Bruce A. Lessey

Expression Profiling of Endometrium from Women with Endometriosis Reveals Candidate Genes for Disease-Based Implantation Failure and Infertility

Endometriosis is clinically associated with pelvic pain and infertility, with implantation failure strongly suggested as an underlying cause for the observed infertility. Eutopic endometrium of women with endometriosis provides a unique experimental paradigm for investigation into molecular mechanisms of reproductive dysfunction and an opportunity to identify specific markers for this disease. We applied paralleled gene expression profiling using high-density oligonucleotide microarrays to investigate differentially regulated genes in endometrium from women with vs. without endometriosis. Fifteen endometrial biopsy samples (obtained during the window of implantation from eight subjects with and seven subjects without endometriosis) were processed for expression profiling on Affymetrix Hu95A microarrays. Data analysis was conducted with GeneChip Analysis Suite, version 4.01, and GeneSpring version 4.0.4. Nonparametric testing was applied, using a P value of 0.05, to assess statistical significance. Of the 12,686 genes analyzed, 91 genes were significantly increased more than 2-fold in their expression, and 115 genes were decreased more than 2-fold. Unsupervised clustering demonstrated down-regulation of several known cell adhesion molecules, endometrial epithelial secreted proteins, and proteins not previously known to be involved in the pathogenesis of endometriosis, as well as up-regulated genes. Selected dysregulated genes were randomly chosen and validated with RT-PCR and/or Northern/dot-blot analyses, and confirmed up-regulation of collagen alpha2 type I, 2.6-fold; bile salt export pump, 2.0-fold; and down-regulation of N-acetylglucosamine-6-O-sulfotransferase (important in synthesis of L-selectin ligands), 1.7-fold; glycodelin, 51.5-fold; integrin alpha2, 1.8-fold; and B61 (Ephrin A1), 4.5-fold. Two-way overlapping layer analysis used to compare endometrial genes in the window of implantation from women with and without endometriosis further identified three unique groups of target genes, which differ with respect to the implantation window and the presence of disease. Group 1 target genes are up-regulated during the normal window of implantation but significantly decreased in women with endometriosis: IL-15, proline-rich protein, B61, Dickkopf-1, glycodelin, N-acetylglucosamine-6-O-sulfotransferase, G0S2 protein, and purine nucleoside phosphorylase. Group 2 genes are normally down-regulated during the window of implantation but are significantly increased with endometriosis: semaphorin E, neuronal olfactomedin-related endoplasmic reticulum localized protein mRNA and Sam68-like phosphotyrosine protein alpha. Group 3 consists of a single gene, neuronal pentraxin II, normally down-regulated during the window of implantation and further decreased in endometrium from women with endometriosis. The data support dysregulation of select genes leading to an inhospitable environment for implantation, including genes involved in embryonic attachment, embryo toxicity, immune dysfunction, and apoptotic responses, as well as genes likely contributing to the pathogenesis of endometriosis, including aromatase, progesterone receptor, angiogenic factors, and others. Identification and validation of selected genes and their functions will contribute to uncovering previously unknown mechanism(s) underlying implantation failure in women with endometriosis and infertility, mechanisms underlying the pathogenesis of endometriosis and providing potential new targets for diagnostic screening and intervention.

Placental apoptosis in preeclampsia.

Objective To determine whether preeclampsia is associated with an increase in placental apoptosis and differential expression of mediators of apoptosis. Methods Placental samples from 31 preeclamptic women and 31 normotensive controls were analyzed using terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling staining. Expression of Fas, Fas ligand, Bcl-2, and Bax was assessed using immunohistochemistry. Results The median percent apoptotic nuclei was significantly higher for the study group than for the controls (0.49 versus 0.19; P = .001), as was the median percent apoptotic nuclei in the trophoblast nuclei (0.33 versus 0.09; P < .01). Fas ligand expression was significantly less and Fas expression significantly greater in the villus trophoblast among the study subjects compared with controls. There was no difference in the expression of Bax or Bcl-2 between groups. Conclusion Placental apoptosis and altered expression of Fas and Fas ligand in trophoblast might influence pathogenesis or sequelae of preeclampsia.

