Simultaneous Measurement of Thirteen Steroid Hormones in Women with Polycystic Ovary Syndrome and Control Women Using Liquid Chromatography-Tandem Mass Spectrometry

Background

The measurement of adrenal and ovarian androgens in women with PCOS has been difficult based on poor specificity and sensitivity of assays in the female range.

Methods

Women with PCOS (NIH criteria; n = 52) and control subjects with 25–35 day menstrual cycles, no evidence of hyperandrogenism and matched for BMI (n = 42) underwent morning blood sampling. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to simultaneously measure 13 steroids from a single blood sample to measure adrenal and ovarian steroids. Androgen and progesterone results were compared in the same samples using RIA.

Results

Testosterone, androstenedione, progesterone and 17OH progesterone levels were higher when measured using RIA compared to LC-MS/MS, although the testosterone RIA demonstrated the best agreement with the LC-MS/MS using a Bland-Altman analysis. Results using LC-MS/MS demonstrated that the concentration of androgens and their precursors were higher in women with PCOS than controls [median (2.5, 97.5th %ile); 1607 (638, 3085) vs. 1143 (511, 4784) ng/dL; p = 0.03]. Women with PCOS had higher testosterone [49 (16, 125) vs. 24 (10, 59) ng/dL], androstenedione [203 (98, 476) vs. 106 (69, 223) ng/dL] and 17OH progesterone levels [80 (17, 176) vs. 44 (17, 142) ng/dL] compared to controls (all P<0.02), but no differences in serum concentrations of the adrenal steroids DHEAS, cortisol, corticosterone and their 11 deoxy precursors. Women with PCOS also had an increase in the product:precursor ratio for 3β-hydroxysteroid dehydrogenase [22% (6, 92) vs. 20% (4, 43); p = 0.009].

Conclusion

LC-MS/MS was superior to RIA in measuring androstenedione, progesterone and 17OH progesterone levels, while testosterone measurements were better matched in the two assays. Androgen levels were higher in women with PCOS in the absence of a difference in adrenal-predominant steroids. These data support previous findings that the ovary is an important source for the androgen excess in women with PCOS.     

Impact of adrenal versus ovarian androgen ratio on signs and symptoms of polycystic ovarian syndrome

The aim of this study was to examine the effect of adrenal versus ovarian androgen (dehydroepiandrosterone sulfate/total testosterone [DHEAS/TT]) on clinical presentation and related metabolic disturbances in Turkish women with polycystic ovarian syndrome (PCOS). Two hundred eighty PCOS cases were taken into the study. For all cases, the DHEAS/TT ratio was calculated. The median value of this ratio was 4.40. Patients with an androgen ratio lower than 4.40 were included in Group 1 and cases with a ratio higher than 4.40 were Group 2. The two groups were compared in terms of hormonal, biochemical and clinical parameters. Body mass index and waist circumference were lower, the Ferriman-Gallwey score was higher and the cycle length was shorter in Group 2. High DHEAS level was associated with better lipid profiles and lower levels of inflammatory markers, meaning good metabolic control in these women, in spite of increased hirsutism rates. In patients with PCOS, both androgens are usually high in proportion to each other. Therefore, it would be more meaningful to use the DHEAS/TT ratio for an assessment of the metabolic and phenotypic effects of PCOS.     

1α,25-Dihydroxyvitamin D3 affects hormone production and expression of steroidogenic enzymes in human adrenocortical NCI-H295R cells

The current study presents data indicating that 1α,25-dihydroxyvitamin D₃ affects the production of hormones and expression of crucial steroidogenic enzymes in the human adrenocortical cell line NCI-H295R. This cell line is widely used as a model for adrenal steroidogenesis. Treatment of the cells with 1α,25-dihydroxyvitamin D₃ suppressed the levels of corticosterone, aldosterone, DHEA, DHEA-sulfate and androstenedione in the culture medium. In order to study the mechanisms behind this suppression of hormone production, we investigated the effects of 1α,25-dihydroxyvitamin D₃ on important genes and enzymes controlling the biosynthesis of adrenal hormones. The mRNA levels were decreased for CYP21A2 while they were increased for CYP11A1 and CYP17A1. No significant changes were observed in mRNA for CYP11B1, CYP11B2 or 3β-hydroxysteroid dehydrogenase (3βHSD). In similarity with the effects on mRNA levels, also the endogenous enzyme activity of CYP21A2 decreased after treatment with 1α,25-dihydroxyvitamin D₃. Interestingly, the two CYP17A1-mediated activities were influenced reciprocally — the 17α-hydroxylase activity increased whereas the 17,20-lyase activity decreased. The current data indicate that the 1α,25-dihydroxyvitamin D₃-mediated decrease in corticosterone and androgen production is due to suppression of the 21-hydroxylase activity by CYP21A2 and the 17,20-lyase activity by CYP17A1, respectively. In conclusion, the current study reports novel findings on 1α,25-dihydroxyvitamin D₃-mediated effects on hormone production and regulation of genes and enzymes involved in steroidogenesis in the adrenocortical NCI-H295R cell line, a model for human adrenal cortex.     

