Histologic classification and pathologic findings for endometrial biopsy specimens obtained from 2964 perimenopausal and postmenopausal women undergoing screening for continuous hormones as replacement therapy (CHART 2 Study).

OBJECTIVE: The aim of this study was to screen for disorders and to histologically classify endometrial biopsy specimens from 2964 perimenopausal and postmenopausal women who were candidates for hormonal replacement therapy. STUDY DESIGN: Endometrial biopsy specimens were obtained with a Vabra aspiration curette, processed by standard methods, and stained by hematoxylin and eosin and special methods to reveal subtle features of the endometrium. RESULTS: Of the endometrial biopsy specimens, 68.7% were atrophic, 23.5% were proliferative, 0.5% were secretory, 0.6% were hyperplastic, 0.07% were adenocarcinoma, and 6.6% were insufficient for classification. Three independent senior microscopists agreed on the classification of each biopsy specimen. CONCLUSION: The number of patients is the largest ever screened for a single hormone replacement therapy study. The low yield of endometrial cancer indicates that biopsies are unnecessary before hormone replacement therapy is initiated in asymptomatic women.

The comparative effect on bone density, endometrium, and lipids of continuous hormones as replacement therapy (CHART study). A randomized controlled trial.

OBJECTIVE: To compare the effect of continuous norethindrone acetate (NA)-ethinyl estradiol (EE2) combinations with matching unopposed EE2 or placebo. DESIGN: A 2-year, double-blind, placebo-controlled, parallel-group clinical trial. SETTING: Outpatients at 65 centers. PATIENTS: Asymptomatic or mildly symptomatic women aged 40 years or older who had undergone the onset of spontaneous menopause within the last 5 years and who had an intact uterus. INTERVENTIONS: Patients were equally randomized to placebo or 1 of 8 treatment groups: 0.2 mg of NA and 1 microg of EE2; 0.5 mg of NA and 2.5 microg of EE2; 1 mg of NA and 5 microg of EE2; 1 mg of NA and 10 microg of EE2; 1 microg of EE2; 2.5 microg of EE2; 5 microg of EE2; or 10 microg of EE2. PRIMARY OUTCOME MEASURES: Bone mineral density (BMD) measured by quantitative computed tomography, serum lipids, and endometrial effects as assessed by rate of hyperplasia and proliferative status. RESULTS: Twelve hundred sixty-five patients entered the study. Bone mineral density increased significantly from baseline (P<.001) in the 1 mg NA-5 microg EE2 and the 1 mg NA-10 microg EE2 treatment groups at each annual assessment. Among the unopposed EE2 groups, only the 10-microg group had increased BMD above baseline, but also was accompanied by an unacceptably high rate of endometrial hyperplasia. The NA-EE2 treatment groups had a significant linear dose-response trend for increasing BMD. Increased endometrial proliferation and hyperplasia occurred with increasing unopposed estrogen doses. The combination of NA and EE2 effectively protected the endometrium against hyperplasia. The percentage of change in the ratio of high-density lipoprotein cholesterol to low-density lipoprotein cholesterol was positive for all treatment groups. The increase in triglyceride levels associated with EE2 was attenuated with NA-EE2 treatment. CONCLUSIONS: Daily treatment with NA-EE2 was well tolerated and protected the endometrium from EE2-induced proliferation and hyperplasia. The NA-EE2 treatments produced a dose-related significant increase in BMD that was not present with unopposed EE2 treatment. The overall effect of NA-EE2 treatments on lipid measures was favorable.

Antiproliferative effects of low-dose micronized progesterone.

