Peroxisome-proliferator activator receptor-gamma activation decreases attachment of endometrial cells to peritoneal mesothelial cells in an in vitro model of the early endometriotic lesion.

The aim of this study was to investigate whether peroxisome proliferator-activated receptor (PPAR)-gamma activation has an effect on the attachment of endometrial cells to peritoneal mesothelial cells in a well-established in vitro model of the early endometriotic lesion. The endometrial epithelial cell line EM42 and mesothelial cell line LP9 were used for this study. EM42 cells, LP9 cells or both were treated with the PPAR-gamma agonist ciglitazone (CTZ) at varying concentrations (10, 20 and 40 microM) x 48 h with subsequent co-culture of EM42 and LP9 cells. The rate of EM42 attachment and invasion through LP9 cells was then assessed and compared with control (EM42 and LP9 cells co-cultured without prior treatment with CTZ). Next, attachment of CTZ-treated and untreated EM42 cells to hyaluronic acid (HA), a cell adhesion molecule (CAM) on peritoneal mesothelial cells, were assessed. Although there was no difference in EM42 attachment when LP9 cells alone were treated with CTZ, treatment of EM42 cells with 40 microM CTZ decreased EM42 attachment to LP9 cells by 27% (P < 0.01). Treatment of both EM42 and LP9 cells with 40 microM CTZ decreased EM42 attachment to LP9 by 37% (P < 0.01). Treatment of EM42 cells with 40 microM CTZ decreased attachment to HA by 66% (P = 0.056). CTZ did not decrease invasion of EM42 cells through the LP9 monolayer. CTZ may inhibit EM42 cell proliferation. In conclusion, CTZ significantly decreased EM42 attachment to LP9 cells and HA in an in vitro model of the early endometriotic lesion.

The vasoactive peptide angiotensin-(1-7), its receptor Mas and the angiotensin-converting enzyme type 2 are expressed in the human endometrium.

Angiotensin (Ang)-(1-7) is one of the major active components of the renin-angiotensin system, produced from cleavage of Ang II by angiotensin-converting-enzyme type 2 (ACE2), which acts through a specific G protein-coupled receptor, Mas. We have investigated whether the human endometrium expresses these components during menstrual cycle. By radioimmunoassay, Ang-(1-7) was detected in endometrial wash fluid at picomolar concentrations. Using immunofluorescence, both the peptide and its receptor were identified in cultured endometrial epithelial and stromal cells. By immunohistochemistry, Ang(1-7) was localized in the endometrium throughout menstrual cycle, being more concentrated in the glandular epithelium of mid- and late secretory phase. This pattern corresponded to the ACE2 mRNA, which was more abundant in epithelial cells than in stromal cells (2-fold increase, p < 0.05) and in the secretory vs. proliferative phase (6.6-fold increase, p < 0.01). The receptor Mas was equally distributed between epithelial and stromal cells and did not change during menstrual cycle. The physiological role of this peptide system in normal and pathological endometrium warrants further investigation.

Activin A increases invasiveness of endometrial cells in an in vitro model of human peritoneum.

The aim of this study was to investigate whether activin A has an effect on the attachment and/or invasion of endometrial cells in a modeled peritoneum in vitro. Cultured endometrial stromal cells (ESCs) and endometrial epithelial cells (EECs) were treated with activin A (6.25-50 ng/ml) and with activin A (25 ng/ml) with and without inhibin A or follistatin. Fluorescent labeled cells were added to confluent peritoneal mesothelial cells (PMCs) and to a monolayer of confluent PMCs grown in a Matrigel invasion assay. The rate of endometrial cell attachment and invasion through PMCs was assessed. The expression of cell adhesion proteins N- and E-cadherin was evaluated with real-time RT-PCR. Activin A (25 ng/ml) promoted invasion of the endometrial cells through the modeled peritoneum (>2-fold versus control) and this effect was partially reversed by inhibin A and follistatin. Activin A had no effect on the rate of attachment of the endometrial cells to the PMCs or in the rate of proliferation. In addition, activin A induced a decreased mRNA expression of E-cadherin in cultured EECs. In conclusion, activin A increases invasion of EECs and ESCs into modeled peritoneum. In EECs, this effect may be related to down-regulation of E-cadherin expression. Further studies are warranted to evaluate the role of activin-A in the genesis of the endometriotic lesion.

Mesothelial cell-associated hyaluronic acid promotes adhesion of endometrial cells to mesothelium.

