Endometrial Transcriptome Research Articles

Transcriptome profiling of buffalo endometrium reveals molecular signature distinct to early pregnancy.

Buffalo reproduction struggles with a high incidence of early embryonic mortality. Effective treatment and prevention strategies for this condition are not available due to lack of understanding of molecular pathways in early pregnancy of this species. In the present study, we have attempted to understand these molecular pathways by characterizing the endometrial transcriptomic profiles of pregnant buffalos during early pregnancy. For the transcriptome profiling, buffalo endometrial tissues of 29-36days of pregnancy and of nonpregnant luteal phase were collected from the local slaughterhouse. We confirmed the status of pregnancy based on the crown vertebral length of the foetus. Total RNA was isolated and sequencing was performed using the Illumina nextseq platform. The raw reads were filtered and mapped to the Bos taurus UMD 3.1 reference genome assembly. An average of 24,597 genes was investigated for differential expression between the two groups. Transcriptome data identified a total of 450 differentially expressed genes (using a cut off value of log2 fold changes >2 and <-2) in early pregnancy in comparison to the nonpregnant group (Padj<0.05). Among these, 270 genes were significantly upregulated and 180 genes were downregulated. The most impacted pathways were related to secretion, transport, ionic homeostasis, mitosis and negative regulation of viral processes. In conclusion, our study characterized a unique set of DEGs, during the early pregnancy of buffalo, which potentially modulate the endometrial environment to establish and maintain a successful pregnancy.

Estradiol-17β-Induced Changes in the Porcine Endometrial Transcriptome In Vivo.

Estradiol-17β (E2) is a key hormone regulating reproductive functions in females. In pigs, E2, as the main conceptus signal, initiates processes resulting in prolonged corpus luteum function, embryo development, and implantation. During early pregnancy the endometrium undergoes morphological and physiological transitions that are tightly related to transcriptome changes. Recently, however, the importance of E2 as a primary conceptus signal in the pig has been questionable. Thus, the aim of the present study was to determine the effects of E2 on the porcine endometrial transcriptome in vivo and to compare these effects with transcriptome profiles on day 12 of pregnancy. Microarray analysis revealed differentially expressed genes (DEGs) in response to E2 with overrepresented functional terms related to secretive functions, extracellular vesicles, cell adhesion, proliferation and differentiation, tissue rearrangements, immune response, lipid metabolism, and many others. Numerous common DEGs and processes for the endometrium on day 12 of pregnancy and E2-treated endometrium were identified. In summary, the present study is the first evidence for the effect of E2 on transcriptome profiles in porcine endometrium in vivo in the period corresponding to the maternal recognition of pregnancy. The presented results provide a valuable resource for further targeted studies considering genes and pathways regulated by conceptus-derived estrogens and their role in pregnancy establishment.

Seminal Plasma Modifies the Transcriptional Pattern of the Endometrium and Advances Embryo Development in Pigs.

Background: Seminal plasma (SP) promotes sperm survival and fertilizing capacity, and potentially affects embryo development, presumably via specific signaling pathways to the internal female genital tract.Objectives: This study evaluated how heterologous SP, infused immediately before postcervical artificial insemination (AI) affected embryo development and the transcriptional pattern of the pig endometria containing embryos.Materials and Methods: Postweaning estrus sows (n = 34) received 40-mL intrauterine infusions of either heterologous pooled SP or Beltsville Thawing Solution (BTS; control) 30 min before AI of semen extended to 10% of homologous SP. Embryos (all sows) and endometrium samples (3 sows/group) were removed during laparotomy 6 days after the infusion of SP or BTS to morphologically evaluate the embryos to determine their developmental stage and to analyze the endometrial transcriptome using microarrays (PORGENE 1.0 ST GeneChip array, Affymetrix) followed by qPCR for further validation.Results: Embryo viability was equal between the groups (~93%), but embryo development was significantly (P < 0.05) more advanced in the SP-treated group compared to control. A total of 1,604 endometrium transcripts were differentially expressed in the SP group compared to the control group. An enrichment analysis showed an overrepresentation of genes and pathways associated with the immune response, cytokine signaling, cell cycle, cell adhesion, and hormone response, among others.Conclusions: SP infusions prior to AI positively impacted the preimplantation embryo development and altered the expression of the endometrial genes and pathways potentially involved in embryo development.

