Previous transcriptome studies of the human endometrium have revealed hundreds of simultaneously up- and down-regulated genes that are involved in endometrial receptivity. However, the overlap between the studies is relatively small, and we are still searching for potential diagnostic biomarkers. Here we perform a meta-analysis of endometrial-receptivity associated genes on 164 endometrial samples (76 from ‘pre-receptive’ and 88 from mid-secretory, ‘receptive’ phase endometria) using a robust rank aggregation (RRA) method, followed by enrichment analysis, and regulatory microRNA prediction. We identify a meta-signature of endometrial receptivity involving 57 mRNA genes as putative receptivity markers, where 39 of these we confirm experimentally using RNA-sequencing method in two separate datasets. The meta-signature genes highlight the importance of immune responses, the complement cascade pathway and the involvement of exosomes in mid-secretory endometrial functions. Bioinformatic prediction identifies 348 microRNAs that could regulate 30 endometrial-receptivity associated genes, and we confirm experimentally the decreased expression of 19 microRNAs with 11 corresponding up-regulated meta-signature genes in our validation experiments. The 57 identified meta-signature genes and involved pathways, together with their regulatory microRNAs could serve as promising and sought-after biomarkers of endometrial receptivity, fertility and infertility.

The transcriptome analysis of whole-blood RNA by sequencing holds promise for the identification and tracking of biomarkers; however, the high globin mRNA (gmRNA) content of erythrocytes hampers whole-blood and buffy coat analyses. We introduce a novel gmRNA locking assay (GlobinLock, GL) as a robust and simple gmRNA reduction tool to preserve RNA quality, save time and cost. GL consists of a pair of gmRNA-specific oligonucleotides in RNA initial denaturation buffer that is effective immediately after RNA denaturation and adds only ten minutes of incubation to the whole cDNA synthesis procedure when compared to non-blood RNA analysis. We show that GL is fully effective not only for human samples but also for mouse and rat, and so far incompletely studied cow, dog and zebrafish.

Targeted next-generation sequencing (NGS) methods have become essential in medical research and diagnostics. In addition to NGS sensitivity and high-throughput capacity, precise biomolecule counting based on unique molecular identifier (UMI) has potential to increase biomolecule detection accuracy. Although UMIs are widely used in basic research its introduction to clinical assays is still in progress. Here, we present a robust and cost-effective TAC-seq (Targeted Allele Counting by sequencing) method that uses UMIs to estimate the original molecule counts of mRNAs, microRNAs, and cell-free DNA. We applied TAC-seq in three different clinical applications and compared the results with standard NGS. RNA samples extracted from human endometrial biopsies were analyzed using previously described 57 mRNA-based receptivity biomarkers and 49 selected microRNAs at different expression levels. Cell-free DNA aneuploidy testing was based on cell line (47,XX, +21) genomic DNA. TAC-seq mRNA profiling showed identical clustering results to transcriptome RNA sequencing, and microRNA detection demonstrated significant reduction in amplification bias, allowing to determine minor expression changes between different samples that remained undetermined by standard NGS. The mimicking experiment for cell-free DNA fetal aneuploidy analysis showed that TAC-seq can be applied to count highly fragmented DNA, detecting significant (p = 7.6 × 10−4) excess of chromosome 21 molecules at 10% fetal fraction level. Based on three proof-of-principle applications we demonstrate that TAC-seq is an accurate and highly potential biomarker profiling method for advanced medical research and diagnostics.

STUDY QUESTION Does cellular composition of the endometrial biopsy affect the gene expression profile of endometrial whole-tissue samples? SUMMARY ANSWER The differences in epithelial and stromal cell proportions in endometrial biopsies modify whole-tissue gene expression profiles, and also affect the results of differential expression analysis. WHAT IS ALREADY KNOWN Each cell type has its unique gene expression profile. The proportions of epithelial and stromal cells vary in endometrial tissue during the menstrual cycle, along with individual and technical variation due to the way and tools used to obtain the tissue biopsy. STUDY DESIGN, SIZE, DURATIONUsing cell-population specific transcriptome data and computational deconvolution approach, we estimated the epithelial and stromal cell proportions in whole-tissue biopsies taken during early secretory and mid-secretory phases. The estimated cellular proportions were used as covariates in whole-tissue differential gene expression analysis. Endometrial transcriptomes before and after deconvolution were compared and analysed in biological context.

No abstract

Pernicious anemia is a rare condition characterized by vitamin B12 deficiency anemia due to lack of intrinsic factor, often caused by autoimmune gastritis. Patients with pernicious anemia have a higher incidence of other autoimmune disorders, such as type 1 diabetes, vitiligo, and autoimmune thyroid issues. Therefore, the disease has a clear autoimmune basis, although the genetic susceptibility factors have thus far remained poorly studied. We conduct a genome-wide association study meta-analysis in 2166 cases and 659,516 European controls from population-based biobanks and identify genome-wide significant signals in or near the PTPN22 (rs6679677, p = 1.91 × 10−24, OR = 1.63), PNPT1 (rs12616502, p = 3.14 × 10−8, OR = 1.70), HLA-DQB1 (rs28414666, p = 1.40 × 10−16, OR = 1.38), IL2RA (rs2476491, p = 1.90 × 10−8, OR = 1.22) and AIRE (rs74203920, p = 2.33 × 10−9, OR = 1.83) genes, thus providing robust associations between pernicious anemia and genetic risk factors.