OBJECTIVE: To study the pharmacodynamic effects of oral micronized P on endometrial maturation. DESIGN: This was a controlled, open, parallel group, pilot study. SETTING: The experiment was performed in an outpatient academic clinical research unit. PATIENTS: Twelve healthy, P-challenged, estrogen-primed, postmenopausal women participated in the study. INTERVENTIONS: Patients were given 300 mg micronized P daily (8:00 A.M.) or twice (8:00 A.M. and 4:00 P.M.) daily from study days 1 through 14 after estrogen priming for 30 days. Blood samples were taken at 0, 0.5, 1, 1.5, 2, 3, 4, 6, and 8 hours after the 8:00 A.M. dose on study day 1 and 14 and again at 8:00 and 9:30 A.M. on days 3 and 5 fasting, days 7 and 9 after a fatty meal, and day 11 after a high fiber meal. Endometrial biopsies were taken on day 1 and 14. MAIN OUTCOME MEASURES: Progesterone concentrations were measured. Endometrial biopsies were studied for effects on histology, glycogen content of glands, ribosomal RNA, and nuclear estrogen receptors in glands, surface epithelium, and stroma. RESULTS: Day 1 and 14 P kinetics were similar for 8 hours. Dose-dependent increases in glandular glycogen, decrease in ribosomal RNA, and decrease in nuclear estrogen receptors were demonstrated. CONCLUSIONS: Oral micronized P can induce antiproliferative changes in the human endometrium at doses lower than those required for transformation of the endometrium to a full secretory state.

Response of leiomyosarcoma to cryosurgery: clinicopathological and ultrastructural study.

Two elderly patients with primary leiomyosarcoma (LMS) of the scalp were treated cryosurgically. Complete involution of both tumours with full epithelialization of the affected sites was achieved. Pretreatment biopsies and sequential biopsies obtained after treatment allowed observation of microscopical changes taking place during tumour involution. Gradual shrinkage of both LMS, closely monitored under the operating microscope, started immediately after the initial freezing. Light and electron microscopic observation of the shrinking LMS revealed a rapid disappearance of the tumoral architecture. Early accumulation of eosinophils and erythrocytes was followed by migration of lymphocytes and plasma cells. Capillary neoformation, fibroblasts and newly formed connective tissue fibres became apparent during the later stages of healing. Two years after treatment, both patients showed no signs of recurrence. These results suggest cryosurgery--performed in an extended protracted fashion--can be a valuable therapeutic choice in the management of LMS, particularly when surgical excision is not feasible.

Pretreatment with the adenosine A1 selective agonist, 2-chloro-N6-cyclopentyladenosine (CCPA), causes a sustained limitation of infarct size in rabbits.

OBJECTIVE: The highly selective adenosine A1 receptor agonist, 2-chloro-N6-cyclopentyl-adenosine (CCPA), has been shown to be as cardioprotective as ischaemic preconditioning when evaluated with an early staining method using tetrazolium. However, tetrazolium-positive tissue measured 3 h after reperfusion may still overestimate the long term salvage. To test for this possible artefact, a 72 h reperfusion rabbit model of myocardial infarction was used, and infarct size was assessed by histology. METHODS: Myocardial infarction was induced by a 30 min coronary occlusion. Rabbits were assigned to a control group receiving no treatment, pretreatment with 0.125 mg.kg-1 CCPA, or 0.25 mg.kg-1 pretreatment with CCPA (0.25 mg.kg-1) followed by an A1 selective antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) 30 min after reperfusion to reverse the haemodynamic side effects. RESULTS: In the 0.125 mg.kg-1 CCPA group, 30.8(SEM 4.2)% of the ischaemic zone was infarcted, which was significantly less than that seen in the control group [46.5(3.0)%; p < 0.01]. Reversing the side effects of CCPA by giving DPCPX soon after reperfusion did not block the protective effects [26.2(1.9)% infarction; p < 0.01 v control]. CONCLUSIONS: This finding confirms a genuine anti-infarct effect of adenosine A1 receptor stimulation when given prior to the onset of ischaemia. Furthermore blocking the A1 receptors soon after reperfusion reverses the side effects but does not block protection.

Tissue response to bioerodible, subcutaneous drug implants: a possible determinant of drug absorption kinetics.

