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Byline: Douglas V. Horbelt, Daniel K. Roberts, Tim H. Parmley, Nola J. Walker Keywords: Human endometrial microvasculature; hyperplasia; ultrastructure; cell-to-cell interactions; extracellular matrix Abstract: OBJECTIVE: Our purpose was to determine the effect of hyperplasia on cell-to-cell communication in the microvasculature of the human endometrium. STUDY DESIGN: Ten biopsy specimens of simple and complex hyperplasia of the human endometrium were investigated by electron microscopy. RESULTS: The microvasculature of the hyperplastic endometrium shows morphologic disorder. The extracellular matrix is altered, and cell death is widespread. Intercellular contact is infrequent or absent. CONCLUSIONS: These results are in marked contrast to the ultrastructure of the normal microvasculature. The complex cell-to-cell interaction observed in normal tissue appears lost. This report, combined with others, indicates that cell-to-cell contact is sparse in hyperplastic endometrium. (AM J OBSTET GYNECOL 1996;174:174-83.) Author Affiliation: Wichita, Kansas, and Little Rock, Arkansas Article History: Received 13 October 1994; Revised 21 February 1995; Accepted 24 April 1995 Article Note: (footnote) [star] From the Department of Obstetrics and Gynecology, University of Kansas School of Medicine - Wichita, Wesley Medical Center,a and the Departments of Obstetrics and Gynecology and Pathology, University of Arkansas for Medical Sciences. b , [star][star] Reprint requests: Nola J. Walker, EMSA, Electron Microscopy Laboratory, Wesley Medical Center, 550 N. Hillside, Wichita, KS 67214-4976., a 0002-9378/96 $5.00 + 0 6/1/65861

Transmission electron microscopy indicates that cell connections may be altered in hyperplastic human endometrium, an abnormal but noncancerous growth of the uterine lining. Ten such endometrial samples showed neither the normal connections between cells nor the normal connections to the underlying cell layers. These types of connections are known to be important in regulating and coordinating normal tissue growth. Such growth is crucial in the cyclic endometrial changes of the menstrual cycle. The basal lamina, a layer of noncellular material just under the top layer of skin, also seems to be changed by endometrial hyperplasia. It was thicker in some samples and broken in others.

In the early proliferative phase endometrial capillary basal lamina is loosely formed and discontinuous. It becomes more distinct in the late proliferative phase and develops whorled extensions that include pericytes in the early secretory to midsecretory phase. Throughout the cycle both endothelial cells and pericytes possess cytoplasmic processes that extend through discontinuities in the basal lamina and make contact with each other. These contacts may be simple or complex. They become more elaborate as the cycle progresses to the midsecretory phase. Endothelial cells show progressive but heterogeneous differentiation into the midsecretory phase, with individual cell hypertrophy. In the late-secretory phase the endometrial stroma and the basal lamina are characterized by widespread degeneration, and the cell-to-cell contacts sharply decrease. However, endothelial cells remain relatively intact. (Am J Obstet Gynecol 1992;166:1393-406.)

Objective:We investigated the relationship between cyclin E mRNA overexpression and p53 protein accumulation in epithelial ovarian cancers.Methods:mRNA was isolated and cDNA was prepared from 36 epithelial ovarian tumors (three adenomas, three low malignant potential tumors, and 30 carcinomas), and six normal ovaries. The cyclin E mRNA expression levels relative to an internal control, β-tubulin, were determined by semiquantitative polymerase chain reaction (PCR). Cyclin E and p53 protein expression in ovarian cancer tissues were examined by immunohistochemistry using the same series of samples. Fisher exact test of significance and an unpaired t test were used for statistical analysis.Results:Considerable levels of cyclin E mRNA were detected in all normal ovaries and ovarian tumor samples examined by semiquantitative PCR amplification. mRNA levels of cyclin E were significantly higher in nine of 30 (30%) ovarian cancers compared with those in normal ovaries. The immunohistochemical expression of cyclin E protein was confirmed in the nuclei of tumor cells in 13 of 30 (43%) ovarian cancers. p53 protein accumulation was detected in 12 of 30 (40%) ovarian cancers examined. There was a significant inverse correlation between cyclin E mRNA overexpression and p53 protein accumulation(P <.01, Fisher exact test).Conclusions:Cyclin E mRNA overexpression frequently occurs in ovarian cancers without p53 protein accumulation. Cyclin E might have an important effect on the development of a limited number of ovarian cancers.

