Study question Does the endometrial transcriptome profile differ between infertile women with or without a recurrent implantation failure (RIF)? Summary answer Although two different clusters emerged from the endometrial transcriptome data, these were not associated with clinical phenotype (RIF vs non-RIF). What is known already Despite the transfer of morphologically ‘good-quality’ embryos in IVF/ICSI, implantation failure often occurs, which may be explained by impaired endometrial receptivity. In order to guide prognosis and use effective therapeutic interventions, identifying a gene expression profile predictive of endometrial receptivity as well as implantation failure, would be of great value. Additionally, transcriptome analysis may also shed light on alterations in biological processes responsible for the implantation failure. Thousands of potential biomarkers for endometrial receptivity have already been identified by transcriptomic approach, however due to differences in study methodology, there is little overlap of markers between studies. Study design, size, duration Endometrial tissue was obtained from a cohort of 141 infertile women undergoing endometrial scratching within a randomised controlled trial (RCT) (SCRaTCH trial, NL5193/NTR5342). Briefly, women aged 18-44 years with failed implantation after one full IVF/ICSI cycle and planning a subsequent IVF/ICSI cycle, were eligible. Participants were followed-up until 12 months after randomisation, with the primary outcome being live birth, defined as the delivery of at least one live foetus after 24 weeks of gestation. Participants/materials, setting, methods Endometrial tissue was obtained with an endometrial biopsy catheter in the midluteal phase of a natural cycle preceding subsequent IVF/ICSI. Biopsies were snap-frozen and stored at -80 °C until use. After thawing, total RNA isolation, library preparation and paired-end RNA-sequencing were performed. Raw data was preprocessed and mapped to GRCh38. Reads (counts per million) were normalised using library size. Differential gene expression (DGE) analysis was conducted using the EdgeR package with significance threshold FDR <0.05. Main results and the role of chance Out of 141 endometrium samples, 107 were included in the RNA-sequencing based on RNA quality. For DGE analysis, data of two groups were compared: the ‘fertile’ group, women with a live birth after ≤3 good quality embryo(s) transfers (n = 23), and the RIF group, women with no live birth after ≥3 good quality embryo(s) transfers (n = 23). Two clusters were visible in the principle component analysis (PCA) plot showing transcriptome data of the fertile and RIF samples (cluster 1, n = 29; cluster 2, n = 10), which was not explained by clinical phenotype, as both clusters contained samples of both the fertile and RIF group. DGE analysis between the fertile and RIF group resulted in respectively 3 significantly upregulated and 0 significantly downregulated genes, whereas DGE analysis between the two clusters resulted in 2,235 significantly upregulated and 2,162 significantly downregulated genes. Enrichment analysis of differentially expressed genes between both clusters demonstrated upregulation of enriched terms mainly annotated to cell migration and downregulation of enriched terms mainly annotated to lipid and mitochondrial metabolism. Limitations, reasons for caution A strength of the study is the large number of samples included. Bulk RNA-sequencing was conducted and there was a variation in LH-based timing of the biopsies (5-8 days after LH surge) for which adjustments of the transcriptome data for tissue cellular composition and menstrual cycle were performed. Wider implications of the findings Future studies investigating underlying biological mechanisms in the endometrium in (in)fertility by a (multi-)omics analysis approach with standardised methodology are required to obtain consistencies in relevant biomarkers/pathways, and in due course create possibilities to improve and personalise care for infertile couples. Trial registration number NL5193/NTR5342
Study question Is there a difference in the endometrial transcriptome and/or reproductive tract microbiota composition when OD versus MVP is used as LPS for fresh embryo transfer? Summary answer Endometrial transcriptome signatures and microbiota composition at the vaginal, cervical or intra-uterine site presented no significant differences following OD versus MVP. What is known already Adequate LPS is crucial to achieve a successful pregnancy following ovarian stimulation (OS) and fresh embryo transfer. OD has been proven to be non-inferior compared to MVP in two phase III clinical RCTs. Additionally, a combined individual participant data and aggregate data meta-analysis showed an odds ratio in favor of OD for live birth. Little information is available on the potential differences at the molecular level of the reproductive organs following the administration of either LPS strategies. Given the potential immunomodulating properties of OD, of main interest is the endometrial functionality and microbiota composition of the female genital tract. Study design, size, duration Thirty oocyte donors were planned to have two OS-cycles followed by one week of LPS (OD or MVP) in a randomized, cross-over, double-blind, double-dummy fashion. An endometrial biopsy, as well as vaginal/cervical/intra-uterine samples were collected. Endometrial RNA-sequencing was performed, raw reads were processed using STAR/htseq-count, differential gene expression was evaluated with EdgeR. Microbiota profiles were obtained by 16S-rRNA-sequencing using the DADA2-pipeline with RDP-classifier. Comparative analysis of genera relative abundances was performed in R. Participants/materials, setting, methods All oocyte donors were <35 years old, had regular menstrual cycles, no intra-uterine contraceptive device, AMH within normal range and BMI≤ 29 kg/m2. OS was performed in a GnRH antagonist protocol followed by dual triggering (1000U hCG + 0.2mg triptorelin) as soon as ≥ 3 follicles of 20mm were present. Following oocyte retrieval, subjects initiated LPS consisting of MVP 200 mg (Utrogestan®) or OD 10 mg (Duphaston®), both three times daily. Main results and the role of chance Subject and stimulation characteristics were comparable between the groups. For endometrial RNA-sequencing, 21 pairs (n = 42 biopsies) were available with the same oocyte donor having a biopsy after OD as well as after MVP (the remaining 9 subjects were excluded due to late follicular progesterone elevation/drop-out after one cycle). After correction for multiple hypothesis testing, no differentially expressed genes could be withheld and the principal component analysis plot showed one mixed OD/MVP cluster (PCA1 25% variance, PCA2 16% variance). The average Euclidean distance between samples of the OD group was significantly lower than for the MVP group (respectively 12.1 vs 18.8, comparison of the two-group wise Euclidean distributions results in p = 6.98e-14 using the Mann-Whitney test). Microbiota profiling was performed before and after OD/MVP (4 collections per subject). Samples with insufficient high-quality reads were excluded, resulting in 42 intra-uterine samplings, 82 cervical and 84 vaginal swabs used for analysis. No difference was seen at any site of the female reproductive tract following OD versus MVP in microbiota diversity, richness, genera abundances (delta contrasts [Mann–Whitney U test], padj > 0.1), nor community composition (dbRDA genus-level Euclidean distances in vagina [R2= 0.22%, padj=0.26], cervix [R2= 0.36%, padj=0.20], uterus [R2= -0.06%, padj=0.45]). Limitations, reasons for caution Sample size was limited. Whole tissue endometrial transcriptomics was performed on individual biopsies without accounting for the potential bias of different tissue compartment compositions among biopsies. As it was a double blind, double dummy study design, the impact of not administering medication via the vaginal route could not be evaluated. Wider implications of the findings This is the first molecular study comparing OD/MVP. Results show that there is no difference between OD/MVP’s ability to give rise to the receptive state, which corresponds with the clinical trial data. As the inter-sample distance of RNA-profiles was smaller following OD, interindividual variations could be lower for this approach. Trial registration number EUDRACT 2018-000105-23
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