4 Sperm, but not seminal plasma, elicit changes in the bovine endometrial transcriptome after natural mating

Studies in mice and pigs have shown that seminal plasma (SP) can modulate the uterine environment, leading to an increase in embryo survival and implantation. However, in cattle, conflicting evidence has resulted from studies in which SP was infused into the uterus. In contrast to mice and pigs, it is questionable whether any SP reaches the bovine uterus during natural mating. However, sperm could act as vehicle for SP proteins that bind to their plasma membrane at ejaculation. Thus, the aim of this study was to analyse the effect of exposure to SP, in the presence or absence of sperm, on the endometrial transcriptome. Beef heifers were oestrus synchronized and mated after being seen in standing oestrus with intact bulls (which ejaculate sperm and SP, n=7) or vasectomized bulls (VX, which only ejaculate SP, n=8) or left unmated (control, n=7). To reduce a potential bull effect, 3 vasectomized and 2 intact Holstein bulls were used. Each heifer was mounted once to one intact or VX bull. Heifers were slaughtered 24h (±6h) after mating, and their reproductive tracts were recovered. Endometrial tissue from the base of the uterine horn ipsilateral to the ovulatory follicle was processed for RNA sequencing. Surprisingly, mating with a VX bull resulted in no differentially expressed genes (DEG) in the endometrium compared with unmated controls. In contrast, mating with intact bulls resulted in 22 DEG (10 down regulated and 12 upregulated) compared with unmated controls and 24 DEG (6 downregulated and 18 upregulated) compared with VX samples. The upregulated genes in intact versus VX samples included several that may have a potential role in implantation: CX3CL1, C4BPA, VCAM1-like, and IL17F. Gene CX3CL1 promotes trophoblast migration in women and has been described as a potential bovine embryokine. In addition, decreased levels of C4BPA have been correlated with implantation failure and unexplained recurrent abortions in women. Further, VCAM1 is involved in bovine conceptus implantation and is downregulated in women with unexplained infertility. In mice, SP regulates inflammation of IL17F in the uterus through the γδT/IL-17 pathway. On the other hand, the immune-related genes PRSS2 and CEBPD were downregulated in intact versus control samples, and BOLA-DQB was downregulated in intact versus VX samples. Gene PRSS2, involved in tissue remodelling, is upregulated in the endometrium of repeated breeder cows. Gene CEBPD, is a transcription factor that enhances IL6 expression, whereas BOLA-DQB is involved in antigen presentation. Interestingly, the gene coding for the α-chain of this protein is also downregulated in high fertility cows. In conclusion, these results do not support a direct effect of SP in regulating pregnancy success in cattle. However, sperm can elicit modest endometrial transcriptomic changes related to implantation and immune system regulation. Further research is needed to elucidate the role of intrinsic sperm proteins, or SP proteins transported by these cells, in the modulation of the uterine environment. This work was supported by EU Horizon 2020 Marie Sklodowska-Curie (No. 792212).

Is the profile of transcripts altered in the eutopic endometrium of infertile women with endometriosis during the implantation window?

Compared to healthy women, is the profile of transcripts altered in the eutopic endometrium of infertile women with endometriosis during the implantation window (IW)? The eutopic endometrium of infertile women with endometriosis seems to be transcriptionally similar to the endometrium of infertile and fertile controls (FC) during the IW. Endometriosis is a disease related to infertility; nevertheless, little is known regarding the ethiopathogenic mechanisms underlying this association. Some studies evaluating the eutopic endometrium of endometriosis patients suggest there is an endometrial factor involved in the disease-related infertility. However, no study to date has evaluated the endometrial transcriptome (mRNA and miRNA) by next generation sequencing (NGS), comparing patients with endometriosis as the exclusive infertility factor (END) to infertile controls (IC; male and/or tubal factor) and FC. From November 2011 to November 2015 we performed a case-control study, where 17 endometrial samples (six END, six IC, five FC) were collected during the IW. All endometrial samples had the RNA extracted. Two libraries were prepared for each one (mRNA and miRNA), which were sequenced, respectively, at HISEQ 2500 (RNA-Seq) and MiSeq System (miRNA-Seq), Illumina. The normalization and differential expression were conducted in statistical R environment using DESeq2 package. qPCR was used for data validation, which were analyzed by Kruskal-Wallis test and Dunn posttest (P< 0.05). RNA-Seq revealed no differentially expressed genes (DEG) among END, IC and FC groups. miRNA-Seq revealed three differentially expressed miRNAs (has-27a-5p, has-miR-150-5p, has-miR-504-5p) in END group compared to FC group. However, none of the miRNAs identified in the sequencing was validated by qPCR. The main limitation of this study was the small sample size evaluated as a result of the restrictive eligibility criteria adopted, limiting the generalization of the results obtained here. On the other hand, strict eligibility criteria, which eliminated factors potentially related to impaired endometrial receptivity, were required to increase the study's internal validity. This study brings new perspectives on the mechanisms involved in endometriosis-related infertility. The present findings suggest the eutopic endometrium of infertile women with endometriosis, without considering the disease's stage, is transcriptionally similar to controls during the IW, possibly not affecting receptivity. Further studies are needed to evaluate endometrial alterations related to endometriosis' stages. This study received financial support from the Sao Paulo Research Foundation (FAPESP-Fundação de Amparo à Pesquisa do Estado de São Paulo; fellowship 2011/17614-6, MGB) and from the National Council for Scientific and Technological Development (CNPq-Conselho Nacional de Desenvolvimento Científico e Tecnológico; INCT-National Institutes of Hormones and Woman's Health, grant 471 943/2012-6, 309 397/2016-2, PAN; fellowship 140 137/2015-7, MGB). The authors have no conflicts of interest. N/A.

