Global Gene Expression Profiling of Individual Human Oocytes and Embryos Demonstrates Heterogeneity in Early Development

Early development in humans is characterised by low and variable embryonic viability, reflected in low fecundity and high rates of miscarriage, relative to other mammals. Data from assisted reproduction programmes provides additional evidence that this is largely mediated at the level of embryonic competence and is highly heterogeneous among embryos. Understanding the basis of this heterogeneity has important implications in a number of areas including: the regulation of early human development, disorders of pregnancy, assisted reproduction programmes, the long term health of children which may be programmed in early development, and the molecular basis of pluripotency in human stem cell populations. We have therefore investigated global gene expression profiles using polyAPCR amplification and microarray technology applied to individual human oocytes and 4-cell and blastocyst stage embryos. In order to explore the basis of any variability in detail, each developmental stage is replicated in triplicate. Our data show that although transcript profiles are highly stage-specific, within each stage they are relatively variable. We describe expression of a number of gene families and pathways including apoptosis, cell cycle and amino acid metabolism, which are variably expressed and may be reflective of embryonic developmental competence. Overall, our data suggest that heterogeneity in human embryo developmental competence is reflected in global transcript profiles, and that the vast majority of existing human embryo gene expression data based on pooled oocytes and embryos need to be reinterpreted

Global gene expression profiling of individual human oocytes and embryos demonstrates heterogeneity in early development

Early development in humans is characterised by low and variable embryonic viability, reflected in low fecundity and high rates of miscarriage, relative to other mammals. Data from assisted reproduction programmes provides additional evidence that this is largely mediated at the level of embryonic competence and is highly heterogeneous among embryos. Understanding the basis of this heterogeneity has important implications in a number of areas including: the regulation of early human development, disorders of pregnancy, assisted reproduction programmes, the long term health of children which may be programmed in early development, and the molecular basis of pluripotency in human stem cell populations. We have therefore investigated global gene expression profiles using polyAPCR amplification and microarray technology applied to individual human oocytes and 4-cell and blastocyst stage embryos. In order to explore the basis of any variability in detail, each developmental stage is replicated in triplicate. Our data show that although transcript profiles are highly stage-specific, within each stage they are relatively variable. We describe expression of a number of gene families and pathways including apoptosis, cell cycle and amino acid metabolism, which are variably expressed and may be reflective of embryonic developmental competence. Overall, our data suggest that heterogeneity in human embryo developmental competence is reflected in global transcript profiles, and that the vast majority of existing human embryo gene expression data based on pooled oocytes and embryos need to be reinterpreted

