What initial laboratory tests should be ordered for a reproductive‑age female with secondary amenorrhea of ≥6 months and a negative pregnancy test?

Initial Laboratory Workup for Secondary Amenorrhea

Order a pregnancy test (urine or serum β-hCG), serum FSH, LH, prolactin, and TSH as your initial laboratory panel for any reproductive-age woman with secondary amenorrhea of ≥6 months and a negative pregnancy test. 1, 2, 3

Essential First-Line Laboratory Tests

Mandatory Initial Panel

• Pregnancy test (urine β-hCG) – Must be performed first to exclude pregnancy, even with a reported negative test, as pregnancy remains the most common cause of amenorrhea in reproductive-age women 1, 2, 4

• Serum follicle-stimulating hormone (FSH) – Distinguishes between ovarian failure (elevated FSH >25-40 IU/L indicating primary ovarian insufficiency) versus hypothalamic/pituitary dysfunction (low or normal FSH) 1, 2, 3

• Serum luteinizing hormone (LH) – An LH:FSH ratio >2:1 strongly suggests polycystic ovary syndrome (PCOS), the most common cause of secondary amenorrhea in this population 1, 5

• Serum prolactin – Hyperprolactinemia accounts for approximately 20% of secondary amenorrhea cases; draw as a single morning resting sample to avoid false elevations from stress or exercise 1, 2, 3

• Serum thyroid-stimulating hormone (TSH) – Thyroid dysfunction is a reversible cause of amenorrhea and must be excluded in all cases 1, 2, 3

Additional Testing Based on Clinical Context

If Clinical Hyperandrogenism Present (Hirsutism, Acne, Male-Pattern Hair Loss)

• Total testosterone – Levels >2.5 nmol/L warrant evaluation for androgen-secreting tumor; modest elevations (1.5-2.5 nmol/L) suggest PCOS 1, 5

• DHEA-sulfate (DHEAS) – Helps exclude non-classical congenital adrenal hyperplasia if markedly elevated (>10.0 nmol/L) 1, 5

If Functional Hypothalamic Amenorrhea (FHA) Suspected

• Estradiol level – Low estradiol (<30 pg/mL or <110 pmol/L) confirms hypoestrogenism and supports FHA diagnosis 1

• Calculate energy availability – Document if patient consumes >30 kcal/kg fat-free mass/day; inadequate energy availability is the hallmark of FHA 1

If Amenorrhea Persists >6 Months

• DXA scan for bone mineral density – Prolonged hypoestrogenic states significantly increase osteoporosis risk; 90% of peak bone mass is attained by age 18, making early assessment critical 1

Imaging Studies (Not Initial Bloodwork, But Often Concurrent)

• Pelvic ultrasound (transvaginal preferred if sexually active) – Indicated when LH:FSH ratio >2, to assess for polycystic ovarian morphology, or to evaluate uterine/ovarian anatomy 1, 2

• Pituitary MRI – Order only if prolactin remains elevated after excluding hypothyroidism, or if patient has headaches, visual changes, or galactorrhea suggesting pituitary pathology 1

Critical Red Flags Requiring Urgent Additional Workup

• Severe hirsutism or virilization – Measure serum testosterone immediately; levels >2.5 nmol/L require evaluation for androgen-secreting tumor 1

• Galactorrhea – Perform nipple expression during physical exam; if present with elevated prolactin, obtain pituitary MRI to exclude prolactinoma 1

• Headaches or visual field defects – Obtain pituitary MRI urgently to rule out pituitary adenoma or other sellar mass 1

• Weight loss >5% body weight in 6 months or BMI <18.5 kg/m² – Screen for eating disorders using validated tools; these patients require multidisciplinary eating disorder team referral 1, 5

Common Pitfalls to Avoid

• Do not draw FSH/LH during oral contraceptive use – Stop hormonal contraceptives for at least 2 months before hormonal assessment, as they suppress normal FSH and LH patterns and render results uninterpretable 5

• Do not draw prolactin post-exercise, post-stress, or post-seizure – These conditions cause transient elevations; ensure the sample is drawn as a morning resting value 1

• Do not assume amenorrhea in athletes is benign – Even when clinical picture suggests FHA, other pathology (prolactinoma, PCOS, primary ovarian insufficiency) must be excluded with laboratory testing 1

• Do not prescribe oral contraceptives before completing workup – OCPs mask the underlying problem without addressing the cause and make subsequent hormonal assessment impossible 1

Interpretation Algorithm Based on Initial Results

If FSH Elevated (>25-40 IU/L)

• Diagnosis: Primary ovarian insufficiency – Confirm with repeat FSH in 4-6 weeks; these patients maintain unpredictable ovarian function and should not be presumed infertile 1, 2

If Prolactin Elevated

• First exclude hypothyroidism by checking TSH; if TSH normal and prolactin persistently elevated, obtain pituitary MRI 1

If LH:FSH Ratio >2:1

• Diagnosis: Polycystic ovary syndrome – Proceed with pelvic ultrasound and consider fasting glucose/insulin to assess metabolic syndrome risk 1, 5

If FSH/LH Both Low or Normal with Low Estradiol

• Diagnosis: Functional hypothalamic amenorrhea – Assess for eating disorders, excessive exercise (>10 hours/week intense training), or significant psychosocial stressors 1

If All Hormones Normal

• Perform progestin challenge test (medroxyprogesterone 10 mg daily for 10 days) – Withdrawal bleeding confirms adequate estrogen and patent outflow tract; absence of bleeding suggests either estrogen deficiency or anatomic obstruction 3, 6