Davide Francomano,1Andrea Lenzi,1 and Antonio Aversa1
1Department of Experimental Medicine, Medical Pathophysiology, Food Science and Endocrinology Section, Sapienza University of Rome, Viale Regina Elena 324, 00161 Rome, Italy
Metabolic and hormonal modifications after long-term testosterone (T) treatment have never been investigated. 20 hypogonadal men (mean T = 241 ng/dL–8.3 nmol/L) with metabolic syndrome (MS, mean age 58) were treated with T-undecanoate injections every 12 weeks for 60 months. 20 matched subjects in whom T was unaccepted or contraindicated served as controls. Primary endpoints were variations from baseline of metabolic and hormonal parameters. In T-group, significant reductions in waist circumference ( -9.6+-3.8cm, P<0.0001), body weight (-15+-2.8 Kg, P<0.0001 ), and glycosylated hemoglobin (-1.6%+-0.5, P<0.0001) occurred, along with improvements in insulin sensitivity (HOMA-I; -2.8+-0.6, P<0.0001 ), lipid profile (total/HDL-cholesterol ratio -2.9+-1.5, P<0.0001), systolic and diastolic blood pressure ( -23+-10 and -16+-8 mm Hg, ,P<0.0001 resp.), and neck and lumbar T-scores (+0.5+-0.15 gr/cm2, ; P<0.0001, +0.7+-0.8 , P<0.0001 resp.). Also, serum vitamin D ( +14.0+-1.3 ng/mL, P<0.01), TSH ( -0.9+-0.3 mUI/mL, P<0.01 ), GH ( 0.74+-0.2 ng/mL, P<0.0001 ), and IGF1 (105+-11 ng/mL, P<0.01 ) levels changed in T-group but not in controls. Normalization of T levels in men with MS improved obesity, glycemic control, blood pressure, lipid profile, and bone mineral density compared with controls. Amelioration in hormonal parameters, that is, vitamin D, growth hormone, and thyrotropin plasma levels, were reported.
Obesity, and particularly visceral fat excess, is associated with insulin resistance, hyperglycemia, atherogenic dyslipidemia, and hypertension as well as prothrombotic and proinflammatory states and with vitamin D deficiency . Several papers have suggested that a significant relationship between low levels of testosterone (T) and the metabolic syndrome (MS) exists . Also, epidemiological studies have found that low T levels are a predictor of mortality in elderly men . In addition, increasing evidence is accumulating regarding inverse associations between the severity of features of the MS and plasma T . An inverse relationship between waist circumference (WC), a surrogate of visceral obesity, and T levels exists , thus leading to hyperinsulinism and reduced levels of sex hormone binding globulin (SHBG) and luteinizing hormone (LH), and all these factors along with increased leptin contribute to the suppression testicular steroidogenesis . Also, in centrally obese individuals, there is an overactivity of the corticotropin-releasing-hormone (CRH)—corticotropin (ACTH)—cortisol axis as speculated by pioneer work of Björntorp and coauthors who demonstrated that this increased activity may result in a suppression of the production of T and growth hormone (GH) .
The European male ageing (EMAS) study is the first epidemiological study suggesting an upper limit of 11 nmol/L (FT 220 pmol/L) as the one correct for treating testosterone deficiency syndrome (TDS) . Despite the fact that in this study the reported prevalence of hypogonadism was low (17%), Corona et al. reported an incidence as high as 29.3% in obese men . This can be explained by the fact that EMAS investigated a relatively healthy sample of the general population, whereas Corona assessed T levels in outpatients presenting with erectile dysfunction (ED). In fact, T substitution in men with such values determines significant improvement in body composition, as reported in several studies [10, 11]. If this may be considered the threshold T level for the appearance of major symptoms like erectile dysfunction or decreased sexual desire, this may not be true for reverting body composition and mineral density changes induced by TDS. As previously demonstrated by other authors, the improvement in metabolic parameters may require achievement of higher and sustained therapeutic levels of testosterone over the time . Moreover, evidence exists suggesting that T regulates adipogenesis and therefore increases lean body mass and reduces fat mass thus regulating body composition . Long-term hormonal and anthropometric variations during T replacement therapy (TRT) in men with metabolic syndrome have not been investigated in controlled studies.
Aim of this study was to evaluate the effects of TRT on metabolic and hormonal parameters in hypogonadal men with MS.
2. Patients and Methods
2.1. Inclusion, and Exclusion Criteria
Forty patients, aged from 45 to 65 years, were enrolled into this prospective study. Patients were included in the study if they were between 45 and 65 years of age, had MS and/or type 2 diabetes mellitus (T2DM) defined by the International Diabetes Federation  and total serum T level below 320 ng/dL (11 nmol/L) or calculated free-T levels below 255 pmol/L (74 pg/mL) on two early morning separate days (between 8:00 and 11:00 a.m.) at least 1 week apart, and had at least two symptoms of hypogonadism. Patients were not included in the study in case of the following: use of TRT or anabolic steroids or any other hormone replacement therapy in the previous 12 months; history of prostate or breast cancer or other tumours; drug or alcohol abuse; blood coagulation alterations; symptomatic obstructive sleep-apnoea syndrome; haematocrit level ≥52% at baseline; age-adjusted elevated prostate-specific antigen (PSA) level or abnormal digital rectal examination (DRE) of prostate suspicious for cancer or severe symptomatic benign prostatic hyperplasia; an International Prostate Symptom Scale (IPSS) 13 at baseline; use of 5–reductase inhibitors; presence of any uncontrolled endocrine disorder including diabetes (HbA1c 9); presence of New York Heart Association III or IV heart failure; hepatic insufficiency; severe neurological and psychiatric disease; and patients requiring or undergoing fertility treatment. We also excluded men who had diseases potentially affecting the skeleton, such as chronic renal disease or malabsorption, or were taking medications or drugs affecting bone turnover including any vitamin supplementation or nutraceutics or more than three alcoholic drinks a day. All concomitant oral hypoglycemic, anti-hypertensive, and lipid-lowering medications were permitted if started within the previous 12 months and continued throughout the study without dose adjustments. Subjects were asked to maintain their usual physical exercise and lifestyle for the duration of the study. Written informed consent was obtained before commencement of the study according to Protocol and Good Clinical Practice on the conducting and monitoring of clinical studies and approved by our University Ethical Committee.
2.2. Primary Outcome Measures
The primary outcomes were variation from baseline of the metabolic, bone, and hormonal parameters. At baseline, every three (within the first year) and six (in the following 4 years) months, the following evaluations were assessed: general physical examination and anthropometric parameters (i.e., body weight (BW), height, BMI, and waist circumference (WC)), systolic and diastolic blood pressure, heart rate, blood samples for biochemical and hormonal analyses, and digital rectal examination (DRE). Every twelve months, BMD was calculated by using a whole-body dual-energy X-ray absorptiometry (DEXA-HOLOGIC QDR-1000) according to the instructions of the manufacturer and standardized procedures, and the individual bone mineral density (BMD) variation has been measured with a T-score . Calibration with the manufacturer’s spine phantom and quality control analysis were performed daily. The long-term precision error in vitro was 0.54% (phantom); short-term precision error in vivo was 1.2% for the lumbar spine and 2% for the femoral neck . BMD was expressed in grams per square centimeter (g/cm2) and result expressed as T-score.
Fasting blood samples were tested for glucose, triglycerides, high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) at the hospital’s clinical laboratories. Hormonal assessment included serum total T (TT) and LH, as measured by chemiluminescent microparticle immunoassay (CMIA, Architect System) (Abbott Laboratories, Abbott Park, IL, USA), with detection limit of 0,28 M, calculated free T (according to http://www.issam.ch/), sex hormone binding globulin (SHBG), estradiol, prolactin, thyroid stimulating hormone (TSH), growth hormone (GH), somatomedin-C (IGF1), insulin, and PSA were analyzed by immunometric assay based on chemiluminescence using an automated clinical chemistry analyzer (Immulite 2000, Diagnostic Product Corp., Los Angeles, CA, USA). To overcome seasonal variability, 25-hydroxy vitamin D (25OHD; ng/mL) was measured by chemiluminescent immunoassay always during the same season and each subject served as an internal control (ARUP Laboratory, Salt Lake City, UT; coefficient of variation (CV) 8.6–10.0%). HbA1c was measured by high performance liquid chromatography (Bio-Rad Laboratories, Hercules, CA, USA). To assess insulin sensitivity, we calculated the HOMA-I using the formula [fasting insulin in mU/L × fasting glucose in mmol/L]/22.5.
2.3. Modality of Treatment
After screening any patient for the presence of hypogonadism, twenty of 72 patients met the inclusion/exclusion criteria and entered into the study. Patients received TU (TRT group) administered intramuscularly at a dose of 1000 mg every 6 weeks for the first two injections and then every 12 weeks, according to recommendations, for a period of 60 months. Twenty patients not fulfilling inclusion/exclusion criteria or refusing TRT for personal reasons and preferring lifestyle changes as the primary treatment were observed throughout the time and served as controls. Due to severe overweight, most patients adhered to comply with a standard hypocaloric diet and slight changes in lifestyle that is, low/moderate walking at least three times per week. Each patient was assigned to a personalized nutritional program, consisting in a hypocaloric diet with a protein of 0.8–1 g/Kg of lean body weight, along with a personalized movement program, with recommendation of at least 60 minutes/week of aerobic exercise of low/moderate intensity (40% of maximum heart rate). Physical activity should have been distributed in at least 3 days/week, and there must be no more than 2 consecutive days without activity . The patients were monitored for compliance with a personal diary indicating “yes” or “no” regarding the lifestyle changes prescriptions.
Safety parameters included DRE, PSA total and free, hemoglobin, hematocrit, liver, and kidney functions were monitored every three (within the first year) and six (in the following 4 years) months, respectively, according to previously published procedures .
Patients with the following clinical laboratory parameters were withdrawn either at the baseline or during the course of study: if hematocrits levels were >52%; PSA level increased >1.0 ng/mL above the baseline PSA if baseline PSA was <2.0 ng/mL; PSA levels increase >50% of the baseline PSA if baseline PSA was >2.0 ng/mL.
2.5. Statistical Analyses
Data were analyzed using -tests (for single between-group comparisons), analysis of covariance (for between-group comparisons at specific time points, using baseline score as a covariate), and a mixed linear regression model on repeated measures data (for between-group comparisons across all time points) to analyze data for an Intent-to-Treat Group (including all subjects enrolled and treated in this trial with values imputed for their Last Observation Carried Forward (LOCF) for any subjects who did not complete the trial) and a Completer’s Group (including only data from subjects who completed the trial per protocol). Data were expressed as means standard deviation when normally distributed, and as median (quartiles) when nonparametric. A value < 0.05 was taken as statistically significant. Statistical analysis was performed using the computer statistical package SPSS 11.0 (SPSS Inc., Chicago, IL, USA).
3.1. Metabolic and Hormonal Parameters
Demographic characteristics of the patients at baseline are shown in Table 1.
Demographic characteristics of patients at baseline.
All patients included were hypogonadal because of metabolic disturbances, that is, metabolic syndrome and/or diabetes, and none had primary/secondary hypogonadism with alteration of gonadotropins (data not shown). As expected, at the end of the study, the values of TT were higher in the TRT compared to the control group (+ 9.1+-1.7nmol/L, P<0.0001 ) while estradiol levels showed a trend to increase (Table 2).
At LOCF, only TRT group showed a significant reduction of BMI (−2.9+-1.4, P<0.0001); also, WC (−9.6+-3.8 cm, P<0.0001; Figure 1(a)) and body weight (−15+-2.8 Kg, P<0.0001; Figure 1(b)) significantly decreased in all men (100%) treated with TU compared with controls, who displayed a trend to increase both parameters over the time. This was mainly due to major compliance of TRT group towards diet and physical exercise compared with controls (90% versus 10% of overall patients, P<0.0001, data not shown). There was a significant reduction of blood glucose as evaluated by mean HbA1c levels during the 60 months study follow-up period (−1.6+-0.5%, P<0.001; Figure 1(c)) for the TRT group only.
Effects of 5-year treatment with long-acting TU on (a) waist circumference (cm), (b) body weight (Kg), and (c) glucose homeostasis (HBA1c) in 40 hypogonadal men (T < 11 nmol/L) with metabolic syndrome (IDF). variations were evaluated yearly in the testosterone treatment (TRT) versus controls (CTRL).
In this latter group, significant reduction in insulin sensitivity as evaluated by HOMA-i (−2.8+-0.6, P<0.0001) and lipid profile (total/HDL-cholesterol: −2.9+-1.5, P<0.0001; and Triglycerides: -41+-25, P<0.0001) was found. Also only TRT group showed a significant reduction in both systolic (− 23+-10 mm Hg, P<0.0001; Figure 2(a)) and diastolic (− 16+-8 mm Hg, P<0.001; Figure 2(b)) blood pressure, heart rate (−15+-5 bpm, P<0.001; Table 2) and a significant increment in neck and lumbar -scores (+0.5+-0.15 gr/cm3, P<0.0001; +0.7+-0.8 gr/cm3, P<0.0001, resp.).
Effects of 5-year treatment with long-acting TU on (a) systolic blood pressure (mm Hg) and (b) diastolic blood pressure (mm Hg) in 40 hypogonadal men (T < 11 nmol/L) with metabolic syndrome (IDF). variations were evaluated yearly in the testosterone treatment (TRT) versus controls (CTRL).
Interestingly, serum vitamin D (+14.0+-1.3 ng/mL, P<0.01 ), TSH ( -0.9+-0.3 mUI/mL, P<0.001), GH (+0.74+-0.2, P<0.0001), and IGF1 (+105+-11, P<0.01 ) levels changed in TRT group only (Table 2).
A significant increase in hematocrit (+2.8+-0.9%, P<0.001) and PSA levels (+0.37+-0.29 ng/mL, P< 0.01) within the normal reference range values was found in TRT group only without any clinical symptom or worsening in voiding function . This increase occurred within the first 12 months of treatment and remained stable throughout the remaining period of study (Table 2).
This is the first long-term controlled, nonsponsored study with T-undecanoate (TU) for a 60-month period in hypogonadal men with MS. Anthropometric, hormonal, and body composition parameters were investigated. Our results clearly demonstrate that TU is able to improve anthropometric measurements in a stepwise yearly manner, that is, WC and total BW; not surprisingly, a significant reduction in blood pressure and heart rate was reported compared to controls. Also, hormonal panel including vitamin D, TSH, GH, and IGF1 circulating levels all improved and these hormonal changes were not described elsewhere in such a population. No serious adverse event related to TU treatment was reported over the time.
Several recent studies have focused on normalizing T levels by using TU injections in obese hypogonadal men with TDS. Saad et al. investigated the effects of TU injection in 110 elderly men with obesity and MS and demonstrated that age, BMI, and C-reactive protein (CRP) levels, in addition to hypogonadism, can be used clinically to predict which men mostly benefit from T supplementation with regard to components of the MS . Aversa et al. demonstrated that three-years TU in middle-aged men with TDS and MS determined a significant increase in both vertebral and femoral BMD that was correlated with the increments in serum T levels, probably independently from estradiol modifications and this was mainly related to CRP reduction . In another study, Saad et al. demonstrated that TU treatment of 255 hypogonadal men determined a weight loss in approximately 95% of all patients, with marked changes in body composition, that is, an increase in lean body mass and a decrease in fat mass . Yassin and Doros confirmed same results in a registry study of 261 hypogonadal men . In all reported studies to date, T treatment consistently showed decreased fat and increased lean body masses. Similarly, Traish et al. reported significant changes in MS components during TRT at physiological levels . Even if obtained in uncontrolled studies, these findings suggest that T may be a physiological modulator of body composition due to its role in promoting myogenesis and inhibiting adipogenesis and its role in carbohydrate, lipid, and protein metabolism. Data obtained in the present controlled study are confirmative of the evidence previously reported in uncontrolled studies that features of the MS present in elderly men must not be a limiting factor in prescribing TU in view of its advantages on metabolic, bone, and hormonal ameliorations as well as on overall improvements in estimated cardiovascular disease (CVD) risk.
T is a well-known regulator of many metabolic functions in liver, adipose tissue, muscles, coronary arteries, and the heart. The TC/HDL-C ratio is another important marker of CVD risk and its modification during treatment may indicate major changes in metabolic function that is, improvement in insulin resistance and decreased ischemic heart disease risk . It is thought that it may represent a better marker than the apoB/apoA1 ratio for identifying insulin resistance and MS in some populations . A recent study demonstrated that patients with peripheral artery disease treated with atorvastatin showed improvement in endothelial function and this was associated with decreased TC/HDL-C ratio, suggesting that this ratio may be related to endothelial damage . The improvement of endothelial function may be the basis for the reduction of blood pressure and heart rate found in the present study. In fact, in previous report from our group we demonstrated that one-year TU is able to improve arterial stiffness and endothelial function in morbidly obese men (unpublished data), thus confirming that a sustained and advantageous effect of TRT on cardiovascular function is present in men with MS, thus leading to reduced CV risk throughout the time. The present data confirm, in a controlled study, that long-term TU reduces the risk of CVD in men with MS as previously described in observational studies .
Morbidly obese patients have been reported to often present with vitamin D insufficiency and secondary hyperparathyroidism. In obese women who undergo weight loss therapy, an abnormal vitamin D metabolism is still reported after 5-year follow-up ; similarly, bariatric surgery does not completely revert preexisting vitamin D deficient states and secondary hyperparathyroidism . The reduction in WC and BW during weight loss program appear to be a common finding in the obese population following controlled weight loss programs; however, in our obese hypogonadal male patients (with MS), the finding of persistent and sustained yearly weight loss over the time was very surprising when compared with control group in whom no modification occurred despite the fact that slight lifestyle changes were recommended to both groups. Hagenfeldt et al. firstly described the improvement in vitamin D plasma levels after TRT in a small group of men with Klinefelter’s syndrome through a possible, indirect action of increased estradiol circulating levels due to aromatization . Other authors have speculated that, in normal conditions, Leydig cell may contribute to the 25-hydroxylation of vitamin D through the CYP2R1 enzyme that catalyzes the hydroxylation of cholecalciferol to 25-hydroxyvitamin D . This enzyme is in turn regulated by insulin-like 3 (INSL3), which has also a role in osteoblast function, through an LH-T related mechanism. Testicular dysfunction determines reduced T levels, along with low INSL3 and 25-hydroxyvitamin D levels, and consequently may lead to an increased risk of osteopenia and osteoporosis. In our patients a mild osteopenia was present, and improvements in bone mineral density were reported despite no modification in estradiol levels. We speculate that the increase in vitamin D obtained by our patients may be partly due to T-induced overall trunk fat mass reduction, since in cross-sectional studies we had previously demonstrated a close relationship between trunk fat mass, vitamin D, osteocalcin, and testosterone levels in obese men . Also, a direct effect of testosterone on renal expression of the l-alpha-hydroxylase gene might be possible, as androgen receptors have been demonstrated in kidney tissue .
On the other hand, other hormones or regulatory factors could mediate the effect on vitamin D indirectly. GH and IGF-I have been reported to influence vitamin D metabolism both in animals and in humans . Previous studies demonstrated that increasing serum T concentrations to the mid-normal range with low-dose T administration for 26 weeks increases nocturnal, spontaneous, pulsatile GH secretion, and morning IGF-I concentrations in healthy older men, supporting the hypothesis that age-related reductions in T may contribute to the concurrent “somatopause” . Accordingly, in the present study, the stimulatory effects obtained after TRT on GH secretion may be interpreted as an indirect effect due to the activation of lipolytic cascade of adipocytes leading to a better insulin sensitization, reduction of abdominal fat, and amelioration of pituitary function. Several reports in the literature consider obesity as a sort of “panhypopituitarism” condition determining a multiendocrine dysfunction. It is well established that caloric restriction applied for a relatively short term usually is able to increase GH release significantly in normal weight subjects ; however, this release results significantly reduced in obese subjects, who exhibit large diet-induced weight losses . The recovery of the GH/IGF-I axis after weight loss suggests an acquired defect, rather than a preexisting pituitary disorder. Noteworthy, in our control group, we hypothesize that the persistent impairment of endocrine axes, that is, GH/IGF-I might have acted toward expansion and maintenance of fat mass and have contributed to perpetuation of the obese state.
Few studies have investigated the effects of controlled weight loss on thyroid hormone axis in male obese subjects. Cross-sectional studies have demonstrated that T3 and TSH correlate positively with adiposity . In a recent study, moderate weight loss intervention resulted in a significant decrease in circulating T3 and only a marginal decrease in TSH and in fT4 . Altogether, these observations indicate that even a moderate weight loss intervention may generate some perturbation in this axis. Our data obtained in TRT group clearly show that the stepwise decrease in fat mass, anthropometric and blood pressure parameters throughout the time may be considered an important factor also impacting on thyroid homeostasis. The fact that these changes were not observed in the control group is in keeping with the failure in achieving a correct weight (and abdominal fat) loss.
A limitation of the study represented by the low number of subjects investigated. We understand that it is difficult to rely on overall changes occurring in a small cohort of patients, but we are aware of the fact that this is a spontaneous, unsponsored study not designed to specifically investigate the effects of T on metabolic and hormonal pattern; thus patients were followed up for their specific comorbidities. Another limitation of this study was that a limited number of plasma hormones was investigated; thus PTH, gonadotropins, osteocalcin, and free fraction of thyroid hormones were not measured in all patients, in part because of financial constraints.
The marked weight loss observed in hypogonadal men with MS replaced with TU is an important finding of the present study and is in agreement with previous in vitro studies where T regulates lineage of mesenchymal pluripotent cells by promoting the myogenic lineage and inhibiting the adipogenic lineage . T also inhibits triglyceride uptake and lipoprotein lipase activity resulting in rapid turnover of triglycerides in the subcutaneous abdominal adipose tissue and mobilizes lipids from the visceral fat depot . Thus, T-induced changes on metabolism and body composition might have been determined by increased motivation, enhancement of mood, and promotion of more energy expenditure; this in turn might be responsible of the multiple endocrine modifications occurred on pituitary function. The changes in vitamin D levels and hormonal status (GH, IGF1, and TSH) are likely to be explained by the reduction of trunk fat mass content. By contrast, in control groups all these changes were not present despite the fact that lifestyle changes were applied.
In conclusion, this study demonstrates that TU in hypogonadal men with MS has favorable effect on body composition and metabolic parameters, after five-years replacement. The present study also provides first evidence that remarkable reduction of blood pressure and heart rate, as well as amelioration of vitamin D, GH/IGF1, and TSH plasma levels, are also attained. This may in turn yield to different overall CVD estimated risk and overall survival rates as well as to different pharmacological management of T2DM, hypertension, and dyslipidemia in men with MS and obesity.
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M. L. Hartman, J. D. Veldhuis, M. L. Johnson et al., “Augmented growth hormone (GH) secretory burst frequency and amplitude mediate enhanced GH secretion during a two-day fast in normal men,” Journal of Clinical Endocrinology and Metabolism, vol. 74, no. 4, pp. 757–765, 1992.
M. H. Rasmussen, A. Juul, L. L. Kjems, and J. Hilsted, “Effects of short-term caloric restriction on circulating free IGF-I, acid-labile subunit, IGF-binding proteins (IGFBPs)-1-4, and IGFBPs-1-3 protease activity in obese subjects,” European Journal of Endocrinology, vol. 155, no. 4, pp. 575–581, 2006.
M. A. Michalaki, A. G. Vagenakis, A. S. Leonardou et al., “Thyroid function in humans with morbid obesity,” Thyroid, vol. 16, no. 1, pp. 73–78, 2006.
R. V. Agnihothri, A. B. Courville, J. D. Linderman et al., “Moderate weight loss is sufficient 10 to affect thyroid hormone homeostasis and inhibit its peripheral conversion,” Thyroid, vol. 24, no. 1, pp. 19–26, 2014.
R. Singh, J. N. Artaza, W. E. Taylor, N. F. Gonzalez-Cadavid, and S. Bhasin, “Androgens stimulate myogenic differentiation and inhibit adipogenesis in C3H 10T1/2 pluripotent cells through an androgen receptor-mediated pathway,” Endocrinology, vol. 144, no. 11, pp. 5081–5088, 2003.
P. Mårin, B. Odén, and P. Björntorp, “Assimilation and mobilization of triglycerides in subcutaneous abdominal and femoral adipose tissue in vivo in men: effects of androgens,” Journal of Clinical Endocrinology and Metabolism, vol. 80, no. 1, pp. 239–243, 1995.
Douglas S. King, PhD; Rick L. Sharp, PhD; Matthew D. Vukovich, PhD; Gregory A. Brown, MS; Tracy A. Reifenrath, MS; Nathaniel L. Uhl; Kerry A. Parsons, MS
Context Androstenedione, a precursor to testosterone, is marketed to increase blood testosterone concentrations as a natural alternative to anabolic steroid use. However, whether androstenedione actually increases blood testosterone levels or produces anabolic androgenic effects is not known.
Objectives To determine if short- and long-term oral androstenedione supplementation in men increases serum testosterone levels and skeletal muscle fiber size and strength and to examine its effect on blood lipids and markers of liver function.
Design and Setting Eight-week randomized controlled trial conducted between February and June 1998.
Participants Thirty healthy, normotestosterogenic men (aged 19-29 years) not taking any nutritional supplements or androgenic-anabolic steroids or engaged in resistance training.
Interventions Twenty subjects performed 8 weeks of whole-body resistance training. During weeks 1, 2, 4, 5, 7, and 8, the men were randomized to either androstenedione, 300 mg/d (n=10), or placebo (n=10). The effect of a single 100-mg androstenedione dose on serum testosterone and estrogen concentrations was determined in 10 men.
Main Outcome Measures Changes in serum testosterone and estrogen concentrations, muscle strength, muscle fiber cross-sectional area, body composition, blood lipids, and liver transaminase activities based on assessments before and after short- and long-term androstenedione administration.
Results Serum free and total testosterone concentrations were not affected by short- or long-term androstenedione administration. Serum estradiol concentration (mean [SEM]) was higher (P<.05) in the androstenedione group after 2 (310  pmol/L), 5 (300  pmol/L), and 8 (280  pmol/L) weeks compared with presupplementation values (220  pmol/L). The serum estrone concentration was significantly higher (P<.05) after 2 (153  pmol/L) and 5 (142  pmol/L) weeks of androstenedione supplementation compared with baseline (106  pmol/L). Knee extension strength increased significantly (P<.05) and similarly in the placebo (770  N vs 1095  N) and androstenedione (717  N vs 1024  N) groups. The increase of the mean cross-sectional area of type 2 muscle fibers was also similar in androstenedione (4703  vs 5307  mm2; P<.05) and placebo (5271  vs 5728  mm2; P<.05) groups. The significant (P<.05) increases in lean body mass and decreases in fat mass were also not different in the androstenedione and placebo groups. In the androstenedione group, the serum high-density lipoprotein cholesterol concentration was reduced after 2 weeks (1.09 [0.08] mmol/L [42 (3) mg/dL] vs 0.96 [0.08] mmol/L [37 (3) mg/dL]; P<.05) and remained low after 5 and 8 weeks of training and supplementation.
Conclusions Androstenedione supplementation does not increase serum testosterone concentrations or enhance skeletal muscle adaptations to resistance training in normotestosterogenic young men and may result in adverse health consequences.
Androgenic-anabolic steroids have been shown to enhance the gains in muscle size and strength associated with resistance training.1-4 Androstenedione, a precursor to testosterone, is normally produced by the adrenal gland and gonads and is converted to testosterone through the action of 17β-hydroxysteroid dehydrogenase, which is found in most body tissues.5-9 Androstenedione is also produced by some plants and has recently been marketed as a product for increasing blood testosterone concentrations to be used as a natural alternative to anabolic steroid use.
However, the interconversions of androstenedione and testosterone to other androgens, as well as to estrogens, are complex. In addition to serving as a precursor to testosterone, androstenedione may be converted into estrogens directly.10,11 Since testosterone is also aromatized to estradiol,11,12 it is also possible that increased production of testosterone following androstenedione administration may also result in increased aromatization, which would further attenuate any increase in the blood testosterone concentration. These considerations raise the question of whether androstenedione supplementation increases the blood testosterone concentration and produces anabolic-androgenic effects.
To date only one study has investigated the effect of oral androstenedione administration on the blood testosterone concentration.13 These authors observed 4- and 7-fold increases in the blood testosterone concentration in 2 healthy women, respectively, after the ingestion of a single dose of 100 mg of androstenedione. The effect of androstenedione administration on blood testosterone levels in healthy men is unknown. Therefore, one purpose of this study was to determine whether short- and long-term administration of oral androstenedione increases the blood testosterone concentration and enhances gains in muscle size and strength when combined with a resistance-training program.
