition of exogenous IGFBP3 to embryo culture media increased the rate of development to the 2-, 4-, 5-, and 8-cell stages. Addition of inhibitors of the IGF1 and EGF receptors prevented this IGFBP3-mediated improvement in developmental rate, but the effect was not cumulative, indicating that both receptors are transactivated downstream of IGFBP3 as part of the same signalling pathway. Acute exposure to IGFBP3 increased phosphorylation of Akt and rps6 in 4-8 cell embryos, suggesting activation of the PI3-kinase/Akt pathway downstream of the IGF1 and EGFR receptors to promote cell proliferation and survival. In conclusion, addition of IGFBP3 to embryo culture media increases early cleavage rates independent of IGF1 signalling and therefore, IGFBP3 addition to IVF culture media should be considered.

Stage specific gene expression of folate mediated one-carbon metabolism enzymes and transporters in buffalo oocytes and pre-implantation embryos

DNA synthesis and methylations are crucial during pre-implantation embryonic development, and are mediated by one-carbon metabolism of folates. Folates, transported into the cells via folate receptors (FOLR1 and FOLR2) and carriers (SLC19A1), are metabolized by various enzymes involved in folate-methionine cycle. However, the variations in temporal expression of folate transporters and folate-methionine cycle enzymes during pre-implantation embryo development is obscure. Thus, the present study aimed to investigate the differential expression of the genes for folate transporters and folate-methionine cycle enzymes. We also examined the expression of folate transport proteins in different pre-implantation development stages. Immature buffalo oocytes were matured in maturation medium followed by in vitro fertilization and culture at standard culture conditions. The temporal pattern of gene expression in buffalo, when compared to previous studies, indicated an inter-specific variation. The transcripts of some enzymes and folate transporters were significantly upregulated after zygotic genome activation. The transcripts as well as proteins for FOLR1, FOLR2 and SLC19A1 were present in oocytes and all the pre-implantation embryo stages. FOLR1 was present in the nuclei of different stages of developing embryos but not in the metaphase (MII) oocytes. As a result, the present study advocates the existence of active folate transport in buffalo oocytes and pre-implantation embryos. The data provided by the analysis of differential gene expression of folate transporters and metabolic enzymes would likely contribute to a better understanding of the role of folates in embryo development as well as advancements in assisted reproductive technologies.

ESHRE survey results and good practice recommendations on managing chromosomal mosaicism.