Transcriptome sequencing of endometrium revealed alterations in mRNAs and lncRNAs after ovarian stimulation.

Using RNA-sequencing analysis, we investigated the relationship between ovarian stimulation and endometrial transcriptome profiles during the window of implantation (WOI) to identify candidate predictive factors for the WOI and to optimize timing for embryo transfer. Twelve women with normal basal hormone levels and regular ovulation were randomly assigned into three groups based on sampling time: late-proliferate phase (P group), and receptive phase in natural cycles (LH+7, N group) and stimulated cycles (hCG+7, S group). Transcriptome profiles of mRNAs and long non-coding RNAs (lncRNAs) were then compared among the three groups. Validation was performed using real-time qPCR. Comparison of transcriptome profiles between the natural and stimulated endometrium revealed 173 differentially expressed genes (DEGs), with a > 2-fold change (FC) and p < 0.05, under the influence of supraphysiological estradiol (E2) induced by ovarian stimulation. By clustering and KEGG pathway analysis, molecules and pathways associated with endometrial receptivity were identified. Of the 39 DEGs common to the three groups, eight genes were validated using real-time PCR. ESR1, MMP10, and HPSE were previously reported to be associated with endometrial receptivity. In addition, three novel genes (IL13RA2, ZCCHC12, SRARP) and two lncRNAs (LINC01060, LINC01104) were new potential endometrial receptivity-related markers. Using mRNA and lncRNA sequencing, we found that supraphysiological E2 levels from ovarian stimulation had a marked impact upon endometrial transcriptome profiles and may result in a shift of the WOI. The precise mechanisms underlying the supraphysiological hormone-induced shift of the WOI require further research. ChiCTR180001453.

Coding RNA Sequencing of Equine Endometrium during Maternal Recognition of Pregnancy.

Equine maternal recognition of pregnancy (MRP) is a process whose signal remains unknown. During MRP the conceptus and endometrium communicate to attenuate prostaglandin F2α (PGF) secretion, sparing the corpus luteum and maintaining progesterone production. Recognition of a mobile conceptus by the endometrium is critical by days 14–16 post-ovulation (PO), when endometrium produces PGF, initiating luteolysis. The objective of this study was to evaluate endometrial gene expression changes based upon pregnancy status via RNA sequencing. This experiment utilized a cross-over design with each mare serving as both a pregnant and non-mated control on days nine, 11, and 13 PO (n = 3/status/day). Mares were randomly assigned to collection day and pregnancy confirmed by terminal uterine lavage at the time of endometrial biopsy. Total RNA was isolated and libraries prepared using Illumina TruSeq RNA sample preparation kit. Reads were mapped and annotated using HISAT2 and Stringtie. Expression values were evaluated with DESEQ2 (P ≤ 0.05 indicated significance). On day nine, 11, and 13 there were 1435, 1435 and 916 significant transcripts, respectively. Multiple genes with splice variants had different expression patterns within the same day. These are the first data to evaluate the endometrial transcriptome during MRP on days nine, 11, and 13.

Global transcriptomic response of bovine endometrium to blastocyst-stage embryos.

The aims of this study were (i) to investigate changes in the global transcriptome of bovine endometrial explants induced by exposure to blastocysts, (ii) to investigate if male and female blastocysts elicit a differential response in the endometrial transcriptome in vitro and (iii) to determine whether bovine endometrium responds to the presence of murine embryos. In Experiment 1, endometrial explants from the same uterus were cultured for 6 h with or without 20 in vitro-produced bovine blastocysts. In Experiment 2, endometrial explants were cultured with male or female bovine blastocysts produced in vitro by IVF either using sex-sorted semen or conventional unsorted semen followed by embryo sexing based on a biopsy. In Experiment 3, endometrial explants were cultured alone or in the presence of bovine blastocysts (n = 25) or murine blastocysts (n = 25). Following culture, explants were snap frozen and stored at -80°C until RNA extraction, qPCR or RNA-Seq. Culture with bovine blastocysts increased endometrial expression of 40 transcripts, all of which were interferon-tau induced. Culture with male or female bovine blastocysts increased transcript abundance of five classic interferon-stimulated genes (MX1, MX2, ISG15, OASY1, RSAD2) in explants; however, there was no difference in abundance of transcripts previously reported to be related to embryonic sex (IFNAR1, IFNAR2, CTGF, ARTN, SLC2A1, SLC2A5). Exposure to murine blastocysts did not elicit any detectable change in transcript abundance. These findings, coupled with our previous data, indicate that very local, interferon-tau-induced changes in endometrial gene expression occur in response to blastocysts; whether such changes play any role in subsequent pregnancy recognition remains to be established.

