The concentrations of steroids in antral fluid, the number of granulosa cells, the status of the oocyte, and the diameter of each follicle were determined in human ovaries so that follicles at each stage of the menstrual cycle could be classified as large (greater than or equal to 8 mm diameter) or small (less than 8 mm diameter) and healthy or atretic. The granulosa cells and thecal-enriched tissue from each follicle and the stromal tissue from each ovary were cultured for 6 days in vitro. The amounts of progesterone (P), <em>androstenedione</em> (delta <em>4</em>), testosterone, dihydrotestosterone, estrone, and estradiol (E2) generated by the different tissues were measured on days 0, 2, <em>4</em>, and 6 of culture. It was found that granulosa cells, thecal tissue, and stromal tissue all have the biosynthetic capacity to produce P, delta <em>4</em>, testosterone, dihydrotestosterone, estrone, and E2. No individual steroid-secreting compartment of the ovaries studied, whether part of the follicle or of the stroma, had the exclusive capability of producing any of the above-named steroids at any stage of the menstrual cycle or at any stage of antral follicle growth or atresia. Although the steroids produced by the human follicle appear not to be unique to any one cell type, the patterns of steroidogenesis by the granulosa and thecal compartments differ from one another and from the stroma throughout follicular maturation and atresia. During follicular development, granulosa cells produce large amounts of E2 and small amounts of delta <em>4</em>. During the preovulatory phase, cells from large follicles (greater than or equal to 8 mm diameter) differentiate from an estrogen-secreting state into a P- and, to a lesser extent, an delta <em>4</em>-secreting one. By contrast, during follicular atresia, granulosa cells continue to synthesize delta <em>4</em>, but their capacity to synthesize estrogen is substantially reduced. Furthermore, granulosa cells from atretic follicles are incapable of transforming from an androgen-secreting state into a P-secreting one in tissue culture. During follicular growth, thecal tissue secretes about 2--3 times more delta <em>4</em> than E2. By contrast, during follicular atresia, thecal tissue retains its capacity to synthesize delta <em>4</em> but loses much of its capacity to synthesize E2. The in vitro capacity of thecal tissue to produce steroids exceeds that of the stroma (on a per weight basis) from 2- to 500-fold. Thecal tissue from healthy but not from atretic follicles is capable of differentiating from an androgen- and estrogen-secreting state to a predominantly P-secreting one in tissue culture. It is postulated that although steroid synthesis may not be rigidly compartmentalized during follicular development, appreciable amounts of the steroids secreted by the granulosa and theca may enter different compartments before leaving the ovary...

Phenylalanine and/or tryptophan scanning mutagenesis was performed at 15 sites within CYP3A<em>4</em> proposed to be involved in substrate specificity or cooperativity. The sites were chosen on the basis of previous studies or from a comparison with the structure of P<em>4</em>50(eryF) containing two molecules of <em>androstenedione</em>. The function of the 25 mutants was assessed in a reconstituted system using progesterone, testosterone, 7-benzyloxy-<em>4</em>-(trifluoromethyl)coumarin (7-BFC), and alpha-naphthoflavone (ANF) as substrates. CYP3A<em>4</em> wild type displayed sigmoidal kinetics of ANF 5,6-oxide formation and 7-BFC debenzylation. Analysis of 12 mutants with significant steroid hydroxylase activity showed a lack of positive correlation between ANF oxidation and stimulation of progesterone 6beta-hydroxylation by ANF, indicating that ANF binds at two sites within CYP3A<em>4</em>. 7-BFC debenzylation was stimulated by progesterone and ANF, and 7-BFC did not inhibit testosterone or progesterone 6beta-hydroxylation. Correlational analysis showed no relationship between 7-BFC debenzylation and either progesterone or testosterone 6beta-hydroxylation. These data are difficult to explain with a two-site model of CYP3A<em>4</em> but suggest that three subpockets exist within the active site. Interestingly, classification of the mutants according to their ability to oxidize the four substrates utilized in this study suggested that substrates do bind at preferred locations in the CYP3A<em>4</em> binding pocket.

To determine whether hyperandrogenism caused by an inborn error of adrenal steroidogenesis could produce insulin resistance, we examined insulin sensitivity in females with 21-hydroxylase deficiency. Minimal modelling was used to analyze the results of tolbutamide-modified, frequently sampled, iv glucose tolerance testing. Insulin sensitivity [Si; (min-1) (microU/mL)-1] was plotted against body mass index (BMI; defined as kilograms per m2). Six patients with nonclassical 21-hydroxylase deficiency (mean age, 27 yr; mean BMI, 23.2) underwent testing. None of these patients was in active puberty, nor was any patient being treated with glucocorticoids at the time of the study. Twelve eumenorrheic nonhyperandrogenic young adult female control subjects (mean age, 27 yr; mean BMI, 22.<em>4</em>) were also tested. The basal 17-hydroxyprogesterone concentration, but not the total serum testosterone level, was significantly different in the two groups (mean +/- SEM, 11,987 +/- 2,761 vs. <em>4</em>,059 +/- 802 pmol/L; P < 0.05). As a group the patients' Si values were significantly lower than those of the controls (mean +/- SEM, <em>4</em>.1 +/- 0.6 vs. 9.7 +/- 1.2; P < 0.05). There was no correlation between Si and basal serum 17-hydroxyprogesterone, testosterone, delta <em>4</em>-<em>androstenedione</em>, or dehydroepiandrosterone. We conclude that chronic hypersecretion of androgen precursors due to an inborn error of metabolism can induce a reduction in insulin sensitivity.