The R453Q and D151A polymorphisms of hexose-6-phosphate dehydrogenase gene (H6PD) influence the polycystic ovary syndrome (PCOS) and obesity

Hexose-6-phosphate dehydrogenase (H6PDH) influences 11β-hydroxysteroid dehydrogenase activity, a key enzyme in the peripheral metabolism of cortisol that modulates insulin sensitivity in adipose tissue. To study the associations of R453Q and D151A polymorphisms in the H6PDH gene (H6PD) with polycystic ovary syndrome (PCOS) and their influence on clinical and metabolic variables, we genotyped 237 patients with PCOS and 135 control women for the R453Q (rs6688832) and D151A (rs34603401) variants in H6PD. The R453Q genotypes were distributed differently in patients and controls (χ(2)=9.55, P=0.002). Genotypes of D151A were distributed evenly in women with PCOS and controls, but showed a different distribution in non-obese and obese women (χ(2)=3.95, P=0.047), especially within the PCOS subgroup (χ(2)=4.65, P=0.031). A backward stepwise likelihood ratio logistic regression model (Nagelkerke's R(2)=0.490; χ(2)=164; P<0.0001) retained free testosterone (OR=1.13; 95% CI: 1.10-1.17) and H6PD Q453 alleles (OR=0.46; 95% CI: 0.27-0.79) as statistically significant predictors for PCOS, whereas homeostasis model assessment of insulin resistance and the H6PD D151A variant were excluded by the model. Both H6PD variants were associated with several phenotypic variables, including fasting insulin, homeostasis model assessment of insulin resistance and androstenedione levels. In summary, the R453Q and D151A variants of the H6PD gene are associated with PCOS and obesity, respectively, and may contribute to the PCOS phenotype by influencing obesity, insulin resistance and hyperandrogenism.     

Evaluation of endothelial dysfunction, lipid metabolism in women with polycystic ovary syndrome: relationship of paraoxonase 1 activity, malondialdehyde levels, low-density lipoprotein subfractions, and endothelial dysfunction

The aim of this study was to assess relationship of insulin resistance, oxidant-antioxidant status, endothelial dysfunction, lipid metabolism, and their contribution to the risks of cardiovascular disease in women with polycystic ovary syndrome (PCOS). Forty-five women with PCOS and 17 healthy women were included in this study. Nitric oxide (NO), endothelin-1 (ET-1), malondialdehyde (MDA), Apo A1, Apo B, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), triglyceride, small, dense LDL cholesterol (sdLDL-C), large buoyant LDL cholesterol (LbLDL-C) levels, and paraoxonase 1 (PON1) activity were measured in serum/plasma obtained from study groups. Insulin resistance [homeostasis model assessment (HOMA) index] and serum sex hormone binding globulin (SHBG), total testosterone (tT), free testosterone (fT), androstenedione, and dehydroepiandrosteronsulfate (DHEAS) levels were also evaluated. Significantly decreased SHBG, NO, HDL-C levels, and PON1 activities, but increased tT, fT, androstenedione, DHEAS, HOMA index, MDA, ET-1, LDL-C, sdLDL-C, and LbLDL-C values were found in PCOS patients compared with those of controls. There was a positive correlation between MDA and fT levels; and a negative correlation between PON1 activity and fT. Our data show that insulin resistance, dyslipidemia, endothelial dysfunction, and oxidative stress might contribute to the excess risk of cardiovascular disease reported in PCOS patients.     

