What hormones and timing should be included in a female hormone panel to evaluate menstrual function, fertility, polycystic ovary syndrome, androgen excess, hyperprolactinemia, thyroid status, and menopausal transition?

Female Hormone Panel: Essential Components and Timing

A comprehensive female hormone panel should include FSH, LH, estradiol, progesterone, TSH, prolactin, and testosterone, with specific timing based on menstrual cycle phase to accurately assess ovarian function, ovulation, PCOS, thyroid status, and menopausal transition. 1, 2

Core Hormonal Assessment

Early Follicular Phase Testing (Cycle Days 3-6)

The early follicular phase represents the optimal window for baseline reproductive hormone assessment when levels are at their nadir and most interpretable. 1

Essential hormones to measure on days 3-6:

• FSH (Follicle-Stimulating Hormone): FSH >10 IU/L indicates diminished ovarian reserve; FSH >35 IU/L suggests ovarian failure and requires repeat testing 4 weeks later for confirmation 1, 2
• LH (Luteinizing Hormone): Baseline LH helps establish etiology of any dysfunction; LH <7 IU/mL may indicate hypothalamic dysfunction 1, 2
• Estradiol: Baseline estradiol <30 pg/mL confirms hypoestrogenism characteristic of functional hypothalamic amenorrhea 2
• Testosterone: Total testosterone >2.5 nmol/L supports PCOS diagnosis or valproate effect; levels >5 nmol/L warrant urgent evaluation for ovarian or adrenal tumor 2

Critical interpretation ratios:

• LH/FSH ratio >2 strongly suggests PCOS, while ratio <1 is seen in approximately 82% of functional hypothalamic amenorrhea cases 1, 2

Mid-Luteal Phase Testing (Day 21 or 7 Days Post-Ovulation)

Progesterone measurement during mid-luteal phase is the most reliable hormonal indicator of ovulation. 1

• Progesterone <6 nmol/L indicates anovulation, commonly seen in PCOS, hypothalamic amenorrhea, or hyperprolactinemia 1, 2
• This single measurement confirms whether ovulation occurred, as normal baseline FSH, LH, and estradiol do not guarantee ovulatory function 1

Additional Essential Hormones (Any Cycle Day)

Thyroid Function

• TSH (Thyroid-Stimulating Hormone): Both hypothyroidism and hyperthyroidism disrupt menstrual cycles; TSH should be measured in all women with menstrual irregularities 2
• Thyroid dysfunction prevalence in PCOS women is similar to controls (approximately 2%), but screening remains essential as it represents a reversible cause of amenorrhea 3, 2

Prolactin Assessment

• Prolactin >20 μg/L indicates hyperprolactinemia and should prompt evaluation for pituitary adenoma or medication effects 2
• Critical timing consideration: Obtain morning resting sample; never draw immediately after seizure, stress, breast examination, or sexual activity as these cause transient elevations 2
• Prolactin >100 μg/L or any elevation with headaches/visual disturbances requires urgent endocrine or neurosurgical referral 2
• The prevalence of hyperprolactinemia in PCOS is not increased compared to controls (1.3% versus 3%) 3

Androgen Excess Evaluation

When clinical signs of hyperandrogenism are present (hirsutism with Ferriman-Gallwey score indicating excess, acne, androgenetic alopecia):

• Androstenedione >10.0 nmol/L warrants investigation for adrenal or ovarian neoplasms 2
• DHEA-S (age-adjusted thresholds): Age 20-29 >3800 ng/mL; age 30-39 >2700 ng/mL screens for non-classical congenital adrenal hyperplasia 2

Menopausal Transition Assessment

For women with irregular cycles suggesting perimenopause:

• FSH and estradiol levels become less reliable during the menopausal transition due to fluctuations 1
• Anti-Müllerian hormone (AMH) may be preferred as it does not vary by menstrual day and is not affected by exogenous estrogen or progesterone 1
• In postmenopausal women, testosterone, free testosterone, SHBG, and DHT are significantly lower than in premenopausal women, concomitant with declines in estradiol and estrone 4

Critical Timing and Collection Pitfalls

Menstrual Cycle Considerations

• For women with regular cycles (21-35 days): Schedule baseline testing on day 2,3,4, or 5 of cycle 1
• For women with irregular or absent periods: Different timing rules apply; FSH, LH, and testosterone can be drawn at any time in amenorrheic patients 2
• Hormonal contraceptives must be discontinued at least 2 months before testing to allow return of normal pituitary-ovarian axis function 1, 2

Sample Collection Accuracy

• FSH and LH baseline measurements should be the average of three estimations taken 20 minutes apart for most accurate assessment 1
• Progesterone must be obtained during the expected mid-luteal phase (approximately day 21 in 28-day cycle or 7 days after suspected ovulation) 2

Algorithmic Approach to Panel Selection

For menstrual irregularity/fertility evaluation:

1. Pregnancy test first (mandatory) 2
2. Day 3-6: FSH, LH, estradiol, testosterone, TSH, prolactin 1, 2
3. Mid-luteal: Progesterone 1, 2

For suspected PCOS:

1. Day 3-6: FSH, LH (calculate ratio), testosterone, TSH, prolactin 2, 5
2. If hyperandrogenic signs present: Add androstenedione, DHEA-S 2
3. Mid-luteal: Progesterone to confirm anovulation 2
4. Consider fasting glucose and insulin for metabolic assessment 2

For suspected hypothalamic amenorrhea:

1. Day 3-6 (or any time if amenorrheic): FSH, LH, estradiol (expect low), TSH, prolactin 2
2. Estradiol <30 pg/mL and LH/FSH ratio <1 support diagnosis 2

For menopausal transition:

1. Consider AMH instead of or in addition to FSH/estradiol due to cycle variability 1
2. If using FSH: Requires two elevated values (>40 mIU/mL) 4 weeks apart for primary ovarian insufficiency diagnosis 2

Common Diagnostic Pitfalls

• Assuming normal baseline hormones guarantee ovulation: Mid-luteal progesterone is essential to confirm actual ovulation occurred 1
• Misdiagnosing functional hypothalamic amenorrhea with polycystic ovarian morphology as PCOS: FHA patients have low LH, low estradiol, thin endometrium (<5 mm), and LH/FSH ratio <1 despite ovarian appearance on ultrasound 2
• Drawing prolactin at wrong time: Post-ictal, post-stress, or after breast examination causes false elevations 2
• Testing while on hormonal contraceptives: Suppresses normal FSH and LH patterns, rendering results uninterpretable 1
• Single FSH measurement in perimenopausal women: Levels fluctuate dramatically; AMH provides more stable assessment 1