New Methods of Pregnancy Testing in Adolescent Girls

Symposium on Pediatric and Adolescent Gynecology

New Methods of Pregnancy Testing in Adolescent Girls Brij B. Saxena, Ph.D., D.Sc. *

It has been estimated that approximately 55 per cent of all women begin sexual activity before they are 19 years old, and that one in 10 of all women become pregnant before their eighteenth birthday. 3 The actual rate of births to adolescents 15 to 19 years of age has dropped from 97.3 per 1000 girls to 58.7 per 1000 girls in the period between 1957 and 1974. However, the birth rate in women over 20 has dropped much more, so that teenage births are still higher in the total percentage of births. In addition, the birth rate in 10 to 14 year olds has actually increased. The combined result is, approximately, one million teenage pregnancies per year, a high percentage of which will result in live births. 3 Besides this fact, the problems lie in a high medical risk and in the emotional and socioeconomic difficulties for both the mother and the child. Klein 9 has described this pattern as a "syndrome of failure." The most obvious reason why more teenagers are getting pregnant is that more teenagers are now sexually active at ages younger than ever before. The adolescents make little use of contraceptives, partly due to ignorance. One of the most common causes of unwanted pregnancy is the refusal to take responsibility for one's actions, that is, to use contraceptives and to acknowledge one's sexual activity. The same rule applies to acknowledgement of the fact of pregnancy; many girls effectively deny to themselves and their parents that they are pregnant until they are well into pregnancy, thereby eliminating the option of abortion and deferring prenatal care until an advanced gestational age. The effects of adolescent pregnancy are unpleasant for nearly all, and can be devastating for some, while fairly easily reparable for others. A girl is faced with four alternatives when she learns that she is pregnant: to marry, to have an abortion, to put the child up for adoption, or to bring up the child herself. The best thing, of course, is to prevent teenagers from becoming pregnant in the first place. There is no one simple way to do this. The best approach would seem to be an all-around frankness about sex from parents and teachers, and a willingness to answer the questions that all adolescents have about sex. Contraceptives should be made available to people of all ages. "Professor of Biochemistry and Endocrinology, Departments of Medicine and Obstetrics and Gynecology, Cornell University Medical College, New York, New York

Pediatric Clinics of North America- Vol. 28, No. 2, May 1981

437

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BRIJ B. SAXENA

Teenagers will not stop having sex if they are denied access to birth control; they will simply have unprotected, instead of protected, sex. Society should provide easily accessible health care services such as sex education, reliable pregnancy testing, safe abortion, and maternal care in order to give teenagers the opportunity to proceed toward a successful career. In the realization that some teenagers will become pregnant in spite of these efforts, it is obvious that the knowledge and use of newer, more sensitive and reliable tests for pregnancy which are easily accessible and are of moderate cost are the first steps in the early diagnosis and management of pregnancy, especially in adolescent girls. Accurate diagnosis of pregnancy soon after conception offers the option of terminating an unwanted pregnancy by simple, effective, and inexpensive procedure (soft cannula vacuum aspiration of the uterine contents performed within six weeks after the last menstrual period) or permits early initiation of prenatal maternity care particularly in the case of abnormal pregnancy and will avoid needless termination procedures due to false positive results.

SYMPTOMS OF PREGNANCY Unprotected intercourse around the time of ovulation, which may occur around the 14th day of a normal28-day cycle, is followed by fertilization of the ovum by the sperm. The implantation of the blastocyst generally occurs around the 22nd day following the last normal menstrual period. The most familiar sign of pregnancy is the missed period. However, a delay in expected menstruation unrelated to pregnancy may occur in subjects with irregular menstrual cycles, rapid loss of weight, stress, and following cessation of contraceptive pills. Other symptoms that provide presumptive evidence of pregnancy are fatigue and lassitude, increased body temperature, and breast fullness or pain particularly in primiparae. The pregnancy is associated with the darkening of the areola, enlargement of the nipple, protusion of the Montgomery's glands, and swelling of the axilla from ectopic mammary tissue. Colostrum may not occur until the sixth to the eighth week of pregnancy. 12 Subjective feelings of nausea and vomiting (morning sickness) caused by distention of the abdomen, constipation, and reduced peristalsis as well as weight gain may also appear after two weeks. Frequency in urination results from relaxation of bladder and urethral muscles. Enlargement of the cystic and uretheral vessels may cause tenesmus and burning micturition. 6 Upon physical examination by a physician, findings on bimanual palpation during the first week following the time of expected menstruation are often normal or inconclusive for pregnancy, and changes such as softening of the uterine isthmus and a violaceous coloration of the vulva, vagina, or cervix do not become definitive for two and a half to three weeks following implantation. Among the objective signs, the uterus is clinically unenlarged until seven to eight weeks. The first change is the softening of the upper cervix (He gar's sign) which is associated with the softening of the cervix itself (Goodwill's sign), and, finally, the cervix and vaginal mucosa acquire a bluish hue (Chadwick's sign) owing to increased venous enlargement or capillary dilation. Quickening or the perception of fetal movement becomes detectable around the 14th to 20th week.

