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  • Pregnancy Tests

    Most pregnancy tests are based on the fact that the placenta secretes human chorionic gonadotropin (hCG), a hormone that has a luteinizing action on ovarian follicles and probably has other functions that are not completely known. Serum hCG levels of about 25 milli-international units (mIU)/ ml (IU/L) are reached about 8-10 days after conception. The hCG levels double approximately every 2 days (various investigators have reported doubling times ranging from 1-3 days) during approximately the first 6 weeks of gestation. Levels of about 500 mIU/ml are encountered about 14-18 days after conception (28-32 days after the beginning of the last menstrual period). Serum levels are generally higher than urine levels for about the first 2 weeks after conception and about the same as urine levels during the third week. Thereafter, urine levels are higher than serum. The serum (and urine) hCG levels peak about 55-70 days (8-10 weeks) after conception (literature range, 40-77 days). Peak serum values are about 30,000 mIU/ml (range, 20,000-57,000 mIU/ml). Serum and urine levels then decline rather rapidly during the last part of the first trimester, with serum levels eventually stabilizing at about 10,000 mIU/ml. These levels are maintained for the remainder of pregnancy, although some investigators describe a brief rise and fall in the third trimester. Urine levels generally parallel serum levels, but the actual quantity of urine hCG obtained in terms of milliinternational units per milliliter is considerably dependent on technical aspects of the kit method being used (discussed later).

    The hCG molecule is composed of two subunits, alpha and beta. The alpha subunit is also a part of the pituitary hormones LH, FSH, and TSH. The beta subunit, however, is different for each hormone. The hCG molecule in serum becomes partially degraded or metabolized to beta subunits and other fragments that are excreted in urine.

    Biologic tests. The first practical biologic test for pregnancy was the Ascheim-Zondek test, published in 1928. Urine was injected into immature female mice, and a positive result was indicated by corpus luteum development in the ovaries. This took 4-5 days to perform. The next major advance took place in the late 1950s when frog tests were introduced. These took about 2 hours to complete. The result was almost always positive by the 40th day after the last menses, although it could become positive earlier.

    Immunologic tests. In the 1960s it was learned that antibodies to hCG could be produced by injecting the hCG molecule into animals. This was the basis for developing immunologic pregnancy tests using antigen-antibody reactions. In the late 1960s and during the 1970s, both latex agglutination slide tests and hemagglutination tube tests became available. The slide tests took about 2 minutes to perform and had a sensitivity of 1,500-3,000 mIU/ml, depending on the manufacturer. The tube tests required 2 hours to complete and had a sensitivity of 600-1,500 mIU/ml. The antibody preparations used at that time were polyclonal antibodies developed against the intact hCG molecule, and they cross-reacted with LH and TSH. This did not permit tests to be sensitive enough to detect small amounts of hCG, because urine LH could produce false positive results.

    Beta subunit antibody tests. In the late 1970s, methods were found to develop antibodies against the beta subunit of hCG rather than against the whole molecule. Antibody specific against the beta subunit could greatly reduce or even eliminate the cross-reaction of hCG with LH. However, the degree of current beta subunit antibody specificity varies with different commercial companies. By 1980, sensitivity of the slide tests using beta hCG antibody had reached 500-1,500 mIU/ml, and sensitivity of the beta hCG tube tests was approximately 200 mIU/ml. Both the slide and the tube tests required a urine specimen. In the 1980s, standard immunoassay methods were developed for beta hCG in serum that provide a sensitivity of 3-50 mIU/ml. These methods detect pregnancy 1-2 weeks after conception. The great majority of current tests use monoclonal antibodies, either alone or with a polyclonal antibody that captures the hCG molecule and a monoclonal antibody that identifies it. Several manufacturers developed abbreviated serum pregnancy immunoassays that compared patient serum with a single standard containing a known amount of beta hCG (usually in the range of 25 mIU/ml). A result greater than the standard means that beta hCG is present in a quantity greater than the standard value, which in usual circumstances indicates pregnancy. Current serum immunoassay procedures take between 5 minutes and 2 hours to perform (depending on the manufacturer). The abbreviated method is much less expensive and is usually quicker. Several urine tests are available that detect 50 mIU/ml of hCG.

    Technical problems with human chorionic gonadotropin. Some (not all) of the kits that detect less than 25 mIU/ml of hCG may have problems with false-positive results of several different etiologies. First, of course, there may be incorrect performance of the test or patient specimen mishandling. The antibodies used in the different manufacturer’s tests have different specificities. Serum hCG molecules may exist in different forms in some patients: whole (“intact”) molecule, free beta subunit, free alpha subunit, or other degraded hCG fragments. Considerable quantities of serum free beta or alpha subunits are more often seen with tumors. Different antibodies may detect different amounts of hCG material depending on whether the antibody detects only the whole molecule, the beta subunit on the whole molecule, or the free beta subunit only (including in urine a partially degraded free beta subunit known as the “core beta fragment”). Most anti-beta antibodies actually detect both whole molecule (because of the structural beta subunit), free beta subunit, and core beta fragments. Therefore, the amount of hCG (in mIU/ml) detected in urine depends on several factors: (1) whether a specific whole-molecule or a beta-hCG method is used. The specific whole-molecule method reports about the same quantity of intact hCG in serum or urine, whereas the beta-specific assay would report higher amounts of hCG in urine than in serum since it detects intact hCG plus the beta subunits and fragments that are present in greater quantities in urine than serum; (2) degree of urine concentration or dilution; (3) stage of pregnancy, since more beta fragments appear in urine after the first few weeks; (4) how the particular kit is standardized (discussed later). Some beta-hCG antibodies have a certain degree of cross-reaction with LH, although theoretically a beta-specific antibody should not do so. The serum of occasional patients contains heterophil-type antibodies capable of cross-reacting with monoclonal test antibodies (HAMA) that were produced in mouse tissue cells and could produce a false positive result. This most often happens with double antibody “sandwich” test methods. Some kits are affected in a similar way by renal failure.

    Another confusing aspect of pregnancy testing relates to standardization of the tests by the manufacturers (that is, adjusting the test method to produce the same result as a standard, which is a known quantity of the material being assayed). In hCG testing, the manufacturers use a standard from the World Health Organization (WHO). The confusion arises because the earliest WHO standard used for this purpose (Second International Standard; second IS) was composed of a mixture of whole-molecule hCG, free beta subunits, and other hCG fragments. When the supply of second IS was exhausted, the WHO developed the first (and then the third) International Reference Preparation (IRP), which is mostly whole-molecule hCG without free beta subunit. However, hCG kits standardized with the original second IS give results about half as high as current kits standardized against the first or third IRP. Also, certain current kits specific for whole-molecule hCG would not detect some of the hCG fragments in the original second IS. This difference in antibody behavior may at least partially explain discrepant reports in the literature of equal quantities of hCG in pregnancy serum and urine and other reports of urine values as high as 10 times serum values. After the first few weeks of pregnancy, maternal serum contains primarily intact hCG; maternal urine contains some intact hCG but much larger quantities of free beta subunits and core beta fragments.

    Finally, it has been reported in several studies that occasionally normal nonpregnant women may have low-level circulating levels of an hCG-like substance, usually less than 25 mIU/ml. This was reported in about 2.5% (range, 0%-14%) of patients in these studies, although most evaluations of hCG test kits have not reported false positive results. When one kit was reactive, sometimes one or more different kits would also be reactive, but usually some kits do not react with these substances. At present, in most laboratories there is no satisfactory way to know immediately whether a positive result is due to pregnancy, is due to hCG-producing tumor, or is false positive, especially when the test is a yes-no method. Although there are ways to investigate possible discrepancies, it usually takes considerable time and retesting to solve the problem or it may necessitate consultation with a reference laboratory.

    Other uses for hCG assay. Pregnancy tests are useful in certain situations other than early diagnosis of normal pregnancy. These conditions include ectopic pregnancy, spontaneous abortion (which occurs in about 15% of all pregnancies; literature range, 12%-31%), and hCG-producing neoplasms. Ectopic pregnancy and neoplasms will be discussed in detail later. When the differential diagnosis includes normal intrauterine pregnancy, ectopic pregnancy, and threatened, incomplete, or complete abortion, the pattern obtained from serum quantitative beta-hCG assays performed every other day may be helpful. During the first 4 weeks of pregnancy (beginning at conception), there is roughly a doubling of hCG every 2 days (range, 1-3 days). As noted earlier, serum beta hCG by immunoassay first detects embryonic placental hCG in titers of 2-25 IU/L between 1 and 2 weeks after conception. Ectopic pregnancy and abortions may demonstrate an increase in their hCG levels at the same rate as in normal pregnancy up to a certain point. In the case of ectopic pregnancy, that point is usually less than 4 weeks (possibly as long as 6 weeks) after conception, since the ectopic location usually limits placental growth or rupture occurs. The typical pattern of ectopic pregnancy is a leveling off (plateau) at a certain time. The usual pattern of abortion is either a decrease in beta-hCG levels as abortion takes place or a considerable slowing in the rate of increase. However, these are only rules of thumb. About 15% of normal intrauterine pregnancies display less increase (decreased rate of increase) than expected, and thus could be mistaken for beginning abortion by this criterion alone. Also, ectopic pregnancy values may sometimes decline rather than plateau if the fetus dies.

