A case-control study of breast cancer and hormonal contraception in Costa Rica.

By 1981, 11% of married women in Costa Rica ages 20-49 years had used depot-medroxyprogesterone acetate (DMPA) and 58% had used oral contraceptives (OCs). Since 1977, the Costa Rican Ministry of Health has maintained a nationwide cancer registry. These circumstances provided an opportunity for a population-based, case-control study of DMPA, OCs, and breast cancer in Costa Rica. Cases were 171 women ages 25-58 years with breast cancer diagnosed between 1982 and 1984; controls were 826 women randomly chosen during a nationwide household survey. Cases and controls were interviewed with the use of a standard questionnaire covering their reproductive and contraceptive histories. Logistic regression methods were used to adjust for confounding factors. While few cases or controls had ever used DMPA, DMPA users had an elevated relative risk (RR) estimate of breast cancer of 2.6 (95% confidence limits = 1.4-4.7) compared with never users. However, no dose-response relationship was found; even the group of women who had used DMPA for less than 1 year had an elevated RR estimate (RR = 2.3; 95% confidence limits = 1.0-5.1). In contrast, OC users had no elevation in RR compared with never users (RR = 1.2; 95% confidence limits = 0.8-1.8). The results of the DMPA analysis are inconclusive. Before decisions are made on whether to continue providing this effective contraceptive method, other ongoing studies will need to confirm of refute these findings.

PIP: Depot-medroxyprogesterone acetate (DMPA) is used by approximately 2 million women world-wide. In Costa Rica, by 1981 11% of the married women had used DMPA. Although no definitive link between the use of DMPA and the development of cancer has been found, the possibility has been recognized as one of the reasons for the U.S food and Drug Administration denying approval of the drug. Since 1977, the Ministry of Health in Costa Rica has sustained a national cancer registry. The accuracy of the reporting of the registry was studied by comparing a sampling of gynecologic cancer cases in the year 1983 to the national registry. In examination of the records, an elevated relative risk of breast cancer for DMPA users was found (2.6), and an elevated relative risk for women who had even used DMPA for a short period of time (2.3). The failure to interview 1/3 of the cases, differences in detection and the misclassification of DMPA use contributed cumulatively to the belief that a positive connection between breast cancer and DMPA use could otherwise be drawn. In contrast to the association found between DMPA ever use and breast cancer, no connection was found between OC use and the incidence of breast cancer. However, due to the small number of DMPA users recorded and the void of complete and accurate recorded information, the study results remain inconclusive.

Enhancement of tumor growth and metastases by medroxyprogesterone acetate in transplanted uterine adenocarcinoma cells of the rat.

PIP: This study describes the effects of medroxyprogesterone acetate on tumor growth and metastases in transplanted uterine adenocarcinoma cells of the rat. High-and-low-tumorigenic cloned cells of Sprague-Dawley rat uterine adenocarcinoma originally induced by 7,12-dimethylbenz (alpha) anthracene in vivo were used. Both were derived from the same parent culture. They were cultured for more than 2 years and both retained almost the same transplantability. Survival rate of cell colonies in vitro was reduced in both lines after progesterone treatment of more than 8 mcg per ml. This reduction was dose dependent. About 1 million cells suspended in .2 ml culture medium were injected sc into the interscapular region of isologous newborn rats. At 5 weeks these rats were given .5 mg medroxyprogesterone acetate twice a week for 2 weeks. At 7 weeks they were killed. High-tumorigenic cells produced growing tumors in all newborn rats. About a third of these rats died of metastases during the 7-week observation period. Tumors produced by low-tumorigenic cells grew slowly and occasionally regressed without metastases to the lung. Tumors in female rats were larger than those in males. Enhancement of tumor growth and metastases by this progesterone compound was observed in rats inoculated with low-tumorigenic cells as compared to controls. The enhancement was not significant in tumors produced by high-tumorigenic cells. The progesterone may act immunosuppressively in vivo, or make alterations in environmental conditions of the tumors.^ieng

Mammary nodules in dogs during four years' treatment with megestrol acetate or chlormadinone acetate.

PIP: A 7 year study of megestrol and chlormadinone in female dogs is in progress. This report characterized histopathologically 60 mammary nodules during the first 4 years of the study. 100 purebred female beagles, 6-12 months of age, were randomly assigned to 5 equal groups. One group was used as a control. Oral doses were .01, .10, and .25 mg/kg/day of megestrol acetate in coconut oil in capsules and of chlormadinone acetate .25 mg/kg/day in lactose tablets. These doses were 1, 10, and 25 times the projected dose of megestrol for humans and about 25 times the human dose of chlormadinone. After 2 years 4 dogs from each group were necropsied. One high-dose megestrol-treated and 1 chlormadinone-treated dog had benign mixed mammary tumors. Palpable nodules were first observed at 16 months in the chlormadinone-treated dogs, at 18 months in dogs given the high dose megestrol and at 27 months in the dogs treated with middle-dose megestrol. Transitory nodules were found in 4 control dogs after 21 months and in low dose megestrol-treated dogs at 26 months. Of 38 grossly detected nodules evaluated microscopically from the megestrol-treated dogs 27 were nodular hyperplasia, 5 were benign mixed mammary tumors, 3 were ductal dialatations, 1 was a lymph node, 1 was fat necrosis and 1 was the umbilicus. Of 22 nodules from the chlormadinone-treated dogs 12 were nodular hyperplasia, 4 benign mixed mammary tumors, 1 chondromucoid degeneration and 1 adenocarcinoma with widespread metastases. 3 nodules were lymph nodes and 1 other had no mammary tissue. Involutions, regression and sclerosis of many areas of nodular hyperplasia were evident at 4 years. Thus of the 60 nodules evaluated during the first 4 years of the study 50 were non-neoplastic and 10 were neoplastic. It is considered that the 1 adenocarcinoma may have been spontaneous and not a treatment-related neoplasm. A precursor stage through nodular hyperplasia apparently did not occur.^ieng

Effect of oral contraceptives on the mammary glands of Rhesus monkeys: a preliminary report.