The fibrous tissue compartments that develop in response to the subcutaneous implantation of bioerodible heat-fused rods of norethindrone and cholesterol (85 and 15%, respectively) were studied by light and electron microscopy at various intervals after implantation to determine whether the biological inflammatory response may play a role in drug absorption. Thirty-five regularly menstruating, sterilized (tubal ligation), healthy females each received four Annuelle rods. The microanatomy of seven of the largest implants (135 mg norethindrone) was studied. A dense fibrous biological compartment was found to surround each rod. By light microscopy no abnormal tissue response was revealed. Scanning and transmission electron microscopy showed that the surfaces of the rods were covered by a cellular matrix of mononuclear cells. The fibrous compartment was composed of a loose cellular bed immediately surrounding the norethindrone rod, a dense fibrous connective tissue envelope containing blood and lymphatic vessels, and an outer fatty connective tissue layer. Transmission electron microscopy confirmed that the cellular tissue immediately surrounding the rods was composed mainly of lipid laden macrophages. Norethindrone levels in tissue capsules at 3 and 10.5 months were 0.05 and 8.4% by weight, respectively. These observations suggest that the local inflammatory response plays a role in the active processing of this delivery system. This picture is qualitatively different from the general view of the fibrous capsule as a simple rate limiting membrane. The effects observed in this study suggest that a more complex, functional biological system develops in response to the subcutaneous introduction of a drug delivery device.

Splenic sequestration associated with sickle cell trait and hereditary spherocytosis.

Coexistence of sickle cell trait and hereditary spherocytosis (HS) is unusual, and only 16 cases have been reported in the literature. These patients have the same clinical and hematological features as individuals having HS alone. We report a serious complication, acute splenic sequestration crisis (ASSC), occurring in two patients with sickle cell trait and HS. One patient experienced four episodes of ASSC during an 11-year span, while the other had two episodes of this complication during a 4-year period. Red blood cell studies and membrane protein analysis confirmed the diagnosis of HS as a consequence of spectrin deficiency. Splenectomy resulted in marked clinical and hematological improvement in both patients. Histological examination of spleens following splenectomy confirmed that significant erythrostasis and sickling had indeed occurred. ASSC can occur in patients with coexistence of sickle cell trait and HS, and this potentially life-threatening complication should be considered in this condition.

Reversal of increased microvascular permeability associated with ischemia-reperfusion: role of cAMP.

Ischemia-reperfusion (IR) is a form of oxidant injury known to increase microvascular permeability in the lung. Agents that increase adenosine 3',5'-cyclic monophosphate (cAMP) levels have been shown to have beneficial effects in several models of oxidant lung injury associated with increased microvascular permeability. We investigated the role of adenylate cyclase activation with isoproterenol (ISO) or forskolin (FSK) in reversing the increased microvascular permeability associated with IR. ISO or FSK administered after 45 min of ischemia and 46 min of reperfusion caused a reduction in the capillary filtration coefficient (Kfc) from 1.25 +/- 0.13 to 0.53 +/- 0.08 and 0.55 +/- 0.10 ml.min-1.cmH2O-1.100 g tissue-1, respectively, at 90 min of reperfusion. This reduction in Kfc was accompanied by a rise in perfusate cAMP levels from 16.5 +/- 4.9 and 31.2 +/- 11.9 pmol/ml at 45 min of reperfusion to 444.2 +/- 147.8 and 276.1 +/- 91.0 pmol/ml at 105 min of reperfusion in lungs treated with ISO or FSK, respectively, at 46 min of reperfusion. Dibutyryl cAMP (DBcAMP), a membrane-permeable cAMP analogue, mimicked the permeability effect by reducing Kfc to 0.67 +/- 0.15 at 90 min of reperfusion. Significant hemodynamic changes occurred but were small and cannot explain the observed effect on Kfc. Photomicrographs from lungs treated with ISO or FSK revealed a reversal of the morphological manifestations of increased microvascular permeability. We conclude that the increased microvascular permeability associated with IR can be reversed by ISO, FSK, and DBcAMP and that cAMP produced by the lung contributes to the observed reversal.

Endometrial morphology in asymptomatic postmenopausal women.

Few data are available regarding endometrial histologic features in asymptomatic perimenopausal and postmenopausal women. This study encompasses endometrial biopsy specimens obtained from 801 such women before enrollment in a multicenter study of estrogen-progestin replacement. One endometrial cancer was found (0.13%); four additional biopsy specimens showed atypia (total 0.63%). The endometrium was atrophic in 373 (46.9%), proliferative in 133 (16.7%), secretory in 54 (6.8%), and hyperplastic in 41 (5.2%). Insufficient tissue for diagnosis was obtained in 195 (24.5%). We conclude that the yield for neoplasia is so low that screening endometrial biopsy is not justified in asymptomatic perimenopausal and postmenopausal women.