Proteases have been implicated in the extracellular modulation required for tumor growth and invasion. In an effort to categorize those proteases contributing to ovarian carcinoma progression, we have utilized redundant primers to conserved amino acid (AA) domains surrounding the catalytic triad of His, Asp and Ser to amplify serine proteases that are differentially expressed in carcinomas. Using this method, we have identified and cloned a serine protease named TADG-15 (tumor-associated differentially expressed gene 15) that is overexpressed in ovarian tumors. TADG-15 is a transmembrane multidomain serine protease which includes ligand binding domains and a serine protease in the extracellular space.

Proteases are known to play important roles in tumor invasion and metastasis. Protease M, which was originally identified by Anisowicz and colleagues in 1996, is a new member of the serine protease family. We also identified the protease M transcript in a differential PCR screen of ovarian tumors and have investigated its expression in 44 ovarian tumors (12 low malignant potential tumors, 32 carcinomas) and 10 normal ovaries using quantitative PCR. The PCR product was labeled with 32P and a phosphoimager was used to determine the relative expression of the protease M gene compared to internal control β-tubulin. mRNA expression levels of protease M were significantly elevated in 9 of 12 low malignant potential tumors and 30 of 32 carcinomas. Northern blot hybridization showed that the 1.7-kb protease M transcript was abundant in carcinoma but not detected in normal ovary. Immunohistochemical staining of normal overy and ovarian tumor tissue sections with antibodies generated to protease M derived peptides corroborated the semi-quantitative PCR and Northern analysis data. Our results suggest that protease M is frequently overexpressed in ovarian tumors and may therefore contribute to the invasive nature or growth capacity of ovarian carcinomas.

Objective:In a continued effort to identify and characterized secreted proteases that are overexpressed in ovarian carcinomas, we discovered the testisin protease as such a candidate. When this discovery was originally made, no data existed in the literature or in the GenBank database that identified such a gene. Our main objective was to determine whether this gene was overexpressed exclusively in ovarian tumor tissues compared with normal ovary and whether it was expressed in any other normal tissues.Methods:mRNA was isolated and cDNA was prepared from 34 ovarian tumors (four adenomas, three low malignant potential tumors, and 37 carcinomas) and seven normal ovaries. The testisin mRNA expression level relative to internal control, β-tubulin, was determined by Northern blot analysis and semiquantitative polymerase chain reaction (PCR).Results:Northern blot hybridization showed that the testisin transcript was abundant in ovarian carcinoma but was not detected in normal ovary. On examination of Northern blots from normal fetal and adult tissues, only adult testis showed abundant transcripts of testisin. Semiquantitative PCR examination showed that the testisin mRNA levels in ovarian tumors of low malignant potential and in ovarian carcinomas were significantly higher than in normal ovaries (P < .01). Testisin mRNA level in ovarian carcinomas was also significantly higher than in ovarian adenomas (P < .05). Testisin overexpression rates in advanced stage (stage 2 or 3) diseases were significantly higher than that in early stage disease (stage 1) in ovarian carcinoma samples (P < .05).Conclusions:The induction of the testisin transcript might contribute to the development, progression, and invasive or metastatic capacity of ovarian carcinomas.

