What laboratory tests should be ordered for a reproductive‑age woman presenting with amenorrhea?

Laboratory Workup for Amenorrhea

All women presenting with amenorrhea require an initial panel of five mandatory tests: pregnancy test (urine or serum β-hCG), serum prolactin, thyroid-stimulating hormone (TSH), follicle-stimulating hormone (FSH), and luteinizing hormone (LH). 1, 2, 3, 4

Mandatory First-Line Laboratory Tests

Core Hormonal Panel

• Pregnancy test – Must be performed first to exclude pregnancy before proceeding with any other hormonal evaluation 1, 3, 4
• Serum prolactin – Draw as a single morning resting sample (avoid post-exercise, post-stress, or post-ictal collection); levels >20 μg/L suggest hyperprolactinemia and warrant evaluation for pituitary adenoma or medication effects 5, 1, 2
• TSH – Identifies thyroid dysfunction as a reversible cause; both hypothyroidism and hyperthyroidism can cause amenorrhea 5, 1, 2
• FSH and LH – Draw between cycle days 3-6 if cycles present, or at any time in amenorrheic patients; calculate LH/FSH ratio to differentiate etiologies 5, 1, 2

Interpretation of FSH/LH Results

• Elevated FSH (>40 mIU/mL) confirms primary ovarian insufficiency; requires repeat testing 4 weeks later for confirmation (two elevated values needed for diagnosis) 1, 2
• LH/FSH ratio >2 strongly suggests polycystic ovary syndrome (PCOS) 5, 1, 2
• Low FSH with low LH indicates hypothalamic or pituitary dysfunction (hypogonadotropic hypogonadism) 1, 2, 6
• LH/FSH ratio <1 is seen in approximately 82% of functional hypothalamic amenorrhea (FHA) cases 1

Additional Testing Based on Clinical Context

When Hyperandrogenism is Present (Hirsutism, Acne, Male-Pattern Hair)

• Total testosterone – Levels >2.5 nmol/L suggest PCOS or valproate use; draw on cycle days 3-6 5, 1, 2
• Androstenedione – Levels >10.0 nmol/L warrant evaluation for adrenal or ovarian tumor 5, 1
• DHEA-S (dehydroepiandrosterone sulfate) – Age-adjusted thresholds (age 20-29: >3800 ng/mL; age 30-39: >2700 ng/mL) screen for non-classical congenital adrenal hyperplasia 5, 1

When Functional Hypothalamic Amenorrhea is Suspected

• Estradiol – Low levels (<30 pg/mL) confirm hypoestrogenism characteristic of FHA 5, 1, 2
• Mid-luteal progesterone – Draw during expected mid-luteal phase; levels <6 nmol/L indicate anovulation (common in PCOS, hypothalamic amenorrhea, or hyperprolactinemia) 5, 1

When Metabolic Concerns Exist (Obesity, PCOS Features)

• Fasting glucose and insulin – Fasting glucose >7.8 mmol/L suggests diabetes; glucose/insulin ratio >4 suggests reduced insulin sensitivity associated with PCOS 5

For Young Women with Primary Ovarian Insufficiency

• Karyotype analysis – Recommended if age <40 years with elevated FSH to identify Turner syndrome or other chromosomal abnormalities 5, 1

Timing Considerations for Blood Draws

• FSH, LH, testosterone should be drawn between cycle days 3-6 (or at any time in amenorrheic patients) 5, 1
• Progesterone must be drawn during the mid-luteal phase according to the menstrual cycle to assess ovulation 5, 1
• Prolactin should be a single morning resting sample, never post-ictal, as seizures cause transient elevation 5, 2

Critical Clinical Context Required Before Ordering Labs

History Elements That Direct Testing

• Weight changes, eating patterns, exercise habits – Screen for Female Athlete Triad or disordered eating, which causes FHA 1, 2, 4
• Galactorrhea – Perform nipple expression during exam; presence mandates prolactin measurement and evaluation for pituitary pathology 5, 2
• Headaches or visual changes – Red flags for pituitary adenoma requiring urgent MRI if prolactin elevated 2
• Medication history – Antipsychotics, antiepileptics (especially valproate), and hormonal contraceptives can cause amenorrhea 5, 1

Physical Examination Findings That Guide Testing

• BMI calculation – Obesity (BMI >25) associated with PCOS; low BMI (<18.5) favors FHA 5, 2
• Waist-to-hip ratio – Ratio >0.9 suggests truncal obesity and PCOS 5, 2
• Ferriman-Gallwey score or inspection for hirsutism – Male escutcheon pattern warrants androgen testing 5, 2
• Tanner staging – Essential in primary amenorrhea to determine pubertal development status 1, 7

Common Pitfalls to Avoid

• Do not skip the pregnancy test – Failing to exclude pregnancy first leads to misinterpretation of all other hormonal results 1, 3, 4
• Do not draw prolactin post-ictally – Seizures cause transient prolactin elevation; wait at least 24 hours after any seizure activity 5, 2
• Do not assume chemotherapy-induced amenorrhea equals menopause – Hormone levels are unreliable during tamoxifen treatment; premenopausal estradiol levels can occur with transient chemotherapy-induced amenorrhea 1
• Do not overlook eating disorders – Adolescents frequently minimize or deny disordered eating behaviors; direct questioning about specific behaviors is necessary 1, 2
• Do not assume amenorrhea in athletes is benign – Other pathology must be excluded even when clinical picture suggests FHA 1, 2

When to Order Imaging Studies

• Pelvic ultrasound is indicated when LH/FSH ratio >2, when clinical features suggest PCOS, or to assess endometrial thickness (thin <5 mm suggests estrogen deficiency; thick >8 mm suggests chronic anovulation with unopposed estrogen) 1, 2
• Pituitary MRI is indicated when prolactin remains elevated after excluding hypothyroidism, or when headaches/visual field deficits are present 2
• DXA scan for bone mineral density should be obtained if amenorrhea extends beyond 6 months, regardless of age, due to increased fracture risk from hypoestrogenism 1, 2