Superoxide dismutase conjugated to polyethylene glycol fails to limit myocardial infarct size after 30 min ischemia followed by 72 h of reperfusion in the rabbit.

We tested whether recombinant human superoxide dismutase conjugated to polyethylene glycol (PEG-SOD) to prolong its plasma retention time could limit myocardial infarct size in an ischemia-reperfusion model in the rabbit. One group of animals received 1000 units/kg of PEG-SOD as an intravenous bolus 15 min before coronary occlusion. A second group received saline only and served as controls. Under pentobarbital anesthesia, a left coronary branch was occluded for 30 min and then reperfused. The surgical wounds were repaired and the animals were allowed to recover. Seventy-two hours after the coronary occlusion, the heart was excised and the size of the area at risk (ischemic vascular bed) was assessed with fluorescent particles and the infarct size was determined by histology (Hematoxylin-eosin, Azan stain). Infarct size as a percentage of the area at risk was similar between the groups, 46.5 + 2.7 in the PEG-SOD group (n = 8) and 48.9 + 3.1 in the control group (n = 8). There were no significant differences between the groups indicating that PEG-SOD did not limit infarct size in this model.

Tetrazolium artifactually indicates superoxide dismutase-induced salvage in reperfused rabbit heart.

We tested the ability of a single dose of superoxide dismutase to induce salvage of reperfused rabbit myocardium. Infarct size was measured by tetrazolium method following 3, 24, or 72 h of reperfusion. In addition, the 24 h reperfused hearts were examined to determine if the drug induced salvage in those hearts was reflected in the histology. A coronary arterial branch was occluded for 45 min and then allowed to reperfuse for 3, 24 or 72 h. At the end of the reperfusion period the hearts were removed, perfusion stained with triphenyl tetrazolium, and fixed in buffered formalin. The hearts were sectioned and infarct size was determined in all groups. In addition, the 24 h heart slices were prepared for histology with H&E staining. The results revealed that 5 mg/kg hSOD treatment was associated with smaller infarcts in the 3 and 24 h groups but that differences were no longer apparent in the 72 h group. The 24 h control hearts showed good correlation between infarct size by TTC and that by conventional histology. In the 24 h treatment hearts, however, infarcts by TTC averaged only about 1/2 the size of those by conventional histology. We conclude that a single dose of hSOD fails to offer a sustained reduction of infarct size. Furthermore, histology from the 24 h reperfused group revealed that hSOD did not delay the onset of necrosis but rather simply caused dead tissue to retain its ability to reduce the tetrazolium salts.

Chronic portal hypertension: effects on gastrointestinal blood flow distribution.

The intramural distribution of blood flow in the gastrointestinal tract was measured in shamoperated control and portal vein-stenosed rats. Total organ blood flow, measured via the radioactive microsphere technique, was elevated in the esophagus (66%), stomach (102%), duodenum (42%), jejunum (52%), ileum (54%), and colon (79%) of portal-hypertensive rats. Histological evaluation of carbonized nonradioactive 15-microns microspheres allowed for fractionation of blood flow within the wall (mucosa, submucosa, and muscularis externa) of each organ. The microsphere distribution pattern indicates that intramural blood flow distribution in all organs was not dramatically affected by chronic portal hypertension. These findings further define the characteristics of the factors responsible for the gastrointestinal hyperemia produced by chronic portal hypertension.

Fungus-host relationship in candidiasis. A brief review.

Candidiasis and its causative agent, Candida albicans, have been under continuous study in our clinics and laboratories for the past 20 years. Cultured cells of C albicans and tissues from natural and experimental infections were used for observations by light microscopy, transmission electron microscopy, scanning electron microscopy and freeze fracture techniques. In cultures, the cells of C albicans revealed a more complex cell wall, plasma membrane, intracellular organelles, and biochemical organization than those described in classic text-books on mycology. In infected tissues, noteworthy characteristics of C albicans were prominent vacuoles and invasion of host cells with subsequent intracellular localization and lysis of tissues surrounding the fungus. These findings are discussed in relation to their importance in the pathogenesis and management of candidiasis and to the mechanism of action of anticandida agents.