Increased concentrations of testosterone in the blood have been associated with an increased risk of cardiovascular disease, due both to a lowering of the serum high-density lipoprotein cholesterol (HDL-C) concentration and an increased serum concentration of low-density lipoprotein (LDL) concentration.3,14-19 Elevated blood testosterone concentrations may also result in significant alterations in liver function.20,21 The effects on blood lipids and liver function appear to be more pronounced in oral anabolic steroids, compared with injectable agents. A second purpose of this study, therefore, was to examine the effect of androstenedione administration on blood lipids and on clinical markers of liver function.
A total of 30 healthy, normotestosterogenic young (aged 19-29 years) men were recruited for this experiment, approved by the Iowa State University Human Subjects Committee. These participants were screened to ensure that they were not consuming androstenedione or any other nutritional supplement prior to enrollment in the study and were not currently engaged in a resistance-training program. Subjects were also not taking illicit drugs or abusing alcohol consumption. All subjects were free of any cardiovascular or orthopedic condition that would contraindicate exercise testing or training.
Short-term Administration of Androstenedione
The effect of short-term administration of androstenedione on the serum concentration of androstenedione, free and total testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) was studied in 10 of the men (mean age [SEM], 23  years). On 2 separate days after an overnight fast, separated by 1 week, subjects ingested 100 mg of androstenedione or placebo (250 mg of rice flour), administered in a randomly assigned double-blind manner. This dose was chosen based on the previous report that 100 mg of androstenedione increases blood testosterone concentration by 4- to 7-fold in women.13 Blood samples were obtained before and every 30 minutes after ingestion for 6 hours. Serum hormone concentrations were determined as described below.
Androstenedione Supplementation During Resistance Training
After screening, 20 of the men were randomly assigned in a double-blind manner to groups that consumed either androstenedione or placebo during weeks 1-2, 4-5, and 7-8, during the 8 weeks of resistance training. One subject in each group reported prior resistance-training experience, although none had performed resistance training during the preceding year. Supplementation was administered in a cyclic fashion as recommended by the manufacturer to simulate the supplementation regimen followed by many athletes. This cycle is believed by athletes to allow for a “washout” period and reduce the likelihood of adverse effects due to anabolic steroid administration. Subjects consumed 300 mg of androstenedione or a placebo (250 mg of rice flour) in capsule form each day. The 300-mg/d dosage was chosen to exceed the maximal dosage typically recommended by manufacturers (100-300 mg/d), as well as the dosage shown to increase blood testosterone concentrations in women.13 Supplements were taken in unmarked white capsules in 3 equal doses before 9 AM, at 3 PM, and at bedtime. The androstenedione was derived from wild yams and was provided by Experimental and Applied Sciences Inc (Golden, Colo). Purity of the androstenedione contained in the capsules was assessed with high-performance liquid chromatography by 2 independent laboratories (Biomedical Laboratories Inc, Petaluma, Calif, and Integrated Biomolecule Corp, Tucson, Ariz). These analyses produced values for purity of 99% and 100%, respectively. To encourage compliance, subjects maintained a record of supplement ingestion and were required to return unused supplements at the completion of the study. At the conclusion of the study, when subjects were asked to identify which supplement they were taking, 2 subjects in the placebo group correctly identified the supplement they were taking.
During the 8-week resistance-training program, subjects performed resistance training 3 days per week on nonconsecutive days. Subjects were instructed on proper lifting technique and supervised by 1 of the investigators (G.A.B., T.A.R., N.L.U., or K.A.P.)during all lifting sessions. The resistance-training program was designed to increase the strength of all major muscle groups. Subjects trained on bench press, shoulder press, knee extension, right and left knee flexion, vertical butterfly, leg press, calf press, biceps curl, triceps extension, and lattisimus dorsi pull-down. Subjects performed 3 sets of 10 repetitions for the first 2 weeks. For the final 6 weeks of training, subjects performed 3 sets of 8 repetitions. Resistance was set at 80% to 85% of 1 repetition maximum (1-RM). Following the determination of 1-RM after 4 weeks of training, the training intensity was adjusted to 80% to 85% of the new 1-RM. All resistance training and 1-RM testing was performed on multistation isotonic resistance equipment (FTX; Paramount Fitness Equipment, Los Angeles, Calif).
Muscle strength was assessed with the measurement of 1-RM before and after 4 and 8 weeks of resistance training. After a brief warm-up, subjects were encouraged to meet their 1-RM within 5 trials of progressing resistance. One repetition maximum was assessed on bench press, shoulder press, knee extension, right and left knee flexion, biceps curl, triceps extension, lattisimus dorsi pull-down, and vertical butterfly.
Body mass and circumference measures were obtained before training and after 4 and 8 weeks of training. All circumference measurements were performed by the same investigator (T.A.R) and were obtained for the following sites: biceps, shoulder, chest, abdomen, waist, hips, gluteal, thigh, and calf. Body density and percent body fat were determined with hydrostatic weighing before and after 8 weeks of training using a computer-interfaced load cell and custom computer program. Body fat percent was calculated using the Siri equation22 after estimation of the residual volume.23
To assess diet, subjects kept a food-intake record for 3 days prior to beginning resistance training and supplementation. Subjects were instructed to maintain their typical dietary intake during the course of the study. Diet records were analyzed for composition using a food analysis software package (Food Comp; Iowa State University, Ames). Mean (SEM) daily energy intake was not different in placebo (9983  kJ/d) and androstenedione (9660  kJ/d) groups prior to supplementation and resistance training. Daily protein intake was also not different in placebo (83  g/d) and androstenedione (98  g/d) groups and exceeded the recommended daily allowance for all subjects, suggesting adequate nitrogen balance. Although it was not possible to directly assess diet compliance during the study, subjects were queried at the end of training, and all indicated that their diet did not change during the 8 weeks.
Clinical Blood Chemistry and Hormonal Analyses
Blood samples were obtained after an overnight fast for a standard blood chemistry and hormonal analyses before training and after 2, 5, and 8 weeks of training. Blood samples were drawn without stasis from a catheter inserted into an antecubital vein. Clinical blood chemistry analyses were performed by a commercial laboratory (Labcorp Inc, Kansas City, Mo). Another sample was centrifuged and serum was frozen at −80°C until analysis. Serum concentrations of free and total testosterone, androstenedione, LH, FSH, estradiol, estrone, and estriol were measured with radioimmunoassay using commercially available kits (Diagnostic Products, Los Angeles, Calif, and Diagnostic Systems Laboratories Inc, Webster, Tex). All samples for each subject were assayed in the same run. The intra-assay coefficients of variation were 7.3%, 7.7%, 6.7%, 4.7%, 3.9%, 6.0%, 8.2%, and 7.3% for free testosterone, total testosterone, androstenedione, LH, FSH, estradiol, estrone, and estriol, respectively.
Muscle samples (about 100 mg) were obtained from the lateral aspect of the vastus lateralis muscle using the needle biopsy technique described by Bergstrom.24 Muscle specimens were placed in mounting medium and immediately frozen in isopentane cooled to the temperature of liquid nitrogen for later sectioning and staining. Frozen transverse sections (about 10 µm) were cut on a cryostat (Histostat Microtome; AO Scientific Instruments, Buffalo, NY ) at −20°C and mounted on cover glasses. Muscle sections were stained for adenosine triphosphatase activity at pH 9.4 after a preincubation at pH 4.3. Samples were then counterstained with eosin Y (Sigma-Aldrich, St Louis, Mo) for color enhancement to aid in image analysis. Muscle-fiber type distribution and muscle-fiber areas were determined using a computer-operated image analysis system (Neosis Visilog Image Analysis Software; SGI-Computer; Sony DXC-3000A-Camera). The system captures the light microscope image, traces the muscle-fiber boundaries, counts the light and dark muscle fibers, and measures the cross-sectional areas. For muscle-fiber type–distribution, all type 1 and type 2 muscle fibers were counted. When the data from the placebo and androstenedione groups before and after training are combined, fiber–type distribution was determined on 337 fibers.25 For determination of mean cross-sectional area of type 1 and type 2 fibers, groupings of clearly delineated fibers were highlighted, and 20 fibers of each type were randomly selected by a technician blinded to the treatments.
Data were analyzed using commercial software (SPSS Inc, Chicago, Ill). Statistical analyses were performed using 2-factor (time and treatment) analyses of variance (ANOVA) with repeated measures. When ANOVA revealed a significant interaction (P<.05), specific mean differences were assessed with t tests, using the Bonferroni α correction for multiple comparisons.
One subject assigned to the androstenedione group for the training study had elevated fasting glucose levels, was referred to a physician, and was diagnosed as having diabetes mellitus. This subject’s data were therefore excluded from the analysis.
Acute Hormonal Response to Androstenedione Administration
Ingestion of 100 mg of androstenedione increased the serum androstenedione concentration by 175% during the first 60 minutes following ingestion (Figure 1; P<.05). Between 90 and 270 minutes after ingestion, the serum androstenedione concentration was increased by 325% to 350% with androstenedione. Although the serum androstenedione concentration tended to decrease from 270 to 360 minutes after ingestion, serum levels remained elevated above baseline for androstenedione. Serum concentrations of LH and FSH did not change during the 360 minutes following ingestion of either androstenedione or placebo (Figure 1). Ingestion of 100 mg of androstenedione did not affect the serum concentrations of either free or total testosterone (Figure 2).
Hormonal Response to Androstenedione Administration During Resistance Training
The serum androstenedione concentration (Figure 3) increased 100% in the androstenedione group after 2 and 5 weeks of training and supplementation (P<.05) and tended to be elevated after 8 weeks (P=.07). Serum concentrations of LH and FSH were unaffected by supplementation and training in either androstenedione or placebo groups (Figure 3).
The serum free testosterone concentration (Figure 4) was significantly higher in the androstenedione group than in the placebo group before and following supplementation (significant main effect, P=.01). The serum free testosterone concentration was not significantly altered by the 8-week period of training and supplementation in either placebo or androstenedione groups. The serum total testosterone concentration was not different in placebo and androstenedione groups prior to supplementation and did not change in either group during the period of training and supplementation.
The calculated effect size for the comparison of the serum free testosterone concentrations between week 0 and week 8 for androstenedione was 0.28. Assuming a power of 80% and P=.05, a sample size of 160 would have been required to detect an effect of this size. These calculations highlight the lack of effect of androstenedione supplementation on serum testosterone concentrations.
The serum estradiol concentration was higher prior to supplementation in placebo, due to a very high initial value for 1 subject (460 pmol/L). Figure 1 shows the serum estradiol concentration, after eliminating the data of this subject. The serum estradiol concentration prior to supplementation was not different in placebo and androstenedione groups. The serum estradiol concentration did not change significantly during the 8-week experimental period for the placebo group (Figure 5). The serum estradiol concentration increased significantly (P<.05) in the androstenedione group after 2 weeks (310  pmol/L), 5 weeks (300  pmol/L), and 8 weeks (280  pmol/L) of supplementation compared with presupplementation values (220  pmol/L). The serum estradiol concentration was significantly higher for the androstenedione group compared with the placebo group after 2 and 5 weeks of training and supplementation (P<.05). The serum estriol concentration did not change during the 8 weeks of training and supplementation in either placebo or androstenedione groups. In contrast, the serum estrone concentration was significantly (P<.05) elevated in the androstenedione group after 2 weeks (153  pmol/L) and 5 weeks (142  pmol/L) of training and supplementation compared with presupplement values (106  pmol/L). The serum estrone concentration did not change during the training and supplementation period in the placebo group. The increases in serum estradiol and estrone concentrations observed after 2 weeks of supplementation were observed in all subjects ingesting androstenedione.
The observed values for serum estradiol concentrations appear to be somewhat (20%) higher than those typically reported in the literature. However, there appears to be considerable variability between laboratories, as well as between and within subjects. In addition, these estradiol values obtained at the lower end of the standard curve create more error in the calculation between defined and calculated dose. Serum estriol concentrations were also somewhat higher than those reported in the literature. However, the levels of estriol found in the current study are below the minimal reportable range as indicated by the manufacturer of the radioimmunoassay kits and, therefore, are considered to be within normal limits for men. Regardless of the explanation for these data, comparisons within these subjects over time are valid, since all samples for each subject were analyzed in the same assay.
Clinical Blood Chemistry
The 8-week period of training and supplementation did not affect serum concentrations of total cholesterol, LDL cholesterol, very LDL cholesterol, or triglycerides (Table 1). The serum HDL cholesterol concentration was significantly reduced by 12% (P<.05) after 2 weeks and remained reduced after 5 and 8 weeks of training and supplementation with androstenedione. Serum concentrations of liver function enzymes were within normal limits for all subjects throughout the study and were unaffected by training or supplementation. Training or supplementation did not significantly affect total iron, hematocrit, and hemoglobin concentrations.
There was no significant difference between placebo and androstenedione groups in the number of repetitions per training session, amount of force produced, or relative intensity expressed as a percentage of maximal force production (1-RM). When the data from all exercises are combined, the total amount of force production (SE) during the resistance-training program was 343.2 (16.9) kN and 317.8 (30.3) kN for the placebo group and the androstenedione group, respectively. During the first 4 weeks of training, the mean exercise intensity (SEM) for all exercises was 85% (1%) and 86% (1%) of 1-RM for placebo and androstenedione groups, respectively. During the final 4 weeks of training, the mean exercise intensity for all exercises was 82% (1%) and 84% (1%) of 1-RM for placebo and androstenedione groups, respectively.
Muscle strength (Table 2) did not differ between placebo and androstenedione groups before training or after 4 and 8 weeks of resistance training and supplementation. The resistance training resulted in significant increases in strength for each exercise after 4 weeks of resistance training (P<.05). The final 4 weeks of training further increased muscle strength for each of these exercises. When the data from placebo and androstenedione groups are combined, gains in strength ranged from 14% (3%) for the biceps curl to 47% (4%) for the left leg curl. Knee extension strength increased (P<.05) by 43% and 42% in androstenedione and placebo groups, respectively.
Due to an accidental thawing of 1 sample from the placebo group and 2 samples from the androstenedione group due to freezer failure, muscle-fiber–type distribution and cross-sectional areas were determined in 9 placebo and 7 androstenedione subjects. The percentage of type 1 fibers prior to resistance training and supplementation was similar in placebo (44% [4%]) and androstenedione (48% [2%]) groups. Muscle fiber–type distribution did not change as a consequence of resistance training and supplementation in either androstenedione (44% [3%]) or placebo (44% [4%]) groups. The mean (SEM) cross-sectional area of type 1 fibers was not altered with resistance training and supplementation in placebo (3980  vs 4102  µm2) or androstenedione (3310  vs 3812  µm2). The mean cross-sectional area of type 2 fibers increased similarly (significant main effect; P<.05) in placebo (5271  vs 5728  µm2) and androstenedione (4703  vs 5307  µm2) subjects.
Although the resistance-training program (Table 3) significantly affected body composition, there were no significant differences between androstenedione and placebo subjects. When the data for both groups are combined, the resistance-training program significantly (P<.05) increased mean body mass (SEM) (80.9 [3.2] vs 82.3 [3.1] kg), mean lean body mass (SEM) (62.2 [1.7 ] vs 65.1 [1.6] kg), and mean reduced fat mass (SEM) (18.6 [1.9] vs 17.2 [2.1] kg). Significant increases in circumferences occurred for the biceps, shoulder, and chest sites (P<.05), while the abdominal, waist, hip, and gluteal circumferences decreased during resistance training in both androstenedione and placebo subjects (P<.05).
A major finding of this study is that short- and long-term androstenedione supplementation did not increase the serum testosterone concentration in young men with normal serum testosterone levels. The only prior report on androstenedione administration in humans demonstrated substantial elevations in the blood testosterone concentration in 2 healthy women.13 In these women, 100 mg of androstenedione produced increases in the blood androstenedione concentration from 0 to 5 nmol/L and increased the blood total testosterone from 3 to 18 nmol/L. The results of the present study are in striking contrast, since the 36-nmol/L increase in the serum androstenedione concentration observed after short-term intake of androstenedione was not accompanied by any increase in the serum testosterone concentration. In the German patent26 for androstenedione, it is claimed that ingestion of androstenedione increases the serum testosterone concentration by as much as 237% within 15 minutes, followed by a secondary increase of 48% to 97% occurring 3 to 4 days later, and persisting for an additional 6 to 7 days. However, interpretation of this claim is impossible, since the subject population was not described with respect to age, sex, or hormonal status, and no data are presented.
The unchanged serum testosterone concentration with androstenedione supplementation in the present study, coupled with significant elevations in the serum estrone and estradiol concentrations, suggests that a significant proportion of the ingested androstenedione underwent aromatization to these estrogens.10,11 Anabolic steroid administration has previously been shown to suppress endogenous testosterone production, secondary to decreased serum levels of LH and FSH.27 In our study, serum concentrations of LH and FSH were unaffected by supplementation, suggesting that hypothalamic-pituitary function was not modified by androstenedione supplementation. Therefore, the unchanged serum testosterone concentration, in spite of the approximately 2.5 times higher androstenedione concentration, appears to be related to an increased formation of estrogens from the exogenous androstenedione.
The quantitative contribution of different tissues to the aromatization of androstenedione is unknown. However, aromatizing activity has been reported in most body tissues, and it is clear that there is ample capacity to support the increased estrone and estradiol concentrations reported in the present study. For example, adipose tissue has a maximal aromatizing activity of 0.072 pmol/g per hour with a Michaelis constant of 25 nmol/L.28 Since serum androstenedione concentrations were increased to approximately 24 nmol/L, aromatizing activity would have been at half the maximum rate (12 Vmax or 0.036 pmol/g per hour). With a fat mass of 19.3 kg, calculated total adipose tissue aromatizing activity is 695 pmol/h. If plasma volume is assumed to equal 20% of body weight, or about 4.0 L, the 47-pmol/L increase in the serum estrone concentration observed from week 0 to week 2 would reflect an increase of 188 pmol in the total increase in circulating estrone concentration. Thus, the aromatizing activity of adipose tissue alone could theoretically account for the increased serum estrone concentration observed with androstenedione supplementation. It has also been reported that muscle converts tritiated androstenedione to estrone at a rate almost as great as adipose tissue.29 Because of its large mass, muscle is also, therefore, a quantitatively significant source of estrogens. Since it has been estimated that muscle and adipose tissue combined account for only 35% to 45% of total extragonadal aromatization to estrogens,30 it is clear that whole-body aromatizing activity is sufficient to account for the observed increase in the serum estrone concentration.
Since many androstenedione users undoubtedly ingest amounts in excess of the 300 mg/d taken in our study, it could be argued that the dose of androstenedione was insufficient to raise serum testosterone levels. This dose exceeds the 100- to 200-mg/d intake recommended by most manufacturers and the dose (100 mg) observed to increase the blood testosterone concentration in women.13 The lack of any significant increase in the serum testosterone concentration, despite the 175% and 100% increases in the serum androstenedione concentration observed with short- and long-term administration of androstenedione, however, suggests than any putative increase in serum testosterone with higher doses would be associated with additional elevations in the serum estrogen concentration and lowering of the serum HDL concentration.
The significantly higher serum free testosterone concentrations observed in androstenedione both before and during resistance training and supplementation were unexpected, and difficult to explain, given the random assignment of subjects to each treatment group. However, values for all subjects were in the normal range, and it is unlikely that the initial differences influenced the response to the supplementation period.
Although androstenedione supplementation did not enhance the serum testosterone concentration in these young normotestosterogenic men, the reported increase in serum testosterone levels in women13 may suggest that androstenedione supplementation increases the serum testosterone concentration in hypotestosterogenic populations, such as women and older men.31,32
The resistance-training program used in this investigation was effective in enhancing lean body mass, the cross-sectional area of type 2 muscle fibers, and muscle strength. Gains in muscle size and strength are markedly enhanced when androgenic-anabolic steroids are taken in conjunction with a resistance-training program.1-4 In our study, the increases in lean body mass, muscle fiber cross-sectional area, and muscle strength were not enhanced with androstenedione supplementation. These results are not surprising, since serum testosterone concentrations were not affected by androstenedione supplementation, and since androstenedione has only weak anabolic-androgenic activity in comparison with testosterone.33 The large increases in strength observed in both experimental groups suggest that the lack of any improvement in strength with androstenedione supplementation is not due to an inadequate training stimulus but instead is due to lack of efficacy of androstenedione as an anabolic-androgenic supplement.
A significant lowering of the serum HDL-C concentration was observed with androstenedione administration, a finding in agreement with prior work demonstrating a lowering of the HDL-C concentration with anabolic steroid use.3,15-19 The reduction in HDL-C appears to be due primarily to a reduction in the HDL2 subfraction, secondary to an induction of hepatic triacylglycerol lipase activity.21,34 The serum HDL-C concentration did not reach a level (<0.91 mmol/L [<35 mg/dL]) typically considered to constitute a risk factor for cardiovascular disease.35 However, the finding that cardiovascular disease risk increases 2% to 3% with every 0.03-mmol/L (1-mg/dL) decrease in HDL-C suggests that the significant reduction in HDL-C observed with androstenedione supplementation is clinically relevant.36 Since serum testosterone concentrations were unaffected by androstenedione supplementation, the decrease in HDL-C may be due to the approximately 2.5 times higher serum androstenedione concentration. Previous research has reported that anabolic steroid administration lowers the HDL-C concentration by as much as 27% to 70%.3,15-19 One possible explanation for the significant, but smaller (12%), decrease in the HDL-C concentration in our study is the lower metabolic potency of androstenedione compared with testosterone.33 In addition, subjects in prior studies typically consumed high doses of more metabolically active anabolic steroids for more prolonged periods.
Elevated serum liver transaminase concentrations are frequently observed during clinical steroid therapy using 17α-alkylated or other oral compounds.37,38 Although the serum concentration of liver transaminases has been reported to be significantly elevated with anabolic steroid administration in athletes,20,21 this is not a universal finding.3,25,39 In our study, serum liver enzyme levels were unaffected by the 8-week period of androstenedione administration. However, significant impairment of liver function following more prolonged androstenedione supplementation or with higher dosages cannot be ruled out.
The hormonal milieu induced by androstenedione supplementation may predispose the user to adverse consequences in addition to those documented in this study. Increased serum estrogen levels have been known for some time to be associated with the development of gynecomastia.40 Increased concentrations of estrogens may also increase the risk of cardiovascular disease.41 Elevated estradiol concentrations have been related to increased risk of breast cancer in women42 and pancreatic cancer in men.43 Furthermore, elevated serum androstenedione concentrations have been observed to increase the risk for prostate cancer in some44 but not all45 previous studies, and increased serum androstenedione concentrations may also be associated with pancreatic cancer.46 Taken together, these previous findings suggest that androstenedione supplementation may predispose the user to additional health risks.
In summary, androstenedione administration during resistance training did not significantly alter the serum testosterone concentration in normotestosterogenic young men. The increased muscle size and strength observed with resistance training were also not augmented with androstenedione administration. The use of androstenedione increased the serum concentrations of estradiol and estrone, suggesting an increased aromatization of the ingested androstenedione and/or testosterone derived from the exogenous androstenedione. The use of androstenedione was associated with decreased levels of HDL-C. These data provide evidence that androstenedione does not enhance adaptations to resistance training and may result in potentially serious adverse health consequences in young men.
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Shalender Bhasin, M.D., Thomas W. Storer, Ph.D., Nancy Berman, Ph.D., Carlos Callegari, M.D., Brenda Clevenger, B.A., Jeffrey Phillips, M.D., Thomas J. Bunnell, B.A., Ray Tricker, Ph.D., Aida Shirazi, R.Ph., and Richard Casaburi, Ph.D., M.D.
Athletes often take androgenic steroids in an attempt to increase their strength. The efficacy of these substances for this purpose is unsubstantiated, however.
We randomly assigned 43 normal men to one of four groups: placebo with no exercise, testosterone with no exercise, placebo plus exercise, and testosterone plus exercise. The men received injections of 600 mg of testosterone enanthate or placebo weekly for 10 weeks. The men in the exercise groups performed standardized weight-lifting exercises three times weekly. Before and after the treatment period, fat-free mass was determined by underwater weighing, muscle size was measured by magnetic resonance imaging, and the strength of the arms and legs was assessed by bench-press and squatting exercises, respectively.
Among the men in the no-exercise groups, those given testosterone had greater increases than those given placebo in muscle size in their arms (mean [±SE] change in triceps area, 424±104 vs. -81±109 mm2; P<0.05) and legs (change in quadriceps area, 607±123 vs. -131±111 mm2; P<0.05) and greater increases in strength in the bench-press (9±4 vs. -1±1 kg, P<0.05) and squatting exercises (16±4 vs. 3±1 kg, P<0.05). The men assigned to testosterone and exercise had greater increases in fat-free mass (6.1±0.6 kg) and muscle size (triceps area, 501±104 mm2; quadriceps area, 1174±91 mm2) than those assigned to either no-exercise group, and greater increases in muscle strength (bench-press strength, 22±2 kg; squatting-exercise capacity, 38±4 kg) than either no-exercise group. Neither mood nor behavior was altered in any group.
Supraphysiologic doses of testosterone, especially when combined with strength training, increase fat-free mass and muscle size and strength in normal men.
Anabolic–androgenic steroids are widely abused by athletes and recreational bodybuilders because of the perception that these substances increase muscle mass and strength,1-9 but this premise is unsubstantiated. Testosterone replacement increases nitrogen retention and fat-free mass in castrated animals and hypogonadal men,10-15 but whether supraphysiologic doses of testosterone or other anabolic–androgenic steroids augment muscle mass and strength in normal men is unknown.1-9 Studies of the effects of such steroids on muscle strength have been inconclusive,16-33 and several reviews have emphasized the shortcomings of the studies.1-5,8-10 Some of the studies were not randomized; most did not control for intake of energy and protein; the exercise stimulus was often not standardized; and some studies included competitive athletes whose motivation to win may have kept them from complying with a standardized regimen of diet and exercise.
We sought to determine whether supraphysiologic doses of testosterone, administered alone or in conjunction with a standardized program of strength-training exercise, increase fat-free mass and muscle size and strength in normal men. To overcome the pitfalls of previous studies, the intake of energy and protein and the exercise stimulus were standardized. Because some previous studies had demonstrated significant increases in muscle strength and hypertrophy in experienced athletes but not in sedentary subjects, we studied men who had weight-lifting experience.
This study was approved by the institutional review boards of the Harbor–UCLA Research and Education Institute and the Charles R. Drew University of Medicine and Science. All the study subjects gave informed written consent. The subjects were normal men weighing 90 to 115 percent of their ideal body weights; they were 19 to 40 years of age and had experience with weight lifting. They were recruited through advertisements in local newspapers and community colleges. None had participated in competitive sports in the preceding 12 months. Men who had ever taken anabolic agents or recreational drugs or had had a psychiatric or behavioral disorder were excluded from the study.
Of 50 men who were recruited, 7 dropped out during the control period because of problems with scheduling or compliance. The remaining 43 men were randomly assigned to one of four groups: placebo with no exercise, testosterone with no exercise, placebo plus exercise, and testosterone plus exercise. The study was divided into a 4-week control period, a 10-week treatment period, and a 16-week recovery period. During the four-week control period, the men were asked not to lift any weights or engage in strenuous aerobic exercise.
Of the 43 men, 3 dropped out during the treatment phase: 1 because of problems with compliance, 1 because illicit-drug use was detected by routine drug screening, and 1 because of an automobile accident. Forty men completed the study: 10 in the placebo, no-exercise group; 10 in the testosterone, no-exercise group; 9 in the placebo-plus-exercise group; and 11 in the testosterone-plus-exercise group.
STANDARDIZATION OF PROTEIN AND ENERGY INTAKE
Two weeks before day 1, the men were instructed to begin following a standardized daily diet containing 36 kcal per kilogram of body weight, 1.5 g of protein per kilogram, and 100 percent of the recommended daily allowance of vitamins, minerals, and trace elements. Compliance with the diet was verified every four weeks by three-day records of food consumption. The dietary intake was adjusted every two weeks on the basis of changes in body weight.
The men received either 600 mg of testosterone enanthate in sesame oil or placebo intramuscularly each week for 10 weeks in the Clinical Research Center. This dose is six times higher than the dose usually given as replacement therapy in men with hypogonadism and is therefore supraphysiologic. Doses as high as 300 mg per week have been given to normal men for 16 to 24 weeks without major toxic effects.34
The men in the exercise groups received controlled, supervised strength training three days per week during the treatment period. All the men trained at equivalent intensities in relation to their strength scores before the training. The training consisted of a cycle of weight lifting at heavy intensity (90 percent of the maximal weight the man lifted for one repetition before the start of training), light intensity (70 percent of the pretraining one-repetition maximal weight), and medium intensity (80 percent of this maximal weight) on three nonconsecutive days each week.35 Regardless of the actual weights lifted, the training was held constant at four sets with six repetitions per set (a set is the number of complete repetitions of an exercise followed by rest). Because previous research had demonstrated increases in strength of approximately 7 percent for the bench-press exercise and 12 percent for the squatting exercise after four to five weeks of training,35 the weights were increased correspondingly during the final five weeks of training in relation to the initial intensity. The number of sets was also increased from four to five, but the number of repetitions per set remained constant. The men were advised not to undertake any resistance exercise or moderate-to-heavy endurance exercise in addition to the prescribed regimen.