PIP: To determine the relationship of oral contraceptive use and breast cancer, 96 rhesus monkeys were administered either Enovid-E (2.5 mg norethynodrel and .1 mg mestranol) or Ovulen (1 mg ethynodiol diacetate and .1 mg mestranol) cyclically for 5 years at doses of 1, 10 and 50 times the human dose. The animals' progress was compared with a control group of 32 monkeys. General physical and mammary gland examinations were conducted before treatment and monthly thereafter. During the 5 year study period none of the treated animals demonstrated clinical evidence of mammary gland lesions, no deaths from breast malignancy occurred, and no palpable breast nodules were found.^ieng

Effects of the antifertility drug enovid in five strains of mice, with particular regard to carcinogenesis.

PIP: The antifertility drug, Enovid, was tested for possible carcinogenicity in female mice of 5 specially selected strains: BALB/c, C3H, C3HfB, A, and C57BI. Enovid was chosen for testing since it is one of the most widely used oral contraceptives. The 5 strains of mice provided maximum genetic variation in the test animals. The drug was fed at 3 dose levels: 5 mcg/gm, 10 mcg/gm and 20 mcg/gm of food. The lowest dose did not prevent reproduction. The 10 mcg dose prevented some females from reproducing. The 20 mcg dose prevented all females from reproducing. The strains of mice differed in their response to Enovid. Weight gain was reduced in all strains. Effect on life-span varied, partly because of the tumors. Cervical and vaginal lesions showed invasion of the epithelium into the stroma but was limited, with few exceptions to the BALB/c females. In the BALB/c strain these lesions occurred in controls as well, but showed more progression and a higher incidence with the highest dose of Enovid. None of these lesions appeared grossly as tumors and none had extended beyond the vaginal wall or metastasized. They were observed only on histologic sections. Neither ovarian nor mammary gland tumors were increased in any strain. In the C3H strain such tumors seemed to be inhibited. In the C3HfB strain there was some inhibition of hepatomas and in the BALB/c strain some inhibition of adrenocortical adenoma. Chromophobe adenomas of the hypophysis were significantly increased in old C57BI females treated with the highest dose of Enovid. Offspring of Enovid-treated females showed no abnormalities. Enovid increased the occurrence and may have advanced the progression of epithelial lesions of the cervix and vagina of old BALB/c females. A study of the lesions in untreated females of this strain might help the understanding of carcinoma in situ in women and possibly the appearance of adeno-carcinoma of the vagina of young women whose mothers had been treated with stilbestrol during the first trimester to maintain pregnancy. Other neoplasms in this strain were not increased by the Enovid therapy. In the C3H strain mammary tumors were reduced by the Enovid and those that did occur were found later than in controls. Results from experimental animals should be applied to humans with care. Such results are of greatest value in directing attention to certain areas for investigation.^ieng

Changes in mammographic densities induced by a hormonal contraceptive designed to reduce breast cancer risk.

BACKGROUND: It has been known for some time that oral contraceptives substantially reduce the risk of endometrial and ovarian cancer, but they do not reduce the risk of breast cancer. A hormonal contraceptive regimen has been developed which uses a gonadotropin-releasing hormone against (GnRHA) to suppress ovarian function, and this regimen includes the administration of very low doses of both estrogen and progestogen. This hormonal contraceptive regimen attempts to minimize exposure of the breast epithelium to these steroids and to preserve the maximum beneficial effects of estrogen, while still preventing endometrial hyperplasia. PURPOSE: Our purpose was to determine whether changes occurred in mammographic densities between baseline and 1 year for women on this hormonal contraceptive regimen with reduced estrogen and progestogen levels compared with women in a control group. METHODS: Twenty-one women were randomly assigned in a 2:1 ratio to the GnRHA-based contraceptive group (14 women) or to a control group (seven women). The contraceptive group received the following: 7.5 mg leuprolide acetate depot by intramuscular injection every 28 days; 0.625 mg conjugated estrogen by mouth for 6 days out of 7 every week; and 10 mg medroxyprogesterone acetate orally for 13 days every fourth 28-day cycle. The control group received no medication. Baseline and 1-year follow-up mammograms of contraceptive and control subjects were reviewed in a blinded fashion by two radiologists. RESULTS: Comparison of the changes between the baseline and 1-year mammograms in the two groups of women showed significant (P = .039) reduction in mammographic densities at 1 year for women on the contraceptive regimen. Assessing the reduction in mammographic densities by noting the fineness of fibrous septae showed a highly significant (P = .0048) difference in the contraceptive regimen group. One of the women on the contraceptive regimen was withdrawn from the study because of poor compliance. CONCLUSION: The reduced estrogen and progestogen exposures to the breast that were achieved by the hormonal contraceptive regimen resulted in substantial reductions in follow-up mammographic densities at 1 year compared with baseline. Although there is no direct evidence that such a reduction in densities will lead to a reduced risk of breast cancer, indirect evidence for a protective effect of this regimen is that early menopause reduces breast cancer risk, and that menopause is associated with a reduction in mammographic densities.

