Investigating cycle-to-cycle variation and the impact of endometrial injury through differential gene expression analysis.

study questionDoes endometrial gene expression change across consecutive cycles and after endometrial injury? SUMMARY ANSWER: Although paired endometrial samples collected from the same woman across two consecutive cycles show greater similarity in molecular profile compared to samples from different women, endometrial injury in the preceding cycle did not result in any significant differential gene expression. WHAT IS KNOWN ALREADY: Uterine receptivity is a crucial factor influencing pregnancy rates in IVF. The potential benefits of endometrial injury in enhancing receptivity, as well as the effectiveness of genomic endometrial receptivity tests in predicting uterine receptivity are subjects of ongoing debate. STUDY DESIGN, SIZE, DURATION: This was a prospective study, involving endometrial biopsies from 20 women at mid-luteal phase over two consecutive menstrual cycles. PARTICIPANTS/MATERIALS, SETTING,

methodsDifferential gene expression analysis was conducted using a paired design and included estimated menstrual cycle time as a covariate. Pairwise distances of samples and principal component analysis were used to examine sample-to-sample variation. MAIN RESULTS AND THE ROLE OF CHANCE: Analysis of paired endometrial samples revealed no significant difference in differential gene expression before and after endometrial injury (P > 0.05). Sub-analyses of samples from infertile women and those with proven fertility also did not reveal significant changes in gene expression. Although the pairwise distance analysis indicated slightly more similarity between intra-patient samples when compared to inter-patient samples (P = 0.09), PCA did not show clear clustering of these pairs. LARGE SCALE DATA: The RNA-seq data generated in this study have been deposited in the Gene Expression Omnibus database under accession number GSE303049. LIMITATIONS, REASONS FOR CAUTION: The sample size for this study is relatively small. WIDER IMPLICATIONS OF THE

findingsOur findings highlight the high degree of cycle-to-cycle variability in endometrial gene expression, even within the same individual. Importantly, the lack of significant changes in gene expression as a result of endometrial injury in the preceding cycle, indicates the molecular variability reflects inherent biological fluctuations rather than the effect of prior injury. This molecular variability may help explain why implantation does not always occur immediately, even with high-quality embryos, and why interventions such as endometrial injury or molecular receptivity tests have yielded inconsistent clinical outcomes. STUDY FUNDING/COMPETING INTEREST(S): This project was supported in part by National Health and Medical Research Council (NHMRC) Medical Postgraduate Scholarship No. 1055814 and The Royal Australian and New Zealand College of Obstetricians and Gynaecologists (RANZCOG) Fellows' Clinical Research Scholarship 2013 (W.T.T.). W.T.T., J.C., and P.R. are named inventors on a patent on the molecular dating of endometrial samples with IP Australia, ID: AU2023287239A1-methods for determining menstrual cycle time point. The patent is held jointly by their employers: University of Melbourne and Royal Women's Hospital. No payments have been received for this patent to date. TRIAL REGISTRATION NUMBER: N/A.