NEW MErnODS OF PREGNANCY TESTING

439

PREGNANCY TESTS Historical Development Ancient Egyptian records describe a pregnancy test in which a woman drank a mixture of pounded watermelon and breast milk from a woman who had borne a son. If she vomited, she was thought to be pregnant. According to Hebrew scriptures, if a woman's feet sank deeply into soft ground, she was assumed to be pregnant. Until the middle of the twentieth century, diagnosis of pregnancy was based on missed menstruation and subjective observations such as turgidity of the veins of the breast and increased pleasure during sexual intercourse. Only within the past 50 years have scientists developed objective tests that detect physiologic changes associated with pregnancy. The historical trend has been toward the development of simpler, more reliable, earlier, and less expensive pregnancy tests, and, more recently, in the development of "do-it-yourself' tests performed by women themselves. During the past half century, new pregnancy tests resolve the question of pregnancy for the modern woman early, effectively, and accurately. Early pregnancy tests are vital in order for women to have more control over their health and lives in an increasingly liberal and permissive society in which women are expected to play more independent roles. The diagnosis of pregnancy by the detection of human chorionic gonadotropin (hCG) was first described 53 years ago by Selmar Aschheim and Bernhardt Zondek. 1 The improvements in the techniques for the measurement ofhCG have been directly related to the progress in the purification and isolation of hCG and elucidation of the amino acid sequence of the hormonenonspecific alpha subunit and hormone-specific beta subunit ofhCG. 2 Human chorionic gonadotropin, a glycoprotein with a molecular weight of 36,000 to 40,000 daltons, is secreted by the trophoblast of the developing blastocyst and later by the chorion and the placenta in pregnant women. Like the similar glycoproteins human luteinizing hormone (hLH), human follicle stimulating hormone (hFSH), and human thyroid stimulating hormone (hTSH), each hCG molecule consists of two noncovalently linked polypeptide chains classified as a and f3 subunits. Individually these subunits are devoid of biological activity and reactivity to specific receptors. The a subunits of all these glycoprotein hormones have almost identical amino acid sequences and are practically indistinguishable immunochemically. In contrast, the f3 subunits have their own distinctive amino acid sequences which differentiate them from the f3 subunits of the other glycoproteins. The f3 subunits of hCG and hLH are considerably more similar to each other than the other glycoprotein f3 subunits. However, even these two similar f3 subunits can be distinguished from each other immunochemically by utilizing appropriate methods. The f3 subunit of hCG possesses a unique sequence of 30 additional amino acids at the carboxyl terminus that is not possessed by hLH. Because of this additional and unique sequence, antisera can be prepared by immunizing rabbits with hCG-[3 and selectively screening the antisera for its hCG-{3 specificity. 16 By this method, it is possible to successfully produce antiserum that will specifically react with hCG and its f3 subunit with low but variable levels of crossreactivity with hLH. Selection from antisera is necessary since the hCG-[3 subunit has antigenic sites common to hLH and hCG as well as

440

BRIJ B. SAXENA

specific hCG determinants that are predominantly conformational in nature. By specific modification, it is possible to destroy the common determinants in the isolated {3 subunit of hCG while maintaining the hCG specific determinants. Antisera raised against such a specific modified {3 subunit thus exhibit minimum crossreactivity with hLH and other glycoprotein hormones. This methodology provides a consistent source of antiserum with minimum reliance on the production of antibodies to hCG-specific determinants by chance in a heterologous species. The use of such antisera in recent years has improved the specificity of hCG detection by both immunoassay and radioimmunoassay procedures.

Basis The history, physical examination, and pregnancy tests will usually reveal sufficient information for a definite diagnosis of pregnancy. The presence of hCG in the urine or blood is the most accurate of all the indications of pregnancy. In view of recent findings of hCG-like material in conditions other than pregnancy, 19 it is essential that physicians correlate all the relevant clinical data to ascertain pregnancy, especially in early stages. The duration of nine months of pregnancy, starting from the last menstrual period in a woman, is divided into three trimesters of three months each. The evaluation of pregnancy tests requires an understanding of the normal production ofhCG during early pregnancy (Fig. 1). With this as background, it then becomes possible to choose the pregnancy test that is appropriately

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NEW METHODS OF PREGNANCY TESTING

