key: cord-0300310-vif8j9a2
authors: Gonzalez, Tania L; Eisman, Laura E; Joshi, Nikhil V; Flowers, Amy E; Wu, Di; Wang, Yizhou; Santiskulvong, Chintda; Tang, Jie; Buttle, Rae A; Sauro, Erica; Clark, Ekaterina L; DiPentino, Rosemarie; Jefferies, Caroline A; Chan, Jessica L; Lin, Yayu; Zhu, Yazhen; Afshar, Yalda; Tseng, Hsian-Rong; Taylor, Kent; Williams, John; Pisarska, Margareta D
title: High-throughput miRNA-sequencing of the human placenta: expression throughout gestation
date: 2021-02-04
journal: bioRxiv
DOI: 10.1101/2021.02.04.429392
sha: 2ca0b530a83f823733391cb7a74375f289dc13d9
doc_id: 300310
cord_uid: vif8j9a2

Background Altered placenta miRNA abundance may impact the maternal-fetal interface and pregnancy outcomes. Understanding miRNA changes across gestation 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 miRNA transcriptome in first (N=113) and third trimester (N=47). Results There are 801 miRNAs expressed in both first and third trimester, including 182 with similar expression across gestation (P≥0.05) and 182 significantly different (FDR<0.05). Of placenta-specific miRNA clusters, C14MC is more upregulated in first trimester and C19MC is more highly expressed overall. Conclusion This work provides a rich atlas of healthy pregnancies to direct functional studies investigating the epigenetic differences in first and third trimester placentae. Lay Abstract The human body produces microRNAs which affect the expression of genes and proteins. This study uses next generation sequencing to identify the microRNA profile of first and third trimester human placentae using a large cohort (N=113 first, N=47 third trimester). All pregnancies resulted in healthy babies. We identify microRNAs with significantly different expression between first and third trimester, as well as stably expressed microRNAs. This work provides a baseline for future studies which may use microRNAs to monitor maternal-fetal health throughout pregnancy.

The placenta plays a critical role in fetal development, forming the interface between the 93

The input miRNAs were reduced with higher expression thresholds so that target gene numbers 246 did not exceed IPA software limitations for Core Analysis. The following definitions were 247 applied for highly expressed miRNAs (baseMean>10,000 in both trimesters), similarly expressed 248 miRNAs (P≥0.05, absolute fold-change≤2, and baseMean>1,000 in both trimesters), and 249 differentially expressed miRNAs (FDR<0.05, absolute fold-change>2, and baseMean>1,000 in 250 both trimesters). Due to their smaller number, no additional miRNA filters were required for 251 Core Analysis of C14MC and C19MC targets. 252

Heatmaps 253

Heatmap and dendrograms of samples versus miRNAs were created with a matrix of 254 log 2 (baseMean) values scaled and centered by rows. The heatmaps and dendrograms were 255 created with hierarchical clustering from R package gplots v3.1.1. Heatmaps of gene enrichment 256 were created with R package pheatmap v1.0.12 with a matrix of -log 10 (P) output from IPA Core 257

Validation with qRT-PCR 259 Expression of 6 selected miRNAs was re-analyzed with an independent cohort by qRT-PCR 260 using the miRCURY LNA miRNA PCR system (QIAGEN). The 6 selected (hsa-miR-144-3p, 261 hsa-miR-24-3p, hsa-miR-126-3p, hsa-miR-145-5p, hsa-miR-143-3p, hsa-miR-126-5p) had high 262 expression (baseMean>1000) and were significantly different in miRNA-sequencing 13 ) between first and third trimester. A highly expressed miRNA with stable expression in first 264 and third trimester placentae was used as a reference gene baseMean>10, 000 265 in both trimesters, P=0.9693, FDR=0.9889). RNA from first trimester (N=10) and third trimester 266 (N=6) placenta samples were extracted, then cDNA synthesized using universal primers in the 267 miRCURY LNA RT Kit (QIAGEN). Expression was quantified by qRT-PCR using the 268 miRCURY LNA SYBR Green PCR Kit (QIAGEN) and a BioRad MyIQ machine, analyzed 269 using the ∆ ∆ Ct method, 60 with hsa-miR-130a-3p as an internal reference. Statistics were 270 performed using the Wilcoxon rank-sum test on 

