YWHAZ loss is associated with endometrial dysfunction in proliferative-phase endometriosis

To identify differentially regulated genes in endometriosis, we performed RNA-sequencing analysis on eutopic endometrial tissue collected during the proliferative phase from women with and without endometriosis ( n  = 4 per group). A total of 44 DEGs were identified between control and endometriosis groups using a threshold value of fold-change > ± 2 and false discovery rate < 0.05. Among these, 13 genes were upregulated and 31 genes were downregulated in the endometriosis group. The expression profiles of these DEGs were visualized in a clustered heatmap, revealing a distinct gene expression pattern between the groups ( Figure 1A ). Transcriptomic analysis revealed differential gene expression in the eutopic endometrium from women with endometriosis during the proliferative phase. (A) Heatmap showing 44 differentially expressed genes in endometrial tissue of women with endometriosis ( n  = 4) compared to women without endometriosis ( n  = 4) during the proliferative phase (fold-change > ± 2.00; false discovery rate < 0.05). Upregulated and downregulated genes are color coded as yellow and blue, respectively. (B) Representative image of YWHAZ expression in human endometrial tissue. (C) H-score of YWHAZ expression in the endometriosis group ( n  = 10) compared to controls ( n  = 10). Results are presented as mean ± SEM; ** p  < 0.01, *** p  < 0.001. Among the downregulated genes, YWHAZ was notable for its robust decrease in expression. To validate this result at the protein level, we performed immunohistochemistry on proliferative-phase eutopic endometrial tissue. A section processed without the primary antibody was used as the negative control for the human endometrium ( supplementary Figure S1A , see online supplementary material ). Consistent with the transcriptomic findings, YWHAZ immunoreactivity was markedly reduced in both stromal and epithelial compartments of endometriosis samples. Quantitative analysis revealed significantly lower YWHAZ staining intensity in the endometriosis group compared with controls (stroma: 46.09 ± 14.12 versus 1.79 ± 0.38, p  < 0.01; and epithelium: 181.26 ± 9.91 versus 7.16 ± 3.83, p  < 0.001) ( Figure 1B and C ). Together, these findings identify YWHAZ as a significantly downregulated gene in the eutopic endometrium of women with endometriosis during the proliferative phase. To characterize the temporal expression pattern of YWHAZ during early pregnancy, we performed immunohistochemistry on uterine horns from wild-type C57BL/6 female mice collected from GD 0.5 to GD 7.5 ( Figure 2 ; n  = 5 per time point). YWHAZ expression was detected in the epithelium at GD 0.5 (111.81 ± 32.85) and GD 2.5 (124.94 ± 38.49). YWHAZ expression increased markedly in the uterine epithelium at GD 3.5 (221.66 ± 4.11; p  = 0.010) and GD 4.5 (218.61 ± 17.80; p  = 0.021) compared with GD 0.5. Stromal YWHAZ expression was lower at GD 0.5 (44.97 ± 20.71) and GD 2.5 (68.54 ± 23.07). Interestingly, a significant increase in YWHAZ expression was observed at GD 3.5 (192.57 ± 3.54, p  < 0.001), GD 4.5 (175.24 ± 13.66; p  < 0.001) in the primary decidual zone (206.67 ± 10.62; p  < 0.001), GD5.5 in the secondary decidual zone (219.7 ± 10.00; p  < 0.001), and in both the primary decidual zone and secondary decidual zone (187.6 ± 7.06 and 150.8 ± 27.19; p  < 0.001 and p  = 0.02, respectively) at GD 7.5 compared to GD 0.5. Together, these