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sensitive to detect the presence ofhCG based on the interval from the onset of the last menstrual period to the time the test is performed. Specificity, accuracy, requirements for special equipment, time to perform the test, availability, and cost are some of the important factors in choosing the most suitable pregnancy test. hCG is detected in the serum of pregnant women as early as 9 to 11 days after the LH peak, or 7 to 9 days after ovulation, by radioimmunoassay 10 and by radioreceptorassay 14 capable of detecting 6 to 10 milliunits (miU) of hCG per mi. hCG levels are expressed in international units (IU). An IU (equivalent to 1000 miU) is the specific amount of gonadotropic activity contained in 0.0013 mg of a dried hCG standard preserved at and distributed from the National Institute for Medical Research, London. If we assume that 28 days is the length of a normal cycle, then 40 miU hCG per ml is reached one week after ovulation or 20 to 22 days following the last menstrual period, which can be detected by quantitative radioimmunoassay and radioreceptorassays. The trophoblastic production ofhCG has been estimated to increase twofold every two days. A level of200 miU ofhCG perml is reached by 26 to 28 days, which is the sensitivity of the qualitative radioreceptorassay for hCG, commercially available as Biocept-G (Wampole Laboratories). At 36 days, the hCG titers are around 800 miU of hCG per ml, which is the sensitivity of the Pregnosticon tube test. The UCG-Wampole, Placentest, and in-home Early Pregnancy Test have the sensitivity to detect a 36 to 38 day pregnancy, which is equivalent to approximately 1000 miU ofhCG per mi. Between the 36th and 40th days, the level of hCG ranges between 1600 and 3200 miU per mi. The Pregnosticon Dri-Dot, DAP, Gravindex slide tests have this level of sensitivity. The maximum levels of hCG, as high as 100,000 miU per ml, are occasionally approached between 60 and 80 days of gestation. In the last or third trimester, at a time when levels of metabolites of estrogen and progesterone are increasing, the urinary levels decline and plateau between 3000 and 20,000 miU ofhCG per mi. It should be emphasized that the levels ofhCG referred to above are estimated for an average pregnancy, and significant individual variations in production, secretion (serum levels), and excretion (urinary levels) of hCG exist. SPECIFIC TYPES

During the last century, four different techniques for the determination of hCG in blood and/or urine have been developed (Table 1). These include: (1) bioassays in intact laboratory animals, (2) immunologic tube or slide methods with heme- or latex-agglutination inhibition, as well as the more recently developed competitive protein-binding method such as (3) radioimmunoassay (RIA) by the use of radioisotope labeled hormone and the specific antiserum against hCG, and (4) radioreceptorassay (RRA) by the use of radioisotope-labeled hormone with biological activity and the specific receptors as the binding proteins.

Biological Pregnancy Tests The bioassay of hCG as a pregnancy test was first described in 1927 by Aschheim and Zondek 1 in immature female mice, by Friedman and Lapham

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Table 1.

Summary of Pregnancy Tests" POSITIVE ON DAY AFTER LAST PERFORM-

COST

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2-5 days

20

40 days

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SENSITIVITYf TYPE OF TEST

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in mice and rabbits, by Frank and Berman, and by Kupperman in rats. 5 • 8 Urine or serum samples, when injected into the assay animals, resulting in ovarian hypertrophy, hyperemia (injected with blood), or hemorrhage, are used as a positive indication for the presence ofhCG. The biological tests are accurate and qualitative but are also time-consuming, complicated, and expensive. They can detect 1 IU of hCG per ml of urine and 0.4 IU of hCG per ml of blood (Delfs assay) and thus can detect pregnancy 14 days or earlier following a missed period. The bioassay may be used in the detection of hydatidiform mole, chorioadenoma, and choriocarcinoma when levels ofhCG are high but fail to indicate ectopic pregnancy, threatened abortion, and other conditions associated with low production ofhCG. Galli-Mainini in the male toad (Bufo arenarum) and Wiltberger and Miller in the male frog (Rana pipiens) developed another rapid assay method involving injection of urine or serum in the male animal. 11 • 15 The "hCG present in the urine or serum stimulates the discharge of sperm in the urine of the animal within one to five hours, indicating pregnancy. This bioassay with toads and frogs has the advantage of speed and the animals may be reused since dissection is not required. These tests can detect 1.5 IU of hCG per ml and can detect pregnancy two to four weeks after the last menstrual period with 92 to 93 per cent accuracy. The availability and maintenance of animal colonies, however, become cumbersome and costly. The animal colonies are frequently infected, which interferes with the accuracy and routine use of the bioassay.