The pre-hatching bovine embryo transforms the uterine luminal metabolite composition in vivo

In cattle, conceptus development after elongation relies on well-characterized, paracrine interactions with the hosting maternal reproductive tract. However, it was unrecognized previously that the pre-hatching, pre-implantation bovine embryo also engages in biochemical signalling with the maternal uterus. Our recent work showed that the embryo modified the endometrial transcriptome in vivo. Here, we hypothesized that the embryo modulates the biochemical composition of the uterine luminal fluid (ULF) in the most cranial portion of the uterine horn ipsilateral to the corpus luteum. Endometrial samples and ULF were collected post-mortem from sham-inseminated cows and from cows inseminated and detected pregnant 7 days after oestrus. We used quantitative mass spectrometry to demonstrate that the pre-hatching embryo changes ULF composition in vivo. Embryo-induced modulation included an increase in concentrations of lipoxygenase-derived metabolites [12(S)-HETE, 15(S)-HETE] and a decrease in the concentrations of amino acids (glycine), biogenic amines (sarcosine), acylcarnitines and phospholipids. The changed composition of the ULF could be due to secretion or depletion of specific molecules, executed by either the embryo or the endometrium, but initiated by signals coming from the embryo. This study provides the basis for further understanding embryo-initiated modulation of the uterine milieu. Early embryonic signalling may be necessary to guarantee optimal development and successful establishment of pregnancy in cattle.

Concurrent and long-term associations between the endometrial microbiota and endometrial transcriptome in postpartum dairy cows

BackgroundFertility in dairy cows depends on ovarian cyclicity and on uterine involution. Ovarian cyclicity and uterine involution are delayed when there is uterine dysbiosis (overgrowth of pathogenic bacteria). Fertility in dairy cows may involve a mechanism through which the uterine microbiota affects ovarian cyclicity as well as the transcriptome of the endometrium within the involuting uterus. The hypothesis was that the transcriptome of the endometrium in postpartum cows would be associated with the cyclicity status of the cow as well as the microbiota during uterine involution. The endometrium of first lactation dairy cows was sampled at 1, 5, and 9 weeks postpartum. All cows were allowed to return to cyclicity without intervention until week 5 and treated with an ovulation synchronization protocol so that sampling at week 9 was on day 13 of the estrous cycle. The endometrial microbiota was measured by 16S rRNA gene sequencing and principal component analysis. The endometrial transcriptome was measured by mRNA sequencing, differential gene expression analysis, and Ingenuity Pathway Analysis.ResultsThe endometrial microbiota changed from week 1 to week 5 but the week 5 and week 9 microbiota were similar. The endometrial transcriptome differed for cows that were either cycling or not cycling at week 5 and cyclicity status depended in part on the endometrial microbiota. Compared with cows cycling at week 5, there were large changes in the transcriptome of cows that progressed from non-cycling at week 5 to cycling at week 9. There was evidence for concurrent and longer-term associations between the endometrial microbiota and transcriptome. The week 1 endometrial microbiota had the greatest effect on the subsequent endometrial transcriptome and this effect was greatest at week 5 and diminished by week 9.ConclusionsThe cumulative response of the endometrial transcriptome to the microbiota represented the combination of past microbial exposure and current microbial exposure. The endometrial transcriptome in postpartum cows, therefore, depended on the immediate and longer-term effects of the uterine microbiota that acted directly on the uterus. There may also be an indirect mechanism through which the microbiome affects the transcriptome through the restoration of ovarian cyclicity postpartum.

New perspective on conceptus estrogens in maternal recognition and pregnancy establishment in the pig†.

The proposed signal for maternal recognition of pregnancy in pigs is estrogen (E2), produced by the elongating conceptuses between days 11 to 12 of pregnancy with a more sustained increase during conceptus attachment and placental development on days 15 to 30. To understand the role of E2 in porcine conceptus elongation and pregnancy establishment, a loss-of-function study was conducted by editing aromatase (CYP19A1) using CRISPR/Cas9 technology. Wild-type (CYP19A1+/+) and (CYP19A1-/-) fibroblast cells were used to create embryos through somatic cell nuclear transfer, which were transferred into recipient gilts. Elongated and attaching conceptuses were recovered from gilts containing CYP19A1+/+ or CYP19A1-/- embryos on day 14 and 17 of pregnancy. Total E2 in the uterine flushings of gilts with CYP19A1-/- embryos was lower than recipients containing CYP19A1+/+ embryos with no difference in testosterone, PGF2α, or PGE2 on either day 14 or 17. Despite the loss of conceptus E2 production, CYP19A1-/- conceptuses were capable of maintaining the corpora lutea. However, gilts gestating CYP19A1-/- embryos aborted between days 27 and 31 of gestation. Attempts to rescue the pregnancy of CYP19A1-/- gestating gilts with exogenous E2 failed to maintain pregnancy. However, CYP19A1-/- embryos could be rescued when co-transferred with embryos derived by in vitro fertilization. Endometrial transcriptome analysis revealed that ablation of conceptus E2 resulted in disruption of a number biological pathways. Results demonstrate that intrinsic E2 conceptus production is not essential for pre-implantation development, conceptus elongation, and early CL maintenance, but is essential for maintenance of pregnancy beyond 30 days .

