Serum progesterone levels as an aid in the diagnosis of ectopic pregnancy.

Progesterone levels in 29 women with ectopic pregnancies and 20 women with early intrauterine pregnancies were evaluated using a new direct radioimmunoassay that offers results within four hours. Patients with normal intrauterine pregnancies had serum progesterone levels greater than 20 ng/mL (mean = 30.9 ng/mL) while all patients with ectopic pregnancies had progesterone levels less than 15 ng/mL (mean = 5.7 ng/mL). The incorporation of the progesterone assay into the workup of a patient with suspected ectopic pregnancy can be a useful clinical adjunct to the conventional methods of evaluation.

Lipoprotein lipid concentrations and cardiovascular risk in women with polycystic ovary syndrome.

Twenty-nine patients with polycystic ovary syndrome (PCOS) and 30 normal women had lipoprotein lipid and androgen profiles compared after a 12-h fast. Both PCOS and normal women were evaluated in the proliferative phase of the cycle. PCOS patients had higher serum LH to FSH ratios [2.0 +/- 1.3 (+/- SEM) vs. 0.6 +/- 0.1), higher testosterone (T; 66 +/- 5 vs. 33 +/- 2 ng/ml), higher free T (1.1 +/- 1 vs. 0.4 +/- 0.02 ng/dl), and higher dehydroepiandrosterone sulfate (291 +/- 28 vs. 140 +/- 12 micrograms/dl) levels, and lower T-estrogen-binding globulin-binding capacity (1.5 +/- 0.2 vs. 2.2 +/- 0.1 micrograms/dl) than normal women (all P less than 0.05). The PCOS patients had higher mean serum triglycerides [122 +/- 11 (+/- SEM) 63 +/- 3 mg/dl] and very low density lipoprotein cholesterol levels (24 +/- 2 vs. 13 +/- 1 mg/dl), but lower high density lipoprotein cholesterol levels (43 +/- 2 vs. 58 +/- 2 mg/dl; P less than 0.05). While PCOS patients were heavier and more sedentary and their diets were higher in saturated fat and lower in fiber (P less than 0.01, respectively), the differences in lipoprotein lipid concentrations could not be attributed to body weight. T-estrogen-binding globulin-binding capacity correlated with high density lipoprotein cholesterol in PCOS patients (r = 0.42; P = 0.025) after adjusting for weight. We conclude that hyperandrogenemia in women may result in a male pattern of lipoprotein lipid concentrations. These findings suggest that PCOS patients may have increased atherogenic potential.

Prognostic indicators including DNA histogram type, receptor content, and staging related to human breast cancer patient survival.

Primary tumors from breast cancer patients were evaluated for the biochemical presence of three steroid cytosolic receptors and by DNA histogram analysis using flow cytometry. These parameters were compared with the histological and staging diagnoses and the patients' survival over a 36-month period. A total of 74 patients with primary breast tumors were evaluated. The breast samples invariably demonstrated a peak population of diploid G0/1 cells which contained 2C amounts of DNA, as determined by mixing experiments using normal human breast tissues or trout erythrocytes as fixed standards. The tumors were classified into five DNA histogram types based on their DNA index distributions established by flow cytometry. These results showed that 21% of the tumors were diploid and indistinguishable from the diploid population of normal breast cells, 8% were hypodiploid, 11% were hypertetraploid, 8% were multiploid, and the remaining 52% were hyperdiploid. The DNA index values varied from 0.78 (hypodiploid) to 2.60 (hypertetraploid). The percentages of S-phase cells were lowest in the diploid and hypertetraploid tumors and highest in the hypodiploid tumors. Among the 24 patients who died during the 36-month follow-up, 92% (22 of 24) were classified in one of the aneuploid groups. Three high-risk groups identified on the basis of survival after 36 months were distinguished: hypodiploid (50% survival); multiploid (43% survival); and hyperdiploid (50% survival). Rates of survival in the diploid and hyperdiploid groups were 87 and 71%, respectively. The hypodiploid group was distinguished by having the lowest mean estrogen cytosolic receptor value [26 +/- 13 (S.D.) fmol/mg], progesterone cytosolic receptor value (13 +/- 15 fmol/ mg), and androgen cytosolic receptor value (less than 1 +/- 1 fmol/mg). In contrast, the diploid tumors had some of the highest receptor values, with mean estrogen cytosolic receptor value equal to 102 +/- 114 fmol/mg, progesterone cytosolic receptor value equal to 74 +/- 110 fmol/mg, and androgen cytosolic receptor value equal to 65 +/- 80 fmol/mg. The lowest survival rates (17% after 36 months) occurred in patients over 67 years of age who had aneuploid tumors, compared to 100% survival in patients over 67 years of age with diploid tumors. Our results demonstrate the value of using flow cytometry and steroid receptor values as supplements to histopathology for the characterization of subgroups of mammary cancer patients. The ability to identify patients with a good prognosis compared to those at high risk of recurrence and death will be valuable in the design of future prospective treatment studies.