Objective: TO examine the cyclin D1 mRNA expression level in ovarian tumor samples as compared with normal ovaries and to determine the relationship between cyclin D1 overexpression and p53 mutation status in ovarian tumors. Methods: mRNA was isolated and cDNA was prepared from 27 epithelial ovarian tumors (3 tumors of low malignant potential (LMP) and 24 cancers) and 6 normal ovaries. The cyclin D1 sequences were amplified by using a thermal cycler in parallel with the ß-tubulin gene as an internal control. The cyclin D1 mRNA expression level relative to ß-tubulin was determined by 32P phosphoimager analysis. To confirm the overexpression of the cyclin D1 protein in ovarian tumor cells, immunostaining was performed. The p53 gene mutation status was examined by direct cDNA sequencing. Results: mRNA levels of cyclin D1 were significantly higher in 21 (78%) of the 27 ovarian tumors than in normal ovaries. Cyclin D1 overexpression was detected in ovarian LMP tumors as well as in ovarian cancer cases. Positive immuno staining of cyclin D1 protein was observed in 10 of 18 (56%) ovarian tumors examined. p53 mutations were found in 11 (61%) of 18 ovarian tumors. Of 11 ovarian tumor cases with p53 mutations, 5 showed overexpression of cyclin D1. All 7 ovarian tumor cases without p53 mutations showed significant cyclin D1 mRNA overexpression. Conclusion: Cyclin D1 overexpression seems to be an early genetic event in ovarian tumor development. Although p53 may be one of the proteins whose function regulates the expression of G1 cyclins, ovarian tumors with no p53 mutation consistently showed cyclin D1 overexpression. Cyclin D1 overexpression may play an important role in the tumorigenesis of epithelial ovarian tumors.

Objectives.Our purpose was to study the ultrastructural morphology of the microvasculature of human endometrial adenocarcinoma and to determine the effect of this malignancy on cell-to-cell communication between the components of the microvasculature and with the other tissue compartments of human endometrium.

Matrix metalloproteases are known to play an important role in tumor invasion by mediating degradation of extracellular matrix. In this study, we have investigated the expression of the matrix metalloprotease pump-1 gene (also referred to as MMP-7, Matrilysin) in 44 ovarian tumors (12 low malignant potential tumors, 32 carcinomas) and 10 normal ovaries using quantitative PCR. The PCR product was labelled with 32P and a phosphoimager was used to determine the relative expression of pump-1 compared to an internal control β-tubulin. mRNA expression levels of pump-1 were significantly elevated in 9 of 12 low malignant potential tumors and 26 of 32 carcinomas. Northern blot hybridization showed that the 1.1-kb pump-1 transcript was abundant in carcinoma but seldom expressed in normal adult tissues including normal ovary. Immunohistochemical localization of the pump-1 protein confirms its expression by ovarian tumor cells. Our results suggest that pump-1 is frequently overexpressed in ovarian tumors and may contribute to its invasive nature or growth capacity, therefore pump-1 may serve as a useful marker for early detection of disease and/or a target for therapeutic intervention in downregulation of tumor progression.

The use of cautery laparoscopic sterilization procedures has two major disadvantages: (1) occasional inadvertent thermal injury to organ structures other than the Fallopian tube1, 2and (2) the inability to limit the injury to the Fallopian tube in a manner that might allow subsequent reconstructive surgery. In an effort to avoid the use of thermal energy to effect female sterilization, the Falope ring laparoscope was developed.3From October, 1973, through September, 1975, 4,390 sterilization procedures were completed employing this approach; 902 cases have been completed at the Woman's Clinic of the Johns Hopkins Hospital, 1,741 cases were completed by gynecologists in various cities in the United States, and 1,747 procedures were completed by physicians in various locations throughout the world sponsored by the International Fertility Research Program.

The effect of intrauterine injection of 1% silver nitrate on pregnancy was studied in 13 cynomolgus monkeys. The monkeys received, in a random manner, intrauterine injections of 1ml of either 1 % silver nitrate (n=7) or 0.9% NaCl solution (n=6) between days 27 and 43 of pregnancy. Silver nitrate injection resulted in vaginal bleeding, beginning 1 or 2days after treatment, that lasted for an average of 5.3days. In all of these cases, pregnancy was terminated. Injection of normal saline had variable sequelae, but four of six monkeys were delivered of healthy offspring. All of the silver nitrate-treated animals recycled and, after remating, two of the seven subsequently became pregnant again and were delivered of normal healthy infants. This study demonstrates the efficacy of intrauterine injection of 1% silver nitrate in terminating early pregnancy.