results suggest that YWHAZ has an important role during early implantation and pregnancy progression. Dynamic YWHAZ expression pattern across early pregnancy. (A) Representative image of YWHAZ expression in uterine tissue from gestational day (GD) 0.5 to GD 7.5 ( n  = 5 per time point), showing the epithelial and stromal compartments, with the primary decidual zone (PDZ) and secondary decidual zone (SDZ) visible at GD5.5 and GD7.5. (B) H-score of YWHAZ expression during early pregnancy. Results are presented as mean ± SEM; * p  < 0.05, ** p  < 0.01, *** p  < 0.001. Progesterone resistance refers to the failure of the endometrium to respond to progesterone. Many studies indicate that disrupted progesterone signaling and features of progesterone resistance are common in endometriosis ( Zhang & Wang, 2023 ). To investigate whether YWHAZ contributes to the regulation of progesterone receptor (PGR) signaling, we examined YWHAZ expression in uteri from wild-type C57BL/6 (control) and PRKO female mice at GD 3.5. PRKO mice were validated by immunohistochemistry detection of PGR in the uterine tissue at GD3.5 ( supplementary Figure S1B ). Immunohistochemistry revealed strong YWHAZ expression in the uteri of controls whereas YWHAZ expression was not detected in the uteri of PRKO mice (epithelium 275.15 ± 10.88 versus 0.00 ± 0.00, p  < 0.01; and stroma 194.02 ± 3.96 versus 0.00 ± 0.00, p  < 0.001) ( Figure 3 ). These findings indicate that YWHAZ expression is dependent on PGR, suggesting that YWHAZ acts downstream of PGR in regulating progesterone signaling. Loss of YWHAZ expression in progesterone receptor knockout (PRKO) mice. (A) Representative image of YWHAZ expression in the uterine tissue of control and PRKO mice. (B) H-score of YWHAZ expression in the uterine epithelium and stroma of control and PRKO mice ( n  = 5). Results are presented as mean ± SEM; *** p  < 0.001. Our previous study demonstrated that STAT3 binds to the progesterone receptor A (PR-A) isoform, which is critical for uterine development and function, whereas no interaction occurs with PR-B ( Lee et al., 2013 ). In addition, STAT3 regulates inflammatory and immune related genes, which are key contributors to the pathophysiology of endometriosis ( Rahman et al., 2025a ). In order to investigate the relationship between STAT3 and YWHAZ, we examined YWHAZ expression in the uteri of control and Pgr cre/+ Stat3 f/f mice at GD 3.5 using immunohistochemistry. Loss of STAT3 in the uteri of Pgr cre/+ Stat3 f/f mice at GD 3.5 was confirmed by immunohistochemistry ( supplementary Figure S1C ). In control mice, YWHAZ was strongly expressed in both stromal and epithelial compartments, whereas Stat3 knockout uteri displayed a striking absence of YWHAZ expression (epithelium 274.99 ± 13.49 versus 0.00 ± 0.00; and stroma 189.27 ± 2.18 versus 0.00 ± 0.00, p  < 0.001 respectively) ( Figure 4 ). These results indicate that STAT3 activity is essential for maintaining YWHAZ levels during the peri-implantation period, suggesting that YWHAZ functions within the STAT3 pathway to regulate progesterone signaling. Loss of YWHAZ expression in signal transducer and activator of transcription 3 ( Stat3) knockout mice. (A) Representative image of YWHAZ expression in the uterine tissue of control and Pgr cre/+ Stat3 f/f mice. (B) H-score of YWHAZ expression in uterine epithelium and stroma of control and Pgr cre/+ Stat3 f/f mice ( n  = 5). Results are presented as mean ± SEM; *** p  < 0.001.

