Poor Endometrial Proliferation After Clomiphene is Associated With Altered Estrogen Action.

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 vs optimal endometrial thickening following clomiphene. METHODS: Translational prospective cohort study from 2018 to 2020 at a university-affiliated clinic. Reproductive age women with unexplained infertility treated with 100 mg of clomiphene on cycle days 3 to 7 who developed optimal (≥8mm; n = 6, controls) or suboptimal (<6mm; n = 7, subjects) endometrial thickness underwent preovulatory blood and endometrial sampling. The main outcome measures were endometrial tissue architecture, abundance and location of specific proteins, RNA expression, and estrogen receptor (ER) α binding. RESULTS: The endometrium of suboptimal subjects compared with optimal controls was characterized by a reduced volume of glandular epithelium (16% vs 24%, P = .01), decreased immunostaining of markers of proliferation (PCNA, ki67) and angiogenesis (PECAM-1), increased immunostaining of pan-leukocyte marker CD45 and ERß, but decreased ERα immunostaining (all P < .05). RNA-seq identified 398 differentially expressed genes between groups. Pathway analysis of differentially expressed genes indicated reduced proliferation (Z-score = -2.2, P < .01), decreased angiogenesis (Z-score = -2.87, P < .001), increased inflammation (Z-score = +2.2, P < .01), and ERß activation (Z-score = +1.6, P < .001) in suboptimal subjects. ChIP-seq identified 6 genes bound by ERα that were differentially expressed between groups (P < .01), some of which may play a role in implantation. CONCLUSION: Women with suboptimal endometrial thickness after clomiphene exhibit aberrant ER expression patterns, architectural changes, and altered gene and protein expression suggesting reduced proliferation and angiogenesis in the setting of increased inflammation.

Genetic and epigenetic changes in the eutopic endometrium of women with endometriosis: association with decreased endometrial αvß3 integrin expression.

About 40% of women with infertility and 70% of women with pelvic pain suffer from endometriosis. The pregnancy rate in women undergoing IVF with low endometrial integrin αvß3 (LEI) expression is significantly lower compared to the women with high endometrial integrin αvß3 (HEI). Mid-secretory eutopic endometrial biopsies were obtained from healthy controls (C; n=3), and women with HEI (n=4) and LEI (n=4) and endometriosis. Changes in gene expression were assessed using human gene arrays and DNA methylation data were derived using 385 K Two-Array Promoter Arrays. Transcriptional analysis revealed that LEI and C groups clustered separately with 396 differentially expressed genes (DEGs) (P<0.01: 275 up and 121 down) demonstrating that transcriptional and epigenetic changes are distinct in the LEI eutopic endometrium compared to the C and HEI group. In contrast, HEI vs C and HEI vs LEI comparisons only identified 83 and 45 DEGs, respectively. The methylation promoter array identified 1304 differentially methylated regions in the LEI vs C comparison. The overlap of gene and methylation array data identified 14 epigenetically dysregulated genes and quantitative RT-PCR analysis validated the transcriptomic findings. The analysis also revealed that aryl hydrocarbon receptor (AHR) was hypomethylated and significantly overexpressed in LEI samples compared to C. Further analysis validated that AHR transcript and protein expression are significantly (P<0.05) increased in LEI women compared to C. The increase in AHR, together with the altered methylation status of the 14 additional genes, may provide a diagnostic tool to identify the subset of women who have endometriosis-associated infertility.

Endometrial BCL6 Overexpression in Eutopic Endometrium of Women With Endometriosis.