Discovery of 2-methyl-1-{1-[(5-methyl-1H-indol-2-yl)carbonyl]piperidin-4-yl}propan-2-ol: A novel,potent and selective type 5 17β-hydroxysteroid dehydrogenase inhibitor

Type 5 17β-hydroxysteroid dehydrogenase (17β-HSD5), also known as aldo-keto reductase 1C3 (AKR1C3), is a member of the aldo-keto reductase superfamily of enzymes and is expressed in the human prostate. One of the main functions of 17β-HSD5 is to catalyze the conversion of the weak androgen, androstenedione, to the potent androgen, testosterone. The concentration of intraprostatic 5α-dihydrotestosterone (DHT) in patients following chemical or surgical castration has been reported to remain as high as 39% of that of healthy men, with 17β-HSD5 shown to be involved in this androgen synthesis. Inhibition of 17β-HSD5 therefore represents a promising target for the treatment of castration-resistant prostate cancer (CRPC). To investigate this, we conducted high-throughput screening (HTS) and identified compound 2, which displayed a structure distinct from known 17β-HSD5 inhibitors. To optimize the inhibitory activity of compound 2, we first introduced a primary alcohol group. We then converted the primary alcohol group to a tertiary alcohol, which further enhanced the inhibitory activity, improved metabolic stability, and led to the identification of compound 17. Oral administration of compound 17 to castrated nude mice bearing the CWR22R xenograft resulted in the suppression of androstenedione (AD)-induced intratumoral testosterone production. Compound 17 also demonstrated good isoform selectivity, minimal inhibitory activity against either CYP or hERG, and enhanced pharmacokinetic and physicochemical properties.     

Lack of association between common polymorphisms in the 17beta-hydroxysteroid dehydrogenase type V gene (HSD17B5) and precocious pubarche

BACKGROUND: 17beta-Hydroxysteroid dehydrogenase (type V; HSD17B5) is a key enzyme involved in testosterone production in females. A single nucleotide polymorphism (SNP) in the promoter region of its gene was recently found to be associated with polycystic ovary syndrome (PCOS) and its related hyperandrogenaemia. Precocious pubarche (PP) is a clinical entity pointing to adrenal androgen excess from mid-childhood onward and is associated with ovarian androgen excess from puberty onward. It is therefore a strong risk factor for PCOS. METHODS: To investigate associations between this promoter SNP along with three exonic SNPs (one non-synonymous and two synonymous) from the same gene, and PP, a case-control study was performed in 190 girls with PP (84 of which were also tested for functional ovarian hyperandrogenism) from Barcelona, Spain and 71 healthy controls. Clinical features and hormone concentrations relevant to hyperandrogenism were compared by HSD17B5 genotype and haplotype. RESULTS: Neither HSD17B5 genotypes nor haplotype were associated with PP, or subsequent androgen excess in girls from Barcelona (all P>0.05). CONCLUSIONS: HSD17B5 SNPs predicted to have functional effects do not appear to be a risk factor for PP in girls from Barcelona, despite these girls being at high risk of developing androgen excess in adulthood.     

Immunohistochemical detection and biological activities of CYP17 (P450c17) in the indifferent gonad of the frog Rana rugosa

Sex steroids play a crucial role in the gonad differentiation in various species of vertebrates. However, little is known regarding the localization and biological activity of steroid-metabolizing enzymes during gonadal sex differentiation in amphibians. In the present study, we showed by real-time RT-PCR analysis that the expression of CYP17, one of the key steroidogenic enzymes, was higher in the indifferent gonad during sex differentiation in male than in female tadpoles of Rana rugosa but that there was no difference detected in the 3betaHSD mRNA level between the male and female gonads. We next examined the localization of CYP17, 3betaHSD and 17betaHSD in the indifferent and differentiating gonads by using three kinds of antibodies specific for CYP17, 3betaHSD and 17betaHSD, respectively. Positive signals for CYP17, 3betaHSD and 17betaHSD were observed in somatic cells of the indifferent gonad of males and in the interstitial cell of the testis. The enzymatic activity of CYP17 was also examined in the gonad during sex differentiation in this species. [(3)H]Progesterone (Prog) was converted to [(3)H]androstenedione (AE) in the indifferent gonad in males and females, but the rate of its conversion was higher in males than in females. Moreover, fluorescence in situ hybridization (FISH) analysis revealed that the CYP17 gene was located on the q arm of chromosome 9, indicating that CYP17 was autosomal in R. rugosa. Taken together, the results demonstrate that the CYP17 protein is synthesized in somatic cells of the indifferent gonad during gonadal sex differentiation in R. rugosa and that it is more active in converting Prog to AE in males than in females. The data suggest that CYP17 may be involved in testicular formation during sex differentiation in this species.     