How should ART/preimplantation genetic testing (PGT) centres manage the detection of chromosomal mosaicism following PGT? Thirty good practice recommendations were formulated that can be used by ART/PGT centres as a basis for their own policy with regards to the management of 'mosaic' embryos. The use of comprehensive chromosome screening technologies has provided a variety of data on the incidence of chromosomal mosaicism at the preimplantation stage of development and evidence is accumulating that clarifies the clinical outcomes after transfer of embryos with putative mosaic results, with regards to implantation, miscarriage and live birth rates, and neonatal outcomes. This document was developed according to a predefined methodology for ESHRE good practice recommendations. Recommendations are supported by data from the literature, a large survey evaluating current practice and published guidance documents. The literature search was performed using PubMed and focused on studies published between 2010 and 2022. The survey was performed through a web-based questionnaire distributed to members of the ESHRE special interest groups (SIG) Reproductive Genetics and Embryology, and the ESHRE PGT Consortium members. It included questions on ART and PGT, reporting, embryo transfer policy and follow-up of transfers. The final dataset represents 239 centres. The working group (WG) included 16 members with expertise on the ART/PGT process and chromosomal mosaicism. The recommendations for clinical practice were formulated based on the expert opinion of the WG, while taking into consideration the published data and results of the survey. Eighty percent of centres that biopsy three or more cells report mosaicism, even though only 66.9% of all centres have validated their technology and only 61.8% of these have validated specifically for the calling of chromosomal mosaicism. The criteria for designating mosaicism, reporting and transfer policies vary significantly across the centres replying to the survey. The WG formulated recommendations on how to manage the detection of chromosomal mosaicism in clinical practice, considering validation, risk assessment, designating and reporting mosaicism, embryo transfer policies, prenatal testing and follow-up. Guidance is also provided on the essential elements that should constitute the consent forms and the genetic report, and that should be covered in genetic counselling. As there are several unknowns in chromosomal mosaicism, it is recommended that PGT centres monitor emerging data on the topic and adapt or refine their policy whenever new insights are available from evidence. Rather than providing instant standardized advice, the recommendations should help ART/PGT centres in developing their own policy towards the management of putative mosaic embryos in clinical practice. This document will help facilitate a more knowledge-based approach for dealing with chromosomal mosaicism in different centres. In addition to recommendations for clinical practice, recommendations for future research were formulated. Following up on these will direct research towards existing research gaps with direct translation to clinical practice. Emerging data will help in improving guidance, and a more evidence-based approach of managing chromosomal mosaicism. The WG received technical support from ESHRE. M.D.R. participated in the EQA special advisory group, outside the submitted work, and is the chair of the PGT WG of the Belgian society for human genetics. D.W. declared receiving salary from Juno Genetics, UK. A.C. is an employee of Igenomix, Italy and C.R. is an employee of Igenomix, Spain. C.S. received a research grant from FWO, Belgium, not related to the submitted work. I.S. declared being a Co-founder of IVFvision Ltd, UK. J.R.V. declared patents related to 'Methods for haplotyping single-cells' and 'Haplotyping and copy number typing using polymorphic variant allelic frequencies', and being a board member of Preimplantation Genetic Diagnosis International Society (PGDIS) and International Society for Prenatal Diagnosis (ISPD). K.S. reported being Chair-elect of ESHRE. The other authors had nothing to disclose. This Good Practice Recommendations (GPR) document represents the views of ESHRE, which are the result of consensus between the relevant ESHRE stakeholders and are based on the scientific evidence available at the time of preparation. ESHRE GPRs should be used for information and educational purposes. They should not be interpreted as setting a standard of care or be deemed inclusive of all proper methods of care, or be exclusive of other methods of care reasonably directed to obtaining the same results. They do not replace the need for application of clinical judgement to each individual presentation, or variations based on locality and facility type. Furthermore, ESHRE GPRs do not constitute or imply the endorsement, or favouring, of any of the included technologies by ESHRE.

P-798 Utilization of Haploid Androgenetic Blastomeres to Characterize Male Genomic Inheritance and as a Male Gamete Substitute

