Optimization of techniques for multiple platform testing in small, precious samples such as human chorionic villus sampling

Pisarska, Margareta D.; Akhlaghpour, Marzieh; Lee, Bora; Xu, Ning; Wang, Erica T.; Mackey, Aaron J.; Farber, Charles R.; Rich, Stephen S.; Rotter, Jerome I.; Chen, Yii‐Der I.; Goodarzi, Mark O.; Guller, Seth; Williams, John

doi:10.1002/pd.4936

Cited by 19 publications

(17 citation statements)

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“…CVS were processed as previously described [ 27 ]. Briefly, CVS tissue was thawed on ice with 600 μl of RTL Plus lysis buffer (QIAGEN) and 1% β-mercaptoethanol added to each sample.…”

Section: Methodsmentioning

confidence: 99%

Sex differences in the late first trimester human placenta transcriptome

et al. 2018

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BackgroundDevelopment of the placenta during the late first trimester is critical to ensure normal growth and development of the fetus. Developmental differences in this window such as sex-specific variation are implicated in later placental disease states, yet gene expression at this time is poorly understood.MethodsRNA-sequencing was performed to characterize the transcriptome of 39 first trimester human placentas using chorionic villi following genetic testing (17 females, 22 males). Gene enrichment analysis was performed to find enriched canonical pathways and gene ontologies in the first trimester. DESeq2 was used to find sexually dimorphic gene expression. Patient demographics were analyzed for sex differences in fetal weight at time of chorionic villus sampling and birth.ResultsRNA-sequencing analyses detected 14,250 expressed genes, with chromosome 19 contributing the greatest proportion (973/2852, 34.1% of chromosome 19 genes) and Y chromosome contributing the least (16/568, 2.8%). Several placenta-enriched genes as well as histone-coding genes were identified to be unique to the first trimester and common to both sexes. Further, we identified 58 genes with significantly different expression between males and females: 25 X-linked, 15 Y-linked, and 18 autosomal genes. Genes that escape X inactivation were highly represented (59.1%) among X-linked genes upregulated in females. Many genes differentially expressed by sex consisted of X/Y gene pairs, suggesting that dosage compensation plays a role in sex differences. These X/Y pairs had roles in parallel, ancient canonical pathways important for eukaryotic cell growth and survival: chromatin modification, transcription, splicing, and translation.ConclusionsThis study is the first characterization of the late first trimester placenta transcriptome, highlighting similarities and differences among the sexes in ongoing human pregnancies resulting in live births. Sexual dimorphism may contribute to pregnancy outcomes, including fetal growth and birth weight, which was seen in our cohort, with males significantly heavier than females at birth. This transcriptome provides a basis for development of early diagnostic tests of placental function that can indicate overall pregnancy heath, fetal-maternal health, and long-term adult health.Electronic supplementary materialThe online version of this article (10.1186/s13293-018-0165-y) contains supplementary material, which is available to authorized users.

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“…CVS were processed as previously described [ 27 ]. Briefly, CVS tissue was thawed on ice with 600 μl of RTL Plus lysis buffer (QIAGEN) and 1% β-mercaptoethanol added to each sample.…”

Section: Methodsmentioning

confidence: 99%

Sex differences in the late first trimester human placenta transcriptome

et al. 2018

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“…For instance, a recent study reported lower DNA yields in chorionic villi collected in RNA-later compared to snap frozen samples. 24 During sample processing we did notice a harder consistency of RNA-later tissues which rendered manual homogenization more difficult, with this difference being more noticeable in larger surgical tumor fragments (compared to biopsy specimens), which may explain their lower DNA and RNA yields. Lower RNA yields observed in RNAlater compared to freshly snap frozen samples are consistent with our previous observations in liver specimens.…”

Section: Discussionmentioning

confidence: 89%

The identification of challenges in tissue collection for biomarker studies: the Q-CROC-03 neoadjuvant breast cancer translational trial experience

et al. 2017

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One of the major challenges in biomarker development is the collection of tumor tissue of adequate quality for analysis. A prospective clinical trial was initiated to collect tissues from triple negative breast cancers prior to and after neoadjuvant chemotherapy in order to study the mechanisms of chemoresistance. Sixty patients had pre-chemotherapy biopsies performed by either a surgeon or a radiologist, while those with residual tumor after chemotherapy had research-only biopsies and/or surgical samples collected in liquid nitrogen, RNA-later and formalin. We examined each core for tumor cellularity, stromal content, and necrosis after which, RNA and DNA extraction was performed. We found that biopsies collected with ultrasound guidance were more likely to contain tumor than those collected by the surgeon. Patient reluctance to undergo research-only biopsies after chemotherapy was not a problem. Pre-chemotherapy tumor biopsies frequently did not contain any tumor cells (15%) or did not have ≥50% tumor content (63%). Indeed, 50% of patients had at least 2 pre-chemotherapy core biopsies with <50% tumor content. After chemotherapy, 30% of biopsy or surgical samples in patients with incomplete response did not contain any tumor. Finally, RNA-later not only made histopathological assessment of tumor content difficult, but yielded less DNA than fresh snap frozen samples. We recommend that high-quality tissue procurement can be best accomplished if at least three image-guided core biopsies be obtained per sample, each of these cores be examined for tumor cellularity and that at least some of them be freshly snap frozen in liquid nitrogen.