The CYP19 gene codes for aromatase, a key steroidogenic enzyme involved in the conversion of androgens to estrogens. A tetranucleotide (TTTA) repeat polymorphism is present in intron <em>4</em> of CYP19; 2 out of <em>4</em> breast cancer case-control studies have reported a greater frequency of 2 specific alleles among affected women. We evaluated associations between CYP19 repeat alleles and breast cancer risk in a case-control study nested within the Nurses' Health Study cohort (incident cases: n=<em>4</em>62; controls: n=618). We observed seven different CYP19 alleles (TTTA(7-13)). Compared to controls, cases had a statistically significant greater frequency of the 10 (TTTA)(10) repeat allele (10 allele: 2.3% vs. 0.7%, p = 0.005) and a nonsignificant increase in the frequency of the 12 (TTTA)(12) allele (12 allele: 3.1% vs. 2.1%, p = 0.11). A higher frequency of the 10 allele was observed in more advanced cancer cases defined as four or more involved nodes or distant metastasis [<em>4</em>+ nodes: 5/36 (13.9%) vs. 0-3 nodes: 13/330 (3.9%), p = 0.02]. Among controls, we found women with the 7 repeat allele to have decreased levels of estrone sulfate (-16.<em>4</em>%, p = 0.02), estrone (-6.1%, p = 0.22) and estradiol (-9.9%, p = 0.10), and a lower estrone/<em>androstenedione</em> ratio (E1/A) (-10.5%, p = 0.08) compared to non-carriers. A higher E1/A ratio and elevated estrogen levels were observed among carriers of the 8 repeat allele; E1/A ratio (+21.0%, p = 0.003), estrone (+7.5%, p = 0.16) and estradiol (+10.8%, p = 0.08). However, we observed no evidence of an association between these alleles and breast cancer risk. We were unable to make inferences regarding the effect of the 10 allele on hormone levels due to the small number of allele carriers in the subgroup with hormone levels. As this repeat polymorphism is not close to the splice sites in intron <em>4</em>, linkage disequilibrium with other functional polymorphisms in CYP19 may explain the findings of an increased association between breast cancer and the 10 allele variant of CYP19. We did not detect any sequence variants in the regulatory region or in the adipose-specific exon I.<em>4</em>. The lack of an established effect on CYP19 function associated with the 10 allele means that these findings should be interpreted with caution.

Anovulation in women with polycystic ovary syndrome (PCOS) is the direct effect of high local androgen concentrations on the ovary. Antiandrogens are substances that prevent androgens from expressing their activity on target tissues. Flutamide is a nonsteroid antiandrogen that has been found effective in hirsute patients, although its mechanism of action is unclear. Eight girls, ranging in age from 16-19 yr, with moderate to severe hirsutism and menstrual irregularities were enrolled in this study. The basal hormonal pattern showed anovulatory cycles; increased concentrations of LH, <em>androstenedione</em>, and testosterone; and increased LH/FSH ratio. A baseline ultrasound scan revealed polycystic ovaries in all patients. All were given 250 mg flutamide twice a day for 6 months. LH, FSH, <em>androstenedione</em>, testosterone, estradiol, and progesterone were evaluated before treatment, every <em>4</em> days during the third month of treatment, and on day 2<em>4</em> of the sixth month of treatment. Hirsutism improved, androgen levels dropped, and ovulatory cycles were restored in all subjects. Ultrasonographic examination in follicular phase showed a significant reduction in ovarian volume and ovaries of normal appearance with one dominant follicle. The most important result of the present study was that flutamide restored ovulation in anovulatory PCOS patients. This finding supports the hypothesis that flutamide reduces androgen synthesis through restoration of ovulation, although a direct block of the steroidogenic enzymes of androgen biosynthesis in ovarian thecal cells cannot be excluded.

Hyperandrogenism, a main clinical feature of polycystic ovary syndrome (PCOS), is thought to result from enhanced ovarian and adrenal androgen generation. To investigate the contribution of peripheral steroidogenesis, we used an oral challenge with dehydroepiandrosterone (DHEA) and analyzed its downstream conversion toward androgens in eight women with PCOS (age, 20-32 yr; body mass index, 20-<em>4</em>1 kg/m(2)) and eight healthy women matched for age and body mass index. They underwent frequent serum sampling and urine collection for 8 h on three occasions: at baseline, and after <em>4</em> d of dexamethasone (Dex; <em>4</em> x 0.5 mg/d), followed by ingestion of 100 mg DHEA or placebo. Dex induced similar significant suppression of circulating steroids in both groups. The oral DHEA challenge led to similar significant increases in the area under the concentration-time curve (0-8 h after Dex) of serum DHEA, DHEA sulfate, <em>androstenedione</em>, and testosterone. However, after oral DHEA, PCOS women had significantly higher increases in serum 5 alpha-dihydrotestosterone (P < 0.01), its main metabolite androstanediol glucuronide (P < 0.05), and the 5 alpha-reduced urinary androgen metabolite androsterone (P < 0.05). PCOS women also had significantly higher baseline excretion of 5 alpha-reduced glucocorticoid (P < 0.01) and mineralocorticoid metabolites (P < 0.05). Taken together, these data indicate enhanced peripheral 5 alpha-reductase activity in PCOS. Thus, not only ovary and adrenal, but also liver and peripheral target tissues, significantly contribute to steroid alterations in PCOS.

OBJECTIVE

It has been proposed that physiologic levels of estrogens stimulate immune responses whereas androgens suppress inflammatory reactions. Thus, prevalence of synovial androgens relative to estrogens would be favorable in rheumatoid arthritis (RA). We investigated synovial fluid (SF) concentrations of several estrogens and androgens and conversion products of the sex steroid precursor dehydroepiandrosterone (DHEA) in supernatants of mixed synoviocytes.

METHODS

SF steroid concentrations were measured by high performance liquid chromotography and mass spectrometry in 12 patients with RA and 8 subjects with traumatic knee injury (noninflammatory controls). Conversion of DHEA to downstream hormones was measured by thin-layer chromatography and phosphorimaging detection in 3 patients with RA and 3 patients with osteoarthritis (OA).

