Bruce R. Carr

Consensus on infertility treatment related to polycystic ovary syndrome

The treatment of infertile women with polycystic ovary syndrome (PCOS) is surrounded by many controversies. This paper describes, on the basis of the currently available evidence, the consensus reached by a group of experts regarding the therapeutic challenges raised in these women. Before any intervention is initiated, preconceptional counselling should be provided emphasizing the importance of life style, especially weight reduction and exercise in overweight women, smoking and alcohol consumption. The recommended first-line treatment for ovulation induction remains the anti-estrogen clomiphene citrate (CC). Recommended second-line intervention, should CC fail to result in pregnancy, is either exogenous gonadotrophins or laparoscopic ovarian surgery (LOS). The use of exogenous gonadotrophins is associated with increased chances for multiple pregnancy and, therefore, intense monitoring of ovarian response is required. LOS alone is usually effective in <50% of women and additional ovulation induction medication is required under those circumstances. Overall, ovulation induction (representing the CC, gonadotrophin paradigm) is reported to be highly effective with a cumulative singleton live birth rate of 72%. Recommended third-line treatment is in vitro fertilization. More patient-tailored approaches should be developed for ovulation induction based on initial screening characteristics of women with PCOS. Such approaches may result in deviation from the above mentioned first-, second- or third-line ovulation strategies in well-defined subsets of patients. Metformin use in PCOS should be restricted to women with glucose intolerance. Based on recent data available in the literature, the routine use of this drug in ovulation induction is not recommended. Insufficient evidence is currently available to recommend the clinical use of aromatase inhibitors for routine ovulation induction. Even singleton pregnancies in PCOS are associated with increased health risk for both the mother and the fetus.

Treatment of severe postmenopausal endometriosis with an aromatase inhibitor

OBJECTIVE To treat an unusually aggressive case of recurrent postmenopausal endometriosis. DESIGN Case report. SETTING University of Texas Southwestern Medical Center (Dallas, Texas). PATIENT(S) A 57-year-old woman who presented with recurrent severe endometriosis after hysterectomy and bilateral salpingo-oophorectomy. INTERVENTION(S) Oral administration of anastrozole (an aromatase inhibitor) (1 mg/d) and elemental calcium (1.5 g/d) for 9 months. Alendronate (a nonestrogenic inhibitor of bone resorption), 10 mg/d, was added to this regimen. MAIN OUTCOME MEASURE(S) Reduction in size of endometriotic lesion, pain relief, tissue levels of aromatase P450 messenger RNA, bone density. RESULT(S) Circulating levels of estradiol-17beta were reduced to approximately 50% of the baseline value after the onset of treatment with anastrozole. Pain rapidly decreased and completely disappeared after the 2nd month of treatment. The 30 x 30 x 20-mm bright red polypoid vaginal lesion was reduced to a 3-mm gray tissue by the end of 9 months of treatment. Markedly high pretreatment levels of aromatase P450 messenger RNA in the endometriotic tissue became undetectable in a specimen obtained from a repeated biopsy after 6 months of treatment. Bone density of lumbar spine decreased by 6.2% after 9 months of treatment. CONCLUSION(S) This is the first description of the use of an aromatase inhibitor in the treatment of endometriosis. The short-term results were extraordinarily successful in elimination of pain and near-complete eradication of implants associated with severe endometriosis not responsive to other therapy. We conclude that the recently developed potent aromatase inhibitors are candidate drugs in the treatment of endometriosis that is resistant to standard regimens.

Dissecting human adrenal androgen production

The human adrenal cortex produces aldosterone, cortisol and the so-called adrenal androgens, dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS). Within the adult adrenal, the zona glomerulosa produces aldosterone, the zona fasciculata cortisol and the zona reticularis both DHEA and DHEAS. The processes regulating aldosterone and cortisol synthesis are well defined; however, the mechanisms regulating the production of DHEA(S) remain elusive. The emphasis of this review is based on increasing evidence that cytochrome b(5), DHEA sulfotransferase and 3 beta-hydroxysteroid dehydrogenase play crucial roles in regulating production of DHEA(S). Insight into the mechanisms that regulate the synthesis of these key components of DHEA(S) synthesis should provide important clues to the regulation of adrenal androgen biosynthesis.

