What laboratory tests should be ordered to evaluate a reproductive‑age woman suspected of polycystic ovary syndrome?

Laboratory Tests for Suspected PCOS

Order total testosterone and free testosterone by liquid chromatography-tandem mass spectrometry (LC-MS/MS), TSH, prolactin, a 2-hour oral glucose tolerance test with 75g glucose, and a fasting lipid panel as your core diagnostic workup. 1

First-Line Hormonal Tests

Androgen Assessment

• Measure total testosterone using LC-MS/MS as the single best initial biochemical marker, demonstrating 74% sensitivity and 86% specificity for PCOS diagnosis. 1
• Measure free testosterone or calculate free testosterone using the Vermeulen equation from high-quality total testosterone and SHBG measurements; free testosterone shows superior sensitivity of 89% with 83% specificity. 1
• LC-MS/MS is mandatory over direct immunoassays because it provides superior specificity (92% versus 78%), with immunoassays producing high false-positive rates. 1

Exclusion of Other Endocrine Disorders

• Measure TSH to rule out thyroid disease as a cause of menstrual irregularity. 1, 2
• Measure morning resting serum prolactin; levels >20 µg/L are abnormal and women with PCOS have a 3.15-fold increased risk of hyperprolactinemia compared to women without PCOS. 1, 2
• If prolactin is elevated, confirm with 2-3 samples at 20-60 minute intervals via indwelling cannula to exclude stress-related spurious elevation. 1

Second-Line Androgen Tests (When Primary Tests Are Normal but Clinical Suspicion Remains High)

• Measure androstenedione if total and free testosterone are normal; it shows 75% sensitivity and 71% specificity, with values >10 nmol/L raising suspicion for an adrenal or ovarian androgen-secreting tumor. 1
• Measure DHEAS to evaluate for non-classical congenital adrenal hyperplasia; it demonstrates 75% sensitivity and 67% specificity, with age-adjusted thresholds (≥3800 ng/mL for ages 20-29, ≥2700 ng/mL for ages 30-39) prompting further evaluation. 1

Ovulatory Function Assessment

• Measure LH and FSH on cycle days 3-6 (if cycles occur), averaging three samples taken 20 minutes apart; an LH/FSH ratio >2 supports PCOS but is present in only 35-44% of affected women, making it a poor standalone diagnostic marker. 1
• Measure mid-luteal phase progesterone (day 21 of a 28-day cycle or 7 days before expected menses); levels <6 nmol/L confirm anovulation. 1

Mandatory Metabolic Screening

• Perform a 2-hour oral glucose tolerance test with 75g glucose load to screen for glucose intolerance and type 2 diabetes, as women with PCOS are four times more likely to develop type 2 diabetes than the general population. 1, 3
• Obtain a fasting lipid panel including total cholesterol, LDL, HDL, and triglycerides; metabolic syndrome is twice as common in PCOS patients compared to the general population. 1, 4
• Calculate BMI and waist-hip ratio; a waist-hip ratio >0.9 indicates truncal obesity and heightened metabolic risk. 1, 2
• Consider measuring fasting glucose and insulin to calculate the glucose/insulin ratio; a ratio >4 suggests reduced insulin sensitivity. 1

Anti-Müllerian Hormone (AMH) Guidance

• Do NOT use AMH as a stand-alone test for polycystic ovarian morphology or PCOS diagnosis due to lack of assay standardization, absent validated cut-offs, and considerable overlap between affected and unaffected women. 1, 2
• Although AMH ≥35 pmol/L shows 92% sensitivity and 97% specificity in research cohorts, this threshold requires further validation across diverse populations before clinical adoption. 1

Critical Diagnostic Considerations

When Ultrasound Is NOT Necessary

• If the patient has both irregular menstrual cycles AND clinical/biochemical hyperandrogenism, ultrasound is not necessary for PCOS diagnosis, though it will identify the complete PCOS phenotype. 1

When to Suspect Alternative Diagnoses

• Rapid onset of symptoms, severe hirsutism, or very high testosterone levels warrant immediate evaluation for androgen-secreting tumors. 1, 2
• Screen for Cushing's syndrome if the patient has buffalo hump, moon facies, hypertension, abdominal striae, central fat distribution, easy bruising, or proximal myopathies; untreated Cushing's carries morbidity and mortality risks far exceeding those of PCOS. 1, 2
• Measure FSH to rule out primary ovarian failure, particularly in women in their third decade presenting with amenorrhea; FSH >50 mIU/mL signifies primary gonadal failure. 1

Special Populations

• Do NOT use ultrasound for PCOS diagnosis in adolescents with gynecological age <8 years (<8 years after menarche) due to high false-positive rates from normal multifollicular ovaries. 1
• In adolescents, require persistent oligomenorrhea 2-3 years beyond menarche plus biochemical hyperandrogenism before diagnosing PCOS. 5

Common Pitfalls to Avoid

• Do NOT measure insulin and C-peptide routinely; physical signs of insulin resistance (elevated BMI, acanthosis nigricans) are sufficient for assessment. 1
• Remember that total testosterone is abnormal in only 70% of women with confirmed PCOS, meaning 30% have normal testosterone levels despite having the condition. 1
• Isolated polycystic ovarian morphology on ultrasound is found in 17-22% of asymptomatic women and should not alone establish a PCOS diagnosis. 1
• If the patient is using hormonal contraception (including Implanon), remove it or allow it to expire before testing, as progestin suppresses the hypothalamic-pituitary-ovarian axis and makes hormone levels unreliable. 2

Long-Term Monitoring

• Repeat lipid panel, blood pressure, and weight assessments every 6-12 months to monitor cardiovascular risk factors. 1, 2
• Recognize the increased lifelong risk of type 2 diabetes, dyslipidemia, and cardiovascular disease in women with PCOS. 1, 4