    Ectopic pregnancy

    Ectopic pregnancy is a common gynecologic problem, either by itself or in differential diagnosis. Symptoms include abdominal pain of various types in about 97% of patients (literature range, 91%-100%), abnormal uterine bleeding in about 75% (54%-80%), delayed menses in about 75% (68%-84%), adnexal tenderness on palpation in about 90%-95%, unilateral adnexal mass in about 50% (30%-76%), and fever (usually lowgrade) in about 5% (3%-9%). Hypovolemic shock is reported as the presenting symptom in about 14%. It is obvious that these signs and symptoms can suggest a great number of conditions. In one study, 31% of patients with ectopic pregnancy in the differential diagnosis had a strongly suggestive triad of abdominal pain, uterine bleeding, and an adnexal mass. Only 14% of these patients were found to have ectopic pregnancy. Some conditions that frequently mimic ectopic pregnancy are pelvic inflammatory disease; threatened, incomplete, or complete abortion; corpus luteum rupture; dysfunctional uterine bleeding; and bleeding ovarian cyst. Among routine laboratory tests, a hemoglobin value less than 10 gm/100 ml is reported in about 40% of ectopic pregnancy cases (28%-55%) and leukocytosis in about 50%. Among other diagnostic procedures, culdocentesis for fresh blood is reported to produce about 10% false negative results (5%-18%). Pregnancy test results vary according to the sensitivity of the test. Urine or serum pregnancy tests with a sensitivity of 500-1,000 mIU/ml result in about 25% false negative results (8%-60%). Tests with a sensitivity of 50 mIU/ml yield about 5%-10% false negative results (0%-13%). Serum tests with a sensitivity of 25 IU/L or better have a false negative rate of about 1%-2% (range, 0%-3%). A positive pregnancy test result is not a diagnosis of ectopic pregnancy but signifies only that the patient has increased levels of hCG, for which there could be several possible causes. Also, some manufacturers’ kits are subject to a certain number of false positive results. Interpretation of a negative test result depends on the sensitivity of the test. If the test is a serum hCG immunoassay with a sensitivity of 25 mIU/ml (IU/L) or better, a negative test result is about 98%-99% accurate in excluding pregnancy. However, there are rare cases in which the specimen might be obtained 2-4 days before the patient hCG reaches detectable levels or there could be a technical laboratory error. A repeat test 48 hours later helps to exclude these possibilities.

    As noted previously, failure to double hCG values in 24 hours at gestational age 4-8 weeks occurs in about 66% of ectopic pregnancies, about 85% of spontaneous abortion cases, and about 15% of normal pregnancies. Such an abnormally slow hCG increase rate would warrant closer followup or possibly other diagnostic tests, such as a quantitative serum hCG assay if the 48-hour increase is considerably low. A substantially low serum hCG level for gestational age suggests abnormal pregnancy. Another use for quantitative hCG assay in appropriate cases is to see if the “discriminatory zone” of Kadar has been reached. Originally, this was the range of 6,000-6,500 mIU/ml (IU/L, IRP standard) above which standard transabdominal ultrasound (TAUS) can visualize a normal pregnancy gestational sac in the uterus in about 94% of cases (although TAUS could detect an intrauterine gestational sac below 6,000 mIU/ml in some cases, failure to do so gives no useful information). With more sensitive ultrasound equipment and use of a vaginal transducer, it has been reported that the discriminatory zone upper limit can be reduced to the area of 1,000-1,500 mIU/ml (IU/L), but the exact value must be established by each institution using its particular pregnancy test and ultrasound equipment. Transvaginal ultrasound is more sensitive than TAUS in detecting an adnexal mass or free cul-de-sac fluid that would suggest ectopic pregnancy.

    Neoplasms producing human chorionic gonadotropin

    Neoplasms arising from chorionic villi, the fetal part of the placenta, are known as gestational trophoblastic neoplasms and include hydatidiform mole (the counterpart in tumor classification of benign adenoma) and choriocarcinoma (chorioepithelioma, the equivalent of carcinoma). Hydatidiform mole also has a subdivision, chorioadenoma destruens, in which the neoplasm invades the placenta but there is no other evidence of malignancy. The major importance of hydatidiform mole is a very high (і 10%) incidence of progression to choriocarcinoma.

    Several hormone assays have been proposed as aids in diagnosis. By far the most important is hCG, which is produced by the trophoblast cell component of fetal placental tissue. Current pregnancy tests using monoclonal antibodies to beta subunit of hCG or to the whole molecule can detect levels of 25 mIU/ml (IU/L), sometimes less, without interference by LH, permitting detection of nearly all gestational tumors (except a very few that predominately secrete the free beta fragment of hCG, which would necessitate an assay that would detect this hCG metabolite). Since normal placental tissue secretes hCG, the problem then is to differentiate normal pregnancy from neoplasm. Suspicion is raised by clinical signs and also by finding hCG levels that are increased more than expected by the duration of pregnancy or that persist after removal of the placenta. Twin or other multiple pregnancies can also produce hCG levels above expected values. Although serum levels of hCG greater than 50,000 mIU/ml (or urine levels > 300,000 mIU/ml) are typically associated with gestational neoplasms, especially if these levels persist, a considerable number of patients with gestational tumors have hCG values less than this level. About 25% of patients in one report had values less than 1,000 mIU/ml. In normal pregnancies, serum hCG levels become nondetectable by about 14 days (range, 3-30 days) after delivery. In one study of elective abortions, it took 23-52 days for hCG levels to become nondetectable. After removal of a hydatidiform mole, hCG levels should become nondetectable in about 2-3 months (range, 21-278 days). Once neoplasm is diagnosed and treated, hCG measurement is a guideline for success of therapy and follow-up of the patient for possible recurrence.

    Other hormones useful in possible gestational neoplasms. Fetal and placental tissue produces other hormones that may be useful. Progesterone (or its metabolite pregnanediol) and estradiol are secreted by the placenta in slowly increasing quantity throughout most of pregnancy. It has been reported that during the first 20 weeks of gestation, hydatidiform moles are associated with serum estradiol-17b values that are increased from values expected in normal pregnancy, with good separation of normal pregnancy from molar pregnancy. Serum progesterone levels were increased in about 75% of nonaborted moles up to the 20th week. Urinary pregnanediol levels, on the other hand, are frequently decreased. Finding increased serum progesterone and estradiol-17b levels during the time that peak hCG values are expected (between the 50th and 80th days after the last menstrual period), accompanied by a decreased urine pregnanediol level, would suggest a hydatidiform mole or possibly a choriocarcinoma. Serum human placental lactogen (hPL), or somatomammotropin, is another placental hormone whose level rises during the first and second trimesters and then reaches a plateau during the last 2-3 months. The association of decreased levels of hPL in the first and second trimesters with increased hCG levels suggests neoplasm. There is, however, a small degree of overlap of hPL level in patients with mole and the normal range for pregnancy. One report suggests a possible inverse ratio between hPL values and degree of malignancy (the greater the degree of malignancy, the less serum hPL produced).

    Production of hCG has been reported to occur in nearly two thirds of testicular embryonal cell carcinomas and in about one third of testicular seminomas. Instances of hCG secretion by adenocarcinomas from other organs and, rarely, from certain other tumors have been reported.

  • Gynecomastia

    Gynecomastia is usually defined as enlargement of the male breast. This may be palpable only or may be grossly visible. Either type may be unilateral or bilateral. A small degree of palpable nonvisible gynecomastia is said to be present in about 30%-40% of clinically normal men. Most etiologies of gynecomastia can produce either unilateral or bilateral effects.

    Etiologies. Major etiologies of gynecomastia are presented in the box. In most cases, estrogen (estradiol) is increased relative to testosterone, even if assay values do not demonstrate this. Considering some of the etiologies, some degree of gynecomastia is present in 20%-40% (range, 4%-70%) of boys during puberty but disappears in 1-2 years. Old age demonstrates increased incidence of gynecomastia, possibly due to testicular failure. Testicular failure, either primary (congenital or testicle damage) or secondary (pituitary FSH decrease) can induce increased pituitary production of LH, which, in turn, may induce increased Leydig cell secretion of estradiol. Obesity may result in enhanced conversion of estrogen precursors to estrogen in peripheral tissues. Adrenal hyperactivity, cirrhosis, and chronic renal failure on dialysis therapy may be associated with gynecomastia in some patients due to increased estrogen formation from circulating androgenic precursors. Ten percent to 40% of patients with hyperthyroidism may develop gynecomastia due to increase in testosterone-binding protein. Breast carcinoma in males is rare but is always a possibility in unilateral gynecomastia.

    Laboratory tests. Tests most commonly ordered in patients with gynecomastia are listed in the box. These tests screen for certain possible etiologies. Elevated serum LH levels suggest testicular failure; elevated testosterone levels, Leydig cell tumor; elevated serum estradiol levels, either an estrogen-producing tumor or elevated androgen precursors that are converted to estrogens; serum hCG, testicular or certain other tumors; liver function tests, cirrhosis. There is considerable disagreement among endocrinologists regarding how many tests to order for initial screen and what tests to include in the screen. The box contains tests and procedures frequently mentioned that might be useful to nonendocrinologists. Serum prolactin is usually normal.

    Selected Etiologies of Gynecomastia (% of this Category With Gynecomastia)
    Physiologic etiologies
    Neonatal (60%-90%)
    Pubertal (adolescence) (4%-70%; 25% of overall gynecomastia cases)
    Old age (40%)
    Increased estrogen secretion
    Testicular Leydig cell tumor (25%; 3% of overall gynecomastia cases)
    hCG-secreting tumor
    Hepatoma
    Adrenal cortex tumor
    Increased estrogen precursors
    Obesity
    Cirrhosis (50%)
    Hyperthyroidism (10%-40%)
    Recovery from severe chronic disease
    Renal failure on hemodialysis (50%)
    “Refeeding” gynecomastia after malnutrition (15%)
    Deficiency in androgens (8% of overall gynecomastia cases)
    Testicular failure (primary or secondary)
    Klinefelter’s syndrome (30%-50%)
    Androgen resistance syndromes
    Breast carcinoma
    Medication-induced etiologies (10%-20% of overall gynecomastia cases)
    Testosterone inhibitors (spironolactone, cimetidine) Estrogens
    Androgens
    Others (methyldopa, isoniazid, various psychotropic medications, cytoxins, digitalis, vitamin E
    in large doses, reserpine, ketoconazole)
    Idiopathic (25% of overall gynecomastia cases)

    Possible Workup for Gynecomastia
    Palpation of breast for tumor and examination of testis for tumor
    Medication history
    Inquire about chronic disease; especially dialysis for chronic renal disease, chronic liver disease, or refeeding conditions
    Initial test screen
    Serum estrogen (estradiol)
    Serum free testosterone
    Serum hCG
    Serum LH
    Additional tests (only if indicated)
    Serum androstenedione
    Thyroxine
    Gamma-glutamyltransferase
    Urine 17-KS

  • Female Hirsitism

    Female hirsutism is a relatively common problem in which the overriding concern of the physician is to rule out an ovarian or adrenal tumor. The type and distribution of hair can be used to differentiate physiologic hair growth from nonphysiologic growth (hirsutism). In females there are two types of hair: fine, thin, nonpigmented vellus hair and coarse, curly, pigmented terminal hair. Pubic and axillary terminal hair growth is induced by androgens in the physiologic range. Growth of such hair on the face (especially the chin), sternum area, and sacrum suggests male distribution and therefore excess androgens. Virilization goes one step further and is associated with definite hirsutism, acne, deepening of the voice, and hypertrophy of the clitoris.

