Publication: Sex differences in microRNA expression in first and third trimester human placenta

Sex differences in microRNA expression in first and third trimester human placenta
Biology of Reproduction, 2021 Dec 7, published online ahead of print

• 4 subanalyses of the miRNA-seq cohort in Gonzalez et al. 2021, Epigenomics
• N=113 late first trimester placenta samples from leftover chorionic villus sampling (a prenatal diagnostic test)
• N=47 third trimester placenta samples collected after delivery
• Bulk small RNA-sequencing with a focus on miRNAs
• Sex differences
• First trimester: male vs female placenta
• Third trimester: male vs female placenta
• Sex-specific analysis of first vs third trimester
• Subanalysis of female samples only: first vs third trimester placenta
• Subanalysis of male samples only: first vs third trimester placenta
• Identification of female exclusive and male exclusive gestational changes (miRNAs that change from first to third trimester, but only in one group)

Authors:

Amy E Flowers, Tania L Gonzalez, Nikhil V Joshi, Laura E Eisman, Ekaterina L Clark, Rae A Buttle, Erica Sauro, Rosemarie DiPentino, Yayu Lin, Di Wu, Yizhou Wang, Chintda Santiskulvong, Jie Tang, Bora Lee, Tianyanxin Sun, Jessica L Chan, Erica T Wang, Caroline Jefferies, Kate Lawrenson, Yazhen Zhu, Yalda Afshar, Hsian-Rong Tseng, John Williams, Margareta D Pisarska

PMID: 35040930; DOI: 10.1093/biolre/ioab221

Cell biomarkers in placenta research

Context is important for cell markers. Before you use these markers, consider that KRT7 is a marker for trophoblast cells in placenta, but you would not say that any particular cell is a trophoblast just because it expresses KRT7. It is also a common marker for lung research even though lungs don't have any trophoblasts. That is because KRT7 is generally expressed by epithelial cells, and in placenta those are trophoblasts, but lungs have other epithelial cells. Sample context is important.

Also consider RNA vs protein, localization, and experiment use. Some markers are useful to identify cell types in single cell RNA-seq results because the RNA is specifically or mostly expressed by specific cell types, but the protein is not expressed at the cell surface so you wouldn't use that marker to sort cells for flow cytometry. 

• KRT7 = CK7 = Cytokeratin 7 = epithelial cells, trophoblast cells
• Expressed by all epithelial cells
• All trophoblast cells in placenta are epithelial, so KRT7 is used as a trophoblast cell marker in placenta tissue
• qRT-PCR marker
• Cell surface marker suitable for flow cytometry
• Beware that fibroblasts also express KRT7 and primary cells in culture will eventually become fibroblast cells

• KRT8  = epithelial cell marker, trophoblast marker
• All trophoblast cells in placenta are epithelial, so KRT8 is used as a trophoblast cell marker in placenta tissue

• CDX2 = marker for early cytotrophoblasts (CTBs)
• Expressed in CTBs around 6 weeks, but diminishes as 1st trimester progresses (Horii et al 2016).
• Indicates stem cell-like phenotypes
• Immunohistochemistry (IHC) and gene expression marker for early cytotrophoblasts

HTR8/SVneo total RNA-seq data (Lee et al 2016)

We performed a knockdown experiment with human placenta cell line HTR8/SVneo and sent N=3 samples per group for total RNA-sequencing. This RNA-seq is a good resource for people interested not only in GATA3 (our gene of interest in this experiment), but also other genes. 

Use the control samples as a resource to see roughly how well other genes are expressed in HTR8/SVneo. Beware that there may be slight expression differences between different subcultures of HTR8/SVneo due to genetic drift and culture conditions.

Citation:

Bora Lee, Lindsay L Kroener, Ning Xu, Erica T Wang, Alexandra Banks, John Williams 3rd, Mark O Goodarzi, Yii-der I Chen, Jie Tang, Yizhou Wang, Vineela Gangalapudi, Margareta D Pisarska. "Function and Hormonal Regulation of GATA3 in Human First Trimester Placentation" Biol Reprod. 2016 Nov;95(5):113. doi: 10.1095/biolreprod.116.141861. Epub 2016 Oct 12.

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

High-throughput miRNA-sequencing of the human placenta: expression throughout gestation
Epigenomics, 2021 July; 13(13):995-1012. doi: 10.2217/epi-2021-0055. Epub 2021 May 25.