Modulation of glucocorticoid hormone receptor levels in chicken lymphoid tissue following treatment with androgens in vivo.

Lymphatic tissues are highly sensitive to androgens and androgens are thought to contribute to sex differences in the immune response. In this study we have examined the effects of androgens on cytosolic glucocorticoid receptor levels in lymphoid tissues. The immature chick was chosen for our experimental model because it allows the separate evaluation of the bursa of Fabricius (primarily B-cells) compared to the thymus (primary T-cells). Treatment with dihydrotestosterone (a potent androgen in chicks) for 3-12 days in vivo reduced the cytosolic glucocorticoid (triamcinolone acetonide-[3H]) receptors in bursa tissue to approximately 42% of control levels after 5 days and less than or equal to 5% of control levels after 7 days of treatment. The chick thymus tissues were still approximately 92% of control triamcinolone acetonide receptor levels after 5 days of androgen treatments. However the thymus levels had dropped to less than or equal to 5% of control values after 12 treatment days. Thus a difference in the rate of decrease in the bursa of Fabricius compared to the thymus was indicated. The blastogenesis index (BI), a measurement of the percentage of cells progressing through the cell cycle, was figured using fluorescent DNA staining with diamidino phenylindole followed by flow cytometry analysis. After 3, 5, or 7 days of androgen treatment, the bursa of Fabricius from dihydrotestosterone treated chicks (2 mg/day/chick) had a mean BI = 11.17 (+/- 3.07 SD) which was significantly lower than the bursa of Fabricius from control chicks which showed a mean BI = 27.33 (+/- 3.42 SD). The thymus from dihydrotestosterone treated chicks had a mean BI = 19.57 (+/- 2.19 SD) which was slightly but not significantly higher than the control thymus CI = 17.38 (+/- 0.89 SD). In summary, androgen treatment in vivo induced a decrease in the cytosolic glucocorticoid hormone receptor levels in both the chick thymus and bursa of Fabricius tissues while decreasing the blastogenesis index in the bursa cells but not in the thymus cells.

Gossypol inhibits testicular steroidogenesis.

Gossypol, a compound isolated from cottonseed, has been used in the People's Republic of China as an antifertility agent in men. It was reported that gossypol is highly effective in reducing sperm count. Our study suggests that gossypol inhibits luteinizing hormone, 8-bromo-adenosine 3',5'-monophosphate-induced testosterone formation, and the conversion of pregnenolone to testosterone in isolated rat interstitial cells. Serum testosterone levels were also significantly reduced in rats after 1 week of treatment with gossypol. Oligospermia and azoospermia in men caused by gossypol may be secondary to decreased testosterone biosynthesis.

Short term metabolic effects of the anti-fertility agent, gossypol, on various reproductive organs of male mice.

In order to evaluate the short term metabolic effects of gossypol on the testes as well as any possible effects on the secondary sex organs, Balb C mice were injected subcutaneously with various doses of gossypol (0.25-25.0 mg/kg body weight) in corn oil for 10 days. Wet weights of several different secondary sex reproductive organs decreased during gossypol treatment. However, wet weights of the testes during treatment remained equal to or greater than control values. Following 10 days of gossypol treatment, incorporation of [3H]thymidine or [3H]amino acids into trichloroacetic acid precipitable macromolecules was inhibited in the seminal vesicles and ventral prostates normalized to either DNA or wet weight. Treatment with gossypol also had an inhibitory effect on epididymal sperm count at the two highest doses. These results demonstrate that gossypol will decrease sperm count at high dose levels after treatment of male mice for as short as 10 days. However, its overall effects are not limited to the testes and spermatogenesis but, in addition, it has dramatic inhibitory effects on protein and nuclei acid metabolism in the secondary sex organs.

Ovalbumin messenger ribonucleic acid accumulation in the chick oviduct during secondary stimulation: influence of combinations of steroid hormones and circannual rhythms.

We have analyzed by hybridization the accumulation of ovalbumin mRNA after the administration of estrogen, progesterone, these two hormones together, or each hormone with testosterone to "withdrawn" chicks (chicks previously stimulated with estrogen but then withdrawn from the hormone). We were interested in determining if the time lag between hormone treatment and a significant increase in ovalbumin mRNA levels could be shortened. After every hormonal treatment we found that there was a 1.5-2.5-h lag period before a significant increase in ovalbumin mRNA levels. This lag period was not simply due to the slow delivery of the hormone to the oviduct. Nuclear estrogen receptor levels had plateaued by 1 h. Rather unexpectedly, the rate of ovalbumin mRNA accumulation after this lag period seemed to depend on the time of the year. Ovalbumin mRNA accumulated faster during the summer and early fall than during the winter and early spring. These seasonal differences were also observed in ovalbumin mRNA levels 24 h after hormone treatment. Such a circannual rhythm may help explain reported differences in the rate of ovalbumin mRNA accumulation during secondary stimulation.

