Pregnancy test:- Part 1 – Normal Pregnancy, Beta-HCG, Human Chorionic Gonadotropin (HCG), Ectopic Pregnancy

Sample
- This is done in the urine of the patient.
- Collect the morning sample which has the maximum concentration of HCG.
- Try to do the test on a fresh urine sample.
- You can collect the urine at any time of the day.
- Urine specimen should be clear, in the case of turbidity or urine sediments requires filtration or centrifuge.
- Instruct the patient not to drink after 2000 hours (8 PM) until the morning collection of the urine sample.
- This test can be done in the serum.
- Perform the test within 48 hours of collection.
- Important: Centrifuge the urine at 900 x g for 10 minutes.
- Can store the sample at 2 to 8 °C for 48 hours.
- Serum for β – HCG is stable up to 7 days at 2 to 8 °C.
- For longer periods freeze at -20 °C.
- Avoid hemolyzed, turbid, or the sample which contains particulate material.
Precautions
- If the test is delayed more than 48 hours, then freeze the samples at -20 °C.
- Don’t repeat thawing and freezing again and again.
- Hemolysis and lipemic serum give a false result.
- Hematuria and proteinuria give a false positive test. I will recommend at least centrifuge the urine.
- This test may be negative in the diluted urine.
- drugs like diuretics lead to dilution of the urine and may give a false-negative result.
- There are drugs which give false positive tests are anticonvulsants, hypnotics, tranquilizers, and antiparkinson drugs.
Indications
- For the diagnosis of pregnancy.
- It can be used during a high-risk pregnancy.
- It can be used for ectopic pregnancy.
- For screening of Down’s syndrome.
- This may be used as a tumor marker in some malignancy.
Pathophysiology of Human chorionic gonadotropin (HCG)
- The placental trophoblastic cells produce an appreciable amount of hormone, human chorionic gonadotropin (HCG).
- HCG is a glycoprotein with a subunit of alpha (α) and beta (β).
- Molecular weight is 37,900 D and has a high carbohydrate proportion than any other hormone.
- This is synthesized in the syncytiotrophoblast of the placenta.
- HCG stimulates the corpus luteum to produce progesterone which maintains the pregnancy.
- This hormone is excreted in the urine.
- HCG is present in the blood and urine.
- HCG appears as early as the 10th day of fertilization or conception.
- In the first few weeks of the pregnancy, HCG rises markedly, and the serum levels are higher than the urine.
- After about one month, the HCG levels are the same in the serum and urine.
- This hormone is negative in the urine of men and nonpregnant women.
- <5% of the female may show a minute amount of the HCG.
- HCG consists of :
- Alpha subunit (α-HCG). This is the same for all the glycoprotein hormones. This is also part of pituitary hormones.
- α-HCG has a molecular weight of 14,900 where protein is 10200 and carbohydrates are 47,000.
- Beta subunit (β-HCG). This is specific to the HCG. This gives immunologic and biologic specificity. The β-HCG has antigenic individuality.
- β-HCG has a molecular weight of 23,000 where the protein portion is 16,000 and carbohydrates are 7000.
- Free β-subunit and intact β-subunit HCG are measured in most of the current methodologies.
- The β-HCG unit is specific for a pregnancy test.
- Alpha subunit (α-HCG). This is the same for all the glycoprotein hormones. This is also part of pituitary hormones.
- This test becomes negative after delivery in 3 to 5 days.
- β-HCG in blood detects pregnancy as early as 6 to 10 days of the implantation of the oocyte.
- This will be positive after 14 days of the last cycle in the urine.
- In a normal pregnancy, can find 25 mIU/mL after 2 to 3 days of implantation and after 8 to 10 days of fertilization.
- The qualitative test detects pregnancy.
- This has less sensitivity (20 to 50 IU/L) than the quantitative test.
- This will be negative in the first week of the menstrual cycle.
Normal pregnancy
- A normal pregnancy lasts approximately 40 weeks, it is measured from the first day of the last normal menstrual cycle.
- Normal pregnancy is divided into three trimesters. Each trimester is slightly longer than 13 weeks.
- The first trimester, o to 13 weeks, begins on the first day of last menses.
- Ovulation occurs on approximately the 14th day of the regular menstrual cycle.
- The fertilization occurs in the fallopian tubes and becomes a zygote, which is then carried down the tube into the uterus.
- The zygote divides and becomes morula.