OBJECTIVE: To evaluate the role of hyaluronic acid in the attachment of endometrial cells to mesothelium. DESIGN: In vitro study of adhesion of endometrial stromal and epithelial cells to mesothelial cells. SETTING: University medical center. PATIENT(S): Reproductive-age women without endometriosis undergoing surgery for benign conditions. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The effect of hyaluronidase treatment of mesothelial cells or endometrial cells on adhesion of (51)Cr labeled endometrial stromal and epithelial cells to monolayers of mesothelium was evaluated. The expression of CD44, the hyaluronate receptor, was evaluated by western blot. RESULT(S): Hyaluronidase pretreatment of mesothelial cells decreased the binding of endometrial stromal and epithelial cells to mesothelium by 39% (P< .02) and 31% (P< .03), respectively. There was no effect on endometrial cell binding to mesothelial cells or to collagen IV when the endometrial cells were pretreated with hyaluronidase. CD44 expression by endometrial stromal and epithelial cells was demonstrated by western blot. CONCLUSIONS: This study demonstrates that mesothelial cell-associated hyaluronic acid is involved in attachment of endometrial stromal and endometrial epithelial cells to the mesothelium. We hypothesize that binding of hyaluronic acid by endometrial cells is involved in the pathogenesis of the early endometriotic lesion.

Composition of the extracellular matrix of the peritoneum.

OBJECTIVE: To localize the extracellular matrix proteins collagen I, collagen IV, fibronectin, and laminin in the peritoneal membrane. STUDY DESIGN: Peritoneal biopsies (n = 13) from the anterior abdominal wall and the uterine serosa (n = 3) were incubated with antibodies to collagen IV, laminin, collagen I, and fibronectin. Specimens were examined using light and confocal laser scanning microscopy. RESULTS: All of the extracellular matrix (ECM) proteins were present immediately under the mesothelium. Collagen (Col) IV and laminin (LM) were seen in the smooth muscle of microvascular structures, in the subendothelial basement membrane, and were present in a fascicular pattern in the peritoneal stroma. Collagen I was distributed diffusely in the peritoneal stroma. Fibronectin was also present in the subendothelial basement membrane. CONCLUSIONS: The resolution of the confocal microscope allowed for localization of extracellular matrix proteins in relation to the mesothelium. The presence of collagen IV, laminin, collagen I, and fibronectin under the mesothelium suggests that cells invading the peritoneum must have the ability to degrade and remodel this matrix.

Short-term culture of peritoneum explants confirms attachment of endometrium to intact peritoneal mesothelium.

OBJECTIVE: To evaluate the initial adhesion of endometrium to the peritoneum. DESIGN: Descriptive study using light and confocal laser-scanning microscopy, immunohistochemistry, and transmission electron microscopy. SETTING: University-based laboratory. PATIENT(S): Women without endometriosis undergoing surgery for benign conditions. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Explants of peritoneum (n = 20), prepared from four patients, were cultured for 1 hour with mechanically dispersed proliferative or secretory endometrium. Peritoneum was cultured with endometrium from the same patient. Specimens were fixed and serially sectioned for hematoxylin and eosin stain, immunohistochemistry using an anti-cytokeratin monoclonal antibody, and transmission electron microscopy. RESULT(S): In 17 of 20 explants, endometrium was adherent to intact mesothelium. There was no evidence of transmesothelial invasion at any sites of attachment. Although in most cases endometrium was adherent to mesothelium via endometrial stroma, there were many sites of endometrial epithelium-mesothelium attachment. Confocal laser scanning microscopy demonstrated an intact monolayer of cytokeratin-positive cells below the sites of endometrial implantation. Transmission electron microscopy demonstrated intact, viable, mesothelial cells below sites of attachment. CONCLUSION(S): This study demonstrates that endometrium rapidly adheres to intact peritoneal mesothelium. In addition, this study demonstrates that endometrial epithelial cells, as well as stroma, can attach to mesothelium. Further studies are needed that characterize the mechanism of endometrial-mesothelial cell adhesion.

Expression of the alpha2beta1 and alpha3beta1 integrins at the surface of mesothelial cells: a potential attachment site of endometrial cells.

