The uterine secretome initiates growth of gynecologic tissues in ectopic locations.

Endosalpingiosis (ES) and endometriosis (EM) refer to the growth of tubal and endometrial epithelium respectively, outside of their site of origin. We hypothesize that uterine secretome factors drive ectopic growth. To test this, we developed a mouse model of ES and EM using tdTomato (tdT) transgenic fluorescent mice as donors. To block implantation factors, progesterone knockout (PKO) tdT mice were created. Fluorescent lesions were present after oviduct implantation with and without WT endometrium. Implantation was increased (p<0.05) when tdt oviductal tissue was implanted with endometrium compared to oviductal tissue alone. Implantation was reduced (p<0.0005) in animals implanted with minced tdT oviductal tissue with PKO tdT endometrium compared to WT endometrium. Finally, oviductal tissues was incubated with and without a known implantation factor, leukemia inhibitory factor (LIF) prior to and during implantation. LIF promoted lesion implantation. In conclusion, endometrial derived implantation factors, such as LIF, are necessary to initiate ectopic tissue growth. We have developed an animal model of ectopic growth of gynecologic tissues in a WT mouse which will potentially allow for development of new prevention and treatment modalities.

Cellular junction and mesenchymal factors delineate an endometriosis-specific response of endometrial stromal cells to the mesothelium.

Endometriosis is a debilitating gynecologic disorder that affects ∼10% of women of reproductive age. Endometriosis is characterized by growth of endometriosis lesions within the abdominal cavity, generally thought to arise from retrograde menstruation of shed endometrial tissue. While the pathophysiology underlying peritoneal endometriosis lesion formation is still unclear, the interaction between invading endometrial tissue and the peritoneal mesothelial lining is an essential step in lesion formation. In this study, we assessed proteomic differences between eutopic endometrial stromal cells (ESCs) from women with and without endometriosis in response to peritoneal mesothelial cell (PMC) exposure, using single-cell cytometry by time-of-flight (CyTOF). Co-cultured primary eutopic ESCs from women with and without endometriosis with an established PMC line were subjected to immunostaining with a panel of Maxpar CyTOF metal-conjugated antibodies (n = 28) targeting cell junction and mesenchymal markers, which are involved in cell-cell adhesions and epithelial-mesenchymal transition. Exposure of the ESCs to PMCs resulted in a drastic shift in cellular expression profiles in ESCs derived from endometriosis, whereas little effect by PMCs was observed in ESCs from non-endometriosis subjects. The transcription factor SNAI1 was consistently repressed by PMC interactions. ESCs from endometriosis patients are unique in that they respond to PMCs by undergoing changes in adhesive properties and mesenchymal characteristics that would facilitate lesion formation.

An intrauterine genomic classifier reliably delineates the location of nonviable pregnancies.

OBJECTIVE: To compare the intrauterine gene expression signatures of women with surgically confirmed ectopic pregnancy (ECT) and those of women with miscarriage to inform the development of a genomic classifier for the reliable delineation of pregnancy location in women with clinically nonviable pregnancies of unknown location (NV-PULs). DESIGN: Discovery-based prospective cohort study. SETTING: Academic medical center. PATIENT(S): Women with clinically nonviable early pregnancy to include abnormal intrauterine pregnancy (AIUP), ECT, or NV-PUL. INTERVENTION(S): Endometrial (EM) pipelle sampling of the uterus was conducted at the time of scheduled surgery for clinically nonviable early pregnancy (dilation and curettage, manual vacuum aspiration, or laparoscopy). All pregnancy locations were surgically and/or histologically confirmed as intrauterine or ectopic. MAIN OUTCOME MEASURE(S): Gene expression profiles as determined by array hybridization, quantitative real-time polymerase chain reaction, and nCounter technology. RESULT(S): Intrauterine samples were obtained by EM pipelle from 27 women undergoing surgery for a clinically nonviable early pregnancy. Comparison of array-based global gene expression signatures from women with histologically confirmed ECT versus AIUP revealed 61 differentially expressed genes from which the 5 most informative were included in the pregnancy location classifier. All 5 genes (C20orf85, LRRC46, RSPH4A, WDR49, and ZBBX) were cilia-associated and showed increased expression in pipelle samples from women with ECT relative to expression in samples from women with AIUP. The 5-gene classifier demonstrated an average area under the receiver operator characteristic curve of 0.97 for the detection of ECT. In an external test set composed of publicly available EM pipelle-based gene expression data from a study with similar ECT and AIUP cohorts (n = 19), the classifier revealed an average area under the receiver operator characteristic curve of 0.84. CONCLUSION(S): Consistently increased expression of cilia-associated genes in the uterine cavity of women with ECT provides a reliable molecular signal for the delineation of pregnancy location in women with clinically assessed NV-PUL. A classifier consisting of the 5 most informative cilia-associated genes demonstrated 91% (42/46) accuracy in predicting the pregnancy location.