The objective of this study was to examine B-cell CLL/lymphoma 6 (BCL6) expression in human eutopic endometrium across the menstrual cycle in women with and without endometriosis and to establish a cutoff for future studies. This design was a series of case-control studies in tertiary University teaching hospitals. We examined BCL6 expression by messenger RNA and immunohistochemically in prospectively collected samples in both the proliferative (P) and the secretory phases. BCL6 is minimally increased in the mid-secretory phase of the menstrual cycle compared to the P phase in normal patients. BCL6 protein expression was significantly higher in the secretory phase of patients with endometriosis (n = 29) versus fertile controls without endometriosis at laparoscopy (n = 20; P < .0001). Normal fertile controls (n = 28) recruited for endometrial biopsy also had low levels of secretory phase BCL6 expression compared to women with unexplained infertility (UI; n = 119). A receiving-operator characteristic analysis of these data revealed an area under the curve of 94% (95% confidence interval 85%-100%; P < .0001) with an HSCORE cutoff of 1.4 to differentiate cases with and without endometriosis. Using this cutoff value, BCL6 was positive in 88% of cases with UI. Laparoscopic examination of a subset of 65 patients confirmed abnormalities in 98% of cases; 61 (93.8%) were found to have endometriosis, 3 (4.6%) with hydrosalpinx, and 1 (1.5%) with a normal pelvis. These data suggest that BCL6 is a promising candidate as a single diagnostic biomarker for detection of endometriosis in women with otherwise UI and may be associated with endometrial dysfunction, including progesterone resistance.

Endometrial CXCL13 expression is cycle regulated in humans and aberrantly expressed in humans and Rhesus macaques with endometriosis.

C-X-C ligand 13 (CXCL13), a regulator of mucosal immunity, is secreted by human endometrial epithelium and may be involved in embryo implantation. However, cyclic expression of human endometrial CXCL13 in health and disease is not well studied. This study examines cycle stage-specific endometrial CXCL13 expression in normal humans when compared to those with biopsy-confirmed, stage 1 to 4 endometriosis using real-time reverse transcriptase, real-time polymerase chain reaction and immunohistochemistry. Eutopic endometrial CXCL13 expression was also compared between normal, control Rhesus macaques, and macaques with advanced endometriosis. In healthy women, CXLC13 messenger RNA expression was minimal in the proliferative phase and maximal in the secretory phase. However, in the presence of endometriosis, proliferative-phase endometrial expression markedly increased in both humans and rhesus subjects (P < .05). The cross-species and cross-stage concordance suggests a pathophysiologic role for CXCL13 in endometriosis and its use as a biomarker for disease.

Prospective assessment of midsecretory endometrial leukemia inhibitor factor expression versus ανß3 testing in women with unexplained infertility.

OBJECTIVE: To evaluate endometrial leukemia inhibitor factor (LIF) expression as a marker of endometrial receptivity in women with unexplained infertility (UI). DESIGN: Prospective case-control study. SETTING: University-associated infertility clinics. PATIENT(S): Women with UI for more than 1 year and healthy control women. INTERVENTION(S): Endometrial biopsy. MAIN OUTCOME MEASURE(S): Time to pregnancy was compared between patients with UI who were evaluated for endometrial LIF protein as well as ανß3 integrin expression. Endometrium was evaluated using immunohistochemistry (IHC) and messenger RNA by real time reverse transcriptase-polymerase chain reaction (PCR) (quantitative real-time reverse transcriptase-PCR) in samples from women with UI as well as healthy control women. RESULT(S): Leukemia inhibitor factor was expressed in epithelial cells in a cyclic fashion in controls, and overall expression in the secretory phase was similar between controls and women with UI, whereas ανß3 integrin expression was reduced. However, using quantitative real-time PCR, LIF messenger RNA abundance was 4.4-fold lower in women with low levels of ανß3 integrin expression compared with samples with normal integrins. By immunohistochemistry, ανß3 integrin expression was always lacking when the histology was out of phase, whereas LIF expression was only negative in a subset of those samples. Reduced endometrial LIF expression was strongly associated with poor reproductive outcomes. CONCLUSION(S): Endometrial LIF expression peaks in the midsecretory phase and is reduced in some women with UI. The use of LIF in combination with ανß3 integrin as biomarkers appears to be superior to integrin testing alone when evaluating endometrial receptivity, primarily because of its earlier pattern of expression during the secretory phase.

Primate-specific melanoma antigen-A11 regulates isoform-specific human progesterone receptor-B transactivation.