RESULTS

Overall, SF concentration of free estrogens tended to be higher in RA patients versus controls (p < 0.06). Molar ratio of free SF estrogens/free SF androgens was elevated in RA compared to controls (1.17 +/- 0.32 vs 0.29 +/- 0.08, without unit; p = 0.017). The free SF concentration of the precursor <em>androstenedione</em> was significantly higher in RA patients than in controls (10<em>4</em>.6 +/- 32.6 vs 30.<em>4</em> +/- 0.<em>4</em> ng/ml; p = 0.011), and SF estrone the aromatase conversion product of <em>androstenedione</em> was also elevated in RA compared to controls (13.6 +/- 2.6 vs 6.6 +/- 0.8 ng/ml; p = 0.035). The biologically active estrogen derivatives, 16a-hydroxyestrone and <em>4</em>-hydroxyestradiol, were both higher in RA compared to controls (p = 0.085 and p = 0.0<em>4</em><em>4</em>, respectively). In mixed RA synoviocytes, DHEA conversion yielded high local levels of 17beta-estradiol (708 pmol/l = 0.193 ng/ml) compared to testosterone (88 pmol/l = 0.026 ng/ml).

CONCLUSIONS

SF levels of estrogens relative to androgens are significantly elevated, while those of androgens are markedly reduced, in patients with RA compared to controls. This imbalance is most probably due to increased aromatase activity. Thus, an available steroid precursor, such as DHEA, may be rapidly converted to proinflammatory estrogens in the synovial tissue, which may in turn stimulate the inflammatory process in patients with RA.

BACKGROUND

Adiponectin is regarded as a possible link between adiposity and insulin resistance. The study aim was to measure serum adiponectin levels in women with polycystic ovary syndrome (PCOS) and to assess possible correlations between adiponectin and the hormonal or metabolic parameters of the syndrome.

METHODS

Eighty-five selected women were classified as: Group I (n = 35) with PCOS + body mass index (BMI) >25 kg/m(2); group II (n = 35) with PCOS + BMI <25 kg/m(2); and group III (controls; n = 15) ovulating without hyperandrogenaemia and BMI <25 kg/m(2). Blood samples were collected between the days 3 and 6 of a spontaneous menstrual cycle, at 09:00, after an overnight fast. Serum levels of FSH, LH, prolactin, 17alpha-OH-progesterone, sex hormone-binding globulin (SHBG), androgens, insulin, adiponectin and glucose were measured.

RESULTS

Adiponectin levels were significantly decreased in group I compared with groups II and III. No significant difference in adiponectin levels was found between groups II and III, despite significant differences in insulin levels and glucose:insulin ratio. Multiple regression analysis showed that Delta(<em>4</em>)-<em>androstenedione</em> levels and BMI values were the only significant determinants of serum adiponectin levels.

CONCLUSIONS

Serum adiponectin levels are reduced in obese women with PCOS. Although adiponectin does not seem to be actively involved in the pathogenesis of PCOS, there seems to be an interaction between adiponectin and steroid synthesis or action.

Glucocorticoid excess frequently results in obesity, insulin resistance, glucose intolerance, and hypertension and may be the product of altered glucocorticoid hormone action. Tissue sensitivity to glucocorticoid is regulated by the expression of glucocorticoid receptor isoforms (GRalpha and GRbeta) and 11beta-hydroxysteroid dehydrogenase type I (11betaHSD1)-mediated intracellular synthesis of active cortisol from inactive cortisone. We have analyzed the expression of GRalpha, GRbeta, and 11betaHSD1 and their hormonal regulation in skeletal myoblasts from men (n = 1<em>4</em>) with contrasting levels of adiposity and insulin resistance. Immunohistochemical, Northern blot, and Western blot analysis indicated abundant expression of GRalpha and 11betaHSD1 under basal conditions. The apparent K(m) and maximum velocity for the conversion of cortisone to cortisol were <em>4</em><em>4</em>0 +/- 1<em>4</em> nmol/L and 75 +/- 7 pmol/mg protein.h and <em>4</em>37 +/- 16 nmol/L and 33 +/- 6 pmol/mg protein.h (mean +/- SEM; n = <em>4</em>) in the presence and absence of 20% serum. Incubation of myoblasts with increasing concentrations of glucocorticoid (50-1000 nmol/L) resulted in a dose-dependent decline in GRalpha expression and a dose-dependent increase in GRbeta expression. 11betaHSD1 activity was sensitively up-regulated by increasing concentrations of glucocorticoid (50-1000 nmol/L: P < 0.05). Abolition of these effects by the GR antagonist, RU38<em>4</em>86, indicates that regulation of GRalpha, GRbeta, and 11betaHSD1 expression is mediated exclusively by the GRalpha ligand-binding variant. In contrast, 11betaHSD1 was down-regulated by insulin (20-100 mU/mL: P < 0.01) in the presence of 20% serum, whereas incubation with insulin under serum-free conditions resulted in a dose-dependent increase in 11betaHSD1 activity (P < 0.05). Incubation with insulin-like growth factor I resulted in a similar pattern of 11betaHSD1 activity. Although neither testosterone nor <em>androstenedione</em> (5-200 nmol/L) affected 11betaHSD1 activity, incubation of myoblasts with dehydroepiandrosterone (500 nmol/L) resulted in a decline in 11betaHSD1 activity (P < 0.05). These data suggest that glucocorticoid hormone action in skeletal muscle is determined principally by autoregulation of GRalpha, GRbeta, and 11betaHSD1 expression by the ligand-binding GRalpha isoform. Additionally, insulin and insulin-like growth factor I regulation of 11betaHSD1 may represent a novel mechanism that maintains insulin sensitivity in skeletal muscle tissue by diminishing glucocorticoid antagonism of insulin action.