Endometriosis in the Mouse: Challenges and Progress Toward a 'Best Fit' Murine Model.

Endometriosis is a prevalent gynecologic condition associated with pelvic pain and infertility characterized by the implantation and growth of endometrial tissue displaced into the pelvis via retrograde menstruation. The mouse is a molecularly well-annotated and cost-efficient species for modeling human disease in the therapeutic discovery pipeline. However, as a non-menstrual species with a closed tubo-ovarian junction, the mouse poses inherent challenges as a preclinical model for endometriosis research. Over the past three decades, numerous murine models of endometriosis have been described with varying degrees of fidelity in recapitulating the essential pathophysiologic features of the human disease. We conducted a search of the peer-reviewed literature to identify publications describing preclinical research using a murine model of endometriosis. Each model was reviewed according to a panel of ideal model parameters founded on the current understanding of endometriosis pathophysiology. Evaluated parameters included method of transplantation, cycle phase and type of tissue transplanted, recipient immune/ovarian status, iterative schedule of transplantation, and option for longitudinal lesion assessment. Though challenges remain, more recent models have incorporated innovative technical approaches such as in vivo fluorescence imaging and novel hormonal preparations to overcome the unique challenges posed by murine anatomy and physiology. These models offer significant advantages in lesion development and readout toward a high-fidelity mouse model for translational research in endometriosis.

The human tubal lavage proteome reveals biological processes that may govern the pathology of hydrosalpinx.

Hydrosalpinx, the blockage of fallopian tubes, can result from pelvic inflammatory disease. Hydrosalpinx is a cause of infertility and negatively impacts in vitro fertilization. To better understand the pathobiology of hydrosalpinx, we compared the proteome of lavages from disease vs. healthy fallopian tubes. Results indicate a disruption of redox homeostasis and activation of the complement system, immune cell infiltration, and phagocytosis; pathways that may drive tubal injury. To our surprise among the most prominent proteins with hydrosalpinx was mesothelin (MSLN), which until now has only been associated with epithelial malignancies. Analogous to mesothelioma and ovarian carcinoma, a significant increase of MSLN was detected in plasma from patients with hydrosalpinx. This finding suggests MSLN may provide clinical diagnosis in lieu of the current approaches that require invasive imaging. Importantly, these findings implicate MSLN in a benign disease, indicating that the activation and role of MSLN is not restricted to cancer.

Microdermal Implants Show No Effect on Surrounding Tissue During Surgery With Electrocautery.

BACKGROUND: Microdermal implants are an increasingly popular form of body jewelry. The potential for electrical conduction burn at the site of metal jewelry left in situ during electrosurgery has prompted surgical societies to recommend routine removal before surgery. To date, however, there is a lack of evidence to support this practice. We assessed in vivo thermal effect and tissue damage around implants during and after electrocautery. MATERIALS AND METHODS: Stainless steel microdermal anchors were surgically implanted into four swine. After allowing for initial healing, negative controls were excised and evaluated. An electrocautery grounding pad was placed 2 cm caudal to the implant. Continuous electrocautery (coagulation/30 W) for 30 s was applied to the skin 2 cm cranial to the implant. Surface skin temperature was recorded during electrocautery using thermal imaging. Tissue damage was assessed by gross examination and histologic evaluation. The same procedure was then performed to the contralateral nonimplanted side as a sham control. RESULTS: Electrocoagulation raised skin temperature around the electrocautery tip 27.7°C (Tmax 64.8°C). Skin temperature around the dermal implant rose 1.58°C (Tmax 38.6°C) compared with 2.03°C (Tmax 39.2°C) in the nonimplanted control skin (P = 0.627). Skin temperatures at implanted and control sites showed no statistical difference at any recorded time interval. Histologic review of excised tissue samples showed no evidence of thermal injury. CONCLUSIONS: Metallic implants appear to have no effect on skin temperature during the use of electrocautery even when in close proximity to both the electrocautery pen and return pad. Aggressive steps to remove microdermal implants before surgery may be unnecessary.

A SMART design to determine the optimal treatment of chronic pain among military personnel.