There were N=113 first trimester placenta samples and N=47 third trimester placenta samples 275 studied, including 3 subjects with both first and third trimester placenta sequenced. Principal components analysis (PCA) shows that first and third trimester placenta segregated into distinct 277 clusters along PC1 (29.27% variability explained) and PC2 (20.22% variability explained) 278 (Supplemental File 1). There were significantly more non-Hispanic, Caucasian parents and 279 fetuses in the first trimester group (Table 1) . However, race and ethnicity groups did not cluster 280 in PCA analyses of the miRNA transcriptome (Supplemental File 2). Maternal pre-pregnancy 281 BMI and thyroid disorders requiring thyroid replacement were significantly different among the 282 groups (Table 1 ). There were more cases of pregnancy-induced hypertension requiring 283 antihypertensives and/or magnesium in the third trimester placenta group, compared to none in 284 the first trimester placenta group. 285

We identified 2503 mature miRNAs with high-throughput sequencing of first and third trimester 287 placentae, with 801 miRNAs reaching 10 normalized counts in both first and third trimester 288 placentae (baseMean>10). First trimester placenta expressed 872 mature miRNAs 289 (baseMean>10), derived from 967 miRNA precursors from all chromosomes with annotated 290 miRNAs (22 autosomes and the X chromosome) ( Figure 1A , Supplemental File 3). The majority 291 of these precursor miRNAs originate from chromosomes 19 (12.7%), 14 (10.8%), X (9.6%), and 292 1 (6.8%). Third trimester placenta expressed 882 mature miRNAs (baseMean>10), derived from 293 985 miRNA precursors from all 22 autosomes and the X chromosome ( Figure 1A , Supplemental 294

File 3). The most represented chromosomes were also 19 (12.4%), 14 (10.5%), X (9.7%), and 1 295 (7.1%). 296

Some miRNAs had expression values several orders of magnitude higher than most miRNAs, 298 with the median at baseMean=123.6 but the mean raised to baseMean=5,635 by these highly 299 expressed miRNAs (Supplemental File 3). A threshold of baseMean>10,000 was selected for the 300 "most highly expressed" miRNAs. There were 75 mature miRNAs (derived from 96 precursors) 301 in first trimester and 77 mature miRNAs (derived from 97 precursors) in third trimester placenta 302 which reached this threshold. The most highly expressed miRNA in first trimester was C19MC 303 member miR-517b-3p (baseMean=218,953). The most highly expressed miRNA in third 304 trimester and overall most highly expressed was miR-126-3p (baseMean=337,399). 305

Chromosome 19 encoded 30 mature miRNAs (derived from 36 precursors) which reached 306 baseMean>10,000 in both first and third trimester ( Figure 1B) , making chromosome 19 the 307 source of over 37% of the most highly expressed precursor miRNAs in human placenta. 308 Specifically, 28 of the 36 precursor miRNAs were C19MC members, and 8 localized elsewhere 309 on chromosome 19. The next chromosomes contributing the most highly expressed miRNAs 310 were chromosome 9, chromosome 1, and chromosome X. 311

We performed pathway enrichment analysis on experimentally confirmed targets of the most 312 highly expressed miRNAs ( Figure 1C , Supplemental File 5Ai). The most significantly enriched 313 canonical pathways in first and third trimester were "Molecular Mechanisms of Cancer", 314 "Hepatic Fibrosis Signaling", "Senescence", "Regulation of the Epithelial Mesenchymal 315

Transition by Growth Factors", and "Pancreatic Adenocarcinoma Signaling." 316 Pathway enrichment analysis with both experimentally confirmed miRNA targets as well as 317 targets predicted with high confidence demonstrated similar patterns, though third trimester 318 showed relatively higher enrichment in "Hepatic Fibrosis/Hepatic Stellate Cell Activation" and 319 "Regulation of the Epithelial Mesenchymal Transition by Growth Factors" compared to first 320 trimester (Supplemental File 4, Supplemental File 5Ai). Additional pathways, including 321 inflammatory pathways such as "Neuroinflammation", "Prolactin", "Systemic Lupus 322 Erythematosus in B Cell", and "IL-6" signaling were also enriched (Supplemental File 5Aii). 323

