Advancing PCOS diagnostics with mass spectrometry

• Polyendocrine Metabolic Ovarian Syndrome (PMOS), previously known as Polycystic Ovary Syndrome (PCOS), is a complex endocrine disorder that can impact metabolic health
• Guided by the Rotterdam Criteria, diagnosing PMOS is complex and often requires blood tests to measure androgen levels
• The use of mass spectrometry in diagnosing PMOS offers superior analytical specificity, allowing for more accurate quantification of steroid hormone levels

Polyendocrine Metabolic Ovarian Syndrome (PMOS), previously known as Polycystic Ovary Syndrome (PCOS), is the most prevalent endocrine and metabolic disorder among women of reproductive age. The new name intends to better reflect the condition by moving the focus away from ovarian cysts, as it is now known that there is no increase in the number of cysts in women with the condition. PMOS more accurately describes this condition as a complex endocrine disorder that can impact metabolic health.¹ In fact, metabolic issues, like insulin resistance and obesity, are present in up to 80% of women with the condition.²  

Although there is no cure, diagnosing PMOS early is key to timely treatment to help improve quality of life and reduce the risk of reproductive or metabolic complications such as diabetes, hypertension, and cardiovascular disease.³

Due to the complexity of the condition and the similarity of symptoms to other clinical conditions, PMOS has historically been difficult to diagnose. Misdiagnosis is common, and women may live with symptoms for years before they are properly diagnosed.⁴ 

The diagnosis of PMOS relies on meeting at least two out of three factors from the Rotterdam Criteria:^5,6 

1. Signs of oligoanovulation: Often manifesting as infrequent or no menstruation, and self-reported by women.
2. Polycystic ovary morphology (PCOM): Determined through transvaginal ultrasound to identify numbers of follicles per ovary. A diagnostic advance came in the updated 2023 Rotterdam guidelines, when a blood test to determine anti-Müllerian Hormone (AMH) levels was introduced as an alternative method to determine PCOM.
3. Clinical and/or biochemical signs of hyperandrogenism: Such as hirsutism, acne, or elevated levels of total or free testosterone.

Symptoms manifest in a variety of ways across individuals meaning diagnostic approaches will vary.⁷

The presence of both hyperandrogenism and oligoanovulation defines the classic phenotype of PMOS, which is associated with a more adverse metabolic profile. Since PMOS patients with androgen excess are at high risk for metabolic consequences like liver diseases and insulin resistance, it is important to identify those patients who have abnormal androgen levels.⁵  

Although hirsutism is considered to be the most common manifestation of hyperandrogenism in women with PMOS, it varies among different ethnic groups and is evaluated using visual scoring methods that are observer dependent such as the Ferriman-Gallwey scoring system.⁸ Therefore, blood tests to identify higher than normal levels of androgen steroids in women without obvious hirsutism are an important part of diagnosis.³

Several androgens may be evaluated as part of the diagnostic process:⁹

• Total testosterone (TT) or free testosterone (FT): In women, testosterone is primarily produced in the ovaries, which are the principal source of hyperandrogenism in patients with PMOS. Almost 90% of patients with the condition have been found to have elevated FT levels, while up to 80% of patients have been found to have elevated TT levels. Therefore, testosterone, particularly in the free form, provides a sensitive biomarker for diagnosis. According to the Rotterdam criteria, biochemical hyperandrogenism should be defined first by measurement of elevated total or free testosterone. Alternatively, calculated estimations of free testosterone have been proposed, including free androgen index (the quotient testosterone/SHBG × 100] and calculated bioavailable testosterone (the concentration of testosterone that is free and weakly bound to albumin). These methods have been found to be relatively reliable markers in the diagnosis. 
  If total or free testosterone are not elevated, androstenedione and dehydroepiandrosterone sulfate (DHEAS) can be considered. 
• Androstenedione: Produced by both the ovaries and the adrenal gland, androstenedione has been found to be elevated in nearly 90% of patients with PMOS and may increase numbers identified as having high androgen levels by 10%
• DHEAS: Produced exclusively by the adrenal gland, DHEAS is elevated in up to 35% of patients with PMOS, and may be the only abnormality in circulating androgens in around 10% of patients.

Although several markers are available for testing, accurate measurement of androgens can be challenging as they are typically found at low levels in women and children. Accurate diagnosis of adolescents is particularly important as early diagnosis enables appropriate treatment and lifestyle adjustments to protect against individual metabolic risks such as type 2 diabetes, cardiovascular disease, sleep disorders, premenopausal endometrial cancer, and adverse outcomes during pregnancy.¹⁰    

Routine methods to measure steroid hormones are based on immunoassays (IAs) as they are quick and easy to use. However, they are limited regarding sensitivity and precision required for PMOS diagnosis. For example, although IAs are accurate for testosterone measurement in men, the reliability of these methods within the female reference range has been questioned as they lack specificity and are subject to matrix effects.^5,11  

The use of mass spectrometry technology, an analytical technique that measures the mass-to-charge ratio (m/z) of molecules, offers an additional approach. When combined with liquid chromatography, tandem mass spectrometry (LC-MS/MS) offers superior analytical specificity for low molecular weight analytes and is growing in popularity as the method of choice for steroid hormone analysis.⁵  

Studies have shown that in adult women, LC–MS/MS demonstrated a higher diagnostic value for testosterone compared with immunoassay for diagnosing PMOS, and the diagnostic accuracy of total testosterone detected by LC–MS/MS was higher. Furthermore, immunoassays demonstrated poor agreement with LC–MS/MS, with 20% of women who demonstrated normal values when using LC–MS/MS methods demonstrating hyperandrogenism with immunoassays, which may have resulted in an overdiagnosis of hyperandrogenism.¹⁰

The high sensitivity and specificity offered by LC-MS/MS allows for more accurate quantification of PMOS-related androgens such as total testosterone, free testosterone, androstenedione, and DHEAS. Since immunoassays demonstrate poor sensitivity and precision for diagnosing hyperandrogenism in PMOS, LC–MS/MS methods are therefore recommended by the 2023 International PCOS Guidelines over immunoassays for the measurement of such steroid hormones. The guidelines specifically advise laboratories to utilize validated LC-MS/MS assays for measuring total testosterone, and further recommend that, when necessary, assays for androstenedione and DHEAS should also be conducted using LC-MS/MS.⁵

Ultimately, the use of mass spectrometry helps advance PMOS diagnostics by reducing diagnostic variability, improving the reliability of diagnosis, and supporting more precise clinical management.¹²