Accuracy of predicting stages of bovine estrous cycle by gross appearance of the corpus luteum.

Four readily identifiable changes in appearance of corpora lutea (Stages I to IV) occur during a bovine estrous cycle. Accuracy of estimating the stage of an estrous cycle by appearance of corpora lutea was determined in a double-blind study. One investigator observed estrus in a group of heifers while another with no prior knowledge of reproductive histories of the heifers estimated stage of the estrous cycle by visual inspection of their corpora lutea. Stage of the estrous cycle (Stage I, days 1 to 4; Stage II, days 5 to 10; Stage III, days 11 to 17; Stage IV, days 18 to 20) was estimated correctly in 41 of 48 heifers. The correlation between estimated and actual days of the estrous cycle was .81. In addition, concentrations of progestins and weights of corpora lutea during estimated stages of the estrous cycle were similar to many other investigations. Stages of the estrous cycle in heifers can be estimated from appearance of corpora lutea.

Estrogen receptors and effects of estrogen on membrane electrical properties of coronary vascular smooth muscle.

The effect of estrogen stimulation in vitro on the electrical properties of vascular smooth muscle (VSM), and the concentration of estrogen receptors in VSM were measured in isolated coronary arteries. Microelectrode measurements of the dog coronary artery membrane potential (Em) showed quiescent values of -51 millivolts (mV) and an input resistance (rin) of 10 megohms. Addition by diethylstilbestrol (DES) at 10(-6) M hyperpolarized the membrane to -64 mV and reduced input resistance (rin) to 5 megohms within 15 minutes. Extrapolation of the Em vs. log [K]o curve to zero potential gave similar values of [K]i of around 170 mM in both normal and DES treated muscles suggesting that the DES induced hyperpolarization is not due to increased Na-K pump activity. The 0.5% ethanol vehicle alone had no effect on the membrane potentials. Tetraethylammonium ion (TEA) induced action potentials in the previously quiescent tissue. When DES was applied in the presence of TEA, the membrane potential increased and the action potentials were abolished. Scatchard analysis of the estrogen receptor binding demonstrated both a high and a low affinity receptor for estrogen in the VSM. These data indicate that DES hyperpolarizes the VSM cells by a mechanism other than an increased Na-K pump activity. The mechanism of this increased Em may be due to factors which increase K+ conductance either mediated directly through estrogen interaction with its cytosolic receptors or through some unidentified second mechanism.

Quantation by flow microfluorometry of total cellular DNA in Acanthamoeba.

The DNA content of five species of Acanthamoeba was determined by flow microfluorometry. Acanthamoeba castellanii (AC-30), acanthamoeba polyphaga (APG and P-23), acanthamoeba rhysodes, acanthamoeba culbertsoni (A-1), and acanthamoeba royreba were grown in a casitone based medium 24-48 HR. The trophozoites were harvested, and evaluated for DNA-bound fluorescence. All species tested has DNA values between 2.0-5.0 pg/cell. These results placed DNA/cell values of Acanthamoeba slightly lower than DNA/cell values of other eucaryotic cells and much lower than Amoeba proteus values. These results indicate that FMF may be a useful adjunct in distinguishing Acanthamoeba cells from either eucaryotic cells or some other amoeba. However, differences in DNA/cell between species of Acanthamoeba are small and would not be useful in identification of species.

Quantitation of cellular deoxyribonucleic acid by flow microfluorometry.

This report characterizes for the first time an easy, reproducible means of standardizing the relative fluorescent units normally reported for flow microfluorometry. Absolute values for deoxyribonucleic acid/cell are obtained by using nucleated red blood cells as references. Cell were selected and characterized for the quantitative analysis of deoxyribonucleic acid per cell over a range from 2 pg/cell to 93 pg/cell using literature values for species having nucleated erythrocytes. Fluorescence staining by either acridine-orange (green wavelength) or propidium iodide (red wavelength) gave linear curves over the entire range investigated only when "gain controls" and current are optimized. The range was equivalent to mammalian cell values from 1 N (=3.5 pg deoxyribonucleic acid/cell) to 28 N (=91 pg deoxyribonucleic acid/cell). The standard curves obtained with nonmammalian erythrocytes were compared to mammalian free-cell preparations of bovine thymus and liver cells which fell at 6.8 and 6.9 pg deoxyribonucleic acid/cell, respectively. The routine use of these easily obtainable red blood cells will allow ready comparisons on the basis of absolute values for deoxyribonucleic acid per cell for work between experiments, work between staining procedures and dye types and work between laboratories.