Reproductive failure is defined as the inability to conceive or maintain a pregnancy till term or near-term. It includes a broad spectrum of clinical manifestations, such as unexplained infertility, repeated implantation failure (RIF) in women undergoing in-vitro fertilization (IVF)/embryo transfer (ET) and/or frozen ET (FET), and recurrent pregnancy loss (RPL). RPL is defined as two or more clinical pregnancy losses (Practice Committee of the Asrm, 2012, Bender Atik et al., 2023). On the other hand, RIF is a condition diagnosed in patients undergoing IVF cycles after three failed IVF-ET or FET cycles with good-quality embryos (Coughlan et al., 2014, Cimadomo et al., 2023). Unfortunately, some patients may suffer from both RPL and RIF. Multiple etiologies have been linked to reproductive failures, including endocrine dysfunction, parental genetic abnormalities, uterine anatomical malformations, immune disorders, infectious etiologies, and thromboembolic conditions. Particularly, a significant proportion of women with RPL have immune disorders such as dysregulation of uterine NK cells, aberrant cytokine profiles, abnormal angiogenesis, and decidualization due to immune imbalance. Despite all those, many cases remain unexplained (Practice Committee of the Asrm, 2012). Proper endometrial decidualization is critical since pregnancy preparation begins even before the embryo reaches the uterus. Increasing attention has been directed toward implantation and its related factors, which are essential for establishing and maintaining pregnancy.

Decidualization refers to the functional and morphological changes of the endometrial cells that form the uterine lining and are suitable for blastocyst implantation. Decidual stromal cells (DSCs) are not simply modified endometrial stromal cells (ESCs) but a distinct cell type resulting from terminal differentiation and the genetic reprogramming of ESCs (Ng et al., 2020). Genetic reprogramming involves downregulating genes associated with pro-inflammatory responses and resistance to tissue invasion, while simultaneously upregulating genes that promote cellular proliferation, immune tolerance, and the facilitation of tissue invasion (Ng et al., 2020).

The decidualization score (DS) was recently established as an evaluation tool for endometrial decidualization during the secretory (luteal) phase endometrium (Dambaeva et al., 2021). It evaluates collectively endometrial expression of immune regulatory factors, such as interleukin 15 (IL-15) and granzyme B (GZMB), and other factors directly related to progesterone signaling and tissue homeostasis (forkhead box protein O1 [FOXO1], solute carrier family 2 member 1 [SCL2A1], serum and glucocorticoid regulated kinase 1 [SGK1], and sodium channel epithelial 1 subunit alpha [SCNN1A]). DS is based on gene expression analysis of endometrial samples using targeted next-generation sequencing (NGS) of RNA material. A combined evaluation of the selected genes is designated as a score. These genes were selected based on their differential expression between patients and normal controls, as identified in the previous study (Dambaeva et al., 2021). A normal DS was defined as a score > 4 (more than 4 genes are within the normal range). Scores ≤ 4 were considered abnormal, with borderline cases defined as exactly 4 and low scores as < 4. Low DS (< 4) has been associated with an increased risk of reproductive failure.

Women with reproductive failure may have abnormal ovulation and hormonal levels, which can affect endometrial decidualization and gene expression important for implantation (Maruyama and Yoshimura, 2008, Wetendorf and Demayo, 2012). Even with confirmed ovulation and timed endometrial biopsy (5–7 days post-ovulation), histologically out-of-phase endometrium, such as proliferative, desynchronized, or mixed endometrial patterns, is frequently observed. Earlier studies reported that histological dating of the endometrium did not discriminate between infertile and fertile women (Coutifaris et al., 2004), leading to the abandonment of routine histological evaluation of the luteal-phase endometrium. In contrast, molecular assays of decidualization markers continued to be developed and applied. However, the absence of histopathologic assessment may compromise the interpretation of molecular results and potentially contribute to suboptimal management of reproductive failure.

During the normal menstrual cycle, the endometrium undergoes tightly coordinated changes in gene families and signaling pathways as it transitions from the proliferative to the secretory phase (Talbi et al., 2006), and these transcriptional programs vary further with advancing age, ethnicity, and cell-type–specific regulatory mechanisms (Mortlock et al., 2022). Consequently, endometrial gene-expression profiles may differ across histological phases and may be further altered in the presence of endometrial pathology. Yet, studies that simultaneously examine endometrial histology and gene-expression patterns remain scarce. Therefore, this study aims to determine whether the histological phase of the endometrium influences gene expression profiles, particularly genes included in the DS test and other markers of decidualization and embryo implantation, in women with reproductive failure who underwent endometrial biopsy after confirmed ovulation.