(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.Thecopyright holder for this preprint . http://dx.doi.org/10.1101/357152 doi: bioRxiv preprint first posted online Jun. 28, 2018; 3 PARTICIPANTS/MATERIAL, SETTING, METHODS Paired early-and mid-secretory 50endometrial biopsies were obtained from thirty-five healthy, regularly cycling, fertile volun-51 teers, aged 23 to 36 years, and analysed by RNA sequencing. Differential gene expression 52 analysis was performed using two approaches. In one of them, computational deconvolution 53 was applied as an intermediate step to adjust for epithelial and stromal cells' proportions in 54 endometrial biopsy. The results were then compared to conventional differential expression 55 analysis. 56 MAIN RESULTS AND THE ROLE OF CHANCE The estimated average proportions of 57stromal and epithelial cells in early secretory phase were 65% and 35%, and during mid-secre-58 tory phase 46% and 54%, respectively, that correlated well with the results of histological eval-59 uation (r=0. 88, p=1.1×10 -6 ). Endometrial tissue transcriptomic analysis showed that approxi-60 mately 26% of transcripts (n=946) differentially expressed in receptive endometrium in cell-61 type unadjusted analysis also remain differentially expressed after adjustment for biopsy cel-62 lular composition. However, the other 74% (n=2,645) become statistically non-significant after 63 adjustment for biopsy cellular composition, underlining the impact of tissue heterogeneity on 64 differential expression analysis. The results suggest new mechanisms involved in endometrial 65 maturation involving genes like LINC01320, SLC8A1 and GGTA1P, described for the first time 66 in context of endometrial receptivity. 67

Background The uterine cervix has an important role in female reproductive health, but not much is known about the genetic determinants of cervical biology and pathology. Genome-wide association studies (GWAS) with increasing sample sizes have reported a few genetic associations for cervical cancer. However, GWAS is only the first step in mapping the genetic susceptibility and thus, the underlying biology in cervical cancer and other cervical phenotypes is still not entirely understood. Here, we use data from large biobanks to characterise the genetics of cervical phenotypes (including cervical cancer) and leverage latest computational methods and gene expression data to refine the association signals for cervical cancer. Methods Using Estonian Biobank and FinnGen data, we characterise the genetic signals associated with cervical ectropion (10,162 cases/151,347 controls), cervicitis (19,285/185,708) and cervical dysplasia (14,694/150,563). We present the results from the largest trans-ethnic GWAS meta-analysis of cervical cancer, including up to 9,229 cases and 490,304 controls from Estonian Biobank, the FinnGen study, the UK Biobank and Biobank Japan. We combine GWAS results with gene expression data and chromatin regulatory annotations in HeLa cervical carcinoma cells to propose the most likely candidate genes and causal variants for every locus associated with cervical cancer. We further dissect the HLA association with cervical pathology using imputed data on alleles and amino acid polymorphisms. Results We report a single associated locus on 2q13 for both cervical ectropion (rs3748916, p=5.1 x 10-16) and cervicitis (rs1049137, p=3.9 x 10-10), and five signals for cervical dysplasia - 6p21.32 (rs1053726, p=9.1 x 10-9; rs36214159, 1.6 x 10-22), 2q24.1 (rs12611652, p=3.2 x 10-9) near DAPL1, 2q13 ns1049137, p=6.4 x 10-9) near PAX8, and 5p15.33 (rs6866294, p=2.1 x 10-9), downstream of CLPTM1L. We identify five loci associated with cervical cancer in the trans-ethnic meta-analysis: 1p36.12 (rs2268177, p= 3.1 x 10-8), 2q13 (rs4849177, p=9.4 x 10-15), 5p15.33 (rs27069, p=1.3 x 10-14), 17q12 (rs12603332, p=1.2 x 10-9), and 6p21.32 (rs35508382, p=1.0 x 10-39). Joint analysis of dysplasia and cancer datasets revealed an association on chromosome 19 (rs425787, p=3.5 x 10-8), near CD70. Conclusions Our results map PAX8/PAX8-AS1, LINC00339, CDC42, CLPTM1L, HLA-DRB1, HLA-B, and GSDMB as the most likely candidate genes for cervical cancer, which provides novel insight into cervical cancer pathogenesis and supports the role of genes involved in reproductive tract development, immune response and cellular proliferation/apoptosis. We further show that PAX8/PAX8-AS1 has a central role in cervical biology and pathology, as it was associated with all analysed phenotypes. The detailed characterisation of association signals, together with mapping of causal variants and genes offers valuable leads for further functional studies.