Box 836.1 Indications for measurement of β human chorionic gonadotropin
• Early diagnosis of pregnancy
• Diagnosis and management of gestational trophoblastic disease
• As a part of maternal triple test screen
Syncytiotrophoblastic cells of conceptus and later of placenta synthesize hCG. Human chorionic gonadotropin supports the corpus luteum of ovary during early pregnancy. Progesterone, produced by corpus luteum, prevents ovulation and thus maintains pregnancy. After 7-10 weeks of gestation, sufficient amounts of progesterone are synthesized by placenta, and hCG is no longer needed and its level declines.
CLINICAL APPLICATIONS OF TESTS FOR HUMAN CHORIONIC GONADOTROPIN
- Early diagnosis of pregnancy: Qualitative serum hCG test becomes positive 3 weeks after last menstrual period (LMP), while urine hCG test becomes positive 5 weeks after LMP.
- Exclusion of pregnancy before prescribing certain medications (like oral contraceptives, steroids, some antibiotics), and before ordering radiological studies, radiotherapy, or chemotherapy. This is necessary to prevent any teratogenic effect on the fetus.
- Early diagnosis of ectopic pregnancy: Trans-vaginal ultrasonography (USG) and quantitative estimation of hCG are helpful in early diagnosis of ectopic pregnancy (before rupture).
- Evaluation of threatened abortion: Serial quantitative estimation of hCG is helpful in following the course of threatened abortion.
- Diagnosis and follow-up of gestational trophoblastic disease (GTD).
- Maternal triple test screen: This consists of measurement of hCG, α-fetoprotein, and unconjugated estriol in maternal serum at 14-19 weeks of gestation. The maternal triple screen identifies pregnant women with increased risk of Down syndrome and major congenital anomalies like neural tube defects.
- Follow-up of ovarian or testicular germ cell tumors, which produce hCG.
In women with normal menstrual cycle, conception (fertilization of ovum to form a zygote) occurs on day 14 in the fallopian tube. Zygote travels down the fallopian tube into the uterus. Division of zygote produces a morula. At 50-60-cell stage, morula develops a primitive yolk sac and is then called as a blastocyst. About 5 days after fertilization, implantation of blastocyst occurs in the uterine wall. Trophoblastic cells (on the outer surface of the blastocyst) penetrate the endometrium and develop into chorionic villi. There are two main forms of trophoblasts—syncytiotrophoblast and cytotrophoblast. Placental development occurs from chorionic villi. After formation of placenta, the conceptus is called as an embryo. When embryo develops most major organs, it is called as fetus (after 10 weeks of gestation).
Box 836.2 Diagnosis of early pregnancy
• Positive serum hCG test: 8 days after conception or 3 weeks after last menstrual period (LMP)
• Positive urine hCG test: 21 days after conception or 5 weeks after LMP
• Ultrasonography for visualization of gestational sac:
– Transvaginal: 21 days after conception or 5 weeks after LMP
– Transabdominal: 28 days after conception or 6 weeks after LMP
Human chorionic gonadotropin is synthesized by syncytiotrophoblasts (of placenta) and detectable amounts (~5 mIU/ml) appear in maternal serum about 8 days after conception (3 weeks after LMP). In the first trimester (first 12 weeks, calculated from day 1 of LMP) of pregnancy, hCG levels rapidly rise with a doubling time of about 2 days. Highest or peak level is reached at 8-10 weeks (about 100,000 mIU/ml). This is followed by a gradual fall, and from 15-16 weeks onwards, a steady level of 10,000-20,000 mIU/ml is maintained for the rest of the pregnancy (Figure 836.1). After delivery, hCG becomes non-detectable by about 2 weeks.
Box 836.2 shows minimum time required for the earliest diagnosis of pregnancy by hCG test and ultrasonography (USG).
Two types of pregnancy tests are available:
- Qualitative tests: These are positive/negative result types that are done on urine sample.
- Quantitative tests: These give numerical result and are done on serum or urine. They are also used for evaluation of ectopic pregnancy, failing pregnancy, and for follow-up of gestational trophoblastic disease.
Ectopic pregnancy refers to the implantation of blastocyst at a site other than the cavity of uterus. The most common of such sites (>95% cases) is fallopian tube. Early diagnosis and treatment of tubal ectopic pregnancy is essential since it can lead to maternal mortality (from rupture and hemorrhage) and future infertility. Ectopic pregnancy is a leading cause of maternal death during first trimester. Diagnosis of ectopic pregnancy can be readily made in most cases by ultrasonography and estimation of β-subunit of human chorionic gonadotropin.
Early diagnosis of unruptured tubal pregnancy can be made by quantitative estimation of serum hCG and ultrasonography. In normal intrauterine pregnancy, hCG titer doubles every 2 days until first 40 days of gestation. If hCG rise is abnormally slow, then an unviable pregnancy (either ectopic or abnormal intrauterine pregnancy) should be suspected.
Transabdominal USG can detect gestational sac in intrauterine pregnancy 6 weeks after LMP. The level of hCG in serum at this stage is >6500 mIU/ml. If gestational sac is not visualized at this level of hCG, then there is a possibility of ectopic pregnancy. Transvaginal ultrasonography can detect ectopic pregnancy average 1 week earlier than abdominal ultrasonography; it can detect gestational sac if β-hCG level is 1000-1500 mIU/ml. Therefore, if gestational sac is not visualized in the presence of >1500 mIU/ml of β-hCG level, an ectopic pregnancy can be suspected.