There were 182 mature miRNAs with similar expression in the first and third trimester placentae 325 (P≥0.05, fold-change≤2 and baseMean>10, Supplemental File 3), suggesting consistent 326 expression throughout gestation ( Figure 1D ). These mature miRNAs are derived from 206 327 precursor miRNAs, with greatest representation from chromosomes 19 (17.0%), 14 (10.2%), X 328 (9.7%), and 1 (7.3%) ( Figure 1E ). The most highly expressed similar miRNA was C19MC 329 member hsa-miR-515-5p with first trimester baseMean=129,659 and third trimester 330 baseMean=129,323, P=0.902 between trimesters. This was followed closely by other C19MC 331 members: hsa-miR-158b, hsa-miR-518f-3p, hsa-miR-1323, and hsa-miR-1283. 332

There were 588 mature miRNAs significantly differentially expressed between first and third 334 trimester placentae (FDR<0.05, baseMean>10) (Figure 2A 202 precursors with highest representation from chromosomes 9 (9.9%), X (9.4%), 1 and 14 338 (8.4% each), and 19 (7.4%) ( Figure 2B ). The most differentially expressed miRNA was hsa-339 miR-4483, with 38.2-fold higher expression in the first trimester placenta (FDR=0) and a 340 baseMean decrease from 984.1 to 25.5 from first to third trimester ( Figure 2C , Supplemental File 341 3). The next most significantly differentially expressed miRNA was hsa-miR-139-5p with 18.1-342 fold higher expression in the third trimester (FDR=3.69x10 -298 ), baseMean increasing from 28.0 343 to 497.5 ( Figure 2C ). The differentially expressed miRNA with the highest overall expression 344 was hsa-miR-126-3p, with a 3.13-fold higher expression (FDR=4.52x10 -97 ) in third trimester 345 placenta (baseMean=337,399) compared to first trimester placenta (baseMean=107,787) ( Figure  346 2D). Of the differentially expressed miRNAs, those with the greatest fold changes had lower to 347 moderate expression around baseMean 100-1,000 and were predominantly elevated in the first 348 trimester, whereas those with the highest expression had lower fold changes and were 349 predominantly elevated in the third trimester ( Figure 2D ). Zero differentially expressed miRNAs 350 with baseMean>1,000 reached fold-change of 4 ( Figure 2D , Supplemental File 3). 351

Six differentially expressed miRNAs identified using NGS were selected for validation ( Figure  353 2E). We performed qRT-PCR using an independent cohort of first (N=10) and third trimester 354 (N=6) placenta samples. The miRNA hsa-miR-130a-3p was selected as an internal reference due 355 to high and stable expression in first and third trimester placentae (P=0.9693, fold-356 change=0.9984 first/third, baseMean=11,097). All six validated miRNAs (hsa-miR-24-3p, hsa-357 miR-144-3p, hsa-miR-145-5p, hsa-miR-126-3p, hsa-miR-126-5p, and hsa-miR-143-3p) were 358 upregulated in third trimester placenta with 2.5 to 3.7-fold changes by sequencing, and all six 359 were confirmed significant by qRT-PCR with P<0.003 ( Figure 2E ). 360

A heatmap of the 182 similarly expressed miRNAs shows no clustering of the first and third 362 trimester samples ( Figure 3A ). The heatmap of 180 differentially expressed miRNAs shows 363 placenta sample clustering by trimester, and miRNAs clustering into two groups by direction of 364 upregulation ( Figure 3B ). There was little subject variability in miRNAs in first and third 365 trimester, but some miRNAs were not consistently expressed (baseMean=0, red). 366

Pathway enrichment analysis was performed for experimentally confirmed miRNA targets to 367 identify potential regulatory roles of the miRNAs expressed in placenta ( Figure 3C , 368

Supplemental File 4B). The most significantly enriched pathways, targeted by both similarly and differentially expressed miRNAs in first and third trimester placenta were "Molecular 370 Mechanisms of Cancer" and "Hepatic Fibrosis Signaling". None of the top 20 pathways were 371 more significantly targeted by similarly expressed miRNAs, suggesting high variability 372 throughout gestation (Supplemental File 5Bi). Differentially expressed miRNAs targeted more 373 significantly by highly expressed miRNAs in the first trimester include "Molecular Mechanisms 374 of Cancer", "Hepatic Fibrosis Signaling", "Senescence", and "Regulation of the Epithelial 375 Mesenchymal Transition by Growth Factors" pathways, suggesting these pathways are distinctly 376 regulated by miRNAs in the first versus third trimester ( Figure 3C , Supplemental File 5Bi). 377

