Joel T. Hargrove

Development of a method to isolate and culture highly purified populations of stromal and epithelial cells from human endometrial biopsy specimens

Appropriate endometrial maturation is of paramount importance to achieve reproductive success. Practical and ethical considerations require that in vitro methods be available to evaluate regulation of human endometrial function. Additionally, tissue complexity requires separation of individual cell populations. This report describes an improved method for isolation of endometrial epithelial and stromal cells, using biopsy specimens as a tissue source. Separated cells were obtained using selective enzymatic digestion in conjunction with physical separation procedures. Isolated populations exhibited over 95% homogeneity, ascertained immunocytochemically. Using this system, isolated cells from normal endometrium can readily be obtained for in vitro studies. Within the defined conditions of a culture system, important areas of current concern in the endometrium such as ectopic endometrial growth and implantation can be addressed.

Epithelial cells from normal human endometrium express a tumor-associated glycoprotein (TAG-72) epitope in vitro

Monoclonal antibody B72.3 identifies a tumor-associated glycoprotein (TAG-72) epitope derived from a human breast carcinoma metastasis. Recently, expression of this epitope was noted in normal endometrium during the secretory, but not proliferative, menstrual interval. In light of known hormonal control of normal endometrial growth and differentiation, we investigated in vitro expression of TAG-72 epitope in purified endometrial epithelium cultured under serum-free conditions on Matrigel biomatrix. Cells from secretory endometrium exhibited homogeneous tumor-associated glycoprotein 72 epitope expression. Unexpectedly, epithelium from the proliferative interval developed expression after 5 to 6 days of culture. Epithelial cells from both intervals maintained expression over 12 days of culture without exogenous estradiol and progesterone. Spontaneous, uniform expression of tumor-associated glycoprotein 72 epitope by normal endometrial epithelial cells in vitro is in marked contrast to the cyclic, heterogeneous expression observed in vivo. Such expression also differs from published in vitro observations of cancer cell lines that express this epitope.

Distribution of tumor-associated glycoprotein-72 (TAG-72) expression throughout the normal female reproductive tract.

SummaryCompared with the degree of investigation of stage-specific expression of tumor-associated glycoprotein-72 (TAG-72) in fundal endometrium, other regions of the female reproductive tract have not received comparable attention. Regional, cell-type-specific, and temporal differences in estrogen receptor and progesterone receptor distribution and concentration among these tissues should make such examination beneficial to our understanding of hormonal regulation of TAG-72 expression. The pattern of monoclonal antibody (MAb) B72.3 recognition in the cervix, uterus, oviduct, and ovary was examined by immunohistochemistry during both the proliferative and secretory intervals of the normal menstrual cycle. Intense immunoreactivity in fundal endometrium was limited to the secretory menstrual interval. Conversely, TAG-72 expression was generally weaker, sporadically distributed, and not stage specific in the lower uterine segment, endocervix, and cervix; no expression was detected in the oviduct or ovary. The disparity in both temporal and spatial distribution of TAG-72 expression throughout the female reproductive tract does not appear to be directly associated with the well-described changes in circulating estradiol or progesterone or the receptors for these steroids. Results suggest that regulation of TAG-72 expression may involve local paracrine/autocrine mechanisms, which in turn may be subject to hormonal influence.

Menopausal Hormone Replacement Therapy with Continuous Daily Oral Micronized Estradiol and Progesterone

The safety and efficacy of a daily combination of micronized estradiol (E2) (0.7-1.05 mg) and progesterone (200-300 mg) were evaluated in ten menopausal women with moderate to severe vasomotor symptoms and/or vaginal atrophy over a 12-month study interval. For comparison, five similar women were placed on conjugated estrogens, 0.625 mg daily, and medroxyprogesterone acetate, 10 mg daily, for the first 10 days of each calendar month for 12 months. Patients were evaluated at 0, 1, 3, 6, and 12 months. Estrogens rose significantly from baseline in both groups (P<.01). Progesterone increased significantly above baseline in the E2 and progesterone group (P<.01), but did not change in the conjugated estrogens and medroxyprogesterone acetate users. All women on E2 and progesterone had a decrease in total cholesterol and an increase in high-density lipoprotein cholesterol from baseline (P<.01). Those on conjugated estrogens and medroxyprogesterone acetate had no significant change from baseline in total cholesterol; however, they did have an increase in high-density lipoprotein cholesterol values (P<.01). In the E2 and progesterone group, the endometrial histology became completely quiescent and there was no uterine bleeding after 6 months of observation. Four of five women on conjugated estrogens and medroxyprogesterone acetate continued regular withdrawal bleeding throughout the study period, but no endometrial hyperplasia was encountered. This study demonstrates that the daily administration of a combination of micronized E2 and progesterone results in symptomatic improvement, minimal side effects, an improved lipid profile, and amenorrhea without endometrial proliferation or hyperplasia in menopausal women. (Obstet Gynecol 73:606, 1989)

Sedative and Hypnotic Effects of Oral Administration of Micronized Progesterone May Be Mediated through Its Metabolites

Progesterone and its metabolites were measured in serum extracts by radioimmunoassay and gas chromatography-mass spectrometry, respectively, after ingestion of micronized progesterone by eight postmenopausal women. One subject received 400 mg of micronized progesterone orally that induced a hypnotic state that lasted for approximately 2 hours. Blood samples were drawn periodically from all subjects for measurement of progesterone and its metabolites in serum. Levels of serum progesterone and its metabolites increased significantly from baseline values and reached a peak between 2 and 6 hours after oral progesterone administration. Significant quantities of five compounds (progesterone, 5 alpha-pregnan-3 alpha-ol-20-one, 5 beta-pregnan-3 alpha-ol-20-one, 5 beta-pregnan-3 alpha,20 beta-diol, and 5 beta-pregnan-3 alpha-ol-11,20-dione) that have been reported to possess anesthetic qualities were identified. The sedative and hypnotic effects of oral administration of progesterone may be mediated through those compounds.