Aspects of embryo-maternal communication in establishment of pregnancy in cattle

Establishment of pregnancy in mammals requires reciprocal molecular communication between the conceptus and endometrium that modifies the endometrial transcriptome and uterine luminal milieu to support pregnancy. Due to the small size of the early embryo and elongating conceptus relative to the volume of the uterine lumen, collection of endometrium adjacent to the developing conceptus is difficult following conventional uterine flushing methods in cattle. Use of endometrial explants in culture can overcome this challenge and reveal information about the dialogue between the developing embryo and the uterus. The aim of this short review is to summarize some of our recent findings in relation to embryo maternal interaction during bovine pregnancy establishment and to put them in the wider context of fertility in cattle.

Supplementation with long-acting progesterone in early diestrus in beef cattle: I. effect of artificial insemination on onset of luteolysis

Progesterone (P4) supplementation in early diestrus advances changes in the endometrial transcriptome, stimulating embryonic development. However, it also induces early onset of luteolysis. Occurrence of luteolysis before D16 postmating can be detrimental to fertility. A potential counteracting role of the elongating conceptus on early luteolysis is understood poorly. The aim of the study was to evaluate the effect of artificial insemination (AI; ie, pregnancy) on the temporal dynamics of luteolysis of cows supplemented with P4. Nonsuckled beef cows were inseminated at 12 h after estrus (D0: ovulation) or were not inseminated (no-AI). On D3, the AI cows were assigned to receive a single dose of 150 mg of injectable long-acting P4 via intramuscular injection (AI + iP4; n = 23), and the no-AI cows were assigned to receive iP4 (iP4; n = 21) or saline (control, n = 22). Corpus luteum (CL) development and regression were determined by ultrasonography (US) between D3 and D21. Plasma P4 concentrations were measured on D3 and every other day from D9 to D21. Pregnancy status was determined by US (D28‒D32). iP4 supplementation reduced luteal development (D5–D10) compared to the control group and increased incidence of luteolysis between D14 and D15. On D15, the proportion of cows that underwent luteolysis and plasma P4 concentrations differed between the iP4 group (47.6; 2.10 ± 0.47) and the control group (13.6; 4.40 ± 0.46) and was intermediate in the AI + iP4 group, respectively (26.1%; 3.70 ± 0.45 ng/mL; P < 0.05). The AI effects were due to the pregnant cows (n = 7). Considering nonpregnant cows only, the proportion of early luteolysis in the AI + iP4 group (37.5%) was similar to the iP4 group. Pregnancy was not established in cows having a shortened luteal lifespan. Indeed, interval to luteolysis in the AI + iP4 group (15.50 ± 0.66 d) was similar to the iP4 group (16.38 ± 0.46 d), but less than the control group (17.38 ± 0.40 d; P = 0.05). In conclusion, the effect of AI on extending luteal lifespan occurred exclusively in cows that maintained pregnancy.

