Gas chromatography-mass spectrometry based steroid metabolomics in women with different phenotypes of polycystic ovarian syndrome and normal body weight

Introduction

To date, there is no single classification of hyperandrogenism (HA). Most researchers distinguish two main forms – tumor and non-tumor or functional, which is divided into ovarian, adrenal, and mixed depending on the genesis of the disorder. In addition, HA is distinguished between true, receptor, and transporter HA [1]. The most common causes of HA are polycystic ovary syndrome (PCOS) and a nonclassic form of congenital adrenal hyperplasia (NCAH), which is primarily caused by 21-hydroxylase enzyme deficiency [2][3]. The clinical presentation of PCOS and NCAH may be similar and requires differential diagnosis [4]. To identify the source of HA, the determination of various hormones is used, and functional tests for stimulation and suppression of ovarian and adrenal function are performed. It is very difficult to determine the localization of the HA source because the spectrum of synthesized hormones and key enzyme systems of androgen synthesis in ovaries and adrenal glands are similar [5].

Immunoassay methods are widely used in the determination of hormones because of their high sensitivity. However, the low specificity of these methods and the presence of cross-reactions lead to an increasing number of false-positive results and thus to overdiagnosis [6]. For example, the content of 17-hydroxyprogesterone (17-OHP) may be within the normal range in women with NCAH [7]. In patients with PCOS, elevated levels of 17-OHP are detected in half of the cases, and 20–30% of patients have increased adrenal androgens such as dehydroepiandrosterone sulfate (DHEA-S). Patients with clinical signs of HA with basal levels of 17-OHP within normal values undergo a stimulation test with a synthetic analog of corticotropin (tetracosactide), which is the "gold standard" for diagnosis of NCAH [8, 9]. Currently, there are no preparations of tetracosactide authorized in Russia. In some heterozygous carriers of mutations in the 21-hydroxylase gene, 17-OHP levels may be the same as in patients with NCAH. The main obstacle in genetic testing is the complexity of molecular genetic analysis and the fact that most available panel screening tests examine the 10–12 most common mutations and may not detect all the mutations that are currently known [10]. Chromatography techniques provide steroid profiles of blood and urine, which are the most valuable diagnostic tests for diseases associated with impaired steroid hormone synthesis and metabolism [11]. According to some authors, the assessment of steroid hormones by tandem chromatography-mass spectrometry is a reliable method for the diagnosis of NCAH, allowing for a significant reduction in the number of false-positive results [12]. Other researchers highlight particular importance in determining the urine steroid profile (USP) by gas chromatography-mass spectrometry (GC-MS), which makes it possible to identify a large number of androgens, glucocorticoids, their precursors, and metabolites [13-15]. There are sporadic studies on steroid metabolomics in obese patients with PCOS. Increased urinary excretion (UE) of pregnenes, dehydroepiandrosterone (DHEA) and its metabolites, androstenedione metabolites, biologically active 5α- and 5β-tetrahydrometabolites of glucocorticoids, and reduced activity of 11β-hydroxysteroid dehydrogenase (11β-HSD) type 1 enzyme were revealed, which lead to the accumulation of inactive glucocorticoids [16][17]. Deng et al. examined normal-weight, overweight, and obese women with PCOS. A comparative analysis showed that normal-weight women had signs of 21-hydroxylase enzyme deficiency in the absence of a mutation in the gene in contrast to overweight and obese women, which also confirms different variants of steroidogenesis [18]. As a result of androgen excess in women, clinical manifestations, such as acne, hirsutism, and alopecia, can be different in severity. As a rule, 5α-reductase, which converts testosterone to the more active androgen dihydrotestosterone, is responsible for the manifestation of androgenic alopecia and acne [16]. There are two isoforms of 5α-reductase: 5α-reductase 1 and 2 (SRD5A1, SRD5A2). 5α-reductase 1 is expressed in the scalp, liver, ovaries, uterus, kidneys, and brain; 5α-reductase 2 is expressed in the liver and, to a lesser extent, in the scalp and skin [19]. When the activity of SRD5A1 is high, women show pronounced signs of androgen-dependent dermopathy without other manifestations. Currently, there is considerable evidence of increased 5α-reductase activity in women with PCOS.

