Functional heterogeneity in trophoblast stem cells derived from recurrent pregnancy loss products of conception

Abstract Recurrent pregnancy loss (RPL), defined as two or more consecutive miscarriages, affects 1–5% of couples attempting to conceive and most frequently occurs between 6 and 8 weeks of gestation, coinciding with critical stages of placental development. Despite this temporal association, placental contributions to RPL pathogenesis remain poorly understood. We hypothesized that trophoblast lineage development, including extravillous trophoblast (EVT) and syncytiotrophoblast (STB) differentiation, are disrupted in RPL. To test this hypothesis, we established and functionally characterized human trophoblast stem (TS) cell lines derived from products of conception (POC) obtained from consenting idiopathic RPL cases. Four TS cell lines were successfully derived from five POC samples, representing both normal and abnormal karyotypes. All established TS cell lines exhibited canonical stem-state trophoblast features, including epithelial morphology, ELF5 promoter hypomethylation, expression of trophoblast-specific microRNAs, and trophoblast marker expression. Despite this preserved identity, TS cell lines displayed marked heterogeneity in proliferation and differentiation capacity. TS cells derived from POC with abnormal karyotypes exhibited prolonged doubling times and impaired differentiation, while those from normal karyotypes more closely resembled cytotrophoblast-derived control TS cells. Although all TS cells formed morphologically comparable STB spheroids, functional deficits in human chorionic gonadotropin β secretion were observed in a cell-line specific manner. EVT differentiation capacity varied substantially across POC-derived TS cell lines. Transcriptomic analyses revealed karyotype-associated transcriptional heterogeneity, including enrichment of senescence- and cell cycle-associated pathways across subsets of POC-derived TS cell lines. Together, these findings demonstrate that patient-specific TS cell lines retain trophoblast identity yet exhibit meaningful functional heterogeneity that becomes evident upon differentiation, underscoring the importance of functional validation in newly established in vitro placental cell lines. Overall, POC-derived TS cells are a powerful platform for investigating molecular mechanisms underlying RPL.