When the pathway enrichment analysis was repeated with both experimentally confirmed 378 miRNA targets as well as targets predicted with high confidence, additional patterns emerge. 379

Differentially expressed miRNAs target the "Hepatic Fibrosis / Hepatic Stellate Cell Activation" 380 pathway more heavily than similar miRNAs when predicted targets are included (Supplemental 381 File 5Bii). Addition of predicted targets highlights specific cytokine and growth factor pathways, 382

including "IL-6" and "IGF-1" signaling which are heavily targeted by similarly expressed 383 miRNAs, and less so by differentially expressed miRNAs (Supplemental File 4, Supplemental 384

File 5Bii). 385

The placenta specific miRNA clusters expressed 42 mature miRNAs similarly expressed 387 between first and third trimester placenta (P≥0.05, baseMean>1), 24 from C14MC and 18 from 388 C19MC ( Figure 4AB , Supplemental File 6). There were 105 mature miRNAs differentially 389 expressed between first and third trimester (FDR<0.05 and baseMean>1), 64 from C14MC and 390 41 from C19MC ( Figure 4AB , Supplemental File 6). The cluster miRNAs with highest fold-391 change came from C14MC: hsa-miR-1197 (6.28-fold, FDR=4.83x10 -118 ), hsa-miR-758-5p (5.59-392 fold, FDR=5.07x10 -101 ), hsa-miR-496 (4.05-fold higher in first, FDR=3.92x10 -109 ), and hsa-miR-393 665 (3.98-fold, FDR=1.75x10 -95 ), all higher in first trimester compared to third trimester 394 placenta. 395

The most significantly upregulated third trimester miRNA was hsa-miR-520c-3p (2.78-fold 396 higher, FDR=2.91x10 -111 ), followed by hsa-miR-181d-5p (3.18-fold higher, FDR=6.38x10 -70 ), 397 both from C19MC. Overall, the C14 miRNA cluster contributed more differentially expressed 398 miRNAs, reaching higher fold-changes in first trimester than C19MC ( Figure 4A ). Although 399 C19MC contributed fewer total miRNAs, and at lower fold-change differences between trimesters, the C19MC baseMean distribution was an order of magnitude higher than C14MC 401 distribution in both overall baseMean median (C19MC=5,696; C14MC=198.7) and mean 402 (C19MC=21,278; C14MC=1,122) ( Figure The placenta is a unique organ that changes function greatly throughout gestation, meeting 427 different challenges and needs at different stages of pregnancy. Placentation in the first trimester 428 sets the groundwork for its functions throughout gestation for fetal development. Placental 429 function is in part epigenetically regulated through miRNAs, including the placenta-specific 430 miRNA clusters, C14MC and C19MC that play critical roles in regulation of this vital organ. This is the first study to our knowledge to use high-throughput sequencing to compare miRNA 432 expression between first and third trimester human placentae of healthy pregnancies resulting in 433 delivery. 434

The miRNA expression profiles in first and third trimester have similar chromosome 435 distributions, with expected peaks at chromosomes 14 and 19, as well as peaks at chromosome 1, 436 the largest human chromosome and chromosome X, which has a higher density of miRNAs 437 compared to autosomes. 61,62 The most highly expressed miRNA, hsa-miR-126-3p, was 438 upregulated in third trimester, and was validated with qRT-PCR using an independent cohort. In 439 a recent study comparing first and second trimester placenta, miR-126-3p was identified to be 440 among the 10 most highly expressed miRNAs and identified in maternal plasma. 63 Although 441 variation among the first and second trimester was not different, 63 our study identified hsa-miR-442 126-3p to be differentially expressed and highest in the third trimester, likely having a unique 443 role in the third trimester compared to earlier in gestation. It is also highly abundant in fetal 444 circulation and human umbilical vein endothelial cells, 64 suggesting a role during parturition and 445 fetal development and may become a potential biomarker for developmental origins of health 446 and disease. 447

