Maintaining physiological testosterone levels by adding dehydroepiandrosterone to combined oral contraceptives: I. Endocrine effects.

OBJECTIVE: To determine whether adding dehydroepiandrosterone to combined oral contraceptives (COCs) maintains physiological levels of free testosterone. STUDY DESIGN: A randomized, double-blind, placebo-controlled, two-way crossover study conducted in 81 healthy women (age range: 20-35 years; Body mass index (BMI) range: 18-35 kg/m2) using oral contraceptives. Androgens, sex hormone-binding globulin (SHBG), estradiol (E2) and estrone (E1) were measured, and free testosterone and the free testosterone index were calculated. Subjects discontinued oral contraceptive use for at least one menstrual cycle before being randomized to receive five cycles of ethinyl estradiol (EE) combined with either levonorgestrel (EE/LNG group) or drospirenone (EE/DRSP group) together with either dehydroepiandrosterone (DHEA) (50 mg/day orally) or placebo. Subsequently, all subjects crossed over to the other treatment arm for an additional five cycles. RESULTS: Both COCs decreased the levels of all androgens measured. Significant decreases (p<.05) were found with EE/LNG and EE/DRSP for total testosterone (54.5% and 11.3%, respectively) and for free testosterone (66.8% and 75.6%, respectively). Adding DHEA to the COCs significantly increased all androgens compared to placebo. Moreover, including DHEA restored free testosterone levels to baseline values in both COC groups and total testosterone levels to baseline in the EE/LNG group and above baseline in the EE/DRSP group. SHBG concentrations were significantly higher with EE/DRSP compared to EE/LNG (p<.0001). The addition of DHEA did not affect the levels of SHBG. CONCLUSIONS: Taking COCs reduces total and free testosterone levels and increases SHBG concentrations. By coadministration with DHEA, physiological levels of total and free testosterone are restored while using EE/LNG. With EE/DRSP, only the free testosterone level is normalized by DHEA coadministration. IMPLICATIONS: A daily oral dose of 50-mg DHEA maintains physiological free and total testosterone levels in women who are using an EE/LNG-containing COC.

On the possible role of mammary-derived growth hormone in human breast cancer.

The incidence of breast cancer has risen worldwide, especially in countries where it used to be low, very probably as a result of economic prosperity and changes in life-style. In women, the available data have resulted in the concept of progression from normal breast development to cancer through precursor lesions sensitive to hormones and growth factors that can be produced locally in the mammary gland, acting as paracrine or autocrine stimulating agents. The local endocrine environment in the breast can be different from the situation in the circulation. In the dog, growth hormone (GH) can be produced locally in the mammary glands and its production can be stimulated by progestins. This GH probably plays a paracrine role in the progesterone-induced proliferation and differentiation of mammary epithelium. There is increasing evidence that the local mammary progestin/GH-axis is operational not only in dogs but also in human breast cancer. No data are yet available on the production of mammary-derived GH in women.

Gene polymorphisms that may influence the biological effects of progestins.

Many of the biological actions of progestins depend on binding to intracellular receptors and through a long chain of events to subsequent stimulation of transcriptional activity and protein synthesis. This process requires at least a few hours in time and many different proteins called coregulators do play a role after binding to the receptor. Evidence for polymorphisms in the gene coding for the PR has been obtained and many studies have already attempted to show associations between particular polymorphisms and human diseases. However, at present no consistent and conclusive picture has emerged on clinically important associations. Studies on links between polymorphisms in genes coding for coregulators are just beginning. The second pathway, the so-called non-genomic actions, is related to rapid effects of progestins that occur within minutes. At this moment a number of different membrane bound receptors have been identified. No data are available yet on polymorphisms in genes coding for these proteins or to link any of these membrane receptors to specific human pathology.

Classification and pharmacology of progestins.

Besides the natural progestin, progesterone, there are different classes of progestins, such as retroprogesterone (i.e. dydrogesterone), progesterone derivatives (i.e. medrogestone) 17alpha-hydroxyprogesterone derivatives (i.e. chlormadinone acetate, cyproterone acetate, medroxyprogesterone acetate, megestrol acetate), 19-norprogesterone derivatives (i.e. nomegestrol, promegestone, trimegestone, nesterone), 19-nortestosterone derivatives norethisterone (NET), lynestrenol, levonorgestrel, desogestrel, gestodene, norgestimate, dienogest) and spironolactone derivatives (i.e. drospirenone). Some of the synthetic progestins are prodrugs, which need to be metabolized to become active compounds. Besides the progestogenic effect, which is in common for all progestins, there is a wide range of biological effects, which are different for the various progestins and have to be taken into account, when medical treatment is considered.