    The major etiologies of hirsutism are listed in the box. Hirsutism with or without other evidence of virilization may occur before or after puberty. When onset is prepubertal, the etiology is more often congenital adrenal hyperplasia or tumor. After puberty, PCO disease, Cushing’s syndrome, late-onset congenital adrenal hyperplasia, and idiopathic causes are more frequent. Findings suggestive of tumor are relatively sudden onset; progressive hirsutism, especially if rapidly progressive; and signs of virilization.

    Laboratory tests

    There is considerable disagreement among endocrinologists regarding which tests are most useful in hirsutism. The tests most often used are urine 17-KS, serum testosterone (total or free testosterone), DHEA-S, serum androstenedione, serum dihydrotestosterone, urine 3-a-androstanediol glucuronide, serum LH and FSH, serum prolactin, serum 17-OH-P, urine free cortisol, an ACTH stimulation (“Cortrosyn”) test, and the low-dose overnight dexamethasone suppression test. Each of these tests is used either to screen for one or more etiologies of hirsutism or to differentiate between several possible sources of abnormality. There is considerable disagreement concerning which tests to use for initial screening purposes, with some endocrinologists ordering only two or three tests and others using a panel of five to seven tests. Therefore, it may be worthwhile to briefly discuss what information each test can provide.

    Some Conditions That Produce Hirsutism
    Ovary
    PCO disease
    (Hyperthecosis)
    Ovarian tumor
    Adrenal
    Congenital adrenal hyperplasia (CAH)
    Cushing’s syndrome (nontumor)
    Adrenal tumor
    Testis
    Leydig cell tumor
    Other
    Idiopathic hirsutism
    Hyperprolactinemia
    Starvation
    Acromegaly (rare)
    Hypothyroidism (rare)
    Porphyria (rare)
    Medications
    Phenytoin (Dilantin)
    Diazoxide (Hyperstat)
    Minoxidil(Loniten)
    Androgenic steroids
    Glucocorticosteroids
    Streptomycin

    Urine 17-KS was one of the first tests in hirsutism or virilization. Elevated urine 17-KS levels suggest abnormality centered in the adrenal, usually congenital adrenal hyperplasia (CAH) or adrenal tumor. Ovarian tumors, PCO disease, or Cushing’s syndrome due to pituitary adenoma usually do not produce elevated urine 17-KS levels (although some exceptions occur). However, some cases of CAH fail to demonstrate elevated 17-KS levels (more often in the early pediatric age group and in late-onset CAH). Serum 17-OH-P in neonatal or early childhood CAH and 17-OH-P after ACTH stimulation in late-onset CAH are considered more reliable diagnostic tests. Also, a significant minority (16% in one series) of patients with adrenal carcinoma and about 50% of patients with adrenal adenomas fail to show elevated 17-KS levels. In addition, 17-KS values from adrenal adenoma and carcinoma overlap, although carcinoma values in general are higher than adenoma values. The overnight low-dose dexamethasone test is more reliable to screen for Cushing’s syndrome of any etiology (including tumor) than are urine 17-KS levels, and the 24-hour urine free cortisol assay is a little more reliable than the overnight dexamethasone test (see Chapter 30). At present most endocrinologists do not routinely obtain 17-KS levels.

    Serum testosterone was discussed earlier. Testosterone levels are elevated (usually to mild degree) in more than one half of the patients with PCO disease and in patients with Leydig cell tumors or ovarian testosterone-producing tumors. Elevated serum testosterone suggests a problem of ovarian origin because the ovaries normally produce 15%-30% of circulating testosterone; and in addition, the ovary produces about one half of circulating androstenedione from adrenal DHEA and this androstenedione is converted to testosterone in peripheral tissues. On the other hand, testosterone is produced in peripheral tissues and even in the adrenal as well as in the ovary. Serum testosterone levels therefore may be elevated in some patients with nonovarian conditions such as idiopathic hirsutism. These multiple origins make serum testosterone the current best single screening test in patients with hirsutism. Several investigators have found that serum free testosterone levels are more frequently elevated than serum total testosterone levels in patients with hirsutism, although a lesser number favor total testosterone. Possible false results in total testosterone values due to changes in testosterone-binding protein is another point in favor of free testosterone. Some investigators have reported that serum androstenedione and DHEA-S levels are elevated in some patients with free testosterone values within reference range and that a test panel (e.g., free testosterone, androstenedione, dihydrotestosterone, and DHEA-S) is the most sensitive means of detecting the presence of excess androgens (reportedly with a sensitivity of 80%-90%).

    Dehydroepiandrosterone-sulfate (DHEA-S) is produced entirely in the adrenal from adrenal DHEA. Therefore, elevated DHEA-S levels suggest that at least some excess androgen is coming from the adrenal. The DHEA-S test is also more sensitive for adrenal androgen excess than the urine 17-KS test. A minor difficulty is that a certain number of patients with PCO disease (in which the ovary is supposed to be the source of excess androgen production) and also certain patients with idiopathic hirsutism have some DHEA-S evidence of adrenal androgen production. For example, one third of PCO disease patients in one series were found to have increased DHEA-S levels. Although this was interpreted to mean that some adrenal factor was present as well as the ovarian component, it tends to confuse the diagnosis. Another difficulty is that some infertile women without hirsutism have been reported to have elevated DHEA-S levels. Androstenedione, as noted previously, is a metabolite of adrenal DHEA that is produced about equally in the adrenal and in the ovaries but then reaches peripheral tissues where some is converted to testosterone. Therefore, elevated serum androstenedione levels indicate abnormality without localizing the source. Dihydrotestosterone (DHT) is a metabolite of testosterone and is formed mainly in peripheral tissues. Therefore, elevated DHT levels suggest origin from tissues other than the ovaries or adrenals. Androstanediol glucuronide is a metabolite of DHT and has the same significance. Some investigators report that it is elevated more frequently than DHT. Serum prolactin levels are usually elevated in prolactin-producing pituitary tumors (prolactinoma). These tumors are said to be associated with hirsutism in about 20% of cases. The mechanism is thought to be enhancement of ACTH effect on formation of DHEA. However, it has also been reported that up to 30% of patients with PCO disease have mildly elevated serum prolactin levels (elevated < 1.5 times the upper limit of the reference range). Prolactinomas are more likely in patients with irregular menstrual periods.

    Luteinizing hormone and FSH are useful in diagnosis of PCO disease, which typically (although not always) shows elevated LH levels, with or without elevated FSH levels.

    Urine free cortisol or low-dose overnight dexamethasone test are both standard tests used for diagnosis of Cushing’s syndrome, which is another possible cause of hirsutism.

    Serum 17-OH-P is used to diagnose CAH. Levels of the 17-OH-P specimens drawn between 7 A.M. and 9 A.M. are elevated in nearly all patients with CAH who have symptoms in the neonatal period or in early childhood. However, in late-onset CAH that becomes clinically evident in adolescence or adulthood, 17-OH-P levels may or may not be elevated. The most noticeable symptom of late-onset CAH is hirsutism. Reports indicate that about 5%-10% (range, 1.5%-30%) of patients with hirsutism have late-onset CAH. The most effective test for diagnosis of late-onset CAH is an ACTH (Cortrosyn) stimulation test. A baseline 17-OH-P blood specimen is followed by injection of 25 units of synthetic ACTH, and a postdose specimen is drawn 30 minutes after IV injection or 60 minutes after intramuscular injection. Exact criteria for interpretation are not frequently stated. However, comparison of test results in the literature suggests that an abnormal response to ACTH consists of 17-OH-P values more than 1.6 times the upper limit of the normal 17-OH-P range before ACTH. An “exaggerated” response appears to be more than 3 times the upper limit of the pre-ACTH normal range. In homozygous late-onset CAH there is an exaggerated 17-OH-P response to ACTH. CAH heterozygotes may have a normal response to ACTH or may have an abnormal response to ACTH that falls between a normal response and an exaggerated response.

    Suppression tests, such as a modified dexamethasone suppression test with suppression extended to 7-14 days, have been advocated in the past to differentiate between androgens of adrenal and ovarian origin. Dexamethasone theoretically should suppress nontumor androgen originating in the adrenal. However, studies have shown that the extended dexamethasone suppression test may be positive (i.e., may suppress androgen levels to < 50% of baseline) in some patients with PCO disease, and the test is no longer considered sufficiently dependable to localize the origin of increased androgen to either the adrenal or the ovary alone.

    Radiologic visualization of abdominal organs is helpful if PCO disease, ovarian tumor, or adrenal tumor is suspected. Ultrasound examination of the ovaries and CT of the adrenals are able to detect some degree of abnormality in most (but not all) patients.