The present study details gross and histologic findings of 79 previously ligated fallopian tubes from 3 groups of patients. Of 20 oviducts removed after documented sterilization failure (group I), 6 revealed a process compatible with endometriosis. Four of nine previously ligated fallopian tubes removed at the Johns Hopkins Hospital (group II) were successfully injected with India ink. In two patients histologic examination demonstrated the India ink in epithelium-lined spaces that lay beyond the muscle of the tubal wall extending from the tubal lumen to the serosal surface.

Histopathologic features, blood chemistry, and hematologic features were studied in cynomolgus monkeys following intravascular or intrauterine administration of a 30-mg solution of quinacrine hydrochloride. Intrauterine quinacrine administration resulted in extensive necrosis of the endometrial surface, and lesions were observed at 24 hours after treatment which obliterated the cornual areas of the uterus. Necrosis was also observed on occasion in the ampulla or isthmic portion of the tube. Evidence of repair of the reproductive tract was seen 7 and 28 days following treatment. No lesions were observed in any nonreproductive organ examined, whether quinacrine was administered by the intrauterine or intravascular route. Blood chemistry data revealed moderate and transient increases in serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), and lactic dehydrogenase (LDH). No other blood chemistry or hematologic changes were noted that could be attributed to quinacrine administration. For the conditions described in these studies, intrauterine administration of quinacrine appears to be a safe procedure. However, the potential toxicity of the drug is discussed.

Recent efforts have been made to develop a chemical oviductal occluding agent. Intrauterine quinacrine has been used in certain areas of the world with moderate success in effected tubal closure. This report presents the pharmacokinetics of a quinacrine solution (30 mg) as administered to cynomolgus monkeys via the intrauterine route, compared with intravascular injection. The data show rapid transfer of the drug from the uterine to the vascular compartment and uptake by almost all tissues examined. Although plasma concentrations disappear within 24 hours, levels can be detected in most tissues for at least 1 week following intrauterine injection. After 28 days, however, tissue levels of the drug are absent or near the limit of detection.

Twenty-five of 48 women (52%) sterilized by unipolar cautery techniques conceived following tubal anastomosis, of whom 17 (36%) had a living child. The overall cumulative probability of conception at the end of follow-up as determined by life-table analysis was 76%. Increasing age, parity, and the duration of the interval from sterilization to reversal did not influence pregnancy success. A decreased pregnancy rate was associated with ampullary-isthmic anastomosis; however, a pregnancy was least likely to occur in women with shortened oviducts of ≤ 4 cm (P < 0.01).

Neoplasms arising from the surface or lining cells of the ovary are classically referred to under such general terms as cystadenocarcinoma, papillary carcinoma, and undifferentiated cancer. Evidence is presented in this discussion to support the thesis that such lesions are mesotheliomas, and that such a thesis can be defended embryologically, histologically, and clinically.

This paper describes recent success in performing hysterosalpingography in cynomolgus monkeys. The animals were anesthesized, and a needle was manipulated through the cervical canal into the endometrial cavity for injection of contrast material. Difficulties in threading the needle through the canal were due to several blind pouches formed by folds in the cervical mucosa. The uterine lumen and both tubes could be visualized in over 75% of the cases. The study demonstrates that the hysterosalpingography technique can be performed satisfactorily in cynomolgus monkeys.

Endometrial biopsies were obtained from 46 hypogonadal patients (44 with gonadal dysgenesis; 2 panhypopituitary) who were receiving estrogen-progestogen therapy. Endometrial abnormalities occurred only in patients receiving a total lifetime conjugated estrogen dose of ≥2500 mg and who had received estrogen treatment for a period longer than 4.2 years. The biopsy outcome was significantly related (P<0.01) to the estrogen dose at time of biopsy and to the total lifetime dose (P<0.05). The progestational drugs administered did not protect against development of endometrial abnormalities. None of the abnormal endometrial patterns were associated with abnormal vaginal bleeding.