Progesterone acting through the progesterone receptor (PR) and its coregulators prepares the human endometrium for receptivity to embryo implantation and maintains pregnancy. The menstrual cycle-dependent expression of melanoma antigen-A11 (MAGE-11) in the mid-secretory human endometrium suggested a novel function in human PR signaling. Here we show that MAGE-11 is an isoform-specific coregulator responsible for the greater transcriptional activity of human PR-B relative to PR-A. PR was recruited to progesterone response regions of progesterone-regulated FK506-binding protein 5 (FKBP5) immunophilin and small Ras family G protein cell growth inhibitor RASD1 genes. Expression of MAGE-11 lentivirus shRNA in human endometrial Ishikawa cells expressing PR-B showed that MAGE-11 is required for isoform-specific PR-B up-regulation of FKBP5. In contrast, MAGE-11 was not required for progesterone up-regulation of RASD1 in endometrial cells expressing the PR-A/B heterodimer. Target gene specificity of PR-B depended on the synergistic actions of MAGE-11 and p300 mediated by the unique PR-B NH(2)-terminal (110)LLXXVLXXLL(119) motif that interacts with the MAGE-11 F-box region in a phosphorylation- and ubiquitinylation-dependent manner. A progesterone-dependent mechanism is proposed in which MAGE-11 and p300 increase PR-B up-regulation of the FKBP5 gene. MAGE-11 down-regulates PR-B, similar to the effects of progesterone, and interacts with FKBP5 to stabilize a complex with PR-B. We conclude that the coregulator function of MAGE-11 extends to isoform-specific regulation of PR-B during the cyclic development of the human endometrium.

G protein-coupled estrogen receptor (GPER) expression in normal and abnormal endometrium.

Rapid estrogen effects are mediated by membrane receptors, and evidence suggests a role for both a membrane-associated form of estrogen receptor alpha (ESR1; ERα) and G-protein coupled receptor 30 (GPER; GPR30). Considering estrogen's importance in endometrial physiology and endometriosis pathophysiology, we hypothesized that GPER could be involved in both cyclic changes in endometrial estrogen action and that aberrant expression might be seen in the eutopic endometrium of women with endometriosis. Using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemical analysis of normal endometrium, endometrial samples demonstrated cycle-regulated expression of GPER, with maximal expression in the proliferative phase. Eutopic and ectopic endometrium from women with endometriosis overexpressed GPER as compared to eutopic endometrium of normal participants. Ishikawa cells, an adenocarcinoma cell line, expressed GPER, with increased expression upon treatment with estrogen or an ESR1 agonist, but not with a GPER-specific agonist. Decreased expression was seen in Ishikawa cells stably transfected with progesterone receptor A. Together, these data suggest that normal endometrial GPER expression is cyclic and regulated by nuclear estrogen and progesterone receptors, while expression is dysregulated in endometriosis.

Cyclic regulation of transcription factor C/EBP beta in human endometrium.

BACKGROUND: The transcription factor CCAAT/enhancer-binding protein (C/EBP) beta is a critical mediator of murine endometrial function during embryo implantation. Our objective is to characterize changes in C/EBP beta mRNA abundance and protein localization over the normal human menstrual cycle. METHODS: Fifty normally cycling volunteers without reproductive disorders were randomized to undergo endometrial sampling on a specific cycle day, with secretory phase samples timed using urinary LH surge. Samples were assessed for relative C/EBP beta mRNA expression using quantitative real-time RT-PCR and for C/EBP beta protein localization using immunohistochemistry. The semiquantitative histologic scoring (HSCORE) system was used to compare staining intensity in each tissue compartment between each cycle phase. RESULTS: C/EBP beta mRNA expression by whole endometrium peaks in the late secretory phase and is significantly higher than that in the proliferative and mid-secretory phases. A marked increase in nuclear C/EBP beta protein immunostaining is seen in stromal cells beginning about cycle day 20, coincident with the start of endometrial receptivity. This increased staining continues for the remainder of the cycle. CONCLUSION: In the normal human menstrual cycle, C/EBP beta mRNA and protein expression also change, with increased nuclear immunostaining in the mid-secretory phase, suggesting a possible role for C/EBP beta in human endometrial receptivity.

Hormone control and expression of androgen receptor coregulator MAGE-11 in human endometrium during the window of receptivity to embryo implantation.