EVALUATION AND OUTCOME MEASURES
The primary end points were fat-free mass, muscle size as measured by magnetic resonance imaging (MRI), and muscle strength as based on the one-repetition maximal weight lifted during the bench-press and squatting exercises before and after the 10-week treatment period. Serum concentrations of total and free testosterone, luteinizing hormone, follicle-stimulating hormone, and sex hormone–binding globulin were measured on days 14 and 28 of the control period and days 2, 3, 7, 14, 28, 42, 56, and 70 of the treatment period. Blood counts, blood chemistry (including serum aminotransferases), serum concentrations of prostate-specific antigen, and plasma concentrations of total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, and triglycerides were measured at the start of the control period and on day 4; on days 28, 56, and 70 of the treatment period; and four months after the discontinuation of treatment. Periodic evaluations to identify adverse effects were performed by examiners unaware of the study-group assignments on days 1 and 28 of the control period; days 28, 56, and 70 of the treatment period; and four months after the discontinuation of treatment. Mood and behavior were evaluated during the first week of the control period and after 6 and 10 weeks of treatment. Sexual function and semen characteristics were not assessed.
ASSESSMENT OF MUSCLE SIZE
Muscle size was measured by MRI of the arms and legs at the humeral or femoral mid-diaphyseal level, the junction of the upper third and middle third of the bone, and the junction of the middle third and lower third. The cross-sectional areas of the arms and legs, the subcutaneous tissue, the muscle compartment, and the quadriceps and triceps muscles were computed, and the areas at the three levels were averaged.
ANALYSIS OF BODY COMPOSITION
Fat-free mass was estimated on the basis of measurements of body density obtained by underwater weighing. During weighing, the men were asked to exhale to the residual volume, as measured by helium dilution.
MEASURES OF MUSCLE STRENGTH
The effort-dependent performance of muscle was assessed on the basis of the maximal weight lifted for one repetition during the bench-press and squatting exercises.36 Each man completed increasingly more difficult lifts with the same weights and bars that he used during training; in each exercise, the maximal weight lifted (the one-repetition maximum) was recorded as a measure of muscle strength.
Serum concentrations of luteinizing hormone and follicle-stimulating hormone were measured by immunofluorometric assays,36 each with a sensitivity of 0.05 IU per liter. Serum testosterone was measured by immunoassay,37 and free testosterone was measured by equilibrium dialysis.37 Serum concentrations of sex hormone–binding globulin and prostate-specific antigen were measured by immunoassays using reagents purchased from Delphia–Wallac (Turku, Finland) and Hybritech (San Diego, Calif.), respectively.
ASSESSMENT OF MOOD AND BEHAVIOR
A standardized Multidimensional Anger Inventory38 that includes 38 questions to measure the frequency, duration, magnitude, and mode of expression of anger, arousal of anger, hostile outlook, and anger-eliciting situations and a Mood Inventory that includes questions pertaining to general mood, emotional stability, and angry behavior were administered before, during (week 6), and after the treatment (unpublished data). For each man a live-in partner, spouse, or parent answered the same questions about the man’s mood and behavior.
The Shapiro and Wilk test was used to test whether the outcome variables had a normal distribution. Changes were computed for each subject as the difference between the values for each variable at the beginning and end of the treatment period (from day 0 to day 70). These values were averaged among the subjects in each group to obtain the group means. Analysis of variance was used to determine whether there were base-line differences among the four groups. Two-tailed, paired t-tests were used to test for changes in each outcome variable in each group. If there was a change, an analysis of variance was used to test for differences between groups in the amount of change, and then Scheffé’s test was used to assess pairwise differences. This test adjusts for multiple comparisons, but it does not yield exact P values for pairwise comparisons between groups.
The four groups were similar with respect to age and weight, height, and body-mass index before treatment (Table 1). Acne developed in three men receiving testosterone and one receiving placebo, and two men receiving testosterone reported breast tenderness, but no other side effects were noted. The serum liver-enzyme concentrations, hemoglobin concentrations, hematocrits, and red-cell counts did not change in any study group (Table 2). Serum creatinine concentrations did not change, except in the testosterone-plus-exercise group, in which the mean (±SE) serum creatinine concentration increased from 1.0 mg per deciliter (88 μmol per liter) to 1.1 mg per deciliter (97 μmol per liter) (P=0.02). Plasma concentrations of total and LDL cholesterol and triglycerides did not change in any study group; plasma HDL cholesterol decreased significantly in the placebo-plus-exercise group. There was no change in the serum concentration of prostate-specific antigen in any group.
Table 3.Serum Concentrations of Endocrine Hormones in the Study Subjects before and after the 10 Weeks of Treatment.
The base-line serum concentrations of total and free testosterone in the four groups were similar. The serum concentrations of total and free testosterone increased significantly in the two testosterone groups, but not in the placebo groups (Table 3). The base-line serum concentrations of luteinizing hormone, follicle-stimulating hormone, and sex hormone–binding globulin were similar in the four groups, and the concentrations decreased significantly in the two testosterone groups.
BODY WEIGHT AND COMPOSITION
Table 4.Body Weight, Fat-free Mass, and Muscle Size and Strength before and after the 10 Weeks of Treatment.
Body weight did not change significantly in the men in either placebo group (Table 4). The men given testosterone without exercise had a significant mean increase in total body weight, and those in the testosterone-plus-exercise group had an average increase of 6.1 kg in body weight — a greater increase than in the other three groups.
Figure 1.Changes from Base Line in Mean (±SE) Fat-free Mass, Triceps and Quadriceps Cross-Sectional Areas, and Muscle Strength in the Bench-Press and Squatting Exercises over the 10 Weeks of Treatment.
Fat-free mass did not change significantly in the group assigned to placebo but no exercise (Table 4 and Figure 1). The men treated with testosterone but no exercise had an increase of 3.2 kg in fat-free mass, and those in the placebo-plus-exercise group had an increase of 1.9 kg. The increase in the testosterone-plus-exercise group was substantially greater (averaging 6.1 kg). The percentage of body fat did not change significantly in any group (data not shown).
The mean cross-sectional areas of the arm and leg muscles did not change significantly in the placebo groups, whether the men had exercise or not (Table 4 and Figure 1). The men in the testosterone groups had significant increases in the cross-sectional areas of the triceps and the quadriceps (Table 4); the group assigned to testosterone without exercise had a significantly greater increase in the cross-sectional area of the quadriceps than the placebo-alone group, and the testosterone-plus-exercise group had greater increases in quadriceps and triceps area than either the testosterone-alone or the placebo-plus-exercise group (P<0.05).
Muscle strength in the bench-press and the squatting exercises did not change significantly over the 10-week period in the group assigned to placebo with no exercise. The men in the testosterone-alone and placebo-plus-exercise groups had significant increases in the one-repetition maximal weights lifted in the squatting exercises, averaging 19 percent and 21 percent, respectively (Table 4 and Figure 1). Similarly, mean bench-press strength increased in these two groups by 10 percent and 11 percent, respectively. In the testosterone-plus-exercise group, the increase in muscle strength in the squatting exercise (38 percent) was greater than that in any other group, as was the increase in bench-press strength (22 percent).
MOOD AND BEHAVIOR
No differences were found between the exercise groups and the no-exercise groups or between the placebo groups and the testosterone groups in any of the five subcategories of anger assessed by the Multidimensional Anger Inventory. No significant changes in mood or behavior were reported by the men on the Mood Inventory or by their live-in partners, spouses, or parents on the Observer Mood Inventory.
Our results show that supraphysiologic doses of testosterone, especially when combined with strength training, increase fat-free mass, muscle size, and strength in normal men when potentially confounding variables, such as nutritional intake and exercise stimulus, are standardized. The combination of strength training and testosterone produced greater increases in muscle size and strength than were achieved with either intervention alone. The combined regimen of testosterone and exercise led to an increase of 6.1 kg in fat-free mass over the course of 10 weeks; this increase entirely accounted for the changes in body weight.
The exercise was standardized in all the men, and therefore the effects of testosterone on muscle size and strength cannot be attributed to more intense training in the groups receiving the treatment. Careful selection of experienced weight lifters, the exclusion of competitive athletes, and close follow-up ensured a high degree of compliance with the regimens of exercise, treatment, and diet, which was verified by three-day food records (data not shown) and the values obtained for serum testosterone, luteinizing hormone, and follicle-stimulating hormone. Except for one man who missed one injection, all the men received all their scheduled injections. It has been argued that studies in which large doses of androgens are used cannot be truly blinded because of the occurrence of acne or other side effects. In this study, neither the investigators nor the personnel performing the measurements knew the study-group assignments. Three men receiving testosterone and one man receiving placebo had acneiform eruptions; these men may have assumed themselves to be receiving testosterone. Thus, it cannot be stated with certainty that the men were completely unaware of the nature of their treatments.
The doses of androgenic steroids used in previous studies were low,1-5,11,12 mostly because of concern about potential toxic effects. In contrast, to our knowledge the dose of testosterone enanthate administered in this study (600 mg per week) is the highest administered in any study of athletic performance. Undoubtedly, some athletes and bodybuilders take even higher doses than those we gave. Furthermore, athletes often “stack” androgenic and anabolic steroids, taking multiple forms simultaneously. We do not know whether still higher doses of testosterone or the simultaneous administration of several steroids would have more pronounced effects. The absence of systemic toxicity during testosterone treatment was consistent with the results of studies of the contraceptive efficacy of that hormone.34
The method used in this study to evaluate muscle performance on the basis of the one-repetition maximal weight lifted is dependent on effort. Although the men receiving testosterone did have increases in muscle size, some of the gains in strength may have resulted from the behavioral effects of testosterone.
The dose dependency of the action of testosterone on fat-free mass and protein synthesis has not been well studied. Forbes39 proposed a single dose–response curve extending from the hypogonadal to the supraphysiologic range. Others have suggested that there may be two dose–response curves: one in the hypogonadal range, with maximal responses corresponding to the serum testosterone concentrations at the lower end of the range in normal men, and the second in the supraphysiologic range, presumably representing a separate mechanism of action — that is, a pathway of independent androgen receptors.1,40
Supraphysiologic doses of testosterone, with or without exercise, did not increase the occurrence of angry behavior by these carefully selected men in the controlled setting of this experiment. Our results, however, do not preclude the possibility that still higher doses of multiple steroids may provoke angry behavior in men with preexisting psychiatric or behavioral problems.
Our results in no way justify the use of anabolic–androgenic steroids in sports, because, with extended use, such drugs have potentially serious adverse effects on the cardiovascular system, prostate, lipid metabolism, and insulin sensitivity. Moreover, the use of any performance-enhancing agent in sports raises serious ethical issues. Our findings do, however, raise the possibility that the short-term administration of androgens may have beneficial effects in immobilized patients, during space travel, and in patients with cancer-related cachexia, disease caused by the human immunodeficiency virus, or other chronic wasting disorders.
Supported by a grant (1 RO1 DK 45211) from the National Institutes of Health, by a General Clinical Research Center grant (MO-00543), and by grants (P20RR11145-01, a Clinical Research Infrastructure Initiative; and G12RR03026) from the Research Centers for Minority Institutions.
We are indebted to Dr. Indrani Sinha-Hikim for the serum hormone assays, to Dr. Paul Fu for the plasma lipid measurements, to the staff of the General Clinical Research Center for conducting the studies, and to BioTechnology General Corporation, Iselin, New Jersey, for providing testosterone enanthate.
From the Department of Medicine, Charles R. Drew University of Medicine and Science, Los Angeles (S.B., C.C., B.C.); the Exercise Science Laboratory, El Camino College, Torrance, Calif. (T.W.S., T.J.B.); the Department of Medicine, Harbor–UCLA Medical Center, Torrance, Calif. (N.B., J.P., R.C.); and the Department of Public Health, Oregon State University, Corvallis (R.T., A.S.).
Address reprint requests to Dr. Bhasin at the Division of Endocrinology, Metabolism and Molecular Medicine, Charles R. Drew University of Medicine and Science, 1621 E. 120th St., MP #2, Los Angeles, CA 90059.
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Biotransformation of organic compounds by using microbial whole cells provides an efficient approach to obtain novel analogues which are often difficult to synthesize chemically. In this manuscript, we report for the first time the microbial transformation of a synthetic anabolic steroidal drug, oxymetholone, by fungal cell cultures.
Incubation of oxymetholone (1) with Macrophomina phaseolina, Aspergillus niger, Rhizopus stolonifer, and Fusarium lini produced 17β-hydroxy-2-(hydroxy-methyl)-17α-methyl-5α-androstan-1-en-3-one (2), 2α,17α-di(hydroxyl-methyl)-5α-androstan-3β,17β-diol (3), 17α-methyl-5α-androstan-2α,3β,17β-triol (4), 17β-hydroxy-2-(hydroxymethyl)-17α-methyl-androst-1,4-dien-3-one (5), 17β-hydroxy-2α-(hydroxy-methyl)-17α-methyl-5α-androstan-3-one (6), and 2α-(hydroxymethyl)-17α-methyl-5α-androstan-3β-17β-diol (7). Their structures were deduced by spectral analyses, as well as single-crystal X-ray diffraction studies. Compounds 2–5 were identified as the new metabolites of 1. The immunomodulatory, and anti-inflammatory activities and cytotoxicity of compounds 1–7 were evaluated by observing their effects on T-cell proliferation, reactive oxygen species (ROS) production, and normal cell growth in MTT assays, respectively. These compounds showed immunosuppressant effect in the T-cell proliferation assay with IC50 values between 31.2 to 2.7 μg/mL, while the IC50 values for ROS inhibition, representing anti-inflammatory effect, were in the range of 25.6 to 2.0 μg/mL. All the compounds were found to be non-toxic in a cell-based cytotoxicity assay.
Microbial transformation of oxymetholone (1) provides an efficient method for structural transformation of 1. The transformed products were obtained as a result of de novo stereoselective reduction of the enone system, isomerization of double bond, insertion of double bond and hydroxylation. The transformed products, which showed significant immunosuppressant and anti-inflammatory activities, can be further studied for their potential as novel drugs.
Microbial regio- and stereo-selective transformations of steroids have been extensively investigated [1, 2]. We have been studying microbial transformation of bioactive steroids with the objectives of producing their novel metabolites and understanding their metabolism [3–8]. Oxymetholone [17β-hydroxy-2-(hydroxymethylene)-17α-methyl-5α-androstan-3-one, C21H32O3 (1), a 17α-alkylated anabolic-androgenic steroid, has been approved by the US Food and Drug Administration for the treatment of blood anemia, osteoporosis, HIV-associated wasting, antithrombin III deficiency, pediatric growth impairment, and damaged myocardium; as well as for stimulating muscle growth in malnourished, and underdeveloped individuals. However, it is also been abused by some athletes for enhancing muscle mass and strength [9, 10]. Compound 1 also known to exhibit immunosuppressant activity in vivo by decreasing the activity of T-cells .
During current study, compound 1 was found to exhibit significant immunomodulatory and anti-inflammatory activities in vitro in T-cell proliferation and reactive oxygen species (ROS) production assay, respectively. T-Lymphocytes play a key role in cell mediated immune response by activating various T-cells and modulating autoimmune response. Thus inhibition of T-cell proliferation can serve as an approach to treat various immune disorders , including organ rejection after transplant . The immune system also utilizes various cellular processes for elimination of pathogens, such as phagocytosis which involves elimination of pathogens by enzyme catalyzed oxidative burst. Ironically prolonged overproduction of ROS can damage body’s own cells and tissues, and lead to chronic inflammation and other autoimmune diseases .
Based on these preliminary findings, we investigated biotransformation of 1 to obtain new analogues. Preliminary experiments showed that Macrophomina phaseolina, Aspergillus niger, Rhizopus stolonifer, and Fusarium lini can efficiently transform 1 into several metabolites. Subsequent large scale fermentation was carried out and four new metabolites 2–5, along with two known metabolites 6 and 7 were obtained. The structures of new metabolites were unambiguously deduced through 1D- and 2D-NMR and by single-crystal X-ray diffraction techniques. Metabolites 2–7 were then evaluated for the inhibition of T-cell proliferation (IC50 values in the range of 31.2 to 2.7 μg/mL), and ROS production (1 and 2 had a strong inhibition (IC50 = 2 to 2.3 μg/mL)), representing their immunosuppressant and anti-inflammatory potential.
Results and discussion
Structure elucidation of metabolites
Incubation of oxymetholone (1) (C21H32O3) with Macrophomina phaseolina, Aspergillus niger, Rhizopus stolonifer and Fusarium lini produced six metabolites 27 (Figure 1). Metabolites 6 and 7 were previously obtained through chemical hydrogenation of 1, however this is the first report of their biomimetic synthesis. The structures of new metabolites 25 were unambiguously deduced largely by single-crystal X-ray diffraction analyses.
The HREI-MS M+ = m/z 332.2333 (calcd 332.2351) (C21H32O3)], IR (1666 cm-1) and UV (236.4 nm) spectra of 2 suggested the C = C isomerization of 1 into 2[8, 16]. The 1H- and 13C-NMR spectra (Table 1) showed a new olefinic methine H/C signal at δ 6.48/δ 152.4, along with a hydroxyl-bearing methylene H2/C at δ 4.78 and 4.76 (J21a,b = 15.0 Hz)/δ 59.8 for HC-1 and H2C-21, respectively. Me (19) protons (δ 0.96) were correlated with C-1 (δ 152.4) in HMBC, while C-21 protons (δ 4.78, 4.76) were correlated with C-1 (δ 152.4) and C-2 (δ 137.5). Additionally, C-1 (δ 6.48) and C-4 protons (δ 2.47, 2.26) were also correlated with C-3 (δ 199.0), and C-2 (δ 137.5) C-1 Proton also showed HMBC correlations with C-21 (δ 59.8). The structure of new metabolite 2 was finally deduced through single-crystal X-ray diffraction techniques (Figure 2). The asymmetric unit contains two independent molecules of metabolite 2. The ORTEP diagrams of 2 (Figure 2) showed four trans fused rings A, B, C, and D with half chair/chair/chair, and envelop conformations, respectively. C-17 -OH and methyl groups exist in pseudoequatorial and pseudoaxial orientations, respectively. The shorter bond lengths of C-2—C-3 single-bond (1.476(8) Å) is due to the conjugation of C-1—C-2 (1.321(7) Å) olefinic bond with the C-3 carbonyl moiety. All bond angles and lengths were within the normal range.
Table 1 1H- and13C-NMR Chemical Shifts of New Compounds 2–5 (δ in ppm;JandW1/2in Hz)
1.63; 1.57, m
2.28, dd (12.5, 4.5); 1.30, m
4.05, m (W1/2 = 20.6 )
4.51, br. s (W1/2 12.0)
3.86, m (W1/2 = 20.6 )
2.47, dd (17.3, 4.2), 2.26, dd (17.3, 4.0)
1.71; 1.60 m
1.87 ddd (13.0, 5.0, 2.5); 1.72, m
1.32; 1.27, m
1.31; 1.25, m
1.28; 1.22, m
2.32, 2.21, m
1.71; 0.85, m
1.65; 0.90, m
1.65; 0.85, m
1.75; 0.85, m
1.63; 1.37, m
1.57; 1.29, m
1.62; 1.33, m
1.65; 1.55, m
1.61; 1.22, m
1.78; 1.49, m
1.58, 1.35, m
1.53; 1.14, m
1.53; 1.24, m
1.57; 1.34, m
1.54; 1.29, m
1.48; 1.30, m
2.14, ddd (13.5, 11.7, 3.5); 1.77, m
2.30, ddd (15.6, 9.2, 6.7); 1.95, m
2.13; 1.76, m
2.14, td (13.0, 3.5); 1.78, m;
4.09; 3.82, d (10.3)
4.78; 4.76, d (15.0)
4.17; 4.03, m
5.06; 4.98, m
Computer–generated ORTEP diagram of metabolite 2(Hydrogens are omitted for clarity).
The UV inactive metabolite 3 showed molecular ion (M+) at m/z 352.2643 (calcd 352.2613), in agreement with the formula C21H36O4, indicating reduction of C = C bond and addition of any oxygen. The 1H- and 13C-NMR spectra of 3 (Table 1) showed two pairs of downfield OH-bearing methylene H2/C signals at δ 4.09, 3.82 (d, J20a,b = 10.3 Hz)/δ 67.3 (H2C-20) and 4.17, 4.03 (m)/δ 66.1 (H2C-21), along with an OH-bearing methine H/C at δ 4.51 (br. s, W1/2 = 12.0 Hz)/δ 67.6 (HC-3). The H2C-21 (δ 4.17, 4.03) and HC-3 (δ 4.51) both showed COSY correlations with the vicinal HC-2 (δ 1.98). Moreover, C-21 protons showed HMBC correlations with C-1 (δ 36.5), C-2 (δ 40.5) and C-3 (δ 67.6) (Figure 3a). Similarly, C-20 methylene protons at δ 4.09, 3.82 exhibited HMBC interactions with C-13 (δ 46.1), C-16 (δ 34.1), and C-17 (δ 83.2). Stereochemistry at C-2 and C-3 was deduced from the NOE correlations of HC-2 (δ 1.98) with Me-19 (δ 0.87), and HC-3 (δ 4.51) with HC-5 (δ 2.01) (Figure 3b). On the basis of above observations, the new metabolite was characterized as 2α,17α-di(hydroxymethyl)-5α-androstan-3β-17β-diol (3).
Important (a) HMBC, and (b) NOESY correlations in metabolite 3.
The formula C20H34O3 for 4 (M+ = m/z 322.2507, calcd 322.2508) indicated four double bond equivalents and loss of oxidative carbon. The UV and IR spectra indicated the absence of enone functionality. The 1H- and 13C-NMR (Table 1) of metabolite 4 was substantially different from that of the substrate 1, first due to the lack of HC-21 olefinic methine signal, and second the appearance of two new hydroxyl-bearing methine H/C signals at δ 4.05 (m, W1/2 = 20.6 Hz)/δ 73.1 and 3.87 (m, W1/2 = 20.6 Hz)/δ 76.7. Vicinal correlations between H2C-1 (δ 2.27, 1.30), HC-2 (δ 4.05), HC-3 (δ 3.87) and H2C-4 (δ 1.88, 1.72) in the COSY 45° spectrum indicated the oxidative loss of C-21 methine. HC-2 (δ 4.05) and HC-3 (δ 3.87) were also correlated to C-1 (δ 46.5), C-4 (δ 37.2), C-5 (δ 45.4), and C-10 (δ 37.6) in the HMBC spectrum. The structure of new metabolite 4 was unambiguously established by single-crystal X-ray diffraction analysis as half water solvate (Figure 4). It showed four trans fused rings A, B, C, and D exist in chair/ chair/chair, and envelop conformations, respectively. The C-2 and C-3 vicinal diols adopt equatorial orientations, whereas hydroxyl and methyl substituents at C-17 were found in pseudoequatorial and pseudoaxial orientations, respectively. All bond angles and lengths were in agreement with other related steroidal structures .
Computer-generated ORTEP diagram of metabolite 4 (Hydrogens are omitted for clarity).
The molecular composition C21H30O3 was deduced from the HREI-MS of metabolite 5 (M+ = m/z 330.2183, calcd 330.2195), which was 2 amu less than the metabolite 2. The UV spectrum of 5 exhibited a λmax at 249.8 nm due to extended conjugation in ring A. The 1H- and 13C-NMR spectra (Table 1) of metabolite 5 was distinctly similar to 2, indicating the presence of two olefinic H/C at δ 7.45 (s)/δ 150.0 (HC-1) and 6.28 (d, J = 1.0 Hz)/δ 124.0 (HC-4) in ring A, along with hydroxy-methylene H2/C signals at δ 5.06, 4.98 (m)/δ 59.5 (H2C-21). The methine signals δ 6.28/δ 124.0 and methylene signals δ 5.06, 4.98/δ 59.5 showed similar HMBC correlations as those of HC-1 and H2C-21 for metabolite 2. Similarly, C-4 proton (δ 6.28) was correlated to C-3 (δ 186.0), C-5 (δ 169.4), C-6 (δ 32.5) and C-10 (δ 43.5) in HMBC spectrum. The structure of metabolite 5 was unambiguously deduced by single-crystal X-ray diffraction studies (Figure 5). The asymmetric unit contains two water solvated independent molecules of metabolite 5. The ORTEP diagrams (Figure 5) showed that compound 5 is consisting of four fused rings A , B, C and D. Trans fused rings B, C and D adopt chair/chair and envelop conformations, respectively, with the pseudo equatorial orientation of hydroxyl substituent at C-17. Ring A was found to be planner in geometry due to extended conjugation. All the bond angles and lengths were within the normal range as observed in previously reported steroids .
Computer–generated ORTEP diagram of metabolite 5(Hydrogens are omitted for clarity).
The structure of known metabolite 6 was unambiguously deduced through single-crystal X-ray diffraction studies. ORTEP diagrams (Figure 6) showed that it consists of trans fused rings A, B, C, and D with chair/chair/chair and envelop conformations, respectively. The hydroxymethylene moiety attached to C-2, and OH at C-17 were found in equatorial and peudo equatorial orientations, respectively. All the bond angles and lengths were found within normal range. Known metabolite 7 was structurally identified by comparing of its spectral data with the one reported earlier.
Computer–generated ORTEP diagram of metabolite 6(Hydrogens are omitted for clarity).
T-Cell proliferation inhibitory activity
Oxymetholone (1) is known to modulate cell-mediated immunity in vivo. This was in part due to a decrease in T-cell activity . This initial observation prompted us to investigate the T-cell proliferation inhibitory potential of 1 and its metabolites.
Compounds 1–7 were evaluated for their effect on T-cell proliferation by employing PHA to activate human peripheral mononuclear cells (PBMC), isolated from the blood sample from healthy human volunteers. The results indicate that metabolite 6 possess more potent T-cell proliferation inhibitory activity (IC50 = 2.7 μg/mL), as compared to the substrate 1 (IC50 = 7.5 μg/mL) and standard prednisolone (IC50 < 3.1 μg/ mL) (Table 2, Figure 7); whereas compounds 7 and 4 showed a moderate inhibitory activity (IC50 = 10.6 ± 0.4 and 11.8 ± 0.9 μg/mL, respectively). Compounds 2–5 showed a weak inhibition of T-cell proliferation as compared to compounds mentioned earlier. Limited SAR indicated that greater flexibility in ring A, due to reduction of C = C or C = O bonds probably contributes in activity.
Table 2 Inhibitory effect of compounds on the T-cell proliferation in comparison to standard
7.5 ± 0.4
17.4 ± 1.2
17.0 ± 1.2
11.8 ± 0.9
31.2 ± 1.9
2.7 ± 0.2
10.6 ± 0.4
a)Each IC50 value represents the mean value of triplicate reading ± SD.
Effect of compounds 1–7 on phytohemagglutinin (PHA) activated T–cell proliferation. The effect of compounds on the T-cell proliferation is compared with control. Each bar represents the mean value of triplicate reading ± SD.
ROS inhibition activity
Compounds 1–7 were also investigated for their effect on ROS production by using whole blood and professional phagocytic PMNs and the detecting probes luminal (Table 3, Figures 8 and 9). Compound 2 showed slightly stronger (p ≤ 0.005) inhibition (IC50 = 2.0 μg/mL) as compared to 1 (IC50 = 2.3 μg/mL). Both compounds were at least five fold more active than the standard ibuprofen (IC50 = 11.2 μg/mL) in whole blood phagocytes. Metabolite 7 showed a moderate inhibitory activity (IC50 = 25.6 μg/mL), while other compounds did not show any activity even at the highest concentration (100 μg/mL). Compounds which showed potent inhibitory activities were further evaluated by using professional phagocytes PMNs. Compound 1 showed a significant inhibition of ROS generation in the PMNs (IC50 = 6.3 μg/mL). Interestingly ROS generation inhibitory activity of compound 1 and its known metabolites 6 and 7 have not been studied before.
Table 3 Inhibitory effects of compounds on ROS production in human whole blood and PMNs
IC50 μg/mLa) (Whole Blood phagocytes)
IC50 μg/mLa) (PMNs)
2.3 ± 0.0
6.3 ± 0.1
2.0 ± 0.8
11.2 ± 1.9
2.5 ± 0.6
a)Each IC50 value represents the mean value of triplicate reading ± SD.