In hyperplastic endometrial stroma, cell-to-cell contacts are infrequent. Those that are present are not well developed and may appear degenerative. Significant amounts of extracellular matrix further impede contact. The cells of hyperplastic endometrial stromal are, for the most part, poorly differentiated and both apoptosis and necrosis are common. Fibroblast-like cells make up most of the increased cellularity, with few endometrial granulocytes or other cells types. Copyright 1994, 1999 Academic Press

This report documents the clinical outcome of 137 consecutive menstrual extractions. The pre- and postprocedural pregnancy testing is correlated with histologic examination of tissue obtained. This report reviews the management of the unsuccessful cases.

Extract: The specific activities of three disease-related lysosomal enzymes were compared in cultured skin fibroblasts from seven normal women (M), their fetuses (F), and cells cultured from corresponding amniotic fluid cells (A). All samples were obtained in weeks 17–21 of pregnancy. Highly significant differences in the mean specific activities of ß-galactosidase (ß-Gal), ß-D-N-acetylglucosaminidase (Hex), and arylsulfatase A (ARA) are evident in control cells developed from each of these sources. In addition, the pattern of differences in the three cell types was dissimilar for each enzyme. The mean activities in nanomoles of product produced per hour per milligram of soluble cell protein were: ß-Gal: F = 248, A = 676, M = 375; Hex: F = 3412, A = 6548, M = 6781; and ARA: F = 143, A = 244, M = 458. The differences in enzyme specific activities between the three cell types studied indicated significant differences in the enzymology of these cells under defined culture conditions. In light of these findings the prerequisites necessary for antenatal diagnosis of inherited metabolic disorders are recommended.Speculation: If quantitative enzymologic data from cultured amniotic fluid cells are to be used for the antenatal diagnosis of specific inherited genetic disorders, it is essential that relative specific activities of the enzymes in question be established in cultured cells from each of the pertinent cell types. This is essential if heterozygous fetuses are to be correctly identified in utero and if fetal tissues are to be used to corroborate intrauterine diagnoses based on amniotic cell analysis.

The specific activities of three disease-related lysosomal enzymes were compared in cultured skin fibroblasts from seven normal women (M), their fetuses (F), and cells cultured from corresponding amniotic fluid cells (A). All samples were obtained in weeks 17–21 of pregnancy. Highly significant differences in the mean specific activities of β-galactosidase (β-Gal), β-D-N-acetylglucosaminidase (Hex), and arylsulfatase A (ARA) are evident in control cells developed from each of these sources. In addition, the pattern of differences in the three cell types was dissimilar for each enzyme. The mean activities in nanomoles of product produced per hour per milligram of soluble cell protein were: β-Gal: F = 248, A = 676, M = 375; Hex: F = 3412, A = 6548, M = 6781; and ARA: F = 143, A = 244, M = 458. The differences in enzyme specific activities between the three cell types studied indicated significant differences in the enzymology of these cells under defined culture conditions. In light of these findings the prerequisites necessary for antenatal diagnosis of inherited metabolic disorders are recommended.

Amniotic fluid was obtained at cesarean section from 5 third-trimester pregnancies and the proportion of orange-staining cells in the amniotic fluid determined using Nile blue sulfate staining. In the freshly obtained fluids 25 to 75 per cent of the cells stained orange. The cells were sedimented and processed for examination in the electron microscope. Virtually all the cells appeared to be squamous epithelial cells of fetal epidermis or mucous membrane. They contained variable numbers of keratin filaments and P.A.S.-positive particles resembling glycogen. No fat-containing cells were found. The presence of the squamous cells appeared to correlate with the thickening of the stratum corneum near term. It is concluded that the orange-staining cell is a cornified fetal squamous cell coated on its exterior surface by lipid material that appears to be intracellular in the light microscope and is removed during processing for electron microscopy.