Abstract Study question Can sperm genomes be replicated by generating androgenetic embryos, and can resulting haploid blastomeres be used as a substitute for male gametes with known genotype? Summary answer Once utilized as male gametes, haploid androgenetic blastomeres can be used for gamete genotyping and can sustain embryo development to live offspring. What is known already Heterozygosity in human spermatozoa may represent a risk to offspring health. This has led to attempts to replicate the gamete genome by generating haploid embryonic stem cells (haESCs) that can be used to typify heterozygosity or as gamete substitutes. Although haESC lines are unstable with a tendency to self-diploidize, utilizing haploid androgenetic cells in the early embryonic stages, such as the blastomere stage, can provide multiple copies of genetically functional pseudo-gametes. Study design, size, duration Haploid androgenetic embryos were generated using spermatozoa from B6 heterozygous mice (B6-EGFP x B6D2F1) and cultured until the 8-cell stage. Individual haploid blastomeres with green fluorescent protein (GFP) expression were fused with activated recipient oocytes. Resulting conceptuses were cultured in a time-lapse system to monitor each stage of preimplantation development. Blastocysts with GFP expression were transferred to pseudo-pregnant surrogates. Pregnancy and offspring reproductive health were evaluated. Piezo-actuated ICSI was performed on unmanipulated oocytes as control. Participants/materials, setting, methods To generate haploid androgenetic embryos, metaphase II oocytes of B6D2F1 mice were treated with cytochalasin B, enucleated under Oosight®, and inseminated by piezo-actuated ICSI. Haploid androgenetic embryos were cultured to the 8-cell stage. Androgenetic blastomeres with GFP expression were coated with inactivated Sendai-virus and grafted to an activated recipient oocyte from another cohort. Cleavage parameters of reconstructed zygotes were compared to controls. Blastocysts were transferred into the uterine cavity of a 2.5-day post-coitus CD-1 surrogate. Main results and the role of chance A total of 421 oocytes were enucleated with a survival rate of 98.6%. The ooplasts underwent ICSI; 351 (84.6%) constructs developed a single male pronucleus 4-6h post-ICSI, and 208 (59.3%) constructs cleaved to the 8-cell stage. Parthenogenic activation by calcium ionophore was successful in 96.4% of the recipient oocytes, confirmed by the appearance of a single female pronucleus. A total of 148 haploid androgenotes with GFP expression were grafted onto corresponding activated oocytes; 145 successfully fused. Up to 2 unused sibling haploid blastomeres per embryo were genotyped using whole exome sequencing by a reference laboratory. A total of 481 untreated oocytes were inseminated, yielding 437 control zygotes. The cleavage of reconstructed embryos into the 2-cell (96.4%), 4-cell (94.3%), and 8-cell stage (91.4%) was comparable to that of control conceptuses (97.5%, 93.8%, and 92.7% respectively). Although morula compaction (80.0%) and blastulation (60.7%) were lower than control (89.2% and 80.8%, P <0.001, respectively), morphokinetic parameters were comparable. A total of 45 blastocysts were transferred into 5 recipient mice; 8 blastocysts implanted (17.8%) in 2 surrogates, yielding 5 live offspring (11.1%) weighing 1.51±0.22g. All live offspring expressed GFP and were confirmed fertile once reaching adulthood. Limitations, reasons for caution Although copies of gamete substitutes were generated that could be genotyped and used to produce live offspring with the desired phenotype in a mouse model, the system needs to be optimized and proven reproducible in humans, specifically in regard to the role of the sperm centrosome. Wider implications of the findings Once our proposed technique is implemented in humans, it may benefit patients to control germline heterozygosity by allowing genotyping of identical copies of the gametes. This technique, when used in conjunction with heritable genomic editing, can obviate the inherently highly condensed sperm genome to provide genome-edited gamete substitutes. Trial registration number N/A

The role of highly dispersed silica nanoparticles in the realization of the effects of granulosa on the maturation and fertilization competence of Sus scrofa domesticus oocytes.

Reproductive technologies are some of the key directions in the context of the need to preserve and select highly productive farmed animals in terms of economically useful traits. Improvements of the existing models of the in vitro oocyte maturation system help to solve the problem of low yield of porcine embryos at the final stages of preimplantation development. In the present study, a model of culture medium for gametes (NCSU-23 with 10 % homologous follicular fluid, 10 IU hCG and 10 IU eCG) modernized by the addition of 1·106 granulosa cells (GCs) per ml and 0.001 % of highly dispersed silica nanoparticles (HDSn) is proposed for use in the IVM and IVF technology of donor porcine oocytes. Analysis of the oocyte chromatin status by the Tarkowsky method and assessment of the level of destructive changes in chromatin (apoptosis, pyknosis) revealed a significant percentage increase in matured oocytes and a decrease in the proportion of granulosa cells with degenerated chromatin when using the original culture system. The positive effects of a joint addition of GCs and HDSn to the maturation system have made it possible to increase the indicators of the meiotic maturation and fertilization of oocytes. Optimal results of developmental competence of oocytes were achieved with the joint use of GCs and HDSn in the maturation system (the proportion of matured cells reached 89 %, the level of oocytes with chromosome degeneration was 12 %, 39 % of embryos reached the final stage of preimplantation development). The positive effect of HDSn on oocyte fertilization was accompanied by an abrupt decrease in destructive processes in GCs during culture in the presence of HDSn. The level of somatic cells with pyknotic nuclei was 32 % and the level of apoptosis (TUNELtest), 21 %, compared with the control (43 and 31 %, p <0.01, respectively). Thus, a high efficiency of the porcine oocyte maturation system in the joint culture of gametes with GCs and HDSn was revealed. It makes it possible to recommend a model of this culture medium at the IVM of female gametes of Sus scrofa domesticus for improving the quality of donor oocytes used in cell and genetic engineering.