- The morula develops a cavity, the primitive yolk sac, and becomes a blastocyst, which implants in the uterine wall about 5 days after fertilization.
- The cells on the exterior wall of the blastocyst become trophoblasts, which invade the uterine endometrium and develop into chorionic villi, creating the placenta.
- Now, these products of conception are referred to as an embryo.
- A cavity called the amnion forms within the embryo and enlarges with the accumulation of liquor amnii, usually called as amniotic fluid.
- From the combination of three primary cells named as:
- Endoderm.
- Mesoderm.
- Ectoderm.
- Now the organs will start to develop, this process is called organogenesis.
- In the 10th week, the embryo is formed, where most major organs are developed and now it is called a fetus.
- In the 13th weeks, the fetus weighs approximately 13 grams and is 8 cms long.
- During the second trimester, 13 to 26 weeks, the growth of the fetus is rapid. The fetus weighs around 700 grams, 30 cms long, and many organs begin to mature.
- During the third trimester, 26 to 40 weeks, maturation of the organs is complete, weight is 3200 grams, and is about 50 coms long.
- Now the term is 37 to 40 weeks, then normal labor starts by the rhythmic contraction of the uterus.
Stages of normal pregnancy and development of fetus:Clinical features First trimester Second trimester Third trimester Time period 0 to 13 weeks 13 to 26 weeks 26 to 40 weeks Weight 13 grams 700 grams 3200 grams Length 8 cms 30 cms 50 cms Organs development Embryo (Fetus), three epithelial layers Organs start maturing Maturation of organs is complete
Complications of the pregnancy:
Most of the pregnancy progress without any complications. The most common causes can arise from the mother, placenta, or fetus.
- Complication arising from the mother are:
- Ectopic pregnancy.
- Hyperemesis graviderum.
- Preeclampsia.
- Liver diseases.
- Isoimmunization by the blood groups is hemolytic disorders.
- Grave’s disease.
- HELLP syndrome (H =hemolysis, EL = elevated liver enzymes, LP = low platelets count).
- Abnormalities of the placenta are:
- Molar pregnancy (Hydatidiform mole).
- 5% of the partial mole transform into choriocarcinoma.
- 20% of the complete mole transform into choriocarcinoma.
- Rarely choriocarcinoma.
- Molar pregnancy (Hydatidiform mole).
- Complications due to the fetus are:
- Neural tube defect.
- Down’s syndrome.
- Trisomy 18.Preterm delivery.
- Preterm delivery.
- Presence of fetal fibronectin.
- Fetal respiratory distress syndrome.
Normal, HCG level During Pregnancy
- Negative in nonpregnant women.
- Positive in pregnant women.
- The blood test is positive after 11 days of conception.
- This test may become positive as early as 4 days after the expected date of menstruation.
- Or pregnancy detected 8 to 14 days after the first missed menstrual cycle and the positivity is 95%.
- The urine test is positive after 12 to 14 days of conception (fertilization).
- The peak level by 8 to 11 weeks of pregnancy, in another reference peak level, is at 60th to 70th day of pregnancy, then starts drops progressively.
- The peak level at 8th to 10th week of gestation in serum and urine is around 30,000mIU/mL.
- The blood test is positive after 11 days of conception.
Normal values of HCG in the pregnancy:
Detectable level | HCG mIU/mL |
6 to 8 days of conception | level around 10 to 15 |
Double every 3 days | 1200 to 6000 |
Double every 4 days | 6000 to peak level |
10 to 12 weeks | 150,000 to 200,000 |
At the end first trimester | around 100,000 |
By the early second trimester | peak level is 10,000 (800ng/mL) |
2n trimester | 10,000 to 50,000 |
3rd trimester | 10,000 to 50,000 |
After delivery until 2 weeks | detectable |
Ectopic pregnancy | no normal dynamics of HCG (Abnormal) |
- To convert into SI units x 1.0 = IU/L
Source 2
HCG
- Qualitative = Negative
- Pregnancy = Positive
- Male and nonpregnant females = <5 mIU/mL
- Quantitation of HCG
Gestation week | Whole HCG mIU/mL |
<1 | 5 to 50 |
2 | 50 to 500 |
3 | 100 to 10,000 |
4 | 1000 to 30,000 |
5 | 3500 to 115,000 |
6 to 8 | 12,000 to 270,000 |
12 | 15,000 to 220,000 |
β – HCG Normal
- <2 ng/mL
- Or <5 mIU/mL
Types of pregnancy diagnostic tests:
- Biologic test on urine.