Further characterization of endometrial integrins during the menstrual cycle and in pregnancy

OBJECTIVE To profile the changes in integrin expression in cycling and pregnant endometrium. DESIGN Immunohistochemistry was performed on endometrium from proliferative, early, and midsecretory phase and early pregnancy using a blinded panel of monoclonal antibodies directed against integrins. Closer examination of the cycle-dependent integrins was performed in 112 patients throughout the cycle and from the first trimester of pregnancy. SETTING An academic teaching hospital. PATIENTS Ovulatory women or women undergoing pregnancy termination. MAIN OUTCOME MEASURE Staining intensity of each antibody in epithelial or stromal cells. RESULTS Certain integrins were present constitutively throughout the menstrual cycle whereas others were expressed only during the luteal phase. Four integrins increased in the decidua of pregnancy. The timing of expression of the two cycle-dependent integrins (alpha 4 beta 1 and alpha v beta 3) framed the putative window of implantation and suggests a role in establishment of uterine receptivity. CONCLUSIONS Our findings confirm the patterns of endometrial integrins and suggest important roles for integrins in the process of implantation and decidualization.

Effect of progestin on the ovarian epithelium of macaques : Cancer prevention through apoptosis?

Objective: The apoptosis pathway is a vital mechanism in vivo that functions to eradicate genetically damaged cells prone to malignancy. The purpose of this study was to determine whether oral contraceptives, which confer significant protection against subsequent epithelial ovarian cancer, induce apoptosis in the ovarian epithelium. Methods: Female cynomologus macaques (N = 75) were randomized to receive a diet for 35 months containing either no hormones, the oral contraceptive Triphasil (Wyeth-Ayerst Laboratories, Philadelphia, PA), the estrogenic component of Triphasil (ethinyl estradiol) alone, or the progestin component of Triphasil (levonorgestrel) alone, each administered in a cyclic fashion. At study termination, the animals underwent ovariectomy and the ovarian epithelium was examined morphologically and immunihistochemically for apoptosis. The percentage of ovarian epithelial cells undergoing apoptosis was measured in each animal and compared between the treatment groups. Results: The median percentage of ovarian epithelial cells undergoing apoptosis by treatment was control (3.8%), ethinyl estradiol (1.8%), Triphasil (14.5%), and levonorgesrel (24.9%). Compared with control and ethinyl estradiol-treated monkeys, a statistically significant increase in the proportion of apoptotic cells was noted in the ovarian epithelium of monkeys treated with the oral contraceptive Triphasil (P ≤ .01) or levonorgestrel (P < .001), with a maximal effect (six-fold) seen in the group treated with levonorgestrel alone. Conclusion: Oral contraceptive progestin induces apoptosis in the ovarian epithelium. Given the importance of the apoptosis pathway for cancer prevention, an effective chemopreventive strategy may be possible using progestins or other agents that selectively induced apoptosis in the ovarian epithelium to prevent the development of ovarian cancer.

Elevated Endometrial Androgen Receptor Expression in Women with Polycystic Ovarian Syndrome

Abstract Androgen receptors (AR) have been identified in human endometrium; however, their role in endometrial cyclic development and function remains poorly understood. The objective of the present study was to investigate the profile of endometrial AR in normal menstrual cycles and in the endometrium of women with polycystic ovarian syndrome (PCOS). This syndrome is characterized by chronic hyperandrogenism and oligo-ovulation, and it is often associated with poor reproductive performance. Using immunohistochemistry and reverse transcription-polymerase chain reaction, we found that women with PCOS exhibited elevated endometrial AR expression compared to normal, fertile controls. This increase was most apparent in glandular and luminal epithelium. Furthermore, when compared to endometrium from fertile women, PCOS endometrium showed other abnormalities in endometrial development, including delay or absence of the αvβ3 integrin, a well-characterized biomarker of uterine receptivity described previously (Lessey et al., JCI 1992; 90:188–195). To better understand and to gain insights regarding these findings, we used in vitro cell-culture models to study the regulation of AR in primary endometrial stromal and the well-differentiated epithelial cell line (Ishikawa). Based on Western blot analysis, epithelial AR is up-regulated by estrogens and androgens and is inhibited by progestins and epidermal growth factor (EGF). On the other hand, EGF significantly induced the expression of αvβ3, whereas estrogen and androgen treatment inhibited its expression. Collectively, these results suggest that the poor reproductive performance observed in women with PCOS may be due, in part, to the concomitant increase in both serum androgens and elevations in endometrial AR. This combination may reduce endometrial receptivity as judged by the down-regulation of αvβ3 integrin.

Luminal and Glandular Endometrial Epithelium Express Integrins Differentially Throughout the Menstrual Cycle: Implications for Implantation, Contraception, and Infertility

PROBLEM: Integrins belong to a family of cell adhesion molecules that are present on virtually all cells. The temporal and spatial expression of these important proteins on the human endometrium suggests that certain integrins may participate in the cascade of molecular events leading to successful implantation.

