At a Glance

Amenorrhea is the absence of menstrual blood flow. Primary amenorrhea should be considered in a 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 absence of breast development, and have not established periodic menstruation by 13 years of age should also be worked up for primary amenorrhea.

Amenorrhea and premature ovarian failure may accompany disorders arising from mutations in enzymes with reproductive effects. This includes such disorders as nonclassical congenital adrenal hyperplasia (CAH; 21-α, 17α-hydroxylase, and 3-β-hydroxysteroid dehydrogenase deficiencies), disorders of sexual development (aromatase, P450scc, and 17-β-hydroxysteroid dehydrogenase type 3 (17βHSD-3) deficiencies), and galactosemia (galactose-1-phosphate uridyltranferase deficiency).

In CAH, defective enzymes in the adrenal hormone synthesis pathway alter the formation and regulation of certain steroids, including androgens. In classical, or prenatal, 21-α hydroxylase deficiency, the most common cause of CAH, mineralocorticoids and glucocorticoid synthesis is impeded, shunting precursors down the androgen pathway. Thus, XX-females with CAH can be virilized in utero and born with ambiguous genitalia. In nonclassic, or late-onset, 21-α hydroxylase deficiency CAH, adult and adolescent women present with signs and symptoms similar to polycystic ovary syndrome (PCOS) and sometimes primary amenorrhea.

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In 17α-hydroxylase deficiency, deoxycorticosterone, progesterone and aldosterone are elevated, but androgens and estrogens are decreased. Thus, adolescent and adult women with 17α-hydroxylase deficiency present with absence of secondary sexual characteristics and primary amenorrhea. In 3-β-hydroxysteroid dehydrogenase deficiency, all adrenal steroid synthesis is impaired, thus, clinical manifestations begin in infancy.

In nonclassic 3-β-hydroxysteroid dehydrogenase deficiency, however, adolescent women present with hirsutism and/or menstrual irregularity. A very rare form of CAH, lipoid CAH, results from a deficiency in P450scc (or 20,22-desmolase), the first enzyme in the steroid synthesis pathway. Individuals with this deficiency demonstrate a host of clinical manifestations from lack of steroids, including lack of virilization. Therefore, females may only undergo limited sexual development and may not menstruate. There are cases of nonclassic lipoid CAH that may present later in life.

Disorders of sexual development can manifest as virilization of females (46,XX) as in aromatase deficiency, or lack of virilization of males (46,XY) as in 17βHSD-3 deficiency. Aromatase catalyzes the formation of estrogens from androgen; thus, without aromatase, androgens accumulate and estrogens are inadequate. Aromatase-deficient 46,XX females, therefore, present in infancy with pseudohermaphroditism, clitoromegaly, and lack of pubertal development (including primary amenorrhea), and in adulthood with severe estrogen deficiency and virilization.

In 17βHSD-3 deficiency, testosterone is not synthesized in the testes, preventing virilization and formation of external genitalia of 46,XY males. Thus, at birth, these infants are often identified and raised as female. However, virilization may initiate at puberty, presenting as hirstutism and amenorrhea. These individuals have abnormal external female genitalia, often including a blind-ending vagina and clitoromegaly. Due to the lack of virilizing hormones, males deficient in P450scc (lipoid congenital adrenal hyperplasia) do not form normal external genitalia, and, therefore, may be raised as female. Thus, at puberty, these individuals may present with lack of menstruation.

Galactosemia is a metabolic disease resulting from a deficiency of galactose-1-phosphate uridyltransferase (GALT). Common clinical manifestations are observed in infants exposed to lactose and include poor growth and feeding, emesis, diarrhea, hepatomegaly, jaundice, lethargy, hypotonia, and cataracts. Although the mechanism is unclear, primary ovarian insufficiency, hypergonadotropinism, and amenorrhea are associated with galactosemia.

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

The diagnostic work-up for primary amenorrhea should begin with a detailed history and measurement of human chorionic gonadotropin (hCG) to rule out pregnancy in patients with secondary sex characteristics. Patients without secondary sex characteristics and/or a negative pregnancy test should have a thorough external physical exam looking for ambiguous genitalia and/or hirsutism and a pelvic exam or ultrasonography to determine if a uterus is present. Patients with a detectable uterus should be evaluated for hypothyroidism and prolactinemia.

A high thyroid stimulating hormone (TSH) result suggests the primary 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). High concentrations of FSH and/or LH are suggestive of primary ovarian failure.

The diagnostic work-up for CAH begins with determination of serum 17-hydroxyprogesterone (17-OHP), which should be elevated in nonclassic 21-α hydroxylase deficiency, along with 17-hydroxypregnenolone, dehydroepiandrosterone (DHEA), DHEA-sulfate, androstenedione, testosterone, and progesterone.

In 17α-hydroxylase deficiency, deoxycorticosterone, corticosterone, and progesterone are elevated, but not 17-OHP. Also, patients with 17α-hydroxylase deficiency may have elevated aldosterone, causing hypertension and hypokalemic alkalosis.