All human uterine tissues used was approved by the Institutional Review Boards of Atrium Health Wake Forest Baptist (Winston-Salem, NC, USA) under IRB00057549. Written informed consent was obtained from all human subjects prior to tissue acquisition. All animal experiments were approved by the University of Missouri Animal Care and Use Committee (protocol number 65323). Mice were housed and bred in a designated animal care facility at the University of Missouri with controlled humidity (30%–70%) and temperature (68–79°F) conditions and a 12 hr light/dark cycle. Eutopic endometrium was obtained by endometrial biopsy from women between the ages of 18 and 45 years, including four with endometriosis and four without the disease, during the proliferative phase. The presence or absence of disease was confirmed in all endometriosis cases, and controls included women who were laparoscopically negative for disease. Use of an intrauterine device (IUD) or hormonal therapies in the 3 months preceding surgery was exclusionary. Histological dating of endometrial samples was performed by a certified pathologist. Eutopic endometrial biopsies from control ( n  = 4) and endometriosis ( n  = 4) groups were used for RNA-sequencing analysis. Total RNA was isolated from each sample using the RNeasy Total RNA Isolation kit (Qiagen; Aarhus, Denmark) following the manufacturer’s instructions. RNA purity and concentration were analyzed by the Genomic Technology Core at the University of Missouri to confirm sample concentration and purity (50–200 ng/µl, RNA integrity number (RIN) > 8.0) before RNA-sequencing. For RNA-sequencing, libraries were prepared from 500 ng of total RNA using the KAPA mRNA Hyperprep kit (Kapa Biosystems; Wilmington, MA, USA). RNA was sheared to 300–400 bp. Prior to PCR amplification, cDNA fragments were ligated to Integrated DNA Technologies (IDT) for Illumina TruSeq UD Indexed adapters (Illumina Inc; San Diego, CA, USA). The quality and quantity of the finished libraries were assessed using a combination of Agilent DNA high-sensitivity chip (Agilent Technologies, Inc.; Santa Clara, CA, USA) and QuantiFluor ® dsDNA system (Promega Corp.; Madison, WI, USA). Individually indexed libraries were pooled, and 50 bp paired-end sequencing was performed on an Illumina NovaSeq6000 sequencer using an S2, 100 bp sequencing kit (Illumina Inc.; San Diego, CA, USA) to an average depth of 50 million reads per sample. Base calling was done by Illumina RTA3, and the output of NextSeq Control Software (NCS) was demultiplexed and converted into FastQ format with Illumina Bcl2fastq software (version 1.9.0). The raw reads were initially trimmed and filtered by removing low-quality reads (fragments that scored < 20) and aligned to the hg38 human genome reference and assembled using STAR (version 2.7.11b) ( Dobin et al., 2013 ). Expression values of RNA-sequencing were expressed as counts. DEGs were identified by the “exact negative binomial test” in the EdgeR software package ( Robinson et al., 2010 ). The DEGs between control and endometriosis groups were set as fold-change >2 (upregulated genes) or <−2 (downregulated genes) and false discovery rate < 0.05. DEG expression values were normalized, scaled, and clustered to generate a heatmap using the ComplexHeatmap package ( Gu et al., 2016 ). The raw and processed RNA‑sequencing data generated in this study have been deposited in the NCBI Gene Expression Omnibus (GEO) under accession number GSE315857 , which is a dataset derived from GSE272606. The data that support the findings of this study are available from the article and supplementary information files (see online supplementary material ). For the early pregnancy study, wild-type C57BL/6 female mice at 8 weeks of age were mated with wild-type C57BL/6 fertile male mice, and uterine samples from pregnant mice were obtained on different gestational days (GDs). The morning of vaginal plug is designated as GD 0.5. Uterine horns were collected at GDs 0.5, 2.5, 3.5, 4.5, 5.5, and 7.5 ( n  = 5 per time-point). Additionally, 8-week old litter-mate control, PRKO, and Pgr cre/+ Stat3 f/f female mice were mated with wild-type C57BL/6 fertile male mice and uterine horn samples were collected at GD 3.5 ( n  = 5 per group) ( Jeong et al., 2005 ; Kim et al., 2015 ; Lee et al., 2013 ; Teasley et al., 2018 ). Uterine horns were fixed in 4% paraformaldehyde in 1× phosphate-buffered saline (PBS), dehydrated through ethanol washes, and embedded in paraffin blocks for immunostaining. Paraffin-embedded human endometrial samples from women with and without endometriosis ( n  = 10 per group) were sectioned at 5 µm thickness, and immunohistochemistry staining and analysis were performed as previously described ( Kim et al., 2015 ; Rahman et al., 2025b ). Briefly, the sections were deparaffinized in xylene and rehydrated through a graded ethanol series. After antigen blocking, the sections were incubated with 10% normal goat serum (Vector Laboratories; Newark, CA, USA) in PBS (pH 7.5) and subsequently incubated with primary antibody diluted in 10% normal goat serum in PBS overnight at 4 °C (anti-14-3-3 zeta, 1:500; CS7413, Cell Signaling Technology; Danvers, MA, USA). On the following day, the sections were washed in PBS and incubated with a secondary antibody for 1 hr at room temperature. Subsequently, they were incubated with horseradish peroxidase (Vector Laboratories) for 45 min. After PBS washes, immunoreactivity was visualized using the Vectastain Elite DAB kit (Vector Laboratories). Slides were counterstained with hematoxylin and saturated lithium carbonate. A semi-quantitative H-score system (0–300) was used to compare staining intensities ( Kim et al., 2025 ). Quantification of H-scores was performed using Visiopharm image analysis software (Visiopharm, Hørsholm, Denmark). For the early pregnancy, PRKO, and Pgr cre/+ Stat3 f/f samples, paraffin blocks were sectioned at 5 μm thickness, and immunohistochemistry analyses were performed in the same way as for human tissue sections. For statistical analysis, Student’s t-test with unequal variance was used to compare data with only two groups. For data containing more than two groups, one-way ANOVA (analysis of variance) followed by Tukey’s post hoc test was used for multiple comparisons. All data are presented as mean ± SEM. The statistical significance was considered when p  < 0.05. All statistical analyses were performed using the Prism version 10.2.0 package from GraphPad (GraphPad Prism; San Diego, CA, USA).