PIP: In California, physicians randomly assigned 21 women aged 25-40 to either the contraceptive group or the control group as part of a study aimed to determine whether or not a hormonal contraceptive regimen with reduced estrogen and progestogen levels affects mammographic densities. Eligibility criteria included premenopausal women with a 5-fold greater than normal risk of breast cancer, no prior cancer, bone mineral density not less than 2 standard deviations below normal, normal cholesterol, and a normal physical and pelvic examination. The contraceptive group received intramuscular injection of 7.5 mg leuprolide acetate depot every 28 days, 0.625 mg oral conjugated estrogen for 6 out of 7 days every week, and 10 mg oral medroxyprogesterone acetate for 13 days every fourth 28-day cycle. The reduction in mammographic densities in women on the contraceptive regimen between baseline and 1 year was significantly different than that of the controls whose mammographic densities remained essentially the same (p = 0.039). Cases had significantly more change in fibrous septae between baseline and 1 year than did controls (+0.82 units vs. -0.07; p = 0.0048). These results indicate that lower levels of estrogen and progestogen reduces mammographic densities, which may reduce the risk of breast cancer since increased mammographic densities are linked to an increased risk of breast cancer. Reduced mitotic activity in breast epithelial cells during menopause and with lower levels of estrogen and progestogen (i.e., reduced mammographic densities) suggest that early menopause may also protect against breast cancer.

Breast cancer among young U.S. women in relation to oral contraceptive use.

BACKGROUND: While most studies have found no association between oral contraceptive use and breast cancer, several studies of younger women have reported an association with long-term oral contraceptive use. PURPOSE. We studied the relationship of patterns of oral contraceptive use to breast cancer risk among younger women. These women have had oral contraceptives available their entire reproductive lives and are now entering the breast cancer-prone years. METHODS: A population-based, case-control study of breast cancer was conducted in three counties in western Washington State among women born in 1945 or later, ages 21-45. Case patients were 747 women with breast cancer diagnosed in 1983-1990 and identified through the Seattle-Puget Sound Surveillance, Epidemiology, and End Results cancer registry. Control subjects were 961 women identified by random-digit telephone dialing. Subjects were interviewed in person, using pictures of brands of oral contraceptives and calendars of life events as recall aids. RESULTS: There was no increased incidence of breast cancer associated with ever having used oral contraceptives. Because only 8% of this cohort had never used oral contraceptives, short-term users (< 1 year) were combined with never users as the reference group for further analyses. A small increased risk of breast cancer was associated with long duration of oral contraceptive use (odds ratio for > or = 10 years = 1.3; 95% confidence interval [CI] = 0.9-1.9; P for trend = .03), particularly among women aged 35 years or younger (odds ratio for > or = 10 years = 1.7; 95% CI = 0.9-3.1). Breast cancer was also modestly related to oral contraceptive use early in reproductive life (odds ratio for use within 5 years of menarche = 1.3; 95% CI = 1.0-1.8; P for trend = .04) and to use of high-progestin-potency oral contraceptives for at least 1 year (odds ratio = 1.5; 95% CI = 1.1-2.1). These associations were adjusted for age, age at menarche, term pregnancy, induced abortion, and family history of breast cancer. The associations were not further confounded by case-control differences in education, religion, breast feeding of offspring, or infertility; in oral contraceptive contraindications, indications, or complications; or in measures of breast cancer detection such as mammography or breast biopsy. CONCLUSIONS: Long-term oral contraceptive use among young women or use beginning near menarche may be associated with a small excess breast cancer risk, possibly due to susceptibility to genetic damage in breast epithelial cells at ages of high breast cell proliferative activity. IMPLICATIONS: Future studies should investigate whether the patterns of risk we reported are present as this cohort ages.

PIP: A case control study was conducted in Washington among 21-45 year old white women from King, Pierce, and Snohomish counties (i.e., Seattle metropolitan area) to examine the relationship between oral contraceptive (OC) use and breast cancer. The 747 cases were diagnosed with invasive breast cancer between January 1983 and April 1990. The researchers combined short term OC users with never users since just 8% of all subjects had never used OCs. They controlled for age, age at menarche, term pregnancy, induced abortion, and family history of breast cancer. Longterm use (i.e., =or 10 years) of OCs was associated with a small increased risk of breast cancer (odds ratio [OR] = 1.3; p for trend = 0.03), especially among women not older than 35 years (OR = 1.7). This finding was consistent with results of other studies. OC use early in reproductive life (i.e., within 5 years of menarche) was also associated with a moderate increase in breast cancer (OR = 1.3; p for trend = 0.04). Breast cancer risk was also elevated among women who used high progestin potency OCs (as defined by the Dickey method for classifying OC potency) for at least 1 year (OR = 1.5). Case control differences in education, religion, breast feeding of children, or infertility; in OC contraindications, indications, complications; or in measures of breast cancer detection (e.g., mammography or breast biopsy) did not confound the associations. An association between breast cancer and long term OC use among young women and OC use beginning close to menarche suggest that puberty, a time when breast epithelial cells are undergoing considerable proliferative activity, are susceptible to genetic damage. Further research is needed to determine whether the aforementioned patterns of breast cancer risk continues as the cohort becomes older.

