Context: Suboptimal endometrial thickening is associated with lower pregnancy rates and occurs in some infertile women treated with clomiphene.

Objective: To examine cellular and molecular differences in the endometrium of women with suboptimal versus optimal endometrial thickening following clomiphene.

Design: Translational prospective cohort study from 2018-2020.

Setting: University-affiliated clinic.

Patients or Participants: Reproductive age women with unexplained infertility treated with 100mg of clomiphene cycle days 3-7 who developed optimal (≥8mm; n=6, controls) or suboptimal (<6mm; n=7, subjects) endometrial thickness.

Interventions: Pre-ovulatory blood and endometrial sampling.

Main outcome measures: Endometrial tissue architecture, abundance and location of specific proteins, RNA expression, ERa binding.

Results: The endometrium of suboptimal subjects compared to optimal controls was characterized by a reduced volume of glandular epithelium (16% vs 24%, P=0.01), decreased immunostaining of markers of proliferation (PCNA, ki67) and angiogenesis (PECAM-1), increased immunostaining of pan-leukocyte marker CD45 and ERb, but decreased ERa immunostaining (all P<0.05). RNASeq identified 398 differentially expressed genes between groups. Pathway analysis of differentially expressed genes indicated reduced proliferation (Z-score= -2.2, P<0.01), decreased angiogenesis (Z-score = -2.87, P<0.001), increased inflammation (Z-score= +2.2, P<0.01), and ERb activation (Z-score= +1.6, P<0.001) in suboptimal subjects. ChIP-seq identified 6 genes bound by ERα that were differentially expressed between groups (P<0.01), some of which may play a role in implantation.

Conclusions: Women with suboptimal endometrial thickness after clomiphene exhibit aberrant estrogen receptor expression patterns, architectural changes and altered gene and protein expression, suggesting reduced proliferation and angiogenesis in the setting of increased inflammation.