    Polycystic ovary (PCO) disease

    PCO disease is considered by some to be the most common cause of female postpubertal hirsutism. It is also an important cause of amenorrhea, oligomenorrhea, and female sterility. PCO disease is defined both clinically and by histopathology. The classic findings at operation are bilaterally enlarged ovaries (about 65%-76% of cases), which on pathologic examination have a thickened capsule and numerous small cysts (representing cystic follicles) beneath the capsule. However, PCO disease is considered to have a spectrum of changes in which there is decreasing ovarian size and a decreasing number of cysts until the ovaries are normal in size (about 25%-35% of cases) with few, if any, cysts but with an increased amount of subcapsular and interstitial stroma. There is also a condition called “hyperthecosis” in which the thecal cells of the stroma are considerably increased and have a luteinized appearance. Some consider hyperthecosis a separate entity; some include it in PCO disease but consider it the opposite end of the spectrum from the polycystic ovary type; and some combine PCO disease and hyperthecosis together under a new name, “sclerocystic ovary syndrome.”

    Clinically, there is considerable variety in signs and symptoms. The classic findings were described by Stein and Leventhal, and the appellation Stein-Leventhal syndrome can be used to distinguish patients with classic findings from patients with other variants of PCO disease. The Stein-Leventhal subgroup consists of women who have bilaterally enlarged polycystic ovaries and who are obese, are hirsute without virilization, have amenorrhea, and have normal urine 17-KS levels. Other patients with PCO disease may lack one or more of these characteristics. For example, only about 70% of patients have evidence of hirsutism (literature range, 17%-95%). Some patients with PCO disease have hypertension, and some have abnormalities in glucose tolerance. A few have virilization, which is said to be more common in those with hyperthecosis.

    Laboratory tests. Laboratory findings in PCO disease are variable, as are the clinical findings. In classic Stein-Leventhal cases, serum testosterone levels are mildly or moderately elevated in about 50% of patients, and other androgen levels are elevated in many patients whose serum testosterone level remains within reference range. Free testosterone levels are elevated more frequently than total testosterone levels. Higher testosterone values tend to occur in hyperthecosis. Most investigators consider the increased androgen values to be derived mainly from the ovary, although an adrenal component has been found in some patients. Although urine 17-KS levels are usually normal, they may occasionally be mildly increased. The most characteristic finding in PCO is an elevated serum LH level with FSH levels that are normal or even mildly decreased. However, not all patients with PCO show this gonadotropin pattern.

    Summary of tests in hirsutism

    Most endocrinologists begin the laboratory investigation of hirsutism with a serum testosterone assay. Many prefer free testosterone rather than total testosterone. The number and choice of additional tests is controversial. Additional frequently ordered screening tests include serum DHEA-S, serum DHT, and the ACTH-stimulated 17-OH-P test. If abnormality is detected in one or more of these tests, additional procedures can help to find which organ and disease is responsible.

  • Secondary Amenorrhea

    Secondary amenorrhea implies ending of menstruation after menstruation has already begun. A list of etiologies of secondary amenorrhea is presented in the box, grouped according to evidence of ovarian function (from tests discussed in the section on primary amenorrhea). If menstruation definitely takes place, especially if it continues for several months or years, this eliminates some of the etiologies of primary amenorrhea such as m;auullerian dysgenesis and most types of male pseudohermaphroditism. Otherwise, the list of possible causes and the workup are similar to that for primary amenorrhea. Even chromosome studies are still useful; in this case, to rule out Turner’s syndrome or its variants. Certain conditions are much more important or frequent in secondary, as opposed to primary, amenorrhea; these include hyperprolactinemia (with or without galactorrhea), early menopause, psychogenic cause, anorexia nervosa, severe chronic illness, and hypothyroidism. Normal pregnancy should also be excluded.
    Hyperprolactinemia is found in 20%-30% of patients with secondary amenorrhea. There may or may not be galactorrhea. The condition may be idiopathic, may be due to pituitary tumor, or sometimes develops when a person taking oral contraceptives stops the medication or in the postpartum period.
    Some cases of periodic amenorrhea or varying time periods without menstruation are due to anovulation. Anovulation is said to cause up to 40% of all female infertility. Some of these patients have enough estrogen production to permit the endometrium to reach the secretory phase, so these women will respond to a dose of progesterone (“progesterone challenge”) by menstruating. These women frequently will ovulate if treated with clomiphene, which has an antiestrogen action on the hypothalamus. Other patients will not respond to progesterone challenge and usually do not respond to clomiphene. Some of these women have hypothalamic function deficiency, pituitary failure, exercise-induced (e.g., rigorous athletic training) or stress-related amenorrhea, anorexia nervosa, or ovarian failure.

    Some Etiologies of Secondary Amenorrhea Classified According to Ovarian Function
    I. Pregnancy
    II. Decreased ovarian hormone production
    A. Primary ovarian failure
    1. Normal menopause
    2. Some cases of gonadal dysgenesis (Turner’s syndrome)
    3. Acquired ovarian failure (idiopathic, postradiation, postchemotherapy, postmumps infection)
    4. PCO disease
    B. Secondary ovarian failure
    1. High pituitary prolactin value (with or without galactorrhea)
    2. Hypothalamic failure to secrete adequate GTRH levels (tumor, trauma, infection, nonintracranial illness)
    3. Pituitary failure (primary pituitary tumor, Sheehan’s syndrome, empty sella syndrome)
    4. Anorexia nervosa or severe malnutrition
    5. Rigorous athletic training
    6. Psychogenic amenorrhea
    7. Severe acute or chronic illness
    8. Increased nonovarian estrogen levels (tumor, obesity, therapy)
    9. Hypothyroidism or hyperthyroidism
    10. Cushing’s disease or Addison’s disease
    11. Poorly controlled diabetes
    III. Increased ovarian estrogen or androgen production
    A. Androgen-secreting ovarian tumor
    B. Estrogen-secreting ovarian tumor
    IV. Normal ovarian hormone production
    A. Local uterine pathology

  • Female Delayed Puberty and Primary Amenorrhea

    Onset of normal puberty in girls is somewhat variable, with disagreement in the literature concerning at what age to diagnose precocious puberty and at what age to suspect delayed puberty. The most generally agreed-on range of onset for female puberty is between 9 and 16 years. Signs of puberty include breast development, growth of pubic and axillary hair, and estrogen effect as seen in vaginal smears using the Papanicolaou stain. Menstruation also begins; if it does not, the possibility of primary amenorrhea arises. Primary amenorrhea may or may not be accompanied by evidence that suggests onset of puberty, depending on the etiology of the amenorrhea. Some of the causes of primary amenorrhea are listed in the box.

    Some Etiologies of Female Delayed Puberty and Primary Amenorrhea
    Mullerian dysgenesis (Mayer-Rokitansky syndrome): congenital absence of portions of the female genital tract, with absent or hypoplastic vagina. Ovarian function is usually normal. Female genotype and phenotype.
    Male pseudohermaphroditism: genetic male (XY karotype) with female appearance due to deficiency of testosterone effect.
    1. Testicular feminization syndrome: lack of testosterone effect due to defect in tissue testosterone receptors.
    2. Congenital adrenal hyperplasia: defect in testosterone production pathway.
    3. Male gonadal dysgenesis syndromes: defect in testis function.
    a. Swyer syndrome (male pure gonadal dysgenesis): female organs present except for ovaries. Bilateral undifferentiated streak gonads. Presumably testes never functioned and did not prevent mьllerian duct development.
    b. Vanishing testes syndrome (XY gonadal agenesis): phenotype varies from male pseudohermaphrodite to ambiguous genitalia. No testes present. Presumably testes functioned in very early embryologic life, sufficient to prevent mьllerian duct development, and then disappeared.
    c. Congenital anorchia: male phenotype, but no testes. Presumably, testes functioned until male differentiation took place, then disappeared.
    Female sex chromosome abnormalities (Turner’s syndrome—female gonadal dysgenesis— and Turner variants): XO karyotype in 75%-80% of patients, mosaic in others. Female phenotype. Short stature in most, web neck in 40%. Bilateral streak gonads.
    Polycystic (Stein-Leventhal) or nonpolycystic ovaries, not responsive to gonadotropins.
    Deficient gonadotropins due to hypothalamic or pituitary dysfunction.
    Hyperprolactinemia: pituitary overproduction of prolactin alone.
    Effects of severe chronic systemic disease: chronic renal disease, severe chronic GI disease, anorexia nervosa, etc.
    Constitutional (idiopathic) delayed puberty: puberty eventually takes place, so this is a retrospective diagnosis.
    Other: Cushing’s syndrome, hypothyroidism, and isolated GH deficiency can result in delayed puberty.

    Physical examination is very important to detect inguinal hernia or masses that might suggest the testicular feminization type of male pseudohermaphroditism, to document the appearance of the genitalia, to see the pattern of secondary sex characteristics, and to note what evidence of puberty exists and to what extent. Pelvic examination is needed to ascertain if there are anatomical defects preventing menstruation, such as a nonpatent vagina, and to detect ovarian masses.

    Laboratory tests

    Basic laboratory tests begin with chromosome analysis, since a substantial minority of cases have a genetic component. Male genetic sex indicates male pseudohermaphroditism and leads to tests that differentiate the various etiologies. Turner’s syndrome and other sex chromosome disorders are also excluded or confirmed. If there is a normal female karyotype and no chromosome abnormalities are found, there is some divergence of opinion on how to evaluate the other important organs of puberty—the ovaries, pituitary, and hypothalamus. Some prefer to perform serum hormone assays as a group, including pituitary gonadotropins (FSH and LH), estrogen (estradiol), testosterone, prolactin, and thyroxine. Others perform these assays in a step-by-step fashion or algorithm, depending on the result of each test, which could be less expensive but could take much more time. Others begin with tests of organ function. In the algorithm approach, the first step is usually to determine if estrogen is present in adequate amount. Vaginal smears, serum estradiol level, endometrial stimulation with progesterone (for withdrawal bleeding), and possibly endometrial biopsy (for active proliferative or secretory endometrium, although biopsy is considered more often in secondary than in primary amenorrhea)—all are methods to detect ovarian estrogen production. If estrogen is present in adequate amount, this could mean intact hypothalamic-pituitary-ovarian feedback or could raise the question of excess androgen (congenital adrenal hyperplasia, Cushing’s syndrome, PCO disease, androgen-producing tumor) or excess estrogens (obesity, estrogen-producing tumor, iatrogenic, self-medication). If estrogen effect is absent or very low, some gynecologists then test the uterus with estrogen, followed by progesterone, to see if the uterus is capable of function. If no withdrawal bleeding occurs, this suggests testicular feminization, congenital absence or abnormality of the uterus, or the syndrome of intrauterine adhesions (Asherman’s syndrome). If uterine withdrawal bleeding takes place, the uterus can function, and when this finding is coupled with evidence of low estrogen levels, the tentative diagnosis is ovarian failure.