43 Effect of early life nutrition on endometrial gland development and endometrial gene expression in heifers

In cattle, uterine gland development (adenogenesis) occurs postnatally, beginning immediately after birth, and is normally complete by approximately 2 months of age. Normal development of uterine glands is essential for subsequent fertility. Early life plane of nutrition can impact age at puberty, but the impact of early postnatal nutrition on adenogenesis is unclear. The aim of this study was to examine the effect of enhanced nutrition during the first 21 weeks of life on heifer reproductive organ growth, endometrial gland development, and endometrial gene expression. To this end, Angus×Holstein-Friesian heifer calves with a mean (±s.d.) age and body weight of 19 (±5) days and 51.2 (±7.8) kg, respectively, were assigned to either a high (HIGH; n=16; targeted average daily gain, ADG, 1.2 kg/d) or a moderate plane of nutrition (MOD; n=16; targeted ADG 0.5 kg/d). Calves were slaughtered at five months of age, and their reproductive tracts were collected and dissected for analysis. To assess endometrial gland number, a cross-section of the midpoint of one horn (n=8 per group) was fixed in 10% neutral buffered formalin, embedded, and processed for histology. Slides were stained with haematoxylin-eosin. Five pictures were randomly taken on each slide using a stereomicroscope at 40× magnification and the number of glands was counted using ImageJ (https://imagej.nih.gov/ij/) software. In addition, endometrial tissue samples were collected and snap frozen for subsequent quantitative RT-PCR analysis. At the time of slaughter, calves on the HIGH and MOD diets presented a body weight of 189.6 and 110.2kg, respectively (P&amp;lt;0.001), indicating an ADG of 1.18 kg/d for the HIGH group and 0.50 kg/d for the MOD group (P&amp;lt;0.001). Calves in the HIGH group had a greater (P&amp;lt;0.001) total reproductive tract (mean±s.d.; 114.01±35.56g v. 52.67±14.29g), isolated cervix (33.17±9.35g v. 16.14±5.27g), uterus (59.35±25.78g v. 27.46±10.61g), and ovarian (14.80±11.38g v. 5.40±1.94g) tissue weights compared with those in the MOD group. This difference was also present when organ weight was expressed on a per unit of body weight basis (P&amp;lt;0.05). However, no differences were found in the number of endometrial glands between groups (HIGH: 453.50±94.18v. MOD: 482.38±124.69). In accordance with this result, transcript abundance of six paracrine mediators of gland development (ESR1, IGF1, IGFBP1, WNT5A, WNT7A, and FGF10) was not affected by diet. In conclusion, enhanced early-life plane of nutrition increased the size of the reproductive tract in heifer calves but did not affect endometrial gland development. Future studies will compare the global endometrial transcriptome between groups to explore other putative effects of diet on uterine development. Research was supported by EU, Horizon 2020 Marie Sklodowska-Curie, REPBIOTECH 675526, Science Foundation Ireland 13/IA/1983, and São Paulo Research Foundation (FAPESP) 2017/21415-5.

56 Spatial analysis of transcriptome changes in porcine endometrium on Day 14 of pregnancy

During the conception cycle, the embryo undergoes a series of developmental processes including cell division, cellular reorganization, and oestrogen secretion before attaching to the uterine epithelium. The uterine endometrium is complex and consists of various layers and cell types [i.e. luminal epithelium (LE), glandular epithelium (GE), blood cells (B), and stromal areas (S)]. The objective of this study was to characterise the complex transcriptome changes in porcine endometrium during the time of conceptus attachment with respect to localization in different endometrial cell types. RNA-sequencing (RNA-Seq) was conducted for LE, GE, B, and S samples isolated from endometrial tissue collected on Day 14 of pregnancy and the oestrous cycle, respectively (each group n=4), by laser capture microdissection (PALM LCM microscope, Zeiss, Jena, Germany). Total RNA was isolated (RNA integrity number&amp;gt;6.5) and used for the preparation of 32 RNA-seq libraries (Ovation SoLo RNA-Seq System, NuGEN Technologies, San Carlos, CA, USA). Multiplexed (barcode-tagged) libraries were run on an Illumina HiSEqn 2500 (Illumina, San Diego, CA, USA). The obtained sequence data were analysed with a RNA-Seq data analysis pipeline on a local Galaxy server installation. The resulting read counts were used for statistical analysis in EdgeR to identify differentially expressed genes (DEG). Furthermore, an RNA-seq dataset for complete Day 14 endometrial tissue samples from a previous study was analysed using the same pipeline. A total of 14297 genes were detectable in complete endometria, and 12000, 11903, 11094, and 11933 genes in LE, GE, B, and S, respectively. Differential expression analysis was performed between the pregnant and the cyclic nonpregnant group for each cell type and the complete tissue. The highest number of DEG was found for LE (1410) when compared with GE, B, and S (800, 1216, and 384, respectively). In total, 3262 DEG were obtained for the complete tissue between pregnant and nonpregnant gilts. The DEG were assigned to Gene Ontology (GO) terms to characterise overrepresented functional categories and pathways specific for the individual endometrial compartments. The GO classification revealed that most DEG in LE were involved in cell communication, such as ‘extracellular exosome’, ‘extracellular vesicle’, ‘homeostatic process’, whereas the ‘response to organic substance’ and ‘regulation of cell migration’ categories were enriched in GE. In blood vessels, categories such as ‘membrane-bounded vesicle’, ‘cell junction’, ‘cell development’, ‘cell adhesion’ and ‘blood vessel morphogenesis’ were found as overrepresented, whereas in stromal regions, most DEG were assigned to ‘cell communication’ and ‘secretion’. These results confirmed the hypothesis that conceptus signals induce specific transcriptomic regulations in the endometrial compartments/cell types related to their functions during recognition of pregnancy adding a new level of spatial gene expression regulation to endometrial transcriptome analysis.

Endometrial receptivity revisited: endometrial transcriptome adjusted for tissue cellular heterogeneity.