In this study, we identified YWHAZ as one of the most markedly reduced genes in RNA-sequencing analysis of proliferative-phase eutopic endometrium, which revealed a distinct transcriptional profile in women with endometriosis compared with controls. Follow-up immunohistochemistry confirmed that YWHAZ protein expression was markedly decreased in both stromal and epithelial compartments of endometriosis samples, consistent with transcript-level changes. Together, these results highlight YWHAZ as a robustly downregulated gene in the eutopic endometrium of women with endometriosis, suggesting its potential relevance to disease-associated molecular alterations. Moreover, we identified YWHAZ as a previously unrecognized gene of uterine function with potential relevance to endometrial receptivity and endometriosis-associated infertility. By integrating transcriptomic analysis of human eutopic endometrium with mechanistic interrogation in genetically engineered models, we demonstrated that YWHAZ expression is significantly reduced in the eutopic endometrium of women with endometriosis and is dynamically regulated during implantation and decidualization stages of early pregnancy in mice. Importantly, we show that PGR and STAT3 are required for uterine YWHAZ expression, positioning YWHAZ at the intersection of hormonal and cytokine-responsive transcriptional pathways critical for implantation. Our transcriptomic analysis of proliferative-phase eutopic endometrium provides important context for understanding early molecular perturbations associated with endometriosis. The proliferative phase is dominated by estrogen-driven epithelial and stromal proliferation and precedes the establishment of progesterone responsiveness required for uterine receptivity. By focusing on this phase, our analysis captures disease-associated transcriptional alterations that are present before the window of implantation, thereby minimizing confounding effects of decidualization and highlighting intrinsic abnormalities of the eutopic endometrium. The identification of a discrete set of DEGs, including the marked downregulation of YWHAZ, suggests that endometriosis is associated with early disruptions in signaling pathways that may compromise subsequent progesterone-mediated differentiation and implantation competence. Importantly, the reduced expression of YWHAZ during the proliferative phase implies that defects in adaptor-mediated signaling networks may be established prior to the receptive window, potentially predisposing the endometrium to progesterone resistance and impaired decidual responses. These findings align with emerging transcriptomic and single-cell studies indicating that molecular abnormalities in the eutopic endometrium of women with endometriosis precede implantation and contribute to infertility independent of ectopic lesions. YWHAZ encodes 14-3-3 zeta, a multifunctional adaptor protein that regulates the stability, subcellular localization, and activity of a broad array of signaling proteins and transcription factors ( Neal & Yu, 2010 ). Through these interactions, 14-3-3 zeta integrates diverse cellular processes, including proliferation, differentiation, survival, and inflammatory signaling, that are tightly regulated in the normal endometrium and frequently disrupted in endometriosis. Thus, reduced YWHAZ expression in the eutopic endometrium of women with endometriosis may reflect impaired coordination of signaling networks necessary for establishing a receptive endometrial environment, thereby contributing to endometriosis-associated infertility. To define the temporal regulation of YWHAZ during early pregnancy, we examined its expression across key stages of implantation and decidualization in the mouse uterus. Early pregnancy is characterized by tightly orchestrated hormonal and cellular transitions: estrogen-driven epithelial proliferation during the pre-receptive phase (GD 0.5–2.5), followed by progesterone-mediated stromal proliferation and differentiation coincident with uterine receptivity at GD 3.5. Blastocyst attachment occurs at GD 4.5, after which stromal cells undergo extensive proliferation and differentiation to form the primary and secondary decidual zones by GD 5.5–7.5. Our findings demonstrate that YWHAZ expression is low during the