Blockade of the αvβ3 Integrin Adversely Affects Implantation in the Mouse

Abstract The role of endometrial and embryonic integrins during implantation remains unresolved although work in animal models and in humans supports their involvement in this process. Temporal and spatial distribution of the αvβ3 integrin on both embryo and endometrium in women and mice coincides with the time of initial attachment during implantation. In mice, the endometrial and embryonic αvβ3 integrin is present at the time of implantation, as shown by reverse transcription-polymerase chain reaction and immunohistochemistry. In situ hybridization demonstrates the presence of the αvβ3 integrin on the subluminal stromal cells of the uterus. Functional blockade of this integrin on the day of implantation by intrauterine injection of neutralizing monoclonal antibodies against αv or β3 integrin subunits, arg-gly-asp (RGD)-containing peptides, or of the disintegrin echistatin, reduced the number of implantation sites compared to controls receiving BSA. These studies demonstrate that, like the human, the murine αvβ3 integrin is expressed at the time of implantation in the endometrium and on the blastocyst, and may play a critical role in the cascade of events leading to successful implantation.

MicroRNA expression profiling of eutopic secretory endometrium in women with versus without endometriosis.

Endometriosis is a common gynecologic disorder characterized by pain and infertility. In addition to estrogen dependence, progesterone resistance is an emerging feature of this disorder. Specifically, a delayed transition from the proliferative to secretory phase as evidenced by dysregulation of progesterone target genes and maintenance of a proliferative molecular fingerprint in the early secretory endometrium (ESE) has been reported. MicroRNAs (miRNAs) are small noncoding RNAs that collectively represent a novel class of regulators of gene expression. In an effort to investigate further the observed progesterone resistance in the ESE of women with endometriosis, we conducted array-based, global miRNA profiling. We report distinct miRNA expression profiles in the ESE of women with versus without endometriosis in a subset of samples previously used in global gene expression analysis. Specifically, the miR-9 and miR-34 miRNA families evidenced dysregulation. Integration of the miRNA and gene expression profiles provides unique insights into the molecular basis of this enigmatic disorder and, possibly, the regulation of the proliferative phenotype during the early secretory phase of the menstrual cycle in affected women.

DNA profiling analysis of endometrial and ovarian cell lines reveals misidentification, redundancy and contamination

OBJECTIVES Cell lines derived from human ovarian and endometrial cancers, and their immortalized non-malignant counterparts, are critical tools to investigate and characterize molecular mechanisms underlying gynecologic tumorigenesis, and facilitate development of novel therapeutics. To determine the extent of misidentification, contamination and redundancy, with evident consequences for the validity of research based upon these models, we undertook a systematic analysis and cataloging of endometrial and ovarian cell lines. METHODS Profiling of cell lines by analysis of DNA microsatellite short tandem repeats (STR), p53 nucleotide polymorphisms and microsatellite instability was performed. RESULTS Fifty-one ovarian cancer lines were profiled with ten found to be redundant and five (A2008, OV2008, C13, SK-OV-4 and SK-OV-6) identified as cervical cancer cells. Ten endometrial cell lines were analyzed, with RL-92, HEC-1A, HEC-1B, HEC-50, KLE, and AN3CA all exhibiting unique, uncontaminated STR profiles. Multiple variants of Ishikawa and ECC-1 endometrial cancer cell lines were genotyped and analyzed by sequencing of mutations in the p53 gene. The profile of ECC-1 cells did not match the EnCa-101 tumor, from which it was reportedly derived, and all ECC-1 isolates were genotyped as Ishikawa cells, MCF-7 breast cancer cells, or a combination thereof. Two normal, immortalized endometrial epithelial cell lines, HES cells and the hTERT-EEC line, were identified as HeLa cervical carcinoma and MCF-7 breast cancer cells, respectively. CONCLUSIONS Results demonstrate significant misidentification, duplication, and loss of integrity of endometrial and ovarian cancer cell lines. Authentication by STR DNA profiling is a simple and economical method to verify and validate studies undertaken with these models.

Medical management of endometriosis and infertility.

OBJECTIVE To review the literature on the use of medical management of endometriosis and infertility. DESIGN Literature review. RESULT(S) Endometriosis is a common finding in women with infertility, but the mechanism by which it renders a woman infertile remains unclear. Despite many years of controversy and debate, there remains a strong bias against medical treatment for endometriosis-associated infertility. A review of the current literature suggests that medical management of endometriosis may be effective in selected patients and in certain settings, including patients undergoing IVF. CONCLUSION(S) A closer look at the question of medical management of endometriosis reveals that much remains to be learned before a final decision can be made about the use of medical therapies, such as GnRH agonists, for endometriosis and associated infertility.