Laboratory investigations (see Table: Laboratory investigations) are necessary to establish the diagnosis, determine aetiology and screen for complications.

The diagnostic criteria for premature ovarian failure (POF) includes greater than 4 months of amenorrhoea and FSH levels >40IU/L on two separate occasions at least one month apart with exclusion of secondary causes of amenorrhoea (see Table: Causes of secondary amenorrhoea).

Measurement of gonadotrophins (LH and FSH) must be performed in the absence of any exogenous hormone therapy (oral contraceptive pill (OCP) should be ceased at least one month prior) otherwise the results will be uninterpretable.

At present there is no test able to predict premature menopause. Studies assessing ovarian reserve during assisted reproduction indicate that women with higher early follicular phase levels of FSH, low serum inhibin B and lower antral follicle count (number of eggs visible on ultrasound) have a poorer response to ovulation induction and an increased risk of premature menopause / early menopause.

Other hormonal and biophysical markers, such as anti-mullerian hormone and ovarian stromal blood flow respectively, remain as research tools. Progesterone withdrawal test, ovarian antibody determination and ovarian biopsy are not routinely performed. Transvaginal or pelvic ultrasound may be useful to exclude outflow obstruction, detect follicles, assess the endometrium and ovarian volume. The presence of ovarian follicles has been reported in 30-50% of women with spontaneous karyotypically normal POF with evidence of intermittent ovarian function. Streak gonads (undifferentiated gonadal tissue) may be observed in patients with gonadal dysgenesis.

Table: Laboratory Investigations
Investigation	Rationale
Initial investigations:
FSH and oestradiol (E2) on 2 occasions at least one month apart Day 3 FSH and E2 if still menstruating.	Diagnostic criteria: FSH > 40 IU/L Assess ovarian reserve and fertility. Adequate ovarian reserve is suggested by FSH<10 IU and E>300pmol/l
Prolactin, LH, thyroid function tests, pregnancy test, DHEAS, androstenedione Sensitive testosterone, sex hormone binding globulin (SHBG), free androgen index or free testosterone	Exclude causes of secondary amenorrhoea including hypogonadotrophic hypogonadism
Transvaginal ultrasound (transabdominal ultrasound if sexually inactive)	Exclude outflow obstruction Detect presence of follicles Assess endometrial thickness and determine ovarian volume
Following diagnosis:
Thyroid function tests, thyroid autoantibodies Adrenal autoantibodies. (If positive antibodies or symptoms of adrenal insufficiency then am cortisol and ACTH +/- short synacthen test) Fasting glucose Liver function tests Full blood examination, serum B12, gastric parietal cell antibodies, intrinsic factor antibodies ESR, ANA, rheumatoid factor Transglutaminase and endomysial antibodies Ovarian antibodies are not helpful	Identify autoimmune disease associated with POF
Karyotype and fragile X assessment	Identify any genetic abnormality
Fasting lipids Dual X-Ray Densitometry (DEXA) scan (If osteopaenic then 25OH Vitamin D, parathyroid hormone, serum calcium, phosphate, albumin and alkaline phosphatase)	Complication screening