Honnonal Withdrawal Pregnancy Tests This form of bioassay is based on the work of Zondek 20 and uses the woman herself. The estrogen and progesterone are believed to stimulate

NEW METHODS OF PREGNANCY TESTING

443

normal hormonal increases that precede menstruation. Withdrawal of the hormones mimics the usual decrease in hormones typical of normal menstrual cycle and results in menstrual bleeding in the nonpregnant woman. This diagnostic test takes at least two weeks for complete results and is occasionally used in women with amenorrhea with suspected pregnancy or positive hCG test without detectable clinical signs of pregnancy. The test involves the administration of synthetic progestational and estrogenic compounds orally or by injection which is followed by observation for vaginal bleeding. In normal nonpregnant women, withdrawal bleeding occurs within flve days. In pregnant women, the endometrium is already primed and maintained by elevated levels of both progesterone and estrogen produced endogenously by the corpus luteum and thus bleeding is obviated despite withdrawal of steroids. The absence of bleeding within 10 days strongly suggests pregnancy. Recent flndings suggest that progestins may have teratogenic effects, thereby causing multiple congenital malformation of the fetus. These anomalies are described by the. acronym VACTEL (vertebral, anal, cardiac, tracheal, esophageal, and limb). The Food and Drug Administration recommended in 1973 that these tests be abandoned due to the risk involved for both the mother and fetus. The availability of other reliable hCG tests has also dampened enthusiasm for this test. The reliability of the hormonal withdrawal test has been questioned; however, owing to the unavailability of sophisticated pregnancy tests, these tests are still popular in urban areas of developing countries, and more so because they are inexpensive, easy to perform, and are believed to be abortifacient, which is not proven.

Immunological Pregnancy Tests The second phase of the development of pregnancy tests in 1960 was possible due to further purification of the hCG and the development of potent anti-hCG serum in rabbits, thus eliminating the need for maintaining an animal colony in each laboratory. This standard immunoassay, based on classic antigen-antibody reactions, is convenient, inexpensive, and relatively accurate, and has gained wide acceptance as the principal method for the detection of hCG. Two groups of investigators, Wide and Gemzell 17 and Brody and Carlstrom, 4 independently developed methods to couple hCG to red cells (sheep erythrocytes) or latex particles, providing the basis of in vitro tube heme- or latex-agglutination inhibition tests. The sensitivity of the agglutination inhibition tests did not improve over that of the bioassays; however, the cost and time of performance were considerably reduced. The biological and immunological tests provided a sensitivity of 500 to 1500 miU per liter, a sensitivity that can detect pregnancy with a 95 per cent accuracy six weeks after the last menstrual period. However, neither bioassays nor heme-agglutination inhibition tests can prove or disprove the existence of pregnancy in patients who are one week late in menstruation. Similarly, a large number of early pregnancies, prior to six weeks, and abnormal pregnancies, missed abortions, and up to 50 per cent of ectopic pregnancies and trophoblastic tumors with levels of hCG less than 500 miU per ml are not detected. Earlier than six to eight weeks of pregnancy, the hemeagglutination inhibition tests may also be susceptible to false results because of cross reaction with luteinizing hormone or nonspecific immunoreactive

444

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material. Long episodes of amenorrhea, delayed ovulation, and variability in urinary extraction of hCG may also complicate the detection of pregnancy by bioassays and heme-agglutination inhibition methods and often result in false negative results even six weeks after the last menstrual period. The hemeagglutination inhibition tests are not precisely quantitative but are as reliable as the most sensitive bioassay. The immunoassays provide useful, and the most frequently used, tools to evaluate pregnancy status in the physician's office. The immunoassay of hCG is performed as an indirect hemeagglutination inhibition test or a direct agglutination test (Figs. 2 and 3). For immunological tests, the first urine in the morning is the optimal specimen. A positive result obtained on a random sample is also meaningfuL Turbid urines should be filtered or centrifuged. Urines can be stored in a refrigerator for up to 72 hours or in a freezer for up to six months. Urine

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RESULTS

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samples should be thoroughly mixed prior to testing for hCG. The use of preservatives in the urine is generally not recommended. Psychotropic drugs and methadone (140 mg per day) may interfere with the immunologic tests. Slide Tests. In 1962, Robbins and associates 7 described the first slide pregnancy test. The slide test is a qualitative in vitro latex-agglutination inhibition test for the presence of hCG in urine. The test is based upon an immunochemical reaction. When hCG antiserum is mixed with a suspension of latex particles coated with hCG, agglutination of latex particles occurs. If, however, the antiserum is first mixed with urine containing sufficient hCG, the antibodies are neutralized, thus inhibiting the latex particles from agglutinating to yield positive results. On the other hand, if the latex particles agglutinate with the antiserum, the urine is then devoid of sufficient hCG and this yields a negative reaction (Fig. 4A). In the direct agglutination slide test (DAT), the anti-hCG is directly absorbed on latex particles, so that hCG either in urine or serum samples will result in agglutination. Unlike other slide tests, agglutination indicates a positive pregnancy test. As a group, the two-minute latex slide tests for the detection of hCG in urine are simple and convenient

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Figure 4. Patterns indicating positive or negative results in A, a heme- or latex agglutinationinhibition slide pregnancy test, and in B, a heme- or latex agglutination-inhibition tube pregnancy test.