Effect of compounds 1–7 on reactive oxygen species (ROS) production using whole blood phagocytes. The compounds activity was compared with the control (C = cells with activator). Each plot and error bar represents reading ± SD of three repeats.
Effect of compounds 1 and3–7 on reactive oxygen species (ROS) production using isolated neutrophils. The compounds activity was compared with the control (C = cells with activator). Each plot and error bar represents reading ± SD of three repeats.
Compounds 1–7 were found to be non-toxic towards the normal mouse fibroblast (3T3) cells, even at the highest concentrations tested (100 μg/mL).
Our study provides an efficient method for the production of new anabolic steroids by the structural transformation of oxymetholone (1) by using fungi. The procedure presented here can also be used for the study of the metabolism of oxymetholone (1), as well as for the production of potential immunomodulatory and anti-inflammatory drugs. In the current study, compounds 2–7 found to posses a strong inhibitory effect on T-cell proliferation, with IC50 values between 31.2 to 2.7 μg/mL, while in case of the ROS production, only compounds 1 and 2 exerted significant inhibition (IC50 ~ 2.0 μg/mL) on whole blood phagocytes. Whereas only 1 showed significant ROS inhibition (IC50 = 6.3 μg/mL) on the isolated PMNs.
General experimental conditions
Silica gel precoated plates (Merck, PF254; 20 × 20, 0.25 mm, Germany) were used for the TLC based separation. Silica gel (70–230 mesh, Merck) was used for column chromatography. Melting points were determined with a Buchi-535, apparatus and are uncorrected. Optical rotations were measured in methanol with a JASCO P-2000 polarimeter. UV Spectra (in nm) were recorded in methanol with a Hitachi U-3200 spectrophotometer. Infrared (IR) spectra (in cm-1) were recorded with an FT-IR-8900 spectrophotometer. 1H- and 13C-NMR spectra were recorded in C5D5N on a Bruker Avance NMR spectrometer, with residual solvent signal as the internal standard. Standard Bruker pulse sequences were used for 1D- and 2D-NMR experiments. The chemical shifts (δ values) are reported in parts per million (ppm), relative to TMS at 0 ppm. The coupling constants (J values) are reported in Hertz. Electron impact (EI-MS), and high-resolution mass spectra (HREI-MS) were recorded on JEOL JMS-600H mass spectrometer (Japan); in m/z (rel.%). Single-crystal X-ray diffraction data were collected on a Bruker Smart APEX II diffractometer with CCD detector . Data reductions were performed by using SAINT program. The structures were solved by direct methods , and refined by full-matrix least squares on F2 by using the SHELXTL-PC package . The figures were plotted with the aid of ORTEP program . The luminometer used was from Luminoskan RS (Labsystem Luminoskan, Helsinki, Finland), and cell harvester and glass fiber filters used were from Inotech (Dottikon, Swetzerland). Liquid scintillation counter used was LS65000 from Beckman Coulter (Fullerton, CA, USA). Microplate reader used was SpectraMax (Molecular Devices, CA, USA).
The chemicals and reagents were purchased from the following sources: Oxymetholone (1) (TCI, Japan), Luminol (Research Organics, OH, USA), Hanks balance salts solution (HBSS), phytohemagglutinin-L (PHA-L), penicillin, and streptomycin (Sigma,St. Louis, USA), lymphocytes separation medium (LSM) (MP Biomedicals, Illkirch, France), zymosan-A (Saccharomyces cerevisiae) (Fluka BioChemika, Buchs, Switzerland), tritiated thymidine (Amersham Pharmacia Biotech, UK) and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) (Amresco, Solon, OH, USA).
Tissue culture plates were obtained from Iwaki (Japan). Mouse fibroblasts (3T3) were obtained from the European American Culture Collection (EACC). Dulbcco’s modified eagle medium (DMEM) and fetal bovine serum were purchased from Gibco-BRL (Grand Island, NY, USA).
Microorganisms and culture medium
The fungi were obtained either from the Northern Regional Research Laboratories (NRRL), or Karachi University Culture Collection (KUCC) or American Type Culture Collection (ATCC).
Macrophomina phaseolina (KUCC 730) and Fusarium lini (NRRL 2204) were grown in a medium composed of the following ingredients and dissolved in distilled H2O (4.0 L): glucose (40.0 g), glycerol (40.0 mL), peptone (20.0 g), yeast extract (20.0 g), KH2PO4 (20.0 g), and NaCl (20.0 g). The media (2.0 L) for Aspergillus niger (ATCC 10549), and Rhizopus stolonifer (ATCC 10404) were the same as above, except addition of glycerol (10.0 mL) for A. niger and yeast extract (6.0 g) for R. stolonifer.
General fermentation and extraction conditions
The fungal medium was distributed into 250 mL conical flasks (100 mL each) and autoclaved at 121°C. Mycelia of the fungi were transferred to flasks and incubated at 26 ± 2°C for two-three days on rotary shaking (120 rpm). Compound 1, dissolved in acetone, was evenly distributed among all the flasks which were placed on the rotary shaker (120 rpm) at 26 ± 2°C for fermentation. Parallel control experiments were conducted which included an incubation of the fungus without substrate 1, and an incubation of 1 in the medium without fungus. The degree of transformation was analyzed on TLC after one day. The culture medium and mycelium were separated by filtration. The mycelium was washed with dichloromethane (CH2Cl2, 1.0 L). The aqueous filtrate was extracted with CH2Cl2 (4 L × 3). The CH2Cl2 extract was dried over anhydrous Na2SO4, evaporated under reduced pressures, and the resulting brown gum was analyzed by thin-layer chromatography. The control flasks were also harvested in the same manner, and compared with the test to assure the presence of biotransformed products.
Fermentation of oxymetholone (1) with Macrophomina phaseolina
Oxymetholone (1; 800 mg) was dissolved in 20 mL acetone, and uniformly distributed to 40 flasks containing 2 days old M. phaseolina culture. Fermentation was carried out for twelve days. The gummy material (2.5 g), obtained after filtration, extraction and evaporation, was loaded onto a silica gel column for fractionation. The mobile phase was composed of pet. ether and acetone with a gradient of 10%. Three main fractions (OX-1 − 3) were obtained on the basis of TLC analysis. Fraction OX-1 yielded metabolites 2 (07 mg) and 6 (43 mg) on elution from silica gel column (pet. ether: acetone = 9:1), while OX-2, when subjected to silica gel column chromatography, yielded metabolite 7 (21 mg, pet. ether: acetone = 8:2). Fraction OX-3 yielded metabolite 3 (276 mg, pet. ether: acetone = 7:3) after elution from silica gel column.
Colorless crystalline solid; m.p. 197–199°C. [lit. 198–200°C] ; [α]25D +13.5 (c 0.04, MeOH) [lit. +19.7]. Crystal data: empirical formula = C21H34O3, Mr = 334.48, orthorhombic, space group P212121, a = 7.3859 (3) Å, b = 20.6898 (9) Å, c = 12.4157 (6) Å, V = 1897.27 (15) Å3, Z = 4, ρcalc = 1.171 mg m-3, F(000) = 736, μ (Mo Kα) = 0.71073 Å, max/min transmission 0.9962/0.9754, crystal dimensions 0.33 x 0.21 x 0.05 mm, 1.64° < θ < 27.50°, 18840 reflections were collected, out of which 4479 reflections were observed (Rint = 0.0422) and 437 parameters were refined. The R-values were; R1 = 0.0534, wR2 = 0.1354 for I > 2σ (I), and R1 = 0.0707, wR2 = 0.1472 for all data, max/min residual electron density; 0.433/-0.214e Å-3. Crystallographic data for compound 6 can be obtained from the Cambridge Crystallographic Data Center (code CCDC 795530) (Additional file 4 and Additional file 5).
Fermentation of oxymetholone (1) with Aspergillus niger and Rhizopus stolonifer
Incubation of 1 (400 mg/10 mL acetone) with 2 days old culture of A. niger in 20 flasks for 6 days produced the previously isolated metabolites 6 (64 mg) and 7 (136 mg), while R. stolonifer (20 flasks) transformed 1 (400 mg/10 mL acetone) into metabolites 3 (86 mg) and 6 (15 mg).
Fermentation of oxymetholone (1) with Fusarium lini
Incubation of 1 (600 mg/15 mL acetone) with 2-day old F. lini culture in 30 flasks for 12 days produced three metabolites which were purified by silica gel column chromatography to obtain metabolites 2 (146 mg), 4 (32 mg, pet. ether: acetone = 7:3) and 5 (15 mg, pet. ether: acetone = 7:3).
Colorless crystalline solid; m.p.: 124–125°C. [α]25D : -29.0 (c = 0.01, MeOH). IR (KBr): νmax 3409, 2927, 1051 cm-1; 1H-NMR: (500 MHz, C5D5N) see Table 3; 13C-NMR: (125 MHz, C5D5N) see Table 3; EI-MS (%): m/z 322 (M+, 98), 307 (100), 304 (43), 264 (39), 249 (95), 229 (30), 215 (53), 181 (54), 171 (58), 169 (51), 123 (55), 109 (33), 95 (41), 81 (40), 57 (30), 43 (42); HREI-MS: m/z 322.2507 (M+ [C20H34O3]+, calcd 322.2508); Crystal data: empirical formula = C20H35O4 [C20H34O3·OH], Mr = 339.48, monoclinic, space group P21, a = 11.5169 (19) Å, b = 6.7843 (12) Å, c = 12.820 (2) Å, V = 945.6 (3) Å3, Z = 2, ρcalc = 1.192 mg m-3, F(000) = 374, μ (Mo Kα) = 0.71073 Å, max/min transmission 0.9912/0.9785, crystal dimensions 0.27 × 0.12 × 0.11 mm, 1.68° < θ < 25.50°, 4,363 reflections were collected, out of which 1,585 reflections were observed (Rint = 0.0493) and 221 parameters were refined. The R-values were; R1 = 0.0452, WR2 = 0.0808 for I > 2σ (I), and R1 = 0.0791, WR2 = 0.0914 for all data, max/min residual electron density; 0.147/-0.141 e Å-3. Crystallographic data for compound 4 can be obtained from the Cambridge Crystallographic Data Center (code CCDC 795528) (Additional file 7 and Additional file 8).
Colorless crystalline solid; m.p. 163–164°C; [α]25D -33.0 (c = 0.01, MeOH); UV (MeOH): λmax nm (log ε): 249.8 (4.0). IR (KBr): νmax 3402, 1664, 1620, 1082, 1033 cm-1. 1H-NMR: (500 MHz, C5D5N) see Table 3. 13C-NMR: (125 MHz, C5D5N) see Table 3. EI-MS (%): m/z 330 (M+, 35), 312 (19), 294 (13), 254 (17), 161 (24), 152 (76), 147 (29), 134 (100), 121 (34), 107 (18), 91 (14). HREI-MS: m/z 330.2183 (M+, [C21H30O3]+; calcd 330.2195). Crystal data: empirical formula = C42H62O8, Mr = 694.92, monoclinic, space group P21, a = 7.7609 (5) Å, b = 13.2141 (8) Å, c = 18.6769 (11) Å, V = 1913.9 (2) Å3, Z = 2, ρcalc = 1.206 mg m-3, F(000) = 756, μ (Mo Kα) = 0.71073 Å, max/min transmission 0.9879/0.9751, crystal dimensions 0.31 × 0.17 × 0.15 mm, 1.09° < θ < 25.00°, 10,983 reflections were collected, out of which 3,534 reflections were observed (Rint = 0.0364) and 458 parameters were refined. The R-values were; R1 = 0.0523, wR2 = 0.1342 for I > 2σ (I), and R1 = 0.0699, wR2 = 0.1544 for all data, max/min residual electron density; 0.320/-0.311 e Å-3. Crystallographic data for compound 5 can be obtained from the Cambridge Crystallographic Data Center (code CCDC 799213) (Additional file 9 and Additional file 10).
T-Cell proliferation inhibition assay
Peripheral blood mononuclear cells (PBMC) were isolated from heparinized venous blood of healthy adult donors by Ficoll–Hypaque gradient centrifugation . Cells were proliferated as reported earlier . Briefly, cells were cultured at a concentration of 2 × 106/mL in a 96-well round bottom tissue culture plate. Cells were stimulated with 5 μg/mL of phytohemagglutinin. Various concentrations of compounds were added to obtain final concentrations of 0.5, 5, 50 μg/mL, each in triplicate. The plate was incubated for 72 h at 37°C in 5% CO2 environment. After 72 h, cells were pulsed with 0.5 μCi/well, tritiated thymidine, and further incubated for 18 h. Cells were harvested onto a glass fiber filter by using cell harvester. The tritiated thymidine incorporation into the cells, which reflects the proliferation level, was measured by a liquid scintillation counter.
Phagocyte chemiluminescence assay
Luminol-enhanced chemiluminescence assay was performed according to the previous reported method . Briefly 25 μL of whole blood or neutrophils (1 × 106/mL), suspended in Hank’s solution, were incubated with 25 μL compounds (1, 10, 100 μg/mL for whole blood and 0.5, 5, 50 μg/mL for neutrophils) for 30 min. Zymosan 25 μL (20 mg/mL), followed by 25 μL (7 × 10-5 M) of luminol was added to make a final volume of 100 μL. A control without the compound was also run. Peak chemiluminescence was recorded using the luminometer. The luminometer was set with repeated scan mode, 50 scans with 30 s intervals and one second point measuring time.
The cytotoxicity of compounds was determined by using the MTT cellular assay [24, 25] against a normal mouse fibroblast (3T3) cell line. Cells were grown in DMEM and MEM (modified Eagle’s medium), containing 10% FBS and 2% antibiotic (penicillin and streptomycin), and maintained at 37°C in 5% CO2 for 24 hours in a flask. Cells were plated (1 × 105 cell/mL) in 96-well flat bottom plates and incubated for 24 hours for cell attachment. Various concentrations of compounds, ranging between 1.25-100 μM, were added into the well and incubated for 48 hours. A 50 μL [2 mg/mL] MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was added to the well, 4 hours before the end of incubation. Medium and reagents were aspirated and 100 μL DMSO was added and mixed thoroughly for 15 minutes to dissolve the formazan crystals. The absorbance was measured at 570 nm by using a microplate reader. Finally, IC50 (μM) values were calculated, and the experiment was repeated at least three times. Cycloheximide was used as the standard for normal fibroblast cell line.
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The effects of anabolic-androgenic steroid (AAS) abuse on the onset of puberty in female adolescents are largely unknown. This study assessed the acute effects of one AAS, stanozolol, on pubertal onset in the female rat. A single injection of stanozolol (5 mg/kg) on Postnatal Day (PN) 21 advanced vaginal opening but did not alter the onset of vaginal estrus. Higher doses of stanozolol treatment (10 and 25 mg/kg) also advanced vaginal opening but had no effect on vaginal estrus. The advancement of vaginal opening by stanozolol (5 mg/kg) was prevented by the concomitant administration of the pure antiestrogen ICI 182,780 (1 mg/kg) on PN20–22. Administration of the androgen receptor antagonist flutamide (10 mg/kg twice daily) on PN20–22 had no effect on the advancement of vaginal opening by stanozolol. Stanozolol treatment also advanced vaginal opening in ovariectomized rats. Perivaginal injections of a low dose of stanozolol (0.05 mg) on PN21 and PN23 also advanced vaginal opening. These results suggest that stanozolol is acting directly at estrogen receptors in the vaginal epithelium to advance vaginal opening and that prepubertal stanozolol treatment does not induce true precocious puberty.
Anabolic-androgenic steroids (AAS) are synthetic derivatives of testosterone that have been abused by athletes in an attempt to improve athletic performance . In contrast to AAS abuse by adolescent males, which has remained at a steady level since 1991, AAS abuse by adolescent females has actually increased during this same time period . AAS abuse by females has been associated with a number of adverse effects, including menstrual abnormalities and reproductive dysfunction [3–5]. Studies have been focused on the adverse effects of AAS abuse in adult females [3, 6]; however; the physiological effects of AAS abuse by adolescent females are largely unknown, and there is concern that some of the effects may be permanent .
In rat models, there is overwhelming evidence that puberty can be advanced by estrogen and aromatizable androgens [7, 8]. In contrast to the advancement of puberty by estrogen and aromatizable androgens, treatments with nonaromatizable androgens delay the onset of puberty [9, 10]. Thirty-day treatment with one AAS, stanozolol, has differential effects on pubertal endpoints in female rats. Chronic stanozolol treatment advanced the day of vaginal opening (VO) but inhibited vaginal cyclicity . Interpretation of the effects of chronic stanozolol administration on the onset of puberty, as defined by VO and the onset of estrous cyclicity, is confounded by the alteration of the feedback loops of the hypothalamus-pituitary-gonadal (HPG) axis coinciding with the time when puberty normally occurs. The present study was conducted to investigate the effects and mechanism(s) of action of the acute administration of one AAS, stanozolol, in prepubertal rats.
Materials and Methods
Timed-pregnancy Long-Evans rats were received in the vivarium of the Department of Psychological and Brain Sciences at Dartmouth College on Embryonic Day 18. The pregnant rats were housed individually in plastic tubs and maintained on a 12L:12D schedule with lights-off at 0900 h, the standard housing conditions in our vivarium. Food and water were freely available. The temperature and humidity were monitored and held constant. The day of birth was recorded as Postnatal Day 0 (PN0). On PN3, the litters were culled to nine pups, keeping as many female pups as possible. When the female pups were weaned on PN20/21, they were housed two per hanging metal cage.
The timed-pregnancy Long-Evans rats used in these experiments were obtained from Harlan Sprague-Dawley (Indianapolis, IN) and Charles River Laboratories (Wilmington, MA). Rats from Harlan were used until sialodacryoadenitis virus (SDAV) appeared in the Harlan stock, at which point the rats were then obtained from Charles River at the recommendation of our veterinary staff. The stock used for each experiment is noted. Investigations were conducted in accordance with the Guide for the Care and Use of Laboratory Animals.
Procedures and Analysis
The following drugs were used: stanozolol (17α-methyl-5α-androstan-17β-olo(3,2-c)pyrazole; Sigma Chemical Co., St. Louis, MO), flutamide (2-methyl-N-[4-nitro-3-(trifluoromethyl)-phenyl]propanamide; Sigma), ICI 182,780 (7α-[9-[(4,4,5,5,5,-pentafluoropentyl]sulphinyl)nonyl]-estra-1,3,5(10)-triene-3,17β-diol; Tocris Cookson, Ballwin, MO), methandrostenolone (17β-hydroxy-17-methyl-androsta-1,4-dien-3-one; Sigma), and dihydrotestosterone propionate (5α-androstan-17β-ol-3-one propionate; Steraloids, Newport, RI). Drugs were administered by s.c. injection in a sesame oil vehicle, with the exception of flutamide, which was administered in a 10% ethanol/propylene glycol vehicle. Drugs were coded, so the experimenter was blind to the treatment conditions.
The female pups were monitored daily for VO between approximately 0800 and 1000 h. Once VO had occurred, the vaginal cytology of the rats was monitored daily until PN65 (see exception for experiment 3A). The criterion for vaginal estrus was vaginal cytology that contained a majority of cornified epithelium cells in the absence of leukocytes and nucleated epithelial cells . In experiment 3A, ovariectomy was carried out under sodium methohexital (Brevital, 50 mg/kg; Henry Schein, Port Washington, NY) anesthesia. Although body weight was monitored throughout the course of the experiments, body weight data are not presented because there were no differences between treatment groups in any of the experiments.
All data sets, with the exception of the data from experiment 1B, met the assumption of a normal distribution and homogeneity of variance. The dependent measures, such as day of VO and day of first vaginal estrus, were evaluated by one-way analysis of variance, and post hoc analysis was performed with a Newman-Keuls test. The data from experiment 1B did not exhibit homogeneous variance and were thus subjected to nonparametric analysis (Kruskal-Wallis followed by Mann-Whitney U-test). Results are expressed as means ± SEM for each group. Differences were considered significant at P < 0.05.
Experiment 1: Acute Effects of Stanozolol Treatmenton Aspects of Pubertal Onset
In experiment 1A, at 0900 h on PN21, female pups from Harlan were weaned, weighed, randomly assigned to one of two groups (n = 6 pups/group), and given a single injection of either stanozolol (5 mg/kg) or the sesame oil vehicle (1 ml/kg) . PN21 was chosen as the age to treat the pups based on previous research [11, 14]. Experiment 1B was conducted to establish the dose response characteristics of a single injection of stanozolol. At 0900 h on PN21, female pups from Charles River were weaned, weighed, randomly assigned to one of five groups (n = 7 or 8 pups/group), and given a single injection of 0, 1, 5, 10, or 25 mg/kg stanozolol in a sesame oil vehicle.
Experiment 2: Stanozolol Action at Androgenand Estrogen Receptors
Experiment 2A was conducted following the same design as experiment 1, with the addition of groups receiving hormone receptor antagonists. Rats from Harlan received 5 mg/kg stanozolol or the sesame oil vehicle in combination with 1) the nonsteroidal androgen receptor (AR) antagonist flutamide (10 mg/kg twice daily), 2) the pure estrogen receptor (ER) antagonist ICI 182,780 (ICI, 1 mg/kg), or 3) the oil vehicle (1 ml/kg). The groups were as follows: stanozolol/flutamide (n = 12), stanozolol/ICI (n = 12), stanozolol/oil (n = 13), and oil/oil (n = 12). The flutamide, ICI, or vehicle was injected on PN20, 21, and 22. Injections were administered at approximately 0900 h, with the exception of the flutamide, which was administered at 0900 h and 2100 h. The doses of flutamide (10 mg/kg twice daily) and ICI (1 mg/kg) were selected based on previous research [15–17]. In pilot studies, we determined that when administered alone at these doses and ages, neither flutamide nor ICI altered the day of VO or vaginal cyclicity as compared with those of animals that received the oil vehicle: the mean (± SEM) day of VO (n = 6 pups/group) was 34.5 ±1.1 for oil/oil, 35.5 ± 1.0 for oil/flutamide, and 35.0 ± 0.5 for oil/ICI.
Two additional experiments assessed androgen effects on pubertal onset. In experiment 2B, rats from Charles River received 5 mg/kg stanozolol (n = 10), 7.5 mg/kg methandrostenolone (n = 10), or the oil vehicle (n = 11) on PN21. In experiment 2C, on PN21, rats from Charles River received either 7.5 mg/kg dihydrotestosterone propionate (DHTP) (n = 7), a nonaromatizable androgen, or the oil vehicle (n = 6). The doses of androgens were selected based on previous studies demonstrating the inhibition of estrogen-induced sexual receptivity in ovariectomized rats .
Experiment 3: Stanozolol Action at Central Versus Peripheral Targets
Two experiments were conducted to examine the site of action of stanozolol on VO. In experiment 3A, 16 female rats from Charles River were ovariectomized on PN20. One day later these rats received a single injection of either stanozolol (5 mg/kg, n = 8) or oil (1 ml/kg, n = 8). Estrous cyclicity was not monitored for this experiment. In experiment 3B, female rats were given perivaginal injections of 0.05 mg stanozolol in 0.01 ml oil (n = 6) or the oil vehicle (n = 6) on PN21 and PN23. The time course and procedure were similar to that used previously , where it was shown that perivaginal s.c. injections of 0.01 mg testosterone advanced VO.
Acute Effects of Stanozolol Treatment on Aspectsof Pubertal Onset
Vaginal opening vs. vaginal estrus
There was a dissociation between the effects of stanozolol on the day of VO and on the day of first vaginal estrus. As shown in Figure 1A, the day of VO was advanced significantly in the stanozolol group as compared with the oil group (F(1, 10) = 6.2, P < 0.05). In contrast to the effects of stanozolol on VO, the treatment groups did not differ for the day of first vaginal estrus. Furthermore, no long-term effects of a single injection of stanozolol on estrous cyclicity (including cycle length) were observed. During the 14 days after the first vaginal estrus, there were no differences in the number of days of vaginal estrus between the stanozolol and oil groups (Fig. 1B).
Experiment 1A: pubertal events of rats that received 5 mg/kg stanozolol or oil on PN21. A) Postnatal day of VO and of first vaginal estrus of rats that received oil (n = 6) or 5 mg/kg stanozolol (n = 6) on PN21. Data points are means + SEM. B) Number of days of vaginal estrus for the 14 days following the first vaginal estrus. *Significantly different from oil group (P < 0.05)
As shown in Figure 2, dose of stanozolol affected day of VO (H = 14.5, P < 0.05). The day of VO for the 10 and 25 mg/kg stanozolol groups was significantly different than that for the oil group (P < 0.05). Although there was a trend toward advanced VO in the 5 mg/kg stanozolol group, the effect was not significant. In subsequent experiments using rats from Charles River, 5 mg/kg stanozolol was sufficient to advance day of VO (experiments 2B and 3A). As seen in experiment 1, stanozolol (1–25 mg/kg) had no significant effect on the day of first vaginal estrus.
Experiment 1B: postnatal day of VO and of first vaginal estrus of rats that received oil or 1, 5, 10, or 25 mg/kg stanozolol (n = 7 or 8 pups/group) on PN21. Data points are means + SEM. *Significantly different from oil group (P < 0.05)
Stanozolol Acts via the ER to Advance VO
Stanozolol appears to advance VO via an ER-dependent mechanism. As shown in Figure 3, the day of VO was advanced significantly in the stanozolol/oil group as compared with the oil/oil group (F(3, 45) = 12.6, P < 0.05). ICI administration blocked the advancement of VO by stanozolol so that the day of VO of the stanozolol/ICI group was not significantly different from that of the oil/oil group. In contrast, AR blockade had no effect on the advancement of VO by stanozolol. Stanozolol advanced VO in rats receiving flutamide relative to the oil/oil group (P < 0.05). The day of VO of the stanozolol/flutamide group was not significantly different from that of the stanozolol/oil group. As seen in experiment 1, treatment conditions did not have a significant effect on the day of first vaginal estrus.
Experiment 2A: postnatal day of VO and of first vaginal estrus of rats that received oil/oil, stanozolol/oil, stanozolol/ICI, or stanozolol/flutamide (n = 12 or 13 pups/group). The stanozolol or oil was administered on PN21, and the ICI, flutamide, and oil were administered on PN21–23. Data points are means + SEM. *Significantly different from oil/oil group (P < 0.05)
In experiment 2B, administration of another synthetic androgen had an effect on day of VO. As shown in Figure 4, in agreement with the results from the previous experiments, the day of VO was advanced significantly in the stanozolol group as compared with the oil group (P < 0.05). In addition, the day of VO was also advanced significantly in the methandrostenolone group as compared with the oil group (P < 0.05). As before, the treatment conditions had no effect on the day of first vaginal estrus. As shown in Figure 5, there were no differences in either the day of VO or the day of first vaginal estrus between the group receiving the nonaromatizable androgen DHTP and the group that received the vehicle.
Experiment 2B: postnatal day of VO and of first vaginal estrus of rats that received 5 mg/kg stanozolol, 7.5 mg/kg methandrostenolone, or oil (n = 10 or 11 pups/group) on PN21. Data points are means + SEM. *Significantly different from oil group (P < 0.05)
Experiment 2C: postnatal day of VO and of first vaginal estrus of rats that received 7.5 mg/kg DHTP (n = 7) or oil (n = 6) on PN21. Data points are means + SEM
Stanozolol Acts in the Periphery, not Centrally, to Advance VO
Experiment 3A tested the effects of stanozolol on VO in ovariectomized rats. As shown in Figure 6, VO was advanced in the ovariectomized rats that received stanozolol as compared with the ovariectomized rats that received the oil vehicle (F(1, 14) = 28.0, P < 0.05), indicating that the ovaries are not necessary for the advancement of VO by stanozolol. As of PN60, in agreement with previous published studies [18, 19], a portion of the ovariectomized/oil rats exhibited VO (five of eight rats). The three rats that did not exhibit VO during the experiment were assigned a value of PN60 in the analysis.
Experiment 3A. A) Postnatal day of VO of ovariectomized (OVX) rats that received 5 mg/kg stanozolol (n = 8) or oil (n = 8) on PN21. Data points are means + SEM. B) Cumulative percentage of rats that achieved VO tabulated by postnatal day. The data for the Intact Oil groups were from the oil group from experiment 2B and were included for illustration purposes only. *Significantly different from oil group (P < 0.05)
In experiment 3B, rats were injected perivaginally with a low dose of stanozolol. As illustrated in Figure 7, VO was advanced in the group that received perivaginal injections of stanozolol as compared with the group that received perivaginal injections of the oil vehicle. As in the previous experiments, there was no difference in the day of first vaginal estrus between the stanozolol group and the vehicle group.
Experiment 3B: postnatal day of VO and of first vaginal estrus of rats that received perivaginal injections of oil (n = 6) or 0.05 mg of stanozolol in 0.01 ml oil (n = 6) on PN21 and PN23. Data points are means + SEM. *Significantly different from oil group (P < 0.05)
Effects of Acute Stanozolol Treatment on the Onsetof Puberty
A single injection of stanozolol advances VO when administered to prepubertal rats. In contrast to effects on VO, acute stanozolol treatment on PN21 did not alter the onset of estrous cyclicity. Both the day of first vaginal estrus and number of days of vaginal estrus that occurred during the 14 days following the first vaginal estrus were similar for rats that received either stanozolol or the oil vehicle. The latent effects of stanozolol on VO can be related to the effects of estrogen administration during a similar time frame. Estrogen administration (estradiol benzoate, 0.05 μg/100 g body weight on PN26–30) advanced VO . Presumably administration of estrogen or stanozolol sets into motion a cascade of physiological events resulting in VO. In contrast to the effects of stanozolol, however, estrogen also advances the onset of vaginal estrus . Thus, stanozolol treatment does not induce true precocious puberty.
There were apparent differences in the onset of puberty between the two source stocks of rats used in these three experiments. Although statistical comparisons between the two control groups were not conducted, the control rats from Charles River appeared to achieve VO and first vaginal estrus at a younger age than did the control rats from Harlan. The ranges of means for the control rats from Charles River were 28.7–31.3 for the day of VO and 30.7–35.6 for the day of first vaginal estrus. For the control rats from Harlan, the ranges were 34.5–37.5 for day of VO and 35.3–39.3 for day of first vaginal estrus. The first ovulation in rats occurs over a range of days that differs between different laboratory stocks . Despite the apparent differences between the stocks used in our experiments, stanozolol advanced VO in both stocks across all three experiments.
Mechanism and Site of Action
Our results suggest that stanozolol is acting at the peripheral ER to advance VO. Specifically, treatment with the ER antagonist ICI prevented the advancement of VO by stanozolol. In pilot experiments, we determined that administration of ICI alone on PN21 had no effect on VO. Because VO is an estrogen-dependent event, administration of the pure antiestrogen ICI would be predicted to delay VO. No studies have been conducted, however, administering ICI at peripubertal time points. The observation that the administration of stanozolol on PN21 advances the day of VO whereas ICI administration on the same day has no effect on day of VO may appear inconsistent with previous studies indicating that VO is an estrogen-dependent event  and the possibility that stanozolol advances VO via actions at the ER. However, a number of possibilities exist that may account for this apparent disparity. For example, the critical time during which an estrogenic stimulation is required may occur later than PN21. Stanozolol, which is designed for enhanced metabolic half-life , may still be active at later ages, whereas the actions of ICI may be limited to the time period just after injection. If this is the case, then injection of ICI at later ages (PN35+) would be predicted to block VO. The possibility remains to be tested, however, because of the vendor’s tight restrictions on the availability of ICI (100 mg per institution per year).
The data collected using ICI alone does not provide sufficient evidence to conclude that stanozolol is acting directly at the vaginal ER to advance VO . Even though ICI, which is thought unable to cross the blood-brain barrier , blocks the advancement of VO by stanozolol, the final common pathway for either direct or indirect actions of stanozolol on VO would be the activation of ER at the vagina, which would be blocked by ICI in either case. More compelling support for the hypothesis that stanozolol is acting in the periphery to advance VO is that if stanozolol were acting centrally to advance VO, e.g., by mimicking estrogen and stimulating LH release , then both VO and estrous cyclicity would be advanced and true precocious puberty would result. The data from the present experiments indicate that this is not happening. Stanozolol treatment only advanced VO and did not have any effect on the onset of vaginal estrus. In the present experiments, we measured vaginal estrus rather than ovulation to follow the cycle of the rats for the 2 wk following the first vaginal estrus and to observe potential long-term effects of stanozolol on estrous cyclicity. Further experiments need to be conducted to determine the effects of stanozolol on ovulation.
Additional data in the present experiments support the hypothesis that stanozolol is acting directly at the vaginal epithelium to advance VO. For example, stanozolol advanced VO in ovariectomized rats. If stanozolol were acting via a central mechanism to advance VO, then it would most likely act indirectly through the ovaries to trigger VO, and yet stanozolol advanced VO in the absence of ovaries. This result is similar to those of previous experiments, where the treatment of ovariectomized prepubertal rats with aromatizable androgens advanced VO, ruling out an effect dependent on the function of the ovary [22–24]. In other experiments conducted in our laboratory, systemic administration of stanozolol induced central effects, as evidenced by changes in behavior [25, 26]. We hypothesize that the lack of central action of stanozolol in the present study is due to the limited time course (a single injection) of stanozolol administration, the weak affinity of stanozolol for the ER, or some combination of these factors. Moreover, these same factors are likely to contribute to the dissociation between the effects of estrogen and stanozolol on VO versus vaginal estrus. The results from localized injections of stanozolol provide additional support for the hypothesis that stanozolol is acting at the vaginal epithelium to advance VO. Perivaginal injections of low doses of stanozolol advanced VO, whereas the same low dose of stanozolol had no effect on VO when administered systemically.
Stanozolol does not appear to be acting at the AR to advance VO; AR blockade by flutamide had no effect on the advancement of VO by stanozolol. The dose of flutamide used was sufficient to block the inhibition of estrogen-induced receptivity by stanozolol, suggesting that the dose was adequate . There is no evidence in the literature that the AR is involved in the advancement of VO. Contrary to the actions of estrogen and aromatizable androgens, nonaromatizable androgens either have no effect on the onset of puberty or delay it [9, 10]. Taken together, the results of the present experiments suggest that stanozolol may be acting at the vaginal ER to advance VO. This result is unexpected given that stanozolol cannot be aromatized .
The data presented here provide only indirect evidence that stanozolol is acting at the ER. Additional analyses of binding of stanozolol to the ER or evaluation of stanozolol action in ER knockout mice are essential to confirm this hypothesis. We plan to test stanozolol in estrogen-response element assays to clarify whether stanozolol is capable of binding to and activating the ER, as has been shown for other synthetic compounds and xenoestrogens [28, 29]. Such studies will provide a clearer picture of the mechanisms underlying the acute effects of AAS on the maturation of reproductive function. These findings will help us understand the influence of AAS use during adolescence on female reproductive health.
We thank Ms. Gillian Jacob, Ms. Jennifer Fulton, Ms. Lauryn Zipse, Mr. Christian Oberle, and Ms. Megan Kelton for technical assistance. We also thank Drs. Fay A. Guarraci and Leslie P. Henderson for their critical reading of the manuscript.
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1Università degli Studi di Parma, Dipartimento di Medicina e Chirurgia, Centro Universitario di Odontoiatria, Parma. Italy. Università degli Studi di Parma, Dipartimento di Medicina e Chirurgia, Centro Universitario di Odontoiatria, Parma. Italy
2Università degli Studi di Parma, Dipartimento di Medicina e Chirurgia, Unità di Patologia Generale, Parma. Italy
3Istituto dei Materiali per l’Elettronica ed il Magnetismo (IMEM) – CNR, Parma. Italy
Stanozolol (ST) is a synthetic androgen with high anabolic potential. Although it is known that androgens play a positive role in bone metabolism, ST action on bone cells has not been sufficiently tested to support its clinical use for bone augmentation procedures. Objective: This study aimed to assess the effects of ST on osteogenic activity and gene expression in SaOS-2 cells. Material and Methods: SaOS-2 deposition of mineralizing matrix in response to increasing doses of ST (0-1000 nM) was evaluated through Alizarin Red S and Calcein Green staining techniques at 6, 12 and 24 days. Gene expression of runt-related transcription factor 2 (RUNX2), vitamin D receptor (VDR), osteopontin (SPP1) and osteonectin (ON) was analyzed by RT-PCR. Results: ST significantly influenced SaOS-2 osteogenic activity: stainings showed the presence of rounded calcified nodules, which increased both in number and in size over time and depending on ST dose. RT-PCR highlighted ST modulation of genes related to osteogenic differentiation. Conclusions: This study provided encouraging results, showing ST promoted the osteogenic commitment of SaOS-2 cells. Further studies are required to validate these data in primary osteoblasts and to investigate ST molecular pathway of action.
Keywords: Osteogenesis; Bone matrix; Calcification; Gene expression; Androgens; Stanozolol
The research for new strategies and materials to enhance bone repair and/or bone regeneration is a major goal for the management of demanding clinical cases in orthopedics and maxillofacial surgery.
Androgens (or androgenic hormones) can be defined as any natural or synthetic steroid that stimulates or controls the development and maintenance of primary and secondary male characteristics in vertebrates by binding to the androgen receptor AR. 1 Androgens also provide anabolic functions, which result in growth and differentiation of cells and increase in body size. 2 Particularly, they play a significant role in regulating skeletal morphogenesis and maintaining bone homeostasis throughout life. 3,4 The most abundant circulating androgen in men is testosterone, whose effect in peripheral tissues not only depends on a direct action, but also results from a local enzymatic conversion in different metabolites. 5α-reductase and aromatase are among the most important enzymes responsible for testosterone transformation in bone tissues. 5α-reductase activity reflects in the formation of the potent androgen dihydrotestosterone, while aromatase catalyzes androgen conversion into the estrogen estradiol. Depending on its peripheral conversion, systemically administered testosterone may bind either to the AR (testosterone itself or dihydrotestosterone) or to the estrogen receptors ERα/ERβ (testosterone converted to estradiol), which results in androgenic or estrogenic effects. 5–7
The anabolic potential of androgens leads to the synthesis of molecules with a low androgenic and high anabolic action, with prolonged activity compared with endogenous androgens: these synthetic testosterone-derivative drugs are generally known as anabolicandrogenic steroids (AAS). One of these agents is stanozolol (ST), a non-aromatizable AAS derived from dihydrotestosterone.
Systemic administration of AAS in animal models provided some encouraging results, showing an overall increase in bone formation and mineralization, as well as improvements in bone density and biomechanical properties. 8–10 Nevertheless, other investigations reported qualitative alterations in the bone geometry and low bone turnover in response to ST treatment. 11 In brief, the overall efficacy and the long-term safety of AAS administration for the osteoporosis therapy and the prevention of fracture risk appears to be atleast questionable. 12 Systemic administration, local applications of ST and other AAS have been tested in animal models to improve bone healing. Such approaches allow the use of relatively low doses of steroid and imply short-term treatment protocols. Intra-articular ST administration showed positive effects on the synovial membrane and cartilage regeneration in osteoarthritis conditions 13 , and ST- soaked deproteinized bone grafts enhanced new bone formation in calvarial critical-size defects. 14
Although some evidence has been provided in human and animal studies, only a limited number of studies investigating ST effects on bone cells are currently available. SaOS-2 (literally “Sarcoma OSteogenic”) cell line represents a validated option for the study of osteoblastic differentiation and responsiveness to exogenous stimuli. In 1987, Rodan, et al. first conducted a study on SaOS-2 characterization and assessed that these cell lines possess several osteoblastic features and could be useful as a permanent line of human osteoblast-like cells and as a source of bone-related molecules. 15
SaOS-2 cells have the advantage of following the main molecular steps of osteoblast differentiation and have the ability “to deposit a mineralization-competent extracellular matrix”. 16 Thus, they have been recently validated as a feasible model to investigate osteoblast activity and maturation. 17 Immunocytochemical assays revealed that SaOS-2 cells express osteoblast-like markers such as osteocalcin (OC or BGLAP) and osteopontin (OPN or SPP1). Expression of genes involved in osteoblast differentiation and function (i.e. runt-related transcription factor 2, RUNX2) has been documented. 18 Also, the literature data provided evidence of SaOS-2 responsiveness to steroid stimulation. 19
The aim of this study was to assess the effects of ST on osteogenic activity and gene expression in SaOS-2 cells. The investigation of ST effects on bone cells may in fact provide evidence to support the clinical use of this steroid in the field of bone healing and regeneration, particularly for developing targeted drug administration protocols applied to orthopedic, maxillofacial and oral surgery.
MATERIALS AND METHODS
ST powder (ACME Srl, Reggio Emilia, Italy) was weighted and dissolved in absolute ethanol (ETOH), preparing 1000X stock solutions. Sequential dilutions of stocks were performed in the osteogenic medium, to obtain final concentrations of 1 nM, 10 nM, 100 nM, 500 nM and 1000 nM, respectively.
We preliminarily assessed ST effects on cell proliferation using resazurin assay up to 12 days of culture.
SaOS-2 cells ranging from 8 to 12 passages were plated at a density of 1×10× cells/cm2 into 6-well and 24-well plates, using respectively 2 mL and 500 μL of DMEM-low glucose with 10% fetal bovine serum (FBS), penicillin (100 μg/mL), streptomycin (100 μg/ mL) and L-glutamine (2 mM). After 24 h, this medium was replaced with an osteogenic medium consisting of DMEM-low glucose completed with 2-Phospho-L-ascorbic acid (100 μM), L-proline (34.8 μM) and β2-glycerol phosphate (5 mM). The day after (day 0), the medium was changed with fresh osteogenic medium containing stanozolol at the described concentrations, while osteogenic medium with 0.1% ETOH was used as a control. The culture medium was changed every two/three days.
After 6, 12 or 24 days, cells lying in 24-well plates were treated either with Alizarin Red S or Calcein Green staining.
Alizarin Red S staining: the cells were washed three times with PBS and fixed by adding 250 μL of 4% formaldehyde solution for 15 min at room temperature and rinsed twice with ddH2O. Then, 500 μL of Alizarin Red S solution in water (40 mM, pH 4,2) were added to each well, and the whole plates were kept at RT for 30 min with gentle shaking. The dye was removed, and cells were rinsed 5 times (5 min each time) with ddH2O.
To measure Alizarin Red S concentration, each well was treated with 200 μL of 10% acetic acid and incubated for 30 min at RT with shaking. Cells were scraped from the plate and transferred to a 1.5 mL microcentrifuge tube and sealed with parafilm. After vortexing vigorously for 30 seconds, the samples were heated to 85°C for 10 min. Then they were transferred on ice for 5 min and centrifuged at 20000 rpm for 15 min. After centrifugation, the slurry was transferred to a new tube, and pH was adjusted to 4.1-4.5 by adding 75 μL of 10% ammonium hydroxide. An Alizarin Red S standard curve was prepared with serial dilutions of Alizarin Red ranging from 10 mM to 10 μM, absorbance was measured at 405 nm with an Enspire microplate reader (Perkin Elmer, Waltham, Massachusetts, USA).
Calcein Green staining: 24 h before the end of the experimental period, 2 μl of Calcein Green (10 mg/mL) were added to each well. At the end of the experimental period, the samples were treated with 500 μl of acetic acid 10% dabbed with ammonium hydroxide pH 7.0. The whole plate was placed under slow oscillation for 20 min and then placed in an ultrasonic bath for 15 min. Each well was then washed three times with PBS. Semi-quantitative analysis of Calcein Green fluorescence was measured with an Enspire microplate fluorescence reader (Perkin Elmer, Waltham, Massachusetts, USA) set to a wavelength of 512 nm, as described elsewhere. 20
Gene expression analysis
RNA extraction and reverse transcription: At 12 and 24 days of culture, total RNA was isolated from cells seeded onto 6 well dishes with GenEluteTM Mammalian Total RNA Miniprep Kit (Sigma-Aldrich) following the manufacturer’s instructions, and 1 μg RNA/sample was reverse transcribed to cDNA (GoScript Reverse Transcription System, Promega Corporation, Madison, Wisconsin, USA). Briefly, RNA on 0.5 μg of random hexamer oligonucleotide primers, in a total volume of 5 μΙ, was heated to 70°C for 5 min, cooled to 4°C for 5 min, and then incubated with 15 μl of a mixture of components to achieve the final concentration of 0.5 mM each dNTPs, 1× first-strand buffer, 3 mM MgCl2, 1 U/μI Recombinant RNasinR Ribonuclease Inhibitor, Improm-II 1 μl/reaction, for 1 h at 42°C. The reaction was stopped by heating to 70°C for 15 min. The RT reaction was then diluted with nuclease free water to a total volume of 200 μl, and a triplicate of 5 μl aliquots was used for gene expression quantification in a 20 μl PCR.
Polymerase chain reaction: The primer set was designed according to the known sequences reported in GenBank with Primer 3 program [Steve Rozen, Helen J. Skaletsky (1998) Primer3. Code available at http://www-genome.wi.mit.edu/genome_software/other/primer3.html .] ( Figure 1 ). cDNA was amplified with 1X GoTaq qPCR Master, 5 pmol specific primers and RNase-free water. PCR was performed in a 36- well Rotor Gene 3000 (Rotor-Gene™ 3000, version 5.0.60, Mortlake, Australia). Each cycle consisted of a denaturation step at 95°C for 15 s, followed by separate annealing (15 s, 57°C or 60°C, depending on the examined gene) and extension (15 s, 72°C) steps. Fluorescence was monitored at the end of each extension step. A no-template, no-reverse transcriptase control was included in each experiment. At the end of the amplification cycles a melting curve analysis was added. The data analysis was performed according to the Relative Standard Curve Method. 21 Data normalization was carried out in relation to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which was found to be expressed uniformly in all the tested conditions.
Figure 1 Sequences of primers used for RT-PCR
Growth curves were analyzed using the Boltzmann sigmoidal function, and a comparison of curve fits was performed to verify the null hypothesis of one curve fitting all data sets and the alternative hypothesis of different curves for each culture condition. Cell differentiation and osteogenic activity was analyzed with one-way ANOVA and Tukey’s post-test. A linear regression analysis was performed to assess variations on different time-points. p<0.05 was considered the level of statistical significance. Graphs were obtained with GraphPad Prism 6.0 software. Data are expressed as mean value ± standard deviation.
Optical microscopy showed a typical polygonal shape of SaOS-2, which tended to become slightly elongated once they reached confluence. The resazurin assay revealed a growth pattern perfectly fitting a Sigmoidal Boltzmann curve up to 10 days of culture (DMEM low: r2=0.94, ETOH 0.1%: r2 =0.96, ST 1nM: r2=0.98, ST 10nM: r2 =0.93, ST 100nM: r2 =0.99, ST1000 nM: r2 =0.98), while at 12 days of culture an overall decrease in cell vitality was recorded independently of the culture conditions. A comparison of curve fits did not allow us to reject the null hypothesis of one curve fitting all data sets (p=0.8), thus indicating a superimposable growth pattern of SaOS-2 under all the tested conditions up to the end of the experimental period. A graphic representation of data is reported in Figure 2 .
Figure 2 (a) Aspect of SaOS-2 cells at confluency. Optical microscopy, 10X magnification; (b) Graphic representation of SaOS-2 growth under different conditions: DMEM-low (red line), ETOH 0.1% (blue line), ST 1-1000 nM (shades of grey). The X axis represents the days of culture, whereas the Y axis reports fluorescence values expressed in arbitrary units (A.U.)
Alizarin Red S staining confirmed the capacity of SaOS-2 to produce calcified extracellular matrix. The apposed matrix was characterized by round-shaped granules which increased progressively both in size and in number depending on the concentration of the administered steroid and extent of the induction ( Figure 3 a). Cells treated with ST revealed the presence of areas with mineralization since the earlier observation time-point, which peaked at 1000 nM concentration (fold change vs control: ST 1 nM: 1.44±0.08, p>0.05, ST 10 nM: 1.47±0.15, p>0.05, ST 100 nM: 1.55±0.15, p>0.05, ST 500 nM: 1.64±0.16, p>0.05, ST 1000 nM: 2.24±0.56, p<0.05). At 12 days, SAOS cell layers cultured with ST appeared consistently more filled with calcified granules compared with the controls at all the tested doses (fold change vs control ST 1 nM: 1.92±0.08; ST 10 nM: 1.95±0.09; ST 100 nM: 2.06±0.11; ST 500 nM: 2.10±0.16; ST 1000 nM: 2.17±0.01, p<0.01). A similar outcome was recorded at 24 days (fold change vs control ST 1 nM: 2.02±0.19; ST 100 nM: 2.13±0.24; ST 500 nM: 2.25±0.01, ST 1000 nM: 2.20±0.57, p<0.05) ( Figure 3 b).
Figure 3 (a) Appearance of SaOS-cell culture treated with different stanozolol (ST) concentrations (0-1000 nM) at 6, 12 and 24 days after Alizarin Red S staining. Optical microscopy, 10X magnification; (b) Alizarin Red S staining quantification with different ST concentrations (0-1000 nM) at 6, 12 days and 24 days. Data are reported as fold change over controls and expressed as mean ± standard deviation. Asterisks indicate statistical significance (*: p<0.05 vs ST 0 nM; ** p<0.005 vs ST 0 nM; ***: p<001 vs ST 0 nM)
Semiquantitative analysis of Calcein Green fluorescence revealed a deposition of calcium phosphates in response to ST administration ( Figure 4 a). At 6 days’ observation, a dose-dependent trend was also evident (fold change vs control ST 1nM: 1.50±0.16, p>0.05; ST 10 nM: 1.84±0.18, p >0.05; ST 100 nM: 3.58±0.54, p <0.005; ST 500 nM: 4.89±0.46, p <0.01; ST 1000 nM: 11.27±1.06, p <0.01). Observations at further time-points revealed a massive calcification in all the samples. All the tested ST doses produced significantly higher Calcein Green fluorescence compared with the controls both at 12 days (fold change vs control ST 1 nM: 2.03±0.14, p <0.05; ST 10 nM: 2.46±0.21, p <0.05; ST 100 nM: 3.11±0.21, p<0.005; ST 500 nM: 3.21±0.21, p<0.005; ST 1000 nM: 4.04±1.06, p<0.05) and 24 days (fold change vs control ST 1 nM: 1.75±0.10; ST 10 nM: 1.80±0.04; ST 100 nM: 2.07±0.04; ST 500 nM: 1.67±0.04; ST 1000 nM: 1.74±0.04; p<0.01) ( Figure 4 b).
Figure 4 (a) Appearance of samples treated with stanozolol (ST) (0-1000 nM) at 24 days observation period using a phase contrast microscopy and fluorescence microscopy to reveal Calcein Green staining (10X magnification); (b) Graph illustrating fluorescence absorbance of Calcein Green staining with different ST concentrations (0-1000 nM) at 6, 12 and 24 days observation period. Data are expressed as mean ± standard deviation. Asterisks indicate statistical significance (*: p<0.05 vs ST 0 nM; ** p<0.005 vs ST 0 nM; ***: p<001 vs ST 0 nM)
Gene expression analysis
The gene expression analysis related to osteogenic differentiation revealed differences depending both on the time-point (either 12 or 24 days) and on the concentration of the steroid ( Figure 5 ).
Figure 5 Gene expression of SaOS-2 treated with different concentrations (0-1000 nM) of stanozolol (ST) at 12 and 24 days observation period; (a) RUNX2: runt-related transcription factor 2; (b) VDR: Vitamin D Receptor; (c) SPP1: Osteopontin; (d) ON: Osteonectin. Data are reported as fold change over 0 nM ST and are expressed as mean ± standard deviation. Asterisks indicate statistical significance (*: p<0.05 vs control; ** p<0.005 vs control; ***: p<001 vs control)
RUNX2: At 12 days’ observation, the Runx2 expression was shown to increase at growing concentrations of ST, with significant differences vs controls for doses ranging from 10 to 1000 nM (fold change vs control ST 10 nM: 1.701±0.182, p <0.05; ST 100 nM: 1.847±0.226, p<0.005; ST 500 nM: 2.061±0.143, p <0.001; ST 1000 nM: 2.535±0.295, p <0.001). A similar pattern was recorded at 24 days (fold change vs control ST 1 nM: 2.025±0.191; ST 100 nM: 2.130±0.240; ST 500 nM: 2.250±0.014, ST 1000 nM: 2.200±0.566, p <0.05). At 24 days, the Runx2 expression showed a significant increase vs control only at the lowest ST concentrations (1 and 10 nM)used (fold change vs control ST 1nM: 1.514±0.234, p <0.05; ST 10 nM: 1.786±0.201, p <0.005). A tendency to decrease at the highest ST concentrations (500, 1000 nM) was also detected, although without any statistical significance ( Figure 5 a).
VDR: The VDR expression showed a consistent increase vs controls with the administration of the highest ST concentrations (fold change vs control ST 10 nM: 2.037±0.543, p <0.05; ST 100 nM: 2.388±0.427, p <0.001; ST 1000 nM: 2.255±0.247, p <0.001) at 12 days. At 24 days, all the tested ST doses were associated with significantly higher VDR expression vs controls (fold change vs control ST 1 nM: 2.158±0.070; ST 10 nM: 2.622±0.179; ST 100 nM: 2.770±0.090, ST 1000 nM: 2.901±0.073, p <0.001) ( Figure 5 b).
SPP1: The expression pattern of SPP1 showed variations depending on the observation period, with no significant differences in test groups vs controls at 12 days ( p >0.05) and a consistent induction observed at 24 days for all the tested concentrations of ST (fold change vs control ST 1 nM: 2.691±0.145; ST 10 nM: 2.401±0.416; ST 100 nM: 2.540±0.197; ST 500 nM: 2.680±0.166, ST 1000 nM: 2.331±0.048, p <0.005) ( Figure 5 c).
ON: The ON gene expression increased in response to the higher ST dose of 100 nM (fold change vs control: 2.645±0.109, p <0.05) and 1000 nM (fold change vs control: 4.175±0.577, p <0.001) at 12 days. At 24 days, no significant differences in test groups vs controls were recorded ( p >0.05) ( Figure 5 d).
This research investigated the effects of different doses of ST on the proliferation and osteogenic response of SaOS-2 cells. Growing evidence suggests androgens act directly on bone cells, playing a complex regulatory role. 22 Androgen effects on osteogenic differentiation are still controversial, nevertheless it has been suggested they may stimulate osteoblastic differentiation and extracellular bone matrix apposition. 23–25 Previous authors observed the effects of androgenic steroids on cell lines and reported positive effects of testosterone at doses of 10-10 M and 10-9 M on the proliferation of SaOS-2 cells after 48 h. 26 However, to the best of our knowledge, only one study reported on ST effects on osteogenic activity of bone cells, concluding that “Stanozolol at a concentration of 10-10 mol/l to 10-6 mol/l consistently stimulated the incorporation of [ 3 H]thymidine into DNA of human bone cells and increased proliferation” up to 15 days of culture. 27
According to our assay, ST treatment at the doses of 1 to 1000 nM did not affect the growth pattern of SaOS-2 cells up to 12 days of culture. This result may be due to the specific characteristics of the steroid used, although a peculiarity of the cells used in our experimental setting cannot be ruled out. Indeed, various SaOS-2 subpopulations that responded differently to proliferative and differentiative stimuli were identified. 28 Moreover, the phenotypic stability of SaOS-2 may be affected by the number of passages they have undergone: it was noticed that a higher passage SaOS-2 demonstrated higher proliferation rates and lower alkaline phosphatase activities, although mineralization was significantly more pronounced in cultures of late passage cells. 29 Such findings are consistent with our results of an overall high proliferation rate of SaOS-2 ranging from 8 to 12 passages as well as a high mineralizing activity.
Alizarin Red S and Calcein Green staining showed ST administration notably increased mineralization. These findings highlighted the advantages of treating cells with androgens compared with the use of a standard differentiation medium. At 12 days’ observation all the tested doses showed a similar effect with Alizarin Red S quantification technique, whereas a different dose- dependent effect was recorded with Calcein Green staining. These differences may point to a greater sensitivity of Calcein Green technique compared with Alizarin Red S. Nevertheless, neither Alizarin Red S nor Calcein Green revealed any differences between the effect of treatment at 24 days’ observation, when all the samples presented abundant uniform calcification.
RT-PCR analysis revealed a modulatory role played by ST on the gene expression related to osteogenic differentiation. RUNX2 represents an early differentiation marker, as its expression is enhanced since the first stages of osteoblast maturation. 30 The detection of RUNX2 mRNA in control samples confirmed previous observations that described a constitutive expression of this gene in SaOS-2 cells. 18 In addition, we found out that RUNX2 expression may be modulated by steroid treatment: according to our results at 12 days, the expression of RUNX2 was increasing with a dose-dependent trend, consistently with the mineralization pattern (Calcein Green staining). We may hypothesize that treatment with higher doses of ST induced a faster activation in terms of osteo-differentiation and mineralization when compared with lower doses. Thus, an overall decrease in RUNX2 expression at 24 days in samples treated with high doses of ST is compatible with a lower mitotic activity and a more mature phenotype. On the other hand, lower doses may produce a similar effect throughout a longer timeframe. It would be interesting to investigate the mineralization pattern occurring between 12 and 24 days, as at 24 days we observed a massive mineralization, which may mask previous differences between samples.