OBJECTIVE: To localize alpha2beta1 and alpha3beta1 integrins in the cell membrane of peritoneal mesothelium in vivo and in vitro. DESIGN: Descriptive study using confocal and two-photon laser-scanning microscopy. SETTING: University-based laboratory. PATIENT(S): Women without endometriosis undergoing surgery for benign conditions. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Peritoneal biopsies (n = 9) and mesothelial monolayer cultures (n = 4) were incubated with antibodies to the alpha2 and alpha3 subunits and to the intact alpha2beta1 and alpha3beta1 integrins. Specimens were examined with laser-scanning microscopy. RESULT(S): The alpha2 and alpha3 subunits and the intact alpha2beta1 and alpha3beta1 integrins were identified at the base of the mesothelial cells (i.e., toward the basement membrane). There was also expression of the alpha2 and alpha3 subunits and the intact alpha2beta1 and alpha3beta1 integrins at the cell surface (i.e., toward the peritoneal cavity). CONCLUSION(S): The resolution of the confocal and two-photon laser-scanning microscope enabled localization of integrins in mesothelial cells. The presence of alpha2beta1 (collagen-laminin receptor) and alpha3beta1 integrins (collagen-laminin-fibronectin receptor) at the base of mesothelial cells suggests a role for these molecules in adhesion to the basement membrane. The presence of these molecules at the cell surface suggests a potential locus for cell adhesion in such processes as endometriosis and cancer metastasis.

Whole explants of peritoneum and endometrium: a novel model of the early endometriosis lesion.

OBJECTIVE: To determine whether whole fragments of endometrium can adhere to peritoneum with intact mesothelium. DESIGN: Tissue culture and immunohistochemical study. SETTING: University medical center. PATIENT(S): Reproductive-age women undergoing surgery for benign conditions. INTERVENTION(S): Explants of human peritoneum from the anterior abdominal wall and the posterior surface of the uterus were cultured with whole fragments of mechanically dispersed endometrium. MAIN OUTCOME MEASURE(S): Adhesion of endometrial fragments to the surface of the peritoneum was evaluated. Adherent endometrium was identified with the use of the dissecting microscope and by the performance of serial sections of the peritoneum explants. Immunohistochemical staining of the mesothelium with antibodies to cytokeratin was used to ensure an intact layer of mesothelium beneath the endometrial implants. Transmission electron microscopy also was used to evaluate this adhesion process. RESULT(S): Endometrium was identified attached to the surface of the peritoneum. Most of the implants did not have identifiable mesothelium beneath them, but most had intact mesothelium running up to the point of attachment. Approximately 10% of the endometrial implants had intact mesothelium at the site of attachment. Endometrial stromal cells, and not epithelium, attached to the mesothelium. CONCLUSION(S): Endometrium can attach to the mesothelial surface of the peritoneum. Endometrial stromal cells are involved in this attachment. Invasion through the mesothelium seems to occur rapidly.

Is there a risk of cytomegalovirus transmission during in vitro fertilization with donated oocytes?

OBJECTIVE: To define the risk of human cytomegalovirus (HCMV) transmission from donated oocytes. DESIGN: Prospective study. SETTING: University IVF program. PATIENT(S): Sixty-seven couples undergoing 72 cycles of IVF-ET. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Serum from both partners (women: n = 71; men: n = 60) was obtained for detection of antibodies to HCMV. Semen before preparation (n = 53), sperm after preparation (Percoll gradient; n = 47), cervical mucus aspirated at the time of oocyte aspiration (n = 70), and uninseminated oocytes and embryos not suitable for cryopreservation (n = 568) were frozen in liquid nitrogen. Polymerase chain reaction was used for detection of HCMV (immediate early 1 gene) in all samples collected. RESULT(S): Serum antibodies to HCMV were found in 62% of the women and 37% of the men tested. Human cytomegalovirus DNA was detected in 25% of the ejaculates and in 19% of the cervical mucus samples. There was no amplification of HCMV DNA from oocytes or embryos. CONCLUSION(S): Because we were unable to amplify HCMV DNA from any of the oocytes or embryos, it seems unlikely that HCMV is transmissible through oocyte or embryo donation.

Endometrial biopsy during hormone replacement cycle in donor oocyte recipients before in vitro fertilization-embryo transfer.

OBJECTIVE: To examine the usefulness of a trial cycle of hormone replacement therapy (HRT) and endometrial biopsy before the actual ET cycle in recipients of donated oocytes. DESIGN: Retrospective review. SETTING: Clinical practice at the South Texas Fertility Center, San Antonio, Texas. PATIENT(S): Thirty-six concurrent patients who underwent a trial cycle of HRT with endometrial biopsy before the ET cycle with donated oocytes fertilized in vitro. INTERVENTION(S): Patients > or =40 years of age received 100 mg of i.m. progesterone in oil daily; patients <40 years of age received 50 mg daily. Endometrial biopsies were performed during the late luteal phase of the trial cycle. MAIN OUTCOME MEASURE(S): Histologic dating of the biopsy specimens was correlated with the chronologic date of the biopsy. RESULT(S): Five of 20 patients > or =40 years of age had out-of-phase biopsies. All 16 patients <40 years of age had in-phase biopsies. All out-of-phase biopsies subsequently were corrected with higher doses of progesterone. Pregnancy rates after fresh and frozen ETs were not significantly different between the two age groups. CONCLUSION(S): Patients > or =40 years of age are at risk of having out-of-phase endometrial biopsies while they are receiving standard HRT despite receiving higher doses of progesterone. Trial HRT cycles with endometrial biopsies are recommended.