    The next step is to differentiate primary ovarian failure from secondary failure caused by pituitary or hypothalamic disease. Hypothalamic dysfunction may be due to space-occupying lesions (tumor or granulomatous disease), infection, or (through uncertain mechanism) effects of severe systemic illness, severe malnutrition, and severe psychogenic disorder. X-ray films of the pituitary are often ordered to detect enlargement of the sella turcica from pituitary tumor or suprasellar calcifications due to craniopharyngioma. Polytomography is more sensitive for sellar abnormality than ordinary plain films. CT also has its advocates. Serum prolactin, thyroxine, FSH, and LH assays are done. It is necessary to wait 4-6 weeks after a progesterone or estrogen-progesterone test before the hormone assays are obtained to allow patient hormone secretion patterns to resume their pretest status. An elevated serum prolactin value raises the question of pituitary tumor (especially if the sella is enlarged) or idiopathic isolated hyperprolactinemia. However, patients with the empty sella syndrome and some patients with hypothyroidism may have an enlarged sella and elevated serum prolactin levels, and the other causes of elevated prolactin levels (see the box) must be considered. A decreased serum FSH or LH level confirms pituitary insufficiency or hypothalamic dysfunction. A pituitary stimulation test can be performed. If the pituitary can produce adequate amounts of LH, this suggests hypothalamic disease (either destructive lesion, effect of severe systemic illness, or malnutrition). However, failure of the pituitary to respond does not necessarily indicate primary pituitary disease, since severe long-term hypothalamic deficiency may result in temporary pituitary nonresponsiveness to a single episode of test stimulation. Hormone therapy within the preceding 4-6 weeks can also adversely affect test results.

    The box lists some conditions associated with primary amenorrhea or delayed puberty and the differential diagnosis associated with various patterns of pituitary gonadotropin values. However, several cautionary statements must be made about these patterns. Clearly elevated FSH or LH values are much more significant than normal or mildly to moderately decreased levels. Current gonadotropin immunoassays are technically more reproducible and dependable at the upper end of usual values than at the lower end. Thus, two determinations on the same specimen could produce both a mildly decreased value and a value within the lower half of the reference range. In addition, blood levels of gonadotropins, especially LH, frequently vary throughout the day. The values must be compared with age- and sexmatched reference ranges. In girls, the levels also are influenced by the menstrual cycle, if menarche has begun. Second, the conditions listed—even the genetic ones, although to a lesser extent—are not homogeneous in regard to severity, clinical manifestations, or laboratory findings. Instead, each represents a spectrum of patients. The more classic and severe the clinical manifestations, the more likely that the patient will have “expected” laboratory findings, but even this rule is not invariable. Therefore, some patients having a condition typically associated with an abnormal laboratory test result may not show the expected abnormality. Laboratory error is another consideration. Also, the spectrum of patients represented by each condition makes it difficult to evaluate reports of laboratory findings due to differences in patient population, severity of illness, differences in applying diagnostic criteria to the patients, variance in specimen collection protocols, and technical differences in the assays used in different laboratories. In many cases, adequate data concerning frequency that some laboratory tests are abnormal are not available.

    Gonadotropin Levels in Certain Conditions Associated With Primary Amenorrhea or Delayed Puberty
    FSH and LH decreased*
    Hypopituitarism
    Hypothalamic dysfunction
    Constitutional delayed puberty
    Some cases of primary hypothyroidism
    Some cases of Cushing’s syndrome
    Some cases of severe chronic illness
    FSH and LH increased†
    Some cases of congenital adrenal hyperplasia
    Female gonadal dysgenesis
    Male gonadal dysgenesis
    Ovarian failure due to nonovarian agents
    LH increased, FSH not increased‡
    Testicular feminization
    Some cases of PCO disease

    Elevated FSH and LH levels or an elevated LH level alone suggests primary ovarian failure, whether from congenital absence of the ovaries or congenital inability to respond to gonadotropins, acquired abnormality such as damage to the ovaries after birth, or PCO disease.

    An elevated estrogen or androgen level raises the question of hormone-secreting tumor, for which the ovary is the most common (but not the only) location. Nontumor androgen production may occur in PCO disease and Cushing’s syndrome (especially adrenal carcinoma).

    As noted previously, there is no universally accepted single standard method to investigate female reproductive disorders. Tests or test sequences vary among medical centers and also according to findings in the individual patients.

    When specimens for pituitary hormone assays are collected, the potential problems noted earlier in the chapter should be considered.

  • Precocious Puberty

    Precocious puberty can be isosexual (secondary sex characteristics of the same sex) or heterosexual (masculinization in girls and feminization in boys). The syndromes have been further subdivided into those that produce true puberty with functioning gonads and those that produce pseudopuberty, in which development of secondary sex characteristics suggests puberty but the gonads do not function. The major causes of precocious puberty are idiopathic early puberty or the hyper secretion of androgens or estrogens from hypothalamic, pituitary, adrenal, gonadal, or factitious (self-medication) sources.

    Hypothalamic precocious puberty is usually associated with a tumor (e.g., a pinealoma) involving the hypothalamus or with encephalitis. The result is always an isosexual true puberty.

    Pituitary precocious puberty is usually idiopathic (“constitutional”). The result is normal isosexual puberty. In girls the condition rarely may be due to Albright’s syndrome (polyostotic fibrous dysplasia). Primary pituitary tumors do not cause precocious puberty.

    Gonadal precocious puberty in boys results from a Leydig cell tumor of the testis that produces testosterone. This is a pseudopuberty, since FSH is not produced and no spermatogenesis occurs. In girls, various ovarian tumors may cause pseudopuberty, which may be either isosexual or heterosexual, depending on whether the tumor secretes estrogens or androgens. Granulosa-theca cell tumors are the most frequent, although their peak incidence is not until middle age. These tumors most frequently produce estrogens. Dysgerminoma and malignant teratoma are two uncommon ovarian tumors that may produce hCG rather than the usual type of estrogens.

    Adrenal precocious puberty is due to either congenital adrenal hyperplasia or adrenal tumor. In early childhood, congenital adrenal hyperplasia is more likely; if onset is in late childhood or prepubertal age, tumor is more likely. In both cases a pseudopuberty results, usually isosexual in boys and heterosexual (virilization) in girls. Congenital adrenal hyperplasia may also produce pseudohermaphroditism (simulation of male genitalia) in infant girls.

    Hypothyroidism has been reported to cause rare cases of true isosexual precocious puberty in both boys and girls.

    Laboratory evaluation

    Laboratory evaluation depends on whether the physical examination indicates isosexual or heterosexual changes. In girls, heterosexual changes point toward congenital adrenal hyperplasia, ovarian tumor, or adrenal carcinoma. The most important test for diagnosis of congenital adrenal hyperplasia is plasma 17-hydroxyprogesterone (17-OH-P) assay. A 24-hour urine specimen for 17-ketosteroids (17-KS) is also very helpful, since the 17-KS values are elevated in 80%-85% adrenal carcinoma, frequently elevated in congenital adrenal hyperplasia, and sometimes elevated in ovarian carcinoma. If test results for congenital adrenal hyperplasia are negative, normal urine 17-KS levels point toward ovarian tumor. Ultrasound examination of the ovaries and adrenals and an overnight dexamethasone screening test for adrenal tumor are helpful. Computerized tomography (CT) of the adrenals is also possible, but some believe that CT is less successful in childhood than in adults. Isosexual changes suggest hypothalamic area tumor, constitutional precocious puberty, ovarian estrogen-secreting tumor, or hypothyroidism. Useful tests include serum thyroxine assay, ultrasound examination of the ovaries, and CT scan of the hypothalamus area. The possibility of exposure to estrogen-containing creams or other substances should also be investigated. Serum hCG levels may be elevated in the rare ovarian choriocarcinomas.

    Female isosexual precocious puberty must be distinguished from isolated premature development of breasts (thelarche) or pubic hair (adrenarche). Female heterosexual precocious puberty must be distinguished from hirsutism, although this is a more common problem during and after puberty.

    In boys, heterosexual precocious pseudopuberty suggests estrogen-producing tumor (testis choriocarcinoma or liver hepatoblastoma) or the pseudohermaphrodite forms of congenital adrenal hyperplasia. Tests for congenital adrenal hyperplasia plus serum hCG are necessary. Isosexual precocious puberty could be due to a hypothalamic area tumor, constitutional (idiopathic) cause, testis tumor (Leydig cell type), hypothyroidism, or congenital adrenal hyperplasia. Necessary tests include those for congenital adrenal hyperplasia and a serum thyroxine determination. If the results are negative, serum testosterone (to detect values elevated above normal puberty levels) and hypothalamic area CT scan are useful. Careful examination of the testes for tumor is necessary in all cases of male precocious puberty.

  • Male Infertility or Hypogonadism

    About 40%-50% of infertility problems are said to be due to dysfunction of the male reproductive system. Male infertility can be due to hormonal etiology (either lack of gonadotropin or suppression of spermatogenesis), nonhormonal factors affecting spermatogenesis, primary testicular disease, obstruction to sperm passages, disorders of sperm motility or viability or presence of antisperm antibodies (see the box on this page). About 30%-40% is reported to be associated with varicocele. Diagnosis and investigation of etiology usually begins with physical examination (with special attention to the normality of the genitalia and the presence of a varicocele), semen analysis (ejaculate quantity, number of sperm, sperm morphology, and sperm motility), and serum hormone assay (testosterone, LH, and FSH).