Does cellular composition of the endometrial biopsy affect the gene expression profile of endometrial whole-tissue samples? The differences in epithelial and stromal cell proportions in endometrial biopsies modify the whole-tissue gene expression profiles and affect the results of differential expression analyses. 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 method and tools used to obtain the tissue biopsy. Using 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. Paired early- and mid-secretory endometrial biopsies were obtained from 35 healthy, regularly cycling, fertile volunteers, aged 23-36 years, and analysed by RNA sequencing. Differential gene expression analysis was performed using two approaches. In one of them, computational deconvolution was applied as an intermediate step to adjust for the proportions of epithelial and stromal cells in the endometrial biopsy. The results were then compared to conventional differential expression analysis. Ten paired endometrial samples were analysed with qPCR to validate the results. The estimated average proportions of stromal and epithelial cells in early secretory phase were 65% and 35%, and during mid-secretory phase, 46% and 54%, respectively, correlating well with the results of histological evaluation (r = 0.88, P = 1.1 × 10-6). Endometrial tissue transcriptomic analysis showed that approximately 26% of transcripts (n = 946) differentially expressed in receptive endometrium in cell-type unadjusted analysis also remain differentially expressed after adjustment for biopsy cellular composition. However, the other 74% (n = 2645) become statistically non-significant after adjustment for biopsy cellular composition, underlining the impact of tissue heterogeneity on differential expression analysis. The results suggest new mechanisms involved in endometrial maturation, involving genes like LINC01320, SLC8A1 and GGTA1P, described for the first time in context of endometrial receptivity. The RNA-seq data presented in this study is deposited in the Gene Expression Omnibus database with accession number GSE98386. Only dominant endometrial cell types were considered in gene expression profile deconvolution; however, other less frequent endometrial cell types also contribute to the whole-tissue gene expression profile. The better understanding of molecular processes during transition from pre-receptive to receptive endometrium serves to improve the effectiveness and personalization of assisted reproduction protocols. Biopsy cellular composition should be taken into account in future endometrial 'omics' studies, where tissue heterogeneity could potentially influence the results. This study was funded by: Estonian Ministry of Education and Research (grant IUT34-16); Enterprise Estonia (EU48695); the EU-FP7 Eurostars program (NOTED, EU41564); the EU-FP7 Marie Curie Industry-Academia Partnerships and Pathways (SARM, EU324509); Horizon 2020 innovation program (WIDENLIFE, EU692065); MSCA-RISE-2015 project MOMENDO (No 691058) and the Miguel Servet Program Type I of Instituto de Salud Carlos III (CP13/00038); Spanish Ministry of Economy, Industry and Competitiveness (MINECO) and European Regional Development Fund (FEDER): grants RYC-2016-21199 and ENDORE SAF2017-87526. Authors confirm no competing interests.

Cell type-specific endometrial transcriptome changes during initial recognition of pregnancy in the mare.

Previous endometrial gene expression studies during the time of conceptus migration did not provide final conclusions on the mechanisms of maternal recognition of pregnancy (MRP) in the mare. This called for a cell type-specific endometrial gene expression analysis in response to embryo signals to improve the understanding of gene expression regulation in the context of MRP. Laser capture microdissection was used to collect luminal epithelium (LE), glandular epithelium and stroma from endometrial biopsies from Day 12 of pregnancy and Day 12 of the oestrous cycle. RNA sequencing (RNA-Seq) showed greater expression differences between cell types than between pregnant and cyclic states; differences between the pregnant and cyclic states were mainly found in LE. Comparison with a previous RNA-Seq dataset for whole biopsy samples revealed the specific origin of gene expression differences. Furthermore, genes specifically differentially expressed (DE) in one cell type were found that were not detectable as DE in biopsies. Overall, this study revealed spatial information about endometrial gene expression during the phase of initial MRP. The conceptus induced changes in the expression of genes involved in blood vessel development, specific spatial regulation of the immune system, growth factors, regulation of prostaglandin synthesis, transport prostaglandin receptors, specifically prostaglandin F receptor (PTGFR) in the context of prevention of luteolysis.

Interferon tau-dependent and independent effects of the bovine conceptus on the endometrial transcriptome†.

This study investigated bovine conceptus-induced modifications to the endometrial transcriptome related to effects of interferon tau (IFNT), conceptus origin (in vivo vs. in vitro), and conceptus sex. In vitro (IVF) or in vivo (superovulation and artificial insemination, AI) produced blastocysts were transferred into recipient heifers on day 7 of the estrous cycle. On day 15, IVF- or AI-derived conceptuses were obtained by uterine flushing and individually placed on endometrial explants in media for 6 h. Explants were also cultured with media alone as a control or media containing 100 ng/mL IFNT. Total explant RNA was analyzed by RNA-Seq. Incubation of endometrium with IFNT or IVF- or AI-derived conceptuses changed (P≤0.001) expression of 491, 498, and 576 transcripts, respectively, compared to the control. Further, 369 differentially expressed genes (DEGs) were common between explants exposed to IFNT or a conceptus. A total of 240 DEGs were uniquely altered by conceptuses (IVF- and AI-derived) but not IFNT. Of these transcripts, 46 were shared between the IVF and AI groups, while 61 and 133 were specific to IVF and AI conceptuses, respectively. Five genes [melanophilin (MLPH), prominin-2 (PROM2), myeloid associated differentiation marker (MYADM), vomeronasal 1 receptor 4 like (VN1R4L) and 5-hydroxytryptamine receptor 1A (HTR1A)] were more abundant in endometrium exposed to female compared to male conceptuses (P<0.001). A single gene [ADP-ribosylation factor like GTPase 4C (ARL4C)] was more abundant in response to male conceptuses (P<0.001) than female conceptuses. These data support the hypothesis that conceptus regulation of gene expression in the endometrium is complex and involves factors other than IFNT that may have a biological role in pregnancy establishment.