Another hypothesis to explain RUNX2 decrease at 24 days is that of a biphasic effect of higher ST doses, which may improve cell differentiation at early time points (12 days) and may not keep this effect at late time points (24 days). A biphasic effect of androgens on cell viability has been described in the literature, with an initial increase in cell proliferation followed by a decrease after prolonged exposure. 31 However, according to our preliminary assay, ST treatment did not affect the growth pattern of SaOS-2 up to 12 days. It would be interesting to investigate whether a different effect on cell viability is observed between 12 and 24 days.
An increase in SPP1 expression in response to ST was recorded respectively at 12 and 24 days of ST treatment, which first demonstrated the modulatory activity of this androgen on genes related to osteogenic function. Interestingly, the expression pattern of RUNX2 and SPP1 was shown to be inversely correlated, with a marked increase of SPP1 observed together with a decrease in RUNX2 expression. This finding may indicate an expression switch from 12 to 24 days, as it was observed that in SaOS-2 cells RUNX2 repressed SPP1 gene expression, and the induction of SPP1 expression during normal human osteoblast differentiation has been previously related to a decrease in RUNX2. 32 Consistently, the ON expression pattern revealed that, at the highest tested concentration, ST promoted the initial phases of osteoblastic commitment (12 days), whereas its action was no more evident at a longer time-point (24 days), when the differentiation was more advanced. Another gene expression that was strongly enhanced by ST treatment in our study was VDR, which encodes the nuclear hormone receptor for vitamin D3 and has been recognized as a key gene for SaOS-2 differentiation elsewhere. 33 It would be relevant to assess changes in the expression of other genes typical of both early and late differentiation phases and to set a more complete differentiation profile of cells in response to growing steroid doses. Moreover, an examination of protein levels would be appropriate to validate our RT- qPCR data, since mRNA expression could not directly correlate to protein translation and activity.
A major limitation of this study is represented by the lack of assessment of ST receptor binding and molecular pathway of action. Since ST is a non- aromatizable androgen, we may suppose its action to be exerted through AR. The expression of AR in SaOS-2 cells has been previously described in the literature. 34 However, the interaction of ST with AR and its influence on cell transcriptional activity is still unclear: previous studies documented an activation of AR in response to ST treatment, 35 but also a variety of other receptors have been reported as ST ligands (including progesterone receptor, estrogen receptor alpha and low-affinity glucocorticoid-binding sites). 36–39 Such differences could be dependent on the cell type, as ST may have tissue-specific binding sites and elicit differential biological responses. According to these considerations, it would be relevant to characterize SaOS-2 receptor profile, to investigate ST binding to AR and to perform blockage tests to verify the activation of different molecular pathways in response to ST administration.
Finally, we recommend considering potential side effects of AAS in further in vivo studies: changes in cholesterol levels (increased low-density lipoprotein and decreased high-density lipoprotein), liver damage, nephropathy, cardiovascular pathologies as well as conditions pertaining to hormonal imbalance have been reported in response to AAS high-dose or prolonged administration. 40
This study provided encouraging results, as it showed ST promoted the osteogenic commitment of SaOS-2 cells, by enhancing the mineralization process and modulating the expression of genes related to osteogenic differentiation.
Nevertheless, further studies are required to validate these data in primary osteoblasts as well as to investigate ST receptor binding and molecular pathway of action.
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15- Rodan SB, Imai Y, Thiede MA, Wesolowski G, Thompson D, Bar- Shavit Z, et al. Characterization of a human osteosarcoma cell line (Saos-2) with osteoblastic properties. Cancer Res. 1987;47(18):4961- 6.
16- McQuillan DJ, Richardson MD, Bateman JF. Matrix deposition by a calcifying human osteogenic sarcoma cell line (SAOS-2). Bone. 1995;16(4):415-26.
17- Prideaux M, Wijenayaka AR, Kumarasinghe DD, Ormsby RT, Evdokiou A, Findlay DM, et al. SaOS2 Osteosarcoma cells as an in vitro model for studying the transition of human osteoblasts to osteocytes. Calcif Tissue Int. 2014;95(2):183-93.
18- Lucero CM, Vega OA, Osorio MM, Tapia JC, Antonelli M, Stein GS, et al. The cancer-related transcription factor Runx2 modulates cell proliferation in human osteosarcoma cell lines. J Cell Physiol. 2013;228(4):714-23.
19- Rao LG, Wylie JN, Kung Sutherland MS, Murray TM. 17 betaoestradiol enhances the stimulatory effect of 1,25-dihydroxyvitamin D3 on alkaline phosphatase activity in human osteosarcoma SaOS-2 cells in a differentiation-dependent manner. J. Endocrinol. 1996;148(1):181-7.
20- Hale LV, Ma YF, Santerre RF. Semi-quantitative fluorescence analysis of calcein binding as a measurement of in vitro mineralization. Calcif Tissue Int. 2000;67(1):80-4.
21- Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001;29(9):e45.
22- Vanderschueren D, Vandenput L, Boonen S, Lindberg MK, Bouilln R, Ohlsson C. Androgens and bone. Endocr Rev. 2004;25(3):389-425.
23- Lin IC, Slemp AE, Hwang C, Sena-Esteves M, Nah HD, Kirschner RE. Dihydrotestosterone stimulates proliferation and differentiation of fetal calvarial osteoblasts and dural cells and induces cranial suture fusion. Plast Reconstr Surg. 2007;120(5):1137-47.
24- Gao K, Wang X, Liu Q, Chen W, Wang G, Zhang D, et al. Evaluation of osteoblast differentiation and function when cultured on mesoporous bioactive glass adsorbed with testosterone. J Cell Biochem. 2018;119(7):5222-32.
25- Costa KJ, Passos JJ, Gomes AD, Sinisterra RD, Lanza CR, Cortés ME. Effect of testosterone incorporation on cell proliferation and differentiation for polymer-bioceramic composites. J Mater Sci Mater Med. 2012;23(11):2751-9.
26- Fohr B, Schulz A, Battmann A. Sex steroids and bone metabolism: comparison of in vitro effects of 17beta-estradiol and testosterone on human osteosarcoma cell lines of various gender and differentiation. Exp Clin Endocrinol Diabetes. 2000;108(6):414-23.
27- Vaishnav R, Beresford JN, Gallagher JA, Russell RG. Effects of the anabolic steroid stanozolol on cells derived from human bone. Clin Sci (Lond). 1988;74(5):455-60.
28- Kasperk CH, Faehling K, Börcsök I, Ziegler R. Effects of androgens on subpopulations of the human osteosarcoma cell line SaOS2. Calcif Tissue Int. 1996;58:376-82.
29- Hausser HJ, Brenner RE. Phenotypic instability of Saos-2 cells in long-term culture. Biochem Biophys Res Commun. 2005;333(1):216-22.
30- Cutarelli A, Marini M, Tancredi V, D’Arcangelo G, Murdocca M, Frank C, et al. Adenosine triphosphate stimulates differentiation and mineralization in human osteoblast-like Saos-2 cells. Dev Growth Differ. 2016;58(4):400-8.
31- Wiren KM, Toombs AR, Zhang XW. Androgen inhibition of MAP kinase pathway and Elk-1 activation in proliferating osteoblasts. J Mol Endocrinol. 2004;32(1):209-26.
32- Lamour V, Detry C, Sanchez C, Henrotin Y, Castronovo V, Bellahcène A. Runx2- and histone deacetylase 3-mediated repression is relieved in differentiating human osteoblast cells to allow high bone sialoprotein expression. J Biol Chem. 2007;282(50):36240-9.
33- Rao LG, Liu LJ, Rawlins MR, McBroom RJ, Murray TM, Reddy GS, et al. The biological activities of 1alpha,25-dihydroxyvitamin D3 and its synthetic analog 1alpha,25-dihydroxy-16-ene-vitamin D3 in normal human osteoblastic cells and human osteosarcoma SaOS-2 cells are modulated by 17-beta estradiol and dependent on stage of differentiation. Biol Pharm Bull. 2001;24(3):242-8.
35- Feldkoren BI, Andersson S. Anabolic-androgenic steroid interaction with rat androgen receptor in vivo and in vitro: a comparative study. J Steroid Biochem. 2005;94:481-7.
36- Ellis AJ, Cawston TE, Mackie EJ. The differential effects of stanozolol on human skin and synovial fibroblasts in vitro: DNA synthesis and receptor binding. Agents Actions. 1194;41(1-2):37-43.
37- Zhu SY, Li YH, Ma HM, Huang TT, Luo HB, Dou J, et al. Stanozolol regulates proliferation of growth plate chondrocytes via activation of ERalpha in GnRHa-treated adolescent rats. J Pediatr Endocrinol Metab. 2011;24(5-6):275-81.
38- Fernández L, Chirino R, Boada LD, Navarro D, Cabrera N, del Rio I, et al. Stanozolol and danazol, unlike natural androgens, Interact with the low affinity glucocorticoid-binding sites from male rat liver microsomes. Endocrinology. 1994;134(3):1401-8.
39- Ucer S, Iyer S, Bartell SM, Martin-Millan M, Han L, Kim HN, et al. The effects of androgens on murine cortical bone do not require AR or ERα signaling In osteoblasts and osteoclasts. J Bone Miner Res. 2015;30(7): 1138-49.
40- Goldman A, Basaria S. Adverse health effects of androgen use. Mol Cell Endocrinol. 2018;464:46-55.
Arri Coomarasamy, M.B., Ch.B., M.D., F.R.C.O.G., Adam J. Devall, B.Med.Sci., Ph.D., Versha Cheed, M.Sc., Hoda Harb, M.B., Ch.B., Ph.D., Lee J. Middleton, B.Sc., Ioannis D. Gallos, D.M.S., M.D., Helen Williams, B.Sc., Abey K. Eapen, M.D., Ph.D., Tracy Roberts, Ph.D., R.G.N., Chriscasimir C. Ogwulu, Ph.D., Ilias Goranitis, Ph.D., Jane P. Daniels, M.Med.Sci., Ph.D.,
Bleeding in early pregnancy is strongly associated with pregnancy loss. Progesterone is essential for the maintenance of pregnancy. Several small trials have suggested that progesterone therapy may improve pregnancy outcomes in women who have bleeding in early pregnancy.
We conducted a multicenter, randomized, double-blind, placebo-controlled trial to evaluate progesterone, as compared with placebo, in women with vaginal bleeding in early pregnancy. Women were randomly assigned to receive vaginal suppositories containing either 400 mg of progesterone or matching placebo twice daily, from the time at which they presented with bleeding through 16 weeks of gestation. The primary outcome was the birth of a live-born baby after at least 34 weeks of gestation. The primary analysis was performed in all participants for whom data on the primary outcome were available. A sensitivity analysis of the primary outcome that included all the participants was performed with the use of multiple imputation to account for missing data.
A total of 4153 women, recruited at 48 hospitals in the United Kingdom, were randomly assigned to receive progesterone (2079 women) or placebo (2074 women). The percentage of women with available data for the primary outcome was 97% (4038 of 4153 women). The incidence of live births after at least 34 weeks of gestation was 75% (1513 of 2025 women) in the progesterone group and 72% (1459 of 2013 women) in the placebo group (relative rate, 1.03; 95% confidence interval [CI], 1.00 to 1.07; P=0.08). The sensitivity analysis, in which missing primary outcome data were imputed, resulted in a similar finding (relative rate, 1.03; 95% CI, 1.00 to 1.07; P=0.08). The incidence of adverse events did not differ significantly between the groups.
Among women with bleeding in early pregnancy, progesterone therapy administered during the first trimester did not result in a significantly higher incidence of live births than placebo. (Funded by the United Kingdom National Institute for Health Research Health Technology Assessment program; PRISM Current Controlled Trials number, ISRCTN14163439. opens in new tab.)
Miscarriage affects one in five pregnancies.1 Miscarriage can cause excessive bleeding, infection, and complications associated with surgical treatment,2 as well as substantial psychological harm, including anxiety, depression, and post-traumatic stress disorder.3,4
Progesterone, which is produced by the corpus luteum in the ovary, is necessary to prepare the endometrium for implantation of the embryo and thus is an essential hormone for a successful pregnancy. Additional progesterone is produced when an embryo implants in the endometrium and during early placental development. Subsequently, beginning at approximately 12 weeks of pregnancy, the placenta becomes the dominant source of progesterone.5
The physiological importance of progesterone has prompted researchers, physicians, and patients to consider progesterone supplementation during early pregnancy to prevent miscarriages. Progesterone supplementation in early pregnancy has been attempted in two contexts: the first is to prevent miscarriages in asymptomatic women who have a history of recurrent miscarriages, and the second is to rescue a pregnancy in women who have started to bleed during early pregnancy.6 We addressed the first scenario in a previous issue of the Journal7 and found no beneficial effect of progesterone in women with a history of unexplained recurrent miscarriages. The current trial focuses on women with vaginal bleeding in early pregnancy.
A Cochrane review (originally published in 2007 and last updated in 2018)6 of 7 randomized trials of progestational agents that involved women with bleeding in early pregnancy showed a significantly lower risk of miscarriages among women who received progesterone than among those who received placebo or no treatment (odds ratio, 0.64; 95% confidence interval [CI], 0.47 to 0.87) but noted that the trials were small (the largest trial had a sample size of 191) and had methodologic weaknesses. Another Cochrane review of 13 trials of progestational agents that involved women with recurrent miscarriages was originally published in 2003 and was last updated in 2018.8 The American College of Obstetricians and Gynecologists reviewed the evidence and concluded, “For threatened early pregnancy loss, the use of progestins is controversial, and conclusive evidence supporting their use is lacking. Women who have experienced at least three prior pregnancy losses, however, may benefit from progesterone therapy in the first trimester.”9 We conducted the multicenter, randomized, parallel-group, double-blind, placebo-controlled PRISM (Progesterone in Spontaneous Miscarriage) trial to investigate whether treatment with progesterone would result in a higher incidence of live births among women with bleeding in early pregnancy than placebo.
The PRISM trial was approved by the United Kingdom Medicines and Healthcare Products Regulatory Agency, the United Kingdom National Research Ethics Service Committee (South Central Oxford), and the National Health Service research and development department at each participating hospital. The trial was conducted at clinics that are part of the trial research network of the Tommy’s National Centre for Miscarriage Research, which is funded by Tommy’s Charity. Progesterone and placebo were purchased from Besins Healthcare. This company had no role in the design of the trial; in the collection, analysis, or interpretation of the data; or in the preparation of the manuscript. Trial oversight and monitoring were provided by a trial steering committee and by an independent data and safety monitoring committee. The first, second, and last authors vouch for the accuracy and completeness of the data and analyses and for the fidelity of the trial to the protocol, available with the full text of this article at NEJM.org.
The participants in the PRISM trial were recruited at 48 hospitals in the United Kingdom. Women were eligible for enrollment in the trial if they were 16 to 39 years of age, if they had completed less than 12 weeks of pregnancy, if they presented with vaginal bleeding, and if they had an intrauterine gestational sac that was visible on ultrasonography. The upper threshold of 39 years for maternal age was chosen because the probability of miscarriages due to chromosomal abnormalities increases with advancing age,10 and progesterone treatment could not be expected to prevent such miscarriages. Participants were excluded if at the time of presentation the fetal crown–rump length was 7 mm or longer with no visible heartbeat; if the gestational sac was a mean of 25 mm or greater in diameter with no visible fetal pole on ultrasonography; if they had evidence of ectopic pregnancy; if they had life-threatening bleeding; if they had current or recent use of progesterone supplementation; if they had contraindications to progesterone therapy (i.e., a history of liver tumors; current genital or breast cancer, severe arterial disease, or acute porphyria; or a history during pregnancy of idiopathic jaundice, severe pruritus, or pemphigoid gestationis); or if they were participating in any other blinded, placebo-controlled trials of medicinal products in pregnancy. All the participants provided written informed consent.
Participants were randomly assigned, in a 1:1 ratio, to administer to themselves vaginal suppositories containing either 400 mg of micronized progesterone (Utrogestan, Besins Healthcare) or matching placebo twice daily, from the time of randomization through 16 completed weeks of gestation (or earlier if pregnancy ended before 16 weeks). If vaginal administration was not preferred, participants could administer the suppositories rectally. Randomization was performed through a secure, centralized Internet facility with the use of minimization to balance the trial-group assignments according to maternal age (<35 years vs. ≥35 years), body-mass index (BMI [the weight in kilograms divided by the square of the height in meters], <30 vs. ≥30), fetal heart activity (present vs. absent), estimated gestation at presentation (<42 days vs. ≥42 days), and amount of vaginal bleeding (pictorial blood-loss assessment chart [PBAC] score of ≤2 vs. score of ≥3; scores range from 1 to 4, with higher scores indicating greater vaginal blood loss).11 The appearance, route, and timing of administration of progesterone and placebo were identical. Participants, physicians, and trial nurses were unaware of the trial-group assignments throughout the course of the trial.
The primary outcome was the birth of a live-born baby after at least 34 completed weeks of gestation. Secondary outcomes included the time from conception to the end date of pregnancy, ongoing pregnancy at 12 weeks of gestation, miscarriage (defined as loss of pregnancy before 24 weeks of gestation), live birth before 34 weeks of gestation, ectopic pregnancy, stillbirth (defined as intrauterine death after at least 24 weeks of gestation), termination of pregnancy, the week of gestation at delivery, birth weight, size (small or large) for gestational age, preeclampsia, Apgar scores, survival at 28 days of neonatal life, and congenital abnormalities, as well as other antenatal, intrapartum, postpartum, and neonatal outcomes. A detailed list of all secondary outcomes is provided in the Supplementary Appendix, available at NEJM.org. We attempted to collect outcome data for all participants who underwent randomization, regardless of adherence to the trial-group assignment.
We calculated that 1972 women would need to be included in each trial group to provide 90% power to detect a minimally important absolute difference of 5 percentage points between the progesterone group and the placebo group in the incidence of live births after at least 34 weeks of gestation (65% vs. 60%), at a two-sided alpha level of 0.05. This minimally important difference was chosen on the basis of a national survey of clinical practitioners in the United Kingdom. We planned to include 4150 women in the trial to account for an expected 5% loss to follow-up.
The analysis of the primary outcome was performed according to the intention-to-treat principle; a main analysis of all available data was supplemented by a sensitivity analysis of the primary outcome that included all participants and took into account any missing data with the use of multiple imputation.12 A Poisson regression model with robust standard errors was used to estimate the relative rates and corresponding 95% two-sided confidence intervals, with adjustment for the minimization variables. This method has been shown to be appropriate and to be less prone to convergence issues than other similar methods.13
For the primary outcome, a P value was generated with the use of a two-sided chi-square test. The statistical analysis plan did not include a provision for correction for multiplicity when the analyses of the secondary outcomes were performed. Therefore, the results are reported as point estimates and 95% confidence intervals, without P values. For continuous outcomes, a linear regression model was used to estimate mean differences, with the same adjustment that was used in the analysis of the primary outcome. The widths of the confidence intervals were not adjusted for multiplicity, so the intervals should not be used to infer definitive treatment effects.
We analyzed the treatment effect on the primary outcome in prespecified subgroups defined according to maternal age (<35 years vs. ≥35 years), BMI (<30 vs. ≥30), fetal heart activity (present vs. absent), estimated gestation at presentation (<6 weeks vs. 6 to <9 weeks vs. ≥9 weeks), amount of vaginal bleeding (PBAC score of ≤2 vs. score of ≥3),11 number of previous miscarriages (0 vs. 1 or 2 vs. ≥3), number of gestational sacs (1 vs. ≥2), race (white, black, south Asian, or other), history of polycystic ovaries (yes vs. no), and previous cervical excision (yes vs. no). The effects of these subgroups were examined by adding the variables for the interaction of subgroup with trial group to the regression model; a chi-square test was used to determine whether the effects of progesterone and placebo differed in the various subgroups.
Interim analyses of principal safety and effectiveness outcomes were performed on behalf of the data and safety monitoring committee by the trial statistician (who remained unaware of the treatment assignments) on two occasions. Because these analyses were performed with the use of the Peto principle,14 no adjustment was made in the final P values to determine significance.
From May 19, 2015, through July 27, 2017, a total of 12,862 women were identified as being eligible for the PRISM trial; of these women, 4153 were randomly assigned to receive either progesterone (2079 women) or placebo (2074 women) (Figure 1). The percentage of women with available data for the primary outcome was 97% (4038 of 4153 women). Demographic and baseline characteristics were similar in the two trial groups (Table 1, and Table S1 in the Supplementary Appendix). Information on the route of administration was available for 88% (3662 of 4153) of the women: 99% (3611 of 3662 women) administered the suppositories vaginally and 1% (51 of 3662 women) administered them rectally.
Enrollment, Randomization, and Follow-up.
The incidence of live births after at least 34 weeks of gestation was 75% (1513 of 2025 women) in the progesterone group and 72% (1459 of 2013 women) in the placebo group (relative rate, 1.03; 95% CI, 1.00 to 1.07; P=0.08). The sensitivity analysis, in which multiple imputation was used for missing data, did not change the findings (relative rate, 1.03; 95% CI, 1.00 to 1.07; P=0.08).
The incidence of ongoing pregnancy at 12 weeks was 83% (1672 of 2025 women) in the progesterone group and 80% (1602 of 2013 women) in the placebo group (relative rate, 1.04; 95% CI, 1.01 to 1.07). The incidence of miscarriage was 20% (410 of 2025 women) in the progesterone group and 22% (451 of 2013 women) in the placebo group (relative rate, 0.91; 95% CI, 0.81 to 1.01). The results of all the other secondary outcomes are presented in Table 2, and in Table S2 in the Supplementary Appendix.
A significant subgroup effect was identified for only 1 of the 10 prespecified subgroups — the subgroup of participants defined according to the number of previous miscarriages. The incidence of live births in the subgroup of women who had no previous miscarriages was 74% in the progesterone group and 75% in the placebo group (relative rate, 0.99; 95% CI, 0.95 to 1.04); the incidence among women who had one or two previous miscarriages was 76% and 72%, respectively (relative rate 1.05; 95% CI, 1.00 to 1.12); and the incidence among women who had three or more previous miscarriages was 72% and 57%, respectively (relative rate, 1.28; 95% CI, 1.08 to 1.51) (P=0.007 for the interaction between trial group and the number of miscarriages) (Figure 2). The results of two post hoc subgroup analyses in which we categorized the number of previous miscarriages differently from the subgroup analysis described here are provided in Figure S1 in the Supplementary Appendix.
There was no significant between-group difference in the percentage of participants who had either a maternal or neonatal serious adverse event (5% [105 of 2025 participants] in the progesterone group and 5% [98 of 2013 participants] in the placebo group), including specifically the percentage of babies who had neonatal congenital abnormalities (3.4% in each group), nor was there any significant between-group difference in the number of maternal or neonatal serious adverse events. A summary of serious adverse events is provided in Table S3 in the Supplementary Appendix.
Demographic and Baseline Characteristics of the Participants.
Primary Outcome and Secondary Outcomes.
Our large multicenter, randomized, double-blind, placebo-controlled trial showed that among women with bleeding in early pregnancy, progesterone therapy administered during the first trimester of pregnancy did not result in a significantly higher incidence of live births after at least 34 weeks of gestation than placebo. There was also no significant difference between the groups in the incidence of miscarriage or stillbirth. Although there appeared to be slightly more ongoing pregnancies at 12 weeks in the progesterone group than in the placebo group, an inference of benefit cannot be drawn because the confidence interval for the relative rate was not adjusted for multiplicity of testing.
The large sample size in our trial allowed investigation of the primary outcome in prespecified subgroups. Among the 10 subgroup analyses, 1 showed differential effects of progesterone: the effect of progesterone in women with bleeding in early pregnancy differed according to the number of previous miscarriages, with a suggestion of benefit among women who had had three or more previous miscarriages. Previous reports have indicated a steep and proportionate increase in the loss of chromosomally normal pregnancies (i.e., euploid miscarriages) with increasing number of previous miscarriages.15 Given that the potential benefit of progesterone therapy would be expected to be specific to euploid pregnancies, an increasing level of benefit in women with increasing number of previous miscarriages is consistent with our understanding of the biologic factors associated with risk of miscarriage. A history of miscarriage is one of only two stratification or prognostic risk factors (the other being maternal age) cited in the 2017 guideline of the European Society of Human Reproduction and Embryology on recurrent pregnancy loss as being useful for identifying high-risk patients.16However, we did not identify this subgroup as one of special interest a priori in our statistical analysis plan,17 and multiple comparisons were performed (without adjustment for multiplicity); thus, this observation requires validation.
Some limitations of our trial should be considered. First, we studied a vaginal preparation of progesterone, at a dose of 400 mg twice daily, and it is possible that the results observed with this regimen are not generalizable to women receiving other doses and preparations by other routes. Micronized vaginal progesterone has an identical molecular structure to natural progesterone, whereas other formulations of progestational agents have a different molecular structure and therefore potentially different mechanisms of action and pharmacologic features. Immunomodulatory effects of progesterone at the trophoblastic–decidual interface have been proposed as a mechanism whereby progesterone might prevent miscarriage.18 The vaginal route delivers a greater proportion of drug to the relevant site (i.e., the uterus) with the use of the “first uterine pass” effect.19,20 Furthermore, trials that have evaluated vaginal progesterone in the prevention of preterm birth have shown its effectiveness when administered by this route.21,22
Second, we started progesterone treatment only in women who had an intrauterine sac; therefore, our trial cannot provide evidence on the effects of earlier use of progesterone, before a pregnancy sac is visible on an ultrasound examination. Third, the participants discontinued progesterone at 16 weeks of gestation; however, we consider it to be unlikely that therapy beyond this time would have affected the outcomes related to miscarriage. Finally, although we found no increase in the risk of congenital abnormalities among babies of women treated with progesterone, the trial was not powered for such rare outcomes.
In conclusion, treatment with progesterone did not result in significant improvement in the incidence of live births among women with vaginal bleeding during the first 12 weeks of pregnancy.
Supported by the United Kingdom NIHR Health Technology Assessment program (project number HTA 12/167/26).
Disclosure forms provided by the authors are available with the full text of this article at NEJM.org.
Dr. Lumsden reports receiving advisory fees from PregLem; and Dr. Norman, receiving consulting fees, paid to the University of Edinburgh, from Dilafor and GlaxoSmithKline. No other potential conflict of interest relevant to this article was reported.
This article presents independent research commissioned by the National Institute for Health Research (NIHR). A monograph reporting the data collected in this trial is planned for publication in the NIHR Journals Library. Further information is available at www.journalslibrary.nihr.ac.uk/hta/. opens in new tab. The views and opinions expressed by the authors in this publication are those of the authors and do not necessarily reflect those of the National Health Service, the NIHR, the Medical Research Council, the NIHR Central Commissioning Facility, the NIHR Evaluation, Trials and Studies Coordinating Centre, the NIHR Health Technology Assessment program, or the Department of Health.
We thank the women who participated in this trial; the investigators for supervising recruitment and randomization at the trial centers (Mr. Samson Agwu, Mrs. Rita Arya, Miss Miriam Baumgarten, Dr. Catey Bass, Miss Sumita Bhuiya, Prof. Tom Bourne, Mr. James Clark, Mr. Samual Eckford, Mr. Zeiad El-Gizawy, Mrs. Joanne Fletcher, Miss Preeti Gandhi, Dr. Mary Gbegaje, Dr. Ingrid Granne, Mr. Mamdough Guirguis, Dr. Pratima Gupta, Dr. Hadi Haerizadeh, Dr. Laura Hipple, Mr. Piotr Lesny, Miss Hema Nosib, Mr. Jonathan Pepper, Mr. Jag Samra, Ms. Jayne Shillito, Dr. Rekha Shrestha, Dr. Jayasree Srinivasan, Dr. Ayman Swidan, and Prof. Derek Tuffnell); the PRISM research nurses who assisted in the collection of data; Leanne Beeson, Mary Nulty, and Louisa Edwards for their support in managing and coordinating the trial; Prof. Siladitya Bhattacharya for chairing the trial steering committee; Prof. Andrew Shennan for chairing the data and safety monitoring committee; Dr. Javier Zamora and Dr. Willem Ankum for participating in the data and safety monitoring committee; and all those not otherwise mentioned above who have contributed to the PRISM trial.
