What are the appropriate initial and follow‑up tests for evaluating amenorrhea in a reproductive‑age woman?

Amenorrhea Evaluation: Initial and Follow-Up Testing

First Step: Exclude Pregnancy

The initial test for any reproductive-age woman with amenorrhea must be a pregnancy test, regardless of sexual history or contraceptive use. 1, 2, 3, 4

Initial Laboratory Panel (Order Simultaneously After Negative Pregnancy Test)

All women with amenorrhea require measurement of serum FSH, LH, prolactin, and TSH as the core initial hormonal assessment. 1, 2, 5, 3

Specific Tests and Timing:

• FSH and LH: Draw between cycle days 3-6 if any cycles present, or at any time in complete amenorrhea 1, 2
• Prolactin: Single morning resting sample; never draw post-exercise, post-stress, or within 24 hours of a seizure 1, 2
• TSH: Any time; identifies reversible thyroid dysfunction 1, 2, 5
• Estradiol: Add when functional hypothalamic amenorrhea (FHA) is suspected based on history of low weight (BMI <18.5), excessive exercise, or stress 1, 5

Physical Examination Priorities

Document these specific findings that direct further testing:

• BMI calculation: Obesity (>25 kg/m²) suggests PCOS; low BMI (<18.5 kg/m²) suggests FHA 1, 2
• Waist-to-hip ratio: >0.9 indicates truncal obesity associated with PCOS 1, 2
• Nipple expression: Check for galactorrhea indicating hyperprolactinemia 1, 2
• Hirsutism assessment: Ferriman-Gallwey scoring; presence triggers androgen testing 1, 2
• Tanner staging: Essential in primary amenorrhea to assess pubertal development 1, 6

Algorithmic Interpretation and Follow-Up Testing

If Prolactin Elevated (>20 μg/L):

1. First, check TSH (if not already done): Hypothyroidism causes secondary prolactin elevation 1, 5
2. If TSH normal and prolactin remains elevated: Order pituitary MRI to evaluate for prolactinoma 1, 5
3. Urgent referral if prolactin >100 μg/L or any headaches/visual changes present 1, 2

If FSH Elevated (>40 mIU/mL):

1. Diagnosis: Primary ovarian insufficiency (POI) 1, 5, 3
2. Confirm with repeat FSH 4 weeks later (two elevated values required for definitive diagnosis) 1, 5
3. Additional testing for POI:
   • Karyotype if age <40 years (screen for Turner syndrome) 1, 5
   • Fragile X premutation screening 1
   • Autoimmune antibodies (21-hydroxylase, adrenal cortex) 1
4. Immediate management: Start transdermal estradiol replacement (100 μg patch twice weekly) with cyclic progesterone to prevent bone loss and cardiovascular complications 1, 5

If LH/FSH Ratio >2:

1. Diagnosis: Strongly suggests PCOS 1, 2, 5
2. Order pelvic ultrasound to identify polycystic ovarian morphology 1, 2, 5
3. Add androgen testing if hirsutism, acne, or androgenetic alopecia present:
   • Total testosterone (>2.5 nmol/L supports PCOS) 1, 2
   • Androstenedione (>10.0 nmol/L warrants tumor evaluation) 1, 2
   • DHEA-S (age-adjusted thresholds for adrenal pathology) 1, 2
4. Metabolic screening: Fasting glucose and lipid panel (PCOS increases diabetes and cardiovascular risk) 1, 3

If LH/FSH Ratio <1 with Low Estradiol (<30 pg/mL):

1. Diagnosis: Functional hypothalamic amenorrhea (FHA) in 82% of cases 1, 5
2. Pelvic ultrasound: Assess endometrial thickness; <5 mm confirms hypoestrogenism 1, 5
3. Critical history elements to document:
   • Weight loss >5% in past 6 months 1, 2
   • Exercise >10 hours/week of intense training 1
   • Calculate energy availability: <30 kcal/kg fat-free mass/day indicates deficit 1
   • Screen for eating disorders using direct questioning about specific behaviors 1
4. DXA scan for bone mineral density if amenorrhea >6 months, regardless of age 1, 5

If All Hormones Normal:

1. Order progesterone challenge test: Give medroxyprogesterone 10 mg daily for 10 days 4, 7
   • Positive withdrawal bleed: Indicates adequate estrogen and patent outflow tract; suggests PCOS with normal initial labs or hypothalamic dysfunction 1, 4
   • Negative (no bleed): Indicates either estrogen deficiency or outflow obstruction 4, 7
2. If no withdrawal bleed: Order pelvic ultrasound to assess endometrial thickness and rule out structural abnormalities 1, 5

Primary Amenorrhea: Additional Considerations

If no breast development by age 13 or no menarche by age 15, evaluate immediately rather than waiting. 1

Specific to Primary Amenorrhea:

• If breast development absent: Measure FSH to distinguish hypogonadotropic hypogonadism (low/normal FSH) from gonadal dysgenesis (elevated FSH) 1, 6
• If breast development present but uterus absent on ultrasound: Measure testosterone to distinguish Müllerian agenesis (female-range testosterone) from androgen insensitivity syndrome (male-range testosterone) 1, 6
• If both breast development and uterus present: Follow secondary amenorrhea algorithm above 1, 6

Critical Follow-Up Testing Based on Duration

If Amenorrhea Persists >6 Months:

Order DXA scan for bone mineral density assessment regardless of age or etiology. 1, 5 Prolonged hypoestrogenism causes irreversible bone loss, particularly critical in adolescents who should achieve 90% of peak bone mass by age 18. 1

If FHA Diagnosed:

• Mid-luteal progesterone testing to confirm ovulation if cycles resume after addressing stressors 1
• Repeat DXA annually if amenorrhea continues despite intervention 1

Common Pitfalls to Avoid

• Never assume amenorrhea in athletes or stressed patients is benign: Other pathology must be excluded even when clinical picture suggests FHA 1
• Do not prescribe oral contraceptives as first-line for FHA: This masks the problem without addressing energy deficit and does not protect bone density as effectively as physiologic estrogen replacement 1
• Do not use ultrasound to diagnose PCOS in girls <8 years post-menarche: Multi-follicular ovaries are normal in this age group, yielding high false-positive rates 1
• Do not rely solely on LH/FSH ratio for PCOS diagnosis: Sensitivity is only 35-44%; clinical hyperandrogenism with elevated testosterone is more reliable 1
• Never draw prolactin post-ictally: Seizures cause transient elevation; wait at least 24 hours 1, 2
• Do not assume chemotherapy-induced amenorrhea equals menopause: Hormone levels are unreliable during tamoxifen treatment, and ovarian function may be unpredictable 1

Tests NOT Needed in Initial Evaluation

Do not order these tests routinely in a healthy reproductive-age woman with amenorrhea: 2

• Pelvic examination (unless placing IUD or fitting diaphragm)
• Pap smear
• Clinical breast examination
• HIV screening (unless specific risk factors)
• Lipid panel, fasting glucose, liver enzymes (unless specific indication from history or BMI)