Temperature of embryo culture for assisted reproduction

BACKGROUND: 'Infertility' is defined as the failure to achieve pregnancy after 12 months or more of regular unprotected sexual intercourse. One in six couples experience a delay in becoming pregnant. In vitro fertilisation (IVF) is one of the assisted reproductive techniques used to enable couples to achieve a live birth. One of the processes involved in IVF is embryo culture in an incubator, where a stable environment is created and maintained. The incubators are set at approximately 37°C, which is based on the human core body temperature, although several studies have shown that this temperature may in fact be lower in the female reproductive tract and that this could be beneficial. In this review we have included randomised controlled trials which compared different temperatures of embryo culture. OBJECTIVES: To assess different temperatures of embryo culture for human assisted reproduction, which may lead to higher live birth rates. SEARCH METHODS: We searched the following databases and trial registers: the Cochrane Gynaecology and Fertility (CGF) Group Specialised Register of Controlled Trials, the Cochrane Central Register of Studies Online, MEDLINE, Embase, PsycINFO, CINAHL, clinicaltrials.gov, The World Health Organization International Trials Registry Platform search portal, DARE, Web of Knowledge, OpenGrey, LILACS database, PubMed and Google Scholar. Furthermore, we manually searched the references of relevant articles and contacted experts in the field to obtain additional data. We did not restrict the search by language or publication status. We performed the last search on 6 March 2019. SELECTION CRITERIA: Two review authors independently screened the titles and abstracts of articles retrieved by the search. Full texts of potentially eligible randomised controlled trials (RCTs) were obtained and screened. We included all RCTs which compared different temperatures of embryo culture in IVF or intracytoplasmic sperm injection (ICSI), with a minimum difference in temperature between the two incubators of ≥ 0.5°C. The search process is shown in the PRISMA flow chart. DATA COLLECTION AND ANALYSIS: Two review authors independently assessed trial eligibility and risk of bias and extracted data from the included studies; the third review author resolved any disagreements. We contacted trial authors to provide additional data. The primary review outcomes were live birth and miscarriage. Clinical pregnancy, ongoing pregnancy, multiple pregnancy and adverse events were secondary outcomes. All extracted data were dichotomous outcomes, and odds ratios (OR) were calculated with 95% confidence intervals (CIs) on an intention-to-treat basis. We assessed the overall quality of the evidence for the main comparisons using GRADE methods. MAIN RESULTS: We included three RCTs, with a total of 563 women, that compared incubation of embryos at 37.0°C or 37.1°C with a lower incubator temperature (37.0°C versus 36.6°C, 37.1°C versus 36.0°C, 37.0° versus 36.5°C). Live birth, miscarriage, clinical pregnancy, ongoing pregnancy and multiple pregnancy were reported. After additional information from the authors, we confirmed one study as having no adverse events; the other two studies did not report adverse events. We did not perform a meta-analysis as there were not enough studies included per outcome. Live birth was not graded since there were no data of interest available. The evidence for the primary outcome, miscarriage, was of very low quality. The evidence for the secondary outcomes, clinical pregnancy, ongoing pregnancy and multiple pregnancy was also of very low quality. We downgraded the evidence because of high risk of bias (for performance bias) and imprecision due to limited included studies and wide CIs.Only one study reported the primary outcome, live birth (n = 52). They performed randomisation at the level of oocytes and not per woman, and used a paired design whereby two embryos, one from 36.0°C and one from 37.0°C, were transferred. The data from this study were not interpretable in a meaningful way and therefore not presented. Only one study reported miscarriage. We are uncertain whether incubation at a lower temperature decreases the miscarriage (odds ratio (OR) 0.90, 95% CI 0.52 to 1.55; 1 study, N = 412; very low-quality evidence).Of the two studies that reported clinical pregnancy, only one of them performed randomisation per woman. We are uncertain whether a lower temperature improves clinical pregnancy compared to 37°C for embryo incubation (OR 1.08, 95% CI 0.73 to 1.60; 1 study, N = 412; very low-quality evidence). For the outcome, ongoing pregnancy, we are uncertain if a lower temperature is better than 37°C (OR 1.10, 95% CI 0.75 to 1.62; 1 study, N = 412; very low quality-evidence). Multiple pregnancy was reported by two studies, one of which used a paired design, which made it impossible to report the data per temperature. We are uncertain if a temperature lower than 37°C reduces multiple pregnancy (OR 0.80, 95% CI 0.31 to 2.07; 1 study, N = 412; very low-quality evidence). There was insufficient evidence to make a conclusion regarding adverse events, as no studies reported data suitable for analysis. AUTHORS' CONCLUSIONS: This review evaluated different temperatures for embryo culture during IVF. There is a lack of evidence for the majority of outcomes in this review. Based on very low-quality evidence, we are uncertain if incubating at a lower temperature than 37°C improves pregnancy outcomes. More RCTs are needed for comparing different temperatures of embryo culture which require reporting of clinical outcomes as live birth, miscarriage, clinical pregnancy and adverse events

Interactome comparison of human embryonic stem cell lines with the inner cell mass and trophectoderm

