Laboratory Workup for Amenorrhea
The initial laboratory evaluation for amenorrhea must include a pregnancy test, followed by serum FSH, LH, prolactin, and TSH—these five tests form the mandatory baseline panel that directs all subsequent diagnostic pathways. 1
Mandatory First-Line Laboratory Tests
Core Hormonal Panel (Order for All Patients)
Pregnancy test (urine or serum β-hCG) is the absolute first step and must be performed before any other hormonal testing, as pregnancy is the most common cause of amenorrhea in reproductive-age women. 1, 2, 3
Follicle-stimulating hormone (FSH) should be drawn between cycle days 3–6 (or at any time in amenorrheic patients) to differentiate primary ovarian insufficiency (FSH >40 mIU/mL) from hypothalamic/pituitary causes (low FSH). 1, 4
Luteinizing hormone (LH) should be drawn simultaneously with FSH; the LH/FSH ratio provides critical diagnostic information—a ratio >2 strongly suggests PCOS, while a ratio <1 is seen in approximately 82% of functional hypothalamic amenorrhea cases. 1, 5
Prolactin must be measured using a single morning resting sample (avoid post-exercise, post-stress, or post-ictal collection); levels >20 μg/L indicate hyperprolactinemia and warrant further evaluation for pituitary adenoma or medication effects. 1, 3
Thyroid-stimulating hormone (TSH) identifies thyroid dysfunction as a reversible cause of amenorrhea; both hypothyroidism and hyperthyroidism can disrupt menstrual cycles. 1, 5
Additional Hormonal Testing Based on Clinical Context
When Hyperandrogenism Is Present (Hirsutism, Acne, Virilization)
Total testosterone should be drawn on cycle days 3–6; levels >2.5 nmol/L suggest PCOS or valproate effect, while levels >5 nmol/L warrant urgent evaluation for ovarian or adrenal tumor. 1, 5
Androstenedione >10.0 nmol/L requires investigation for adrenal or ovarian neoplasms. 1
DHEA-S using age-adjusted thresholds (age 20–29: >3800 ng/mL; age 30–39: >2700 ng/mL) screens for non-classical congenital adrenal hyperplasia. 1
When Functional Hypothalamic Amenorrhea Is Suspected
Estradiol <30 pg/mL confirms hypoestrogenism characteristic of functional hypothalamic amenorrhea and distinguishes it from PCOS (which typically shows normal or elevated estradiol). 1, 6
Mid-luteal progesterone (measured during expected mid-luteal phase) <6 nmol/L indicates anovulation, commonly seen in PCOS, hypothalamic amenorrhea, or hyperprolactinemia. 1
When Primary Ovarian Insufficiency Is Suspected
Repeat FSH measurement 4 weeks after initial elevated value (>40 mIU/mL) is required for definitive diagnosis—two elevated values are mandatory. 1, 6
Karyotype analysis is recommended for women <40 years with elevated FSH to detect Turner syndrome or other chromosomal abnormalities. 1, 2
Metabolic Testing (When PCOS Is Suspected)
Fasting glucose >7.8 mmol/L suggests diabetes; a glucose/insulin ratio >4 points to reduced insulin sensitivity associated with PCOS. 1
Lipid panel should be obtained in patients with PCOS due to increased risk of dyslipidemia and metabolic syndrome. 2
Critical Timing Considerations
FSH, LH, and testosterone must be drawn between cycle days 3–6 of the menstrual cycle; in amenorrheic patients, these can be drawn at any time. 1
Progesterone must be obtained during the expected mid-luteal phase (approximately day 21 of a 28-day cycle) to accurately assess ovulation. 1
Prolactin requires a single morning resting draw and must never be performed post-ictally, as seizures cause transient elevation; wait at least 24 hours after any seizure. 1
Common Pitfalls to Avoid
Do not draw prolactin immediately after stress, breast examination, or sexual activity, as these cause transient elevations; obtain a morning resting sample instead. 1
Do not measure hormones while the patient is on hormonal contraception, as this affects accuracy and interpretation of results. 5
Do not rely on a single FSH measurement, as levels fluctuate significantly; timing during the menstrual cycle is crucial, and two elevated values are required to diagnose primary ovarian insufficiency. 1, 5
Do not assume amenorrhea in athletes or stressed adolescents is benign—other pathology must be excluded even when the clinical picture suggests functional hypothalamic amenorrhea. 6
Do not order LH and FSH in early post-menarchal adolescents (gynecologic age <8 years) with irregular bleeding, as results are difficult to interpret and often lead to unnecessary investigations. 6
Diagnostic Algorithm by Hormone Pattern
Elevated FSH (>40 mIU/mL)
- Confirms primary ovarian insufficiency; repeat FSH in 4 weeks (two elevated values required). 1, 6
- Order karyotype if age <40 years to identify Turner syndrome. 1
- Check for fragile-X premutation and autoimmune antibodies (21-hydroxylase, adrenal cortex). 6
Low FSH with Low Estradiol
- Indicates hypothalamic amenorrhea or pituitary failure. 6
- Assess for weight loss, excessive exercise, eating disorders, or psychological stress. 6, 4
- Consider DXA scan for bone mineral density if amenorrhea >6 months. 6
Normal FSH with LH/FSH Ratio >2
- Strongly suggests PCOS. 1, 5
- Order pelvic ultrasound to assess for polycystic ovarian morphology. 1
- Measure testosterone if clinical hyperandrogenism is present. 1
- Screen for metabolic syndrome with fasting glucose and lipid panel. 2
Elevated Prolactin (>20 μg/L)
- First exclude hypothyroidism by checking TSH. 1
- If prolactin remains elevated and TSH is normal, obtain pituitary MRI to evaluate for adenoma. 1
- Refer for endocrine or neurosurgical assessment if prolactin >100 μg/L or if accompanied by headaches or visual disturbances. 1
When to Refer to Specialist
Prolactin >100 μg/L or any elevation with headaches or visual disturbances suggests pituitary adenoma. 1
Testosterone >5 nmol/L or rapid virilization indicates potential ovarian or adrenal tumor. 1
Persistently abnormal hormone levels despite treatment or signs of pituitary pathology. 6
Primary amenorrhea with no signs of puberty by age 13 years and elevated FSH, or no menarche by age 16 despite secondary sexual characteristics. 6