Long-term effects of progestins on bone quality and fractures.

The effects of progestins on the quality of bone and their influence on the risk of fractures are reviewed. Data discussed are based on experimental studies in vivo that generally lasted for longer than one year. Information is given on the background of osteoporosis and on several means of inducing changes in bone quality. In young women who start using oral contraceptives based on progestins alone shortly after pubertal development, a significant decrease in bone quality has been documented. World Health Organization experts have concluded that this is not a real argument for restrictions on the use of these contraceptives. In postmenopausal women, no evidence has been found for a bone-protective or an estrogen-antagonistic effect of progestins. A wide range of estrogens have been used that have shown positive effects on bone, which are not antagonized by progestins. The therapeutic use of high-dose megestrol acetate may result in marked negative effects on bone, leading to severe osteoporosis, possibly due to the inherent glucocorticoid activity of this progestin. Other pharmacotherapeutic agents that can be used in postmenopausal therapy, and that clearly have beneficial effects on bone, are discussed.

Gene polymorphisms involved in the regulation of bone quality.

Osteoporotic fractures in subjects at advanced age constitute a tremendous and growing problem. Established lifestyle risk factors can explain only a modest proportion of the liability to osteoporotic fractures. Bone mineral density (BMD) is considered the best established risk factor for osteoporotic fractures. The importance of genetic factors in the quality of bone is substantial, but no consensus exists yet on the genes that are involved. However, concomitant diseases, balance disorders and lifestyle habits are more important for fractures in elderly subjects. The abundance of common sequence variations, so-called polymorphisms, in the human genome and their high frequency in the population have made them targets to explain variation in the risk. Some genes have been identified that appear to be involved in the regulation of bone mass and in the pathogenesis of osteoporosis. Among these are those coding for the two estrogen receptors (ERalpha and ERbeta), the androgen receptor (AR) and the vitamin D receptor (VDR). In addition, enzymes involved in the biogenesis of estrone and estradiol have attracted attention as well as polymorphisms in the regulatory region of the type I collagen gene, COLIA1, affecting the binding site for the transcription factor Specificity protein 1 (Sp1). Although evidence suggests that the quality of bone is determined to a large extent by genetic factors, research so far has not been able to unequivocally identify genes involved in this matter. Over the last years a large number of studies have pointed to the variability in many genes and their relation with BMD, bone-related symptoms or specific therapies. The findings emphasize the complexity of the genetics of bone mass and bone loss.

Feasibility study of new calibrators for thyroid-stimulating hormone (TSH) immunoprocedures based on remodeling of recombinant TSH to mimic glycoforms circulating in patients with thyroid disorders.

BACKGROUND: Differences between the glycosylation patterns of a pituitary thyroid-stimulating hormone calibrator (pitTSH) and serum samples have been shown to be responsible for nonidentical epitope expression and for introducing discrepancies in TSH measurements. We studied the feasibility of developing new candidate reference materials by remodeling recombinant TSH (recTSH) to generate potential mimics of serum TSH. METHODS: Terminal sialylation and/or inner fucosylation of recTSH were remodeled by a combination of enzyme treatments followed (or not) by lentil lectin-Sepharose affinity chromatography. The resulting TSH preparations were screened for epitope similarity in 23 immunoassays mapping 3 antigenic clusters common to the pitTSH 2nd International Reference Preparation (IRP) and the recTSH 1st IRP and then challenged against a pool of 63 patients with increased serum TSH (>60 mIU/L). RESULTS: pitTSH was poorly correlated with serum TSH, with a mean (SD) slope of 2.124 (0.001), in contrast to recTSH [slope, 1.178 (0.056)]. Comparison of variably sialylated preparations with recTSH gave slopes of 0.860 (0.057) for desialylated TSH, 1.064 (0.057) for alpha2,3/6-oversialylated recTSH, and 0.953 (0.033) for alpha2,6-resialylated recTSH, indicating that TSH forms enriched in sialic acid closely resemble serum TSH. Further testing against serum TSH showed satisfactory agreement with both TSH preparations containing alpha2,6-sialic acid [slopes, 1.064 (0.057) and 0.953 (0.033)], particularly in the absence of nonfucosylated forms [0.985 (0.044)]. CONCLUSIONS: Glyco-engineered recTSH preparations enriched in sialic acid and inner fucose are promising candidates for future reference materials. These preparations may have advantages over existing preparations used for standardizing TSH measurements.