We obtained blood samples from 88 women <em>4</em>5-58 yr old who were having cyclic menses every 1-2 mth (37 women, 133 samples) or were amenorrheic for greater than 3 mth (51 women, 310 samples). Samples were obtained at intervals of 3-<em>4</em> mth and analyzed for estrogens, androgens and gonadotropins using radioimmunoassay techniques. There was a gradual decline in the concentrations of estrone (E1), estradiol (E2), estrone sulfate (E1SO<em>4</em>) and progesterone (P) as the time from the last menses increased. A relatively stable concentration was reached in 12 mth for E1, E2, and E1SO<em>4</em> and in 2 mth for P. The concentrations of testosterone, dihydrotestosterone, <em>androstenedione</em>, dehydroepiandrosterone and dehydroepiandrosterone sulfate remained relatively constant as the time from the last menses increased. There was no apparent difference in the mean values of any of these hormones for any time interval from the last menses. The concentrations of both luteinizing hormone (LH) and follicle stimulating (FSH) were noted to increase initially but they appeared to become stable after 12 mth for FSH and after only 6 mth for LH. Using only the measurements made on the initial blood samples obtained in all patients, we found significant correlations between FSH concentrations and the concentrations of E1, E2 and E1SO<em>4</em> for women who were less than 3 mth from a menses as well as those whose last menses had occurred 3 or more mth previously. The correlations were generally not significant for LH in either groups of women.(ABSTRACT TRUNCATED AT 250 WORDS)

Bone loss associated with menopause leads to an increase in skeletal fragility and fracture risk. Relevant animal models can be useful for evaluating the impact of ovarian failure on bone loss. A chemically induced model of menopause in which mice gradually undergo ovarian failure yet retain residual ovarian tissue has been developed using the chemical <em>4</em>-vinylcyclohexene diepoxide (VCD). This study was designed to compare skeletal effects of VCD-induced ovarian failure to those associated with ovariectomy (OVX). Young (28 day) C57Bl/6Hsd female mice were dosed daily with vehicle or VCD (160 mg/kg/d, IP) for 15 days (n = 6-7/group) and monitored by vaginal cytology for ovarian failure. At the mean age of VCD-induced ovarian failure (approximately 6 wk after onset of dosing), a different group of mice was ovariectomized (OVX, n = 8). Spine BMD (SpBMD) was measured by DXA for 3 mo after ovarian failure and OVX. Mice were killed approximately 5 mo after ovarian failure or OVX, and bone architecture was evaluated by microCT ex vivo. In OVX mice, SpBMD was lower than controls 1 mo after OVX, whereas in VCD-treated mice, SpBMD was not lower than controls until 2.9 mo after ovarian failure (p < 0.05). Both VCD-induced ovarian failure and OVX led to pronounced deterioration of trabecular bone architecture, with slightly greater effects in OVX mice. At the femoral diaphysis, cortical bone area and thickness did not differ between VCD mice and controls but were decreased in OVX compared with both groups (p < 0.05). Circulating <em>androstenedione</em> levels were preserved in VCD-treated mice but reduced in OVX mice relative to controls (p < 0.001). These findings support that (1) VCD-induced ovarian failure leads to trabecular bone deterioration, (2) bone loss is attenuated by residual ovarian tissue, particularly in diaphyseal cortical bone, and (3) the VCD mouse model can be a relevant model for natural menopause in the study of associated bone disorders.

BACKGROUND

Because precocious pubarche (PP) reveals late-onset congenital adrenal hyperplasia (LO-CAH) in 5 to 20% of cases, an adrenal stimulation test is recommended in all patients presenting with it. This test is stressful and expensive, and results are normal in more than 80% of cases.

OBJECTIVE

Our objective was to identify clinical and plasma predictors of LO-CAH among patients presenting with PP.

METHODS

We conducted a retrospective cohort study that included all patients seen for PP at our hospital between 1999 and 2006 (n = 238). All had undergone an ACTH test.

METHODS

LO-CAH was defined by a post-ACTH 17-hydroxyprogesterone (17-OHP) plasma level greater than 10 ng/ml and confirmed by mutational analysis of the CYP21 gene. The association of standard clinical and laboratory indicators with LO-CAH was assessed.

RESULTS

Ten (<em>4</em>%) of 238 patients had LO-CAH. Basal 17-OHP, Delta<em>4</em>-<em>androstenedione</em>, and testosterone plasma levels were significantly higher in these patients. A 2-ng/ml threshold for basal 17-OHP plasma levels offered 100% (95% CI, 69-100) sensitivity for the diagnosis of LO-CAH and 99% (95% CI, 96-100) specificity.

CONCLUSIONS

We identified three plasma predictors of LO-CAH in patients presenting with PP. A selective strategy based on a 2-ng/ml basal 17-OHP plasma level threshold would have safely avoided 99% of the unnecessary ACTH tests among our patients.

OBJECTIVE

Does fibrin introduced into the extracellular matrix affect the growth and maturation of individual primate follicles during encapsulated three-dimensional (3D) culture?

CONCLUSIONS

While not altering follicle survival, fibrin-alginate (FIBRIN) improves macaque primary, but not secondary, follicle development during encapsulated 3D culture in terms of growth, steroidogenesis, anti-Müllerian hormone (AMH)/vascular endothelial growth factor (VEGF) production and oocyte maturation.

BACKGROUND

Efforts to grow non-human primate ovarian follicles from the secondary to the antral stage during encapsulated 3D culture have been successful. However, the growth and maturation of primary follicles in vitro has not been reported in primates, especially in chemically defined conditions.

METHODS

In vitro follicle maturation was investigated using the rhesus macaque (Macaca mulatta). Ovaries (n = 7 pairs) were obtained during the early follicular phase of the menstrual cycle (cycle day 1-<em>4</em>). Primary (80-120 µm diameter) and secondary (125-225 µm diameter) follicles were isolated mechanically, randomly assigned to experimental groups, encapsulated into alginate (0.25% w/v) or FIBRIN (25 mg/ml fibrinogen-0.25% alginate) and cultured for 13 and 5 weeks, respectively.