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“…Tissue samples (5-15 mg) were stored with 250 μl RNA later RNA Stabilization Reagent (QIAGEN, Hilden, Germany) at −80°C in the CSMC Prenatal Repository until further processing. Tissue was processed as previously described [58]. Briefly, tissue was thawed on ice with 600 μl of RLT Plus lysis buffer (QIAGEN) and 1% β-mercaptoethanol added to each sample.…”

Section: Methodsmentioning

confidence: 99%

The maternal-fetal interface of successful pregnancies and impact of fetal sex using single cell sequencing

Sun

Gonzalez

Deng

et al. 2019

Preprint

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2 2 3 1 identified five major cell types (trophoblasts, stromal cells, hofbauer cells, antigen 3 2 presenting cells and endothelial cells) with unique crosstalk at the maternal-fetal 3 3 interface. We identified seven unique trophoblast subclusters, including new subtypes 3 4 that transition into the terminal cell types, extra-villous trophoblasts and 3 5syncytiotrophoblasts. As fetal sex impacts pregnancy, we analyzed sex differences in 3 6 each cell type and identified differences in immune cell function. TGFβ1, β -estradiol, 3 7 and dihydrotestosterone emerge as upstream regulators of sexually dimorphic genes in 3 8 a cell type specific manner. Thus, the fetal contribution at the maternal-fetal interface is 3 9 cell and sex specific.4 0 Key words 4 1 Maternal-fetal interface, first trimester, ongoing pregnancy, human early pregnancy, 4 2 placenta, single cell RNA sequencing, sexual dimorphism, placenta cell types, receptor, 4 3 ligand 4 4 placental development that might impact maternal-fetal communication and contribute 6 9 to overall outcomes. 7 0 Results 7 1 7 2 RNA-sequencing of matched decidua and placenta tissue from first trimester 7 3 To identify normal early gene expression at the maternal-fetal interface, total RNA-7 4 sequencing for matched decidua (maternal) and chorionic villi (fetal placenta) was 7 5 performed. Principal component 1 shows that tissue type explains most of the difference 7 6 between samples. Principal component 2 corresponds to fetal sex (Figure S1). 7 7 RNA-sequencing identified 16,035 genes reaching threshold FPKM>1 and Z-score>0 in 7 8 decidua, placenta or both. Of these, 12,479 (77.8%) are protein-coding genes, including 7 9 3002 significantly differentially expressed genes (DEGs) with FDR<0.10 (Figure S2A, 8 0 Table S1). Over a quarter of protein-coding DEGs are tissue-unique at the maternal-8 1 fetal interface, meaning they are expressed in either decidua or placenta, but not both. 8 2 There are 2223 protein-coding DEGs upregulated in decidua compared to placenta 8 3 ("decidua-upregulated"), including 827 only expressed in decidua ("decidua-unique"). 8 4 There are 779 protein-coding DEGs upregulated in placenta compared to decidua 8 5 ("placenta-upregulated"), including 60 only expressed in placenta tissue ("placenta-8 6 unique").8 7 Receptors and ligands differentially expressed at the maternal-fetal interface 8 8To investigate maternal-fetal communication, we focused on receptor and ligand DEGs. 8 9We identified 818 receptors expressed at the maternal-fetal interface (TableS2) [7][8][9] 9 0 with 356 receptors significantly differentially expressed (FDR<0.10) between decidua 9 1 5 and placenta, including 290 decidua-upregulated receptors and 66 placenta-9 2 upregulated receptors (Figure S2B). Half of decidua-upregulated receptors are also 9 3 decidua-unique (146/290; 50.3%). The 5 decidua-unique receptors with highest fold-9 4 change over placenta are PIGR (FC = 215.3), GABRP (FC = 165.4), CDHR1 (FC = 9 5 156.5), TSPAN1 (FC = 149.1), and SCARA5 (FC = 125.4) (Table S2). Of t...

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Optimization of techniques for multiple platform testing in small, precious samples such as human chorionic villus sampling

Cited by 19 publications

References 49 publications

Sex differences in the late first trimester human placenta transcriptome

Sex differences in the late first trimester human placenta transcriptome

The identification of challenges in tissue collection for biomarker studies: the Q-CROC-03 neoadjuvant breast cancer translational trial experience

The maternal-fetal interface of successful pregnancies and impact of fetal sex using single cell sequencing

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