Relationships between sex hormones assessed in amniotic fluid, and maternal and umbilical cord serum: what is the best source of information to investigate the effects of fetal hormonal exposure?

Levels of testosterone (T) (total and free), androstenedione (A4), dehydroepiandrosterone sulphate (DHEAS), sex hormone-binding globulin (SHBG), and estradiol (E2) were measured by radioimmunoassay (RIA) in 156 normal pregnancies (77 male and 79 female fetuses). Samples were obtained from amniotic fluid, 2nd and 3rd trimester maternal serum, and umbilical cord serum at birth. During the critical period of brain differentiation, at the beginning of the second trimester of pregnancy, sex differences in T and A4 were found in amniotic fluid and not in maternal serum. This finding adds to the fact that mostly low and nonsignificant correlations were found for the different androgenic hormones between levels assessed in amniotic fluid and maternal plasma at this particular and very sensitive period of fetal brain development. On the other hand, high correlations were found for the same hormones between the samples of maternal serum in the 2nd and the 3rd trimester. Our data show that, of all available sources, amniotic fluid seems to be the best candidate to investigate the effects of early fetal androgen exposure.

Local biosynthesis and metabolism of oestrogens in the human breast.

The origin of oestrogens at the level of the breast itself is discussed. In particular in postmenopausal women an accumulation of oestradiol at the site of breast tumours has been documented by a number of independent studies. The mechanism behind the high local oestrogens concentrations is thought to be the in situ production of these steroids by local processes with androstenedione as the main precursor. The presence of all enzymes required for this production has been demonstrated in a large proportion of breast tumours, with probably aromatase, hydroxysteroid dehydrogenase type 1 and sulfatase as the most important enzymes leading to the biologically highly active oestradiol. The individual enzymes that are relevant for the biosynthesis and the metabolism of oestrogens are discussed. The conclusion is reached that a number of these local processes may be involved in the promotion of premalignant lesions and in stimulation of growth of malignant tumours in the human breast.

CYP17 and CYP19 genetic polymorphisms in endometrial cancer: association with intratumoral aromatase activity.

Excessive estrogenic influence is known to be associated with initiation/promotion of endometrial cancer (EC). Allelic polymorphisms of the genes involved in steroidogenesis/steroid metabolism may contribute to EC susceptibility. It is important to know endocrine mechanisms by which such susceptibility is acquired. Here, we compared CYP19 (aromatase) and CYP17 (17alpha-hydroxylase/17,20-lyase) gene polymorphisms correspondingly in 136 and 165 EC patients and in 116 and 188 non-affected women primarily of postmenopausal age. In these expanded studies we confirmed our previous observations that genotypes with longest alleles of CYP19 (A6 or A7) are over-represented (64.7+/-4.0 vs. 49.1+/-4.6%, P = 0.04, and 11.0+/-2.7 vs. 1.7+/-1.2%, P = 0.01)) and A2/A2 CYP17 genotype is under-represented (12.1+/-2.5 vs. 25.0+/-3.2%, P = 0.001) in patients as compared to controls. Additionally, aromatase activity was studied by tritiated water release assay in tumor tissues of 32 EC patients. In carriers of A2/A2 CYP17 genotype this activity was significantly lower than in carriers of A1/A1 genotype or in combined group of A1/A1 and A1/A2 CYP17 carriers (P = 0.04 in both cases). On the other side, intratumoral aromatase activity demonstrated tendency to higher values in carriers of longest CYP19 alleles (A6A6 and A6A7) than in carriers of all other CYP19 allele variants (P = 0.066). Thus, specific set of genetic polymorphisms (carrying of CYP17 A1 allele and combination of longest A6 or A7 CYP19 alleles) may predispose to the induction of higher rate of local estrogen biosynthesis in malignant endometrium, that in its turn may support growth of the latter. Further studies are warranted to connect revealed regularities with the type I or II of EC.

Endogenous sex hormones in men aged 40-80 years.