Effects of 17 beta-estradiol and medroxyprogesterone acetate upon MtTW15 mammosomatotropic pituitary tumor growth and hormone production in male and female rats.

The purpose of this study was to characterize the effects of two functionally diverse steroids, 17 beta-estradiol and medroxyprogesterone acetate (MPA), on MtTW15 rat mammosomatotropic pituitary tumor growth and hormone production. Steroid responsiveness, as well as the hormonally autonomous nature of the tumor, was studied by treating both male and female tumor-bearing rats for 7 weeks with weekly injections of either 17 beta-estradiol (600 ng/g body weight/week) or MPA (200 microgram/g body weight/week) and, subsequently, comparing both the tumor weights and the in vivo production of growth hormone (GH) and prolactin (PRL) among the treatment groups. Large tumors (6 to 20 gm) were obtained in all treatment groups, indicating hormonal autonomy; however, tumors were markedly smaller, on the average, in untreated males an ovariectomized females. Treatment of such rats with 17 beta-estradiol stimulated tumor growth. Radioimmunoassay of tumor and serum GH and PRL levels in all treatment groups indicated the following: (a) tumors from untreated male or female hosts did not favor the production of one hormone over the other to any great extent; (b) MPA, however, promoted significant increases (p less than 0.05) in GH production in both male and female tumor-bearing rats while having little effect on the production of PRL; and (c) 17 beta-estradiol significantly inhibited (p less than 0.05) GH production and promoted PRL production by tumors borne by either sex. Selected studies utilizing multiple doses of MPA (1 to 500 microgram per gm body weight per week) and 17 beta-estradiol (10 to 800 ng per gm body weight per week) were accomplished and demonstrated that hormone production can be influenced in a dose-related manner. These results indicated that the estrogen-induced MtTW15 rat pituitary tumor is hormonally autonomous, yet divergently responsive to two different classes of steroidal compounds, thus making this tumor line an appropriate model for the study of hormonally responsive pituitary tumor cells.

PIP: The purpose of this study was to characterize the effects of 2 functionally diverse steriods, 17beta-estradiol and (MPA) medroxyprogesterone acetate on MtTW15 rat mammosomatotropic pituitary tumor growth and hormone production. Steroid responsiveness, as well as the hormonally autonomous nature of the tumor, was studied by treating both male and female tumor-bearing rats for 7 weeks with weekly injections of either 17beta-estradiol (600 ng/g body weight/week) or MPA (200 mcg/g body weight/week) and, subsequently, comparing both the tumor weights and the in vivo production of (GH) growth hormone and (PRL) prolactin among the treatment groups. Large tumors (6 to 20 gm) were obtained in all treatment groups, indicating hormonal autonomy; however, tumors were markedly smaller, on the average, in untreated males and ovariectomized females. Treatment of such rats with 17beta-estradiol stimulated tumor growth. Radioimmunoassay of tumor and serum GH and PRL levels in all treatment groups indicated the following: (a) tumors from untreated male or female hosts did not favor the production of 1 hormone over the other to any great extent; (b) MPA, however, promoted significant increases (p 0.05) in GH production in both male and female tumor-bearing rats while having little effect on the production of PRL; and (c) 17-estradiol significantly inhibited (p 0.05) GH production and promoted PRL production by tumors borne by either sex. Selected studies utilizing multiple doses of MPA (1 to 500 mcg/gm body weight/week) and 17 beta-estradiol (10 to 800 ng/gm body weight/week) were accomplished and demonstrated that hormone production can be influenced in a dose-related manner. There results indicated that the estrogen-induced MtTW15 rat pituitary tumor is hormonally autonomous, yet divergently responsive to 2 different classes of steroidal compounds, thus making this tumor line an appropriate model for the study of hormonally responsive pituitary tumor cells.

On the epidemiology of oral contraceptives and disease.

PIP: Adverse and beneficial effects, especially with regard to mortality rates, of oral contraceptives (OC) are reviewed. In 1980 approximately 80 million women used OCs worldwide. OCs were first marketed in the United States in the 1960's, but by the 1980's low-dose combination pills with less estrogen and progesterone content became widespread along with the minipill, injectable preparations depo- medroxyprogesterone DMPA, and norethindrone containing capsules. Relative disease risk estimates are based on cohort studies and case- control studies. The Royal College of General Practitioners RCGP Oral Contraceptive Study of 1974 involved 46,000 women aged over 15 (50% were OC users, 50% were nonusers) the Oxford Family Planning Association Contraceptive Study of 1976 recruited 17,032 women aged 25-39, 56% of whom used OCs, and the Walnut Creek Contraceptive Drug Study of 1981 studied 16,638 women aged 18-54 of whom 28% were OC users and 33% were former users. A somewhat elevated mortality among ever-users of OCs in the order of 20% seems to be indicated by these studies mostly attributable to diseases of the circulatory system. Current OC use is also a risk factor in thrombotic stroke of the order of 4 or 5, but former use of OCs lowers the risk to 2. The effect of OC dose and formulation, duration of use, and predisposing factors on hemorrhagic and thrombotic stroke appears to be inconclusive with varying data from different studies. There is evidence for some increase in ischemic heart disease among current OC users, and also a 2-fold increase of myocardial infarction (MI) when smoking, serum cholesterol, and hypertension is taken into account, moreover higher estrogen dosage also contributes to a higher incidence of MI. There is also a 5-fold increase of venous thromboembolism among OC users induced by duration of use and estrogen potency, as OCs seem to promote atherogenesis, although the roles of progesterone and estrogen are conflicting. combination pills reduce the rate of endometrial cancer, provided protection against ovarian cancer, and do not seem to increase breast cancer incidence, although the relative risk of cervical cancer is elevated. Mortality risks with older OCs outweigh the benefits.^ieng

Contraception for the insulin-dependent diabetic woman: the view from one clinic.

