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. 2021 Jul;13(13):995-1012.
doi: 10.2217/epi-2021-0055. Epub 2021 May 25.

High-throughput miRNA sequencing of the human placenta: expression throughout gestation

Tania L Gonzalez  1 , Laura E Eisman  1 , Nikhil V Joshi  1   2 , Amy E Flowers  1 , Di Wu  3 , Yizhou Wang  3 , Chintda Santiskulvong  4 , Jie Tang  5 , Rae A Buttle  1 , Erica Sauro  1 , Ekaterina L Clark  1 , Rosemarie DiPentino  1 , Caroline A Jefferies  6 , Jessica L Chan  1   2 , Yayu Lin  1 , Yazhen Zhu  7 , Yalda Afshar  2 , Hsian-Rong Tseng  7 , Kent Taylor  2   8 , John Williams 3rd  1   2 , Margareta D Pisarska  1   2   5
Affiliations

High-throughput miRNA sequencing of the human placenta: expression throughout gestation

Tania L Gonzalez et al. Epigenomics. 2021 Jul.

Abstract

Aim: To understand miRNA changes across gestation in healthy human placentae. This is essential before miRNAs can be used as biomarkers or prognostic indicators during pregnancy. Materials & methods: Using next-generation sequencing, we characterize the normative human placenta miRNome in first (n = 113) and third trimester (n = 47). Results & conclusion: There are 801 miRNAs expressed in both first and third trimester, including 182 with similar expression across gestation (p ≥ 0.05, fold change ≤2) and 180 significantly different (false discovery rate <0.05, fold change >2). Of placenta-specific miRNA clusters, chromosome 14 miRNA cluster decreases across gestation and chromosome 19 miRNA cluster is overall highly expressed. Chromosome 13 clusters are upregulated in first trimester. This work provides a rich atlas of healthy pregnancies to direct functional studies investigating the epigenetic differences in first and third trimester placentae.

Keywords: chorionic villi; gestational differences; human transcriptome; miRNA; miRNA atlas; miRNome; placenta; pregnancy; stable miRNAs; uncomplicated pregnancies.

Plain language summary

Lay abstract The human body produces miRNAs which affect the expression of genes and proteins. This study uses next-generation sequencing to identify the miRNA profile of first and third trimester human placentae using a large cohort (n = 113 first trimester; n = 47 third trimester). All pregnancies resulted in healthy babies. We identify miRNAs with significantly different expression between first and third trimester, as well as stably expressed miRNAs. This work provides a baseline for future studies which may use miRNAs to monitor maternal–fetal health throughout pregnancy.

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Conflict of interest statement

Financial & competing interests disclosure

This work was supported by the National Institute of Health grants: R01 HD091773, R01 HD074368, T32 DK007770 and U01 EB026421. The funding agency was not involved in the design, analysis or interpretation of the data reported. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Figures

Figure 1.
Figure 1.. Expressed miRNAs in first and third trimester placenta.
(A) Chromosome distribution of precursor miRNAs expressed at baseMean >10 in first or third trimester placenta. Barplot: frequency per chromosome. Diamond points and right y-axis: percentage of placenta-expressed miRNAs per total chromosome miRNAs. (B) Distribution of the most highly expressed precursor miRNAs at baseMean >10,000. (C) Pathway enrichment analysis with experimentally confirmed target genes of the most highly expressed miRNAs. (D) The expression distribution of miRNAs similarly expressed in first and third trimester at p ≥ 0.05 and fold change ≤2. The red line (baseMean = 10) is the threshold selected for stable expression. (E) Counts of similarly expressed miRNAs with p ≥ 0.05, fold change ≤2 and baseMean >10 in both trimesters.
Figure 2.
Figure 2.. Differentially expressed miRNAs between first and third trimester placenta.
(A) Scatter plot of absolute fold change distribution across chromosomes for all DE miRNAs at FDR <0.05 and baseMean >10. The dotted line represents FC = 2. (B) Chromosome frequency of 180 DE miRNA precursors at FDR <0.05, FC >2, baseMean >10. (C) Volcano plot of all miRNAs with baseMean >10. Key as in (A), with addition of open black squares for nonsignificant miRNAs (FDR ≥0.05). (D) Expression versus absolute fold change for 180 DE miRNAs. (E) Six DE miRNAs (green) were selected for validation via quantitative real-time-PCR in an independent cohort. The bar plot shows qRT-PCR results normalized to an internal reference, hsa-miR-130a-3p (blue). The superimposed line shows fold changes in miRNA-seq. All six miRNAs were validated significantly different between first and third trimester with p < 0.003. DE: Differentially expressed; FC: Fold change; FDR: False discovery rate.
Figure 3.
Figure 3.. Heatmaps showing sample miRNA variability.
Heatmaps: rows = scaled and centered miRNA log2(baseMean), columns = hierarchically clustered samples. BaseMean = 0 samples are highlighted red. (A) One hundred eighty two similarly expressed miRNAs with p ≥ 0.05, fold change ≤2 and baseMean >10. The miRNAs are listed alphabetically. (B) One hundred eighty differentially expressed miRNAs with FDR <0.05, fold change >2 and baseMean >10. The miRNAs are hierarchically clustered. (C) Pathway enrichment analysis for experimentally confirmed targets of S and D expressed miRNAs. D: Differentially; FDR: False discovery rate; S: Similarly.
Figure 4.
Figure 4.. Placenta-specific chromosome 14 miRNA cluster & chromosome 19 miRNA cluster.
(A & B) Expression versus absolute fold change plots for cluster miRNAs at baseMean >1. Pink = upregulated in first trimester at false discovery rate <0.05. Purple = upregulated in third trimester at FDR <0.05. Blue = similarly expressed with p ≥ 0.05. Point labels are the miRNA names minus the ‘hsa-miR-’ prefix. (A) C14MC miRNAs. (B) C19MC miRNAs. (C & D) Pathway enrichment analysis with experimentally confirmed targets of the similarly expressed or differentially expressed miRNAs in (C) C14MC or (D) C19MC. C14MC: Chromosome 14 miRNA cluster; C19MC: Chromosome 19 miRNA cluster; D: Differentially expressed; S: Similarly expressed.
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