    Some Important Causes of Male Infertility, Classified According to Primary Site of the Defect

    Hormonal
    Insufficient hypothalamic gonadotropin (hypogonadotropic eunuchoidism)
    Insufficient pituitary gonadotropins (isolated LH deficiency [“fertile eunuch”] or pituitary
    insufficiency)
    Prolactin-secreting pituitary adenoma
    Excess estrogens (cirrhosis, estrogen therapy, estrogen-producing tumor)
    Excess androgens
    Excess glucocorticosteroids
    Hypothyroidism
    Nonhormonal factors affecting testis sperm production
    Varicocele
    Poor nutrition
    Diabetes mellitus
    Excess heat in area of testes
    Stress and emotion
    Drugs and chemicals
    Febrile illnesses
    Cryptochism(undescended testis, unilateral or bilateral)
    Spinal cord injuries
    Primary testicular abnormality
    Maturation arrest at germ cell stage
    “Sertoli cell only” syndrome
    Klinefelter’s syndrome and other congenital sex chromosome disorders
    Testicular damage (radiation, mumps orchitis, inflammation, trauma)
    Myotonic dystrophy
    Posttesticular abnormality
    Obstruction of sperm passages
    Impaired sperm motility
    Antisperm antibodies

    Testicular function tests. Male testicular function is based on formation of sperm by testicular seminiferous tubules under the influence of testosterone. FSH is necessary for testicular function because it stimulates seminiferous tubule (Sertoli cell) development. Testosterone secretion by the Leydig cells (interstitial cells) of the testis is necessary for spermatogenesis in seminiferous tubules capable of producing sperm. LH (in males sometimes called “interstitial cell-stimulating hormone”) stimulates the Leydig cells to produce testosterone. Testosterone is controlled by a feedback mechanism whereby testosterone levels regulate hypothalamic secretion of GnRH, which, in turn, regulates pituitary secretion of LH. The adrenals also produce androgens, but normally this is not a significant factor in males. In classic cases, serum levels of testosterone and gonadotropins (LH and FSH) can differentiate between primary testicular abnormality (failure of the testis to respond to pituitary gonadotropin stimulation, either congenital or acquired) and pituitary or hypothalamic dysfunction (either congenital or acquired). In primary gonadal (testis) insufficiency, pituitary gonadotropin levels are usually elevated. Of the various categories of primary gonadal insufficiency, the Klinefelter’s xxy chromosome syndrome in its classic form shows normal FSH and LH gonadotropin levels during childhood, but after puberty both FSH and LH levels are elevated and the serum testosterone level is low. Klinefelter’s syndrome exists in several variants, however, and in some cases LH levels may be within reference range (single LH samples also may be confusing because of the pulsatile nature of LH secretion). Occasionally patients with Klinefelter’s syndrome have low-normal total serum testosterone levels (due to an increase in SHBG levels) but decreased free testosterone levels. Elevated pituitary gonadotropin levels should be further investigated by a buccal smear for sex chromatin to elevate the possibility of Klinefelter’s syndrome. Some investigators may perform a chromosome analysis because of possible inaccuracies in buccal smear interpretation and because some persons with Klinefelter’s syndrome have cell mosaicism rather than the same chromosome pattern in all cells, or a person with findings suggestive of Klinefelter’s syndrome may have a different abnormal chromosome karyotype. In the “Sertoli cell only syndrome,” testicular tubules are abnormal but Leydig cells are normal. Therefore, the serum FSH level is elevated but LH and testosterone levels are usually normal. In acquired gonadal failure (due to destruction of the testicular tubules by infection, radiation, or other agents), the FSH level is often (but not always) elevated, whereas LH and testosterone levels are often normal (unless the degree of testis destruction is sufficient to destroy most of the Leydig cells, a very severe change). In secondary (pituitary or hypothalamic) deficiency, both the pituitary gonadotropin (FSH and LH) and the testosterone levels are low.

    Although serum testosterone is normally an indicator of testicular Leydig cell function and, indirectly, of pituitary LH secretion, other factors can influence serum testosterone levels. The adrenal provides much of the precursor steroids for testosterone, and some types of congenital adrenal hyperplasia result in decreased levels of testosterone precursors. Cirrhosis may decrease serum testosterone levels. Increase or decrease of testosterone-binding protein levels may falsely increase or decrease serum testosterone levels, since the total serum testosterone value is being measured, whereas assay of free (unbound) testosterone is not affected.

    Stimulation tests. Stimulation tests are available to help determine which organ is malfunctioning in equivocal cases.

    1. HCG directly stimulates the testis, increasing testosterone secretion to at least twice baseline values.
    2. Clomiphene stimulates the hypothalamus, producing increases in both LH and testosterone levels. The medication is given for 10 days. However, clomiphene does not stimulate LH production significantly in prepubertal children. Serum testosterone and LH values must be close to normal pubertal or adult levels before the test can be performed, and the test can be used only when there is a mild degree of abnormality in the gonadal-pituitary system.
    3. The GnRH stimulation test can directly evaluate pituitary capability to produce LH and FSH and can indirectly evaluate testicular hormone-producing function that would otherwise depend on measurement of basal testosterone levels. Normally, pituitary LH stimulates testosterone production from Leydig cells of the testis, and the amount of testosterone produced influences hypothalamic production of GnRH, which controls pituitary LH. When exogenous (test) GnRH is administered, the pituitary is stimulated to produce LH. The release of testosterone from the testis in response to LH stimulation inhibits further LH stimulation to some degree. In most male patients with primary gonadal failure, basal serum LH and FSH become elevated due to lack of inhibitory feedback from the testis. However, some patients may have basal serum LH and FSH levels in the lower part of the population reference range, levels that are indistinguishable from those of some normal persons; although preillness values were higher in these patients, their preillness normal levels are rarely known. In these patients, a GnRH stimulation test may be useful. Some investigators have found that the degree of inhibition of LH production in response to GnRH administration is more sensitive in detecting smaller degrees of testicular hormone production insufficiency than basal LH levels. The decreased testosterone levels decrease feedback inhibition on the hypothalamus and administration of GnRH results in an exaggerated LH or FSH response (markedly elevated LH and FSH levels compared to levels in normal control persons). The test is performed by obtaining a baseline serum specimen for LH and FSH followed by an intravenous (IV) bolus injection of 100 µg of synthetic GnRH. Another serum specimen is obtained 30 minutes later for LH and FSH. Certain factors can influence or interfere with GnRH results. Estrogen administration increases GnRH effect by sensitizing the pituitary, and androgens decrease the effect. Patient sex and age (until adult pulsatile secretion schedule is established) also influence test results. When used as a test for pituitary function, some patients with clinically significant degrees of failure show a normal test result; therefore, only an abnormal result is definitely significant.

    Semen analysis. Semen analysis is an essential part of male infertility investigation. Semen findings that are highly correlated with hypofertility or infertility include greatly increased or decreased ejaculate volume, very low sperm counts, greatly decreased sperm motility, and more than 20% of the sperm showing abnormal morphology. However, the World Health Organization (WHO) recently changed its definition of semen morphologic abnormality, and now considers a specimen abnormal if more than 70% of the sperm have abnormal morphology. One particular combination of findings is known as the “stress pattern” and consists of low sperm count, decreased sperm motility, and more than 20% of the sperm being abnormal in appearance (especially with an increased number of tapering head forms instead of the normal oval head). The stress pattern is suggestive of varicocele but can be due to acute febrile illness (with effects lasting up to 60 days), some endocrine abnormalities, and antisperm agents. An abnormal semen analysis should be repeated at least once after 3 weeks and possibly even a third time, due to the large variation within the population and variation of sperm production even within the same person as well as the temporary effects of acute illness. Most investigators recommend that the specimen be obtained 2-3 days after regular intercourse to avoid artifacts that may be produced by prolonged sperm storage. The specimen should be received by the laboratory by 1 hour after it is obtained, during which time it should be kept at room temperature.

    Semen analysis: sperm morphology

    Fig. 31-1 Semen analysis: sperm morphology. a, acrosome; b, nucleus (difficult to see); c, post-acrosomal cap; d, neckpiece; e, midpiece (short segment after neckpiece); f, tail. A, normal; B, normal (slightly different head shape); C, tapered head; D, tapered head with acrosome deficiency; E, acrosomal deficiency; F, head vacuole; G, cytoplasmic extrusion mass; H, bent head (bend of 45° or more); I, coiled tail; J, coiled tail; K, double tail; L, pairing phenomenon (sperm agglutination); M, sperm precursors (spermatids); N, bicephalic sperm.

    Testicular biopsy. Testicular biopsy may be useful in a minority of selected patients in whom no endocrinologic or other cause is found to explain infertility. Most investigators agree that complete lack of sperm in semen analysis is a major indication for biopsy but disagree on whether biopsy should be done if sperm are present in small numbers. Biopsy helps to differentiate lack of sperm production from sperm passage obstruction and can suggest certain possible etiologies of testicular dysfunction or possible areas to investigate. However, none of the histopathologic findings is specific for any one disease. The report usually indicates if spermatogenesis is seen and, if so, whether it is adequate, whether there are normal numbers of germ cells, and whether active inflammation or extensive scarring is present.