• Next generation sequencing of small RNAs in human placenta, with emphasis on microRNAs (miRNAs)
• N=113 late first trimester placenta samples from leftover tissue after chorionic villus sampling ("CVS", a prenatal diagnostic test)
• This is the earliest timepoint that we can directly study human placenta in continuing (non-termination) pregnancies.
• Chorionic villi are stringy tissue that grow on the outside of the placenta to maximize surface area with maternal tissue. They share the same genetics as the fetus.
• N=47 third trimester placenta samples collected after delivery
• miRNA profiles of:
• All expressed miRNAs, normalized counts over 10
• We considered lower expression thresholds, but expression was inconsistent across all patients
• Similarly expressed miRNAs, P>=0.05, fold-change<=2, normalized counts>10
• These miRNAs are fairly consistently expressed between first and third trimester
• They are not significantly different between first and third trimester
• Their expression may be altered by other variables (e.g. patient health, environmental factors, any developmental differences that affect pregnancy outcomes) - for future research!
• Differentially expressed miRNAs (still significant after adjusting for multiple comparisons), FDR<0.05, fold-change>2, normalized counts>10
• Note that all FDR<0.05 miRNAs are also P<0.05. The Benjamini-Hochberg false discovery rate P value adjustment is a stricter criteria than P<0.05.
• These miRNAs change expression as the pregnancy progresses
• Chromosome 14 and 19 microRNA cluster (C14MC, C19MC) members
• These clusters are known to be expressed in placenta, per prior microarray studies
• We identified many C14MC and C19MC expressed in first and third trimester, some similarly expressed and some significantly different between trimesters
• C14MC produced some miRNAs very upregulated in first trimester
• C19MC members were generally more highly expressed in both first and third trimester
• Some of these miRNAs also enter maternal circulation, per prior research
• Chromosome 13 has two small miRNA clusters significantly upregulated in first trimester placenta, with lower expression in third trimester. 
• Two C13MC regions in human placenta identified for the first time

Authors:

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

Links:

• Preprint full text at bioRxiv (February 2021)
• https://doi.org/10.1101/2021.02.04.429392
• Peer-reviewed and published at Epigenomics (May 2021)
• https://doi.org/10.2217/epi-2021-0055
• Added additional supplemental information on within-chromosome hot spots including two clusters at chromosome 13 that show increased expression in first trimester, compared to third trimester placenta.
• Added additional principal components analysis supplemental figures to show that samples clustering was explained by trimester, not fetal sex or race or ethnicity or paternal ages or maternal BMI.
• Additional clarifications to methods and results, per our reviewer feedback.
• Otherwise very similar to the pre-print version. Same dataset, analysis, and conclusions.
• PubMed ID: 34030457
• PubMed Central ID (PMCID): PMC8244582
• Raw data and counts: NCBI GEO accession GSE184860 (160 samples) = Bulk small RNA-seq
• Counts include miRNAs and piRNAs, but only miRNAs were used in the differential expression analysis.
• ResearchGate - full text available for free
• Also see our follow up manuscript focusing on miRNA sex differences in this cohort, Flowers et al. 2021, using the same data.

Publication: Sexually dimorphic crosstalk at the maternal-fetal interface

Tianyanxin Sun*, Tania L Gonzalez*, Nan Deng, Rosemarie DiPentino, Ekaterina L Clark, Bora Lee, Jie Tang, Yizhou Wang, Barry R Stripp, Changfu Yao, Hsian-Rong Tseng, S Ananth Karumanchi, Alexander F Koeppel, Stephen D Turner, Charles R Farber, Stephen S Rich, Erica T Wang, John Williams, III, Margareta D Pisarska. 

"Sexually dimorphic crosstalk at the maternal-fetal interface". The Journal of Clinical Endocrinology & Metabolism, 105(12): e4831-e4847, 2020, https://doi.org/10.1210/clinem/dgaa503

*Co-first authors.

Links

• PubMed ID: 32772088
• Journal of Clinical Endocrinology & Metabolism research article
• Receptor-ligand gene expression
• Cell markers of placenta cell types
• Cell markers of trophoblast cell types within placenta (subanalysis)
• Gene expression differences due to fetal sex
• Preprint at bioRxiv
• Includes additional information about the bulk RNA-seq of first trimester maternal-fetal interface using matched maternal decidua and placenta (CVS) RNA-seq
• Includes additional details about the single cell RNA-seq tSNE plots and cell developmental trajectories
• Sequencing data deposited into NCBI GEO superseries GSE131875
• GSE131696: Single cell RNA-sequencing of CVS samples
• N=6 placenta at late first trimester, 3 female and 3 male
• See preprint for extended details on the cell differentiation trajectory of cytotrophoblasts (stem cell-like) into extravillous trophoblasts (migratory/invasive cells) and syncytiotrophoblasts (multi-nuclei fused cells that act as a protective barrier in placenta).
• GSE131874: Total RNA-sequencing of matched maternal decidua and CVS tissue
• N=4/group, 2 pregnancies with female fetus, 2 pregnancies with male fetus
• N=8 samples total, 4 placenta and 4 decidua from 4 pregnancies
• See preprint for matched CVS vs decidua differential gene expression analysis
• We did not focus on this as much in the published version
• All pregnancies continued to full term delivery & live births
• Supplemental Figures deposited into FigShare.com
• Collection: https://doi.org/10.6084/m9.figshare.c.4741742.v2
• Figure S1 = PCA plot of matched chorionic villi and maternal decidua (bulk RNA-seq)
• Figure S2 = Heatmaps of differentially expressed genes from pairwide comparison of placental cell clusters (single cell analysis)
• Table S1 = Patient demographics and pregnancy outcomes
• 4 pregnancies for bulk RNA-seq
• 6 pregnancies for single cell RNA-seq
• 7 pregnancies for immunohistochemistry to validate single cell RNA-seq
• Table S2 = Placental cell markers from single cell RNA sequencing
• Table S3 = Placental cell type-specific sexually dimorphic genes
• Table S4 = Placenta receptors and ligands sexually dimorphic in both bulk and single cell RNA sequencing
• Table S5 = Trophoblast subcluster markers in human first trimester
• Links to figshare.com are correct in the final published version, but not the pre-proof PubMed Central version.

