Effects of RNA integrity on transcript quantification by total RNA sequencing of clinically collected human placental samples.

RNA degradation is a ubiquitous process that occurs in living and dead cells, as well as during handling and storage of extracted RNA. Reduced RNA quality caused by degradation is an established source of uncertainty for all RNA-based gene expression quantification techniques. RNA sequencing is an increasingly preferred method for transcriptome analyses, and dependence of its results on input RNA integrity is of significant practical importance. This study aimed to characterize the effects of varying input RNA integrity [estimated as RNA integrity number (RIN)] on transcript level estimates and delineate the characteristic differences between transcripts that differ in degradation rate. The study used ribodepleted total RNA sequencing data from a real-life clinically collected set (n = 32) of human solid tissue (placenta) samples. RIN-dependent alterations in gene expression profiles were quantified by using DESeq2 software. Our results indicate that small differences in RNA integrity affect gene expression quantification by introducing a moderate and pervasive bias in expression level estimates that significantly affected 8.1% of studied genes. The rapidly degrading transcript pool was enriched in pseudogenes, short noncoding RNAs, and transcripts with extended 3' untranslated regions. Typical slowly degrading transcripts (median length, 2389 nt) represented protein coding genes with 4-10 exons and high guanine-cytosine content.-Reiman, M., Laan, M., Rull, K., Sõber, S. Effects of RNA integrity on transcript quantification by total RNA sequencing of clinically collected human placental samples.

Microdeletions and microduplications linked to severe congenital disorders in infertile men.

Data on the clinical validity of DNA copy number variants (CNVs) in spermatogenic failure (SPGF) is limited. This study analyzed the genome-wide CNV profile in 215 men with idiopathic SPGF and 62 normozoospermic fertile men, recruited at the Andrology Clinic, Tartu University Hospital, Estonia. A two-fold higher representation of > 1 Mb CNVs was observed in men with SPGF (13%, n = 28) compared to controls (6.5%, n = 4). Seven patients with SPGF were identified as carriers of microdeletions (1q21.1; 2.4 Mb) or microduplications (3p26.3, 1.1 Mb; 7p22.3-p22.2, 1.56 Mb; 10q11.22, 1.42 Mb, three cases; Xp22.33; 2.3 Mb) linked to severe congenital conditions. Large autosomal CNV carriers had oligozoospermia, reduced or low-normal bitesticular volume (22-28 ml). The 7p22.3-p22.2 microduplication carrier presented mild intellectual disability, neuropsychiatric problems, and short stature. The Xp22.33 duplication at the PAR1/non-PAR boundary, previously linked to uterine agenesis, was detected in a patient with non-obstructive azoospermia. A novel recurrent intragenic deletion in testis-specific LRRC69 was significantly overrepresented in patients with SPGF compared to the general population (3.3% vs. 0.85%; χ2 test, OR = 3.9 [95% CI 1.8-8.4], P = 0.0001). Assessment of clinically valid CNVs in patients with SPGF will improve their management and counselling for general and reproductive health, including risk of miscarriage and congenital disorders in future offspring.

Parent-of-origin-specific allelic expression in the human placenta is limited to established imprinted loci and it is stably maintained across pregnancy.