Studies to confirm the source of 11ß-hydroxyandrostenedione

In a longitudinal study of 82 children we found a gradual rise in median plasma concentrations of 11ß-hydroxyandrostenedione (11ß-OH-A4) from 2.5 to 6.4 nmol/1 during childhood which was similar in both sexes. This could reflect changes in adrenal function during the adrenarche and sexual maturation. Plasma concentrations of 11ß-OH-A4 in adults follow the patterns of cortisol secretion. In patients with diseases of the adrenal cortex, the plasma concentrations of 11ß-OH-A4 were consistent with the pathology of each condition. In women with polycystic ovaries (PCO) undergoing gonadotrophic stimulation for in vitro fertilization and embryo transfer, 11ß-OH-A4 (median = 3.8 nmol/l), testosterone and androstenedione, were raised when compared to women with normal ovaries (11ß-OH-A4 median = 2.6 nmol/l). Follicular fluid has concentrations of 11ß-OH-A4 six to twelve times greater than plasma levels and in women with PCO, 11ß-OH-A4 concentrations were lower than in women with normal ovaries, which is consistent with an inhibition of ovarian 11ß-hydroxylase. Granulosa cells in vitro demonstrated the production of 11ß-OH-A4 by side chain cleavage of cortisol. These data support an adrenal source for 11ß-OH-A4 but the raised plasma concentrations in women with polycystic ovary syndrome (PCOS) may reflect the excess androgen output from the ovary. 11ß-OH-A4 may therefore be an additional marker for ovarian dysfunction.     

Biotransformation of pregnenolone - 7alpha-3H, progesterone - 7alpha-3H and dehydroepiandrosterone - 7alpha-3H by porcine fetal and maternal adrenal homogenate preparations.

The biotransformation of pregnenolone-7alpha-3H and of progesterone-7alpha-3H by porcine fetal and maternal adrenal homogenates at 56 and 112 days of pregnancy and of dehydroepiandrosterone-7alpha-3H by fetal adrenal homogenates has been investigated in vitro. Both pregnenolone-7alpha-3H and progesterone-7alpha-3H were metabolized extensively by maternal adrenal preparations, the principal radioactive metabolites isolated being cortisol, corticosterone, 11-deoxycortisol, deoxycorticosterone, 11beta-hydroxyprogesterone and androstenedione. In addition, 17alpha-hydroxyprogesterone, 20alpha-dihydroprogesterone and cortisone were formed from both substrates and 17alpha-hydroxypregnenolone and progesterone were formed from pregnenolone. Although essentially the same radioactive metabolites were isolated after incubation of fetal adrenal glands with pregnenolone-7alpha-3H or progesterone-7alpha-3H, a greater proportion of the radioactivity was associated with corticosteroids at 112 days of pregnancy than at 56 days. 11beta-Hydroxyandrostenedione and androstenedione were isolated and identified together with an unknown polar metabolite, after incubation of fetal adrenal tissue with dehydroepiandrosterone-7alpha-3H. These results are discussed in relation to feto-placental steroid biosynthesis and metabolism and the role of the fetal adrenal in the initiation of parturition in the pig.     

The identification of androstenedione and androstenedione 3-enol glucuronide in the blood of a woman with an interstitial cell ovarian tumor after 4-14C-testosterone

After the administration of 4-¹⁴C-testosterone to a woman bearing an interstitial cell ovarian tumor, the presence of two metabolites were identified in the blood. ¹⁴C-androstenedione comprised 3% of the radioactivity in the free fraction and androstenedione 3-enol glueuronide comprised 0.5% of the radioactivity in the glucuronide fraction.     

Analysis of the binding energies of testosterone, 5alpha-dihydrotestosterone,androstenedione and dehydroepiandrosterone sulfate with an antitestosterone antibody

