Sex Hormone Testing: Timing, Methods and Interpretation

Sex hormones are among the most frequently tested and most frequently misinterpreted laboratory measures. A progesterone taken on the wrong day, an oestradiol measured in a perimenopausal woman, a testosterone drawn in the afternoon or a result obtained while taking the contraceptive pill can all lead to wrong conclusions. The key to interpreting sex hormones is knowing when to test, how to test and when testing will not help.

Testing in menstruating women

Early follicular phase

Follicle stimulating hormone, luteinising hormone and oestradiol are usually measured on days 2 to 5 of the menstrual cycle, counting the first day of bleeding as day one. In this phase, hormone levels are low and relatively stable, allowing assessment of ovarian function. A raised FSH suggests reduced ovarian reserve; a high LH to FSH ratio has been associated with polycystic ovary syndrome but is not part of diagnostic criteria.

Mid luteal progesterone

Progesterone rises after ovulation. Measuring it about 7 days before the expected period, day 21 in a 28 day cycle, confirms whether ovulation has occurred. In longer or irregular cycles, the test should be timed relative to the next expected period, and repeated if needed. A single low value in an irregular cycle may simply reflect wrong timing.

Anti Müllerian hormone

AMH is produced by small ovarian follicles and reflects ovarian reserve. It can be measured at any point in the cycle. It is used in fertility assessment, and the 2023 international guideline allows raised AMH as an alternative to ultrasound for the polycystic ovarian morphology criterion in adults. AMH does not predict natural fertility in the short term in women without infertility, and should not be used as a fertility test for the general population.

Testosterone and androgens in women

Female testosterone levels are low, and many automated immunoassays are inaccurate at these concentrations. Liquid chromatography with tandem mass spectrometry is more accurate and is recommended where available. Measuring sex hormone binding globulin allows calculation of free testosterone or the free androgen index. Very high testosterone levels, or rapidly progressive virilisation, warrant urgent assessment for an androgen secreting tumour.

Prolactin

Prolactin can be raised by stress, recent breast stimulation, some medicines such as antipsychotics and some antidepressants, hypothyroidism and pituitary tumours. Mildly raised values may need repeating. Macroprolactin, a large inactive form, can cause raised results without clinical significance and can be checked by the laboratory.

When testing does not help

• During hormonal contraception: combined oral contraceptives suppress the natural cycle, so FSH, LH, oestradiol and progesterone results do not reflect natural function. Contraceptives also raise SHBG, lowering free testosterone.
• In perimenopause: hormone levels fluctuate widely, so a single FSH or oestradiol result can be misleading. Guidelines recommend diagnosing perimenopause and menopause clinically in women over 45 without blood tests.
• During acute illness, which suppresses reproductive hormones.
• During breastfeeding, when high prolactin and suppressed ovarian function are normal.

Testing in men

Testosterone should be measured in the morning, ideally fasting, and confirmed on a separate occasion before a diagnosis of deficiency is made. SHBG helps interpret total testosterone, especially in obesity, when SHBG is low. LH and FSH distinguish primary from secondary hypogonadism. Oestradiol in men requires a sensitive assay and is rarely needed routinely. Men using anabolic steroids or testosterone may have suppressed LH and FSH, and results after stopping reflect a recovery period.

Salivary and urinary testing

Salivary testing is sometimes used to guide dosing of compounded bioidentical hormones. National scientific reviews have concluded that this practice is not supported by evidence.

Monitoring hormone therapy

For women using menopausal hormone therapy, dosing is usually guided by symptom response rather than blood levels, although oestradiol levels may be checked in specific situations, such as poor absorption with transdermal preparations. For men on testosterone therapy, testosterone and haematocrit are monitored according to guidelines. Gender affirming hormone therapy is monitored according to specialist protocols.

Worked cases

The mistimed progesterone

A 32 year old woman trying to conceive has a day 21 progesterone that is low, and she is told she is not ovulating. Her cycles are 35 days long. When the test is repeated 7 days before her next expected period, day 28, her progesterone confirms ovulation.

The contraceptive confusion

A 26 year old woman on the combined pill has blood tests showing low FSH, LH and oestradiol and a low free testosterone. She has been told her hormones are imbalanced. These results are exactly what the pill produces and are not interpretable as natural hormone status.

The perimenopausal snapshot

A 47 year old woman with hot flushes and irregular periods has a normal FSH on one test and is told she is not perimenopausal. Given her age and symptoms, perimenopause is a clinical diagnosis, and a single FSH during fluctuation is uninformative. Her symptoms are managed accordingly.

The afternoon testosterone

A 45 year old man has a low testosterone on a test taken at 4 pm after lunch. A repeat morning fasting test is within the normal range.

The raised prolactin

A 30 year old woman with irregular periods has a mildly raised prolactin. She takes an antipsychotic. Her doctor considers the medicine as a cause, checks thyroid function and macroprolactin, and arranges further assessment only if indicated.

Right test, right time

Sex hormone testing is only as good as its timing, method and context. Knowing when to test, choosing reliable methods and recognising when a result cannot be interpreted protects patients from misdiagnosis and unnecessary treatment.