From Tommy’s National Centre for Miscarriage Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham (A.C., A.J.D., V.C., H.H., L.J.M., I.D.G., H.W., A.K.E., T.R., C.C.O.), the Faculty of Medicine and Health Sciences, University of Nottingham (J.P.D.), and Nottingham University Hospitals NHS Trust (S.D.), Nottingham, City Hospitals Sunderland NHS Foundation Trust, Sunderland (A.A., K.H.), the Miscarriage Association, Wakefield (R.B.A.), East Lancashire Hospitals NHS Trust, Burnley (K.B.), Tommy’s Charity (J.B.), Guy’s and St. Thomas’ NHS Foundation Trust (T.H.), King’s College Hospital NHS Foundation Trust (J.J., J.R.), University College London Hospitals NHS Foundation Trust (K.K., D.J.), West Middlesex Hospital, Chelsea and Westminster NHS Foundation Trust (N.N., C.B.), and Barts and the London NHS Trust (A.S.), London, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle (M.C.), Lancashire Teaching Hospitals NHS Foundation Trust, Preston (F.C.), the MRC Centre for Reproductive Health, University of Edinburgh, Edinburgh (W.C.D., J.E.N., A.W.H.), Liverpool Women’s NHS Foundation Trust (L.W.) and St. Helens and Knowsley NHS Trust (S.R.), Liverpool, University Hospitals Coventry and Warwickshire NHS Trust, Coventry (F.I.), the Department of Medicine, University of Glasgow, Glasgow (M.-A.L.), South Tees Hospitals NHS Foundation Trust, Middlesbrough (P.M.), University Hospitals Bristol NHS Foundation Trust, Bristol (C.E.O.), the Biomedical Research Unit in Reproductive Health, University of Warwick, Warwick (S.Q.), Shrewsbury and Telford NHS Trust, Telford (M.U.), Portsmouth Hospitals NHS Trust, Portsmouth (N.V.), and Surrey and Sussex Healthcare NHS Trust, Redhill (C.W.) — all in the United Kingdom; the Melbourne School of Population and Global Health, University of Melbourne, Melbourne, VIC, Australia (I.G.); and the Carver College of Medicine, University of Iowa Health Care, Iowa City (A.E.).
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20.Cicinelli E, Cignarelli M, Sabatelli S, et al. Plasma concentrations of progesterone are higher in the uterine artery than in the radial artery after vaginal administration of micronized progesterone in an oil-based solution to postmenopausal women. Fertil Steril 1998;69:471-473.
21.Fonseca EB, Celik E, Parra M, Singh M, Nicolaides KH. Progesterone and the risk of preterm birth among women with a short cervix. N Engl J Med 2007;357:462-469.
22.Romero R, Conde-Agudelo A, Da Fonseca E, et al. Vaginal progesterone for preventing preterm birth and adverse perinatal outcomes in singleton gestations with a short cervix: a meta-analysis of individual patient data. Am J Obstet Gynecol 2018;218:161-180.
The shorter and lower dose regime saves money but there is an increased risk of follicular cyst development, which may occur at doses below 13mg/day.
Control of the oestrous cycle in the pig can be achieved through the predictable suppression of the follicular phase using a progestogen. Altrenogest (also called allyl trenbolone) is an orally active progestogen which can be fed to pigs to achieve this suppression and thus allow control of the porcine reproductive cycle. During the feeding of altrenogest the original corpora lutea will regress normally but, due to suppression of LH, follicle growth is stopped at the medium sized stage. The effect is an artificially extended luteal phase. From about 8 to 12 hours after the last altrenogest treatment on day 18, pulsatile LH activity increases and the natural follicular phase will commence allowing females to exhibit oestrus 5 to 8 days later, as illustrated in Figure 1. If an eCG and hCG combination is administered the day after removal of the altrenogest more of the gilts will cycle around day 5.
Figure 1. Percentage of gilts exhibiting oestrus following removal of 18 days of altrenogest
How is altrenogest used on farms?
It is essential to read the data sheets for your country. In general, there are two recommended programmes:
18 days at 20mg/day – most of the world
14 days and 15 mg/day – USA
The shorter and lower dose regime saves money but there is an increased risk of follicular cyst development, which may occur at doses below 13mg/day.
How to administer altrenogest?
Pigs readily consume the altrenogest solution but need to become familiar with dosing. Therefore, to minimise the risk of under-dosing and of wasting product, for 4 days prior to the altrenogest dosing regimen individually dose gilts with canola oil or apple juice to get them used to being dosed; then switch to altrenogest. If an individual doser is not available the altrenogest may also be administered individually via toast or to a group with top-dressing onto feed, but note accurate dosing is a major key to success.
How to use altrenogest in your farm production cycle
There is a role for altrenogest in all three breeding female classes: the gilts, sows and returns.
The classical role of altrenogest has been to synchronise a group of gilts into the breeding programme. Altrenogest results in a pool of gilts ready for breeding on their 2nd observed oestrus at 230-240 days of age and ≥130 kg liveweight. This reduces the need for a large gilt pool with the obvious saving in feed and manpower. However, it is essential to plan and manage your gilt pool to ensure breeding targets are met and that intra-batch variation is minimised to less than 10%.
When should the group of gilts cycle within a batch?
There are two thoughts:
With the sows – to enhance breeding managementThis can reduce the time taken to achieve the mating within a batch. In this case the altrenogest is last given the day before the batch of sows are weaned. For example, if weaning on a Monday, the last altrenogest dose is administered on the Sunday and the gilts and sows will be mated the following Friday and Saturday.
Before the main group of sows – to enhance gilt farrowing management.The gilts are mated 2-3 days before the sows. In this case (with a Monday weaned sow) the last altrenogest dose is administered on the Friday and the gilts mated on Wednesday and Thursday. This provides a number of advantages:
The gilts farrow first so more time can be devoted to them
They farrow in a cleaner farrowing house
The parity 1 piglets are then the oldest at weaning and so not the smallest and weakest!
The parity 1 lactation is slightly longer than the sows allowing for more food to be eaten and a reduction in wean to service interval. This can be especially beneficial in 3-week lactation batch models as parity 1 sows have a reduced farrowing rate and litter size if weaned below 19 days of lactation.
There are two significant uses of altrenogest in sows: to delay parturition (farrowing); to assist in the development of a batching programme.
To delay parturition. In some pathogen control programmes it may be desirable to both synchronise farrowing and to extend the period without piglets. A case in point is during farm attempts to eliminate Porcine Epidemic Diarrhoea virus (PED). In this case sows are administered altrenogest from day 112 of pregnancy to day 115 (for the main group). On day 115 an injection of PGF2α is administered to assist parturition.
To assist in the creation of a batching programmes. Many producers around the world want to capitalise on the health and discipline benefits of batching programmes. The use of altrenogest can ensure the success of these programmes. For example, see table 1.
Table 1. Moving from a one-week batch to a 3 or 4-week batch programme using altrenogest
Development of a three week batch programme from a weekly batch programme
Wean sows and place on altrenogest for 1 week
Batch is created
Wean sows one week early
To develop a 4 week batch programme from a weekly batch programme
Wean sows and place on altrenogest for 2 weeks
Batch is created
Wean sows and place on altrenogest for 1 week
Wean sows one week early
Occasionally, and more often with batching programmes, sows may return to oestrus out of sequence. These animals can either be culled or efficiently replaced into the breeding pool by the judicious use of altrenogest. Simply feed altrenogest from 12 days after the out-of-sequence oestrus until 5 days before desired breeding date. This concept can also be adapted for weaned parity 1 sows to create a skip-a-heat concept allowing sows to recover from lactation.
Paul E. Goss, M.D., Ph.D., James N. Ingle, M.D., José E. Alés-Martínez, M.D., Ph.D., Angela M. Cheung, M.D., Ph.D., Rowan T. Chlebowski, M.D., Ph.D., Jean Wactawski-Wende, Ph.D., Anne McTiernan, M.D., John Robbins, M.D., Karen C. Johnson, M.D., M.P.H., Lisa W. Martin, M.D., Eric Winquist, M.D., Gloria E. Sarto, M.D.,
Tamoxifen and raloxifene have limited patient acceptance for primary prevention of breast cancer. Aromatase inhibitors prevent more contralateral breast cancers and cause fewer side effects than tamoxifen in patients with early-stage breast cancer.
In a randomized, placebo-controlled, double-blind trial of exemestane designed to detect a 65% relative reduction in invasive breast cancer, eligible postmenopausal women 35 years of age or older had at least one of the following risk factors: 60 years of age or older; Gail 5-year risk score greater than 1.66% (chances in 100 of invasive breast cancer developing within 5 years); prior atypical ductal or lobular hyperplasia or lobular carcinoma in situ; or ductal carcinoma in situ with mastectomy. Toxic effects and health-related and menopause-specific qualities of life were measured.
A total of 4560 women for whom the median age was 62.5 years and the median Gail risk score was 2.3% were randomly assigned to either exemestane or placebo. At a median follow-up of 35 months, 11 invasive breast cancers were detected in those given exemestane and in 32 of those given placebo, with a 65% relative reduction in the annual incidence of invasive breast cancer (0.19% vs. 0.55%; hazard ratio, 0.35; 95% confidence interval [CI], 0.18 to 0.70; P=0.002). The annual incidence of invasive plus noninvasive (ductal carcinoma in situ) breast cancers was 0.35% on exemestane and 0.77% on placebo (hazard ratio, 0.47; 95% CI, 0.27 to 0.79; P=0.004). Adverse events occurred in 88% of the exemestane group and 85% of the placebo group (P=0.003), with no significant differences between the two groups in terms of skeletal fractures, cardiovascular events, other cancers, or treatment-related deaths. Minimal quality-of-life differences were observed.
Exemestane significantly reduced invasive breast cancers in postmenopausal women who were at moderately increased risk for breast cancer. During a median follow-up period of 3 years, exemestane was associated with no serious toxic effects and only minimal changes in health-related quality of life. (Funded by Pfizer and others; NCIC CTG MAP.3 ClinicalTrials.gov number, NCT00083174. opens in new tab.)
Estrogens contribute to normal breast development but can also promote breast cancer in preclinical models and in women with high circulating plasma estrogen levels.1-4 To date, chemoprevention of breast cancer has focused on the selective estrogen-receptor modulators (SERMs) tamoxifen and raloxifene, which exert antiestrogenic effects on the breast, as well as agonist or antagonist effects on other organs. In the National Surgical Adjuvant Breast and Bowel Project P-1 trial, tamoxifen significantly reduced the number of invasive breast cancers, by 49% (P<0.001) as compared with placebo.5 A meta-analysis of trials comparing tamoxifen with placebo showed that tamoxifen reduced the incidence of breast cancer by 38% with no effect on mortality.6 On the basis of these collective data on tamoxifen, the estimated number needed to treat to prevent one breast cancer after 5 years is about 95 and is reduced to 56 after 10 years.7 Similar risk reductions occur with raloxifene.8-10 Tamoxifen increases the risks of endometrial cancers and venous thromboembolism; raloxifene does not increase the risk of endometrial cancers but does cause similar toxic effects.
The acceptance of tamoxifen or raloxifene for reducing the risk of breast cancer has been poor, in part because they are both associated with rare but serious toxic effects.11-13 Of the approximately 2 million U.S. women who could potentially benefit from treatment with tamoxifen, only 4% of those at increased risk for breast cancer and only 0.08% of all U.S. women 40 to 79 years of age have accepted the use of this drug for chemoprevention.13-15 A 2002 expert assessment concluded that tamoxifen lacks overall health benefits and recommended that future trials be conducted with placebo controls.16 Novel, less toxic interventions are needed that will reduce the threshold of risk yet provide a net benefit.17
Aromatase inhibitors profoundly suppress estrogen levels in postmenopausal women and inhibit the development of breast cancer in laboratory models.18-21 In early trials of breast cancer therapy, both nonsteroidal and steroidal aromatase inhibitors reduced contralateral primary breast cancers more than did tamoxifen; after 5 years of tamoxifen therapy, letrozole resulted in a further reduction of 46%, as compared with placebo.22-27 Preclinical models and clinical studies suggest that because of exemestane’s antiestrogenic effects, such as those on bone resorption due to this drug’s mild androgenic activity, it is a good candidate for study in a breast-cancer prevention trial.28-30
The NCIC Clinical Trials Group Mammary Prevention.3 trial (NCIC CTG MAP.3) is an international, randomized, double-blind, placebo-controlled trial conducted in Canada, the United States, Spain, and France. The trial was approved by the health regulatory authorities and institutional review boards at the participating centers, and enrollment began in September 2004. After stratification according to current use of low-dose aspirin (≤100 mg per day) (yes or no) and Gail risk score (for calculation of this score, see www.cancer.gov/bcrisktool. opens in new tab and the Supplementary Appendix, available with the full text of this article at NEJM.org) (≤2.0% or >2.0%), subjects were randomly assigned to one of three treatment groups with the use of a dynamic minimization algorithm: 25 mg of exemestane plus placebo, 25 mg of exemestane plus celecoxib, or placebo plus placebo pills, administered daily after a morning meal.31,32 After 31 patients were enrolled, 10 patients discontinued treatment with celecoxib because of concern for cardiovascular safety.33 Before enrollment and during the study, written informed consent and reconsent in all the participating countries included counseling about the risks and benefits of treatment with tamoxifen and raloxifene. The trial was event-driven, with a planned maximum duration of therapy of 5 years or until a breast event, a neoplastic disease, or a cardiovascular event was diagnosed or unacceptable toxicity developed.
Women 35 years of age or older were eligible if they were postmenopausal (older than 50 years of age with no spontaneous menses for at least 12 months; or 50 years of age or younger either with no spontaneous menses [amenorrheic] within 12 months of randomization [e.g., spontaneous or secondary to hysterectomy] and a follicle-stimulating hormone level within the postmenopausal range or with prior bilateral oophorectomy). In addition, women had at least one of the following risk factors: age 60 years or older; Gail risk score greater than 1.66%; prior atypical ductal or lobular hyperplasia or lobular carcinoma in situ on breast biopsy or prior ductal carcinoma in situ treated with mastectomy. Prior menopausal hormone therapies (estrogen with or without progestin), luteinizing hormone–releasing hormone analogues, prolactin inhibitors, antiandrogens, or selective estrogen-receptor modulators were allowable, but not within 3 months of randomization. Women were ineligible if they were premenopausal, had prior invasive breast cancer or prior ductal carcinoma in situ treated with lumpectomy, were known carriers of the BRCA1 or BRCA2 gene, had a history of other malignancies (except nonmelanoma skin cancer, treated in situ cancer of the cervix, or other solid tumors treated with no evidence of disease for 5 years), had uncontrolled hypothyroidism or hyperthyroidism, or had chronic liver disease.
END POINTS AND ASSESSMENT
The primary outcome was incidence of invasive breast cancer. Secondary end points included a combined incidence of invasive and noninvasive (ductal carcinoma in situ) breast cancer; incidence of receptor-negative invasive breast cancer; incidence of combined atypical ductal hyperplasia, atypical lobular hyperplasia, and lobular carcinoma in situ; number of clinical breast biopsies; clinical fractures; adverse cardiovascular events, including myocardial infarction or coronary heart disease that resulted in death; overall incidence of other cancers; the side-effect profile and safety; and health-related and menopause-specific qualities of life (assessed by means of the Medical Outcomes Study 36-Item Short-Form Health Survey [SF-36] and the Menopause-Specific Quality of Life [MENQOL] questionnaire, respectively34-36).
At baseline, each patient’s history of prior diseases and treatment, family history of cancer, and reproductive history were obtained. Physical examination was performed, including height, weight, blood pressure, pulse, and clinical breast examination to confirm no suspicious breast abnormalities. Other requirements included complete blood count, liver-function tests, renal-function tests, and normal results on bilateral mammography and bone-mineral-density measurements within the past year. Symptoms at baseline were graded according to the National Cancer Institute’s Common Terminology Criteria for Adverse Events (CTCAE), version 3.0.37 Quality of life (QOL) was measured within 1 week before randomization. Clinical assessments occurred at 6 and 12 months after randomization and yearly thereafter and included physical examination with clinical breast examinations, recording of concomitant medications, symptoms and adverse events, and QOL assessments. Women discontinuing the study drug continued to undergo clinical assessments and follow-up for clinical outcomes and adverse events.
Mammography was required within 12 months before randomization and every 12 months from the time of the initial mammogram during and after the treatment. Breast cancers could be detected on clinical breast examination during the clinic visits or on annual mammography. All mammograms and radiographic reports of fractures were reviewed centrally, and all evidence of disease on breast-biopsy specimens was reviewed by an adjudication committee. Study accrual and safety data were reviewed every 6 months by an independent data and safety monitoring committee. The original protocol and subsequent amendments are available at NEJM.org.
The planned final analyses presented here include invasive breast cancer incidence and other secondary breast cancer end points, estimated on the basis of time from randomization to when an end point was reached. The sample-size estimate was based on an assumption of a rate of invasive breast cancer of 0.60% per year in the group given placebo, as compared with 0.21% in those treated with exemestane, with a relative reduction of 65% with exemestane. To detect this with a two-sided 5% level and 90% power, a total of 38 cases of invasive breast cancer were required, projected to occur when 4560 women were randomly assigned to treatment groups in a 3-year period and then followed for an additional 1.2 years. No interim analyses were planned. Accrual was completed on March 23, 2010; the protocol target-event rate was met on November 5, 2010. All data queries were resolved, and the database was locked on March 1, 2011.
Comparisons of time-to-event primary and secondary end points were based on the stratified log-rank test, adjusting for the two stratification factors at randomization. Cox proportional-hazards models were used to derive hazard ratios and associated 95% confidence intervals. Fisher’s exact test was used to compare adverse events between the treatment and placebo groups. Mean change scores from baseline to each assessment were calculated for all SF-36 and MENQOL subscales and summaries. Changes measuring 5 to 10% of the scale breadth or 0.5 SD were considered to be potentially clinically meaningful.38-40 Scores measuring changes in QOL were considered worsened if they decreased by 5 or more points (out of a total of 100) from baseline on the SF-36 and if they increased by 0.5 or more points (out of a total of 8) on the MENQOL. A chi-square test was used to compare the differences in proportions of patients found to have potentially clinically meaningful changes in QOL.
The study drug, exemestane, and funding support were provided by Pfizer, but this sponsor had no role in the design of the study or in the accrual, management, or analysis of the data. The decision to publish and the drafting of the manuscript were undertaken entirely by the first author, coauthors, and staff at the NCIC CTG central office, who vouch for the fidelity of the study to the protocol and for the accuracy and completeness of the data.
Between February 11, 2004, and March 23, 2010, 4560 women were randomly assigned to either exemestane (2285 patients) or placebo (2275 patients). After randomization, 15 women (6 taking exemestane and 9 taking placebo) were considered to be ineligible to continue with the study but are included in the primary intention-to-treat analysis as randomly assigned. The exemestane and placebo groups were well balanced for race, body-mass index, and breast cancer risk factors (Table 1, and Table 1 in the Supplementary Appendix).
Table 1. Baseline Characteristics of Patients Randomly Assigned to Exemestane or Placebo.
Major risk factors among the women who were enrolled included age of at least 60 years (49%); 5-year risk of breast cancer developing (Gail risk score >1.66%) (40%), and prior atypical ductal hyperplasia, atypical lobular hyperplasia, lobular carcinoma in situ, or prior ductal carcinoma in situ treated with mastectomy (11%). Prior menopausal hormone therapy use was recorded in 1310 women in the exemestane group (57.3%; range from 1 to 588 months) and 1327 women in the placebo group (58.3%; range from 1 to 360 months). Pretreatment bone mineral density and prior history of clinical fractures, cardiovascular risk factors, and the concomitant use of bisphosphonates, lipid-lowering drugs, and cardiovascular drugs were similar in the two study groups.
On November 5, 2010, at the time of the clinical data cutoff, 735 women (32.8%) assigned to exemestane and 646 women (28.7%) assigned to placebo were no longer taking the study medication. About 5% in each group had discontinued the protocol treatment because of treatment completion. The major reasons for early discontinuation of the protocol treatments were toxic effects (15.4% in the exemestane groups vs. 10.8% in the placebo group, P<0.001) and patient refusal (6.9% vs. 6.0%, P=0.22). The median time from randomization to off-protocol treatment was 10.2 months (range, 0.1 to 61.5) for exemestane and 14.2 months (range, 0.1 to 62.9) for placebo. Approximately 85% of women were compliant and 15% were noncompliant with the protocol guidelines for the study treatments. Scheduled annual mammography was performed equally in the two groups, with 7.2% and 7.7% of women having missed at least one scheduled mammography appointment in the exemestane and placebo groups, respectively.
At a median of 35 months of follow-up (range, 0 to 63.4), 43 invasive breast cancers were diagnosed: 11 in the exemestane groups and 32 in the placebo group (annual incidence, 0.19% with exemestane vs. 0.55% with placebo; hazard ratio, 0.35 with exemestane; 95% confidence interval [CI], 0.18 to 0.70) (Table 2). Figure 1 shows the cumulative incidence of invasive breast cancer in these two groups. There were 37 ductal (10 in the exemestane groups and 27 in the placebo group) and 6 lobular (1 in the exemestane groups and 5 in the placebo group) cancers. The majority of cancers in each group were estrogen-receptor–positive, HER2/neu–negative, and node-negative (Table 2).
Table 2. Incidence of Invasive and Preinvasive Breast Events by Treatment Group.
Figure 1. Cumulative Incidence of Invasive Breast Cancer.
Exemestane appeared to be superior to placebo in all prespecified subgroups defined by concurrent use of low-dose aspirin, Gail risk score, age, body-mass index, prior atypical ductal hyperplasia, atypical lobular hyperplasia, or lobular carcinoma in situ and prior ductal carcinoma in situ treated with mastectomy (Figure 2). Exemestane also appeared to be superior in unplanned subgroups: invasive breast cancers according to prior use of menopausal hormone therapy (hazard ratio, 0.30 for prior users; 95% CI, 0.11 to 0.81; hazard ratio, 0.41 for prior nonusers; 95% CI, 0.16 to 1.05) and continent of residence (hazard ratio, 0.34 for North America; 95% CI, 0.16 to 0.71; hazard ratio, 0.39 for Europe; 95% CI, 0.07 to 1.99). The annual incidence of invasive breast cancer plus ductal carcinoma in situ (20 in the exemestane group and 44 in the placebo group) was 0.35% and 0.77% in the exemestane and placebo groups, respectively (hazard ratio, 0.47; 95% CI, 0.27 to 0.79). Combined lobular carcinoma in situ, atypical ductal hyperplasia, and atypical lobular hyperplasia occurred in 4 women (0.2%) in the exemestane group and 11 (0.5%) in the placebo group (hazard ratio, 0.36; 95% CI, 0.11 to 1.12). The number needed to treat to prevent one case of invasive breast cancer with exemestane therapy was 94 in 3 years and 26 in 5 years, but few women completed 5 years of therapy.
Table 3. Side Effects during Treatment, According to Severity.
Table 3 (and Table 2 in the Supplementary Appendix) shows adverse events that occurred in 5% or more of women, with a difference between groups of 1% or more and prespecified secondary end points of toxicity. Symptoms and adverse events (all grades) occurred in 88% of women in the exemestane group versus 85% in the placebo group (P=0.003). Arthritis (P=0.01) and hot flashes (P<0.001) were more common in the exemestane group, but differences between the groups in the frequency of those with grade 2 or higher symptoms were modest (arthritis, 6.5% vs. 4.0%; hot flashes, 18.3% vs. 11.9%). Table 3 (and Table 2 in the Supplementary Appendix) shows no significant differences between the two groups in prespecified secondary end points, including new diagnoses of osteoporosis or cardiovascular events. Clinical fracture rates were also similar in the two groups, and the proportion of women in each group who were prescribed bisphosphonate therapy during the trial was also similar (24.5% for exemestane and 24.1% for placebo). There was no significant difference in the number of cancers other than breast cancer (50 [2.2%] vs. 44 [2.0%]) or time to detection of these cancers (1.8 yr vs. 1.6 yr). No significant differences were detected between the two groups with respect to hypercholesterolemia, hypertriglyceridemia, abnormal liver-function tests, acne, alopecia, rash, weight gain, or hair loss (data not shown). Table 3 in the Supplementary Appendix shows health-related and menopause-specific QOL results. Compliance in completing the QOL questionnaire at each follow-up visit was 92.9 to 97.4% for the exemestane group and 94.3 to 97.5% for the placebo group. No between-group differences in overall health-related QOL responses were found when distributions of worsened, stable, and improved scores on the SF-36 (Physical and Mental Component Scores) were compared despite worsened menopause-specific QOL among those taking exemestane (7% more overall). There were 38 deaths during the study (19 in each group). Causes of death in the exemestane and placebo groups, respectively, were breast cancer, 1 and 0; other malignancies, 10 and 12; cardiovascular events, 5 and 4; and other causes, 3 and 3. None were adjudicated as treatment-related.
In this randomized, placebo-controlled trial in healthy postmenopausal women, exemestane reduced the relative incidence of invasive breast cancers by 65%, from 0.55% to 0.19%. Exemestane also reduced the risk of known breast-cancer precursor lesions — ductal carcinoma in situ, lobular carcinoma in situ, atypical ductal hyperplasia, and atypical lobular hyperplasia — suggesting possible further reductions in invasive cancers during long-term follow-up. Most tumors in these study patients were estrogen-receptor–positive. HER2-positive tumors, which have a poor prognosis, were also reduced with exemestane. Future studies to corroborate this finding would be important.
Menopausal symptoms such as hot flashes, fatigue, sweating, insomnia, and arthralgia were frequent among all the women in the study but were predictably somewhat more common in those taking exemestane. Also of potential clinical importance, more women in the exemestane group self-reported that menopause-related vasomotor and sexual symptoms had worsened. However, these symptoms did not appear to affect self-reports of overall health-related QOL among those taking exemestane because summary measures of physical and mental components of the SF-36 did not differ between the two study groups. (Full QOL results are not reported here.) Unlike the rare endometrial cancers and thromboemboli associated with tamoxifen, particularly in postmenopausal women, no serious adverse events or end-organ toxic effects, including fractures, were attributable to exemestane. Mild loss of bone mineral density with the aromatase inhibitors is well documented, but the annual excess incidence of fractures in trials comparing aromatase inhibitors and tamoxifen are probably due mainly to the bone-protective effects of tamoxifen.41,42 After the cessation of therapy in several large trials comparing aromatase inhibitors and tamoxifen in early breast cancer, bone mineral density improved and the difference in fracture rates was reduced.43-45 In these trials, adverse events also attenuated rapidly after cessation of treatment and correlated with recovery of estrogen levels to their normal postmenopausal range. The absence of excess clinical fractures in patients treated with exemestane in this study is reassuring. This occurred despite similar baseline bone mineral density in the two groups and the use of bisphosphonate therapy both before and during the study. Although differences in the occurrence of colorectal, lung, and endometrial cancers and malignant melanomas have been reported in tamoxifen comparator trials, no differences were seen in this trial or in the placebo-controlled MA.17 trial.26,46 Small numerical, but not significant, differences in the number of cardiovascular events have also been reported in trials comparing aromatase inhibitors with tamoxifen,22,23,46 with more events among the patients treated with aromatase inhibitors, and these differences may have been due to the slightly protective effect of tamoxifen, as suggested by Mouridsen et al.47 It is reassuring that when the aromatase inhibitors were compared with placebo, these differences were not seen either in this prevention trial or in our early breast cancer MA.17 trial.
This trial has some limitations. The median follow-up of 3 years is relatively short, and although consistent with our projections, the total number of breast events (66) was small. The optimal duration of endocrine therapy for breast-cancer prevention is not known, but in a previous placebo-controlled trial of early breast cancer, we found that prolonged aromatase-inhibitor therapy was associated with continued reductions in the incidence of contralateral breast cancers even after the aromatase inhibitor was discontinued.48 The number of women needed to treat in MAP.3 to prevent one case of breast cancer is 94 with 3 years of exemestane therapy, but is projected to be 26 at 5 years, although the number of women who received treatment for a full 5 years was low. By identifying subgroups of participants in the MAP.3 trial who would benefit most or who would be most vulnerable to toxic effects, one might be able to reduce the number needed to treat.
Despite these limitations we found a favorable risk-to-benefit ratio with a strong preventive effect of exemestane and, with a limited median follow-up of 3 years, an excellent safety profile across a spectrum of women at average to high risk for breast cancer. We reached our protocol-specified number of events for this final analysis; after unblinding, women taking the active drug will be offered exemestane to complete 5 years of therapy, and MAP.3 sites will have the option of offering 5 years of exemestane treatment to those initially assigned to placebo. We and others are conducting placebo-controlled trials in healthy women and patients with early breast cancer to evaluate prolonged aromatase-inhibitor therapy in postmenopausal women of similar age. The results of these ongoing trials should contribute to our understanding of the long-term efficacy and toxicity of aromatase inhibitors.
Supported by the Canadian Cancer Society Research Institute, the Canadian Institutes for Health Research, Pfizer, and the Avon Foundation.