BACKGROUND: Genomic imprinting, mediated by parent-of-origin-specific epigenetic silencing, adjusts the gene expression dosage in mammals. We aimed to clarify parental allelic expression in the human placenta for 396 claimed candidate imprinted genes and to assess the evidence for the proposed enrichment of imprinted expression in the placenta. The study utilized RNA-Seq-based transcriptome and genotyping data from 54 parental-placental samples representing the three trimesters of gestation, and term cases of preeclampsia, gestational diabetes, and fetal growth disturbances. RESULTS: Almost half of the targeted genes (n = 179; 45%) were either not transcribed or showed limited expression in the human placenta. After filtering for the presence of common exonic SNPs, adequacy of sequencing reads and informative families, 91 genes were retained (43 loci form Geneimprint database; 48 recently proposed genes). Only 11/91 genes (12.1%) showed confident signals of imprinting (binomial test, Bonferroni corrected P < 0.05; > 90% transcripts originating from one parental allele). The confirmed imprinted genes exhibit enriched placental expression (PHLDA2, H19, IGF2, MEST, ZFAT, PLAGL1, AIM1) or are transcribed additionally only in the adrenal gland (MEG3, RTL1, PEG10, DLK1). Parental monoallelic expression showed extreme stability across gestation and in term pregnancy complications. A distinct group of additional 14 genes exhibited a statistically significant bias in parental allelic proportions defined as having 65-90% of reads from one parental allele (e.g., KLHDC10, NLRP2, RHOBTB3, DNMT1). Molecular mechanisms behind biased parental expression are still to be clarified. However, 66 of 91 (72.5%) analyzed candidate imprinted genes showed no signals of deviation from biallelic expression. CONCLUSIONS: As placental tissue is not included in The Genotype-Tissue Expression (GTEx) project, the study contributed to fill the gap in the knowledge concerning parental allelic expression. A catalog of parental allelic proportions and gene expression of analyzed loci across human gestation and in term pregnancy complications is provided as additional files. The study outcome suggested that true imprinting in the human placenta is restricted to well-characterized loci. High expression of imprinted genes during mid-pregnancy supports their critical role in developmental programming. Consistent with the data on other GTEx tissues, the number of human imprinted genes appears to be overestimated.

RNA sequencing of chorionic villi from recurrent pregnancy loss patients reveals impaired function of basic nuclear and cellular machinery.

Recurrent pregnancy loss (RPL) concerns ~3% of couples aiming at childbirth. In the current study, transcriptomes and miRNomes of 1st trimester placental chorionic villi were analysed for 2 RPL cases (≥6 miscarriages) and normal, but electively terminated pregnancies (ETP; n = 8). Sequencing was performed on Illumina HiSeq 2000 platform. Differential expression analyses detected 51 (27%) transcripts with increased and 138 (73%) with decreased expression in RPL compared to ETP (DESeq: FDR P < 0.1 and DESeq2: <0.05). RPL samples had substantially decreased transcript levels of histones, regulatory RNAs and genes involved in telomere, spliceosome, ribosomal, mitochondrial and intra-cellular signalling functions. Downregulated expression of HIST1H1B and HIST1H4A (Wilcoxon test, fc≤0.372, P≤9.37 × 10-4) was validated in an extended sample by quantitative PCR (RPL, n = 14; ETP, n = 24). Several upregulated genes are linked to placental function and pregnancy complications: ATF4, C3, PHLDA2, GPX4, ICAM1, SLC16A2. Analysis of the miRNA-Seq dataset identified no large disturbances in RPL samples. Notably, nearly 2/3 of differentially expressed genes have binding sites for E2F transcription factors, coordinating mammalian endocycle and placental development. For a conceptus destined to miscarriage, the E2F TF-family represents a potential key coordinator in reprogramming the placental genome towards gradually stopping the maintenance of basic nuclear and cellular functions.

Extensive shift in placental transcriptome profile in preeclampsia and placental origin of adverse pregnancy outcomes.

One in five pregnant women suffer from gestational complications, prevalently driven by placental malfunction. Using RNASeq, we analyzed differential placental gene expression in cases of normal gestation, late-onset preeclampsia (LO-PE), gestational diabetes (GD) and pregnancies ending with the birth of small-for-gestational-age (SGA) or large-for-gestational-age (LGA) newborns (n = 8/group). In all groups, the highest expression was detected for small noncoding RNAs and genes specifically implicated in placental function and hormonal regulation. The transcriptome of LO-PE placentas was clearly distinct, showing statistically significant (after FDR) expressional disturbances for hundreds of genes. Taqman RT-qPCR validation of 45 genes in an extended sample (n = 24/group) provided concordant results. A limited number of transcription factors including LRF, SP1 and AP2 were identified as possible drivers of these changes. Notable differences were detected in differential expression signatures of LO-PE subtypes defined by the presence or absence of intrauterine growth restriction (IUGR). LO-PE with IUGR showed higher correlation with SGA and LO-PE without IUGR with LGA placentas. Whereas changes in placental transcriptome in SGA, LGA and GD cases were less prominent, the overall profiles of expressional disturbances overlapped among pregnancy complications providing support to shared placental responses. The dataset represent a rich catalogue for potential biomarkers and therapeutic targets.