At a Glance

Amenorrhea is the absence of menstrual blood flow. Primary amenorrhea should be considered in any patient with secondary sex characteristics who has not experienced periodic menstruation by 15 years of age or 5 years after breast development. Patients who have not developed secondary sex characteristics, especially the absence of breast development, and have not established periodic menstruation by 13 years of age should also be worked up for primary amenorrhea.

Hypothalamic dysfunction is the most common cause of amenorrhea and is characterized by hypogonadotropic hypogonadism. Patients with primary amenorrhea due to hypothalamic abnormalities often have delayed development of secondary sex characteristics. These patients are phenotypic females with prepubertal external and internal genitalia. Depending on the etiology of the hypothalamic dysfunction, these women are typically normal height, but some (if not treated) may develop short stature.

Primary hypothalamic amenorrhea may be caused by gonadotropin releasing hormone (GnRH) deficiency and/or dysregulation of the hypothalamic-pituitary-gonadal (HPG) axis. Functional hypothalamic amenorrhea, although rare in adolescents prior to onset of menses, is caused by excessive emotional stress, frequent exercise, significant weight loss and/or eating disorders, malnutrition, and chronic disease, such as type 1 diabetes, End stage renal disease, or HIV. In these women, GnRH secretion is disrupted, leading to decreased gonadotropin and sex steroid secretion. Functional hypothalamic amenorrhea is usually reversible with diet and/or lifestyle changes or treatment of chronic disease.

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Primary hypothalamic amenorrhea in women with anosmia may be due to Kallmann’s syndrome or other disease associated with complete congenital GnRH deficiency.

What Tests Should I Request to Confirm My Clinical Dx? In addition, what follow-up tests might be useful?

The work-up for primary amenorrhea begins with a careful history and physical exam to look for anatomical defects, development of secondary sexual characteristics, and/or a personal or family history of delayed puberty, mental retardation, short stature, cancer, autoimmune disease, infertility, and/or amenorrhea.

A personal history of galactorrhea, traumatic brain injury, anosmia, chronic disease, significant weight loss, and/or emotional or physical trauma may also assist in the diagnostic work-up. Physical exam of patients with suspected hypothalamic dysfunction may reveal a phenotypic woman of normal or short stature and normal/prepubescent internal and external genitalia with minimal development of secondary sex characteristics.

The presence of virilization may suggest polycystic ovary syndrome (see chapter on Primary Amenorrhea due to Polycystic Ovary Syndrome) or an androgen secreting adenoma.

Because functional hypothalamic primary amenorrhea is usually a transient disease, conception is possible. Thus, the laboratory work-up should include measurement of human chorionic gonadotropin (hCG) to rule out pregnancy. If patient is not pregnant, the patient should undergo imaging studies to detect the presence and absence of uterus and gonads.

Patients with hypothalamic primary amenorrhea have a normal prepubescent uterus, but, prior to further work-up for hypothalamic primary amenorrhea, patients with a detectable uterus should be evaluated for hypothyroidism and prolactinemia. A high thyroid stimulating hormone (TSH) result suggests the amenorrhea is due to primary hypothyroidism and should be followed with fT4 analysis. A reduced fT4 confirms hypothyroidism. An elevated prolactin result should prompt a physician to perform an MRI in search of a pituitary adenoma. Prolactin inhibits gonadotropin function, thus, causing amenorrhea in nursing mothers and patients with prolactinomas.

Patients with normal TSH and prolactin should be evaluated for gonadotropic function by measuring luteinizing hormone (LH) and follicle stimulating hormone (FSH). Patients with GnRH deficiencies and/or dysregulation of their HPG axis will have normal or low FSH and LH (hypogonadotropic). Although suggested by the lack of secondary sex characteristics, estrogen measurements may also be helpful. Low estrogen concentrations (hypogonadism) are expected in patients with primary hypothalamic amenorrhea.

Because of the day-to-day variability in estrogen concentrations, the progestin challenge test may be helpful to establish estrogen reserves and/or etiology of primary amenorrhea in patients with an intact uterus and some secondary sex characteristics.

Theoretically, if progesterone is given to an estrogen primed uterus, withdrawal bleeding (menses) will occur. Progesterone is given orally for up to 1 week. Bleeding should occur within 1 week of progesterone withdrawal if the woman’s ovaries have produced enough estrogen (>40 pg/mL serum) to prime her uterus. A woman with hypothalamic amenorrhea should not respond appropriately to progesterone withdrawal; however, about 40% of women with functional hypothalamic amenorrhea bleed 1 week after stopping progesterone.

Low concentrations of FSH, LH, and estradiol or a failed progesterone challenge test are suggestive of hypothalamic amenorrhea. Hypogonadotropic hypogonadism (HH) is confirmed with at least 2 decreased FSH and 2 decreased estrogen measurements. GnRH stimulation testing may help differentiate between pituitary and hypothalamic causes of HH. Patients with hypothalamic disease will respond to GnRH injections by secreting LH and FSH, whereas patients with pituitary disease will not secrete gonadotropins in response to releasing hormone.

Once pituitary causes are excluded, correlation of laboratory testing with patient history (e.g., stress, excessive exercise, significant weight loss, chronic disease) is essential for the diagnosis of hypothalamic primary amenorrhea. (Table 1)

Table 1.
FSH Estradiol LH
low to normal low low

Are There Any Factors That Might Affect the Lab Results? In particular, does your patient take any medications – OTC drugs or Herbals – that might affect the lab results?

