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

Sample

1. This is done in the urine of the patient.
2. Collect the morning sample which has the maximum concentration of HCG.
   1. Try to do the test on a fresh urine sample.
   2. You can collect the urine at any time of the day.
   3. Urine specimen should be clear, in the case of turbidity or urine sediments requires filtration or centrifuge.
3. Instruct the patient not to drink after 2000 hours (8 PM) until the morning collection of the urine sample.
4. This test can be done in the serum.
5. Perform the test within 48 hours of collection.
6. Important: Centrifuge the urine at 900 x g for 10 minutes.
7. Can store the sample at 2 to 8 °C for 48 hours.
8. Serum for β – HCG is stable up to 7 days at  2 to 8 °C.
   1. For longer periods freeze at -20 °C.
   2. Avoid hemolyzed, turbid, or the sample which contains particulate material.

Precautions

1. If the test is delayed more than 48 hours, then freeze the samples at -20 °C.
2. Don’t repeat thawing and freezing again and again.
3. Hemolysis and lipemic serum give a false result.
4. Hematuria and proteinuria give a false positive test. I will recommend at least centrifuge the urine.
5. This test may be negative in the diluted urine.
6. drugs like diuretics lead to dilution of the urine and may give a false-negative result.
7. There are drugs which give false positive tests are anticonvulsants, hypnotics, tranquilizers, and antiparkinson drugs.

Indications

1. For the diagnosis of pregnancy.
2. It can be used during a high-risk pregnancy.
3. It can be used for ectopic pregnancy.
4. For screening of Down’s syndrome.
5. This may be used as a tumor marker in some malignancy.

Pathophysiology of Human chorionic gonadotropin (HCG)

1. The placental trophoblastic cells produce an appreciable amount of hormone, human chorionic gonadotropin (HCG).
2. HCG is a glycoprotein with a subunit of alpha (α)  and beta (β).
   1. Molecular weight is 37,900 D and has a high carbohydrate proportion than any other hormone.
   2. This is synthesized in the syncytiotrophoblast of the placenta.
      

      HCG formation from placental cells

      

      Placental hormones and HCG

   3. HCG stimulates the corpus luteum to produce progesterone which maintains the pregnancy.

3. This hormone is excreted in the urine.
   1. HCG is present in the blood and urine.
   2. HCG appears as early as the 10th day of fertilization or conception.
   3. In the first few weeks of the pregnancy, HCG rises markedly, and the serum levels are higher than the urine.
   4. After about one month, the HCG levels are the same in the serum and urine.
4. This hormone is negative in the urine of men and nonpregnant women.
   1. <5% of the female may show a minute amount of the HCG.
5. HCG consists of :
   1. Alpha subunit (α-HCG). This is the same for all the glycoprotein hormones. This is also part of pituitary hormones.
      1. α-HCG has a molecular weight of 14,900 where protein is 10200 and carbohydrates are 47,000.
   2. Beta subunit (β-HCG). This is specific to the HCG. This gives immunologic and biologic specificity. The β-HCG has antigenic individuality.
      1. β-HCG has a molecular weight of 23,000 where the protein portion is 16,000 and carbohydrates are 7000.
         

         HCG molecular structure

   3. Free β-subunit and intact β-subunit HCG are measured in most of the current methodologies.
   4. The β-HCG unit is specific for a pregnancy test.
      

      HCG secretion in the urine

6. This test becomes negative after delivery in 3 to 5 days.
7. β-HCG in blood detects pregnancy as early as 6 to 10 days of the implantation of the oocyte.
8. This will be positive after 14 days of the last cycle in the urine.
9. In a normal pregnancy, can find 25 mIU/mL after 2 to 3 days of implantation and after 8 to 10 days of fertilization.
10. The qualitative test detects pregnancy.
11. This has less sensitivity (20 to 50 IU/L) than the quantitative test.
12. This will be negative in the first week of the menstrual cycle.

Normal pregnancy

1. A normal pregnancy lasts approximately 40 weeks, it is measured from the first day of the last normal menstrual cycle.
2. Normal pregnancy is divided into three trimesters. Each trimester is slightly longer than 13 weeks.
3. The first trimester, o to 13 weeks, begins on the first day of last menses.
4. Ovulation occurs on approximately the 14th day of the regular menstrual cycle.
5. The fertilization occurs in the fallopian tubes and becomes a zygote, which is then carried down the tube into the uterus.

6. The zygote divides and becomes morula.
   1. The morula develops a cavity, the primitive yolk sac, and becomes a blastocyst, which implants in the uterine wall about 5 days after fertilization.
   2. The cells on the exterior wall of the blastocyst become trophoblasts, which invade the uterine endometrium and develop into chorionic villi, creating the placenta.
   3. Now, these products of conception are referred to as an embryo.
7. A cavity called the amnion forms within the embryo and enlarges with the accumulation of liquor amnii, usually called as amniotic fluid.
8. From the combination of three primary cells named as:
   6. Endoderm.
   7. Mesoderm.
   8. Ectoderm.
9. Now the organs will start to develop, this process is called organogenesis.
10. In the 10th week, the embryo is formed, where most major organs are developed and now it is called a fetus.
11. In the 13th weeks, the fetus weighs approximately 13 grams and is 8 cms long.
12. 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.
13. During the third trimester, 26 to 40 weeks, maturation of the organs is complete, weight is 3200 grams, and is about 50 coms long.
14. 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.