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BRIJ B. SAXENA

and are the tests of choice for use in the physician's office for the confirmation of uncomplicated pregnancy. These tests are, however, relatively insensitive (1000 to 2500 miU per ml) and approach 90 per cent accuracy only, at least 45 days after the last menstrual period. The sources of error in a slide test include the use of dilute urine caused by, for example, excessive liquid intake, proteinuria, intake of methadone or psychotropic drugs in high dosage, and, of course, technical error which is largely the result of improper mixing of reagents and sample. In a good laboratory, a slide and tube test should be performed on all specimens. If both agree, the results should be sent to the physician. If the tube test is positive and the slide test is negative, further testing is recommended (such as a serum test) or another specimen should be requested. Tube Test. In 1960, Wide and Gemzell 17 introduced the first hemeagglutination inhibition tube pregnancy test, Pregnosticon. These tests are based on an in vitro (indirect) heme-agglutination inhibition test for the detection of hCG in urine. This test can be modified to detect hCG in systematic dilutions of the urine, thus providing quantitative estimates (see Fig. 6). In this test, when the antiserum is mixed with erythrocytes coated with hCG, the erythrocytes agglutinate. The urine of a pregnant woman, which contains hCG, is mixed with the antiserum; the antiserum is neutralized by the hCG in the urine and the agglutination of the hCG-coated erythrocytes is inhibited. The nonagglutinated erythrocytes, because they are not clumped together, will roll down the sides of the test tube to rest on the slope of the hemispherical bottom of the test tube in a characteristic ring pattern indicating a positive test (Fig. 4B). Agglutinated cells, implying no hCG and therefore no pregnancy, will remain in suspension. These tests require an incubation period of 90 to 120 minutes and require urinary hCG levels of 500 to 1200 miU per ml for positive results. These tests are thus reliable beginning the eighth day following a missed period. Prior to that, these tests have only 77 per cent accuracy. The tube test can be affected by proteinuria (2+ to 4+ by Labstix). Due to increased production of pituitary luteinizing hormone in menopausal women, in patients with ovarian failure and polycystic ovaries, in patients following removal of ovaries, as well as in patients with choriocarcinoma and hydatidiform mole, these tests may yield false results. Urine samples from these patients could be diluted two to three times prior to testing in order to distingush pregnancy-related hCG which is many times higher in concentration than the pituitary luteinizing hormone. Such observations should be correlated with clinical manifestations and interpreted with caution. Drugs directly do not affect either direct or indirect tube tests whereas proteinuria does. Technical errors such as improper filtration or centrifugation, accidental vibration, or exposure of tubes to heat or direct sunlight during incubation may also cause false results. Because of slightly improved sensitivity, this test can be performed with urine samples obtained within two weeks of possible implantation. The tube test is associated with fewer false positive results than the slide test, but a woman suspected to be pregnant with a negative result is advised to repeat the test several days later. It is especially useful when diagnostic curettage or hysterectomy is planned, when elective radiologic or surgical procedures are planned, or when low

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NEW METHODS OF PREGNANCY TESTING

447

levels of hCG are anticipated as in ectopic pregnancy or spontaneous abortion. In ectopic pregnancy, false negative results are greater than 50 per cent by the rapid slide test and up to 35 per cent with the tube test. Thus a negative test by either the slide or tube test does not exclude ectopic pregnancy. In fact, the diagnosis is often made from clinical findings (palpation of an adnexal mass, demonstration of blood in the cul-de-sac, laparoscopy, sonography). In the face of the threatened or attempted abortion, positive biological or immunological tests do not always indicate the survival of the fetus. Following spontaneous abortion, positive results up to one to two weeks may occur to indicate the clearance of hCG from the body. Positive responses to immunological tests may also be obtained in men with testicular carcinoma (chorioepithelioma) and removal of testes, or in men and women with ectopic tumors including undifferentiated carcinoma of the lung; the latter cases are associated with gynecomastia in men. However, owing to relative insensitivity of the immunological hCG test, a negative test does not rule out early occurrence of the disease and more sensitive radioimmunoassays or radioreceptorassays should be performed.

Do-it-yourself Pregnancy Tests For the women of the twentieth century, the science of pregnancy has been refined to the point where they may be able to diagnose pregnancy using a do-it-yourself test purchased at the pharmacy. Such tests are of special interest to women with irregular periods, those whose periods have not returned after stopping oral contraceptives, those with infertility problems, those who do not wish to become pregnant, and among adolescents who are suspicious of pregnancy or in whom amenorrhea results from excessive weight loss by dieting. Detection of early pregnancy by self-test permits simpler, quicker, and less traumatic means of termination such as menstrual extraction or miniabortion for those who do not wish to remain pregnant. For women who desire pregnancy, a self-test can help to protect the baby by prompting the mother to start proper prenatal care during the first month of pregnancy, when the risk of damage to the fetus is the greatest. In spite of these advantages, the advisability, accuracy, and economy of self-tests for pregnancy have been debated, and in many cases the need to follow the result of a test with a visit to a physician has been deemed necessary. Most do-it-yourself pregnancy tests are based on the modifications of the original heme-agglutination inhibition principle except claim different degrees of sensitivity and specificity. These can be used by women without a doctor's prescription or an intervening laboratory. These tests have some advantages but require considerable care in performance, interpretation of results, and in dealing with the question of "what to do next?". If the laboratory tests, which use test tubes or slides, are performed about two weeks after a missed period, they can give, within a short time, a highly accurate determination of the presence or absence of pregnancy. There are, of course, limitations to a self-test for pregnancy. Most would be doing the test for the first time and would be less likely to do it correctly. Pregnancy testing is an emotional event and people may have difficulty in following instructions or be impatient. One survey in Canada, where do-it-yourself pregnancy tests have been sold for ye.ars, showed that few women trust the results of tests they I