Experience in a large diabetic clinic has confirmed the suspicion that insulin-dependent diabetic women are at considerably increased risk of thromboembolic disease if they take combined estrogen/progestogen oral contraceptive preparations. The most obvious alternative, an intrauterine device, is associated with an unexpectedly high failure rate, probably because of an unusual metabolic interaction with the diabetic endometrium. In a small group of diabetic women the progestogen-only pill was found to be a successful form of contraception not associated with any side effects except for menstrual irregularities. For most diabetic women the choice of contraceptive should therefore be between a progestogen-only pill and a mechanical method. Female sterilization and injectable progesterone each have their place in particular circumstances. Careful counseling of each patient is essential to ensure the best choice of contraceptive and correct application of the chosen method.

PIP: Experience in a clinic for diabetics is recounted in terms of successful methods of contraception for the insulin-dependent woman. Earlier reports of increased risk to side effects (especially thromboembolic disease) and failure in women with diabetes using combined (estrogen/progestin) oral contraception were confirmed. The failure rate could be lowered by allowing the women to adjust their insulin dose, but the incidence of thrombotic disorder remains high. Of 120 insulin-dependent women taking the combined pill (compared with 156 nonuser diabetics) 6 patients had thrombotic episodes, whereas none of the controls did. The use of IUDs is discouraged among diabetic women because of an extremely high failure rate, probably caused by an unusual metabolic interaction with the diabetic endometrium. In this clinic, a small (n=45) group of women was given progesten-only contraceptives (norethisterone, .35 mg orally) and, of the 29 completing over a year on the preparation, 15 have had fairly regular bleeding and 14 have experienced very irregular cycles. Aside from the menstrual irregularity, the progesten-only pill proved successful; no pregnancies have occurred. This method is the recommended one for diabetic women. Equally successful with proper fitting and instruction were mechanical methods. Sterilization is only indicated when the family is completed or pregnancy is absolutely contraindicated.

Grwoth hormone, prolactin, and cortisol in dogs developing mammary nodules and an acromegaly-like appearance during treatment with medroxyprogesterone acetate.

PIP: Concentrations of (GH) growth hormone, (PRI) prolactin, cortisol, progesterone, and (MPA) medroxyprogesterone acetate were determined by RIA in blood sera collected from beagle bitches 17 months after initiating treatment with MPA (75 mg/kg.3 months; n = 12) MPA vehicle (controls; n = 12), or progesterone implants which produced physiological levels of progesterone (13.8 + or - 2.1 ng/ml; n = 12). In the MPA-treated bitches, mean MPA levels were 104 + or - 6 ng/ml, mean GH levels were elevated (9.5 + or - 3.0 vs. 0.4 + or - 0.1 ng/ml; P 0.01); mean PRL levels were unchanged (13.7 + or - 2.8 vs. 12.6 + or - 1.2 ng/ml); and mean cortisol levels were suppressed (1.7 + or - 0.2 vs. 13.7 + or - 1.4 ng/ml; P 0.01) in comparison to those in control animals. None of these parameters was significantly affected by progesterone treatment. External signs of an acromegaly-like condition and large mammary gland nodules (diameters, 5 mm) were noted in, and limited to, 9 bitches with elevated ( 2.5 ng/ml) GH levels (12.8 + or - 3.0 ng/ml). These were 8 MPA-treated bitches which developed the acromegal-like condition during treatment and 1 progesterone-treated bitch which appeared acromegalic before treatment and in which the condition was considered to have developed spontaneously. The data suggest that the acromegaly-like changes and large mammary nodules in dogs administered the contraceptive progestin MPA occurred as a result of MPA-induced elevations in GH. The results do not preclude the possibility that the MPA-induced suppression of cortisol and/or the direct action of MPA on the mammary glands also contributed to mammary nodule formation. MPA-treated dogs may also provide a unique experimental model for studying chronic elevations in endogenous GH levels and for testing compounds for their ability to suppress GH levels.^ieng

Aromatization of norethindrone to ethinyl estradiol by human placental microsomes.

The interaction of 19-norethindrone [4-estren-17 alpha-ethinyl-17 beta-ol,3-one (NET)] with human placental microsomes was investigated using enzymatic and spectral techniques. The incubation of [6,7-3H]norethindrone with human placental microsomes, NADPH, and molecular oxygen resulted in the production of ethinyl estradiol [1,3,5-(10)estratrien-17 alpha-ethinyl-3,17 beta-diol (EE)]. The reaction was linear with respect to time and protein concentration. Androstenedione inhibited the enzymatic aromatization of NET to EE. The product was identified by thin layer chromatography, recrystallization to constant specific activity, and derivative formation. No acid or base was used in any step of product identification. To ensure that spontaneous aromatization of metabolites of NET did not contribute to our results, representative samples were treated with sodium borohydride before processing. Sodium borohydride reduces the 4-en-3-one grouping of the A-ring, thereby preventing chemical aromatization. Sodium borohydride treatment did not reduce our observed yields of EE from NET. The addition of NET to a preparation of solubilized, partially purified placental microsomal cytochrome P-450 yielded a type I cytochrome P-450 binding spectrum. The apparent spectral dissociation constant for NET binding to cytochrome P-450 was 28 microM. These results suggest that NET is enzymatically aromatized to EE by human placental microsomes.

