Craig A. Witz

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.

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.

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.

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.

Pathogenesis of Endometriosis

Various theories have been promulgated to explain the pathogenesis of endometriosis. Interest in the genesis of the endometriotic lesion has been a focus since the earliest investigations. More recently, investigators have addressed aspects of the immune system and local peritoneal factors that may be involved with both the histogenesis of endometriosis as well as its sequelae. This review will consider evidence for different theories of histogenesis and will discuss our current understanding of the contribution of the immune system to the etiology of endometriosis. Data will be presented regarding recently described models of the early endometriotic lesion. The interaction of endometrial cells with the peritoneal mesothelium seems critical to our understanding the formation of the early endometriotic lesion. Evidence of rapid transmesothelial migration and invasion of the peritoneum will be considered. As well, candidate adhesion molecules that may facilitate the initial binding of endometrium to the peritoneum will be discussed.

Time Series Analysis of Transmesothelial Invasion by Endometrial Stromal and Epithelial Cells Using Three-Dimensional Confocal Microscopy

OBJECTIVE To evaluate endometrial adhesion and invasion of peritoneal mesothelium. DESIGN Descriptive study using confocal laser-scanning microscopy. SETTING University-based laboratory. PATIENT(S) Women undergoing surgery for benign conditions. INTERVENTION(S) Fluorescence-labeled peritoneal mesothelial cells (PMCs) were grown on coverslips. Fluorescence-labeled endometrial stromal cells (ESCs) and epithelial cells (EECs) and myometrial cells (Myos) were plated on the PMCs. Cultures were examined at 1, 6, 12, and 24-27 hours with differential interference contrast and confocal laser-scanning microscopy. MAIN OUTCOME MEASURE(S) Demonstration of adherence and invasion of endometrial cells through peritoneal mesothelium. RESULT(S) At 1 hour, there was adherence of the ESCs, EECs, and Myos on the perimeter of PMCs. There was no invasion by the Myos. By 6 hours, ESCs and EECs spread over the surface of the PMCs and extended cell processes through PMC junctions. Extension of pseudopodia under the PMCs followed. By 12 hours, there was vacuolization and lifting of PMCs that had been undermined by endometrial cells. CONCLUSION(S) This is the first time-phase study to demonstrate adherence and the process of invasion of endometrial cells through the mesothelium. The application of three-dimensional confocal laser-scanning microscopy is a novel technique that can be used to further examine mechanisms 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.

DNA Microarray Reveals That High Proportions of Human Blastocysts from Women of Advanced Maternal Age Are Aneuploid and Mosaic

ABSTRACT Trophectoderm (TE) biopsy and DNA microarray have become the new technologies for preimplantation genetic diagnosis in humans. In this study, we comprehensively examined aneuploid formation in human blastocysts produced in vitro with microarray and investigated the clinical outcome after transfer of euploid embryos. Biopsied cells from either TE or inner cell mass (ICM) were processed for microarray to examine the errors in 23 pairs of chromosomes and the consistency between TE and ICM. It was found that 56.6% of blastocysts were aneuploid. Further analysis indicated that 62.3% of aneuploid blastocysts had single and 37.7% had multiple chromosomal abnormalities. Chromosome errors could occur in any chromosome, but errors in chromosome 21 accounted for the most (11.3%) among the 23 pairs of chromosomes. Transfer of array-screened blastocysts produced high pregnancy (70.2%) and implantation (63.5%) rates. Microarray of TE and ICM cells in the same blastocysts revealed that high proportions of aneuploid blastocysts (69.2%) were mosaic, including aneuploid TE and euploid ICM, inconsistent anomalies between ICM and TE, or euploid TE cells and aneuploid ICM in the same blastocyst. These results indicate that high proportions of human blastocysts produced in vitro from women of advanced maternal age are aneuploid and mosaic. Errors can occur in any of the 23 pairs of chromosomes in human blastocysts. Biopsy from TE in blastocysts does not exactly predict the chromosomal information in ICM if the embryos are aneuploid. Some mosaic blastocysts have euploid ICM, which may indicate important differentiate mechanism(s) of human preimplantation embryos.

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.

Mesothelium expression of integrins in vivo and in vitro.

To characterize the expression of α subunits of integrin adhesion molecules in peritoneal tissue in vivo and in vitro. 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 of peritoneum from the anterior abdominal wall, the uterine serosa, mesothelial monolayer cultures, and peritoneum explants from the abdominal wall using monoclonal antibodies to α subunits of integrin adhesion molecules. Electron microscopy was performed to localize these adhesion molecules in the mesothelium. The mesothelial expression of α integrin subunits was identical in the anterior peritoneum and uterine serosa. In vivo the mesothelium strongly expressed α2 and α3 and variably expressed α6. In the monolayer cultures there was moderate/strong staining for α2, α3, and α5; and αv; α6 was variably expressed. The ultrastructure of the mesothelium was unique in the anterior peritoneum, uterine serosa, and the nonolayer cultures. The integrin subunits were distributed throughout the cytoplasm, were expressed in the plasma membrane, and were present on the surface (ie, towards the peritoneal cavity) of the mesothelium. 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.