Chronic pain is a leading cause of disability among active duty service members in the U.S. armed forces. Standard rehabilitative care and complementary and integrative health therapies are used for chronic pain rehabilitation. However, the optimal sequence and duration of these therapies has yet to be determined. This article describes a sequential multiple assignment randomized trial (SMART) protocol being used to identify the optimal components and sequence of standard rehabilitative care and complementary and integrative health therapies for reducing pain impact and improving other patient outcomes. Active duty service members referred to Madigan Army Medical Center for treatment of chronic pain are being recruited to the Determinants of the Optimal Dose and Sequence of Functional Restoration and Integrative Therapies study. Study participants are randomized to either standard rehabilitative care (physical and occupational therapy and psychoeducation) or complementary and integrative health therapies (chiropractic, acupuncture, yoga and psychoeducation). Those participants who do not respond to the first 3 weeks of treatment are randomized to receive an additional 3 weeks of either (1) the alternative treatment or (2) the first-stage treatment plus the alternative treatment. This study will also determine factors associated with treatment response that can support clinical decision making, such as baseline fitness, pain catastrophizing, kinesiophobia, post-traumatic stress, pain self-efficacy, and biological indicators. The information gained from this research will be applicable to all integrative chronic pain rehabilitation programs throughout the U.S. Department of Defense and the U.S. Department of Veterans Affairs, and the broader rehabilitation community.

The Gynecologic Health Consequences of Chlamydia trachomatis Infection in Military Servicewomen.

Chlamydia trachomatis is the most common sexually transmitted bacterial infection in the United States. Within the U.S. military, the age- and race-adjusted chlamydia infection rates among female service members are consistently higher than civilian rates, with a 20% annual acquisition rate among young active-duty women. The sequelae of chlamydia disproportionately impact women in terms of severity and cost. Untreated chlamydia progresses to pelvic inflammatory disease in 40% of cases, and is a leading cause of fallopian tube damage and pelvic adhesive disease resulting in ectopic pregnancy, tubal infertility, and acute and chronic pelvic pain. Tubal infertility is among the leading indications for in vitro fertilization (IVF) nationally and rates among couples undergoing IVF at military treatment centers are double the national average. Collectively, chlamydia infection represents a significant resource burden to the military health care system and, in view of the serious gynecologic health sequelae, a significant threat to the readiness of servicewomen. In this review, we discuss the gynecologic impact of chlamydia infection within the military, the critical gaps for research funding, and opportunities for intervention.

Update on Biomarkers for the Detection of Endometriosis.

Endometriosis is histologically characterized by the displacement of endometrial tissue to extrauterine locations including the pelvic peritoneum, ovaries, and bowel. An important cause of infertility and pelvic pain, the individual and global socioeconomic burden of endometriosis is significant. Laparoscopy remains the gold standard for the diagnosis of the condition. However, the invasive nature of surgery, coupled with the lack of a laboratory biomarker for the disease, results in a mean latency of 7-11 years from onset of symptoms to definitive diagnosis. Unfortunately, the delay in diagnosis may have significant consequences in terms of disease progression. The discovery of a sufficiently sensitive and specific biomarker for the nonsurgical detection of endometriosis promises earlier diagnosis and prevention of deleterious sequelae and represents a clear research priority. In this review, we describe and discuss the current status of biomarkers of endometriosis in plasma, urine, and endometrium.

Dexamethasone attenuates the embryotoxic effect of endometriotic peritoneal fluid in a murine model.

PURPOSE: The in vitro fertilization (IVF) pregnancy rate of women with advanced stage endometriosis is nearly half that of the general population, suggesting incomplete targeting of the pathophysiology underlying endometriosis-associated infertility. Compelling evidence highlights inflammation as the etiologic link between endometriosis and infertility and a potential target for adjunctive treatment. The objective of this study was to examine the effect of dexamethasone on murine embryos exposed to human endometriotic peritoneal fluid (PF) using the established murine embryo assay model. METHODS: PF was obtained from women with and without severe endometriosis. Murine embryos were harvested and randomly allocated to five groups of culture media conditions: (1) human tubal fluid (HTF), (2) HTF and 10 % PF from women without endometriosis, (3) HTF and 10 % PF from women with endometriosis (PF-E), (4) HTF with PF-E and 0.01 mcg/mL dexamethasone, and (5) HTF with PF-E and 0.1 mcg/mL dexamethasone. Embryos were cultured in standard conditions and evaluated for blastocyst development. RESULTS: A total of 266 mouse embryos were cultured. Baseline blastulation rates were 63.6 %. The addition of peritoneal fluid from women with endometriosis decreased the blastocyst development rate to 38.9 % (P = 0.008). The addition of 0.1 mcg/mL of dexamethasone to the culture media restored the blastulation rate to near baseline levels (61.2 %; P = 0.019). CONCLUSIONS: The results of our in vitro study demonstrate the capacity of dexamethasone to mitigate the deleterious impact of endometriotic PF on embryo development. If confirmed in vivo, dexamethasone may prove a useful adjunct for the treatment of endometriosis-associated infertility.