Clinical evaluation of an improved injectable microcapsule contraceptive system.

Pharmacokinetics and pharmacodynamics of a long-acting injectable microcapsule, poly(DL-lactide-co-glycolide), delivery system were tested in 10 women. Two doses (75 or 100 mg of norethindrone) were administered by intramuscular injection. Treatment suppressed ovarian function and inhibited ovulation for 3 months in all subjects. Levels of norethindrone in subjects who received the 100 mg dose were proportionately higher than those in subjects who received the 75 mg dose. Subsequent to the injection, there was a rapid rise in the serum levels of norethindrone followed by a gradual decline until 8 to 10 weeks. Between 10 and 20 weeks after treatment, there was a secondary rise and fall in the serum levels of norethindrone. Treatment caused suppression of the endometrium for 3 months, and, except for spotting and irregular menstrual cycles, there were no adverse side effects. Treatment had no significant effect on serum lipids.

Scanning electron microscopy of human spermatozoa after incubation with the spermicide nonoxynol-9.

The most frequently utilized spermicide in vaginal contraceptives is No-9. Schill and Wolff used TEM to demonstrate the focal effects of No-9 on human sperm and reported that No-9 damaged cell membranes and acrosomal membrane complexes. The present study by SEM was made to assess the extent of membrane damage due to the direct action of No-9 during an incubation period of only 5 minutes. SEM revealed that No-9 caused loosening and detachment of acrosomal, neck, and midpiece membranes of all sperm even at the lowest concentration tested (0.05%). The severity of membrane alterations observed in any of these regions would render sperm immotile and unable to penetrate the ovum. The fact that these alterations are produced within 5 minutes after exposure to No-9 attests to the effectiveness of No-9 as a vaginal contraceptive.

PIP: Scanning electron microscopy was used to assess the extent of membrane damage due to the direct action of nonoxynol-9 during an incubation period of 5 minutes. Nonoxynol-9 is the most frequently utilized spermicide in vaginal contraceptives. Nonoxynol-9 disrupted sperm membranes in all regions except the postacrosomal region and tail. It caused loosening and detachment of acrosomal, neck, and midpiece membranes of all sperm even at the lowest concentration tested (0.05%). Damage to all membranes was 1st evident as vesiculations, then membranes became loose and detached. The severity of membrane alterations observed in any of these regions would render sperm immotile and unable to penetrate the ovum. A dose-response relationship was not apparent, and there was no variation in response among sperm donors. The fact that these alterations are produced within 5 minutes confirms the effectiveness of nonoxynol-9 as a vaginal contraceptive. Scanning electron microscopy proved to be well suited for evaluating the extent of damage caused by nonoxynol-9 and provided more information than transmission electron microscopy.

Mechanisms of uterine bleeding in postmenopausal patients receiving estrogen alone or with a progestin.

The response of postmenopausal endometrium to cyclic estrogen and progestin and cyclic estrogen alone was studied in 75 biopsies and over 2000 preparations using standard histologic, histochemical, and scanning and transmission electron microscopic techniques. Estrogen and a progestin caused the atrophic endometrium to assume normal proliferative and secretory phases and to develop nucleolar channel systems. Cyclic unopposed estrogen produced unphysiologic responses in the glands, stromal cells, and vessels. The concept of a progestin or progesterone producing a "medical curettage" should be reappraised. Cyclic estrogen and progestin therapy do not cause all the endometrium to desquamate to the basalis layer. The combination therapy is associated with increased glycoprotein production in the gland and stromal cells, and an orderly regression and remodeling of the endometrium upon hormonal withdrawal. Cyclic estrogen alone causes irregular and unpredictable breakdown, which may or may not extend to the basalis. The stimulation of the endometrium by estrogen alone may allow the endometrium to use the majority of its energy for growth, which may lead to hyperplasia and neoplasia.