    Serum antisperm antibody studies. In patients with no evidence of endocrine, semen, or other abnormality, serum antisperm antibody titers can be measured. These studies are difficult to perform and usually must be done at a center that specializes in reproductive studies or therapy. Serum antisperm antibodies are a common finding after vasectomy, being reported in about 50% of cases (literature range, 30%-70%). The incidence and significance of serum antisperm antibodies in couples with infertility problems are somewhat controversial. There is wide variance of reported incidence (3.3%-79%), probably due to different patient groups tested and different testing methods. In men, one report suggests that serum antibody titer is more significant than the mere detection of antibody. High titers of antisperm antibody in men were considered strong evidence against fertility; low titers were of uncertain significance, and mildly elevated titers indicated a poor but not hopeless prognosis. In women, serum antisperm antibodies are said to be present in 7%-17% of infertility cases but their significance is rather uncertain, since a considerable percentage of these women can become pregnant. Antisperm antibody detected in the cervical mucus of women is thought to be much more important than that detected in serum. A test showing ability of sperm to bind to mannose may be available.

  • Tests of Gonadal Function

    The most common conditions in which gonadal function tests are used are hypogonadism in males and menstrual disorders, fertility problems, and hirsutism or virilization in females. The hormones currently available for assistance include lutropin (luteinizing hormone; LH), follitropin (follicle-stimulating hormone; FSH), testosterone, human chorionic gonadotropin (hCG), and gonadotropin-releasing hormone (GnRH).

    Gonadal function is regulated by hypothalamic secretion of a peptide hormone known as gonadotropin-releasing hormone (GnRH; also called luteinizing hormone-releasing hormone, LHRH). Secretion normally is in discontinuous bursts or pulses, occurring about every 1.5-2.0 hours (range, 0.7-2.5 hours). GnRH half-life normally is about 2-4 minutes (range, 2-8 minutes). There is little if any secretion until the onset of puberty (beginning at age 8-13 years in females and age 9-14 years in males). Then secretion begins predominately at night but later extends throughout the night into daytime, until by late puberty secretion takes place all 24 hours. GnRH is secreted directly into the circulatory channels between the hypothalamus and pituitary. GnRH causes the basophilic cells of the pituitary to secrete LH and FSH. In males LH acts on Leydig cells of the testis to produce testosterone, whereas FSH stimulates Sertoli cells of the testis seminiferous tubules into spermatogenesis, assisted by testosterone. In females, LH acts on ovarian thecal cells to produce testosterone, whereas FSH stimulates ovarian follicle growth and also causes follicular cells to convert testosterone to estrogen (estradiol). There is a feedback mechanism from target organs to the hypothalamus to control GnRH secretion. In males, testosterone and estradiol inhibit LH secretion at the level of the hypothalamus, and testosterone also inhibits LH production at the level of the pituitary. A hormone called inhibitin, produced by the Sertoli cells of the testis tubules, selectively inhibits FSH at both the hypothalamic and pituitary levels. There appears to be some inhibitory action of testosterone and estradiol on FSH production as well, in some circumstances. In females, there is some inhibitory feedback to the hypothalamus by estradiol.

    Luteinizing hormone and follicle-stimulating hormone

    Originally FSH and LH were measured together using bioassay methods, and so-called FSH measurements included LH. Immunoassay methods can separate the two hormones. The LH cross-reacts with hCG in some RIA systems. Although this ordinarily does not create a problem, there is difficulty in pregnancy and in patients with certain neoplasms that secrete hCG. Several of the pituitary and placental glycopeptides (TSH, FSH, hCG, LH) are composed of at least two immunologic units, alpha and beta. All of these hormones have the same immunologic response to their alpha fraction but a different response to each beta subunit. Antisera against the beta subunit of hCG eliminate interference by LH, and some investigators have reported success in producing antisera against the beta subunit of LH, which does not detect hCG. There is some cross-reaction between FSH and TSH, but as yet this has not seemed important enough clinically in TSH assay to necessitate substitution of TSH beta subunit antiserum for antisera already available. Assay of FSH in serum is thought to be more reliable than in urine due to characteristics of the antibody preparations available.

    Serum luteinizing hormone

    LH is secreted following intermittent stimulation by GnRH in pulses occurring at a rate of about 2-4 every 6 hours, ranging from 30% to nearly 300% over lowest values. Therefore, single isolated LH blood levels may be difficult to interpret and could be misleading. It has been suggested that multiple samples be obtained (e.g., four specimens, each specimen collected at a 20-minute interval from another). The serum specimens can be pooled to obtain an average value. FSH and testosterone have a relatively constant blood level in females.

    Urine luteinizing hormone

    In contrast to serum LH, urine LH is more difficult to obtain, since it requires a 24-hour specimen with the usual problem of complete specimen collection. It also has the disadvantage of artifact due to urine concentration or dilution. The major advantage is averaging of 24-hour secretion, which may prevent misleading results associated with serum assays due to LH pulsatile secretion. Another role for urine LH assay is detection of ovulation during infertility workups. LH is rapidly excreted from plasma into urine, so that a serum LH surge of sufficient magnitude is mirrored in single urine samples. An LH surge precedes ovulation by about 24-36 hours (range, 22-4 hours). Since daily urine specimens are obtained beginning 9-10 days after onset of menstruation (so as not to miss the LH surge preceding ovulation if ovulation should occur earlier than the middle of the patient cycle). It has been reported that the best time to obtain the urine specimen is during midday (11 A.M.-3 P.M.) because the LH surge in serum usually takes place in the morning (5 A.M.-9 A.M.). In one study, the LH surge was detected in 56% of morning urine specimens, 94% of midday specimens, and 88% of evening specimens. In contrast, basal body temperature, another method of predicting time of ovulation, is said to be accurate in only 40%-50% of cases. Several manufacturers have marketed simple immunologic tests for urine LH with a color change endpoint that can be used by many patients to test their own specimens. Possible problems include interference by hCG with some of the LH kits, so that early pregnancy could simulate a LH surge. Since the LH surge usually does not last more than 2 days, positive test results for more than 3 consecutive days suggest some interfering factor. Similar interference may appear in some patients with elevated serum LH levels due to ovarian failure (polycystic ovary [PCO] disease, early menopause, etc.).

    Finally, an estimated 10% of patients have more than one urine LH spike, although the LH surge has the greatest magnitude. It may be necessary to obtain serum progesterone or urine pregnanediol glucuronide assays to confirm that elevation of LH values is actually the preovulation LH surge.

    Urine pregnanediol

    The ovarian corpus luteum formed shortly after ovulation secretes increasing amounts of progesterone. Progesterone or its metabolite pregnanediol glucuronide begins to appear in detectable quantities about 2-3 days after ovulation (3-5 days after the LH surge) and persists until about the middle of the luteal phase that ends in menstruation. Conception is followed by progesterone secretion by the placenta. A negative baseline urine pregnanediol assay before the LH surge followed by a positive pregnanediol test result on at least 1 day of early morning urine specimens obtained 7, 8, and 9 days after the LH surge confirm that the LH surge did occur and was followed by ovulation and corpus luteum formation. Urine specimens are collected in the morning rather than at the LH collection time of midday. At least one manufacturer markets a simple kit for urine pregnanediol assay.

    Problems with interpretation of a positive urine pregnanediol glucuronide assay include the possibility that early pregnancy may be present. Also, 5%-10% of patients throughout their menstrual cycle nonovulatory phase have slightly or mildly increased pregnanediol levels compared to the reference range. A urine sample collected before the LH surge can be tested to exclude or demonstrate this phenomenon.

    Serum androgens

    The most important androgens are dehydroepiandrosterone (DHEA), a metabolite of DHEA called DHEA-sulfate (DHEA-S), androstenedione, and testosterone. DHEA is produced in the adrenal cortex, ovary, and testis (in the adrenal cortex, the precursors of DHEA are also precursors of cortisol and aldosterone, which is not the case in the ovary or testis). DHEA is the precursor of androstenedione, and androstenedione is a precursor both of testosterone and of estrogens (see Fig. 30-2). About 50%-60% of testosterone in normal females is derived from androstenedione conversion in peripheral tissues, about 30% is produced directly by the adrenal, and about 20% is produced by the ovary. DHEA blood levels in females are 3 times androstenedione blood levels and about 10 times testosterone blood levels. In normal males after onset of puberty, testosterone blood levels are twice as high as all androgens combined in females. Androstenedione blood levels in males are about 60% of those in females and about 10%-15% of testosterone blood levels.

    Serum testosterone

    About 60%-75% of circulating serum testosterone is bound to a beta globulin variously known as sex hormone-binding globulin (SHBG) or as testosterone-binding globulin (TBG, a misleading abbreviation because of its similarity to the more widely used abbreviation representing thyroxine-binding globulin). About 20%-40% of testosterone is bound to serum albumin and 1%-2% is unbound (“free”). The unbound fraction is the only biologically active portion. Serum assays for testosterone measure total testosterone (bound plus unbound) values. Special techniques to assay free testosterone are available in some reference laboratories and in larger medical centers. Circulating androstenedione and DHEA are bound to albumin only.

    Several conditions can affect total serum testosterone levels by altering the quantity of SHBG without affecting free testosterone. Factors that elevate SHBG levels include estrogens (estrogen-producing tumor, oral contraceptives, or medication), cirrhosis, hyperthyroidism, and (in males) decreased testis function or testicular feminization syndrome. Conditions that decrease SHBG levels include androgens and hypothyroidism.

    There is a relatively small diurnal variation of serum testosterone in men, with early morning levels about 20% higher than evening levels. There is little diurnal change in women. Serum levels of androstenedione in men or women are about 50% higher in the early morning than in the evening.

    In males, testosterone production is regulated by a feedback mechanism with the hypothalamus and pituitary. The hypothalamus produces gonadotropin-releasing hormone (GnRH; also called LHRH), which induces the pituitary to secrete LH (and FSH). LH, in turn, stimulates the Leydig cells of the testis to secrete testosterone.