Mesothelium expression of integrins in vivo and in vitro.

OBJECTIVE: To characterize the expression of alpha subunits of integrin adhesion molecules in peritoneal tissue in vivo and in vitro. METHOD: Peritoneum from the anterior abdominal wall (n = 22) and the serosa of the posterior uterus (n = 11) was obtained from women of reproductive age without endometriosis who were undergoing surgery for benign conditions. Immunohistochemical studies were performed on serial sections of peritoneum from the anterior abdominal wall, the uterine serosa, mesothelial monolayer cultures, and peritoneum explants from the abdominal wall using monoclonal antibodies to alpha subunits of integrin adhesion molecules. Electron microscopy was performed to localize these adhesion molecules in the mesothelium. RESULTS: The mesothelial expression of alpha integrin subunits was identical in the anterior peritoneum and uterine serosa. In vivo the mesothelium strongly expressed alpha 2 and alpha 3 and variably expressed alpha 6. In the monolayer cultures there was moderate/strong staining for alpha 2, alpha 3, and alpha 5; there was minimal expression of alpha v. In the explants there was moderate/strong expression of alpha 2, alpha 3, alpha 5, and alpha v; alpha 6 was variably expressed. The ultrastructure of the mesothelium was unique in the anterior peritoneum, uterine serosa, and the monolayer cultures. The integrin subunits were distributed throughout the cytoplasm, were expressed in the plasma membrane, and were present on the surface (i.e., towards the peritoneal cavity) of the mesothelium. CONCLUSION: Integrins are expressed by the mesothelium of the peritoneum. The mesothelium expression of integrins in vivo differs from that of the mesothelium integrin expression in monolayer culture and explant culture.

Pathogenesis.

There are many hypotheses concerning the pathogenesis of endometriosis, though no single theory can explain all cases. It is likely that several mechanisms are involved. Early studies concentrated on the histogenesis of the endometriotic lesion. Recent evidence has implicated components of the immune system in the pathogenesis of endometriosis. This review considers the evidence for different theories of the histogenesis of endometriosis and discusses possible immune factors that may be involved in the pathophysiology of the disease.

Abnormal uterine bleeding.

Concerns about abnormal menstrual bleeding are a common reason for women to consult a primary care physician. The first step in the evaluation is to determine the patient's ovulatory status. Women with heavy bleeding but normal ovulatory cycles should be evaluated for coagulopathies, structural lesions, and hypothyroidism. In the absence of a systemic or structural cause, menorrhagia can be treated with OCPs or NSAIDs. Intermenstrual bleeding in OCP users may be due to noncompliance or the use of low-dose pills. Encouraging patient compliance and adjustment of the estrogen dose can often solve the problem. If the patient is not on OCPs, intermenstrual bleeding is usually due to a structural or inflammatory lesion. The differential diagnosis for anovulatory bleeding is extensive. Pregnancy, systemic illnesses, and structural lesions should be ruled out by history, physical examination, and laboratory evaluation. Endometrial biopsy is indicated in patients over age 35 and younger patients with risk factors for endometrial cancer, such as chronic anovulation and obesity. Dysfunctional uterine bleeding is a nonspecific term for abnormal uterine bleeding in the absence of systemic or structural disease. It is usually associated with anovulation. Adolescents frequently have dysfunctional uterine bleeding owing to immaturity of the hypothalamic-pituitary-ovarian axis. Perimenopausal women have an increased incidence of irregular bleeding secondary to decreased estrogen production by the ovary. Obesity, polycystic ovary syndrome, stress, crash diets, and vigorous exercise can all disrupt normal ovulatory function. Treatment options for dysfunctional uterine bleeding include oral contraceptives, cyclic progesterone, or hormone replacement with estrogen and progesterone. Patients with structural lesions or those who do not resume normal withdrawal bleeding patterns on hormone therapy should be referred to a gynecologist for further evaluation and treatment.

Characterization of lymphocyte subpopulations and T cell activation in endometriosis.