METHODS

Individual follicles were cultured in alpha minimum essential medium supplemented with FSH. Follicle survival and growth were assessed by microscopy. Follicles that reached the antral stage were treated with recombinant hCG. Metaphase II (MII) oocytes were inseminated via ICSI. Follicle morphology was evaluated by hematoxylin and eosin (H&E) staining. Immunohistochemistry was performed for cytochrome P<em>4</em>50 family 17 subfamily A polypeptide 1 (CYP17A1) and 19 subfamily A polypeptide 1 (CYP19A1). Culture medium was analyzed for estradiol (E2) and progesterone by chemiluminescence, androstenedione (A<em>4</em>) by radioimmunoassay, as well as anti-Müllerian hormone (AMH) and vascular endothelial growth factor (VEGF) by enzyme-linked immunosorbent assay.

RESULTS

A total of 105 primary and 133 secondary follicles were collected. The presence of fibrin in the alginate matrix had no effect on either primary or secondary follicle survival. Growing primary and secondary follicles formed an antrum at Weeks 9 and 3, respectively. The percentage of growing follicles was higher (P < 0.05) for primary follicles cultured in FIBRIN than alginate at Week 13. The diameters were larger for the growing secondary follicles cultured in alginate than FIBRIN at Week 5 (P < 0.05). H&E staining revealed the typical morphology for small antral follicles. CPY17A1 immunostaining was detected in theca cells, while CYP19A1 was observed in granulosa cells. E2 increased (P < 0.05) during antrum formation in growing follicles at Week 9 for primary and Week 3 for secondary follicles. AMH levels in medium from growing primary follicles increased (P < 0.05) after Week <em>4</em> with peak levels at Weeks 9-11. AMH increased (P < 0.05) in growing secondary follicles at Weeks 3-5. VEGF levels in medium were elevated (P < 0.05) in growing primary follicles at Week 9. VEGF increased (P < 0.05) in medium from growing secondary follicles at Weeks 3-5. E2, AMH and VEGF production was higher (P < 0.05) in primary follicle culture with FIBRIN than alginate alone. One primary follicle cultured in FIBRIN (1 of 5 follicles harvested) and a secondary follicle cultured in alginate alone (1 of 15 follicles harvested) yielded an MII oocyte. The fertilized oocyte from primary follicle culture arrested without cell division after fertilization, while the oocyte from secondary follicle culture cleaved and reached the morula stage.

CONCLUSIONS

The study reports on in vitro development and function of individual macaque follicles, that is limited to the interval from the primary and secondary stage to the small antral stage. The findings await translation to human ovarian follicles.

CONCLUSIONS

The 3D model for primate follicle development offers a unique opportunity to investigate the growth and regulation of primate primary, as well as secondary follicles, and their enclosed oocytes, as they grow to the antral stage by monitoring and manipulating factors or signaling pathways in vitro. Since primate primary follicles, in addition to secondary follicles, can be cultured to the antral stage to provide mature oocytes, they represent an additional source of pre-antral follicles for in vitro follicle maturation with the potential to provide gametes for assisted reproductive technology as an option for fertility preservation in women, including patients with cancer.

BACKGROUND

This work was supported by The Oncofertility Consortium (NIH U5<em>4</em> RR02<em>4</em>3<em>4</em>7-HD05829<em>4</em>, PL1-EB0085<em>4</em>2), NIH U5<em>4</em>-HD18185 (Eunice Kennedy Shriver Specialized Cooperative Centers Program in Reproduction and Infertility Research), NIH ORWH/NICHD 2K12HD0<em>4</em>3<em>4</em>88 (BIRCWH), Oregon National Primate Research Center 8P51OD011092. There are no conflicts of interest.

Aldo-keto reductase (AKR) 1C3 (type 5 17beta-hydroxysteroid dehydrogenase and prostaglandin F synthase), may stimulate proliferation via steroid hormone and prostaglandin (PG) metabolism in the breast. Purified recombinant AKR1C3 reduces PGD(2) to 9alpha,11beta-PGF(2), Delta(<em>4</em>)-<em>androstenedione</em> to testosterone, progesterone to 20alpha-hydroxyprogesterone, and to a lesser extent, estrone to 17beta-estradiol. We established MCF-7 cells that stably express AKR1C3 (MCF-7-AKR1C3 cells) to model its over-expression in breast cancer. AKR1C3 expression increased steroid conversion by MCF-7 cells, leading to a pro-estrogenic state. Unexpectedly, estrone was reduced fastest by MCF-7-AKR1C3 cells when compared to other substrates at 0.1muM. MCF-7-AKR1C3 cells proliferated three times faster than parental cells in response to estrone and 17beta-estradiol. AKR1C3 therefore represents a potential target for attenuating estrogen receptor alpha induced proliferation. MCF-7-AKR1C3 cells also reduced PGD(2), limiting its dehydration to form PGJ(2) products. The AKR1C3 product was confirmed as 9alpha,11beta-PGF(2) and quantified with a stereospecific stable isotope dilution liquid chromatography-mass spectrometry method. This method will allow the examination of the role of AKR1C3 in endogenous prostaglandin formation in response to inflammatory stimuli. Expression of AKR1C3 reduced the anti-proliferative effects of PGD(2) on MCF-7 cells, suggesting that AKR1C3 limits peroxisome proliferator activated receptor gamma (PPARgamma) signaling by reducing formation of 15-deoxy-Delta(12,1<em>4</em>)-PGJ(2) (15dPGJ(2)).