Changes in physiologically free circulating estradiol and testosterone during exposure to levonorgestrel.

PIP: The biological activity of circulating sex steroids is dependent upon the relative binding to (SHBG) sex hormone-binding globulin. Only that fraction which is not specifically bound to the high affinity binding protein is available to the receptors or for metabolism. (D-Ng) Levonorgestrel, a synthetic gestagen used for contraception decreases the hepatic production of SHBG. Volunteers wearing contraceptive vaginal rings containing (E2) estradiol and d-Ng were studied to determine the changes in sex steroid binding which occur when d-Ng is administered in a sustained release fashion. The % of steroids not specifically bound to SHBG was measured by fractionating the serum proteins with ammonium sulfate precipitation. A binding capacity assay was used to quantitate SHBG. During the normal menstrual cycle in control subjects, the unbound fractions of both E2 and (T) testosterone remained constant at about 25% of total E2 and 10% of total T. During treatment with d-Ng, however, the % of unbound E2 increased to about 80% of the total, and that of T increased to about 55% of the total, significantly greater than that in the control cycles (p 0.01). SHBG was constant during the normal menstrual cycle, averaging 85.9 + or - 1.92 nM but was suppressed during the administration of d-Ng to 10.0 + or - 2.6 nM. When SHBG concentration was greater than 50 nM, the % binding of both E2 and T were independent of the concentration of this binding protein. When SHBG was suppressed below 50 nM, the % binding of E2 and T was directly related to the concentration of the binding protein. The affinity of SHBG for d-Ng allows competition with E2 and T for SHBG-binding sites at concentrations of SHBG below 50 nM. The increase in physiologically free E2 and T, therefore, may be a result of both the suppression of SHBG concentration by d-Ng as well as the competition between d-Ng and endogenous sex steroids for the decreased number of available binding sites on SHBG.^ieng

The effect of medroxyprogesterone acetate on the pituitary-adrenal axis.

Twelve cancer patients and one patient with diabetes mellitus were treated with medroxyprogesterone acetate (MPA) by intramuscular injection in a total weekly dose of 400, 700, or 1200 mg. The treatment reduced the plasma cortisol concentration by 76% in the AM hours (21 leads to 5.0 mug/dl) and by 75% in the PM hours (12.8 leads to 3.2 mug/dl). Cortisol production rate decreased by 67% (19 leads to 6.2 mg/24 hrs). The 24 hour profile of plasma cortisol concentration measured in 3 patients showed zero secretion over this period. Low plasma ACTH values prevailed during treatment, and a blunted response to maximal ACTH stimulation was found. No evidence of adrenal insufficiency was observed in any patient, even though in some patients the plasma cortisol concentration remained at zero for many weeks. MPA has cortisol-like effects and the suppression of adrenal function is probably mediated by a negative feedback action on the hypothalamus or pituitary.

PIP: The effects of medroxyprogesterone acetate (MPA), administered im in a total weekly dose of 400, 700, or 1200 mg, on the pituitary-adrenal axis were studied in 12 cancer patients and 1 patient with diabetes mellitus. MPA reduced mean A.M. plasma cortisol concentrations by 76% and mean P.M. cortisol concentrations by 75%. The 24-hour production rate of cortisol was reduced by 67%. 41% of the individual A.M. cortisol determinations and 61% of the individual P.M. determinations were not significantly (p greater than .001) different from 0. Plasma ACTH levels were low throughout treatment, and the response to maximal ACTH stimulation was not pronounced. Evidence of adrenal insufficiency was not observed in any of the patients. The results demonstrate the corticosteroid replacement properties of MPA, and it is suggested that the suppression of adrenal function is most likely mediated by a negative feedback action on the hypothalamus or pituitary.

The circadian pattern of melatonin and its positive relationship with progesterone in women.

To investigate the possible role of melatonin in the regulation of the human menstrual cycle, the circadian pattern of melatonin was determined in the follicular and luteal phases of 10 normal women. Four-hourly sampling was used to derive a melatonin index which described the total exposure to melatonin for 24 h. This sampling procedure adequately represented the circadian melatonin output and demonstrated that pulses of melatonin secretion, inconsistent with a measured half-life of 47 min, did not exist. A significant increase (P less than 0.001) in the melatonin index was found in the luteal phase compared to that in the follicular phase. To investigate the influence of exogenous progestins on the melatonin pattern, repeated 24-h profiles were measured in 8 women taking the 3-phase contraceptive pill. There was a significant increase (P less than 0.01) in the melatonin index associated with an increase in the dose of progestin. These results are consistent with a positive relationship between melatonin and progesterone and suggest that changes in the circadian pattern of melatonin secretion, rising during the luteal phase with a fall before ovulation, may act as a modulator of cyclicity.