Molecular dynamics simulations and molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) free energy calculations were used to study the binding of testosterone (TES), 5alpha-dihydrotestosterone (5ADHT), androstenedione (AND), and dehydroepiandrosterone sulfate (DHEAS) to the monoclonal antitestosterone antibody 3-C(4)F(5). The relative binding free energy of TES and AND was also calculated with free energy perturbation (FEP) simulations. The antibody 3-C(4)F(5) has a relatively high affinity (3 x 10(8) M(-1)) and on overall good binding profile for testosterone but its cross-reactivity with DHEAS has been the main reason for the failure to use this antibody in clinical immunoassays. The relative binding free energies obtained with the MM-PBSA method were 1.5 kcal/mol for 5ADHT, 3.8 kcal/mol for AND, and 4.3 kcal/mol for DHEAS, as compared to TES. When a water molecule of the ligand binding site, observed in the antibody-TES crystal structure, was explicitly included in MM-PBSA calculations, the relative binding energies were 3.4, 4.9, and 5.4 kcal/mol for 5ADHT, AND, and DHEAS, respectively. The calculated numbers are in correct order but larger than the corresponding experimental energies of 1.3, 1.5, and 2.6 kcal/mol, respectively. The fact that the MM-PBSA method reproduced the relative binding free energies of DHEAS, a steroid having a negatively charged sulfate group, and the neutrally charged TES, 5ADHT, and AND in satisfactory agreement with experiment shows the robustness of the method in predicting relative binding affinities. The 800-ps FEP simulations predicted that the antibody 3-C(4)F(5) binds TES 1.3 kcal/mol tighter than AND. Computational mutagenesis of selected amino acid residues of the ligand binding site revealed that the lower affinities of AND and DHEAS as compared to TES are due to a combined effect of several residues, each contributing a small fraction to the tighter binding of TES. An exception to this is Tyr99H, whose mutation to Ala lowered the binding of DHEAS 0.7 kcal/mol more than the binding of TES. This is probably due to the hydrogen bonding interaction formed between the OH group of Tyr99H and the sulfate group of DHEAS. Computational mutagensis data also showed that the affinity of the steroids to the antitestosterone antibody 3-C(4)F(5) would be enhanced if Trp47H were repositioned so that it would make more extensive contacts with the bound ligands. In addition, the binding of steroids to antitestosterone, antiprogesterone, and antiestradiol antibodies is discussed.     

Mouse 17alpha-hydroxysteroid dehydrogenase (AKR1C21) binds steroids differently from other aldo-keto reductases: identification and characterization of amino acid residues critical for substrate binding

The mouse 17alpha-hydroxysteroid dehydrogenase (m17alpha-HSD) is the unique known member of the aldo-keto reductase (AKR) superfamily able to catalyze efficiently and in a stereospecific manner the conversion of androstenedione (Delta4) into epi-testosterone (epi-T), the 17alpha-epimer of testosterone. Structural and mutagenic studies had already identified one of the residues delineating the steroid-binding cavity, A24, as the major molecular determinant for the stereospecificity of m17alpha-HSD. We report here a ternary complex crystal structure (m17alpha-HSD:NADP(+):epi-T) determined at 1.85 A resolution that confirms this and reveals a unique steroid-binding mode for an AKR enzyme. Indeed, in addition to the interactions found in all other AKRs (van der Waals contacts stabilizing the core of the steroid and the hydrogen bonds established at the catalytic site by the Y55 and H117 residues with the oxygen atom of the ketone group to be reduced), m17alpha-HSD establishes with the other extremity of the steroid nucleus an additional interaction involving K31. By combining direct mutagenesis and kinetic studies, we found that the elimination of this hydrogen bond did not affect the affinity of the enzyme for its steroid substrate but led to a slight but significant increase of its catalytic efficiency (k(cat)/K(m)), suggesting a role for K31 in the release of the steroidal product at the end of the reaction. This previously unobserved steroid-binding mode for an AKR is similar to that adopted by other steroid-binding proteins, the hydroxysteroid dehydrogenases of the short-chain dehydrogenases/reductases (SDR) family and the steroid hormone nuclear receptors. Mutagenesis and structural studies made on the human type 3 3alpha-HSD, a closely related enzyme that shares 73% amino acids identity with the m17alpha-HSD, also revealed that the residue at position 24 of these two enzymes directly affects the binding and/or the release of NADPH, in addition to its role in their 17alpha/17beta stereospecificity.     

Novel homozygous p.Y395X mutation in the CYP11B1 gene found in a Vietnamese patient with 11β-hydroxylase deficiency

Context

The deficiency of steroid 11β-hydroxylase is caused by mutations in the CYP11B1 gene and is the second major form of congenital adrenal hyperplasia associated with hypertension.

Objective

The objective of this study was to screen the CYP11B1 gene for mutations in one Vietnamese male suffering from congenital adrenal hyperplasia.

Patient

The patient (46,XY) had congenital adrenal hyperplasia. The clinical manifestations presented precocious puberty, hyper-pigmentation and high blood pressure at 4 years.

Results

The patient was a homozygous carrier of a novel mutation located in exon 7 containing a premature stop codon instead of tyrosine at 395 (p.Y395X).