Maternal Dppa2 and Dppa4 are dispensable for zygotic genome activation but important for offspring survival.

ABSTRACTZygotic genome activation (ZGA) represents the initiation of transcription following fertilisation. Despite its importance, we know little of the molecular events that initiate mammalian ZGA in vivo. Recent in vitro studies in mouse embryonic stem cells have revealed developmental pluripotency associated 2 and 4 (Dppa2/4) as key regulators of ZGA-associated transcription. However, their roles in initiating ZGA in vivo remain unexplored. We reveal that Dppa2/4 proteins are present in the nucleus at all stages of preimplantation development and associate with mitotic chromatin. We generated conditional single and double maternal knockout mouse models to deplete maternal stores of Dppa2/4. Importantly, Dppa2/4 maternal knockout mice were fertile when mated with wild-type males. Immunofluorescence and transcriptome analyses of two-cell embryos revealed that, although ZGA took place, there were subtle defects in embryos that lacked maternal Dppa2/4. Strikingly, heterozygous offspring that inherited the null allele maternally had higher preweaning lethality than those that inherited the null allele paternally. Together, our results show that although Dppa2/4 are dispensable for ZGA transcription, maternal stores have an important role in offspring survival, potentially via epigenetic priming of developmental genes.

Identical twins carry a persistent epigenetic signature of early genome programming

Monozygotic (MZ) twins and higher-order multiples arise when a zygote splits during pre-implantation stages of development. The mechanisms underpinning this event have remained a mystery. Because MZ twinning rarely runs in families, the leading hypothesis is that it occurs at random. Here, we show that MZ twinning is strongly associated with a stable DNA methylation signature in adult somatic tissues. This signature spans regions near telomeres and centromeres, Polycomb-repressed regions and heterochromatin, genes involved in cell-adhesion, WNT signaling, cell fate, and putative human metastable epialleles. Our study also demonstrates a never-anticipated corollary: because identical twins keep a lifelong molecular signature, we can retrospectively diagnose if a person was conceived as monozygotic twin.

Genomic imprinting in mouse blastocysts is predominantly associated with H3K27me3

In mammalian genomes, differentially methylated regions (DMRs) and histone marks including trimethylation of histone 3 lysine 27 (H3K27me3) at imprinted genes are asymmetrically inherited to control parentally-biased gene expression. However, neither parent-of-origin-specific transcription nor imprints have been comprehensively mapped at the blastocyst stage of preimplantation development. Here, we address this by integrating transcriptomic and epigenomic approaches in mouse preimplantation embryos. We find that seventy-one genes exhibit previously unreported parent-of-origin-specific expression in blastocysts (nBiX: novel blastocyst-imprinted expressed). Uniparental expression of nBiX genes disappears soon after implantation. Micro-whole-genome bisulfite sequencing (µWGBS) of individual uniparental blastocysts detects 859 DMRs. We further find that 16% of nBiX genes are associated with a DMR, whereas most are associated with parentally-biased H3K27me3, suggesting a role for Polycomb-mediated imprinting in blastocysts. nBiX genes are clustered: five clusters contained at least one published imprinted gene, and five clusters exclusively contained nBiX genes. These data suggest that early development undergoes a complex program of stage-specific imprinting involving different tiers of regulation.