PIP: 10 normal women participated in a study designed to investigate the possible role of melatonin in the regulation of the human menstrual cycle. The circadian pattern of melatonin was determined in the follicular and luteal phases of the study subjects who had conventional life styles and normal sleep/wake rhythms. Also investigated was the influence of exogenous steroids on the circadian pattern of melatonin, using subjects who were taking a 3-phase oral contraceptive (OC). Initially, 2 studies were performed to determine the frequency of sampling necessary to define the circadian profile of melatonin and to determine the relevance of that sampling frequency to the half-life of melatonin. 3 routes of administration were employed: 2 mg melatonin as a 0.04% solution in corn oil containing 2% ethanol, taken orally as a suspension in 50 ml milk; 2 mg melatonin in an 8% solution of ethanol in a nasal spray; and 2 mg melatonin in 250 mg ascorbic acid, taken orally in a gelatin capsule. A program of 4-hourly sampling was adopted. The 10 healthy women ranged in age from 20-40 years and had normal menstrual cycles. A circadian pattern of serum melatonin concentrations was observed in all study subjects. Figure 1 shows the 24-hour profiles of melatonin in 1 woman studied in the follicular and luteal phases of her menstrual cycle. The concentrations of melatonin in samples taken at intervals of 15 minutes for a 24-hour period indicated that episodic release did not take place. The increase in the melatonin index from the follicular to the luteal phase was highly significant for the group as a whole. The investigation of other aspects of the circadian pattern of melatonin showed a significant increase in the luteal phase compared to the follicular phase in peak serum melatonin concentrations, estimated from data at 4-hourly intervals. The mean duration of elevation was 11.6 +- 0.8 h in the luteal phase and 10.7 +- 0.7 h in the follicular phase. All subjects had a single peak concentration, at either 2400 or 0400 h. A change in the melatonin index was found in the 8 women using the 3-phase OC pill when sampled at 2 times during the cycle. A significant increase in the melatonin index was associated with an increase in the dose of progestin, either from no progestin intake to a low-dose or from a low-dose to a higher dose.

Desogestrel- and levonorgestrel-containing oral contraceptives have different effects on urinary excretion of prostacyclin metabolites and serum high density lipoproteins.

Prostacyclin synthesis is stimulated in vitro by high density lipoproteins (HDL), which themselves are differently affected by desogestrel (DG)- and levonorgestrel (LN)- containing oral contraceptives. In this study we measured the urinary excretion of the metabolites of prostacyclin [6-keto-prostaglandin F 1 alpha(6-keto) and 2,3-dinor-6-keto-prostaglandin F1 alpha (dinor)] and of thromboxane A2 [thromboxane B2 (TxB2)] as well as serum HDL- and HDL2 cholesterol concentrations before and during DG and LN administration alone or in combination with ethinyl estradiol (EE) in 26 women. Before the trial, urinary dinor excretion correlated with serum total HDL cholesterol (r = 0.499; P less than 0.01) and HDL2 cholesterol levels (r = 0.668; P less than 0.001; n = 26). Administration of DG (150 micrograms/day; 14 women) or LN (150 micrograms/day; 12 women) for 2 weeks caused no changes in the excretion of these prostanoids, but LN administration decreased serum HDL cholesterol levels. After that, the women underwent a monophasic regimen of 150 micrograms DG or LN plus 30 micrograms EE for 3 months and thereafter polyphasic regimens of the same steroids for a further 3 months. The DG-containing pills increased urinary dinor excretion by 25-40%, but caused no changes in 6-keto and TxB2 excretion, as measured on days 19-21 of the cycles. LN-containing pills reduced urinary 6-keto excretion by 22% at the end of polyphasic treatment, but caused no changes in dinor and TxB2 output. DG plus EE, but not LN plus EE, increased serum total HDL and HDL2 cholesterol concentrations by a maximum of 25%. Thus, a DG plus EE combination may stimulate PGI2 synthesis by increasing the levels of HDL/HDL2. Theoretically, this stimulation protects against occlusive vascular disorders.

PIP: The effects of desogestrel or levonorgestrel alone and in combination with ethinyl estradiol on the urinary excretion of metabolites of antiaggregatory prostacyclin (PGI2) and thromboxane A2 (TxA2) and on serum high density lipoprotein (HDL) and HDL2 cholesterol concentrations were investigated in 26 women. Baseline urinary 6-keto, dinor, and TxB2 excretion and serum HDL or HDL2 cholesterol concentrations did not differ between study groups. Administration of desogestrel and levonorgestrel alone for 2 weeks caused no changes in PG excretion, but it reduced serum HDL and HDL2 cholesterol concentrations. The desogestrel-estradiol combination was accompanied by 40% and 25% rises in urinary dinor excretion and 20% and 15% rises in urinary 6-keto and dinor excretion at the end of the monophasic and polyphasic regimens, respectively, but no significant changes in urinary TxB2 excretion. The levonorgestrel-estradiol combination produced a 22% decrease in urinary 6-keto excretion during polyphasic treatment, but led to no change in the excretion of the other prostanoids. Both monophasic and polyphasic levonorgestrel and estradiol administration lowered serum HDL and HDL2 cholesterol levels, while monophasic desogestrel plus estradiol increased serum HDL2. The mean relative changes in serum HDL2 cholesterol and urinary dinor excretion were parallel in users of desogestrel plus estradiol, suggesting that a serum HDL cholesterol increase induced by this regimen could be related to increased vascular PGI2 production. Although the mechanism that causes increases in PGI2 and HDL during desogestrel-estradiol administration remains unknown, such a combination has the potential to reduce the risk of occlusive-thrombotic vascular disorders--currently the most serious side effect of oral contraceptive use.