There are four phenotypes of PCOS (A, B, C, and D) [20]. Phenotype A is the classic combination of three diagnostic criteria, namely HA (clinical, biochemical, or combined), oligo- and/or anovulation, and polycystic ovarian changes (based on ultrasound examination). Phenotype B is a combination of HA and oligoanovulation syndrome without echographic signs of polycystic ovarian changes. Phenotype C includes HA syndrome and polycystic ovaries according to ultrasound findings in the absence of oligo/anovulation (ovulatory PCOS). Phenotype D is a combination of oligoanovulation and polycystic ovaries according to echography without signs of HA (nonandrogenic PCOS). Different studies on the prevalence of PCOS phenotypes in women of reproductive age show that phenotype A occurs in 44–65% of women, phenotype B – in 8–33%, phenotype C – in 3–29%, and phenotype D – in 23% [21][22]. The search for additional biochemical markers for the differential diagnosis of various phenotypes of PCOS using chromatography methods seems relevant.

Materials and Methods

A total of 84 women aged 24 to 29 years old (mean age 25±0.3 years) with a body mass index (BMI) in the reference range 18.5–24.9 kg were examined. The control group (CG) consisted of 25 healthy women aged 26 (23–30) years old with normal BMI. PCOS was diagnosed according to ASRM/ESHRE (2003), International PCOS Network (2018). According to these guidelines, the presence of two out of the three basic criteria determines the presence of a certain type (phenotype) of PCOS. Patients with PCOS were divided into four groups: 15 patients with clinical and biochemical signs of HA, anovulation and signs of polycystic ovaries, according to ultrasound examination (phenotype A); 11 patients with PCOS and without signs of polycystic ovaries with anovulation and HA (phenotype B); 9 patients with PCOS, ovulation, HA, and polycystic ovaries (phenotype C); and 13 patients with anovulation and signs of polycystic ovaries but without HA made the group with phenotype D. Luteinizing hormone (LH), follicle-stimulating hormone (FSH), free testosterone, 17-OH progesterone (17-OHP), dehydroepiandrosterone sulfate (DHEA-S), Δ-4-androstenedione, and serum sex hormone-binding globulin (SHBG) were determined by immunoassay methods. The authors studied USP by GC-MS with optimization of the sample preparation procedure, which included the liquid extraction method. The optimal amounts of derivatizing agents (methoxyamine and trimethylsilylimidazole) were determined, and the chromatographic analysis conditions were selected [17][23][24]. A total of 69 steroids were identified. The data were statistically processed using the STATISTICA for WINDOWS software system (version 10). The main quantitative characteristics of patients were presented as median (Me), 25th percentile, and 75th percentile (Q25-Q75). The nonparametric Mann-Whitney test was used to compare the results obtained in the study groups. The data was statistically significant at p<0.05.

The study was conducted in accordance with GCP (Good Clinical Practice) international standards.

Results

The immunoassay method revealed a decrease in the SHBG level and an increase in the serum-free testosterone level in patients with PCOS phenotypes A, B, and D. The common feature of those was anovulation. An increase in the serum LH level and LH/FSH ratio by more than 2-fold in comparison with the CG was obtained only in patients with PCOS phenotypes A and B (Table 1). The serum levels of 17-OHP and androstenedione were elevated in patients with PCOS phenotypes A, B, C, and D. The DHEA-S level was increased only in patients with PCOS phenotype B compared to the CG (Table 1).

Different USPs were obtained by GC-MS in patients with PCOS phenotypes A, B, C, and D. UE of DHEA was increased in all examined patients with PCOS compared to the CG (Table 2). It should be noted that UE of DHEA was higher in patients with PCOS phenotype B (p=0.017) and in patients with PCOS phenotype C (p=0.028) compared to patients with PCOS phenotype D. UE of DHEA metabolites (androstenediol-17β(dA2-17β) and 16α-ON-DHEA-2) was increased in patients with PCOS phenotypes A, B, and C. An increase in androstentriol UE (dA3) was detected only in patients with phenotypes A and B (Table 2).

Patients with phenotype A had increased UE of androstenedione metabolites androsterone (An), ethiocholanolone (Et), and 11-OH-An. Patients with phenotype B had increased UE of 5α-metabolites androstenedione An and 11-OH-An in comparison with the CG. An increased 11-OH-An/11-OH-Et ratio was obtained in patients with PCOS phenotypes A, B, C, which is one of the signs of increased 5α-reductase enzyme activity (Table 3).