Among the most highly expressed miRNAs, over 37% were encoded in chromosome 19 (and 448 28/36 or 77.8% were specifically in C19MC), whereas none localized to chromosome 14. This 449 supports an earlier miRNA-seq study which profiled 25 human placentae at delivery and 450 identified higher expression from C19MC than C14MC miRNAs 65 , and additionally we show the 451 same pattern in first trimester. The C19MC miRNAs with high expression in the placenta may 452 potentially be used for targets, as C19MC miRNAs have been identified in maternal circulation, 453 as early as the first trimester, with elevations throughout gestation. 66-68 The most highly 454 expressed miRNA that was similarly expressed was hsa-miR-515-5p, which is a member of the 455 C19MC. Placental expression of hsa-miR-515-5p has been identified to play a key role in human 456 trophoblast differentiation with aberrant up-regulation contributing to pathogenesis of 457 preeclampsia. 35,69 It has also been associated with preterm birth 70 and fetal growth restriction. 43 458

Although, it has been detected in maternal circulation, both in plasma and whole blood fractions, 459 it has also been detected in whole blood fractions of healthy nonpregnant women, 71 and may not 460 be used solely as a biomarker of disease, but may be incorporated with other miRNAs with stable expression across gestation that change with disease using a bivariate biomarker disease 462 approach described by Laurent. 45 The most highly differentially expressed miRNA in first trimester placenta, hsa-miR 205 has 486 been implicated in the epithelial to mesenchymal transition and the maintenance of the epithelial 487 phenotype. [75] [76] [77] [78] [79] In human trophoblast cell lines it has been identified to silence MED1 under 488 hypoxic conditions. 75 suggesting it has a role in the first trimester regulating trophoblast 489 differentiation during physiologic hypoxic conditions. 75,81,82 "Regulation of the Epithelial 490

Mesenchymal Transition by Growth Factors Pathway" was also one of the most significantly 491 targeted pathway by differentially expressed miRNAs and most highly expressed in the first trimester, highlighting the differences between placentation when the placenta is invading 493 maternal tissue and establishing itself in states of low oxygen tension versus time of delivery 494 when the placenta has completed its purpose. 495

Two additional significantly targeted pathways by differentially expressed miRNAs and highly 496 expressed in the first trimester included the "Hepatic Fibrosis Signaling" and the "Senescence" 497 pathways. Hepatic Fibrosis Signaling is classically associated with extracellular matrix 498 deposition, 83 consistent with first trimester placental function when extravillous trophoblasts 499 degrade and induce ECM remodeling to enable migration. 2,5,84,85 500 Cellular senescence is programmed cell-cycle arrest that restricts the propagation of cells, which 501 is induced by various forms of cellular stress, including oxidative stress. Cell fusion, has also 502 been identified to trigger cellular senescence and has been described in the placenta, with the 503 placental expressed fusogen, Syncitin-1 (ERVWE1), which mediates cell-fusion-induced 504 senescence of the syncitiotrophoblast. 86-88 These senescent cells secrete inflammatory cytokines, 505 chemokines and matrix metalloproteinases, known as the senescence associated secretory 506 phenotype (SASP). SASP proteins promote EMT and the degradation of basement membranes, 507 increasing migration and invasion for appropriate placentation. 89 508

Our findings also support the importance of the placenta-specific miRNA clusters throughout 509 gestation, with 42 miRNAs similarly expressed and 105 differentially expressed across first and 510 third trimester. This indicates that while they are placenta-specific miRNAs, the majority have 511 varying roles throughout pregnancy. Differentially expressed canonical pathways targeted by the 512 C14 and C19 clusters were more significant than those of all similarly expressed miRNAs 513 suggesting these clusters have significantly different roles throughout gestation. 66 Similar to 514 other studies using whole villous tissue and primary cytotrophoblasts, 20,48,63 we identified a 515 decrease in C14MC expression from first to third trimester. However, a recent study did not 516 identify a decrease throughout gestation, but their study only focused on the first and second 517 trimester of presumably normal pregnancies. 63 518