early stage but markedly induced in both epithelial and stromal compartments during the window of receptivity and throughout implantation progression. This dynamic, stage-specific pattern suggests that YWHAZ may contribute to the cellular remodeling and signaling integration required for successful implantation and decidualization. However, while our data establish a strong association between YWHAZ expression and these developmental events, direct functional studies will be required to define its precise mechanistic role. Our results further highlight the importance of hormonal and signaling crosstalk in regulating YWHAZ expression. Previous work has shown that progesterone induces another 14-3-3 isoform, 14-3-3τ, in uterine tissue, indicating that this protein family is responsive to progesterone signaling ( Ito et al., 2012 ). Endometriosis patients who failed to improve with progestin-based treatments exhibited markedly reduced PGR expression compared with those who responded, indicating that PGR levels are closely associated with treatment responsiveness ( Marquardt et al., 2019 ; Zhang & Wang, 2023 ). We observed a complete loss of YWHAZ expression in the uteri of PRKO mice, demonstrating that YWHAZ expression is strictly dependent on functional PGR signaling during the peri-implantation period and suggesting its potential as a therapeutic target to overcome progesterone resistance. Therefore, future studies should directly investigate the relationship between YWHAZ and PGR in implantation, decidualization, and endometriosis, as well as identify downstream molecular targets regulated by the PGR/YWHAZ axis in specific uterine cell types. These findings are particularly relevant in the context of endometriosis, where progesterone resistance is a defining feature of the eutopic endometrium. In addition to progesterone signaling, STAT3 emerged as an essential regulator of YWHAZ expression. STAT3 is a key mediator of cytokine and growth factor signaling and has been implicated in uterine receptivity, decidualization, and the inflammatory milieu characteristic of endometriosis. Previous studies have shown that STAT3 can directly interact with YWHAZ through phosphorylation-dependent mechanisms, influencing transcriptional and signaling outputs ( Han et al., 2015 ). Our results showing that YWHAZ expression is significantly reduced in uterine tissue of Pgr cre/+ Stat3 f/f mice provide in vivo evidence that STAT3 is required to maintain YWHAZ expression during early pregnancy. Together, these findings support a model in which progesterone receptor signaling and STAT3 converge to regulate YWHAZ expression, thereby integrating hormonal and inflammatory cues essential for normal uterine function. Although this study provides compelling evidence that YWHAZ is hormonally and transcriptionally regulated in the uterus and disrupted in endometriosis, several limitations should be acknowledged. We did not directly assess the functional consequences of YWHAZ loss on implantation, decidualization, or fertility outcomes, nor did we define its downstream molecular targets in uterine cell types. Additionally, we did not evaluate whether YWHAZ functions within the STAT3 pathway to regulate progesterone signaling as a downstream effector of PGR in women with endometriosis. Future studies employing uterine cell-specific loss- and gain-of-function approaches, as well as mechanistic interrogation of YWHAZ-interacting proteins, will be critical to establish its causal role in endometrial dysfunction. In summary, we identify YWHAZ as a downregulated gene in the eutopic endometrium of women with endometriosis and demonstrate that it exhibits dynamic, stage-specific expression during implantation and decidualization in the mouse uterus. The complete loss of YWHAZ expression in PGR and Stat3 knockout models indicates that its expression is tightly regulated by progesterone receptor and STAT3. Collectively, these findings identify YWHAZ as a hormonally and transcriptionally regulated endometrial gene with potential relevance to uterine receptivity and endometriosis-associated infertility. Elucidating the functional role of YWHAZ may uncover new molecular mechanisms underlying progesterone resistance and provide novel therapeutic opportunities for reproductive disorders.