False-positive and false-negative results in urine pregnancy tests can occur and may result in a misdiagnosis. False-negative results occur in urine that is too dilute. To ensure an appropriate urine specimen, perform urine pregnancy testing on first morning voids and check the protein concentration by measuring the urine specific gravity and/or urine creatinine. False-negative results may also be caused by the variant effect.

This phenomenon occurs when high concentrations of hCG isoforms in urine (hCG beta core fragment) are not recognized by both antibodies in the assay. Instead, they interfere with one antibody and cause a false-negative result. One can test for the variant effect by diluting the urine sample and repeating the testing.

Prolactin is mildly elevated by stress, herpes simplex virus (HSV) infections in the chest wall, and numerous drugs, including dopamine agonists, proton pump inhibitors, antipsychotics (risperidone, phenothiazines, haloperidol), antihypertensives (methyldopa, reserpine, verapamil), estrogens, and illicit drugs (amphetamines, cannabinoids, opiates, etc.). Any of these may lead to dysregulation of gonadotropins, amenorrhea, and infertility.

LH and FSH are episodically released from the pituitary, and concentrations may vary, depending on when they are measured. First morning specimens are recommended. LH and FSH concentrations change throughout the menstrual cycle, even in amenorrheic patients. LH and FSH should be measured early in the follicular phase of the cycle, if possible.

Concentrations of LH and FSH change dramatically during puberty. Results should be evaluated in the context of age and tanner stage specific reference intervals. Drugs, such as anticonvulsants, clomiphene, and naloxone, may falsely elevate LH, whereas smoking, cimetidine, clomiphene, digitalis, and levodopa may elevate FSH. Artificially low LH and FSH results may occur in patients taking oral contraceptives and hormone treatments. Phenothiazines reduce FSH concentrations, whereas digoxin decreases LH.

Estrogen can be measured by immunoassay or LC/MS/MS. Because the immunoassays are imprecise at low concentrations, it is recommended that estrogen be measured by LC/MS/MS in men, children (early tanner stages), and postmenopausal women.

Estrogen concentrations vary throughout the menstrual cycle, even in amenorrheic women, and should be measured in the early follicular phase of the cycle, if possible. Chronic illnesses, such as anemia, hypertension, and liver and kidney diseases, affect estrogen concentrations. Several estrogen-containing drugs, such as birth control pills and hormone replacements, may interfere with the assays causing falsely elevated results. Glucocorticoids, antibiotics (ampicillin and tetracycline), and phenothiazines may also cause false elevations in estrogen measurements. Clomiphene and oral contraceptives may reduce estrogen concentrations.

Patients with excess androgen concentrations may not respond to the progestin challenge test. Further, patients with obesity and/or severe stress may respond to the progestin challenge testing with withdrawal bleeding despite gonadal failure (false-negative results).

As is the case with many immunoassays, heterophilic antibodies can cause false-positive results. Therefore, caution should be taken when elevated hCG, TSH, prolactin, estradiol, LH, and/or FSH results do not match the clinical picture.

What Lab Results Are Absolutely Confirmatory?

Work-up of a patient with primary amenorrhea due to hypothalamic causes is diagnosis of exclusion. There is no confirmatory test for most causes of functional hypothalamic amenorrhea. Identification of one or more mutations in a member of the KAL gene family confirms a diagnosis of Kallmann’s syndrome. Identification of mutations in several other genes by DNA sequencing analysis helps confirm other congenital GnRH deficiencies. The most common of these includes FGFR1, PROKR2, CHD7, and GNRHR.

What Tests Should I Request to Confirm My Clinical Dx? In addition, what follow-up tests might be useful?

Although diagnosis of hypogonadotropic hypogonadism is fairly simple, elucidation of the cause of disease in patients is challenging. Various causes of disease are identified by patient history. A recent history of stress, significant weight loss, excessive exercise, or chronic disease, such as diabetes or Cushing’s disease, identifies functional hypothalamic primary amenorrhea. A recent history of headaches, visual field obstruction, and/or galactorrhea should prompt doctors to perform an MRI to identify a pituitary lesion.

A family history significant for delayed puberty should prompt a genetic work-up. In the absence of anosmia (Kallmann’s syndrome) in these patients, it is often difficult to differentiate between congenital GnRH deficiency and constitutional delay. Measurement of the free alpha subunit may differentiate between these two populations of women. In particular, in response to GnRH therapy, women with congenital hypothalamic amenorrhea showed significantly increased free alpha subunit compared to women with constitutional delay or acquired hypothalamic amenorrhea. FSH and LH responses were similar in both sets of women.

Are There Any Factors That Might Affect the Lab Results? In particular, does your patient take any medications – OTC drugs or Herbals – that might affect the lab results?

Free alpha subunit is elevated in up to 30% of pituitary adenomas. Elevations of free alpha-subunit may also occur within 24 hours of ovulation, in end stage renal disease, in hypothyroidism due to elevated TSH, and in women undergoing assisted reproduction or IVF. Baseline measurements are important for detecting nonstimulated secretion. Free alpha subunit has a short half-life and should be measured within 45 minutes of GnRH injection.