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.