448

BRIJ B. SAXENA

do themselves. Many go back to the pharmacist to have the test repeated. It is a false economy. A doctor can diagnose pregnancy on the basis of a physical examination, without a pregnancy test, about two weeks after a missed period. Infrequently, a pregnancy test will give a false positive result which may result from contamination of urine (such as detergent in the container), presence of blood or excessive protein in the urine, or, more seriously, from the presence of a hydatidiform mole or choriocarcinoma. A self-test may give a negative result early in an ectopic pregnancy, giving the woman false assurance; somewhat later it may have a positive result, leading the woman to think that she has a normal pregnancy or that she may need an abortion (which of course cannot remove an ectopic pregnancy). Any woman with a positive pregnancy test who has an intrauterine device in place or who has had a sterilization operation should see a physician promptly to check the possibility of an ectopic pregnancy.

Radioimmunoassay Radioimmunoassay 18 is based upon the ability of a limited quantity of antibody to bind a fixed amount of radiolabeled antigen and the inhibition of this reaction by an unlabeled antigen. The woman's blood sample is mixed with a predetermined amount of antibody to hCG; the labeled hCG is then added. IfhCG is not present in the blood, the unreacted antibody is available to bind with the radioactive hCG. If hCG is present in the woman's blood, there is inhibition of binding oflabeled hCG to the antibody. The percentage of bound radiolabeled antigen decreases as a function of the concentration of unlabeled antigen in the test sample. Separation of the bound and free radiolabeled antigen is necessary in order to determine the quantity of unlabeled antigen. This can be accomplished by insolubilization of the antigen-antibody complexes either by chemical means, such as polyethylene glycol precipitation, or by the addition of a second antibody directed toward the immunoglobulin present in the original antiserum, or by a combination of these two methods. The quantity of antigen in an unknown sample is then determined by comparing the radioactivity of the precipitate, after centrifugation, with values established with known standards in the same assay system. The standard hCG radioimmunoassay is nonspecific in that pituitary hormones, particularly LH, react with antiserum against hCG. The crossreaction with LH limits the usefulness of the test, especially in patients with trophoblastic disease, in whom hCG levels must then be followed below the circulating endogenous levels of pituitary LH. The antisera raised against the beta subunit ofhCG or of chemically modified 13 subunit, or use of chemically synthesized C-terminal fragments of the hCG-{3 subunit, provide antigens with sites specific for each hormone and which produce hormone-specific antibodies. Such antibodies in the native state or after immuno-affinity purification can be used to measure hCG without cross-reaction with LH in the radioimmunoassay. These antisera are highly specific and provide a sensitivity as high as 6 miU of hCG per ml. Such radioimmunoassays are useful in the determination oflower levels ofhCG and assist in the diagnosis of early or ectopic pregnancy and in the management of all phases of trophoblastic disease. These assays, however, require 24 to 72 hours. A recent

449

NEW METHODS OF PREGNANCY TESTING

modification of the beta subunit hCG radioimmunoassay, consisting of the use of dioxane to separate bound and free hormone, has reduced performance time to three hours without loss of sensitivity, specificity, or precision. More recently, the use of hCG-specific antisera, raised to chemically modified, hormone specific hCG-,8 subunit, coupled to solid phase has further improved the performance and speed of the radioimmunoassay.

Radioreceptorassay 13 Circulating hCG is known to consist of several molecular species, some of which are of low or negligible in vivo biological activity (desialylated hCG, beta subunit, alpha subunit and hCG-like peptides) but still retain considerable immunological activity. For this reason, time-consuming, expensive, and cumbersome bioassays are required if immunological tests are not compatible with the biological activity of the hCG. Radioreceptorassay provides a unique blending of the sensitivity of the radioimmunoassay and the specificity of the bioassay. This is a test that reliably detects pregnancy as early as one to two weeks after conception, and is 98 to 100 per cent accurate at the time of expected menstruation in women with normal pregnancy. The radioreceptorassay is based on the same principle of competitive protein binding as the radioimmunoassay, except that "receptor" is substituted for hCG antibodies as the binding protein (Fig. 5). In contrast to the antigen-antibody reaction, which is immunological, the hormone-receptor interaction is biological,