Conclusion

We have identified a novel mutant of the CYP11B1 gene in one Vietnamese family associated with phenotypes of congenital adrenal hyperplasia. The mutant gene p.Y395X produces a truncated form of the polypeptide and abolishes the enzyme activities, leading to a severe phenotype of congenital adrenal hyperplasia.     

Polycystic ovary syndrome and endothelial dysfunction: A potential role for soluble lectin-like oxidized low density lipoprotein receptor-1

The aims of this study were to investigate whether serum soluble lectin-like oxidized low-density lipoprotein receptor-1 (sLOX-1), oxidized LDL (oxLDL), paraoxonase-1(PON-1) and hydroperoxide (LOOH) levels are altered in women with polycystic ovary syndrome (PCOS) and also to determine if hyperandrogenism, insulin resistance (IR) and Anti-Müllerian Hormone (AMH) are associated with endothelial dysfunction in PCOS. A total of 46 women with PCOS and 46 non-PCOS healthy controls were recruited. Women with PCOS had significantly higher sLOX-1, oxLDL and LOOH concentrations than non-PCOS women [6.16 (3.92−13.95) vs 1.37 (0.63−4.43) ng/mL, p < 0.001; 6.48 ± 1.03 vs 3.16 ± 1.02 μU/L, p < 0.001; 2.45 (1.45−3.45) vs 1.06 (0.64−1.56) μmol/L, p < 0.001]. The mean PON-1 level of PCOS group was lower than non-PCOS group (69.47 ± 10.75 vs 104.08 ± 21.43 U/mL, p < 0.001). There was no significant difference in terms of the sLOX-1, oxLDL, LOOH and PON-1 levels between normal weight and overweight PCOS women. On univariate logistic regression analysis, Ferriman-Gallwey scale (FGS), HOMA-IR and AMH were an independent predictors of high risk group of endothelial dysfunction markers (HR-EDm). Age and BMI were not associated with HR-EDm. When incorporated into the multivariate model, endotelial dysfunction markers independently correlated with clinical hyperandrogenism (FGS) but not with AMH. In conclusion, our results indicated that an increased concentration of sLOX-1 might be an early predictor of endothelial damage in patients with PCOS. Women with PCOS have elevated sLOX-1, oxLDL, LOOH and decreased PON-1 levels, independent of BMI. Endothelial dysfunction in women with PCOS is associated with hyperandrogenism. Further studies are required to confirm our findings.     

Blood production rates of estrogens in women with differing ratios of urinary estrogen conjugates.

On the basis of the ratios of the estrogen conjugates in their urine (estriol/estrone + estradiol: E3/[E1+E2]), 19 women were divided into two groups: 9 women had ratios less than 0.6 and 10 women had ratios greater than 1.3. All women had measurements made of endogenous estrogens in their plasma by radioimmunoassay. They were then given constant infusions of 3H-estrone, 3H-estradiol and 14C-estriol during days 5-7 and days 20-22 of their cycles, and metabolic clearance rates (MCR) and blood production rates (PB) of estrone, estradiol and estriol were determined. Despite the wide disparity in their ratios of urinary estrogens, no differences could be found between the groups for the MCR's and PB's for all estrogens at either time of the cycle. The mean ratios of PB's (PB3/[PB2+PB1]) of estrone, estradiol and estriol ranged from 0.07 to 0.10 for each group during the cycle. The amounts of estriol entering the blood are small compared to the amounts of estrone and estradiol and do not correlate with the ratios of their urinary conjugates.     

Cholesterol side chain cleavage and aromatase activities in the corpus luteum of the pregnant rhesus monkey.

Cholesterol side-chain cleavage (CSCC) and aromatase activities were measured in luteal mitochondria and tissue pieces, respectively, from rhesus monkeys on days 22, 49, 128 and 160 of gestation. CSCC activity did not vary significantly during gestation and thus probably does not respond to chorionic gonadotropin which is elevated on day 22 of pregnancy. It is not known, however, whether CSCC can be stimulated prior to day 22 when the corpus luteum is steroidogenically more active. Both 3H-pregnenolone and 3H-progesterone were synthesized from [1,2-3/]cholesterol. Aromatase activity declined from high levels on days 22 and 49 to a nadir on day 128 of pregnancy. Utilizing either [1beta-3H]androstenedione or [1beta-3H]testosterone as substrate yielded comparable results throughout gestation.