- These tests are not used now. These are of historic importance.
- In this test, the urine of the suspected lady is injected into an animal like a rabbit, mouse, or frog. These animals develop corpus luteum.
- Then these animals were sacrificed and search done for the corpus luteum.
- Immunologic tests.
- These are agglutination inhibition test done on urine and blood.
- In this test antibodies against HCG are produced and the test can be done in 2 min or in some kit 2 hours.
-
- These have a high false-positive rate so the test should be done after 28 days of the last menstrual cycle.
- The false-negative test may be seen when the HCG level is less than 25 to 50 IU/L.
- The false-negative test may be seen if the urine contains:
- Protein.
- Drugs.
- Bacteria contamination.
- White blood cells or RBCs.
- The false-negative result may be seen if the reagents:
- Kept at extreme of the temperature.
- Extreme urine pH.
- Expired reagents.
- Now some of the improved kits can detect after 18 days.
- Monoclonal antibody-based kits can detect pregnancy after 3 to 7 days of conception.
- Limitations:
- These are not quantitative tests and may miss, not find early pregnancy or any other abnormality.
- Because of the monoclonal antibody against HCG can detect a small amount of the HCG even 3 to 7 days of the conception.
- Always run a positive control. The standard usually contains a small amount of the HCG.
- These have a high false-positive rate so the test should be done after 28 days of the last menstrual cycle.
- Radioimmunoassay.
- This is a highly sensitive and reliable test.
- RIA beta-HCG can be detected in the maternal blood.
- RIA can also be done on the urine sample as well.
- RIA is so sensitive that pregnancy can be detected before the missed menstrual cycle.
- The detection limit is around 5 IU/L. But may detect 1 to 2 IU/L.
Routine tests needed in the normal pregnancy for the evaluation of the fetus survival and abnormality:
Test needed in pregnancy | Value in pregnancy | Interpretations/Complications |
HCG level | >10 IU/L | It should double after every 2 days for the first 8 weeks |
Screening in the first trimester (free β-HCG, Pregnancy-associated plasma protein) | Depends upon various factors | To rule out trisomy 21 |
Quad screening in 2nd trimester (HCG, AFP, Estriol, and Inhibin) |
Depends upon various factors | To assess for:
|
Hematocrit (Hct) | 36 to 48% | To assess for the anemia |
Blood grouping | Type A, B, AB, and O | To prevent the hemolytic disease of the newborn |
Rh typing | To prevent the hemolytic disease of the newborn (HDN) | |
Antibody detection | It should be negative | May harm the fetus |
Toxoplasmosis IgG | Negative | To prevent the damage to the baby brain and other organs |
Rubella IgG | If positive indicate immunity | Rubella causes damage to various organs of the fetus (congenital rubella syndrome) |
Treponema pallidum testing | Should be negative | To prevent damage to the fetus like liver, brain, and anemia |
Cervical smear and culture for gonorrhea | Should be negative | To prevent miscarriage, premature birth, and premature rupture of membranes |
Cervical smear and culture for Chlamydia | Should be negative | Tp prevent premature rupture of the membranes, low birth weight, eye, and lung infection |
HB surface antigen (HBS-Ag) | Should be negative | To assess for active disease |
HB surface antibody (HBS-Ab) | Positive (Immune) status | To assess for exposure to vaccine |
HIV | Should be negative | To prevent ectopic pregnancy, early abortion, UTI, bacterial pneumonia, oral and vaginal thrush |
Group B streptococcus | Should be negative | It causes severe infection, pneumonia, and meningitis |
Effects of Pregnancy on different biochemical parameters:
Lab tests | Effect of pregnancy | Explanation |
Hematocrit | It is decreased | It is due to an increase in the plasma volume |
Coagulation factors |
|
|
BUN | It has a mild decrease | There is an increase in glomerular filtration rater |
Creatinine | There is a mild decrease | There is an increase in the glomerular filtration rate |
Alkaline phosphatase (ALK) | It is increased | It is due to an increase in the production of placental heat-stable ALK |
Triglycerides | It is increased | |
Cholesterol | It is increased | |
1,25 dihydroxy vitamin D | It is increased | It is due to increased calcium and transfer of Ca++ to the fetus |
Parathyroid hormone | It is increased | In this case, ionized Ca++ remain normal |
T3 and T4 | These are increased | But the patient is euthyroid |
Thyroxin binding globulin | it is increased | Patient is euthyroid |
Interpretations, Differential Diagnosis, of Positive pregnancy test:
- HCG is present in the pregnancy.