Thanatophoric dwarfism is the result of a generalized failure of endochondral bone formation. It may be diagnosed in utero and is almost uniformly fatal. Hydramnios, prematurity, breech presentation, and cephalopelvic disproportion are clinical features frequently associated with these infants. The early induction of labor in these cases is suggested.

Permeability of fetal skin to isotopic water (THO) was measured between 12 and 24 week's gestational age. Unkeratinized skin obtained from human fetuses between 12 and 18 cm. crown-rump length gave large permeability values similar to amnion and chorion laeve. Partially and well keratinized skin from 18 to 22.5 cm. crown-rump length fetuses showed little and no permeability, respectively, to THO diffusion. These data suggest that fetal skin may function as a site of significant water and solute exchange between fetus and amniotic fluid during early pregnancy.

The amino acid composition of amniotic fluid samples obtained from 51 patients at various stages of fetal gestation was determined by column chromatography. There is a decreasing concentration of most of the amino acids with increasing gestational age. The maximum rate of this decrease occurs between the twenty-second and the twenty-fourth week of pregnancy. As it is well established that the offspring of women with phenylketonuria (PKU) will have in most cases intrauterine and postnatal growth retardation, mental retardation, and/or structural abnormalities, amniotic fluid samples from 2 pregnancies of a woman affected with PKU were also analyzed. In both pregnancies (eighth and fortieth weeks of gestation) the amniotic fluid phenylalanine concentration was elevated to about 35 times that of normal.

Hypertonic urea has been shown to be an effective midtrimester abortifacient. Although safer than hypertonic saline when injected intravascularly, it has not been compared to saline in the case of inadvertent intramyometrial injection. This report document that intramyometrial injection of hypertonic urea will result in the same type of muscle necrosis as that produced by saline.

Leiomyomatosis peritonealis disseminata (LPD) is a rare clinical entity which is currently the object of serious study. Electron microscopic studies to further elucidate the ultrastructural features of the pathology are described here.

Gestational trophoblastic disease, most commonly hydatidiform mole, is an unusual condition within the United States. The incidence of hydatidiform mole has been reported to vary from 1 in 1,200 to 1 in 2,000 pregnancies. This report described eight cases of hydatidiform mole among 4,829 patients presenting for elective first-trimester abortion. Factors which might account for the frequency of hydatidiform mole of 1 in 600 are discussed. Although the clinical course of patients with hydatidiform mole appears benign, gross examination of tissue obtained at suction curettage and the liberal use of histologic evaluation in questionable cases is required to make the diagnosis.

The possibility of preventing erythroblastosis fetalis has made it desirable to identify those patients at risk with regard to sensitization. We have therefore looked for tranplacental hemorrhage in patients undergoing therapeutic abortions. Our results suggest that all of the more common methods of artificially terminating a pregnancy may result in transplacental hemorrhage.

We have compared the specific activities of several lysozomal enzymes in skin fibroblasts cultures from 7 normal women (MAT), their fetuses (FET), and fibroblasts developed form corresponding amniotic fluid cells (AMN). All samples were obtained between the 17th–21st week of pregnancy. Highly significant differences in the mean specific activities of ß-galactosidase, ß-D-N-acetylglucosaminidase, and arylsulfatase. A are evident between fibroblasts developed from each of these sources. In addition, the pattern of differences in the 3 fibroblast types is dissimilar for each enzyme:ß-galactosidase: AMN > MAT > FETß-D-N-acetylglucosaminidase: AMN = MAT > FETArylsufatase A: MAT > AMN > FETBoth the pattern and relative differences in enzyme specific activites are consistent in each of the 7 groups of cultures studied. If quantitative enzymologic data from cultured amniotic fluid cells is to be used for the antenatal diagnosis of specific inherited genetic disorders, it is essential that relative specific activities of the enzymes in question be established in fibroblasts developed from each of the pertinent cell types. These relationships are also critical if hererozygous fetuses are to be correctly identified in utero and if fetal tissue is to be used to corroborate intrauterine diagnoses based on amniotic cell analysis.