Ultrastructure of endometrium of postmenopausal patients on two modes of hormonal therapy: estrone sulfate with or without MPA.

The response of human postmenopausal endometrium to standard, oral doses of an estrogen alone (estrone sulfate) and to an estrogen plus a progestin (medroxyprogesterone acetate; MPA) was evaluated by scanning electron microscopy and transmission electron microscopy. Endometrial biopsies were obtained over a 4-year period from 12 patients with well-established ovarian failure. Biopsies were taken before the initiation of therapy and during each mode of hormonal treatment. The ability of estrone sulfate alone to stimulate growth of the endometrium was shown during the 1st treatment cycle when the atrophic epithelial cells transformed into tall columnar cells which synthesized and released some secretory products. Unopposed estrogen therapy led to excessive ciliation and breakthrough bleeding. Addition of MPA to the estrone sulfate regimen produced a progestational endometrium. The epithelial cells had ultrastructural features of those in normal postovulatory endometrium, including nucleolar channel systems. MPA elicited deciliation and transformation of ciliated cells into secretory cells. Stromal cells hypertrophied, the vascular endothelium thickened, and bleeding subsided. Estrogen-progestin therapy as tremendously more physiological than unopposed estrogen therapy.

Differences in the ultrastructure of the submandibular glands of baboon and Rhesus monkey revealed by the use of different fixatives.

Submandibular glands of adult baboon and Rhesus monkeys were compared after different methods of fixation. In both species, serous acinar cells outnumber mucous acinar cells. In the baboon, serous cells contain secretory granules showing dense cores, moderately dense crescents, and flocculent material. In mucous cells, secretory granules vary in appearance from amorphous to highly ordered depending on fixation. In the Rhesus monkey, serous cells contain the same 3 components of secretory granules as in the baboon. Additionally, a fourth component is represented by a layer of electron-dense material between the crescent and flocculent material. Mucous cells contain electron-dense granules when fixed in Millonig's buffered fixatives, but when Clarke's or Sorensens' buffer is used the granules resemble more typical mucous granules. Duct systems of the two species are similar, but differ mainly in that large foci of glycogen are present in the striated duct cells of the Rhesus monkey.

Ultrastructure of the lung after ventilation.

We looked at the ultrastructure of the lung of rabbits ventilated with air moistened with nebulized water ("nebulized group"), and humidified air ("humidified group") compared to a group of controls. Both groups of ventilated rabbits had plasmalemmal vesicles that were larger than in controls. They were fewer in number (P less than 0.001) and less well defined. Early degenerative changes were noted in 6 of 9 rabbits in the nebulized group and 6 of 9 rabbits in the humidified group, while none of the rabbits in the control group were similarly affected. Interstitial oedema of varying severity was seen in 6/9 rabbits from each of the ventilated groups.

Functional and structural changes in veins in spontaneous hypertension.

Six month old spontaneously hypertensive rats (SHR) and age and sex matched normotensive Wistar-Kyoto rats (WKY) were examined to evaluate the existence of functional and structural changes in the portal vein (PV), inferior vena cavae (IVC) and pulmonary arteries (PA). PV, central nervous and systolic right ventricular pressures did not differ in the anesthetized SHR when compared with WKY, despite elevated systolic arterial pressure. PV, IVC and PA obtained from SHR were less extensible, developed more tension, exhibited an enhanced sensitivity to serotonin and a thromboxane-like prostanoid, accumulated more of the protein precursors 2-14C-leucine, 2-14C-glucosamine, 7-3H-fucose, exhibited a normal rate of uptake of 2-14C-thymidine, and increased protein content and a decreased concentration of DNA. When examined under light and electron microscopy, the veins and PA obtained from SHR demonstrated medial smooth muscle hypertrophy, and increased density of PAS-Schiff positive stain, enlarged and prominent Golgi apparati and an increased cell diameter through the region of the nucleus. These changes were not due to water-logging since the water content of the veins and PA from SHR and WKY did not differ. These data support the conclusion that PV, IC and PA obtained from SHR exhibit functional and structural changes independent of increases in intravenous pressure. These changes may relate to the increase in blood vessel protein.