Methodological Pitfalls in Endometriosis Biospecimen Research: Lessons From MAP4K4 Expression Studies

Endometriosis is a chronic inflammatory condition affecting 10% of reproductive-age women. Current treatments remain limited and non-curative. While its biology is largely informed by human biospecimens, sample heterogeneity and methodological constraints often limit interpretability. Using mitogen-activated protein kinase 4 (MAP4K4), a cell motility and invasion regulator within the MAPK network, as a model molecule, we examined its expression in endometriosis to highlight challenges in biospecimen-based research. Public endometrial microarray datasets were re-analyzed using a standard limma pipeline to evaluate MAP4K4 expression across tissue and disease states. MAP4K4 mRNA and protein were then assessed in eutopic endometrium from women with and without endometriosis and in surgically excised ectopic lesions using quantitative polymerase chain reaction (qPCR), immunohistochemistry (IHC), RNA in situ hybridization (ISH), and laser capture microdissection (LCM) coupled with qPCR. In silico analyses revealed small effect sizes and inconsistent directional change in MAP4K4 dysregulation across public datasets. MAP4K4 mRNA levels differed significantly between pipelle and full-thickness eutopic biopsies. Bulk-qPCR showed no significant differences in the eutopic endometrium between groups, whereas IHC demonstrated increased MAP4K4 protein in mid-secretory eutopic endometrium from women with endometriosis. Whole-lesion qPCR suggested higher MAP4K4 expression in ectopic versus matched eutopic tissue; however, LCM-qPCR and ISH did not identify consistent compartment-specific differences. Histology frequently revealed non-endometrial tissue in excised ectopic lesions. These findings highlight the methodological challenges in endometriosis biospecimen research and support the need for rigorous histological validation, spatially resolved approaches, and multi-modal analysis to improve the reliability and reproducibility of tissue-based studies in endometriosis.