Effects of lower doses of conjugated equine estrogens and medroxyprogesterone acetate on plasma lipids and lipoproteins, coagulation factors, and carbohydrate metabolism

OBJECTIVE To determine the effects of lower doses of conjugated equine estrogens (CEE) alone or CEE and medroxyprogesterone acetate (MPA) on lipoproteins, carbohydrate metabolism, and coagulation/fibrinolytic factors. DESIGN Randomized, double-blind, placebo-controlled study. SETTING Multicenter substudy of the Womens HOPE trial. PATIENT(S) Seven hundred and forty-nine healthy, postmenopausal women. INTERVENTION(S) Women were randomized to receive the following doses in milligrams per day: 0.625 CEE; 0.625 CEE/2.5 MPA; 0.45 CEE; 0.45 CEE/2.5 MPA; 0.45 CEE/1.5 MPA; 0.3 CEE; 0.3 CEE/1.5 MPA; or placebo. MAIN OUTCOME MEASURE(S) Assessment of lipids, lipoproteins, glucose tolerance, and coagulation/fibrinolytic factors at baseline, cycle 6, and year 1. RESULT(S) One year of treatment with any of the CEE or CEE/MPA regimens studied increased high-density lipoprotein cholesterol (HDL-C); the 10% increase in HDL-C for the CEE 0.45/MPA 1.5 group was similar to the CEE 0.625/MPA 2.5 group. Low-density lipoprotein cholesterol was significantly reduced in all of the active treatment groups except the CEE 0.3/MPA 1.5 group at cycle 13. Apolipoprotein A-I and triglyceride levels increased and apolipoprotein B levels decreased in all groups. The lipoprotein (a) level was reduced in the CEE 0.45/MPA 2.5, CEE 0.45/MPA 1.5, and CEE 0.625/MPA 2.5 groups. Minimal changes were observed in carbohydrate metabolism for all groups. Fibrinogen and PAI-1 activity decreased and plasminogen activity increased in all groups. Decreases in antithrombin III and protein S activities were significant for all active treatment groups except the CEE 0.3/MPA 1.5 group. CONCLUSION(S) Lower doses of CEE and CEE/MPA induce favorable changes in lipids, lipoproteins, and hemostatic factors with minimal changes in carbohydrate metabolism.

Developmental changes in steroidogenic enzymes in human postnatal adrenal cortex: immunohistochemical studies

Adrenarche is considered to occur as a result of intra‐adrenal changes in steroidogenic enzymes involved in C19 steroid production. The present study was conducted because developmental changes in steroidogenic enzymes have not been examined well in human postnatal adrenal.

Profiling transcript levels for steroidogenic enzymes in fetal tissues

Cytochrome P450 (CYP) and hydroxysteroid dehydrogenase enzymes are involved in the conversion of cholesterol to steroid hormones. These enzymes are primarily expressed in the placenta, adrenal and gonads. Interestingly, some of these enzyme activities have been demonstrated in non-endocrine tissues, where they may be involved in important paracrine and autocrine actions. This is particularly the case in the human fetus where steroid precursors circulate at high levels and could be metabolized within tissues to produce active steroid hormones. Herein, we tested the hypothesis that transcripts for steroidogenic enzymes are expressed in fetal tissues other than the classical steroidogenic organs. To test this hypothesis, real-time reverse transcription polymerase chain reaction (RT-RTPCR) assays were developed that quantify mRNA levels for steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage (CYP11A), 3beta-hydroxysteroid dehydrogenase types 1 and 2 (HSD3B1 and HSD3B2), 17alpha-hydroxylase (CYP17), 21-hydroxylase (CYP21), 11beta-hydroxylase (CYP11B1), aldosterone synthase (CYP11B2) and aromatase (CYP19). The use of RT-RTPCR allows the specific detection of these transcripts at levels that would not be detectable using northern analysis. In addition, this method can detect levels of transcript that would not lead to sufficient protein for detection of enzymatic activity of protein by western analysis. Thus, this methodology can detect low levels of expression that could play a role in regulating intra-tissue concentrations of steroid hormone. Total RNAs used for RT-RTPCR analysis were isolated from several human fetal tissues, including adrenal, testis, ovary, placenta, aorta, brain, liver, kidney, heart, lung, pancreas, prostate, stomach, and thymus. Our findings suggest that RT-RTPCR is a powerful tool for the examination of steroidogenic enzyme mRNA expressions. Using this approach, we have identified and quantified transcript levels of StAR and steroidogenic enzymes in several endocrine and non-endocrine fetal tissues. Even though some of the mRNA levels measured in these peripheral tissues are extremely lower in respect to the steroidogenic tissues, they could be sufficient to produce local (i.e. autocrine and paracrine) effects because produced steroids are not diluted into the entire circulation. These findings open new perspectives on the role of steroid hormones synthesized locally as probable regulatory factors of the development of several organ systems.

