Diagnostic Tests for Amenorrhea
Begin with a urine or serum pregnancy test in all women of reproductive age presenting with amenorrhea, followed immediately by measurement of FSH, LH, prolactin, and TSH—this core hormonal panel identifies the vast majority of underlying causes and directs all subsequent evaluation. 1, 2
Initial Mandatory Laboratory Tests
Every patient requires this baseline panel:
Pregnancy test (urine β-hCG or serum β-hCG) – Must be performed first because pregnancy is the most common cause of amenorrhea, and failing to exclude it leads to misinterpretation of all subsequent hormonal results 1, 2, 3, 4
Serum FSH and LH – Drawn between cycle days 3-6 if cycles present, or any time in amenorrheic patients; these distinguish primary ovarian insufficiency (FSH >40 mIU/mL) from hypothalamic/pituitary dysfunction (low-normal FSH) and help identify PCOS (LH/FSH ratio >2) 1, 5, 2, 3
Serum prolactin – Obtain as a single morning resting sample; never draw immediately after stress, breast examination, sexual activity, or seizure (wait ≥24 hours post-ictally); levels >20 μg/L indicate hyperprolactinemia and warrant pituitary imaging 1, 5, 2, 4
Serum TSH – Identifies thyroid dysfunction as a reversible cause; both hypothyroidism and hyperthyroidism disrupt menstrual cycles 1, 5, 2, 3
Additional Hormonal Testing Based on Clinical Context
Add these tests when specific clinical features are present:
Serum estradiol – Measure when functional hypothalamic amenorrhea (FHA) is suspected; levels <30 pg/mL confirm hypoestrogenism and help differentiate FHA (low estradiol) from PCOS (normal/elevated estradiol) 1, 5, 2
Total testosterone – Indicated when hirsutism, acne, or androgenetic alopecia are present; levels >2.5 nmol/L suggest PCOS or valproate effect, while levels >5 nmol/L warrant urgent referral for possible androgen-secreting tumor 1, 5
Androstenedione and DHEA-S – Order when hyperandrogenic signs are present to screen for adrenal or ovarian tumors and non-classical congenital adrenal hyperplasia; androstenedione >10.0 nmol/L or age-adjusted DHEA-S thresholds (age 20-29 >3800 ng/mL; age 30-39 >2700 ng/mL) require further investigation 1, 5
Mid-luteal progesterone – Drawn during expected mid-luteal phase to assess ovulation; levels <6 nmol/L indicate anovulation seen in PCOS, hypothalamic amenorrhea, or hyperprolactinemia 1, 5
Imaging Studies
Pelvic ultrasound is the primary imaging modality:
Transvaginal pelvic ultrasound (or transabdominal if virginal) – Indicated when LH/FSH ratio >2, any palpable adnexal mass, or concern for structural abnormality; assesses endometrial thickness (<5 mm suggests estrogen deficiency; >8 mm suggests chronic anovulation with unopposed estrogen), evaluates for polycystic ovarian morphology, and identifies Müllerian abnormalities in primary amenorrhea 1, 5, 2
Pelvic MRI – Reserved for cases where ultrasound findings are equivocal or when detailed anatomic assessment of congenital anomalies is needed in primary amenorrhea 1
Pituitary MRI with gadolinium – Mandatory when prolactin remains elevated after excluding hypothyroidism, when prolactin >100 μg/L, or when headaches or visual field deficits are present; evaluates for pituitary adenoma 1, 5
Specialized Testing for Specific Scenarios
Order these tests only when indicated by initial results:
Karyotype analysis – Required for women <40 years with elevated FSH (>40 mIU/mL on two occasions ≥4 weeks apart) to detect Turner syndrome, Turner variants, or other chromosomal abnormalities causing primary ovarian insufficiency 1, 5, 3
Fragile X premutation testing – Screen women with confirmed primary ovarian insufficiency, as this is a common genetic association 1
Autoimmune antibody panel (21-hydroxylase, adrenal cortex antibodies) – Screen women with primary ovarian insufficiency for associated autoimmune conditions 1
Fasting glucose and insulin – Indicated when PCOS is suspected (BMI >25 kg/m², waist-to-hip ratio >0.9, or LH/FSH ratio >2); glucose >7.8 mmol/L suggests diabetes, and glucose/insulin ratio >4 indicates reduced insulin sensitivity 1, 5
DXA scan for bone mineral density – Mandatory if amenorrhea extends beyond 6 months regardless of age, as prolonged hypoestrogenism significantly increases osteoporosis risk; 90% of peak bone mass is attained by age 18, making early assessment critical in adolescents 1, 2
Algorithmic Interpretation of FSH Results
FSH level directs the diagnostic pathway:
Elevated FSH (>40 mIU/mL) – Confirms primary ovarian insufficiency; repeat FSH in 4 weeks (two elevated values required for diagnosis), then proceed with karyotype, fragile X testing, and autoimmune screening 1, 5, 6
Low or low-normal FSH with low estradiol – Indicates hypothalamic or pituitary dysfunction; assess for eating disorders, excessive exercise, weight loss >5% in past 6 months, BMI <18.5 kg/m², psychological stress, and calculate energy availability (target >30 kcal/kg fat-free mass/day) 1, 2
Normal FSH with LH/FSH ratio >2 – Strongly suggests PCOS; proceed with pelvic ultrasound, androgen profile, and metabolic screening (fasting glucose, lipids) 1, 5
Critical Diagnostic Pitfalls to Avoid
Never assume amenorrhea in athletes or stressed adolescents is benign – Other pathology must be excluded even when clinical picture suggests functional hypothalamic amenorrhea 1
Do not use ultrasound to diagnose PCOS in girls <8 years post-menarche – Multi-follicular ovaries are common in this developmental stage (17-22% of normal adolescents), making sonographic findings unreliable 1
Do not rely on LH/FSH ratio alone for PCOS diagnosis – The ratio >2 has poor sensitivity (only 35-44% of PCOS patients), so prioritize clinical hyperandrogenism and testosterone measurement 1
Never draw prolactin post-ictally – Seizures cause transient elevation; wait ≥24 hours to avoid false-positive results 1
Do not delay hormonal assessment – Time is critical for fertility preservation options if primary ovarian insufficiency is confirmed 2
Distinguishing FHA-PCOM from True PCOS
When ultrasound shows polycystic ovarian morphology but clinical picture is unclear, use this comparison:
| Feature | FHA-PCOM | PCOS |
|---|---|---|
| BMI | Low or normal | Usually >25 kg/m² |
| Energy availability | Low (<30 kcal/kg FFM/day) | Normal or excess |
| Stress/exercise history | Present | Often absent |
| LH/FSH ratio | <1 (82% of cases) | >2 |
| Estradiol | Low (<30 pg/mL) | Normal or elevated |
| Endometrial thickness | Thin (≤5 mm) | Usually normal |
| Testosterone | Lower | Higher |
Tests NOT Recommended in Initial Evaluation
Avoid these unnecessary tests in healthy adolescents and young women: