Guidelines for biomarker discovery in endometrium: correcting for menstrual cycle bias reveals new genes associated with uterine disorders

Transcriptomic approaches are increasingly used in reproductive medicine to identify candidate endometrial biomarkers. However, it is known that endometrial progression in the molecular biology of the menstrual cycle is a main factor that could affect the discovery of disorder-related genes. Therefore, the aim of this study was to systematically review current practices for considering the menstrual cycle effect and to demonstrate its bias in the identification of potential biomarkers. From the 35 studies meeting the criteria, 31.43% did not register the menstrual cycle phase. We analysed the menstrual cycle effect in 11 papers (including 12 studies) from Gene Expression Omnibus: three evaluating endometriosis, two evaluating recurrent implantation failure, one evaluating recurrent pregnancy loss, one evaluating uterine fibroids and five control studies, which collected endometrial samples throughout menstrual cycle. An average of 44.2% more genes were identified after removing menstrual cycle bias using linear models. This effect was observed even if studies were balanced in the proportion of samples collected at different endometrial stages or only in the mid-secretory phase. Our bias correction method increased the statistical power by retrieving more candidate genes than per-phase independent analyses. Thanks to this practice, we discovered 544 novel candidate genes for eutopic endometriosis, 158 genes for ectopic ovarian endometriosis and 27 genes for recurrent implantation failure. In conclusion, we demonstrate that menstrual cycle progression masks molecular biomarkers, provides new guidelines to unmask them and proposes a new classification that distinguishes between biomarkers of disorder or/and menstrual cycle progression.