Xiulian Ren

The DNA methylation landscape of human early embryos

DNA methylation is a crucial element in the epigenetic regulation of mammalian embryonic development. However, its dynamic patterns have not been analysed at the genome scale in human pre-implantation embryos due to technical difficulties and the scarcity of required materials. Here we systematically profile the methylome of human early embryos from the zygotic stage through to post-implantation by reduced representation bisulphite sequencing and whole-genome bisulphite sequencing. We show that the major wave of genome-wide demethylation is complete at the 2-cell stage, contrary to previous observations in mice. Moreover, the demethylation of the paternal genome is much faster than that of the maternal genome, and by the end of the zygotic stage the genome-wide methylation level in male pronuclei is already lower than that in female pronuclei. The inverse correlation between promoter methylation and gene expression gradually strengthens during early embryonic development, reaching its peak at the post-implantation stage. Furthermore, we show that active genes, with the trimethylation of histone H3 at lysine 4 (H3K4me3) mark at the promoter regions in pluripotent human embryonic stem cells, are essentially devoid of DNA methylation in both mature gametes and throughout pre-implantation development. Finally, we also show that long interspersed nuclear elements or short interspersed nuclear elements that are evolutionarily young are demethylated to a milder extent compared to older elements in the same family and have higher abundance of transcripts, indicating that early embryos tend to retain higher residual methylation at the evolutionarily younger and more active transposable elements. Our work provides insights into the critical features of the methylome of human early embryos, as well as its functional relation to the regulation of gene expression and the repression of transposable elements.

Tracing the expression of circular RNAs in human pre-implantation embryos

BackgroundPolyA– RNAs have not been widely analyzed in human pre-implantation embryos due to the scarcity of materials. In particular, circular RNA (circRNA), a novel type of polyA– RNA, has not been characterized during human pre-implantation development.ResultsWe systematically analyze polyA+ messenger RNAs (mRNAs) and polyA– RNAs in individual human oocytes and pre-implantation embryos using SUPeR-seq. We de novo identify 10,032 circRNAs from 2974 hosting genes. Most of these circRNAs are developmentally stage-specific and dynamically regulated. Many of them are maternally expressed, implying their potentially important regulatory functions in oogenesis and the formation of totipotent zygotes. Comparison between human and mouse embryos reveals both high conservation and clear distinction between these two species. Human pre-implantation embryos generate more types of circRNA compared with mouse embryos and this is associated with a striking increase of the length of the circRNA flanking introns in humans. We also perform RNA de novo assembly and identify novel transcript units, many of which are potentially novel long non-coding RNAs.ConclusionsThis study reports the first analysis of the whole transcriptome comprising both polyA+ mRNAs and polyA– RNAs including circRNAs during human pre-implantation development. It provides an invaluable resource for analyzing the unique function and complex regulatory mechanisms of circRNAs during this process.

Synchronization between embryo development and endometrium is a contributing factor for rescue ICSI outcome