The UE of corticosterone tetrahydrometabolites (5β-THB and 5α-THB) and 11-deoxycortisol (THS) were increased in patients with PCOS phenotypes A, B, and C. The UE of 5α-tetrahydrocortisone (5α-THE) and cortolones was increased in patients with PCOS phenotypes A and B, and only 5α-THB underwent UE in patients with PCOS phenotype D (Table 2).

Reduced ratios of 5β-THF/5β-THE and (5β-THF+5α-THF+cortols)/(5β-THE+5α-THE+cortolones) in patients with PCOS phenotype A indicated a decreased 11β-hydroxysteroid dehydrogenase (11β-HSD) type 1 activity, which contributes to increased UE of inactive glucocorticoids (Table 3). Functional hypercortisolism associated with activation of the hypothalamic-pituitary adrenal system leads to the synthesis of adrenal androgens, thereby further disrupting the process of folliculogenesis. In addition, a decrease in the activity of 11β-HSD1 increases cortisol metabolism, which leads to a compensatory increase in ACTH secretion and stimulation of adrenal steroidogenesis, which also confirms the mixed character of hyperandrogenemia in women with PCOS.

Signs of an increase in 5α-reductase activity of varying degrees were obtained in all examined patients with PCOS. Three signs of increased 5α-reductase activity were observed in patients with phenotypes A and B: increased ratios of 11-OH-An/11-OH-Et, 5α-THB/5β-THB, and 5α-THF/5β-THF. Two signs in patients with PCOS phenotype C: increased 11-OH-An /11-OH-Et, and 5α-THF/5β-THF ratios. One sign in patients with PCOS phenotype D: increased UE of 5α-THB and 5α-THB/5β-THB ratios (Table 3). Clinical signs of androgenic dermopathy were more pronounced in women with PCOS and its phenotypes A and B, which was manifested by more pronounced hirsutism and acne located on the face, back, and chest.

Increased UE of pregnantriol (P3) and pregnanetriol (dP3) were common signs of progestogen metabolomics abnormalities in patients with PCOS phenotypes A, B, and C. The UE of 17-OH-pregnanolone (17-OHP), 11-OH-P3, and 6-OH-pregnanolone (6-OHP) were additionally elevated in patients with PCOS phenotype C, and the UE of 17-OH-P in patients with PCOS phenotypes A and B. In patients with PCOS phenotype D, the UE of P3 alone was increased compared to the CG (Table 2). The ratios of (5β-THF+5α-THF+THE)/P3 were less than 3.0, (5β-THF+5α-THF+THE)/17-OHP were less than 12, and (5β-THF+5α-THF+THE)/11-oxo-P3 were less than 20 in combination with increased UE of P3; 11-oxo-P3 and 17-OHP may indicate a 21-hydroxylase deficiency in patients with PCOS and HA, ovulation, and polycystic ovaries (phenotype C) (Table 2). In patients with PCOS phenotype C, 21-deoxy-THF 108 (75-218) μg/24 h and 5-ene-pregnenes (21-OH-pregnenolone 40 (30-42) μg/24 h, 11-OH-pregnentriol 66 (37-104)μg/24 h), not detectable in healthy subjects, may also indicate 21-hydroxylase enzyme deficiency.

In addition, there were two signs of decreased 3β-hydroxysteroid dehydrogenase-2 (3β-HSD2) activity in patients with PCOS phenotypes A, B, and C: decreased (5β-THF+5α-THF+THE)/DHEA and (5β-THF+5α-THF+THE)/dP3 ratios, confirming mixed HA in this group of women (Table 3).