PROBLEM: Numerous studies have characterized the lymphocyte subpopulations in normal eutopic endometrium and suggested a role for the cytokine secretory products of these lymphocytes in regulating endometrial cell proliferation and differentiation. Recent studies have shown that ectopic endometrium contains a greater concentration of scattered stromal lymphocytes than does eutopic endometrium. However, the lymphocyte subpopulations and their activation status have not been characterized in ectopic endometrium. METHODS: We performed immunohistochemical studies on serial sections of proliferative and secretory phase eutopic endometrium and ectopic endometrium obtained during the proliferative phase using monoclonal antibodies to CD4 (T helper-inducer cells), CD8 (T cytolytic-suppressor cells), CD22 (B-cells), CD56 (natural killer cells), and VLA-1 (T-cell activation marker). RESULTS: Ectopic endometrium contained significantly more scattered stromal CD4, CD8, and activated T cells than did proliferative and secretory eutopic endometrium. There were more activated T-cells in proliferative than in secretory eutopic endometrium. Ectopic endometrium contained significantly fewer NK cells than proliferative and secretory endometrium. CONCLUSIONS: These results demonstrate that (1) the increased lymphocyte population in ectopic endometrium is due to increased numbers of CD4 and CD8 cells, and (2) a greater number of activated T cells are present in ectopic endometrium as compared to eutopic endometrium. Increased concentration of stromal T cells and enhanced VLA-1 expression in ectopic endometrium suggest that cytokine products of the activated T-cells may be involved in regulating cellular processes of endometriosis tissue.

Serum progesterone concentrations on the day after human chorionic gonadotropin administration and progesterone/oocyte ratios predict in vitro fertilization/embryo transfer outcome.

PURPOSE: In gonadotropin-releasing hormone analogue-pretreated in vitro fertilization-embryo transfer cycles, pregnancy rates are inversely related to serum progesterone levels on the day of administration of human chorionic gonadotropin. The relationship of the progesterone concentration on other days in the periovulatory period to pregnancy rates in such cycles is little studied. We therefore retrospectively analyzed the relationship between progesterone concentrations on the day after human chorionic gonadotropin and pregnancy in 114 cycles, 28 and 23 of which produced clinical and ongoing/delivered pregnancies, respectively. To assess the effect of the extent of follicular luteinization on success, we also studied the relationship between the progesterone concentration per oocyte retrieved and pregnancy for the day of and day after human chorionic gonadotropin. RESULTS: Progesterone concentrations on the day after human chorionic gonadotropin were inversely associated with clinical pregnancy by multiple logistic regression analysis (P < 0.05). Progesterone/oocyte ratios were inversely associated with clinical pregnancy (P < 0.05) and ongoing/delivered pregnancy (P < 0.02) for both the day of and the day after human chorionic gonadotropin. CONCLUSION: The study results extend the window of time during which elevated progesterone concentration is associated with poor outcome to at least 2 days. This finding is consistent with hypothetical mechanisms attributing the link between progesterone concentration and outcome to either endometrial or follicle/oocyte events. The association of lack of follicular luteinization (low progesterone per oocyte ratios) and favorable outcome suggests a predominant effect of progesterone on follicle/oocyte quality. Further studies are needed to clarify the mechanisms underlying the association between progesterone and in vitro fertilization-embryo transfer outcome.

Complications associated with the absorption of hysteroscopic fluid media.

OBJECTIVES: To review the literature concerning complications resulting from absorption of hysteroscopic fluid distension media and to describe methods to treat and prevent these complications. DESIGN: All pertinent literature on fluid distension media used for endoscopy, as well as relevant reports concerning the management of fluid and electrolyte imbalance, was reviewed. RESULTS: The absorption of large volumes of electrolyte-free, low-viscosity fluid may result in volume overload with water intoxication. Volume overload may cause pulmonary edema, and water intoxication may lead to hyponatremia, hypo-osmolarity, and cerebral edema. In contrast, the absorption of dextran-70 may cause volume overload secondary to the oncotic effect of intravascular dextran. Dextran-70 has been associated with anaphylaxis and coagulation disorders. TREATMENT: The use of diuretics is advocated. Urine output must be closely monitored. Judicious correction of electrolyte imbalance will prevent morbidity. PREVENTION: Meticulous attention to intraoperative fluid balance is imperative. A multichannel hysteroscope is necessary to keep intrauterine pressure low. Extensive surgical procedures may need to be performed in stages. CONCLUSIONS: Severe volume overload and electrolyte imbalance may result from fluid absorption during operative hysteroscopy. Most complications may be avoided by closely monitoring fluid balance intraoperatively.