The aim of this study was to obtain evidence for the genetic basis of polycystic ovaries (PCO) and premature male pattern baldness (PMPB) by screening first-degree relatives of women affected by polycystic ovary syndrome (PCOS). Because of the high prevalence of PCO in the general population, we also studied first-degree relatives of ten asymptomatic control volunteers of reproductive age. The probands were recruited prospectively from infertility and endocrine clinics, where they presented with various clinical symptoms of PCOS. Each had PCO, on transvaginal ultrasound scan. The families of 29 probands and 10 volunteers agreed to take part in the study. Clinical, ultrasound, and biochemical parameters were used to define PCO/PCOS. All female relatives had an ovarian ultrasound scan and hormone profile performed. History was used to assign status in postmenopausal women. All male relatives were assessed for early onset (<30 yr old) male pattern baldness, by photographs. All relatives were assigned affected (PCO/PMPB) or nonaffected status, and segregation analysis was performed. Of the relatives of 29 PCOS probands, 15 of 29 mothers (52%), 6 of 28 fathers (21%), 35 of 53 sisters (66%), and <em>4</em> of 18 brothers (22%) were assigned affected status. First-degree female relatives of affected individuals had a 61% chance of being affected. Of the first-degree male relatives, 22% were affected. Of a total of 71 siblings of PCOS probands, 39 were affected, giving a segregation ratio of 39/32 (55%), which is consistent with autosomal dominant inheritance for PCO/PMPB. In the control families, 1 of 10 probands (10%), 1 of 10 mothers (10%), no fathers, 2 of 13 sisters (15%), and 1 of 11 brothers (9%) were affected. Of a total of 2<em>4</em> siblings, 3 were affected (13%), giving a segregation ratio (observed/expected) of 3/12, which was significantly different from autosomal dominant inheritance. The inheritance of PCO and PMPB is consistent with an autosomal dominant inheritance pattern in PCOS families, perhaps caused by the same gene. There was no such genetic influence in families of women without PCOS. Sisters of PCOS probands with polycystic ovarian morphology were more likely to have menstrual irregularity and had larger ovaries and higher serum <em>androstenedione</em> and dehydroepiandrosterone-sulfate levels than sisters without PCO. This suggests a spectrum of clinical phenotype in PCOS families. Men with PMPB had higher serum testosterone than those without. Collectively, these data are consistent with a role for genetic differences in androgen synthesis, metabolism, or action in the pathogenesis of PCOS.

Using NADPH-cytochrome P-<em>4</em>50 reductase as electron donor the homogeneous pig 17 alpha-hydroxylase-17,20-lyase (CYP17) was shown to catalyse the conversion of delta 5, as well as delta <em>4</em>, steroids (pregnenolone and progesterone respectively) predominantly into the corresponding 17 alpha-hydroxylated products. The latter were then cleaved by the lyase (desmolase) activity of the enzyme into androgens. Cytochrome b5 stimulated both these activities, but its most noticeable effect was on the formation of delta 16-steroids, which compulsorily required the presence of cytochrome b5. These results on the pig enzyme confirm the original findings [Nakajin, Takahashi, Shinoda and Hall (1985) Biochem. Biophys. Res. Commun. 132, 708-713]. The human CYP17 expressed in Escherichia coli [Imai, Globerman, Gertner, Kagawa and Waterman (1993) J. Biol. Chem. 268, 19681-19689] was also purified to homogeneity and was found to catalyse the hydroxylation of pregnenolone and progesterone without requiring cytochrome b5. Like the pig CYP17, the human CYP17 also catalysed the cytochrome b5-dependent direct cleavage of pregnenolone into the delta 5,16-steroid, but unlike it the human enzyme did not cleave progesterone at all. 17 alpha-Hydroxypregnenolone was, however, cleaved into the corresponding androgen but only in the presence of cytochrome b5. 17 alpha-Hydroxyprogesterone was a poor substrate for the human CYP17; although it was converted into <em>androstenedione</em> in the presence of cytochrome b5 its K(m) was 5 times higher and Vmax. 2.6 times lower than those for the hydroxylation of progesterone. The endocrinological and mechanistic implications of these results are discussed.

The high concentrations of estradiol (E2) found in breast tumors of postmenopausal women could be the result of enhanced uptake from plasma or in situ aromatization of androgens to estrogens. To test the relative importance of these two mechanisms, a model system allowing precise distinction between each is required. Such a model was established using aromatase (A+)- and sham (A-)-transfected MCF-7 cells inoculated into ovariectomized (OVX) nude mice. To validate the model, the confounding effect of peripheral aromatization was first excluded experimentally. A- cells were inoculated into OVX mice as homoimplants (A- cells on both flanks) or heteroimplants (A- cells on one flank and A+ cells on the other), and growth of A- cells in response to exogenous aromatase substrate, <em>androstenedione</em> (delta<em>4</em>A), was evaluated. A- cells did not grow in either group during the 8 weeks of observation, indicating the lack of peripheral aromatization in OVX mice. The biological effects of in situ aromatization were then directly examined. We found that A+ cells in the heteroimplant group grew rapidly, and that the average weight of A+ tumor was 7.6-fold larger and tissue E2 concentration was 3-<em>4</em>-fold higher than A- tumors grown in the same animals. These results demonstrate that in situ aromatization rather than uptake can be a determinant of tumor E2 content and growth stimulation. An additional experiment was then designed to evaluate the relative importance of in situ synthesis versus uptake under conditions reflecting postmenopausal physiology. Groups of OVX mice bearing A+ cells received E2 Silastic implants to clamp plasma levels at 5, 7, 10, and 20 pg/ml or delta<em>4</em>A by injection. The highest tumor E2 concentration and growth rate were found in the group receiving delta<em>4</em>A. E2 delivered by Silastic implants always produced lower tissue E2 levels and tumor growth rates than resulted from in situ synthesis. These data provide direct evidence that under physiological conditions reflecting those in postmenopausal women, in situ aromatization in breast tumor makes a major contribution to tissue E2 content. As further validation that our experimental paradigm models the postmenopausal state, we studied OVX animals not given delta<em>4</em>A as substrate. A+ cells also grew under these conditions, and the aromatase inhibitor <em>4</em>-hydroxy<em>androstenedione</em> reduced both tumor E2 level and growth rate, providing additional evidence of the importance of in situ synthesis. These studies provide the first direct evidence that in situ synthesis of E2 in breast tumors, as opposed to peripheral aromatization and uptake from plasma, can enhance tissue E2 levels and stimulate tumor growth.