    Serum estrogens

    There are three major estrogens: estrone ((E1), estradiol (estradiol-17b; E2), and estriol (E3). All of the estrogens are ultimately derived from androgenic precursors (DHEA and androstenedione), which are synthesized by the adrenal cortex, ovaries, or testis. The adrenal is unable to convert androgens to estrogens, so estrogens are directly produced in the ovary or testis (from precursors synthesized directly in those organs) or are produced in nonendocrine peripheral tissues such as the liver, adipose tissue, or skin by conversion of androgenic precursors brought by the bloodstream from one of the organs of androgen synthesis. Peripheral tissue estrogens are derived mainly from adrenal androgens. Estradiol is the predominant ovarian estrogen. The ovary also secretes a much smaller amount of estrone. The primary pathway of estradiol synthesis is from androstenedione to estrone and then from estrone to estradiol by a reversible reaction. This takes place in the ovary and to a much lesser extent in peripheral conversion of testosterone to estradiol. Estrone is produced directly from androstenedione (mostly in peripheral tissues) and to a lesser extent in the ovary from already formed estradiol by the reversible reaction. Estriol in nonpregnant women is formed as a metabolite of estradiol or estrone. In pregnancy, estriol is synthesized by the placenta from DHEA (actually from DHEA-S) derived from the fetal adrenals. This is a different pathway from the one in nonpregnant women. Estriol is the major estrogen of the placenta. The placenta also produces some estradiol, derived from both fetal and maternal precursors. Nonendocrine peripheral tissues (liver, skin, fat) synthesize a small amount of estrone and estradiol in pregnancy, mainly from adrenal precursors.

    In females, there is a feedback mechanism for estradiol production involving the ovaries, hypothalamus, and pituitary. As already mentioned, the hypothalamus produces GnRH. The GnRH is secreted in a pulsatile manner about every 70-90 minutes and is excreted by glomerular filtration. The GnRH stimulates FSH and LH secretion by the pituitary. Some investigators believe there may be a separate (undiscovered) factor that regulates FSH secretion. The FSH acts on the ovarian follicles to stimulate follicle growth and development of receptors to the action of LH. The LH stimulates the follicles to produce estradiol.

    Estradiol values can be measured in serum, and estriol values can be measured in serum or urine by immunoassay. Total estrogen values are measured in urine by a relatively old but still useful procedure based on the Kober biochemical reaction. All of the estrogens can be assayed by gas chromatography. The DHEA and androstenedione values can also be measured by special techniques, but these are available only in large reference laboratories.

  • Acromegaly

    Acromegaly is produced in adults by increase of GH, usually from an eosinophilic adenoma of the pituitary. About two thirds of these patients are female. Signs and symptoms include bitemporal headaches, disturbances of the visual fields, optic nerve atrophy, and physical changes in the face and hands. About 25% of patients have fasting hyperglycemia, and about 50% have decreased carbohydrate tolerance when the oral glucose tolerance test is performed. Serum prolactin is elevated in 25%-40% of patients (literature range, 0%-83%). About 65%-75% of patients with acromegaly display sella turcica enlargement on skull x-ray films. GH assay usually reveals levels above the upper limit of 5 ng/ml. The standard method of confirming the diagnosis is a suppression test using glucose. Normal persons nearly always respond to a large dose of glucose with a decrease in GH levels to less than 50% of baseline, whereas acromegalic patients in theory have autonomous tumors and should show little, if any, effect of hyperglycemia. One study, however, noted relatively normal suppression in a significant percentage of acromegalics. In acromegaly (GH excess), a high somatomedin-C result is diagnostic and is found in nearly all such patients. Other causes of elevated somatomedin-C levels include the adolescent growth spurt and the last trimester of pregnancy. Some investigators believe that somatomedin-C assay is the screening test of choice for acromegaly and that a clear-cut blood level elevation of somatomedin-C might be sufficient for the diagnosis. The major problem is the surge during adolescence.

  • Prolactin Secretion Abnormalities

    Prolactin is another peptide pituitary hormone. It stimulates lactation (galactorrhea) in females, but its function in males is less certain. The major regulatory mechanism for prolactin secretion is an inhibitory effect exerted by the hypothalamus, with one known pathway being under control of dopamine. There is also a hypothalamic stimulating effect, although a specific prolactin-stimulating hormone has not yet been isolated. TRH stimulates release of prolactin from the pituitary as well as release of TSH. Dopamine antagonists such as chlorpromazine or reserpine block the hypothalamic inhibition pathway, leading to increased prolactin secretion. Serum prolactin is measured by immunoassay. Prolactin secretion in adults has a diurnal pattern much like that of GH, with highest levels during sleep.

    Some Conditions Associated With Generalized Retardation or Acceleration of Bone Maturation Compared to Chronologic Age (as Seen on Hand-Wrist X-ray Films
    Bone age retarded
    Hypopituitarism with GH deficiency
    Constitutional growth delay
    Gonadal dysgenesis (Turner’s syndrome)
    Primary Hypothyroidism (20%-30% of patients)
    Cushing’s syndrome
    Severe chronic disease (renal, inflammatory gastrointestinal [GI] disease, malnutrition, chronic
    anemia, cyanotic congenital heart disease)
    Poorly controlled severe type I diabetes mellitus
    Bone age accelerated
    Excess androgens (adrenogenital syndrome; tumor; iatrogenic)
    Excess estrogens (tumor; iatrogenic)
    Albright’s syndrome (polyostotic fibrous dysplasia)
    Hyperthyroidism

    Prolactin assay

    Prolactin-secreting pituitary tumors. Prolactin assay has aroused interest for two reasons. First, about 65% of symptomatic pituitary ad enomas (literature range, 25%-95%), including both microadenomas (<1 cm) and adenomas, produce elevated serum levels of prolactin. The pituitary cell type most often associated with hyperprolactinemia is the acidophil cell adenoma, but chromophobe adenomas (which are by far the most frequent adenoma type) are also involved. In addition, about 20%-30% of women with postpubertal (secondary) amenorrhea (literature range, 15%-50%) have been found to have elevated serum prolactin levels. The incidence of pituitary tumors in such persons is 35%-45%. Many patients have been cured when pituitary adenomas were destroyed or when drug therapy directed against prolactin secretion was given. Hyperprolactinemia has also been reported to be an occasional cause of male infertility.

    Some reports indicate that extension of a pituitary adenoma outside the confines of the sella turcica can be detected by assay of cerebrospinal fluid (CSF) prolactin. Prolactin in CSF rises in proportion to blood prolactin levels but is disproportionately elevated when tumor extension from the sella takes place. A CSF/plasma prolactin ratio of 0.2 or more suggests suprasellar extension of a pituitary tumor. Simultaneously obtained CSF and venous blood specimens are required.

    Not all pituitary tumors secrete prolactin. Autopsy studies have demonstrated nonsymptomatic pituitary adenomas in 2.7%-27% of patients. Theoretically, nonfunctional tumors should have normal serum prolactin levels. Reports indicate, however, that some tumors that do not secrete prolactin may be associated with elevated serum prolactin levels, although the values are usually not as high as levels found with prolactin-secreting tumors.

    Prolactin assay drawbacks. Elevated serum prolactin levels may be associated with conditions other than pituitary tumors, idiopathic pituitary hyperprolactinemia, or hypothalamic dysfunction. Some of these conditions are listed in the box. Especially noteworthy are the empty sella syndrome, stress, and medication. The empty sella syndrome is associated with an enlarged sella turcica on x-ray film. Serum prolactin is elevated in some of these patients, although the elevation is most often not great; and the combination of an enlarged sella plus elevated serum prolactin level could easily lead to a false diagnosis of pituitary tumor. Stress is important since many conditions place the patient under stress. In particular, the stress of venipuncture may itself induce some elevation in serum prolactin levels, so some investigators place an indwelling heparin lock venous catheter and wait as long as 2 hours with the patient resting before the sample is drawn. Estrogens and other medications may contribute to diagnostic problems. In general, very high prolactin levels are more likely to be due to pituitary adenoma than to other causes, but there is a great deal of overlap in the low- and medium-range elevations, and only a minority of pituitary adenomas display marked prolactin elevation. Statistics depend on the diagnostic cutoff level being used. The level of 100 ng/ml (100 µg/L) is most frequently quoted; the majority (45%-81%) of pituitary adenomas are above this level, but only 25%-57% of patients with prolactin levels above 100 ng/ml are reported to have a pituitary adenoma. A value of 300 ng/ml gives clear-cut separation but includes only about one third of the adenomas (literature range, 12%-38%).

    Conditions Associated With Increased Serum Prolactin (% With Elevation Varies)
    Sleep
    Stress (including exercise, trauma, illness)
    Nursing infant
    Pregnancy and estrogens
    Pituitary adenoma
    Hypothalamic space-occupying, granulomatous, or destructive diseases
    Hypothyroidism
    Chronic renal failure
    Hypoglycemia
    Certain medications
    Postpartum amenorrhea syndrome
    Postpill amenorrhea-galactorrhea syndrome
    Empty sella syndrome
    Addison’s disease (Nelson’s syndrome)
    Polycystic ovary (PCO) disease
    Ectopic prolactin secretion (nonpituitary neo-plasms)

    Prolactin stimulation and suppression tests Several stimulation and suppression tests have been used in attempts to differentiate pituitary adenoma from other causes of hyperprolactinemia. For example, several investigators have reported that pituitary adenomas display a normal response to levodopa but a blunted response to chlorpromazine. Normally there is a considerable elevation of prolactin level (at least twofold) after TRH or chlorpromazine administration and a decrease of the prolactin level after levodopa administration. In pituitary insufficiency there typically is failure to respond to TRH, chlorpromazine, or levodopa. In hypothalamic dysfunction there typically is normal response to TRH (which directly stimulates the pituitary), little, if any, response to chlorpromazine, and blunted response to levodopa. Pituitary adenomas are supposed to give a blunted response to TRH and chlorpromazine but a normal decrease with levodopa. Unfortunately, there are sufficient inconsistent results or overlap in adenoma and nonadenoma response that most investigators believe none of these tests is sufficiently reliable. There have also been some conflicting results in differentiating hypothalamic from pituitary disease. Diseases such as hypothyroidism and other factors that affect pituitary function or prolactin secretion may affect the results of these tests.