Apposition to endometrial epithelial cells activates mouse blastocysts for implantation.

How do interactions between blastocyst-stage embryos and endometrial epithelial cells regulate the early stages of implantation in an in vitro model?Mouse blastocyst apposition with human endometrial epithelial cells initiates trophectoderm differentiation to trophoblast, which goes on to breach the endometrial epithelium.In vitro models using mouse blastocysts and human endometrial cell lines have proven invaluable in the molecular characterisation of embryo attachment to endometrial epithelium at the onset of implantation. Genes involved in embryonic breaching of the endometrial epithelium have not been investigated in such in vitro models.This study used an established in vitro model of implantation to examine cellular and molecular interactions during blastocyst attachment to endometrial epithelial cells.Mouse blastocysts developed from embryonic day (E) 1.5 in vitro were hatched and co-cultured with confluent human endometrial adenocarcinoma-derived Ishikawa cells in serum-free medium. A scale of attachment stability based on blastocyst oscillation upon agitation was devised. Blastocysts were monitored for 48 h to establish the kinetics of implantation, and optical sectioning using fluorescence microscopy revealed attachment and invasion interfaces. Quantitative PCR was used to determine blastocyst gene expression. Data from a total of 680 mouse blastocysts are reported, with 3-6 experimental replicates. T-test and ANOVA analyses established statistical significance at P < 0.05, P < 0.01 and P < 0.001.Hatched E4.5 mouse blastocysts exhibited weak attachment to confluent Ishikawa cells over the first 24 h of co-culture, with intermediate and stable attachment occurring from 28 h (E5.5 + 4 h) in a hormone-independent manner. Attached embryos fixed after 48 h (E6.5) frequently exhibited outgrowths, characterised morphologically and with antibody markers as trophoblast giant cells (TGCs), which had breached the Ishikawa cell layer. Beginning co-culture at E5.5 also resulted in intermediate and stable attachment from E5.5 + 4 h; however, these embryos did not go on to breach the Ishikawa cell layer, even when co-culture was extended to E7.5 (P < 0.01). Blastocysts cultured from E4.5 in permeable transwell inserts above Ishikawa cells before transfer to direct co-culture at E5.5 went on to attach but failed to breach the Ishikawa cell layer by E6.5 (P < 0.01). Gene expression analysis at E5.5 demonstrated that direct co-culture with Ishikawa cells from E4.5 resulted in downregulation of trophectoderm transcription factors Cdx2 (P < 0.05) and Gata3 (P < 0.05) and upregulation of the TGC transcription factor Hand1 (P < 0.05). Co-culture with non-endometrial human fibroblasts did not alter the expression of these genes.None.The in vitro model used here combines human carcinoma-derived endometrial cells with mouse embryos, in which the cellular interactions observed may not fully recapitulate those in vivo. The data gleaned from such models can be regarded as hypothesis-generating, and research is now needed to develop more sophisticated models of human implantation combining multiple primary endometrial cell types with surrogate and real human embryos.This study implicates blastocyst apposition to endometrial epithelial cells as a critical step in trophoblast differentiation required for implantation. Understanding this maternal regulation of the embryonic developmental programme may lead to novel treatments for infertility.This work was supported by funds from the charities Wellbeing of Women (RG1442) and Diabetes UK (15/0005207), and studentship support for SCB from the Anatomical Society. No conflict of interest is declared

At age 9, the methylome of assisted reproductive technology children that underwent embryo culture in different media is not significantly different on a genome-wide scale