Disclosure forms provided by the authors are available with the full text of this article at NEJM.org.
This article (10.1056/NEJMoa1103507) was published on June 4, 2011, and updated on June 23, 2011, at NEJM.org.
We thank the 4560 women (2824 from the United States, 1285 from Canada, 432 from Spain, and 19 from France) who agreed to participate in this study; the trial committee; the many investigators, pharmacists, and clinical research associates involved in the trial; Drs. Joe Pater and Lois Shepherd, Dianne Johnston, and Andrea Hiltz for their enthusiastic and unwavering support; the members of the NCIC CTG Data Safety Monitoring Committee; the Central Office staff of the NCIC CTG who contributed to the conduct of the trial; and Pfizer Pharmaceuticals for support and for providing exemestane and placebo.
From Massachusetts General Hospital Cancer Center (P.E.G.) and Dana–Farber Cancer Institute (J.E.G.) — both in Boston; Mayo Clinic, Rochester, MN (J.N.I.); Hospital Nuestra Señora De Sonsoles, Ávila, Spain (J.E.A., on behalf of the Spanish Group for Breast Cancer Research); Los Angeles Biomedical Research Institute, Harbor–UCLA Medical Center, Torrance, CA (R.T.C.); University at Buffalo, Buffalo, NY (J.W.-W.); Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle (A.M.); University of California, Davis, Sacramento (J.R.); University of Tennessee Health Science Center, Memphis (K.C.J.); George Washington University School of Medicine, Washington, DC (L.W.M.); University of Wisconsin School of Medicine and Public Health, Madison (G.E.S.); Kansas University Medical Center, Kansas City (C.J.F.); Centre Hospitalier Universitaire Arnaud de Villeneuve, Montpellier, France (P.P., on behalf of the National Federation of French Cancer Centers); University Health Network, Toronto (A.M.C.), London Health Sciences Centre, London, ON (E.W.), Unité de recherche en santé des populations de l’Université Laval, Quebec (E.M.), Queen’s University Pathology and Molecular Medicine, Kingston, ON (P.F.), British Columbia Cancer Agency, Vancouver, BC (K.A.G.), and NCIC Clinical Trials Group, Kingston, ON (D.T., H.R.) — all in Canada.
Address reprint requests to Dr. Goss at Massachusetts General Hospital Cancer Center, Lawrence House, LRH-302, Boston, MA 02114, or at [email protected].
The NCIC Clinical Trials Group MAP.3 (NCIC CTG MAP.3) investigators are listed in the Supplementary Appendix, available at NEJM.org.
1. Clemons M, Goss P. Estrogen and the risk of breast cancer. N Engl J Med 2001;344:276-285[Erratum, N Engl J Med 2001;344:1804.]
2.Eliassen AH, Missmer SA, Tworoger SS, Hankinson SE. Circulating 2-hydroxy- and 16alpha-hydroxy estrone levels and risk of breast cancer among postmenopausal women. Cancer Epidemiol Biomarkers Prev 2008;17:2029-2035
3.Ahlgren M, Melbye M, Wohlfahrt J, Sorensen TIA. Growth patterns and the risk of breast cancer in women. N Engl J Med 2004;351:1619-1626
4.The Endogenous Hormones and Breast Cancer Collaborative Group. Endogenous sex hormones and breast cancer in postmenopausal women: reanalysis of nine prospective studies. J Natl Cancer Inst 2002;94:606-616
5.Fisher B, Costantino JP, Wickerham DL, et al. Tamoxifen for prevention of breast cancer: report of the National Surgical Adjuvant Breast and Bowel Project P-1 Study. J Natl Cancer Inst 1998;90:1371-1388
6.Cuzick J, Powles T, Veronesi U, et al. Overview of the main outcomes in breast-cancer prevention trials. Lancet 2003;361:296-300
7.Cuzick J. Long-term follow-up in cancer prevention trials (It ain’t over till it’s over). Cancer Prev Res (Phila) 2010;3:689-691
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Sas T.C.J.a, b· Gault E.J.f· Zeger Bardsley M.g· Menke L.A.c· Freriks K.d· Perry R.J.f· Otten B.J.e· de Muinck Keizer-Schrama S.M.P.F.b· Timmers H.d· Wit J.M.c· Ross J.L.g· Donaldson M.D.C.f
There has been no consensus regarding the efficacy and safety of oxandrolone (Ox) in addition to growth hormone (GH) in girls with Turner syndrome (TS), the optimal age of starting this treatment, or the optimal dose. This collaborative venture between Dutch, UK and US centers is intended to give a summary of the data from three recently published randomized, placebo-controlled, double-blind studies on the effects of Ox. The published papers from these studies were reviewed within the group of authors to reach consensus about the recommendations. The addition of Ox to GH treatment leads to an increase in adult height, on average 2.3-4.6 cm. If Ox dosages <0.06 mg/kg/day are used, side effects are modest. The most relevant safety concerns are virilization (including clitoromegaly and voice deepening) and a transient delay of breast development. We advise monitoring signs of virilization breast development and possibly blood lipids during Ox treatment, in addition to regular follow-up assessments for TS. In girls with TS who are severely short for age, in whom very short adult stature is anticipated, or in whom the growth rate is modest despite good compliance with GH, adjunctive treatment with Ox at a dosage of 0.03-0.05 mg/kg/day starting from the age of 8-10 years onwards can be considered.
While growth hormone (GH) has been shown to improve final height (FH) in Turner syndrome (TS), such treatment can only partially overcome the growth failure observed in affected girls. It is for this reason that adjunctive therapy in TS has been tried, notably with oxandrolone (Ox), a synthetic anabolic steroid derived from dihydrotestosterone by replacing the carbon atom in position 2 with an oxygen atom, and methylating the carbon atom in position 17. Ox treatment, when combined with GH, has been shown to increase height velocity in TS  and to improve FH [2,3], but its use has not become standard for two main reasons. Firstly, the patient numbers involved in previous studies reporting increased FH with GH and Ox have been relatively small. For example, Nilsson et al.  found FH to be greatest in girls receiving GH and Ox compared with GH ± ethinyl estradiol (EE2) and GH, Ox and EE2, but there were 15 or fewer girls in each of the three groups, while in the study of Stahnke et al.  only 7 girls treated with GH alone and 15 girls who had received GH and consistent Ox treatment were at FH. Secondly, features of virilization with clitoromegaly and voice deepening have been reported with Ox doses of 0.1 mg/kg/day or more, requiring a reduction in dosage [1,3]. Given these concerns, and because none of the above studies were placebo (Pl)-controlled, there has to date been insufficient information about both the efficacy and safety of Ox and the optimal dose and age at starting this treatment.
The recent publication of three randomized Pl-controlled, double-blind studies on the effects of Ox in addition to GH in girls with TS has generated more insight into the benefit-risk ratio of Ox [4,5,6].
In this paper we summarize the findings of the three recent studies [4,5,6] in terms of efficacy and safety, and present recommendations concerning the use of Ox in TS. We also draw attention to areas where our knowledge remains insufficient. Whilst a meta-analysis of the data from the three studies was carefully considered and would have the advantages of increasing sample size thus decreasing the confidence intervals, there are difficulties in applying this approach to our situation. Firstly, pooled analysis is usually more suited to situations where more than three studies are available for analysis. Secondly, there are considerable differences in Ox dosage and in timing of GH, Ox and estrogen treatment between the treatment regimens of the three studies. Finally, meta-analysis of the results of these three studies could obscure the influence of the various treatment regimens. For these reasons we have chosen to simply compare and contrast the results from each study.
Efficacy of Ox in GH-Treated Girls with TS
Table 1 outlines the characteristics while table 2 gives the results of the three Pl-controlled studies on the effect of Ox in girls with TS who were treated with GH.
Characteristics of three Pl-controlled studies on the effect of Ox in girls with TS treated with GH 1.33-1.43 mg/m2/day
Results of three Pl-controlled studies on the effect of Ox in girls with TS treated with GH 1.33-1.43 mg/m2/day
In 2010, Menke et al.  reported the data of a Dutch randomized, Pl-controlled, double-blind, dose-response study performed in ten centers in the Netherlands. A total of 133 patients with TS were grouped according to age as follows: group 1 (2-7.99 years), group 2 (8-11.99 years), or group 3 (12-15.99 years). Patients were treated with GH, maintaining a dose of 1.33 mg/m2/day, equivalent to 46 µg/kg/day for a body surface of 1 m2, from baseline throughout the study. They were also randomized to receive either Pl or Ox in a low (0.03 mg/kg/day) or conventional (0.06 mg/kg/day) dose from the age of 8 years. Capsules of Ox were made by one pharmacist in predetermined strengths for daily use and the dose of Ox was rounded off to the nearest 0.5 mg. The maximum daily Ox dose was 3.75 mg. Ox or Pl was continued for as long as GH was prescribed. Estrogen therapy was given from the age of 12 years; 17β-estradiol was prescribed in age groups 1 and 2, and EE2 in age group 3 (5 and 0.05 μg/kg/day orally, increasing to 10 and 0.1 μg/kg/day, respectively, after 2 years). Adult height gain was calculated as attained adult height minus predicted adult height using a modification of the Lyon method [5,6]. Compared with Pl, Ox 0.03 mg/kg/day increased adult height gain in the intention-to-treat analysis (mean ± SD, 9.5 ± 4.7 vs. 7.2 ± 4.0 cm, p = 0.02) and per-protocol analysis (9.8 ± 4.9 vs. 6.8 ± 4.4 cm, p = 0.02). By contrast, adult height gain on GH and Ox 0.06 mg/kg/day was not significantly different from that on GH and Pl (8.3 ± 4.7 vs. 7.2 ± 4.0 cm, p = 0.3) . Concerning the difference in height gain between the group receiving Ox 0.03 mg/kg/day and the group receiving Pl, this is not attributable to the former starting GH 0.9 year and Ox 0.7 year earlier since adjustments for age were made in the statistical analysis. The lack of incremental effect of the higher Ox dose of 0.06 mg/kg/day on adult height can be explained partly by an acceleration in bone maturation (p = 0.001) and also by the relatively high numbers of earlier termination of treatment owing to virilization.
In 2011, Zeger et al.  reported the results of a randomized Pl-controlled, double-blind prospective trial carried out in two centers in the USA, addressing the effect of Ox at a dosage of 0.06 mg/kg/day in addition to GH in girls with TS. Patients received combinations of 2.5- and 1.25-mg Ox tablets in order to achieve the desired weekly dose. The dosage of Ox was reduced by 50% if there were signs of virilization and/or bone age advancement. Ox or Pl was added to GH for 4 years, and EE2 was started in all girls after 2 years of treatment with GH+Pl/Ox: in year 3 50 ng/kg/day, and in year 4 100 ng/kg/day. Height gain was assessed as change in absolute height and height SD score (SDS) from baseline using US National Center for Health Statistics and TS-specific standards. Seventy-six girls aged 10-14.9 years with TS were randomized to receive Ox (0.06 mg/kg/day, maximum 3.75 mg/day) or Pl in combination with GH (maintaining a dose of 0.35 mg/kg/week = 50 μg/kg/day throughout the study). At year 4, 21 out of 24 girls in the GH/Ox group and 20 out of 23 in the GH/Pl group had reached near-adult height (bone age ≥13.5 years). For those who had reached near-adult height, the change in height from baseline between the Ox and Pl groups was nearly significant, those having received Ox having grown an average of 4 cm more (25.4 ± 6.7 vs. 21.8 ± 5.3 cm, p = 0.07) .
In 2011, Gault et al.  reported the data of a randomized, double-blind, Pl-controlled trial performed in 36 pediatric endocrinology departments in the UK. A total of 106 girls with TS aged 7-13 years at recruitment received GH therapy, maintaining a dose of 10 mg/m2/week (1.43 mg/m2/day, equivalent to 49 μg/kg/day for a body surface area of 1 m2) throughout the study, and were randomized to Ox (0.05 mg/kg/day with a maximum daily dose of 2.5 mg/day) or Pl from 9 years of age. Ox was administered as either a full or a half 2.5-mg tablet, or taken on alternate days to achieve the desired weekly dose. Those girls with evidence of ovarian failure at 12 years were further randomized to oral EE2 (year 1: 2 μg daily; year 2: 4 μg daily; year 3: 4 months each of 6, 8, and 10 μg daily) or Pl. Participants who received Pl as well as those recruited after the age of 12.25 years started the EE2 protocol at age 14 years. Growth data were analyzed according to FH, defined as height velocity <1 cm/year and bone age at least 15.5 years, and by SITAR (SuperImposition by Translation And Rotation), a method of growth curve analysis which transforms individual growth curves, which can then be superimposed, thus defining an average summary curve for specific groups . Ox increased adult height by 4.6 cm (95% confidence interval 1.9-7.2, p = 0.001, n = 82) and late pubertal induction (14 years) by 3.8 (0.0-7.5) cm (p = 0.05, n = 48). However, mean FHs for Pl/late induction and Ox/induction at 12 years were similar (153.1 and 154.4 cm) indicating that the effects of Ox therapy and late induction were not additive so that there was little benefit of both giving Ox and delaying pubertal induction . The reason for this negative interaction, which nearly achieved statistical significance, is unknown.
In all three studies, Ox directly increased height velocity in girls with TS who were on standard GH treatment (1.33-1.43 mg/m2/day, equivalent to 45-50 μg/kg/day). Although the effect on adult height and adult height gain was calculated in different ways in the three studies, Ox co-treatment was associated with a greater adult height and/or adult height gain in all when compared to girls treated with GH therapy and Pl. Due to the Pl-controlled randomized design, it is unlikely that the observed differences are caused by one or more of the other factors which may influence adult height in GH-treated girls with TS, such as age at start of GH, years of GH treatment, compliance, GH dose, estrogen therapy and genetic factors.
The average effect of Ox on adult height gain varied between 2.3 and 4.6 cm in the three studies. Differences in the magnitude of the effect of Ox between the three studies may be explained by differences in the patient characteristics, dosage regimen, and limitations of the studies (table 1, 2).
Safety of Ox in GH-Treated Girls with TS
In previous studies as well as in the three recent studies, various aspects of safety were assessed. Here we discuss virilization, delay of breast development, body proportions and composition, cardiovascular risk, bone mineral density, circulating IGF-1, and psychosocial aspects.
The US study using Ox 0.06 mg/kg/day (with a maximum daily dose of 3.75 mg) showed subjective clitoral changes (2 girls on Ox and 2 on Pl), acne (1 on Ox) and mild hirsutism (3 on Ox) in patients after which the Ox/Pl dose was reduced . The Dutch study showed that while there were sporadic complaints about virilization in the Pl group (5%), more girls in the Ox groups reported features of mild virilization (subjective voice deepening, hirsutism, and mild clitoromegaly). This was particularly evident in girls receiving Ox 0.06 mg/kg/day (with a maximum of 3.75 mg) (42%), and to a lesser extent in the group receiving Ox 0.03 mg/kg/day (16%) . In addition, the objective deepening of the voice was greater in the groups receiving GH+Ox compared to those receiving GH+Pl . One girl on Ox 0.03 and 7 on Ox 0.06 (vs. zero on Pl, p = 0.005) discontinued Ox because of virilization . By contrast, the UK study (using 0.05 mg/kg/day with a maximum daily dose of 2.5 mg) did not report any virilizing effects .
It is at present unclear as to how the discrepancies in virilizing effects between the three studies can be explained. In the two studies using 0.06 mg/kg/day, virilization was noted in a substantial number of the girls [4,7]. In the US study, in which the study design included dose reduction, the Ox dose was reduced in about 40% of the girls because of virilization . The absence of virilization in the UK study (using Ox 0.05 mg/kg/day) appears to show that this dose regimen does not lead to virilization. However, it should be noted that no systematic and specific assessment of possible virilization was carried out in this study in which investigators were simply asked to record any adverse event/reaction at each visit . Another explanation may be the use of a relatively low maximal Ox dose in the UK study (2.5 mg/day) in comparison to maximum daily dose of 3.75 mg in the US and Dutch studies [4,7]. However, most girls reported virilization before they were receiving a daily dose >2.5 mg [unpubl. data from the Dutch study].
At present, long-term follow-up data are only available in the women who participated in the Dutch study. This follow-up study showed that even after an average period of 8.7 years, the number of girls who still subjectively experienced virilization was higher in the group that had received Ox compared to Pl . There was also a trend towards a higher Ferriman-Gallwey hirsutism score with Ox (though the score was subclinical for most girls). Three girls who had received Ox still had objective clitoromegaly. In addition, the dose-dependent effect of Ox on lowering voice frequency, which was reported in the pediatric Dutch study , was also seen in the follow-up study . However, only a few [7 of the 43 (16%) Ox-treated versus 2 of the 23 (9%) Pl-treated] patients had developed a voice frequency <-2 SDS; in most instances the voice frequency remained within the normal range. The percentage of patients reporting subjective voice deepening was similar between the GH+Ox and GH+Pl groups .
In conclusion, the addition of Ox (0.03-0.06 mg/kg/day) shows mild dose-dependent subjective and objective virilizing effects in girls with TS, which leads in some individuals to dose reduction or premature discontinuation of treatment. Although hirsutism and acne seem to regress after discontinuation of Ox, clitoromegaly and voice deepening appear irreversible.
Delay of Breast Development
In all three studies, estrogens were started approximately 2 years later than the average age of onset of puberty in the non-TS population (12.5 years – and in one arm of the UK study even 14 years – compared with 10.5 years in population studies) . In the Dutch follow-up study, about 50% of the women had experienced their breast development as delayed, while in 24% the delay of breast development was accompanied by negative emotions or unhappy feelings . In line with expectations based on previous observations , Ox therapy was associated with delayed breast development in the US study as well as in the Dutch studies [7,4] while this parameter was not monitored in the UK study . In the US study, mean Tanner breast stage in the Ox group was approximately one stage behind that of the Pl group after 4 years. At follow-up, more patients who had received Ox had not achieved Tanner breast stage 5 than Pl-treated women . In the Dutch study the increase in breast stage SDS was less with Ox 0.03 and 0.06 mg/kg/day treatment than with Pl in the first 2 years or with discontinuation of Ox/Pl . However, the Dutch follow-up study showed no difference in the subjective experience of delay in breast development during puberty and adolescence between the treatment groups. In addition, the effect of Ox on breast development was transient since final breast size, measured as subtraction of the smallest chest circumference (under the breasts) from the widest chest circumference (at the level of the nipples) with the patient in supine position and Tanner breast stage, was similar in the Ox and Pl groups .
In conclusion, girls with TS receiving Ox undergo a delay in breast development, which disappears after several years on an adult estrogen replacement dose. The consequences of this transient side effect on the patients’ well-being in adolescence and adulthood are unknown.
Body Proportions and Body Composition
In the Dutch study there was a trend towards a higher sitting height using higher Ox doses compared to Pl. In addition, shoulder width was somewhat larger and hip width smaller in the Ox groups compared with the Pl group . These findings were confirmed in the follow-up study, although the numerical difference did not reach statistical significance .
Regarding foot and hand length, previous reports have shown that the already relatively large foot and hand length of TS patients increase during GH treatment, with a possible additional effect in girls receiving a higher dose of GH [15,16]. There are no longitudinal data on the effect of Ox on hand and foot length. However, the Dutch follow-up study showed that the addition of Ox to the standard GH dose does not further increase the disproportion in foot or hand length compared with height. Head circumference was greater in both Ox-treated groups in comparison with Pl-treated girls, but the head circumference/height ratio was not different .
The US study found no differences in terms of change in weight SDS, body mass index SDS, and waist-to-hip ratio from baseline throughout the study between Ox and Pl . In the Dutch study a reduction in fat mass and an increment in muscle mass during Ox treatment were found . In the follow-up study, however, this effect of Ox on fat mass and muscle mass, measured using DEXA, was no longer observed . Thus, the initial beneficial effects of Ox on body composition are transient. The UK study did not record data on body proportions or body composition .
In conclusion, the effects of Ox on body proportions (higher sitting height, broader shoulders) are mild and the effects on body composition appear to be transient.
In the US study, high-density lipoprotein (HDL) cholesterol levels were lower in the Ox group from study visits at 6 months to 2 years, but then increased in the Ox group once estrogen replacement was begun so that there was less of a difference by year 4. There was no significant difference between groups in low-density lipoprotein (LDL) cholesterol measurements for any visit. Triglycerides were lower in the Ox group than in the Pl group at years 3 and 4 . The Dutch study did not present longitudinal data on lipids , but lipid data were obtained at the follow-up study. Eight years after discontinuation of Ox/Pl, mean HDL cholesterol was (although within the normal range) significantly lower in the Ox-treated groups than in the Pl group . This finding suggests that the effect of Ox on lipids is not transient and may be considered as sufficient reason to monitor these during treatment.
The Dutch study showed that at start of treatment, during treatment and after discontinuation of GH+Pl/Ox, mean systolic and diastolic blood pressure were significantly higher than in healthy girls, without significant differences between Ox dosage groups .
In the US and Dutch studies in which oral glucose tolerance tests were performed, the glucose and insulin levels were not significantly different between the Ox and Pl groups [7,11,17].
Bone Mineral Density
Previous studies in women with TS have shown that cortical bone mineral density is lower and the fracture risk is higher than in the normal population [18,19]. Bone health was not reported in the Dutch or UK paper. In the US study, Ox had no effect on bone mineral density SDS . Bone health data have been collected as part of the Dutch follow-up study and are being analyzed at the present time. Such long-term data will be of importance in showing whether or not Ox increases bone density as an androgen-related effect.
In the US study, IGF-1 levels were similar between the Ox and Pl groups at baseline, lower in the Ox group from 6 months to 2 years, and again similar thereafter (when estrogen was added). Serum IGFBP-3 levels were similar in the Ox and Pl groups except for the baseline and 6-month visits . In the Dutch study, IGF-1 SDS values were stable over time, although levels were more frequently increased on Ox than on Pl, whereas the increase in IGF-1 levels and IGF-1 to IGFBP-3 ratio (an indicator of free IGF-1) was not significantly different between the dosage groups . Thus, there was no evidence for higher or lower IGF-1 levels following estrogen induction in these two studies. In the UK study, IGF-1 levels were not routinely assessed as part of the protocol.
Cognitive and Psychosocial Development
In the US study a small decrease in frequency of severe arithmetic learning disability after 4 years of Ox was observed . In the Dutch study, behavior problems, frequently present in untreated girls with TS, decreased during therapy, but total and internalizing problem behavior remained increased, without differences between the Ox and Pl groups . Ox treatment was not associated with any obvious psychological symptoms which could be attributable to virilizing side effects.
Optimal Age to Start Ox
The lowest age at which Ox was started in the three studies was 8 , 9  and 10  years, respectively. In view of the possible virilizing effect, starting Ox before the age of 8 years in doses between 0.03 and 0.06 mg/kg/day is not recommended. However, the effect of even lower Ox doses at <8 years of age has not been reported. Since many patients in the three studies were older than 8-10 years at the start of treatment, age at the start of Ox ranged from 8 to 16 years (for details, see table 1). In the Dutch study, no statistically significant difference in the effect of Ox on adult height gain was found between the three age groups . Thus, in contrast to GH treatment, starting Ox therapy as young as 8 years does not appear to be more effective than starting later. Furthermore, it might be expected that possible virilizing side effects from Ox would result in a greater psychosocial burden in a younger girl with TS than in an older girl. In conclusion, adjunctive treatment with Ox appears effective in the age range of 8-16 years. However, starting Ox relatively young, e.g. before 8 years of age, does not seem to be more favorable than starting in the early pubertal age range.
Optimal Dose of Ox
In the US  and Dutch [4,11] studies, an Ox dose of 0.06 mg/kg/day with a relatively high maximum dose of 3.75 resulted in more virilization and increased skeletal maturation, and the efficacy was less than that of the lower dose (0.03 mg/kg/day) in the Dutch study . Consequently, we believe that for most girls an Ox dose of 0.06 mg/kg/day is too high . A dose of 0.05 mg/kg/day with a maximum of 2.5 mg/day appears to be effective and safe, as is the case with a dose of 0.03 mg/kg/day. The slightly higher adult height gain on Ox 0.05 mg/kg/day in the UK study  than on 0.03 mg/kg/day in the Dutch study  may be explained, at least partially, by the differences in baseline characteristics between the Ox and Pl groups (lower age at start in the Ox group in the UK study), and the later start of estrogens in 50% of the girls in the UK study . An even lower Ox dose than 0.03 mg/kg/day has not yet been studied and could theoretically still have a positive effect on height with even fewer signs of virilization. However, the currently available evidence suggests that an Ox dose between 0.03 and 0.05 mg/kg/day offers the best benefit-risk ratio. As even on a dose of 0.03 mg/kg/day some virilization has been observed, patients and parents should be informed about these possible side effects before starting Ox. When these side effects occur, the dose should be decreased.
There is no scientific evidence to prove that a maximum daily dose for Ox should be set (2.5 mg in the UK study, 3.75 mg in the US and Dutch studies) [4,7,8]. In most girls the complaints about virilization started before they were receiving a daily dose of >2.5 mg [unpubl. data from the Dutch study]. Furthermore, if a dose of 0.03 mg/kg is given, most girls will never reach a dose >2.5 mg (given that few will surpass a body weight of 83 kg). However, we suggest that if an Ox dose of 0.03-0.05 mg/kg/day is prescribed, a pragmatic ceiling dose of 2.5 mg can be used to prevent overdosing, particularly in older and heavier girls.
Conclusions on the Possible Role of Ox in TS
The three recent controlled studies have shown that the addition of Ox to GH treatment (starting at an age between 8 and 16 years) leads to an increase in height velocity and a modest increase of adult height, on average 2.3-4.6 cm, confirming the results of previous clinical trials. The effect of an adequate dose of GH alone on adult height is particularly dependent on the age at the start of GH ranging from approximately 6 cm in girls older than 8 years up to 10-12 cm in younger girls. Therefore, the additional effect of Ox can be estimated at 25-50%. The cost of 5 years of Ox treatment at an average dose of 2.5 mg is calculated at USD 6,000-7,000, although in practice many girls will receive lower doses than this. This cost is more than outweighed by the shorter duration of GH treatment if Ox is added, estimated at approximately EUR 10,000 (USD 13,700) when Ox 0.03 mg/kg/day is used , while the efficacy in terms of adult height is approximately 3 cm greater. Two possible additional benefits (as yet not proven) of adjunctive treatment with Ox may be an increase of cortical thickness and a redress of the relative androgen deficiency in adolescent girls with TS. Side effects are modest if dosages are <0.06 mg/kg/day. The most relevant safety concerns are virilization (including clitoromegaly and voice deepening), transient delay of breast development, and a decrease of HDL cholesterol, but side effects in the very long term are unknown. We advise that during Ox treatment subjective and objective signs of virilization, breast development, and blood lipids should be monitored. The laboratory costs of this are significant, estimated at USD 500 per year, but could be viewed as part of good practice in the monitoring of TS, whether or not adjunctive Ox is used. We believe that in girls with TS who are severely short for age, in whom very short adult stature is anticipated, or in whom the growth rate is modest despite good compliance with GH (as evidenced by normal/high IGF-1 levels), adjunctive treatment with Ox at a dosage of 0.03-0.05 mg/kg/day started from the age of 8-10 years onwards can be considered and discussed with the girl and her family.
H. Timmers received a research grant from Pfizer for this research. T.C.J. Sas received lecture fees from Novo Nordisk and Pfizer and did advisory work for Novo Nordisk. J.M. Wit has served on the advisory boards of Pfizer, Ipsen, Versartis, Prolor, and Biopartners and received fees from Pfizer, Ipsen, and Ferring. L.A. Menke received an honorarium for her thesis from Pfizer, Eli Lilly & Co., ACE Pharmaceuticals, Ferring, Novo Nordisk, Ipsen, and Sandoz. M.D.C. Donaldson received travel expenses from the British Society for Paediatric Endocrinology and Diabetes to attend study Steering Group meetings and royalties from endocrine textbook, consultancy fees for medicolegal reports, and lecture fees from endocrine symposia. E.J. Gault received financial support from the Scottish Executive Chief Scientist Office, the British Society for Paediatric Endocrinology and Diabetes and the Child Growth Foundation, travel expenses to attend an international meeting and a departmental honorarium for presenting preliminary results at a specialist nurse workshop, and travel expenses from the British Society for Paediatric Endocrinology and Diabetes to attend study Steering Group meetings. J.L. Ross has received grant support from Eli Lilly & Co., Pfizer, and Novo Nordisk and has served as a consultant for Eli Lilly & Co., Pfizer, and Novo Nordisk. M. Zeger Bardsley, R.J. Perry, K. Freriks, B.J. Otten and S.M.P.F. de Muinck Keizer-Schrama have nothing to disclose.
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