Features of DNA Repair in the Early Stages of Mammalian Embryonic Development.

Cell repair machinery is responsible for protecting the genome from endogenous and exogenous effects that induce DNA damage. Mutations that occur in somatic cells lead to dysfunction in certain tissues or organs, while a violation of genomic integrity during the embryonic period often leads to death. A mammalian embryo’s ability to respond to damaged DNA and repair it, as well as its sensitivity to specific lesions, is still not well understood. In this review, we combine disparate data on repair processes in the early stages of preimplantation development in mammalian embryos.

Understanding Chromosome Structure During Early Mouse Development by a Single-Cell Hi-C Analysis.

Over the past two decades, the development of chromosome conformation capture technologies has allowed to intensively probe the properties of genome folding in various cell types. High-throughput versions of these C-based assays (named Hi-C) have released the mapping of 3D chromosome folding for the entire genomes. Applied to mammalian preimplantation embryos, it has revealed a unique chromosome organization after fertilization when a new individual is being formed. However, the questions of whether specific structures could arise depending on their parental origins or of their transcriptional status remain open. Our method chapter is dedicated to the technical description on how applying scHi-C to mouse embryos at different stages of preimplantation development. This approach capitalized with the limited amount of material available at these developmental stages. It also provides new research avenues, such as the study of mutant embryos for further functional studies.

Партеногенетическое развитие эмбрионов Bos taurus из ооцитов, созревших в различных системах культивирования

Abstract. Identification of the factors determining of donor’s oocyte competence to parthenogenetic development will allow developing an effective method for obtaining parthenotes to solve fundamental problems of regulating gene activity in ontogenesis, creating homozygous embryonic stem cell lines, improving the stages of cloning technology, and modeling of in vitro oocyte maturation media. The purpose of study is to evaluate the potencies of Bos taurus oocytes matured in different culture systems to cold shock-induced parthenogenesis. Methods. For oocyte maturation, culture systems of the following composition were used: 1 – TC-199 with 10 % fetal bovine serum (FBS), 50 μg/ml estradiol, 10 μg/ml luteinizing hormone, 10 μg/ml follicle-stimulating hormone; 2 – TC-199 with 10 % estrous serum of cows; 3 – TC-199 with 50 % fluid from follicles (Ø 3–8 mm); 4 – TC-199 with 50 % protein of follicular fluid (molecular weight of 65 kDa); 5 – TC-199 with 10 % FBS, 1×106 granulosa cells/ml medium; 6 – TC-199 with 10 % FBS and walls of follicles (Ø 6–8 mm); 7 – TC-199 with 10 % FBS, 1×106 granulosa cells/ml medium and walls of follicles (Ø 6–8 mm). After 24 hours of cultivation, the oocytes were activated by cold shock (exposure time 20 minutes, temperature 0…–4 °C. Results. The proportion of embryos at the stages of late morula and blastocysts from oocytes matured in system 7 was 45 % (58/129), which was significantly higher than in other systems: 1 – 28 % (39/141), P &lt; 0.05; 2 – 31 % (42/137), P &lt; 0.05; 3 – 25 % (33/133), P &lt; 0.01; 4 – 18 % (25/139), P &lt; 0.001; 5 – 31 % (41/132), P &lt; 0.05; 6 – 33 % (43/129). The introduction of estradiol or structural components of antral follicles into the oocytes maturation medium contributed to an increase in the proportion of parthenotes at the preimplantation stages of development, including blastocysts, and a decrease in the level of degenerated embryos. Scientific novelty. A comparative morphological analysis of the potentials for parthenogenesis of bovine oocytes matured in various culture systems and activated by cold shock was carried out for the first time. Optimal systems for in vitro maturation of female gametes were proposed. Based on the analysis of the results, we recommend before induction to parthenogenesis bovine oocytes culture in media supplemented with 50 ng/ml estradiol or structural components of antral follicles producing estradiol.