Molecular classification of endometriosis and disease stage using high-dimensional genomic data.

Endometriosis (E), an estrogen-dependent, progesterone-resistant, inflammatory disorder, affects 10% of reproductive-age women. It is diagnosed and staged at surgery, resulting in an 11-year latency from symptom onset to diagnosis, underscoring the need for less invasive, less expensive approaches. Because the uterine lining (endometrium) in women with E has altered molecular profiles, we tested whether molecular classification of this tissue can distinguish and stage disease. We developed classifiers using genomic data from n = 148 archived endometrial samples from women with E or without E (normal controls or with other common uterine/pelvic pathologies) across the menstrual cycle and evaluated their performance on independent sample sets. Classifiers were trained separately on samples in specific hormonal milieu, using margin tree classification, and accuracies were scored on independent validation samples. Classification of samples from women with E or no E involved 2 binary decisions, each based on expression of specific genes. These first distinguished presence or absence of uterine/pelvic pathology and then no E from E, with the latter further classified according to severity (minimal/mild or moderate/severe). Best performing classifiers identified E with 90%-100% accuracy, were cycle phase-specific or independent, and used relatively few genes to determine disease and severity. Differential gene expression and pathway analyses revealed immune activation, altered steroid and thyroid hormone signaling/metabolism, and growth factor signaling in endometrium of women with E. Similar findings were observed with other disorders vs controls. Thus, classifier analysis of genomic data from endometrium can detect and stage pelvic E with high accuracy, dependent or independent of hormonal milieu. We propose that limited classifier candidate genes are of high value in developing diagnostics and identifying therapeutic targets. Discovery of endometrial molecular differences in the presence of E and other uterine/pelvic pathologies raises the broader biological question of their impact on the steroid hormone response and normal functions of this tissue.

Biomarker development in endometriosis.

Endometriosis is a common gynecologic disorder histologically characterized by the displacement of endometrial tissue to extra-uterine locations. A significant cause of infertility and pelvic pain, the global socioeconomic burden of endometriosis is staggering. Laparoscopy remains the gold standard for the diagnosis of the condition. However, the invasive nature of surgery, coupled with the lack of a laboratory biomarker for the disease, results in a mean latency of 6-7 years from onset of symptoms to definitive diagnosis. Unfortunately, the delay in diagnosis may have significant consequences in terms of disease progression. The discovery of a sufficiently sensitive and specific biomarker for the non-surgical detection of endometriosis promises earlier diagnosis and prevention of deleterious sequelae, and remains a top research priority. The enigmatic pathophysiology of endometriosis presents unique challenges to biomarker development that are now well outlined. Within the past decade, significant advancements in understanding the molecular hallmarks of endometriosis have occurred, and promising biomarker candidates are emerging.

Premenstrual spotting of ≥2 days is strongly associated with histologically confirmed endometriosis in women with infertility.

OBJECTIVE: The purpose of this study was to assess the prevalence of endometriosis in women with premenstrual spotting and to determine the predictive value of this symptom in the diagnosis of endometriosis. STUDY DESIGN: We conducted a retrospective cohort study of 80 consecutive women who presented to the infertility clinic for evaluation and who subsequently underwent laparoscopic assessment for infertility with or without pelvic pain. Our main outcome measure was the presence or absence of histologically confirmed endometriosis in women with and without premenstrual spotting. RESULTS: Endometriosis was significantly more prevalent in subfertile women who reported premenstrual spotting for ≥2 days relative to women without this symptom (89% [34/38 women] vs 26% [11/42 women]; P < .0001). Multinomial logistic regression analysis demonstrated the presence of premenstrual spotting for ≥2 days to be associated significantly with the presence of endometriosis (odds ratio, 16; 95% confidence interval, 3.9-65.4; P < .01) and red vesicular lesion type (odds ratio, 52.6; 95% confidence interval, 8.6-323.1; P < .001). CONCLUSION: In this cohort of women with infertility, premenstrual spotting of ≥2 days was associated strongly with histologically confirmed endometriosis and a better predictor than dysmenorrhea or dyspareunia of finding endometriosis at laparoscopy. Premenstrual spotting of at least 2 days was also associated strongly with both higher stage disease and the red vesicular peritoneal endometriosis phenotype.