Networks of interacting co-regulated genes distinguish the inner cell mass (ICM) from the differentiated trophectoderm (TE) in the preimplantation blastocyst, in a species specific manner. In mouse the ground state pluripotency of the ICM appears to be maintained in murine embryonic stem cells (ESCs) derived from the ICM. This is not the case for human ESCs. In order to gain insight into this phenomenon, we have used quantitative network analysis to identify how similar human (h)ESCs are to the human ICM. Using the hESC lines MAN1, HUES3 and HUES7 we have shown that all have only a limited overlap with ICM specific gene expression, but that this overlap is enriched for network properties that correspond to key aspects of function including transcription factor activity and the hierarchy of network modules. These analyses provide an important framework which highlights the developmental origins of hESCs

Going to extremes : The Goldilocks/Lagom principle and data distribution

Numerical data in biology and medicine are commonly presented as mean or median with error or confidence limits, to the exclusion of individual values. Analysis of our own and others' data indicates that this practice risks excluding € Goldilocks' effects in which a biological variable falls within a range between € too much' and € too little' with a region between where its function is € just right'; a concept captured by the Swedish term € Lagom'. This was confirmed by a narrative search of the literature using the PubMed database, which revealed numerous relationships of biological and clinical phenomena of the Goldilocks/Lagom form including quantitative and qualitative examples from the health and social sciences. Some possible mechanisms underlying these phenomena are considered. We conclude that retrospective analysis of existing data will most likely reveal a vast number of such distributions to the benefit of medical understanding and clinical care and that a transparent approach of presenting each value within a dataset individually should be adopted to ensure a more complete evaluation of research studies in future

Characterisation of Osteopontin in an In Vitro Model of Embryo Implantation

At the onset of pregnancy, embryo implantation is initiated by interactions between the endometrial epithelium and the outer trophectoderm cells of the blastocyst. Osteopontin (OPN) is expressed in the endometrium and is implicated in attachment and signalling roles at the embryo–epithelium interface. We have characterised OPN in the human endometrial epithelial Ishikawa cell line using three different monoclonal antibodies, revealing at least nine distinct molecular weight forms and a novel secretory pathway localisation in the apical domain induced by cell organisation into a confluent epithelial layer. Mouse blastocysts co-cultured with Ishikawa cell layers served to model embryo apposition, attachment and initial invasion at implantation. Exogenous OPN attenuated initial, weak embryo attachment to Ishikawa cells but did not affect the attainment of stable attachment. Notably, exogenous OPN inhibited embryonic invasion of the underlying cell layer, and this corresponded with altered expression of transcription factors associated with differentiation from trophectoderm (Gata2) to invasive trophoblast giant cells (Hand1). These data demonstrate the complexity of endometrial OPN forms and suggest that OPN regulates embryonic invasion at implantation by signalling to the trophectoder

Osmotic stress induces JNK-dependent embryo invasion in a model of implantation

In vitro culture during assisted reproduction technologies (ARTs) exposes pre-implantation embryos to environmental stressors, such as non-physiological nutritional, oxidative and osmotic conditions. The effects on subsequent implantation are not well understood but could contribute to poor ART efficiency and outcomes. We have used exposure to hyperosmolarity to investigate the effects of stress on the ability of embryos to interact with endometrial cells in an in vitro model. Culturing mouse blastocysts for 2 h in medium with osmolarity raised by 400 mosmol induced blastocoel collapse and re-expansion, but did not affect subsequent attachment to, or invasion of, the endometrial epithelial Ishikawa cell line. Inhibition of stress-responsive c-Jun N-terminal kinase (JNK) activity with SP600125 did not affect the intercellular interactions between these embryos and the epithelial cells. Four successive cycles of hyperosmotic stress at E5.5 had no effect on attachment, but promoted embryonic breaching of the epithelial cell layer by trophoblast giant cells in a JNK-dependent manner. These findings suggest that acute stress at the blastocyst stage may promote trophoblast breaching of the endometrial epithelium at implantation and implicates stress signalling through JNK in the process of trophectoderm differentiation into the invasive trophoblast necessary for the establishment of pregnancy. The data may lead to increased understanding of factors governing ART success rates and safety

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