Steroid hormone analysis of adolescents and young women with polycystic ovarian syndrome and adrenocortical dysfunction using UPC2-MS/MS

Abstract

Background: We recently identified 35 women with polycystic ovarian syndrome (PCOS) who exhibited features of micronodular adrenocortical hyperplasia. Steroid hormone analysis can be more accurate using state-of-the-art ultra-performance convergence chromatography-tandem mass spectrometry (UPC²-MS/MS). We hypothesized that UPC²-MS/MS may be used to better define hormonally this distinct subgroup of patients with PCOS.

Methods: Plasma from PCOS patients (n = 35) and healthy volunteers (HVs, n = 19) who all received dexamethasone testing was analyzed. Samples were grouped per dexamethasone responses and followed by UPC²-MS/MS analysis. When insufficient, samples were pooled from patients with similar responses to allow quantification over the low end of the assay.

Results: The C11-oxy C₁₉ (11β-hydroxyandrostenedione, 11keto-androstenedione, 11β-hydroxytestosterone, 11keto-testosterone):C₁₉ (androstenedione, testosterone) steroid ratio was decreased by 1.75-fold in PCOS patients compared to HVs. Downstream steroid metabolites 11β-hydroxyandrosterone and 11keto-androsterone were also measurable. The C11-oxy C₂₁ steroids, 11-hydroxyprogesterone and 11keto-dihydroprogesterone levels, were 1.2- and 1.7-fold higher in PCOS patients compared to HVs, respectively.

Conclusions: We hypothesized that UPC²-MS/MS may accurately quantify steroids, in vivo, and identify novel metabolites in a subgroup of patients with PCOS and adrenal abnormalities. Indeed, it appears that adrenal C11-oxy steroids have the potential of being used diagnostically to identify younger women and adolescents with PCOS who also have some evidence of micronodular adrenocortical hyperplasia.

Impact: Adrenal C11-oxy steroids may be clinically important in identifying young patients with PCOS and adrenal abnormalities. The steroids presented in our manuscript have not yet been considered in the clinical setting so far, and we believe that this study could represent a first focused step towards the characterization of a distinct subgroup of women with PCOS who may in fact be treated differently than the average patient with PCOS. This paper can change the understanding of PCOS as one disorder: it is in fact a heterogeneous condition. In addition, for the subgroup of patients with PCOS associated with adrenocortical dysfunction, our paper provides novel hormonal markers that can be used diagnostically. Finally, the paper also adds to the basic pathophysiological understanding of adrenocortical-ovarian interactions in steroidogenesis of young women and adolescent girls with PCOS.

Figure 1:

Analysis of circulating classic C₁₉ and C11-oxy C₁₉ steroids in PCOS women and healthy controls. DHEA, dehydroepiandrosterone A4, androstenedione; T, testosterone; 11OHA4, 11 β-hydroxyandrostenedione; 11OHT, 11 β-hydroxytestosterone; 11KA4, 11-ketoandrostenedione; 11KT, 11-ketotestosterone. Statistical significance was determined by multiple t tests of two-way ANOVA (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

Figure 2:

Comparison of significantly different circulating C19 steroids (nM) in PCOS women and healthy controls. PCOS women: HR, high responders, n=15; NR, normal responders, n=20; and HV, healthy sex- and age-matched volunteers, n=20. (a) dehydroepiandrosterone (DHEA); (b) A4(androstenedione):T(testosterone) ratio; (c) A4; (d) T; (e) 11-ketotestosterone (11KT); (f) 5α-androstane-3α,17β-diol (3αDIOL). Statistical significance was determined by One-way ANOVA and unpaired t-tests. (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns= not significant).

Figure 3:

Comparison of significantly different circulating C21 steroids (nM) in PCOS women and healthy controls. PCOS women: HR, high responders, n=15; NR, normal responders, n=20; and HV, healthy sex- and age-matched volunteers, n=20. (a) cortisol; (b) progesterone (PROG); (c) cortisone (d) 17α-hydroxyprogesterone (17OHPROG); (e) corticosterone (CORT); (f) 16α-hydroxyprogesterone (16OHPROG). Statistical significance was determined by One-way ANOVA and unpaired t-tests. (*P<0.05, **P<0.01, ***P<0.001)

Figure 4:

PC2 plot versus PC1 plot, visually indicating relationships between steroids in the three groups. HV - healthy volunteers; NR - PCOS normal responders; HR, PCOS high responders.