1. Complication arising from the mother are:
   1. Ectopic pregnancy.
   2. Hyperemesis graviderum.
   3. Preeclampsia.
   4. Liver diseases.
   5. Isoimmunization by the blood groups is hemolytic disorders.
   6. Grave’s disease.
   7. HELLP syndrome (H =hemolysis, EL = elevated liver enzymes, LP = low platelets count).
2. Abnormalities of the placenta are:
   1. Molar pregnancy (Hydatidiform mole).
      1. 5% of the partial mole transform into choriocarcinoma.
      2. 20% of the complete mole transform into choriocarcinoma.
   2. Rarely choriocarcinoma.
3. Complications due to the fetus are:
   1. Neural tube defect.
   2. Down’s syndrome.
   3. Trisomy 18.Preterm delivery.
   4. Preterm delivery.
   5. Presence of fetal fibronectin.
   6. 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.

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:

1. 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.
2. 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.

2. • 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:
       1. Protein.
       2. Drugs.
       3. Bacteria contamination.
       4. White blood cells or RBCs.
     • The false-negative result may be seen if the reagents:
       1. Kept at extreme of the temperature.
       2. Extreme urine pH.
       3. 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.
3. Radioimmunoassay.
   1. This is a highly sensitive and reliable test.
   2. RIA beta-HCG can be detected in the maternal blood.
   3. RIA can also be done on the urine sample as well.
   4. RIA is so sensitive that pregnancy can be detected before the missed menstrual cycle.
   5. 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: Neural tube defect Trisomy 21 Other fetal abnormalities
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	Variable changes, few increases and few have no change Factor XI decreases
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:

1. HCG is present in the pregnancy.
2. HCG may also be seen in 65% of the Ectopic pregnancy.
   1. 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.
3. Hydatidiform mole.
4. Choriocarcinoma.
5. Germ cell tumors of the ovary and testes.
6. HCG may be produced in primary liver cell carcinoma.

False-positive pregnancy test:

1. This can happen in 2 to 5% of the cases.
2. This may be seen due to interfering substances like:
   1. Proteins (proteinuria).
   2. Drugs like chlorpromazine, phenothiazine, and methadone.
   3. Bacteria infection.
   4. RBCs or WBCs (hematuria and pyuria).
3. Cross-reactivity with pituitary gonadotropins e.g. a high level of LH in the postmenopausal women.

Negative pregnancy test:

1. Dead fetus.
2. Threatened Abortion.
   1. There is a sudden drop in the level of the plateau.
3. Incomplete abortion.

False-negative tests:

1. These are common because usually, the kits detect HCG level at the concentration of 1000 to 2000 mIU/L.
   1. Therefore, these qualitative tests will not be positive until 8 to 14 days after the first missed menstrual cycle.
2. These qualitative tests may not detect normal pregnancy even after the second trimester (when HCG levels are low).
3. These tests may be negative in the ectopic pregnancy (when HCG levels are low).
4. Use of the old reagents.
5. In case if the antiserum-HCG is denatured by the temperature or change in the pH.
6. May be negative in the diluted urine with low specific gravity.
7. The sample was taken too early in the pregnancy.

Low HCG level indicate:

1. There may be a miscarriage.
2. Maybe blighted ovum.

Ectopic pregnancy

1. 1. Definition:
      1. When fertilized ovum implants in a location other than the uterus.
      2. The most common site is the fallopian tubes.
   2. Signs and symptoms:
      1. There is abdominal pain.
      2. There is amenorrhea.
      3. The patient may vaginal bleeding.
      4. The patient will have other S/S of pregnancy.
   3. Predisposing factors are:
      1. If there is a previous H/O ectopic pregnancy.
      2. Patients with H/O infertility are more prone to ectopic pregnancy.
      3. If there is the H/O gonorrhreal  or chlamydial infection.
      4. If there is the H/O intrauterine device application.
      5. In case of the H/O endometriosis.
      6. More chances in cases of in-vitro fertilization pregnancy.
      7. If it is tubal damage from infections or disease.
      8. In case there is H/O smoking.
      9. In the case of previous H/O miscarriage.
   4. Diagnosis:
      1. Three important symptoms are:
         1. Lower abdominal pain.
         2. Vaginal bleeding.
         3. An adnexal mass.
      2. β-HCG doubling time in early pregnancy ranges from 48 to 72 hours.
      3. A rise in the β-HCG level of at least 66% in 2 days is generally indicative of intrauterine pregnancy.
      4. If there is an abnormal rising in the β-HCG level <66% higher than the original values, should need further evaluation.
      5. β-HCG level varies from undetectable to 200,000 IU/L, depending upon the size and viability of the trophoblastic cells mass.
      6. Now advise the ultrasound and hematocrit will help to guide the correct diagnosis in suspected cases.
      7. The level is not like pregnancy, it will not double as it does in normal pregnancy.
      8. Progesterone also helps with an abnormal pregnancy.
         1. Progesterone levels of >25 ng/mL are found in the normal intrauterine pregnancy  (nearly in 97%).
         2. Values <5 ng/mL are associated with an abnormal pregnancy.
         3. While values between 5 to 25 ng/mL are intermediate.
         4. Progesterone values are limited because in 85% of the pregnancy the values are between 5 to 25 ng/mL.

Positive HCG  test can be seen in:

1. Pregnancy.
2. Ectopic pregnancy (it is positive in 65% of the cases).
3. Hydatidiform mole.
4. In males with testicular germ cell tumors (choriocarcinoma and embryonal cell carcinoma).
5. In a female with ovarian germ cell tumors (choriocarcinoma and embryonal cell carcinoma).
6. Liver cell carcinoma (hepatoma) can also make HCG.

Pregnancy test interpretation for the layman:

1. 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.
2. If it is negative, and no menstruation then repeats the pregnancy test after 6 to 7 days.
3. If still negative then advise for the ultrasound of the abdomen including the uterus.

NOTE: Please see more details on the beta-HCG level.

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