Genome-wide oxidative bisulfite sequencing identifies sex-specific methylation differences in the human placenta.

DNA methylation is an important regulator of gene function. Fetal sex is associated with the risk of several specific pregnancy complications related to placental function. However, the association between fetal sex and placental DNA methylation remains poorly understood. We carried out whole-genome oxidative bisulfite sequencing in the placentas of two healthy female and two healthy male pregnancies generating an average genome depth of coverage of 25x. Most highly ranked differentially methylated regions (DMRs) were located on the X chromosome but we identified a 225 kb sex-specific DMR in the body of the CUB and Sushi Multiple Domains 1 (CSMD1) gene on chromosome 8. The sex-specific differential methylation pattern observed in this region was validated in additional placentas using in-solution target capture. In a new RNA-seq data set from 64 female and 67 male placentas, CSMD1 mRNA was 1.8-fold higher in male than in female placentas (P value = 8.5 × 10-7, Mann-Whitney test). Exon-level quantification of CSMD1 mRNA from these 131 placentas suggested a likely placenta-specific CSMD1 isoform not detected in the 21 somatic tissues analyzed. We show that the gene body of an autosomal gene, CSMD1, is differentially methylated in a sex- and placental-specific manner, displaying sex-specific differences in placental transcript abundance

The RNA landscape of the human placenta in health and disease

AbstractThe placenta is the interface between mother and fetus and inadequate function contributes to short and long-term ill-health. The placenta is absent from most large-scale RNA-Seq datasets. We therefore analyze long and small RNAs (~101 and 20 million reads per sample respectively) from 302 human placentas, including 94 cases of preeclampsia (PE) and 56 cases of fetal growth restriction (FGR). The placental transcriptome has the seventh lowest complexity of 50 human tissues: 271 genes account for 50% of all reads. We identify multiple circular RNAs and validate 6 of these by Sanger sequencing across the back-splice junction. Using large-scale mass spectrometry datasets, we find strong evidence of peptides produced by translation of two circular RNAs. We also identify novel piRNAs which are clustered on Chr1 and Chr14. PE and FGR are associated with multiple and overlapping differences in mRNA, lincRNA and circRNA but fewer consistent differences in small RNAs. Of the three protein coding genes differentially expressed in both PE and FGR, one encodes a secreted protein FSTL3 (follistatin-like 3). Elevated serum levels of FSTL3 in pregnant women are predictive of subsequent PE and FGR. To aid visualization of our placenta transcriptome data, we develop a web application (https://www.obgyn.cam.ac.uk/placentome/).</jats:p

The RNA landscape of the human placenta in health and disease.

Funder: Department of HealthThe placenta is the interface between mother and fetus and inadequate function contributes to short and long-term ill-health. The placenta is absent from most large-scale RNA-Seq datasets. We therefore analyze long and small RNAs (~101 and 20 million reads per sample respectively) from 302 human placentas, including 94 cases of preeclampsia (PE) and 56 cases of fetal growth restriction (FGR). The placental transcriptome has the seventh lowest complexity of 50 human tissues: 271 genes account for 50% of all reads. We identify multiple circular RNAs and validate 6 of these by Sanger sequencing across the back-splice junction. Using large-scale mass spectrometry datasets, we find strong evidence of peptides produced by translation of two circular RNAs. We also identify novel piRNAs which are clustered on Chr1 and Chr14. PE and FGR are associated with multiple and overlapping differences in mRNA, lincRNA and circRNA but fewer consistent differences in small RNAs. Of the three protein coding genes differentially expressed in both PE and FGR, one encodes a secreted protein FSTL3 (follistatin-like 3). Elevated serum levels of FSTL3 in pregnant women are predictive of subsequent PE and FGR. To aid visualization of our placenta transcriptome data, we develop a web application ( https://www.obgyn.cam.ac.uk/placentome/ )

The RNA landscape of the human placenta in health and disease.