Therapeutic controversy: The safety of third-generation oral contraceptives.

PIP: This paper presents the contributions of Drs. Barbieri, Speroff, Walker, and McPherson to the debate on the safety of third-generation oral contraceptives (OCs). In the overview by Barbieri, the issues of how to assess the quality of the evidence and developing a meaningful framework for integrating an analysis of the benefits and risks of a contraceptive hormone are discussed. He also reflects on the leadership role the endocrinologist must assume in better defining the pharmaco-endocrinology of gestodene and desogestrel. Speroff concludes that modern low-dose OCs are very safe. It was noted that the use of OCs yields an overall improvement in individual health, and from a public health point of view the collection of effects associated with OCs leads to a decrease in the cost of health care. In contrast, Walker argues that low-estrogen OCs containing desogestrel and gestodene carry a modestly elevated risk of venous thromboembolism in comparison to other low-estrogen OCs in widespread use. This statement is supported by McPherson, who emphasizes that the risk associated with OCs is real and should be avoided.^ieng

Nuclear progestin receptors in normal and malignant human endometrium.

PIP: The use of medroxyprogesterone acetate (MPA) was incorporated into a nuclear receptor assay for progestin receptor in human endometrium. The assay was developed because MPA is a better ligand than progesterone since it does not bind to corticosteroid-binding globulin and gives greater kinetic stability to the nuclear complex. The MPA nuclear receptor complex for malignant endometrium dissociated at a faster rate than did the complex obtained from normal endometrium, an alteration in binding kinetics which could not be explained by instability of the receptors from malignant endometrium. Factors, including radiation therapy, plasma proteins, endogenous steroids, receptor degradation, tissue heterogeneity, and limited sample size, which influence the interpretation of receptor assay were systematically evaluated. In spite of these controls, it would be premature to conclude that the clinical observations indicate altered receptor from malignant tissue. Further studies are required on endometrial carcinoma which is free of normal tissue fragments. Clinically, nuclear receptor levels were highest in normal endometrium but decreased in samples of malignant endometrium as tumors became more anaplastic. The lowest nuclear binding activity was detected in samples of metastatic endometrial tissue (carcinoma). Hopefully, this nuclear receptor assay (which uses MPA because its dissociation was slower than progesterone) will provide data for correlating clinical response to therapy.^ieng

Influence of medroxypregesteroneacetate on testosterone metabolism by cultured human fibroblasts: a model for drug-steroid interaction.

PIP: Medroxyprogesteroneacetate (MPA) was used to study drug-steroid interaction in an in-vitro cell culture system of human skin fibroblasts from prepubertal children. MPA did not alter testosterone utilization in 9 of the 10 cell lines studed. The addition of MPA inhibited the formation of androstanediol by nearly 53% suggesting an inhibition of 3 alpha-hydroxysteroid dehydrogenase. In 3 cell lines, dihydrotestosterone increased.^ieng

Serum medroxyprogesterone acetate (MPA) concentrations and ovarian function following intramuscular injection of depo-MPA.

A sensitive radioimmunoassay measuring serum medroxyprogesterone acetate (MPA) has been developed in order to measure and correlate serum MPA concentrations and ovarian function in women following im administration of deop-MPA (DMPA), employing goat anti-MPA-3-(O-carboxymethyl) oxime-bovine serum albumin and MPA-3-(O-carboxymethyl) imino-125I-iodohistamine. In the 3 women studied, im injection of 150 mg of DMPA yielded brief initial serum MPA concentrations ranging from 1.5 to 3 ng/ml for a few days. Serum MPA concentrations gradually declined and remained relatively constant at about 1 ng/ml for 2 to 3 months, declined gradually thereafter reaching 0.2 ng/ml during the 6th month and became undetectable (less than 0.02 ng/ml) about 7-1/2 to 9 months following administration. Serum estradiol remained at early to midfollicular phase levels for 4 to 6 months after DMPA injection and rose to preovulatory levels when serum MPA levels fell below 0.5 to 0.25 ng/ml. Ovulation, however, as evidenced by serum progesterone concentrations did not occur, apparently due to suppression of the LH peak by positive feedback inhibition. Prolonged inhibition of cyclic ovarian function following DMPA injection is caused by slow MPA absorption and persists until serum MPA levels have decreased below 0.1 ng/ml or become undetectable about 7 to 9 months after DMPA administration.

PIP: Serum medroxyprogesterone acetate (MPA) concentrations were measured by a newly developed, highly sensitive, radioimmunoassay technique, and ovarian function was evaluated in 3 women after a single im injection of MPA (150 mg). Initial MPA concentrations ranged from 1.5 to 3 ng/ml for the 1st few days, and then gradually declined to a relatively constant level of about 1 ng/ml for 2-3 months. During the 6th month, MPA concentrations had declined to about .2 ng/ml, and became undetectable between 7!-9 months. Serum estradiol levels remained at early or midfollicurlar phase values for 4-6 months following injection, and then increased to preovulatory values (.25-.5 ng/ml). However, ovulation did not occur as determined by serum progesterone concentrations. Prolonged suppression of ovarian function by depo-MPA is due to the slow release of MPA; from the injection site.