Adrenarche results from development of a 3β-hydroxysteroid dehydrogenase-deficient adrenal reticularis

Adrenarche is the increased adrenal production of dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) that occurs during the prepubertal period. To date, the exact mechanism initiating adrenarche is unknown, although many factors have been postulated. In the present study, we examined the hypothesis that alterations in intra-adrenal expression of 3beta-hydroxysteroid dehydrogenase (3betaHSD) or 21-hydroxylase (CYP21) within the inner reticularis zone leads to the increased production of 19-carbon (C19) steroids. After conversion of cholesterol to pregnenolone, 17alpha-hydroxylase/17,20-lyase (CYP17) can metabolize pregnenolone through to DHEA. The enzyme 3betaHSD competes for substrate with CYP17 and effectively removes steroid precursor from the pathway leading to DHEA. On the other hand, deficiency in CYP21 expression is known to cause excessive production of adrenal C19 steroids, suggesting that CYP21 could play a role in adrenarche. Thus, a decrease in 3betaHSD or CYP21 expression would allow substrate to flow toward the synthesis of DHEA. To determine whether adrenarche results from a decreased expression of 3betaHSD or CYP21 in the reticularis, immunohistochemical localization of 3betaHSD and CYP21 was performed, and staining intensities compared using adrenal glands from children ages 4 months to 4 yr (n = 12), ages 5-7 yr (n = 9), ages 8-13 yr (n = 9), and adults ages 25-56 yr (n = 8). There were no differences in the zonal expression of CYP21. No difference in 3betaHSD staining was observed between the glomerulosa and fasciculata from any age group. However, children age 8 yr and older show a significant decrease in 3betaHSD expression in reticularis as compared with the fasciculata. No significant difference was noted for 3betaHSD levels between the fasciculata and reticularis for children age 7 yr or younger. The level of 3betaHSD expression in the reticularis continued to decrease in the adult adrenals examined. These findings suggest that as children mature there is a decreased level of 3betaHSD in the adrenal reticularis that may contribute to the increased production of DHEA and DHEAS seen during adrenarche.

Total testosterone assays in women with polycystic ovary syndrome: Precision and correlation with hirsutism

