Pregnancy Diagnostic Tests: Principle, Procedure, and Results 

Most of the laboratory tests for pregnancy are based on detecting the presence of human chorionic gonadotropins (hCG) in the woman’s urine. The hCG (human chorionic gonadotropin) is secreted by syncytiotrophoblast cells of the placenta. hCG appears as early as the sixth day after fertilization in blood and the eighth day after fertilization in urine, the peak is reached at the ninth week of pregnancy. The level of HCG sharply decreases during the fourth and the fifth months.

In weeks since the last menstrual period (LMP)hCG level
3 weeks LMP5 – 50 mIU/ml
4 weeks LMP5 – 426 mIU/ml
5 weeks LMP18 – 7,340 mIU/ml
6 weeks LMP1,080 – 56,500 mIU/ml
7-8 weeks LMP7,650 – 229,000 mIU/ml
9-12 weeks LMP27,500 – 288,000 mIU/ml
13-16 weeks LMP13,300 – 254,000 mIU/ml
17-24 weeks LMP4,060 – 165,400 mIU/ml
25-40 weeks LMP3,640 – 117,000 mIU/ml
Non-pregnant55 – 200 ng/ml
hCG levels during pregnancy (data source: American Pregnancy Association)

HCG hormone prevents the degeneration of the corpus luteum (which occurs about two weeks after ovulation) and maintains its viability. This allows continued secretion of estrogens and progesterone by the corpus luteum, which are required for the prevention of menstruation and continued attachment of the fetus to the uterine endometrium. 

Some pregnancy tests are so sensitive that the earliest pregnancy diagnosis can be made within a few days of conception, i.e., even before the next missed period. These tests can be grouped into biological, immunological, and radiological.

Biological Diagnostic Tests 

Biological tests for pregnancy detection involve the injection of urine into various animals. Though these test methods are 99% accurate, they are not routinely performed in clinical practice as they are time-consuming and expensive. Another major limitation is the requirement of knowledge of the histological study of gonadal tissues for test result interpretation. 

Some popular biological pregnancy detection tests are the Aschheim-Zondek mouse test, Friedman rabbit test, Galli Mainini frog test, and Hogben test. 

Immunological Tests 

Immunological tests are used routinely in all hospitals and clinics to detect pregnancy. Compared to biological tests, they are less costly, simpler, easy to carry out, take very little time for reporting, and can confirm pregnancy within ten days of conception. 

Happy woman holding pregnancy test stick in the bathroom.
Happy woman holding pregnancy test stick in the bathroom.

Principle 

The HCG (human chorionic gonadotropins) secreted by syncytiotrophoblast cells of the placenta is antigenic. Antibodies against this hormone are available commercially and are employed to detect the presence of hCG in the serum or urine of pregnant women. HCG can be detected using various immunological methods such as precipitation, hemagglutination, complement fixation test, etc. 

The sensitivity level of hCG urine is 1.5-3.5 IU/ml in the slide test and 0.2-1. ml in test tube test. This concentration is reached 10th day of fertilization (i.e., even before the missed period). A pregnancy test can be performed anytime after the eighth day of pregnancy and is ideally performed after 14 days of missed period.

Procedure 

Women undergoing pregnancy tests are advised to restrict water intake for 12-14 hours. They can restrict water intake from the evening and collect the first urine sample the following day in a clean container.

A pregnancy test should be performed within 12 hours of urine collection. The specific gravity of urine should be at least 1.015 and free from protein and blood. 

Latex Agglutination Inhibition (LAI) Test (Gravindex test) 

Small globules of latex (rubber particles coated with pure hCG), and antiserum to hCG, are available commercially in ‘kit’ form. A urine sample is treated with antiserum on a glass slide placed against a black background. 