Recent evidence shows that the outcome of rescue intracytoplasmic sperm injection (ICSI) is unsatisfactory on account of a poor clinical pregnancy rate. These outcomes may be due to either the in-vitro ageing of cultured oocytes before ICSI or the asynchrony between the embryo developmental stage and the endometrial secretory pattern. To address the latter issue, this study performed a retrospective analysis of 534 fresh cycles after rescue ICSI and 64 frozen-thawed cycles in subsequent treatment. Rescue ICSI cycles were divided into three groups: group I included 469 fresh embryo-transfer (FET) cycles; group II included 74 FET cycles in which supernumerary good-quality embryos were also cryopreserved; and group III included 64 frozen-thawed transfer cycles. Group III was considered to have achieved better synchronization than group II. As a result, significantly higher clinical pregnancy (29.69%, 19/64 versus 10.81%, 8/74) and implantation (13.33%, 22/165 versus 5.13%, 8/156) rates were achieved in group III compared with group II (both P<0.05). Therefore, synchronization of embryo development with the endometrium is considered a contributing factor for rescue ICSI outcome. It is recommended that embryos derived from rescue ICSI cycles should be cryopreserved and subsequently used in frozen-thawed cycles. Intracytoplasmic sperm injection (ICSI) of unfertilized 1-day-old oocytes, called rescue ICSI, has frequently been performed in some infertility centres, when fertilization failure sometimes occurs in conventional IVF cycles. Recent studies showed that the outcome of rescue ICSI was unsatisfactory due to poor clinical pregnancy rates. One reason could be asynchrony between the embryo developmental stage and the endometrial secretory pattern. To address this issue, we performed a retrospective analysis of 534 fresh cycles after rescue ICSI (from January 2006 to January 2011) and 64 frozen-thawed transfer cycles in subsequent treatment (from January 2006 to May 2011) in our infertility centre. In this study, rescue ICSI cycles were divided into three groups. As there was no significant difference in womens age (31.22 ± 3.38 versus 31.11 ± 3.27 years) between groups II and III, we principally compared these two groups. Group II included 74 fresh embryo transfer cycles, in which supernumerary good-quality embryos were cryopreserved, and group III included 64 frozen-thawed transfer cycles. Group III was considered to have better synchronization than group II. As a result, significantly higher clinical pregnancy (29.69% versus 10.81%) and implantation (13.33% versus 5.13%) rates were achieved in group III compared with group II. Therefore, endometrial synchronization is considered a contributing factor for rescue ICSI outcome and embryos derived from rescue ICSI cycles should be cryopreserved and subsequently used in frozen-thawed cycles.

Single-cell DNA methylome sequencing of human preimplantation embryos

DNA methylation is a crucial layer of epigenetic regulation during mammalian embryonic development1–3. Although the DNA methylome of early human embryos has been analyzed4–6, some of the key features have not been addressed thus far. Here we performed single-cell DNA methylome sequencing for human preimplantation embryos and found that tens of thousands of genomic loci exhibited de novo DNA methylation. This finding indicates that genome-wide DNA methylation reprogramming during preimplantation development is a dynamic balance between strong global demethylation and drastic focused remethylation. Furthermore, demethylation of the paternal genome is much faster and thorough than that of the maternal genome. From the two-cell to the postimplantation stage, methylation of the paternal genome is consistently lower than that of the maternal genome. We also show that the genetic lineage of early blastomeres can be traced by DNA methylation analysis. Our work paves the way for deciphering the secrets of DNA methylation reprogramming in early human embryos.An analysis of single-cell DNA methylome sequencing data from human preimplantation embryos finds evidence for de novo methylation. Methylation reprogramming at this stage is a balance between global demethylation, which is faster in the paternal genome, and focused remethylation.

Characteristics of embryo development in Robertsonian translocations' preimplantation genetic diagnosis cycles

To explore the embryo development characteristics in Robertsonian translocations (RTs) in their preimplantation genetic diagnosis (PGD) cycles.

Influence of embryo culture medium on incidence of ectopic pregnancy in in vitro fertilization

OBJECTIVE To explore the effect of type of media used to culture embryos for IVF on the incidence of ectopic pregnancy (EP). DESIGN Retrospective analysis. SETTING University-affiliated IVF center. PATIENT(S) The retrospective analysis involved 23,481 women who underwent IVF-ET cycles between 2011 and 2013. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) There was an association between EP and the culture medium. RESULT(S) During 23,481 fresh transfer cycles, 364 patients were diagnosed with EP. The EP to clinical pregnancy rate was 3.01% in the G5 group, 3.89% in the G5 Plus group, and 4.04% in the Global group. The EP to clinical pregnancy rates were significantly higher in the G5 Plus and Global groups than in the G5 group. After adjusting for confounding factors, the incidence of EP was significantly associated with the G5 Plus and Global media. CONCLUSION(S) Our results showed that there is an association between incidence of EP and the culture medium. The rates of EP to clinical pregnancy were significantly higher in the G5 Plus and Global media than in the G5 medium.

Erratum to: The safety of long-term cryopreservation on slow-frozen early cleavage human embryos

Erratum to: J Assist Reprod Genet (2014) 31:471–475 DOI 10.1007/s10815-014-0197-0 The original version of this article unfortunately contained a mistake. The word “equally” is missing in the article note, and should read “Qinli Liu and Ying Lian contributed equally to this work.”