Laboratory Evaluation for Secondary Amenorrhea
The initial laboratory workup for secondary amenorrhea should include a pregnancy test (beta-hCG), followed by measurement of FSH, LH, prolactin, and TSH levels—these five tests form the essential first-line panel that will guide further evaluation. 1, 2, 3
First-Line Laboratory Tests (Order These First)
Pregnancy test (beta-hCG): Must be performed first to exclude pregnancy in all patients with secondary amenorrhea, regardless of sexual history 1, 2, 3
FSH and LH levels: Should be measured between days 3-6 of the menstrual cycle when possible; for accurate assessment, obtain three measurements taken 20 minutes apart and calculate the average 4, 5
Prolactin level: Measure morning resting serum level (not postictal); levels >20 μg/L are abnormal and suggest hyperprolactinemia 4, 1, 6
TSH level: Essential to exclude thyroid dysfunction, which commonly presents with menstrual irregularity 4, 5, 1, 6
Second-Line Tests Based on Clinical Presentation
If Hyperandrogenism Present (Hirsutism, Acne, Male Escutcheon)
Testosterone: Measure on days 3-6 of cycle; levels >2.5 nmol/L are abnormal and suggest PCOS or valproate effect 4, 5
Androstenedione: Levels >10.0 nmol/L warrant evaluation for adrenal or ovarian tumors 4, 5
If Anovulation Suspected
- Mid-luteal phase progesterone: Measure approximately day 21 of a 28-day cycle; levels <6 nmol/L indicate anovulation, commonly seen in PCOS, hypothalamic amenorrhea, or hyperprolactinemia 4, 5
If Primary Ovarian Insufficiency Suspected (High FSH)
Karyotype analysis: Recommended when FSH is elevated to identify chromosomal abnormalities such as Turner syndrome variants 1, 7
Anti-Müllerian hormone (AMH): Provides additional information on ovarian reserve 5
If Metabolic Concerns or PCOS Suspected
- Fasting glucose and insulin levels: Screen for insulin resistance and metabolic syndrome in patients with suspected PCOS 5
Imaging Studies
Transvaginal ultrasound: Recommended to evaluate endometrial thickness (indicator of estrogen status), assess ovarian morphology, and identify polycystic ovarian morphology (≥20 follicles of 2-9mm per ovary or ovarian volume >10ml) 8, 5, 1
Pituitary MRI: Indicated if prolactin is elevated or clinical features suggest hypothalamic-pituitary abnormalities (e.g., galactorrhea, visual field defects) 4, 5, 1
Additional Assessments for Specific Populations
DXA scan for bone mineral density: Recommended if amenorrhea has lasted >6 months, BMI <18.5 kg/m², history of eating disorders, or late menarche (≥16 years) 8, 1
Progesterone challenge test: Can determine estrogen status—withdrawal bleeding indicates adequate estrogen; no bleeding suggests low estrogen or outflow tract obstruction 1
Critical Timing Considerations
Discontinue hormonal contraception before testing for accurate hormone assessment 5
Avoid postictal prolactin measurement in patients with epilepsy, as seizures cause transient prolactin elevation 4
Single FSH measurements are unreliable due to significant fluctuation; repeat testing or use averaged measurements 5
Common Pitfalls to Avoid
Do not rely on a single FSH measurement for diagnosis, particularly in perimenopausal-aged women where levels fluctuate widely 5
Do not measure hormones at incorrect cycle times—FSH/LH should be measured days 3-6, progesterone mid-luteal phase 4, 5
Do not overlook thyroid dysfunction—it commonly causes menstrual irregularity and is easily treatable 5, 6
Do not forget to account for medications (especially antiepileptic drugs like valproate, which can cause PCOS-like syndrome), stress, and weight changes that affect hormone levels 4, 5
Do not assume infertility in patients with primary ovarian insufficiency—they can maintain unpredictable ovarian function 2, 3