Steroids in brain arise from the peripheral endocrine glands and local synthesis. In traumatic brain injury (TBI), the endogenous circulating hormones at the time of injury are important for neuroprotection. In particular, pseudopregnant females recover better than males from TBI. We investigated the effect of pseudopregnancy and TBI on steroid levels in plasma and in three brain regions (within, adjacent, and distal to the lesion site), 6 and 2<em>4</em> h after prefrontal cortex injury. The following steroids were analyzed by gas chromatography/mass spectrometry: pregnenolone, progesterone, 5alpha-dihydroprogesterone, 3alpha,5alpha-tetrahydroprogesterone, 3beta,5alpha-tetrahydroprogesterone, dehydroepiandrosterone, Delta(<em>4</em>)-<em>androstenedione</em>, testosterone, 5alpha-dihydrotestosterone, 3alpha,5alpha-tetrahydrotestosterone, 3beta,5alpha-tetrahydrotestosterone, and 17beta-estradiol. Corticosterone was assayed in plasma to account for stress in the rats. We found different steroid profiles in brain and plasma of male and pseudopregnant female rats and specific profile changes after TBI. In sham-operated pseudopregnant females, much higher levels of progesterone, 5alpha-dihydroprogesterone, 3alpha,5alpha-tetrahydroprogesterone, and 3beta,5alpha-tetrahydroprogesterone were measured in both brain and plasma, compared with sham-operated males. Plasma levels of corticosterone were high in all groups, indicating that the surgeries induced acute stress. Six hours after TBI, the levels of pregnenolone, progesterone, and 5alpha-dihydroprogesterone increased, and those of testosterone decreased in male brain, whereas levels of 5alpha-dihydroprogesterone and 3beta,5alpha-tetrahydroprogesterone increased in brain of pseudopregnant female rats. Plasma levels of 5alpha-dihydroprogesterone did not change after TBI, suggesting a local activation of the 5alpha-reduction pathway of progesterone in both male and pseudopregnant female brain. The significant increase in neurosteroid levels in the male brain after TBI is consistent with their role in neuroprotection. In pseudopregnant females, high levels of circulating progestagens may provide protection against TBI.

Cytochrome P<em>4</em>50c17 catalyzes both 17alpha-hydroxylation and 17,20-lyase conversion of 21-carbon steroids to 19-carbon precursors of sex steroids. P<em>4</em>50c17 can mediate testosterone biosynthesis via the conversion of pregnenolone to dehydroepiandrosterone (the delta(5) pathway) or via conversion of progesterone to <em>androstenedione</em> (the delta(<em>4</em>) pathway). In many species, the 17, 20-lyase activity of P<em>4</em>50c17 for one pathway dominates, reflecting the preferred steroidogenic pathway of that species. All studies of recombinant human P<em>4</em>50c17 and of human adrenal microsomes have found high 17, 20-lyase activity only in the delta(5) pathway. Because the 17, 20-lyase activities in both the delta(<em>4</em>) and delta(5) pathways for testicular P<em>4</em>50c17 have not been directly compared, however, it is not known if the delta(5) pathway dominates in the human testis. To resolve this issue, we assayed the conversion of 17alpha-hydroxypregnenolone to dehydroepiandrosterone (delta(5) 17, 20-lyase activity) and of 17alpha-hydroxyprogesterone to <em>androstenedione</em> (delta(<em>4</em>) 17, 20-lyase activity) by human fetal testicular microsomes. We obtained apparent Michaelis constant (K(m)) and maximum velocity (V(max)) values of 1.0 microM and 0.73 pmol.min(-1). microg(-1) for delta(5) 17, 20-lyase activity and of 3.5 microM and 0.23 pmol.min(-1). microg(-1) for delta(<em>4</em>) 17, 20-lyase activity. Catalytic efficiencies, expressed as the ratio V(max)/K(m), were 0.73 and 0.066 for the delta(5) and delta(<em>4</em>) reactions, respectively, indicating 11-fold higher preference for the delta(5) pathway. We conclude that the majority of testosterone biosynthesis in the human testis proceeds through the conversion of pregnenolone to dehydroepiandrosterone via the delta(5) pathway.

BACKGROUND

Anti-Mullerian hormone (AMH) is expressed in pre- and small-antral follicles. High serum levels are found in women with polycystic ovaries (PCO), accordant with their increased content of small follicles. To evaluate the relationship between AMH, folliculogenesis and hyperandrogenism, we compared serum AMH levels between women with PCO with and without hyperandrogenism and normal controls during controlled ovarian hyperstimulation (COH).

METHODS

Nineteen women with PCO and hyperandrogenism (group A), 10 women with PCO but no hyperandrogenism (group B) and 23 ovulatory women with normal ovarian morphology (group C, controls) underwent COH with the long protocol. Serum levels of AMH, estradiol, <em>androstenedione</em> and follicular tracking were determined before gonadotropins treatment (day 0) and every 2-<em>4</em> days up to the day of HCG administration.

RESULTS

AMH levels declined gradually throughout COH in the three groups, but remained higher in groups A and B compared with the controls. Significantly higher levels were found in group A compared with group B, despite comparable numbers of small follicles. Multiple regression analysis revealed that both the number of small follicles and serum androgens were correlated to AMH.

CONCLUSIONS

Women with PCO have higher serum AMH levels during COH than controls. Hyperandrogenism is associated with an additional increase in AMH. It is conceivable that hyperandrogenism may reflect more severe disruption of folliculogenesis in women with PCO or may affect AMH secretion.

The high ratio of women with systemic lupus erythematosus (SLE) has remained unexplained, despite the recent description of metabolic abnormalities of estrogen and androgen metabolism. Alterations of steroid metabolism in patients with SLE could be important in the pathogenesis of this disease, since it has been reported that gonadal steroids modulate the immune system. Moreover, research with inbred lupus mice has shown that estrogens have adverse effects on the disease in both sexes, whereas androgen therapy or oophorectomy is protective in females. Recently, the finding of elevated testosterone oxidation at C-17 in females with SLE suggested that plasma androgen levels in males and females with SLE should be examined more closely. We studied the varying degrees of clinical activity, with regard to plasma levels of <em>4</em> significant androgens: testosterone, <em>androstenedione</em>, dehydroepiandrosterone, and dehydroepiandrosterone sulfate in a series of 5 male and <em>4</em>2 female SLE patients. Decreased levels of all androgens were observed in women with SLE. The lowest levels of plasma androgens were found in female patients who had active disease, as determined by laboratory and clinical assessment. These data support the fact that specific abnormalities of androgen metabolism in the female are associated with SLE, and may contribute in some way to morbidity and mortality. More importantly, these data may have implications for future therapeutic regimens based on male hormone replacement.

OBJECTIVE

To isolate intact preantral follicles from the human ovary for in vitro studies.

METHODS

Premenopausal human ovary was digested by a mixture of collagenase and deoxyribonuclease (DNase) for 1 hour at 37 degrees C and for 36 hour at <em>4</em> degrees C. Intact preantral follicles at classes 1 and 2 were isolated and cultured for up to 120 hours in a serum-free Dulbecco's modified Eagle's medium (GIBCO, Grand Island, NY) with ITS+ (insulin, transferrin, selenium, linoleic acid, and bovine serum albumin; Collaborative Research, Bedford, MA) with or without FSH. Follicular DNA synthesis was measured by [3H]thymidine incorporation, whereas steroidogenic capacity was assessed by P, <em>androstenedione</em> (A), and 17 beta-E2 RIA. The morphology of long-term cultured follicles was studied by routine histologic procedure.

RESULTS

A large number of intact, preantral follicles was efficiently dissociated from a portion of ovary. Morphologically, follicles were healthy and did not have any thecal layer. Follicle-stimulating hormone, in vitro, induced follicular DNA synthesis by 2<em>4</em> hours and antrum formation in class 2 follicles after 120 hours. Both class 1 and 2 follicles were able to produce a small basal amount of P and A, but they did not produce any measurable E2. However, both classes synthesized increasing amounts of P, A, and E2 after FSH exposure.

CONCLUSIONS

This is a first report of a successful enzymatic isolation of human preantral follicles and their long-term culture in vitro. The growth and differentiation of preantral follicles by FSH clearly indicate the importance of FSH in human follicular development. These results provide an opportunity for quantitative studies on factors regulating folliculogenesis in the human and a novel future direction for human IVF.

Stimulation of androgen-sensitive hair follicles is mediated by dihydrotestosterone (DHT), which is formed in these tissues by 5 alpha-reduction of testosterone. A possible mechanism for increased body hair in some human populations might, therefore, be an increase in 5 alpha-reductase activity, resulting in elevated tissue levels of DHT. If present, this finding could have other important clinical implications, since the 5 alpha-reductase enzyme is pivotal in the pathophysiology of prostatic disease. To explore differences in clinical and biochemical parameters of androgen action, we conducted a study of 18<em>4</em> caucasian and Chinese subjects in whom we evaluated chest hair density and serum levels of androgen precursors and 5 alpha-reduced androgen metabolites. Differences in chest hair density were most notable in the men, in whom comparative mean chest hair scores (using a scale of 0-<em>4</em>) were 3.0 vs. 0.8 (P less than 0.0001), caucasian vs. Chinese. Levels of 5 alpha-reduced androgen products were also strikingly higher in the caucasian vs. Chinese subjects. Serum 3 alpha-androstanediol glucuronide levels (nanomoles per L) were 3<em>4</em>.7 +/- 2.<em>4</em> vs. 19.7 +/- 0.9 (P less than 0.001) for the men and 21.5 +/- 3.2 vs. 9.<em>4</em> +/- 0.6 (P less than 0.001) for the women, and serum levels of androsterone glucuronide (nanomoles per L) were 179 +/- 26 vs. 107 +/- 7 (P less than 0.01) for the caucasian vs. Chinese men and 173 +/- 23 vs. 81 +/- 9 (P less than 0.001) for the women. Serum levels of total and bioavailable testosterone did not differ between the racial groups, but serum levels of the precursor androgens, dehydroepiandrosterone sulfate and <em>androstenedione</em>, were significantly higher in the caucasian vs. Chinese men, but not in the women. We conclude that increased serum levels of 5 alpha-reduced androgen metabolites in caucasians vs. Chinese subjects provide circumstantial evidence for a racial difference in 5 alpha-reductase activity and suggest a mechanism for the increased body hair observed in the caucasian men. Increased levels of precursor androgens may also play a role.

BACKGROUND

Anti-Müllerian hormone (AMH) is produced by the granulosa cells and reflects follicular development. Adult women with polycystic ovary syndrome (PCOS) have increased levels of AMH associated with an excessive number of growing follicles. However, it is not known whether these abnormalities are present before the clinical onset of PCOS.

OBJECTIVE

Our objective was to investigate whether prepubertal daughters of women with PCOS have increased AMH levels.

METHODS

Fourteen female infants (2-3 months old) and 25 prepubertal girls (<em>4</em>-7 yr old) born to PCOS mothers were studied. As a control group, we studied 21 female infants and 2<em>4</em> prepubertal girls born to mothers with regular menses and without hyperandrogenism. The group with PCOS mothers and the control group had normal birth weight and were born from spontaneous singleton pregnancies. Circulating concentrations of gonadotropins, testosterone, <em>androstenedione</em>, estradiol, 17-OH-progesterone, SHBG, inhibin B, and AMH were determined by specific assays.

RESULTS

Serum concentrations of AMH were significantly higher in the PCOS group compared with the control group during early infancy (20.<em>4</em> +/- 15.6 vs. 9.16 +/- 8.6 pmol/liter; P = 0.02<em>4</em>) and during childhood (1<em>4</em>.8 +/- 7.7 vs. 9.61 +/- <em>4</em>.<em>4</em> pmol/liter; P = 0.007). Gonadotropin and serum sex steroid concentrations were similar in both groups during the two study periods, except for FSH, which was lower during childhood in girls born to PCOS mothers.

CONCLUSIONS

We conclude that serum AMH concentrations are increased in prepubertal daughters of PCOS women, suggesting that these girls appear to show evidence of an altered follicular development during infancy and childhood.