Early diagnosis of ectopic pregnancy provides the option of administration of intramuscular methotrexate (rather than surgery), which causes dissolution of conceptus. This improves the chances of patient’s future fertility. Serial measurements of hCG after surgical removal of ectopic pregnancy can help in detecting persistence of trophoblastic tissue.
Termination of pregnancy before fetus becomes viable (i.e. before 20 weeks) is called as abortion.
In threatened abortion, vaginal bleeding is present but internal os is closed and process of abortion, though started, is still reversible. It is possible that pregnancy will continue.
Serial quantitative titers of hCG showing lack of expected doubling of hCG level and USG are helpful in diagnosis and management of abortion.
Gestational Trophoblastic Disease (GTD)
It is characterized by proliferation of pregnancyassociated trophoblastic tissue. The two main forms of GTD are hydatidiform (vesicular) mole (benign) and choriocarcinoma (malignant). Clinical features of GTD are as follows:
- Short history of amenorrhea followed by vaginal bleeding.
- Size of uterus larger than gestational age; uterus is soft and doughy on palpation with no fetal parts and no fetal heart sounds.
- Excessive nausea and vomiting due to high hCG.
- Characteristic snowstorm appearance on pelvic USG.
Quantitative estimation of hCG is helpful in diagnosis and management of GTD.
Trophoblastic cells of GTD produce more hCG as compared to the trophoblasts of normal pregnancy for the same gestational age. Concentration of hCG parallels tumor load. Also, hCG continues to rise beyond 10 weeks of gestation without reaching plateau (as expected at the end of first trimester).
After evacuation of uterus, weekly estimation of hCG is advised till subsequent three (weekly) results are negative; following evacuation of vesicular mole, hCG becomes undetectable (after 2-3 months) on follow-up in 80% of cases. Plateau or rising hCG indicates persistent GTD. In such cases, chemotherapy is indicated.
Negative results for hCG after therapy should be regularly followed up every 3 months for 1-2 years.
LABORATORY TESTS FOR HUMAN CHORIONIC GONADOTROPIN
These are classified into two main groups:
- Biological assays or bioassays
- Immunological assays
In bioassay, effect of hCG is tested on laboratory animals under standardized conditions. There are several limitations of bioassays like need for animal facilities, need for standardization of animals, long time required for the test results, low sensitivity, and high cost. Therefore, bioassays have been replaced by immunological assays.
In Ascheim-Zondek test, urine from pregnant woman is injected into immature female mice. Formation of hemorrhagic corpora lutea in ovaries (after 4 days) is a positive test. Friedman test is similar except that urine is injected into female rabbit. In rapid rat test, injection of urine containing hCG into female rats is followed by hyperaemia and hemorrhage in ovaries. Yet another test measures release of spermatozoa from male frog after injection of urine containing hCG.
These are rapid and sensitive tests for detection and quantitation of hCG. Variable results are obtained by different immunological tests with the same serum sample; this is due to differences in specificity of different immunoassays to complete hCG, β-subunit, and β-core fragment. A number of immunological tests are commercially available based on different principles like agglutination inhibition assay, enzyme immunoassay including enzyme linked immunosorbent assay or ELISA, radioimmunoassay (RIA), and immunoradiometric assay.
A commonly used qualitative urine test is agglutination inhibition assay. Early morning urine specimen is preferred because it contains the highest concentration of hCG. Causes of false-positive test include red cells, leukocytes, bacteria, some drugs, proteins, and excess luteinizing hormone (menopause, midcycle LH surge) in urine. Some patients have anti-mouse antibodies (that are used in the test), while others have hCG-like material in circulation, producing false-positive test. Anti-mouse antibodies also interfere with other antibody-based tests and are known as ‘heterophil’ antibodies. Fetal death, abortion, dilute urine, and low sensitivity of a particular test are causes of false-negative test. Renal failure leads to accumulation of interfering substances causing incorrect results.
In latex particle agglutination inhibition test (Figure 836.2), anti-hCG antibodies are incubated with patient’s urine. This is followed by addition of hCGcoated latex particles. If hCG is present in urine, anti-hCG serum is neutralized, and no agglutination of latex particles occurs (positive test). If there is no hCG in urine, there is agglutination of latex particles (negative test). This is commonly used as a slide test and requires only a few minutes.
Sensitivity of agglutination inhibition test is >200 units/liter of hCG.
Radioimmunoassay, enzyme immunoassay, and radioimmunometric assay are more sensitive and reliable than agglutination inhibition assay.
Quantitative tests are employed for detection of very early pregnancy, estimation of gestational age, diagnosis of ectopic pregnancy, evaluation of threatened abortion, and management of GTD.
- Serum human chorionic gonadotropin:
– Non-pregnant females: <5.0 mIU/ml
– Pregnancy: 4 weeks after LMP: 5-100 mIU/ml
– 5 weeks after LMP: 200-3000 mIU/ml
– 6 weeks after LMP: 10,000-80,000 mIU/ml
– 7-14 weeks: 90,000-500,000 mIU/ml
– 15-26 weeks: 5000-80000 mIU/ml
– 27-40 weks: 3000-15000 mIU/ml
Further Reading: SEMEN ANALYSIS FOR INVESTIGATION OF INFERTILITY