CONTEXT There is no standardized assay of testosterone in women. Liquid chromatography mass spectrometry (LC/MS) has been proposed as the preferable assay by an Endocrine Society Position Statement. OBJECTIVE The aim was to compare assay results from a direct RIA with two LC/MS. DESIGN AND SETTING We conducted a blinded laboratory study including masked duplicate samples at three laboratories--two academic (University of Virginia, RIA; and Mayo Clinic, LC/MS) and one commercial (Quest, LC/MS). PARTICIPANTS AND INTERVENTIONS Baseline testosterone levels from 596 women with PCOS who participated in a large, multicenter, randomized controlled infertility trial performed at academic health centers in the United States were run by varying assays, and results were compared. MAIN OUTCOME MEASURE We measured assay precision and correlation and baseline Ferriman-Gallwey hirsutism scores. RESULTS Median testosterone levels were highest with RIA. The correlations between the blinded samples that were run in duplicate were comparable. The correlation coefficient (CC) between LC/MS at Quest and Mayo was 0.83 [95% confidence interval (CI), 0.80-0.85], between RIA and LC/MS at Mayo was 0.79 (95% CI, 0.76-0.82), and between RIA and LC/MS at Quest was 0.67 (95% CI, 0.63-0.72). Interassay variation was highest at the lower levels of total testosterone (≤50 ng/dl). The CC for Quest LC/MS was significantly different from those derived from the other assays. We found similar correlations between total testosterone levels and hirsutism score with the RIA (CC=0.24), LC/MS at Mayo (CC=0.15), or Quest (CC=0.17). CONCLUSIONS A testosterone RIA is comparable to LC/MS assays. There is significant variability between LC/MS assays and poor precision with all assays at low testosterone levels.

The orphan nuclear receptor NGFIB regulates transcription of 3β-hydroxysteroid dehydrogenase: Implications for the control of adrenal functional zonation

3β-Hydroxysteroid dehydrogenase type 2 (HSD3B2) is a steroid-metabolizing enzyme that is essential for adrenal production of mineralocorticoids and glucocorticoids. Thus, HSD3B2 is expressed at high levels in the glomerulosa and fasciculata, where these steroids are produced. In contrast, the production of dehydroepiandrosterone (DHEA) and DHEA sulfate in the adrenal reticularis is inversely correlated with the expression of HSD3B2. The reasons for the zonal expression of HSD3B2 are not known but represent an important aspect in the biochemical zonation of the adrenal. Using microarray, real time reverse transcriptase-PCR, immunohistochemistry, and HSD3B2 promoter analysis, we demonstrate that the NGFIB family of nuclear hormone receptors plays a critical part in the regulation of HSD3B2 transcription and may play an important role in the functional zonation of the adrenal gland. Microarray analysis of cortisol- versus DHEA sulfate-producing adrenal tissue demonstrated that NGIFB paralleled expression of HSD3B2 with expression much higher in cortisol-producing adrenal tissue; this observation was also demonstrated using real time reverse transcriptase-PCR analysis. In addition, immunohistochemistry confirmed that within adult and fetal adrenal gland NGFIB expression paralleled expression of HSD3B2. Transient transfections into H295R adrenal cells demonstrated that NGFIB family members enhanced HSD3B2 reporter activity but had no effect on a 17α-hydroxylase (CYP17) promoter construct. Deletion and mutational analyses of the 5′-flanking region of the HSD3B2 gene identified a consensus NGFIB response element that bound NGFIB in mobility shift assays. Infection of cultured human adrenal cells with adenovirus-containing NGFIB increased cortisol production by 8-fold and increased expression of HSD3B2 mRNA 26-fold over that observed in mock-infected cells. In primary cultures of adrenal cells, ACTH, an activator of HSD3B2, rapidly induced expression of NGFIB. These results suggest that NGFIB plays a crucial role in adrenal zonation by regulating HSD3B2 gene transcription.

A prospective randomized study of pregnancy rates following intrauterine and intracervical insemination using frozen donor sperm

Cryopreserved sperm have lowered fertility when compared with fresh sperm in artificial insemination by donor programs. The purpose of this study was to compare pregnancy rates following intrauterine insemination (IUI) and intracervical insemination (ICI) with cryopreserved sperm in a prospective trial using the patient as her own control. A total of 154 patients were randomized into alternating treatment cycles and underwent 238 cycles of IUI and 229 cycles of ICI. The pregnancy rate per treatment cycle was 9.7% following IUI and 3.9% following ICI. Treatment outcome was influenced by patient age, ovulatory status, and endometriosis. Pregnancy success correlated well with the post-thaw survival of sperm and the number of motile cells inseminated. In spite of having normal semen parameters, some donors were found to have markedly reduced sperm fecundity. We conclude that IUI with cryopreserved sperm can be an effective treatment for couples with infertility, genetic indications, or other reasons.