Effect of mifepristone on the transcriptomic signature of endometrial receptivity.

How does a single dose of mifepristone on Day 2 after the LH peak (LH + 2) affect the endometrial receptivity transcriptome as assessed by the receptive signature established by the endometrial receptivity analysis (ERA)? A single dose of mifepristone on day LH + 2 renders the endometrium non-receptive by altering the transcriptome associated with endometrial receptivity. Mifepristone is a progesterone receptor modulator that has been shown to alter endometrial receptivity. The ERA is a computational predictor that utilizes gene expression data of 248 genes from next generation sequencing to identify endometrial receptivity status. Endometrial biopsies were collected on day LH + 7 from controls (n = 11) and from women treated with mifepristone (n = 7). For further comparative analysis, samples were also obtained from women in the proliferative phase (n = 7). Mifepristone treatment consisted of 200 mg administered on day LH + 2. Endometrial biopsies were treated for RNA isolation and cDNA conversion and sequencing. Endometrial receptivity status was assessed by the ERA computational predictor. Differential gene expression between groups was also assessed. Ingenuity Pathway Analysis was used for network analysis. Validation of gene expression results was done by qPCR. Control samples were all staged around 'receptive' as would be clinically expected for LH + 7. Treatment samples were all staged as non-receptive (all but one was classified as 'proliferative' and the last as 'pre-receptive'). Differential gene expression analysis yielded 60 differentially expressed genes between the control and treatment groups. Bioinformatic pathway analysis for differential expression showed inactivation of the progesterone and glucocorticoid receptors, consistent with mifepristone action. The primary limitations are the relative small number of subjects and the use of a limited gene panel. This study sheds further light on the endometrial receptivity altering effects of mifepristone and on progesterone action. It further shows the capacity of the ERA to identify pharmacologically induced non-receptive endometrium, which expands its possible use clinically and in research. C.v.G. and N.R.B. have no conflicts of interest. P.G.L. reports honorarium from University of HK/Shenzhen, other from NIF, India, outside the submitted work. K.G.D. reports consultancy for Bayer AG, Exelgyn, HRA-Pharma, Gedeon Richter, MSD, Mithra, Exeltis and Natural cycles, payment for lectures from Bayer AG, NSD, Ferring, HRA-Pharma, Exelgyn and Exeltis and clinical trials for Bayer AG, MSD, Exeltis, Mithra, HRA-Pharma and Sun Pharma. C.S. has a patent gene expression profile (ERA) issued to Igenomix and is scientific director of Igenomix S.L. M.R., R.N. and J.M.V. are employees of Igenomix S.L. N/A.

Do differences in the endometrial transcriptome between uterine horns ipsilateral and contralateral to the corpus luteum influence conceptus growth to day 14 in cattle?

Embryo transfer to the uterine horn contralateral to the ovary containing the corpus luteum (CL) negatively impacts pregnancy establishment in cattle. Our aim was to compare the transcriptome and ability of the ipsilateral and contralateral uterine horns to support preimplantation conceptus survival and growth to day 14. In experiment 1, endometrial samples from both horns were collected from synchronized heifers slaughtered on day 5, 7, 13, or 16 post-estrus (n=5 per time) and subjected to RNA sequencing. In experiment 2, 10 day 7 in vitro produced blastocysts were transferred into the uterine horn ipsilateral (n=9) or contralateral to the CL (n=8) or into both horns (i.e., bilateral, n=9) of synchronized recipient heifers. Reproductive tracts were recovered at slaughter on day 14, and the number and dimensions of recovered conceptuses were recorded for each horn. A total of 217, 54, 14, and 18 differentially expressed genes (>2-fold change, FDR P<0.05) were detected between ipsilateral and contralateral horns on days 5, 7, 13, and 16, respectively, with signaling pathways regulating pluripotency of stem cells, ErbB signaling pathway, and mTOR signaling pathway amongst the top canonical pathways. Site of embryo transfer did not affect recovery rate (48.0%, 168/350) or length of conceptuses (mean±SE 2.85±0.27 mm). Although differences in gene expression exist between the endometrium of uterine horns ipsilateral and contralateral to the CL in cattle, they do not impact conceptus survival or length between day 7 and 14.