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450

BRIJ B. SAXENA

hence the radioreceptorassay yields the estimates of the biological activity of the hormones. The "receptors," macromolecular proteins of approximately 200,000 daltons in molecular weight, are lodged in the lipid bilayer of the plasma membrane of the cells of the target organs. The receptors have binding affinity as high as antisera, that is, Ka of 10'12 M, and approach equilibrium within 15 min at 37o C. The high affinity and specificity of the receptor appear to be selected through evolution to concentrate rapidly the hormones that circulate in the blood at low concentrations. The radioreceptorassay is therefore a sensitive and specific test which detects hCG or luteinizing hormone owing to the structural and biological similarity of the two hormones. However, two- to five-fold more LH than hCG would have to be present to match detection in the radioreceptorassay. The hCG-LH receptor does not detect follicle stimulating hormone, prolactin, or human growth hormone. hCG and LH receptors are present in the ovaries and testes of rats, pigs, sheep, cows, and humans. Corpora lutea of the bovine ovaries obtained from the slaughter house, however, provide a rich and easily available source of the receptor. In the radioreceptorassay ofhCG labeled with 125 I, a constant amount of membrane receptor and a sample of the patient's serum are required. As shown in Figure 5, the test is based on competition for binding to the receptor sites between labeled and unlabeled hCG. In pregnant women, hCG from the serum displaces labeled hCG from the receptor site, resulting in lower residual radioactivity in plasma membrane receptors. In nonpregnant women, labeled hCG is not displaced from receptor sites and maximal radioactivity remains bound to the plasma membrane receptor. A graded increase in hCG in the standard provides a proportional decrease in the radioactive hCG bound to the receptor. This inverse relationship provides the basis for the standard curve for the quantitative receptor assay. The quantitative receptorassay was first developed in our laboratory in 1974. A sensitivity of 5 to 10 miU of hCG per ml allowed the diagnosis of pregnancy as early as six days after conception. The test was repeated in order to rule out interference with the delayed ovulatory LH surge. The radioreceptorassay has also been useful in the diagnosis of ectopic pregnancy and spontaneous abortion, and in the follow-up of patients with trophoblastic disease and infertility problems. In the commercially available qualitative radioreceptorassay Biocept-G Kit (Fig. 6), 40 J-tg of protein equivalent bovine corpus luteal membranes, 100 ~-tl of 1251-hCG, and 100 ~-tl of serum are mixed in a test tube and incubated for 30 min at 37o C, after which 1 ml of cold water is added to stop the reaction. The tube is then centrifuged for 15 min at 2000 to 5000 x g and the supernate is aspirated. The amount of radioactivity in the pellet from the patient's sample and from a reference control serum containing 200 miU of hCG per ml are compared. Results are interpreted as positive if the radioactivity is less in the sample than in the control and as negative if the radioactivity is greater in the sample than in the control. When the radioactivity is within 2.5 per cent of that of the control, the results are inconclusive and another specimen on a subsequent day is examined. The radioreceptorassay is rapid enough to obtain results in just one hour, and is also a reliable screen for new hospital admissions. The possibility of a pregnancy is always present in women during the childbearing years. Many medical and surgical procedures are contraindi-

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Assuming that the first test of undiluted urine is positive and the 1:10 dilution is negative; titer is above 0.75 IU and below 7.5 IU per ml. Eight additional tests of dilutions 1:2 to and including 1:9 are indicated. If the 1:100 dilution is negative, but 1:10 is positive, titer is above 7.5 IU and below 75 IU per ml. This range could be normal at certain stages of pregnancy. Performance of additional tests would depend on clinical indications and the physician's preference. If the 1:500 dilution is positive, titer is above 375 IU per ml, While values up to nearly 500 I,U, per ml have been found during the peak secretion of days 50-90 in pregnancy, nevertheless such a value could occur with hydatidiform mole or choriocarcinoma. Additional tests of dilutions of 1;500 to 1;1500 are indicated. Figure 6.

Quantitative tube test.