OBJECTIVE: To determine the relationship between aging, life-style factors and health-related factors and endogenous sex hormone levels. DESIGN: Cross-sectional study of 400 independently living men between 40 and 80 Years of age. METHODS: After exclusion of subjects who were not physically or mentally able to visit the study center, 400 men were randomly selected from a population-based sample. Total testosterone (TT), bioavailable testosterone (BT) (i.e. not bound to sex hormone-binding globulin (SHBG)), SHBG, estradiol (E(2)) and dehydroepiandrosterone-sulfate (DHEA-S) were investigated for their relationship with age, body mass index (BMI), waist circumference, smoking, physical activity and general health status. Multivariate models using ANCOVA analyses were used to examine the contribution of life-style factors to sex hormone variability. RESULTS: TT, BT and DHEA-S decreased with age; 0.2, 0.7 and 1.2%/Year respectively. SHBG showed an increase with age of 1.1%/Year. No changes with age were found for E(2). General health status modified the association of TT and SHBG with age (P interaction 0.10 and 0.002 respectively). Increased BMI and waist circumference were associated with decreased TT, BT, SHBG and DHEA-S and increased E(2) (all P<0.01). Current smoking, lower alcohol intake and a higher physical activity score were associated with higher TT and SHBG levels. CONCLUSION: This study showed the important determinants of sex hormones were age, BMI, waist circumference, smoking, general health status and physical activity. Furthermore, it can be concluded that general health status modified the effect between sex hormones and age. For future observational studies it should be taken into account that the above-mentioned determinants may alter the association between sex hormones and diseases and related conditions.

The relationship between aromatase in primary breast tumors and response to treatment with aromatase inhibitors in advanced disease.

Aromatase inhibitors are proving to be more effective than tamoxifen for postmenopausal patients with breast cancer. Estrogen concentrations in the breast are similar in both premenopausal and postmenopausal women, and several fold higher than circulating levels in postmenopausal women. In order to investigate the importance of intratumoral aromatase in stimulating the proliferation of the tumor, we used immunocytochemistry to determine the extent of aromatase expression in relationship to the response of the patient to aromatase inhibitor treatment. The relationship between positive staining for aromatase in the primary tumor and response to treatment with an aromatase inhibitor was investigated in a retrospective study of 102 patients with advanced breast cancer. Immunohistochemical staining using a monoclonal antibody against aromatase was performed on paraffin embedded tumor tissue. Response was evaluated using UICC criteria. Nine out of 13 patients with objective response to treatment stained positive and 49 of 89 patients with stable or progressive disease stained positive. No significant relationship between positive staining and objective response to treatment could be found. When patients with 'clinical benefit' (i.e. objective response plus prolonged stable disease of at least 6 months) were considered, also no relationship could be found. Further analysis of subgroups with positive hormone receptors, treatment with newer generation aromatase inhibitors, single metastatic site, non-visceral metastases and previous treatment only with tamoxifen did not show any relationship. Tumor aromatase expression did not correlate with response of patients with advanced breast cancer to aromatase inhibitor treatment. Most patients had relapsed from other treatments before receiving an aromatase inhibitor. It seems likely that many of these patients had tumors that may have progressed to hormone independence at this stage of the disease. Research in patients who have received treatment with aromatase inhibitors in earlier stages of disease (first line and adjuvant treatment) may provide further information on the relationship between tumor aromatase, steroid receptors and response to inhibitor treatment.

Classification and pharmacology of progestins.

Besides the natural progestin, progesterone, there are different classes of progestins, such as retroprogesterone (i.e. dydrogesterone), progesterone derivatives (i.e. medrogestone) 17alpha-hydroxyprogesterone derivatives (i.e. chlormadinone acetate, cyproterone acetate, medroxyprogesterone acetate, megestrol acetate), 19-norprogesterone derivatives (i.e. nomegestrol, promegestone, trimegestone, nesterone), 19-nortestosterone derivatives norethisterone (NET), lynestrenol, levonorgestrel, desogestrel, gestodene, norgestimate, dienogest) and spironolactone derivatives (i.e. drospirenone). Some of the synthetic progestins are prodrugs, which need to be metabolized to become active compounds. Besides the progestogenic effect, which is in common for all progestins, there is a wide range of biological effects, which are different for the various progestins and have to be taken into account, when medical treatment is considered.

Overview on the effects of progestins on bone.