Funder: Department of HealthThe placenta is the interface between mother and fetus and inadequate function contributes to short and long-term ill-health. The placenta is absent from most large-scale RNA-Seq datasets. We therefore analyze long and small RNAs (~101 and 20 million reads per sample respectively) from 302 human placentas, including 94 cases of preeclampsia (PE) and 56 cases of fetal growth restriction (FGR). The placental transcriptome has the seventh lowest complexity of 50 human tissues: 271 genes account for 50% of all reads. We identify multiple circular RNAs and validate 6 of these by Sanger sequencing across the back-splice junction. Using large-scale mass spectrometry datasets, we find strong evidence of peptides produced by translation of two circular RNAs. We also identify novel piRNAs which are clustered on Chr1 and Chr14. PE and FGR are associated with multiple and overlapping differences in mRNA, lincRNA and circRNA but fewer consistent differences in small RNAs. Of the three protein coding genes differentially expressed in both PE and FGR, one encodes a secreted protein FSTL3 (follistatin-like 3). Elevated serum levels of FSTL3 in pregnant women are predictive of subsequent PE and FGR. To aid visualization of our placenta transcriptome data, we develop a web application ( https://www.obgyn.cam.ac.uk/placentome/ )

Placental Streptococcus agalactiae DNA is associated with neonatal unit admission and foetal pro-inflammatory cytokines in term infants.

Streptococcus agalactiae (Group B Streptococcus; GBS) is a common cause of sepsis in neonates. Previous work detected GBS DNA in the placenta in ~5% of women before the onset of labour, but the clinical significance of this finding is unknown. Here we re-analysed this dataset as a case control study of neonatal unit (NNU) admission. Of 436 infants born at term (≥37 weeks of gestation), 7/30 with placental GBS and 34/406 without placental GBS were admitted to the NNU (odds ratio (OR) 3.3, 95% confidence interval (CI) 1.3-7.8). We then performed a validation study using non-overlapping subjects from the same cohort. This included a further 239 cases of term NNU admission and 686 term controls: 16/36 with placental GBS and 223/889 without GBS were admitted to the NNU (OR 2.4, 95% CI 1.2-4.6). Of the 36 infants with placental GBS, 10 were admitted to the NNU with evidence of probable but culture-negative sepsis (OR 4.8, 95% CI 2.2-10.3), 2 were admitted with proven GBS sepsis (OR 66.6, 95% CI 7.3-963.7), 6 were admitted and had chorioamnionitis (inflammation of the foetal membranes) (OR 5.3, 95% CI 2.0-13.4), and 5 were admitted and had funisitis (inflammation of the umbilical cord) (OR 6.7, 95% CI 12.5-17.7). Foetal cytokine storm (two or more pro-inflammatory cytokines >10 times median control levels in umbilical cord blood) was present in 36% of infants with placental GBS DNA and 4% of cases where the placenta was negative (OR 14.2, 95% CI 3.6-60.8). Overall, ~1 in 200 term births had GBS detected in the placenta, which was associated with infant NNU admission and morbidity

Fetus-derived DLK1 is required for maternal metabolic adaptations to pregnancy and is associated with fetal growth restriction.

Pregnancy is a state of high metabolic demand. Fasting diverts metabolism to fatty acid oxidation, and the fasted response occurs much more rapidly in pregnant women than in non-pregnant women. The product of the imprinted DLK1 gene (delta-like homolog 1) is an endocrine signaling molecule that reaches a high concentration in the maternal circulation during late pregnancy. By using mouse models with deleted Dlk1, we show that the fetus is the source of maternal circulating DLK1. In the absence of fetally derived DLK1, the maternal fasting response is impaired. Furthermore, we found that maternal circulating DLK1 levels predict embryonic mass in mice and can differentiate healthy small-for-gestational-age (SGA) infants from pathologically small infants in a human cohort. Therefore, measurement of DLK1 concentration in maternal blood may be a valuable method for diagnosing human disorders associated with impaired DLK1 expression and to predict poor intrauterine growth and complications of pregnancy.M.A.M.C. was supported by a PhD studentship from the Cambridge Centre for Trophoblast Research. Research was supported by grants from the MRC (MR/J001597/1 and MR/L002345/1), the Medical College of Saint Bartholomew's Hospital Trust, a Wellcome Trust Investigator Award, EpigeneSys (FP7 Health-257082), EpiHealth (FP7 Health-278414), a Herchel Smith Fellowship (N.T.) and NIH grant RO1 DK89989. The contents are the authors' sole responsibility and do not necessarily represent official NIH views. We thank G. Burton for invaluable support, and M. Constância and I. Sandovici (University of Cambridge) for the Meox2-cre mice. We are extremely grateful to all of the participants in the Pregnancy Outcome Prediction study. This work was supported by the NIHR Cambridge Comprehensive Biomedical Research Centre (Women's Health theme) and project grants from the MRC (G1100221) and Sands (Stillbirth and Neonatal Death Charity). The study was also supported by GE Healthcare (donation of two Voluson i ultrasound systems for this study) and by the NIHR Cambridge Clinical Research Facility, where all research visits took place.This is the author accepted manuscript. The final version is available from Nature Publishing Group via https://doi.org/10.1038/ng.369