Expression patterns of progesterone receptor membrane components 1 and 2 in endometria from women with and without endometriosis.

Endometriosis is a hormone-dependent inflammatory condition associated with pain and infertility. A growing body of evidence supports attenuated secretory-phase progesterone responsiveness in women with this disease. Herein, we compare the expression of progesterone receptor membrane components (PGRMC) 1 and 2 in eutopic endometrium from 11 women with laparoscopically and/or histologically proven stage III/IV endometriosis and 23 disease-free women. Menstrual cycle phase was determined using a combination of reported cycle day, serum hormone profile, and endometrial histologic dating. The PGRMC-1 (fold change -3.3; P < .05) and PGRMC-2 (fold-change -8.8; P < .05) gene expression were significantly downregulated in secretory phase, eutopic endometrium from women with endometriosis. Immunohistochemistry demonstrated decreased PGRMC-1 and PGRMC-2 protein expression in the secretory phase endometrial stroma cells of women with endometriosis. Consistent with the preclinical work of others, our results reflect downregulation of endometrial PGRMC-1 and PGRMC-2 expression in secretory phase endometrium from women with advanced stage endometriosis. Understanding the molecular mechanisms of attenuated progesterone action in endometriosis has important diagnostic and therapeutic implications.

The genetics and biochemistry of endometriosis.

PURPOSE OF REVIEW: Endometriosis is a common gynecologic disorder characterized by the displacement of endometrial tissue to ectopic locations. Although predisposition to endometriosis is likely multifactorial, a genetic component is evident. The biochemistry of the disorder is an area of active investigation with translational potential. This review synopsizes recent developments regarding the molecular underpinnings of endometriosis. RECENT FINDINGS: Significant advancements in understanding the molecular hallmarks of endometriosis have occurred in recent years. Inflammation, attenuated progesterone action, and neuroangiogenesis constitute emerging themes in the pathophysiology of endometriosis. SUMMARY: Delineation of the biochemical processes involved in endometriosis has important implications for clinical care. The discovery of a sufficiently sensitive and specific biomarker for the nonsurgical detection of endometriosis promises earlier diagnosis and prevention of deleterious sequelae. Understanding the inflammatory cause, attenuated progesterone action at the level of the endometrium, and neuronal sensitization of endometriotic lesions has facilitated development of novel therapeutic approaches for associated pain and infertility.

Effect of cigarette smoking on human oviductal ciliation and ciliogenesis.

OBJECTIVE: To investigate the effect of cigarette smoke exposure on ciliation and ciliogenesis in human oviductal epithelium. DESIGN: Molecular analysis using human tubal segments. SETTING: Academic medical center. PATIENT(S): Twenty women undergoing elective tubal sterilization procedure. INTERVENTION(S): Expression of ciliated cell-specific markers was compared in tubal segments from smokers and nonsmokers using quantitative immunohistochemistry and Western blot analysis. The expression of transcription factors in the motile ciliogenesis program was compared using quantitative polymerase chain reaction and quantitative immunohistochemistry. MAIN OUTCOME MEASURE(S): Oviductal ciliation and expression of transcription factors involved in ciliogenesis. RESULT(S): No significant differences were detected in density of ciliation between groups. Neither number of years of smoking nor pack-year history correlated with density of ciliation. Expression of ciliogenic transcription factors FOXJ1, RFX2, and RFX3 was consistent between groups. CONCLUSION(S): Few studies have evaluated the relationship between smoking and ciliated epithelium in human oviducts. Cigarette smoking does not seem to result in quantitative differences in the density of ciliation nor expression of ciliogenesis factors. Our findings suggest that pathophysiologic mechanisms other than ciliation account for the increased risk of ectopic pregnancy in women who smoke.

Pathogenesis and pathophysiology of endometriosis.

Originally described over three hundred years ago, endometriosis is classically defined by the presence of endometrial glands and stroma in extrauterine locations. Endometriosis is an inflammatory, estrogen-dependent condition associated with pelvic pain and infertility. This work reviews the disease process from theories regarding origin to the molecular basis for disease sequelae. A thorough understanding of the histopathogenesis and pathophysiology of endometriosis is essential to the development of novel diagnostic and treatment approaches for this debilitating condition.