What laboratory tests should be ordered for a patient presenting with amenorrhea (absence of menstruation)?

Laboratory Workup for Amenorrhea

All patients presenting with amenorrhea require an initial panel of five mandatory tests: pregnancy test (urine or serum β-hCG), FSH, LH, prolactin, and TSH. 1, 2, 3 This core panel identifies the most common and treatable causes while guiding further diagnostic steps.

First-Line Mandatory Laboratory Tests

Essential Initial Panel

• Pregnancy test (urine β-hCG or serum) must be performed first in all reproductive-age women, as pregnancy is the most common cause of amenorrhea and failure to exclude it leads to misinterpretation of all subsequent hormonal results 1, 3, 4, 5
• FSH and LH levels differentiate between hypothalamic/pituitary dysfunction (low/normal FSH) versus primary ovarian insufficiency (FSH >40 mIU/mL) 1, 2, 3, 4
• Prolactin level identifies hyperprolactinemia (>20 μg/L), which may indicate pituitary adenoma or medication effect 1, 2, 3, 4
• TSH level detects thyroid dysfunction as a reversible cause of amenorrhea 1, 2, 3, 4

Interpreting the LH/FSH Ratio

• LH/FSH ratio >2 strongly suggests PCOS, while a ratio <1 is seen in approximately 82% of functional hypothalamic amenorrhea (FHA) cases 6, 1, 2
• Draw FSH and LH between cycle days 3-6 if any cycles are present, or at any time in amenorrheic patients 1

Second-Line Hormonal Testing (Based on Clinical Context)

When to Add Estradiol

• Measure estradiol when FHA is suspected (history of weight loss, excessive exercise, stress, low BMI), as low levels indicate hypoestrogenism requiring bone health monitoring 1, 3
• Estradiol assessment helps differentiate FHA (low estradiol) from PCOS (normal/elevated estradiol) 6, 1

When to Add Androgens

• Measure total testosterone and androstenedione when clinical hyperandrogenism is present (hirsutism, acne, male-pattern hair loss) 1, 2
• Testosterone >2.5 nmol/L suggests PCOS or medication effect (particularly valproate) 1

When to Add Progesterone

• Mid-luteal phase progesterone (day 21 of 28-day cycle) determines ovulatory status: levels <6 nmol/L indicate anovulation 1
• However, progesterone testing has limited utility in amenorrheic patients who are not cycling 1

Imaging Studies

Pelvic Ultrasound Indications

• Perform transvaginal ultrasound when hormonal tests or clinical features suggest ovarian pathology, ideally between cycle days 3-9 1, 2
• Assess endometrial thickness: thin endometrium (<5 mm) indicates estrogen deficiency (FHA), while thick endometrium (>8 mm) suggests chronic anovulation with unopposed estrogen (PCOS) 6, 1, 3
• Transvaginal approach is more sensitive than transabdominal for identifying structural abnormalities 1

Special Population Considerations

Female Athletes

• Document weight changes, eating patterns, exercise habits, and calculate energy availability (energy intake minus exercise energy expenditure) to screen for Female Athlete Triad or RED-S 1, 2, 3
• Order DXA scan for bone mineral density if amenorrhea ≥6 months, BMI <18.5 kg/m², history of disordered eating/excessive exercise, or menarche ≥16 years 3

Primary Ovarian Insufficiency Confirmation

• If FSH >40 mIU/mL, repeat FSH measurement 4 weeks later—two elevated values are required for diagnosis 1, 3
• Order karyotype testing if age <40 years to identify Turner syndrome or other chromosomal abnormalities 1
• Note that patients with primary ovarian insufficiency can maintain unpredictable ovarian function and should not be presumed infertile 1, 4

Cancer Survivors

• Measure FSH and estradiol in cancer survivors with menstrual dysfunction, but discontinue hormone replacement therapy prior to laboratory evaluation 1, 2
• Anti-Müllerian hormone (AMH) and inhibin may provide additional information on ovarian reserve 1

Critical Diagnostic Pitfalls to Avoid

Common Errors

• Failing to rule out pregnancy first is the most frequent mistake—always perform pregnancy testing before interpreting other hormonal results 1, 3
• Misdiagnosing FHA with polycystic ovarian morphology (FHA-PCOM) as PCOS: FHA patients typically have low LH, low estradiol, thin endometrium (<5 mm), and LH/FSH ratio <1, despite ovarian appearance on ultrasound 6, 1, 3
• Neglecting bone health assessment in patients with prolonged amenorrhea and hypoestrogenism leads to missed osteoporosis risk 1, 3
• Assuming chemotherapy-induced amenorrhea equals menopause: hormone levels and absence of menses are unreliable indicators during tamoxifen treatment, and premenopausal estradiol levels can occur with transient chemotherapy-induced amenorrhea 6

Timing Considerations

• For patients age ≤60 years with prior hysterectomy and intact ovaries, measure LH, FSH, and estradiol prior to initiating any systemic therapy to accurately assess menopausal status 6
• Draw progesterone only during mid-luteal phase (approximately day 21) if cycles are present; it has limited utility in amenorrheic patients 1