The androgen receptor (AR) is a ligand-activated transcription factor of the male and female reproductive tracts whose activity is modulated by coregulator binding. We recently identified melanoma antigen gene protein-11 (MAGE-11) of the MAGEA gene family that functions as an AR coregulator by binding the AR N-terminal FXXLF motif. Here we report that MAGE-11 is expressed in a temporal fashion in endometrium of normally cycling women. Highest levels of MAGE-11 mRNA and protein occur in the mid-secretory stage, coincident with the window of uterine receptivity to embryo implantation. Studies in human endometrial cell lines together with the hormone profile of the menstrual cycle and pattern of estrogen receptor-alpha expression in cycling endometrium suggest the rise in MAGE-11 mRNA results from down-regulation by estradiol during the proliferative phase and up-regulation by cyclic AMP signaling in the early and mid-secretory stage. In agreement with its coregulatory function, MAGE-11 localizes with AR in glandular epithelial cell nuclei in the mid-secretory stage. The increase in AR protein in the mid-secretory endometrium without an increase in AR mRNA suggests MAGE-11 stabilizes AR in glandular epithelial cell nuclei. This was supported by expression studies at low androgen levels indicating AR stabilization by MAGE-11 dependent on the AR N-terminal transactivation domain. The results suggest that MAGE-11 functions as a coregulator that increases AR transcriptional activity during the establishment of uterine receptivity in the human female.

Gene expression in the rectus abdominus muscle of patients with and without pelvic organ prolapse.

OBJECTIVE: The objective of the study was to compare gene expression in a group of actin and myosin-related proteins in the rectus muscle of 15 patients with pelvic organ prolapse and 13 controls. STUDY DESIGN: Six genes previously identified by microarray GeneChip analysis were examined using real-time quantitative reverse transcriptase-polymerase chain reaction analysis, including 2 genes showing differential expression in pubococcygeus muscle. Samples and controls were run in triplicate in multiplexed wells, and levels of gene expression were analyzed using the comparative critical threshold method. RESULTS: One gene, MYH3, was 3.2 times overexpressed in patients with prolapse (P = .032), but no significant differences in expression were seen for the other genes examined. An age-matched subset of 9 patients and controls showed that MYH3 gene expression was no longer significantly different (P = .058). CONCLUSION: Differential messenger ribonucleic acid levels of actin and myosin-related genes in patients with pelvic organ prolapse and controls may be limited to skeletal muscle from the pelvic floor.

Skeletal muscle heavy-chain polypeptide 3 and myosin binding protein H in the pubococcygeus muscle in patients with and without pelvic organ prolapse.

OBJECTIVE: The purpose of this study was to compare gene expression of skeletal muscle heavy-chain polypeptide 3 (MYH3) and myosin binding protein H (MyBP-H) in the pubococcygeus muscle of patients with pelvic organ prolapse and controls. STUDY DESIGN: Genes previously identified by microarray genechip analysis of pubococcygeus muscle biopsies were examined using real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis. Specimens were obtained from 17 patients with stage III or IV pelvic organ prolapse and 23 controls with minimal to no prolapse. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the housekeeping gene. Samples and controls were run in triplicate in separate wells, and the levels of gene expression were analyzed quantitatively using the comparative critical threshold (Ct) method. Differences in gene expression were analyzed using Wilcoxon rank-sum testing. RESULTS: Significant differences in gene expression were observed between patients with prolapse and controls for both genes. Skeletal muscle myosin heavy-chain polypeptide 3 was 6.5 times underexpressed in patients with pelvic organ prolapse compared to controls (P = .028). Similarly, myosin binding protein H was 3.2 times underexpressed in patients with prolapse (P = .042). Overall, patients had a mean age of 62.4 +/- 6.5 years compared with controls with a mean age of 45.3 +/- 7.4 years (P < .001), so analysis was also performed on an age-matched subset of 8 patients and controls (mean ages of 58.1 +/- 5.4 years and 53.3 +/- 5.0 years, respectively, P = .02) with similar results. Prolapse patients in this subset were similar in parity and race to controls but had lower body mass index (23.2 vs 29.9, P = .04). MYH3 was 10.9 times underexpressed in patients with pelvic organ prolapse compared to controls (P = .027). MyBP-H was 10.4 times underexpressed in patients with prolapse (P = .036). CONCLUSION: These findings suggest that the differences between patients with advanced pelvic organ prolapse and controls may be related to differential gene expression of structural proteins related to myosin. Specifically, advanced pelvic organ prolapse may be related to down-regulation of skeletal muscle heavy-chain polypeptide 3 and myosin binding protein H.

Differential gene expression in pubococcygeus muscle from patients with pelvic organ prolapse.

OBJECTIVE: This study was undertaken to compare differential gene expression in the pubococcygeus muscle in patients with pelvic organ prolapse and controls. STUDY DESIGN: We performed microarray analysis on individual pubococcygeus muscle biopsy specimens from five patients with stage III or IV pelvic organ prolapse and five control subjects without prolapse. This study received full Institutional Review Board approval. Total RNA was extracted, purified, and probed on the Human Genome U95A Array for each individual sample. RNA from patients and controls was not pooled. For microarray analysis, 7 microg of total RNA was used to synthesize complementary DNA that was then biotinylated. Arrays were hybridized for 16 hours in the GeneChip Fluidics Station 400 and were washed and scanned with the Hewlett-Packard GeneArray Scanner. Affymetrix GeneChip 5.0 software was used for scanning and data analysis. RESULTS: Of the 12626 total genes compared, 257 genes were more than 2-fold underexpressed, 20 genes were more than 5-fold underexpressed, and 3 genes were more than 10-fold underexpressed in patients with pelvic organ prolapse compared with control subjects. Myosin-binding protein H was 24.7 times underexpressed in patients with prolapse (normalized signal intensity [NSI]: 0.46 [0.2-0.6]) compared with controls (NSI: 11.4 [0.2-31.3]). Skeletal muscle myosin heavy polypeptide 3 was 17.4 times underexpressed in patients with prolapse (NSI: 0.85 [0.7-0.9]) compared with controls (NSI: 14.8 [1.5-38.3]). Of the 12,626 genes compared, 479 genes were more than 2-fold overexpressed, 18 genes were more than 5-fold overexpressed, and 2 genes were more than 10-fold overexpressed in patients with pelvic organ prolapse compared with controls. Many of these overexpressed genes were related to actin and myosin proteins. Smooth muscle myosin heavy chain was 11.8 times overexpressed in patients (NSI: 5.21 [0.25-22.71]) compared with controls (NSI 0.44 [0.11-0.71]). Myosin light-chain kinase was 5.8 times overexpressed in patients (NSI: 7.9 [0.5-36.1]) compared with controls (NSI: 1.37 [0.38-1.8]). Extracellular matrix proteins were also differentially regulated. Cartilage oligomeric matrix protein precursor was found to be 6.0 times underexpressed, whereas tenascin-C (hexabrachion) was 5.1 times overexpressed in prolapse patients. CONCLUSION: These data suggest that the differences between patients with advanced pelvic organ prolapse and controls may be related to differential gene expression of structural proteins that are related to actin and myosin as well as extracellular matrix proteins in the pubococcygeus muscle. Studies are ongoing to confirm these findings and to further characterize the role of these genes in prolapse.

Changes in gene expression during the early to mid-luteal (receptive phase) transition in human endometrium detected by high-density microarray screening.

High density cDNA microarray screening was used to determine changes in gene expression occurring during the transition between the early luteal (prereceptive) and mid-luteal (receptive) phases in human endometrium. Of approximately 12,000 genes profiled, 693 (5.8%) displayed >2-fold differences in relative levels of expression between these stages. Of these, 370 genes (3.1%) displayed decreases ranging from 2- to >100-fold while 323 genes (2.7%) displayed increases ranging from 2- to >45-fold. Many genes correspond to mRNAs encoding proteins previously shown to change in a similar manner between the proliferative and mid-luteal phases, serving as one validation of the microarray screening results. In addition, novel genes were identified. Genes encoding cell surface receptors, adhesion and extracellular matrix proteins and growth factors accounted for 20% of the changes. Several genes were studied further by Northern blot analyses. These results confirmed that claudin-4/Clostridium perfringens enterotoxin (CPE) receptor and osteopontin (OPN) mRNA increased approximately 4- and 12-fold respectively, while betaig-H3 (BIGH3) decreased >80% during the early to mid-luteal transition. Immunostaining also revealed strong specific staining for claudin-4/CPE, EP(1) and prostaglandin receptor in epithelia, and leukotriene B4 receptor in both epithelia and stroma, at the mid-luteal stage. Collectively, these studies identify multiple new candidate markers that may be used to predict the receptive phase in humans. Some of these gene products, e.g. OPN, may play direct roles in embryo-uterine interactions during the implantation process.