Placenta research 101

Quick primer for new lab members. Placenta genetics, cell lines, and vocabulary you'll hear in lab meetings.

Publication: epigenetics and infertility, infertility treatments, and pregnancy outcomes (review)

Genetics and Epigenetics of Infertility and Treatments on Outcomes.
J Clin Endocrinol Metab. 2019 Jun 1;104(6):1871-1886. doi: 10.1210/jc.2018-01869.
Pisarska MD, Chan JL, Lawrenson K, Gonzalez TL, Wang ET.

Links

• PubMedID: 30561694
• PubMed Central ID: PMC6463256
• The Journal of Clinical Endocrinology & Metabolism journal article
• Request the full-text via ResearchGate.net

Publication: Early placenta gene expression from pregnancies with vs without infertility treatments

Differential gene expression during placentation in pregnancies conceived with different fertility treatments compared with spontaneous pregnancies.

Lee B, Koeppel AF, Wang ET, Gonzalez TL, Sun T, Kroener L, Lin Y, Joshi NV, Ghadiali T, Turner SD, Rich SS, Farber CR, Rotter JI, Ida Chen YD, Goodarzi MO, Guller S, Harwood B, Serna TB, Williams J 3rd, Pisarska MD.

Fertility and Sterility. 2019 Jan 2. pii: S0015-0282(18)32200-3. doi: 10.1016/j.fertnstert.2018.11.005.

Links

• PubMed ID: 30611556
• Fertility and Sterility journal article

Layman's summary

One big question in maternal-fetal health research is: how do fertility treatments affect the baby's health and biology? There's some mixed evidence that babies born with the help of fertility treatments may have lower birth weights (though we didn't find this), higher risk of pregnancy complications, and greater risk of metabolic issues later in life. Are these differences due to the parents' innate biology (whatever led to the infertility in the first place) or due to the medical treatments?

To help figure this out, we compared placenta tissue from ongoing pregnancies to see if there were RNA differences between three groups: spontaneous (no infertility), NIFT (non-IVF fertility treatments, e.g. medicine to help stimulate ovulation), and IVF pregnancies. We got RNA from placenta tissue leftover after a late first trimester test called "chorionic villus sampling" (CVS). It is a prenatal diagnostic test that takes a very small biopsy of the placenta to indirectly check the baby's genetics and make sure the pregnancy is healthy. Using CVS tissue gives us a unique opportunity to study first trimester gene expression in pregnancies that lead to live births.

Results and discussion: Only a few individual genes were significantly different between groups, probably because there was a lot of variability within the patient groups. This is good news since it means that gene expression in late first trimester was pretty similar among all patients. We next looked for trends in pathways (groups of genes). Many immune-related pathways were significantly different in IVF vs spontaneous pregnancies, and some also different in the NIFT vs spontaneous pregnancies. Our lab is continuing to study what this means for patients.

Abstract

Publication: Sex differences in the late first trimester human placenta transcriptome

Data: Total RNA-seq of first trimester trimester placenta

Tania L. Gonzalez, Tianyanxin Sun, Alexander F. Koeppel, Bora Lee, Erica T. Wang, Charles R. Farber, Stephen S. Rich, Lauren W. Sundheimer, Rae A. Buttle, Yii-Der Ida Chen, Jerome I. Rotter, Stephen D. Turner, John WilliamsIII, Mark O. Goodarzi and Margareta D. Pisarska. Biology of Sex Differences 2018 Jan 15. Vol 9: issue 4.

Links

• PubMed ID: 29335024
• Biology of Sex Differences journal article [open access!]
• NCBI GEO accession for RNA-seq data: GSE109082
• Spreadsheet for genes above our FPKM threshold is "Additional file 2" (placenta transcriptome at time of chorionic villus sampling)
• Spreadsheet for genes reaching our False Discovery Rate threshold is "Additional file 4" (genes significantly sex different)

Publication: Up-regulation of microRNA-202-3p in first trimester placenta of pregnancies destined to develop severe preeclampsia, a pilot study (Singh et al. 2017)

Singh K, Williams J 3rd, Brown J, Wang ET, Lee B, Gonzalez TL, Cui J, Goodarzi MO, Pisarska MD. Pregnancy Hypertension 2017 Oct; 10: 7–9.

Links

• PubMed ID: 29153693
• PubMed Central ID:  PMC5970562
• mirBase link for hsa-miR-202-3p 
• Target searches:
• miRDB
• TargetScanHuman
• TargetMiner