The major strengths of this study are the use of first and third trimester tissue from healthy 519 pregnancies resulting in delivery, the cohort size, the availability of detailed demographic 520 information, and the use of high-throughput sequencing. NGS, as opposed to other techniques 521 such as array, allows for greater confidence in the conclusions regarding differential expression, 522 since all known miRNA species previously annotated in the human genome are considered, and 523 bias is not introduced by eliminating certain RNAs. Previous studies analyzing miRNA 524 expression in first and/or third trimester placentae have used microarray technology and most 525 examined very few samples (N=2-6 in each group). 25, 47, 48, 90 There are currently few NGS 526 miRNA profiles of the placenta, and our study is the first to profile both first and third trimester 527 placentae with NGS and a large sample size. We successfully validated all six selected miRNAs 528 using qRT-PCR and an independent cohort. 529

Our study has some limitations. There were some differences in the demographics between the 530 groups from the first and third trimester placenta samples. This includes race, ethnicity, maternal 531 BMI, thyroid disorders, and pregnancy complications, specifically hypertension. However, the 532 overall differences were small. In addition, PCA analysis did not demonstrate outliers. 533 Furthermore, we performed pathway enrichment analysis using only experimentally confirmed 534 targets. When performed using both experimentally confirmed and predicted with high 535 confidence targets, although overall pathways and patterns remained consistent, when we only 536 included experimentally confirmed targets, immune mediated pathways were not represented. 537

Overall, we intended to identify and compare the normative miRNA signatures in the first and 538 third trimester placentae. Our study shows many stably expressed miRNAs throughout gestation 539 as well as significant differences between the miRNA signatures. This work provides a rich atlas 540 to direct functional studies investigating the epigenetic differences in first and third trimester 541 placentae and development of disease related biomarkers or prognostic indicators that are 542 gestational age specific. 543 544

As we improve our understanding of miRNA profiles in placenta and across gestation, miRNAs 546 may be useful biomarkers for non-invasive prenatal diagnostic testing. Our knowledge of 547 miRNA profiles is still in its infancy relative to our knowledge of the protein coding 548 transcriptome. Until recently, most miRNA profiling papers of placenta used arrays with limited 549 samples. However, protocols to capture small RNAs, synthesize cDNA, and perform high-550 throughput NGS are improving rapidly. In 5-10 years' time, we expect that the knowledge of 551 human miRNA profiles in different tissue and extracellular locations will greatly improve as 552

well. This will provide opportunities for biomarker discovery and diagnostic test development, 553 since miRNAs are smaller, more stable RNAs than protein coding transcripts. Currently, the 554 knowledge pool of miRNA targets has limited confirmed miRNA-RNA interactions, but this will 555 improve as the miRNA field continues to evolve. Our work to profile miRNAs in first and third 556 trimester provides a foundation for biomarker discovery during pregnancy and future 557 advancements in maternal-fetal health. 558 559

• This work creates an atlas of the miRNA expression profiles of first and third trimester 561 human placenta from patients who delivered healthy babies. 562

• Chromosome 19 contributes approximately 37% of the most highly expressed miRNAs in 563 both first and third trimester placenta. Most of these miRNAs are localized to the pregnancy-564 associated miRNA cluster, C19MC. 565

• There are 182 miRNAs with similar expression across gestation. Other patient variables may 566 affect the abundance of these miRNAs. 567

• There are 180 miRNAs with significant differences in expression between first and third 568 trimester placenta. These miRNAs may contribute to changes in placental function or be 569 markers of different placental stresses throughout gestation. 570

• Six miRNAs were successfully validated with qRT-PCR in an independent cohort. 571

• The placenta-specific miRNA clusters (C14MC and C19MC) contain both similarly and 572 differentially expressed miRNAs. 573

• C14MC expressed miRNAs with greater fold-change differences across gestation than 574 C19MC miRNAs, though C14MC miRNAs are not among the most highly expressed 575 miRNAs in placenta. 576

• For both similarly and differentially expressed miRNAs, C19MC miRNA placenta 577 expression was overall higher than C14MC expression. Note: Values shown as mean (standard deviation) or n (%). P values were adjusted for fetal sex. 581 an independent cohort. The bar plot shows qRT-PCR results normalized to an internal reference, 600 hsa-miR-130a-3p (blue). The superimposed line shows fold-changes in miRNA-seq. All six 601 miRNAs were validated significantly different between first and third trimester with P<0.003. 602 

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