Methylome-wide analysis of IVF neonates that underwent embryo culture in different media revealed no significant differences.

A growing number of children born are conceived through in vitro fertilisation (IVF), which has been linked to an increased risk of adverse perinatal outcomes, as well as altered growth profiles and cardiometabolic differences in the resultant individuals. Some of these outcomes have also been shown to be influenced by the use of different IVF culture media and this effect is hypothesised to be mediated epigenetically, e.g. through the methylome. As such, we profiled the umbilical cord blood methylome of IVF neonates that underwent preimplantation embryo development in two different IVF culture media (G5 or HTF), using the Infinium Human Methylation EPIC BeadChip. We found no significant methylation differences between the two groups in terms of: (i) systematic differences at CpG sites or regions, (ii) imprinted sites/genes or birth weight-associated sites, (iii) stochastic differences presenting as DNA methylation outliers or differentially variable sites, and (iv) epigenetic gestational age acceleration.

Comparing the cumulative live birth rate of cleavage-stage versus blastocyst-stage embryo transfers between IVF cycles: a study protocol for a multicentre randomised controlled superiority trial (the ToF trial).

INTRODUCTION: In vitro fertilisation (IVF) has evolved as an intervention of choice to help couples with infertility to conceive. In the last decade, a strategy change in the day of embryo transfer has been developed. Many IVF centres choose nowadays to transfer at later stages of embryo development, for example, transferring embryos at blastocyst stage instead of cleavage stage. However, it still is not known which embryo transfer policy in IVF is more efficient in terms of cumulative live birth rate (cLBR), following a fresh and the subsequent frozen-thawed transfers after one oocyte retrieval. Furthermore, studies reporting on obstetric and neonatal outcomes from both transfer policies are limited. METHODS AND ANALYSIS: We have set up a multicentre randomised superiority trial in the Netherlands, named the Three or Fivetrial. We plan to include 1200 women with an indication for IVF with at least four embryos available on day 2 after the oocyte retrieval. Women are randomly allocated to either (1) control group: embryo transfer on day 3 and cryopreservation of supernumerary good-quality embryos on day 3 or 4, or (2) intervention group: embryo transfer on day 5 and cryopreservation of supernumerary good-quality embryos on day 5 or 6. The primary outcome is the cLBR per oocyte retrieval. Secondary outcomes include LBR following fresh transfer, multiple pregnancy rate and time until pregnancy leading a live birth. We will also assess the obstetric and neonatal outcomes, costs and patients' treatment burden. ETHICS AND DISSEMINATION: The study protocol has been approved by the Central Committee on Research involving Human Subjects in the Netherlands in June 2018 (CCMO NL 64060.000.18). The results of this trial will be submitted for publication in international peer-reviewed and in open access journals. TRIAL REGISTRATION NUMBER: Netherlands Trial Register (NL 6857).

Comparison of DNA methylation patterns of parentally imprinted genes in placenta derived from IVF conceptions in two different culture media.

STUDY QUESTION: Is there a difference in DNA methylation status of imprinted genes in placentas derived from IVF conceptions where embryo culture was performed in human tubal fluid (HTF) versus G5 culture medium? SUMMARY ANSWER: We found no statistically significant differences in the mean DNA methylation status of differentially methylated regions (DMRs) associated with parentally imprinted genes in placentas derived from IVF conceptions cultured in HTF versus G5 culture medium. WHAT IS KNOWN ALREADY: Animal studies indicate that the embryo culture environment affects the DNA methylation status of the embryo. In humans, birthweight is known to be affected by the type of embryo culture medium used. The effect of embryo culture media on pregnancy, birth and child development may thus be mediated by differential methylation of parentally imprinted genes in the placenta. STUDY DESIGN, SIZE, DURATION: To identify differential DNA methylation of imprinted genes in human placenta derived from IVF conceptions exposed to HTF or G5 embryo culture medium, placenta samples (n = 43 for HTF, n = 54 for G5) were collected between 2010 and 2012 s as part of a multi-center randomized controlled trial in the Netherlands comparing these embryo culture media. Placenta samples from 69 naturally conceived (NC) live births were collected during 2008-2013 in the Netherlands as reference material. PARTICIPANTS/MATERIALS, SETTING, METHODS: To identify differential DNA methylation of imprinted genes, we opted for an amplicon-based sequencing strategy on an Illumina MiSeq sequencing platform. DNA was isolated and 34 DMRs associated with well-defined parentally imprinted genes were amplified in a two-step PCR before sequencing using MiSeq technology. Sequencing data were analyzed in a multivariate fashion to eliminate possible confounding effects. MAIN RESULTS AND THE ROLE OF CHANCE: We found no statistically significant differences in the mean DNA methylation status of any of the imprinted DMRs in placentas derived from IVF conceptions cultured in HTF or G5 culture medium. We also did not observe any differences in the mean methylation status per amplicon nor in the variance in methylation per amplicon between the two culture medium.groups. A separate surrogate variable analysis also demonstrated that the IVF culture medium was not associated with the DNA methylation status of these DMRs. The mean methylation level and variance per CpG was equal between HTF and G5 placenta. Additional comparison of DNA methylation status of NC placenta samples revealed no statistically significant differences in mean amplicon and CpG methylation between G5, HTF and NC placenta; however, the number of placenta samples exhibiting outlier methylation levels was higher in IVF placenta compared to NC (P < 0.00001). Also, we were able to identify 37 CpG sites that uniquely displayed outlier methylation in G5 placentas and 32 CpG sites that uniquely displayed outlier methylation in HTF. In 8/37 (G5) and 4/32 (HTF) unique outliers CpGs, a medium-specific unique outlier could be directly correlated to outlier methylation of the entire amplicon. LIMITATIONS, REASONS FOR CAUTION: Due to practical reasons, not all placentas were collected during the trial, and we collected the placentas from natural conceptions from a different cohort, potentially creating bias. We limited ourselves to the DNA methylation status of 34 imprinted DMRs, and we studied only the placenta and no other embryo-derived tissues. WIDER IMPLICATIONS OF THE FINDINGS: It has often been postulated, but has yet to be rigorously tested, that imprinting mediates the effects of embryo culture conditions on pregnancy, birth and child development in humans. Since we did not detect any statistically significant effects of embryo culture conditions on methylation status of imprinted genes in the placenta, this suggests that other unexplored mechanisms may underlie these effects. The biological and clinical relevance of detected outliers with respect to methylation levels of CpGs and DMR require additional analysis in a larger sample size as well. Given the importance and the growing number of children born through IVF, research into these molecular mechanisms is urgently needed. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the March of Dimes grant number #6-FY13-153. The authors have no conflicts of interest. TRIAL REGISTRATION NUMBER: Placental biopsies were obtained under Netherlands Trial Registry number 1979 and 1298.