In view of the fact that fractures are the clinically relevant events, risk factors for fractures are discussed first. Bone mineral density (BMD) appears to be a much less important risk factor for the most severe hip fractures than the risk of falling. No results of experimental studies on hormones and fractures at advanced age are available. An overview of the effects of progestins on bone is given. Effects of progestins on bone have been studied by in vitro experiments using cell lines and by more relevant clinical observations. Prospective studies have been conducted following the use of progestins contained in oral contraceptives, alone or in combination with oestrogens; long-term contraception by injection of depot preparations; so-called "add-back" hormonal therapy attempting to reverse the adverse effects of gonadotropin releasing hormone agonists on bone and after different regimens of hormone replacement therapy (HRT) in postmenopausal women. From the data there are no indications that the various progestins, used in clinical practice, have either a bone-protective or an oestrogen antagonistic activity. Progestins do not add or subtract much of the protective action of oestrogens on the bones.

Endogenous sex hormones and cardiovascular disease in men.

Unlike women, men do not experience an abrupt reduction in endogenous sex hormone production. It has, however, become clear that an age-associated decrease in the levels of (bioactive) sex hormones does occur. Whether endogenous sex hormones have an impact on cardiovascular disease has for many years remained largely unknown, but during the last decade more attention has been drawn to the importance of testosterone, estrogens, and adrenal androgens in etiology, prevention, and treatment of male cardiovascular disease. The purpose of this article is to summarize the evidence currently available on the association between endogenous sex hormones and cardiovascular disease in males. Published studies dealing with the relationship between circulating levels of sex hormones and cardiovascular disease in males were reviewed. The studies reviewed in this article suggest that circulating endogenous sex hormones and estrogens have a neutral or beneficial effect on cardiovascular disease in men.

Sex hormones and male health: effects on components of the frailty syndrome.

Physicians are seeing an increasing number of older male patients with chronic diseases and conditions. However, the potential relevance of low levels of circulating endogenous androgens in connection with these diseases and conditions is generally poorly understood. Research findings have suggested that androgens play a distinct role in bone metabolism, body composition such as muscle and fat mass and fat distribution, cognitive functioning, mood and well being. The aim of this paper is to summarize the currently available data on the association between endogenous androgens and the intermediate or clinically manifest indicators of chronic conditions in men that might contribute to the phenomenon "frailty". The evidence that reductions in endogenous androgens play a role in age-related health problems is circumstantial. Therefore, large-scale randomized trials are needed to establish whether aging males with low serum androgen levels benefit from androgen supplementation.

Plasma and urinary sex hormones are differently related to lipids in healthy postmenopausal women.

OBJECTIVE: Endogenous sex hormones can be measured in plasma and urine. We determined the extent to which these two methods provide different information on hormonal status by relating them to lipid profile in postmenopausal women. METHODS: Thirty healthy postmenopausal women collected one 24-h urine sample and a blood sample was taken. Urinary estrone (UE), plasma estrone (PE) and serum lipids were measured. Sex hormone levels were measured with specific radioimmunoassays. Linear regression analysis was used to determine associations between estrone levels and lipids. Results are presented as beta-coefficients in mmol/l per standard deviation (SD) of endogenous estrone levels, adjusted for body mass index (BMI) and smoking (95% confidence interval). A stratified analysis for obese (BMI> or =27 kg/m(2)) versus lean women was performed. RESULTS: Mean levels of endogenous sex hormones were (SD): PE, 90.1 pmol/l (37.3); and UE, 7757 pmol/24 h (2659). PE showed significant associations with HDL-cholesterol (0.18 mmol/l, 95% CI: 0.06; 0.30), triglycerides (-0.25 mmol/l, 95% CI: -0.49; -0.009) and very-low-density-lipoprotein (VLDL-cholesterol) (-0.11 mmol/l, 95% CI: -0.22; -0.003), but not with total and low-density-lipoprotein (LDL-cholesterol). UE was inversely associated with total (-0.41 mmol/l, 95% CI: -0.85; 0.02) and LDL-cholesterols (-0.42 mmol/l, 95% CI: -0.83; -0.005), but not with HDL-cholesterol, triglycerides and VLDL-cholesterol. All associations appeared to be stronger in lean women. CONCLUSION: Both plasma and UE levels appear to be associated to serum lipids in healthy postmenopausal women. However, this relation appears to be different for estrone levels in plasma and urine. Depending on the research question, either blood samples or urine samples may be preferred.