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.

Anatomical defects resulting from abnormal development of external and/or internal genitalia is a common cause of primary amenorrhea. Mullerian agenesis, androgen insensitivity, vaginal septum, and imperforate hymen are the most common developmental defects leading to primary amenorrhea. Patients with normal breast development and/or pubic hair growth, but primary amenorrhea, should be evaluated for an anatomical defect.

Primary amenorrhea is caused by the absence of the vagina and/or uterus in Mullerian agenesis and androgen insensitivity syndrome, whereas patients with an imperforate hymen or transverse vaginal septum are amenorrheic, because the menstrual flow is physically blocked. Mullerian agenesis is thought to be caused by activation of anti-mullerian hormone during embryonic development.

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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 due to anatomical defect 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 short stature, infertility, and/or amenorrhea.

A personal history of normal onset of puberty with cyclic pelvic pain but no menses is common in patients with congenital malformations of the vagina, cervix, and/or uterus.

Physical exam of patients with anatomical defects may reveal a vaginal blind pouch (common in Mullerian agenesis, vaginal septum, and androgen insensitivity syndrome). Cervix should be located on speculum examination. Pelvic exam or ultrasonography should be used to determine if a uterus is present.

The laboratory work-up should begin with measurement of human chorionic gonadotropin (hCG) to rule out pregnancy. Patients with anatomical defects are unlikely to become pregnant; however, it is the most common cause of amenorrhea and should be the initial screening test in any patient with primary amenorrhea.

Nonpregnant patients with an anatomical abnormality and an absent uterus detected by pelvic exam or ultrasonography should have testosterone measured to differentiate mullerian agenesis from androgen insensitivity syndrome. Total testosterone should be measured by LC/MS/MS and sex hormone binding globulin (SHBG) by immunoassay and then free testosterone calculated using a predetermined mathematical model. Elevated total and/or free testosterone suggests androgen insensitivity, whereas patients with mullerian agenesis have normal testosterone.

For patients with secondary sex characteristics, cyclic abdominal pain, a visible anatomic defect on pelvic exam, and a uterus present, detection of a bulging hymen confirms imperforate hymen. The presence of thickened tissue and/or blood within the vagina and visual detection of transverse vaginal septum confirms outflow tract obstruction as the cause for primary amenorrhea. Both can be cured with a simple surgery.

Although not usually necessary, in cases of mild malformations, physicians may wish to measure prolactin, thyroid stimulating hormone (TSH), follicle stimulating hormone (FSH), and luteinizing hormone (LH), and estradiol to rule out other causes of primary amenorrhea. Each should be normal if an anatomical defect has caused the primary amenorrhea.

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 negative-results of 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.

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 measured. First morning specimens are recommended. LH and FSH concentrations change throughout the menstrual cycle, sometimes even in amenorrheic patients. 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 society recommends measurement of testosterone by a quantitative high performance liquid chromatography-Tandem Mass Spectrometry assay. Further 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.

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.

Sex hormone binding globulin is affected by age, sex, and pubertal status and should be evaluated with age, sex, and tanner stage specific reference. Obesity and/or insulin resistance, along with hypothyroidism, androgens, and Cushing’s Disease, decreases SHBG synthesis. Hyperthyroidism, anorexia, oral contraceptives, and liver disease increase SHBG concentrations in serum.

Calculation of free testosterone with mathematical modeling requires accurate measures of total testosterone and SHBG and is site specific. Appropriate validation of the % free testosterone calculation requires analysis of several hundred patients.

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, FSH, and SHBG immunoassay results do not match the clinical picture.

What Lab Results Are Absolutely Confirmatory?

Work-up of a patient with primary amenorrhea due anatomical defects is confirmed by visual identification of a specific defect on pelvic exam and/or ultrasonography. In patients with an absent uterus and/or vagina and elevated testosterone, the diagnosis of androgen insensitivity syndrome is confirmed by karyotyping. These patients have a 46, XY karyotype, whereas patients with mullerian agenesis have a 46, XX karyotype.

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

Partial mosaicisms of the Y chromosome may be present in patients with mullerian agenesis or other disorders. The presence of Y chromosome material increases a patient’s risk for gonadoblastoma, thus, gonadectomy is recommended for all patients with Y mosaicism. The presence of Y chromosome mosaicism should be confirmed in any patient with an anatomical defect. Y chromosome material can be detected by identifying the SRY region via Fluorescence in situ hybridization (FISH) or DNA analysis.

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?

Chemotherapy and/or radiation may affect the yield of metaphase chromosomes, reducing the ability to perform karyotyping analyses.