Discussion

HA is a syndrome caused by impaired androgen secretion and metabolism. In addition to its high prevalence in the population, HA is associated with metabolic disorders, type 2 diabetes, cardiovascular diseases, and reproductive dysfunction. Although HA syndrome includes diseases with different etiologies, its clinical manifestations are mostly the same: acne, hirsutism, menstrual disorders, and androgen-dependent alopecia. The problem of diagnostics and treatment of diseases associated with HA is currently one of the most urgent problems in gynecological endocrinology [25][26]. PCOS is the most common cause of HA syndrome. Its prevalence among women of reproductive age ranges from 8 to 21% [27]. In patients with PCOS and HA (phenotypes A and B), UE of androsterone (5α-metabolite of androstenedione) is increased, which leads to elevated levels of 5α-dihydrotestosterone (DHT) in blood, which has a higher biological activity than testosterone. Blood levels of DHT depend on circulating androgens and cellular 5α-reductase activity and are responsible for the development of androgenic dermopathy. Signs of increased 5α-reductase activity in varying degrees, according to GC-MS data, were obtained in all PCOS phenotypes in women with normal weight: three signs in patients with HA and anovulation (phenotypes A and B), two signs in patients with PCOS phenotype C, and one sign in women with polycystic ovaries without HA (phenotype D). Clinical signs of androgenic dermopathy were more pronounced in women with PCOS and phenotypes A and B. According to GC-MS data, patients with PCOS with phenotypes A, B, and C had increased UE of DHEA and P3 and decreased ratios of the sum of cortisol and cortisone tetrahydro derivatives to these steroids compared to the CG, which is a sign of 3β-NSD2 deficiency. The enzyme 3β-HSD2 is required to convert Δ5-steroids (pregnenolone, 17-hydroxypregnenolone, and dehydroepiandrosterone) into their corresponding Δ4-steroids (progesterone, 17-hydroxyprogesterone, and androstenedione) [28]. These results confirm impaired steroidogenesis of both ovarian and adrenal genesis in this group of women. To assess the intracellular concentration of glucocorticoids, it is necessary to determine not only their plasma levels but also the activity of enzymes that are involved in their metabolism, such as 11β-HSD activity. 11β-HSD1 is an enzyme that catalyzes the conversion of functionally inactive cortisone into the most active glucocorticoid hormone cortisol. According to GC-MS data, the signs of 11β-β-HSD1 insufficiency were found in patients with PCOS phenotype A, which coincides with the data of other researchers who showed the presence of reduced activity of 11β-HSD1 in obese women with the classic PCOS phenotype. Phenotype C in patients with PCOS (ovulatory) is the least studied and difficult to diagnose in contrast to the classic phenotype. The study revealed increased UE of 17-OH-progesterone metabolites: 11-oxo-pregnantriol, pregnantriol, and 17-hydroxypregnenolone, and decreased ratios of the sum of tetrahydro derivatives of cortisol and cortisone to these steroids. In addition, 21-deoxytetrahydrocortisol and non-classical 5-ene-pregnenes were detected in patients with phenotype C, indicating a 21-hydroxylase enzyme deficiency that requires further investigation. The presented data indicate excessive androgen production in many women with PCOS, both in the ovaries and in the adrenal cortex. GC-MS studies of USP allow the researchers to study the metabolomics of steroid hormones and determine the differences in their metabolism in different phenotypes of PCOS.

Conclusions

1. UE of androstenedione metabolites is increased in PCOS patients with HA and anovulation (phenotype A and B), and that of DHEA metabolites is increased in PCOS patients with HA (phenotype A, B and C).
2. Increased UE of 11-oxo-pregnantriol, pregnantriol, and 17-hydroxypregnenolone, reduced ratios of the sum of cortisol and cortisone tetrahydro derivatives to these steroids, detection of 21-deoxytetrahydrocortisol and nonclassic 5-ene-pregnenes in PCOS patients with phenotype C suggest a deficiency of 21-hydroxylase enzyme.
3. Type 1 11β-hydroxysteroid dehydrogenase deficiency was detected in patients with polycystic ovaries, HA, and anovulation (phenotype A), indicating the presence of functional hypercortisolism as a result of an excess of glucocorticoids with biologically low activity.
4. Patients with PCOS with HA (phenotypes A, B, and C) have increased UE of DHEA and pregnetriol, decreased ratios of tetrahydro derivatives of cortisol and cortisone to DHEA and pregnetriol compared to controls, indicating 3β-hydroxysteroid dehydrogenase-2 deficiency.
5. Signs of increased 5α-reductase activity by GC-MS were obtained in all PCOS phenotypes: three signs in patients with HA and anovulation, with polycystic ovaries, and without polycystic ovaries (phenotypes A and B), two signs in patients with phenotype C, and one sign in women with polycystic ovaries without HA (phenotype D).