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452

BRIJ B. SAXENA

cated in pregnancy (extensive radiologic or diagnostic curettage). In trophoblastic disease, serial hCG assays are indicated for evaluation of therapeutic response. The radioreceptorassay has the sensitivity of the radioimmunoassay and the specificity of the bioassay, and therefore has greater sustained accuracy over the existing immunologic and biologic tests for pregnancy. A more accurate measurement of biologically active hCG and hence a significant contribution to improved patient care is offered by the radioreceptorassay. The Biocept-G (Wampole) is extremely useful in the detection of early pregnancy, and, therefore, in seeking termination by so-called "miniabortion" or menstrual extraction procedures that are simple, inexpensive, and could be performed within 15 minutes without anesthesia in the physician's office. Menstrual extraction is illegal in some countries such as Canada in which the conditions permitting legal abortion are contained in the Criminal Code and the procedure is restricted to public hospitals after being approved by an abortion committee. Immediately after the expected period, conventional pregnancy tests are so inaccurate that up to 30 to 50 per cent of menstrual extractions are performed in women who are not pregnant. If a woman chooses to wait until conventional tests become reliable (10 to 15 days after the missed period), more complicated procedures are necessary. The early detection of pregnancy by the radioreceptorassay is beneficial in high-risk pregnancy, in diabetes, or when a patient has been treated for infertility by ovulatory drugs and/or has been artificially inseminated. The radioreceptorassay has been very useful in the differential diagnosis of ectopic pregnancy when only low levels of hCG are present in either the blood or urine. Radioreceptorassay can help to diagnose 93 to 98 per cent of ectopic pregnancies prior to rupture in all patients reporting to the physician soon after the first missed period or even earlier if prompted by other symptoms. Other current uses of the radioreceptorassay are in the detection of multiple gestation, in the diagnosis and management of missed or threatened abortions, in trophoblastic disease, and as a screening test for all women of childbearing age upon admission to the hospital or before undergoing investigative procedures or therapy as outpatients. 14 Such screening will protect the fetus from exposure to radiation and harmful drugs. The usefulness of pregnancy screening is obvious in adolescents prior to prescription of oral contraceptives, insertion of intrauterine devices, administration of rubella vaccine, and use of diagnostic x-rays or drugs which may have deleterious side effects.

REFERENCES 1. Aschheim, S., and Zondek, B.: Hypophysenvordarlapen hormon und ovarialhormon in Ham von Schwangeren. Klin. Wochenschr., 6:1322, 1927. 2. Birken, S., and Canfield, R. E.: Structural and immunochemical properties of human choriogonadotropin. In McKerns, K. W., (ed.): Structure and Function of the Gonadotropins. New York, Plenum Press, 1978, pp. 47-80. 3. Brandt, C. L., Kane, F. J., Jr., and Moan, C. A.: Pregnant adolescents: Some psychosocial factors. Psychosomatics, 19:790-793, 1978. 4. Brody, S., and Carlstrom, G.: Estimation of human chorionic gonadotropin in biological fluids by complement fixation. Lancet, 2:99, 1960. 5. Cabrera, H. A.: A comprehensive evaluation of pregnancy tests. Am. J. Obstet. Gynecol., 103:32-38, 1969.

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6. Danforth, D. N.: Obstetrics and Gynecology. Hagerstown, Maryland, Harper and Row, 1977. 7. Hon, E. H.: A Manual of Pregnancy Testing. Edition 1. Boston, Little, Brown and Co., 1961. 8. Hunt, W. R.: Pregnancy Tests: The Current Status. Population Reports (Series J. Family Planning Program), November 1975, pp. J109-J124. 9. Klein, L.: Antecedents of teenage pregnancy. Clin. Obstet. Gynecol., 21:1151-1159,1978. 10. Kosasa, T. S., Levesque, L.A., Goldstein, D.P., eta!: Clinical use of a solid phase radioimmunoassay specific for human chorionic gonadotropin. Am. J. Obstet. Gynecol., 119:784-791, 1974. 11. Mayo, R. W., and Thompson, R. B.: Comparison of pregnancy tests. Obstet. Gynecol., 25:699704, 1965. 12. McWarder, K. R.: Obstetrics: Essentials of Clinical Practice. Boston, Little, Brown and Co., 1976. 13. Saxena, B. B., Hasan, S. H., Haour, F., et al.: Radioreceptor assay of human chorionic gonadotropin: Early detection of pregnancy. Science, 184:793, 1974. 14. Saxena, B. B., and Landesman, B. B.: Diagnosis and management of pregnancy by the radioreceptorassay of human chorionic gonadotropins. Am. J. Obstet. Gynecol., 131:97-107, 1978. 15. Tietz, N. W.: Comparative study of immunologic and biologic pregnancy tests in early pregnancy. Obstet. Gynecol., 25:197-200, 1965. 16. Vaitukaitis, J. L., Braunstein, G. D., and Ross, G. T.: A radioimmunoassay which measures human chorionic gonadotropin in the presence of luteinizing hormone. Am. J. Obstet. Gynecol., 113:751, 1972. 17. Wide, L., and Gemzell, C. A.: An immunological pregnancy test. Acta Endocrinol., 35:261, 1960. 18. Yalow, R. S., and Berson, S. A.: Immunoassay of endogenous plasma insulin in man. J. Clin. Invest., 39:1157, 1960. 19. Yoshimoto, Y., Wolfsen, A. R., Hirose, F., et al.: Human chorionic gonadotropin-like material: Presence in normal tissues. Am. J. Obstet. Gynecol., 134:729, 1979. 20. Zondek, B.: Simplified hormonal treatment of amenorrhea. J.A.M.A., 118:705-708, 1942. Department of Medicine Cornell University Medical College 1300 York Avenue New York, New York 10021