  • If the urine contains hCG (i.e., if the woman is pregnant), the antibodies in the antiserum are all “used up.” Then if the coated latex particles are added, they do not get agglutinated.
    Urine (hCG present) + Antiserum + Latex particles = No agglutination.
    Therefore, “No agglutination” means a positive result, i.e., the woman is pregnant.
Latex agglutination inhibition test for pregnancy detection
Latex agglutination inhibition test for pregnancy detection
  • If the urine sample does not contain HCG, then the antibodies in the antiserum “remain free.” Then if latex particles are added, an antigen-antibody reaction occurs, and the particles get agglutinated.
    Urine (HCG absent) + Antiserum + Latex particles = Agglutination.
    Therefore, “agglutination” means a negative result, i.e., the woman is not pregnant. 

Hemagglutination Inhibition (HAI) Test (Prognostican test) 

In this test, sheep’s RBCs coated with hCG are employed in place of latex particles. The test is done in a test tube, and observations are made after 2 hours. The principle is the same as LAI. A positive pregnancy test is indicated by the formation of a sharply demarcated brown ring at the bottom of the tube; the absence of the ring suggests a negative test.

One-step Immunoassay test 

This test is based on combining monoclonal antibody-dye conjugate with polyclonal solid-phase antibodies to qualitatively detect hCG in the urine.  A urine sample is applied to the test zone of the card or strip, and if it contains hCG, a pink-purple-colored band develops. A control is provided to check the potency of the test reagents. Several test kits under different proprietary names are available. 

ICT-based test for the detection of pregnancy (Image source: Andy Brunning)

A urine pregnancy test can be performed at home or in a clinic. Depending on the test format, the test kit can be dipped into the urine (dipstick urine pregnancy kit), or urine is applied using an eyedropper in the sample window. Depending on the test format, a color change, the appearance of the test line, a symbol (like a plus or minus), or pregnant or non-pregnant words denote the test result.

Radioimmunoassay (RIA) 

Radioimmunoassay is a more sensitive pregnancy test method and can detect the presence of hCG in the serum as early as 7-10 days following fertilization. 

HCG radiolabeled with iodine (iodine135) is treated with fixed amounts of antibodies and the urine/serum sample. RIA can detect as little as 0.003 IU/ml of the β subunit and 0.001 IU/ml of the α subunit of HCG in the urine or serum.

ELISA (Enzyme-linked immunosorbent Assay) 

ELISA has been widely used to detect a variety of antigens and antibodies. The principle of the test is the same as that of RIA, except that an enzyme is used in place of a radioactive substance. The enzyme acts on the substrate to produce a blue color, a positive pregnancy test.

Although immunological pregnancy tests are sensitive and accurate, false-positive and false-negative results can occur. 

False Positive Results

False positive pregnancy results (pregnancy test is positive, but the woman is not pregnant) may be obtained due to excessive protein or blood in the urine sample, at menopause, or at the time of ovulation due to increased secretion of luteinizing hormone (LH). Such results can also be seen at the beginning or with malignant tumors of the placenta in which hCG is produced from other sources like choriocarcinoma and hydatidiform mole. Thiazide diuretics, steroids, and thyroid drugs may affect the outcome of early pregnancy tests. 

False Negative Results

False negative pregnancy results (pregnancy test is negative, but the woman is pregnant) may be seen when the concentration of hCG in urine or serum is very low or testing too soon, or due to an ectopic pregnancy. 

Ultrasonography 

Ultrasonography (ultrasound) is a real-time, non-invasive pregnancy detection method and the most reliable method of detecting pregnancy. Pulses of ultrasonic waves at high frequency are generated from a piezo-electrical crystal transducer that also acts as a receiver to detect waves reflected from various parts of the uterus. The echoes (reflected waves) are displayed on the ultrasound screen. 

Pregnant woman on ultrasound

The gestational ring is evident as early as the 5th week of pregnancy, cardiac pulsations by the 10th week, and fetal movements by the 11th week. Ultrasonography is particularly useful in detecting fetal viability and position, site of the placenta, multiple pregnancies, amount of liquor present,  fetal-maternal abnormalities, etc. The method is also used for the determination of the sex of the fetus. 

References and further readings

  • What is HCG? American Pregnancy Association (URL: https://americanpregnancy.org/getting-pregnant/hcg-levels/)

Dr. Poonam Acharya

I am a gynecologist currently working at Nepalgunj Medical College. I have done MD in Obstetrics and Gynaecology from Kathmandu University.

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