STUDY QUESTION: Can we detect DNA methylation differences between ART children that underwent embryo culture in different media? SUMMARY ANSWER: We identified no significant differences in site-specific or regional DNA methylation between the different culture medium groups. WHAT IS KNOWN ALREADY: Embryo culture in G3 or K-SICM medium leads to differences in embryonic, neonatal and childhood outcomes, including growth and weight. The methylome may mediate this association as the period of in vitro culture of ART treatments coincides with epigenetic reprogramming. STUDY DESIGN, SIZE, DURATION: This study was conducted as a follow-up to a previous culture medium comparison study in which couples were pseudo-randomized to embryo culture in G3 or K-SICM medium. Of the resultant singletons, 120 (n = 65 G3, n = 55 K-SICM), were recruited at age 9. PARTICIPANTS/MATERIALS, SETTING, METHODS: The ART children provided a saliva sample from which the methylome was analysed using the Infinium MethylationEPIC array. After quality and context filtering, 106 (n = 57 G3, n = 49 K-SICM) samples and 659 708 sites were retained for the analyses. Differential methylation analyses were conducted using mixed effects linear models corrected for age, sex, sample plate and cell composition. These were applied to all cytosine-guanine dinucleotide (CpG) sites, various genomic regions (genes, promoters, CpG Islands (CGIs)) and as a targeted analysis of imprinted genes and birth weight-associated CpG sites. Differential variance was assessed using the improved epigenetic variable outliers for risk prediction analysis (iEVORA) algorithm and methylation outliers were identified using a previously defined threshold (upper or lower quartile plus or minus three times the interquartile range, respectively). MAIN RESULTS AND THE ROLE OF CHANCE: After correcting for multiple testing, we did not identify any significantly differentially methylated CpG sites, genes, promoters or CGIs between G3 and K-SICM children despite a lenient corrected P-value threshold of 0.1. Targeted analyses of (sites within) imprinted genes and birth weight-associated sites also did not identify any significant differences. The number of DNA methylation outliers per sample was comparable between the culture medium groups. iEVORA identified 101 differentially variable CpG sites of which 94 were more variable in the G3 group. LARGE SCALE DATA: Gene Expression Omnibus (GEO) GSE196432. LIMITATIONS, REASONS FOR CAUTION: To detect significant methylation differences with a magnitude of <10% between the groups many more participants would be necessary; however, the clinical relevance of such small differences is unclear. WIDER IMPLICATIONS OF THE FINDINGS: The results of this study are reassuring, suggesting that if there is an effect of the culture medium on DNA methylation (and methylation-mediated diseases risk), it does not differ between the two media investigated here. The findings concur with other methylome studies of ART neonates and children that underwent embryo culture in different media, which also found no significant methylome differences. STUDY FUNDING/COMPETING INTEREST(S): Study funded by March of Dimes (6-FY13-153), EVA (Erfelijkheid Voortplanting & Aanleg) specialty programme (grant no. KP111513) of Maastricht University Medical Centre (MUMC+) and the Horizon 2020 innovation (ERIN) (grant no. EU952516) of the European Commission. The authors do not report any conflicts of interest relevant to this study. TRIAL REGISTRATION NUMBER: Dutch Trial register-NL4083

Liquid biopsy:state of reproductive medicine and beyond

Liquid biopsy is the process of sampling and analyzing body fluids, which enables non-invasive monitoring of complex biological systems in vivo. Liquid biopsy has myriad applications in health and disease as a wide variety of components, ranging from circulating cells to cell-free nucleic acid molecules, can be analyzed. Here, we review different components of liquid biopsy, survey state-of-the-art, non-invasive methods for detecting those components, demonstrate their clinical applications and discuss ethical considerations. Furthermore, we emphasize the importance of artificial intelligence in analyzing liquid biopsy data with the aim of developing ethically-responsible non-invasive technologies that can enhance individualized healthcare. While previous reviews have mainly focused on cancer, this review primarily highlights applications of liquid biopsy in reproductive medicine

Embryo tracking system for high-throughput sequencing-based preimplantation genetic testing