- HCG may also be seen in 65% of the Ectopic pregnancy.
- A level of 20 IU/L or less subunit within the first week of pregnancy indicates ectopic pregnancy because there is an insufficient number of trophoblastic cells.
- Hydatidiform mole.
- Choriocarcinoma.
- Germ cell tumors of the ovary and testes.
- HCG may be produced in primary liver cell carcinoma.
False-positive pregnancy test:
- This can happen in 2 to 5% of the cases.
- This may be seen due to interfering substances like:
- Proteins (proteinuria).
- Drugs like chlorpromazine, phenothiazine, and methadone.
- Bacteria infection.
- RBCs or WBCs (hematuria and pyuria).
- Cross-reactivity with pituitary gonadotropins e.g. a high level of LH in the postmenopausal women.
Negative pregnancy test:
- Dead fetus.
- Threatened Abortion.
- There is a sudden drop in the level of the plateau.
- Incomplete abortion.
False-negative tests:
- These are common because usually, the kits detect HCG level at the concentration of 1000 to 2000 mIU/L.
- Therefore, these qualitative tests will not be positive until 8 to 14 days after the first missed menstrual cycle.
- These qualitative tests may not detect normal pregnancy even after the second trimester (when HCG levels are low).
- These tests may be negative in the ectopic pregnancy (when HCG levels are low).
- Use of the old reagents.
- In case if the antiserum-HCG is denatured by the temperature or change in the pH.
- May be negative in the diluted urine with low specific gravity.
- The sample was taken too early in the pregnancy.
Low HCG level indicate:
- There may be a miscarriage.
- Maybe blighted ovum.
Ectopic pregnancy
-
- Definition:
- When fertilized ovum implants in a location other than the uterus.
- The most common site is the fallopian tubes.
- Signs and symptoms:
- There is abdominal pain.
- There is amenorrhea.
- The patient may vaginal bleeding.
- The patient will have other S/S of pregnancy.
- Predisposing factors are:
- If there is a previous H/O ectopic pregnancy.
- Patients with H/O infertility are more prone to ectopic pregnancy.
- If there is the H/O gonorrhreal or chlamydial infection.
- If there is the H/O intrauterine device application.
- In case of the H/O endometriosis.
- More chances in cases of in-vitro fertilization pregnancy.
- If it is tubal damage from infections or disease.
- In case there is H/O smoking.
- In the case of previous H/O miscarriage.
- Diagnosis:
- Three important symptoms are:
- Lower abdominal pain.
- Vaginal bleeding.
- An adnexal mass.
- β-HCG doubling time in early pregnancy ranges from 48 to 72 hours.
- A rise in the β-HCG level of at least 66% in 2 days is generally indicative of intrauterine pregnancy.
- If there is an abnormal rising in the β-HCG level <66% higher than the original values, should need further evaluation.
- β-HCG level varies from undetectable to 200,000 IU/L, depending upon the size and viability of the trophoblastic cells mass.
- Now advise the ultrasound and hematocrit will help to guide the correct diagnosis in suspected cases.
- The level is not like pregnancy, it will not double as it does in normal pregnancy.
- Progesterone also helps with an abnormal pregnancy.
- Progesterone levels of >25 ng/mL are found in the normal intrauterine pregnancy (nearly in 97%).
- Values <5 ng/mL are associated with an abnormal pregnancy.
- While values between 5 to 25 ng/mL are intermediate.
- Progesterone values are limited because in 85% of the pregnancy the values are between 5 to 25 ng/mL.
- Three important symptoms are:
- Definition:
Positive HCG test can be seen in:
- Pregnancy.
- Ectopic pregnancy (it is positive in 65% of the cases).
- Hydatidiform mole.
- In males with testicular germ cell tumors (choriocarcinoma and embryonal cell carcinoma).
- In a female with ovarian germ cell tumors (choriocarcinoma and embryonal cell carcinoma).
- Liver cell carcinoma (hepatoma) can also make HCG.
Pregnancy test interpretation for the layman:
- Keep in mind if there are missed menstruation cycles, and delayed more than one week. Then ask for the morning sample for a pregnancy test.
- If it is negative, and no menstruation then repeats the pregnancy test after 6 to 7 days.
- If still negative then advise for the ultrasound of the abdomen including the uterus.
NOTE: Please see more details on the beta-HCG level.