In nonclassic 3-β-hydroxysteroid dehydrogenase deficiency, DHEA and DHEA-sulfate are elevated and androstenedione is low. A standard ACTH-stimulation test in a patient with 3-β-hydroxysteroid dehydrogenase deficiency should generate abnormally elevated concentrations of delta-5-17-hydroxypregnenolone.

In lipoid congenital adrenal hyperplasia, all steroids (mineralocorticoids, glucocorticoids, androgens, and all metabolites) are markedly low or undetectable and unresponsive to any stimulation.

The laboratory assessment of disorders of sexual development, specifically aromatase, 17βHSD3, and P450scc deficiencies, should begin with measuring LH and FSH. In pubertal females with aromatase deficiency, basal and GnRH-stimulated LH and FSH are both elevated, as are testosterone and androstenedione. Serum estradiol concentrations are extremely low. 17βHSD-3 deficiency is suggested by a low testosterone:delta-4-androstenedione ratio at baseline and following hCG stimulation, however, variable results may be obtained. Therefore, any suspected case of 17βHSD-3 deficiency should be confirmed by karyotyping or fluorescent in situ hybridization (FISH) of the SRY gene to determine genotypic gender and sequencing of the HSD17B3 gene.

Diagnosis of galactosemia is generally made in infancy in follow-up to an abnormal newborn screen. Laboratory tests diagnostic of galactosemia include elevated plasma galactose and plasma and urine galactitol. Other findings consistent with galactosemia include high amino acids concentrations (particularly phenylalanine, tyrosine, and methionine), hyperbilirubinemia, coagulopathies, abnormal liver function tests, galactosuria, glycosuria, aminoaciduria, and albuminuria. Elevated LH and FSH have been reported for some females with galactosemia.

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 is 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 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.

Premature infants may have 17-hydroxyprogesterone concentrations above the normal range, causing a false-positive screen for CAH on newborn screening. Determination at a later age should generate accurate results. Rare adrenocortical tumors may secrete hormones, such as 17-hydroxyprogesterone, producing elevated concentrations not associated with CAH.

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. It is recommended to measure LH and FSH 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.

Because of lack of precision and correlation with standards in women and children, the Androgen Excess and PCOS Society recommends measurement of testosterone by a quantitative high performance liquid chromatography-Tandem Mass Spectrometry assay. Markedly elevated DHEA-S (>800 mcg/dL) and total testosterone (>200 ng/dL) suggest a virilizing tumor. Furthermore, testosterone values are affected by age, race, body mass index (BMI), and time of day and menstrual cycle. Thus, results should be evaluated in the context of age-, race-, and BMI-specific reference intervals and collected in the morning and early in the menstrual cycle if this can be determined.

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

What Lab Results Are Absolutely Confirmatory?

In cases of amenorrhea with any abnormal findings on physical exam, cytogenetics (FISH and/or karyotyping) may be employed to determine the patient’s genotypic gender (e.g., 46,XY or 46,XX).

A full steroid metabolite profile measuring 17-hydroxyprogesterone, 17-hydroxypregnenolone, androstenedione, cortisol, DHEA, testosterone, deoxycorticosterone, 11-deoxycortisol, and corticosterone is instrumental in diagnosing patients with all forms of CAH.

Suspected aromatase deficiency may be confirmed by in vitro aromatase activity assays and/or sequencing of the CYP19 gene. Likewise, sequencing of the HSD17B3 gene is recommended to confirm a diagnosis of 17βHSD-3 deficiency.

Galactosemia can be confirmed by testing GALT enzyme activity in red blood cells and/or GALT gene sequencing.

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

In patients with primary amenorrhea, enzyme-deficiencies must be differentiated from other causes of hypergonadotropic (high FSH and LH) hypogonadism (low estrogen). Infectious and iatrogenic causes of primary ovarian failure should be ruled out by checking for a history of ovarian surgery, chemotherapy, or irradiation or past viral infection, such as AIDS or mumps. Personal or family history of autoimmune disease, such as Addison’s, thyroid, DM I, SLE, rheumatoid arthritis, vitiligo, Crohn’s disease, and Sjogren’s syndrome, may suggest autoimmune destruction of the ovaries. Detection of antiovarian antibodies in serum of patients with primary amenorrhea and ovarian failure is not recommended because of lack of correlation with disease state or onset.

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?

Concentration of adrenal steroids and metabolites can be affected by use of drugs and supplements. Androstenedione concentration may be elevated by use of androstenedione and DHEA-S supplements. Testosterone concentrations are elevated by many drugs, including danazol, androgenic progestins, valproic acid, acetazolamide, minoxidil, and oral contraceptives. Testosterone concentrations may be reduced in patients taking anabolic steroids. 17-hydroxyprogesterone and other metabolites may be suppressed by use of exogenous glucocorticoids. Thus, glucocorticoid use may mask late-onset CAH.