STUDY QUESTION: Can the embryo tracking system (ETS) increase safety, efficacy and scalability of massively parallel sequencing-based preimplantation genetic testing (PGT)? SUMMARY ANSWER: Applying ETS-PGT, the chance of sample switching is decreased, while scalability and efficacy could easily be increased substantially. WHAT IS KNOWN ALREADY: Although state-of-the-art sequencing-based PGT methods made a paradigm shift in PGT, they still require labor intensive library preparation steps that makes PGT cost prohibitive and poses risks of human errors. To increase the quality assurance, efficiency, robustness and throughput of the sequencing-based assays, barcoded DNA fragments have been used in several aspects of next-generation sequencing (NGS) approach. STUDY DESIGN, SIZE, DURATION: We developed an ETS that substantially alleviates the complexity of the current sequencing-based PGT. With (n = 693) and without (n = 192) ETS, the downstream PGT procedure was performed on both bulk DNA samples (n = 563) and whole-genome amplified (WGAed) few-cell DNA samples (n = 322). Subsequently, we compared full genome haplotype landscapes of both WGAed and bulk DNA samples containing ETS or no ETS. PARTICIPANTS/MATERIALS, SETTING, METHODS: We have devised an ETS to track embryos right after whole-genome amplification (WGA) to full genome haplotype profiles. In this study, we recruited 322 WGAed DNA samples derived from IVF embryos as well as 563 bulk DNA isolated from peripheral blood of prospective parents. To determine possible interference of the ETS in the NGS-based PGT workflow, barcoded DNA fragments were added to DNA samples prior to library preparation and compared to samples without ETS. Coverages and variants were determined. MAIN RESULTS AND THE ROLE OF CHANCE: Current PGT protocols are quality sensitive and prone to sample switching. To avoid sample switching and increase throughput of PGT by sequencing-based haplotyping, six control steps should be carried out manually and checked by a second person in a clinical setting. Here, we developed an ETS approach in which one step only in the entire PGT procedure needs the four-eyes principal. We demonstrate that ETS not only precludes error-prone manual checks but also has no effect on the genomic landscape of preimplantation embryos. Importantly, our approach increases efficacy and throughput of the state-of-the-art PGT methods. LIMITATIONS, REASONS FOR CAUTION: Even though the ETS simplified sequencing-based PGT by avoiding potential errors in six steps in the protocol, if the initial assignment is not performed correctly, it could lead to cross-contamination. However, this can be detected in silico following downstream ETS analysis. Although we demonstrated an approach to evaluate purity of the ETS fragment, it is recommended to perform a pre-PGT quality control assay of the ETS amplicons with non-human DNA, such that the purity of each ETS molecule can be determined prior to ETS-PGT. WIDER IMPLICATIONS OF THE FINDINGS: The ETS-PGT approach notably increases efficacy and scalability of PGT. ETS-PGT has broad applicative value, as it can be tailored to any single- and few-cell sequencing approach where the starting specimen is scarce, as opposed to other methods that require a large number of cells as the input. Moreover, ETS-PGT could easily be adapted to any sequencing-based diagnostic method, including PGT for structural rearrangements and aneuploidies by low-pass sequencing as well as non-invasive prenatal testing. STUDY FUNDING/COMPETING INTEREST(S): M.Z.E. is supported by the EVA (Erfelijkheid Voortplanting & Aanleg) specialty program (grant no. KP111513) of Maastricht University Medical Centre (MUMC+), and the Horizon 2020 innovation (ERIN) (grant no. EU952516) of the European Commission. TRIAL REGISTRATION NUMBER: N/A

Prevalence of chromosomal alterations in first-trimester spontaneous pregnancy loss

Pregnancy loss is often caused by chromosomal abnormalities of the conceptus. The prevalence of these abnormalities and the allocation of (ab)normal cells in embryonic and placental lineages during intrauterine development remain elusive. In this study, we analyzed 1,745 spontaneous pregnancy losses and found that roughly half (50.4%) of the products of conception (POCs) were karyotypically abnormal, with maternal and paternal age independently contributing to the increased genomic aberration rate. We applied genome haplarithmisis to a subset of 94 pregnancy losses with normal parental and POC karyotypes. Genotyping of parental DNA as well as POC extra-embryonic mesoderm and chorionic villi DNA, representing embryonic and trophoblastic tissues, enabled characterization of the genomic landscape of both lineages. Of these pregnancy losses, 35.1% had chromosomal aberrations not previously detected by karyotyping, increasing the rate of aberrations of pregnancy losses to 67.8% by extrapolation. In contrast to viable pregnancies where mosaic chromosomal abnormalities are often restricted to chorionic villi, such as confined placental mosaicism, we found a higher degree of mosaic chromosomal imbalances in extra-embryonic mesoderm rather than chorionic villi. Our results stress the importance of scrutinizing the full allelic architecture of genomic abnormalities in pregnancy loss to improve clinical management and basic research of this devastating condition