HCG(Human chorionic gonadotrophin)

HCG, or human chorionic gonadotropin, is a glycoprotein hormone produced by

syncytial trophoblast cells of the placenta during pregnancy.

by trophoblast cells in (trophoblast diseases), hydatidiform mole and choriocarcinoma

in patients with ovarian germ cell carcinomas

in patients with testicular tumors (60% of the patients with nonseminomas and 10% to 30% with seminomas)

in patients with pituitary gland tumors

ectopic HCG production occurs sometimes in other malignancies including Breast,colon,pancreas and urothelial cancers.

During pregnancy HCG is secreted by the syncytial trophbast cells into the fluids of the mother and can be detected in the mother's blood or urine . This hormone is what we look for with a "pregnancy test".

HCG is first detectable in the blood as early as 7-8 days after ovulation,shortly after the blastocyst implants in the endometrium by sensitive HCG assays. 85% of normal pregnancies will have the HCG level double every 72 hours and reach a maximum level in about 10 to 12 weeks. It decreases to a much lower value by 16 to 20 weeks after ovulation and continues at this level for the remainder pregnancy.

HCG is a glycoprotein having a molecular weight of about 39,000 and has pretty similar molecular structure and function as LH secrted by the pituitary. During pregnancy the most important function is to prevent the normal involution of the corpus luteum at the end of cycle. Insead it causes the corpus luteum to secrete larger quantities of its usual sex hormones , estrogen and progesterone. These sex hormone prevent menstruation and cause the endometrium to continue growing and store large amount of nutrients. Under the influemnce of HCG the corpus luteum grows to about twice the size of its intial size by four weeks after the pregnanacy begins and its continued secretion of estrogen and and progesterone maintains the decidual nature of the endometrium which is necessary for the early development of fetus. If the corpus luteum is removed before 7^thweek of pregnancy, spontaneous abortion almost always occur. After 12 weeks placenta itself secretes sufficient quantities of progeserone and estrogen to maintain pregnancy for remainder of he gestation period, the corpus luteum slowly involutes after the 13^th – 17^th week of gestation.

PREGNANCY STATUS SERUM hCG LEVELS

An hCG level of less than 5 mIU/ml generally indicates you are not pregnant.

from conception	from LMP	(mIU/ML or IU/L)
7 days	3 weeks	0 to 5
14 days	28 days	3 to 426
21 days	35 days	18 to 7,340
28 days	42 days	1080 to 56,500
35 - 42 days	49 - 56 days	7,650 to 229,000
43 - 64 days	57 - 78 days	25,700 to 288,000
57 - 78 days	79 - 100 days	13,300 to 253,000
17 - 24 weeks	2nd trimester	4060 to 65,400
25 wks to term	3rd trimester	3640 to 117,000
After several days postpartum		nonpregnant levels (<5)"

"Results in Twin Gestation"

"Quanitative serum hCG tests detect multiple pregnancy approximately 9 weeks earlier than ultrasound. One small study (Jovanovic, 1977) found hCG levels in 15 singleton and 9 twin pregnancies were as follows:

DAYS FROM LMP	hCG in SINGLTON AVERAGE	GESTATION RANGE	hCG in AVERAGE	TWIN GESTATION RANGE
28	64.7	9.4 to 120	64.7	9.5 to 120
33	450	300 - 600	1,500	200 to 1,800
36	1,500	1,200 - 1,800	19,200	2,400 to 36,000
40	3,600	2,400 - 4,800	58,344	8,700 to 108,000
45	36,000	12,000 - 60,000	126,000	72,000 to 180,000
70	120,000	96,000 - 144,000	414,000	348,000 to 480,000"

There is a large variation in a "normal" HCG level for any given time in pregnancy but commonly

Greater-than-normal levels may indicate:

· choriocarcinoma of the uterus

· ectopic pregnancy

· hydatidiform mole of the uterus

· normal pregnancy

· ovarian cancer

· testicular cancer

Lower-than-normal levels may indicate:

· dead fetus

· incomplete miscarriage

· threatened spontaneous abortion

hCG is a heterodimeric glycoprotein with a total molecular weight of 39,000 consist of two subunits, alpha and beta, held together by ionic and hydrophobic forces. The alpha-subunit is a glycopeptide of 92 amino acids, with asparagine-linked (N-linked) sugar moieties attached at residues 52 and 78. It is stabilized by 5 disulfide linkages.

The ß-subunit of hCG is a glycopeptide of 145 amino acids, stabilized by 6 disulfide linkages. It has N-linked sugar moieties attached at residues 13 and 30, and 4 serine-linked (O-linked) sugar moieties attached to the C-terminal peptide (ß-subunit residues 121-145). The ß-subunits of the glycoprotein hormones are unique, giving them their different biological characteristics.

Process 1

Normal trophoblast hCG molecules (normal pregnancy) have mono and biantennary type N-linked sugar moieties and simple disaccharide-core O-linked sugar units. Trophoblast disease hCG molecules (persistent mole and choriocarcinoma) and germ cell and other cancer hCG molecules can be made with more-complex sugar side chains, triantennary N-linked sugar moieties and tetrasaccharide core O-linked sugar moieties . This type of hCG we call hyperglycosylated hCG or ITA (Invasive Trophoblast Antigen). The N-linked sugar side chains on pituitary hCG have unusual sulfated sugar moieties.

In addition to intact alpha-beta dimmer hCG, free hCG subunits (free alpha-subunit and free Beta-subunit) are produced in pregnancy, trophoblast disease and cancer. These can be detected in serum, plasma and urine samples.

Free subunits can derive from the excess synthesis of alpha or beta subunit or incomplete combination of subunits in cells. Excess alpha subunit which fails to get incorporated in hCG may become hyperglycosylated (like trophoblast disease and cancer hCG) with extra sugar residues on the N-linked sugar moieties . This is called large free alpha-subunit. This can also be detected in plasma, serum and urine samples.

Free subunits also derive from the slow dissociation of hCG, dissociation half-time >40 days at 37 deg, and from the more rapid dissociation of nicked, damaged or hyperglycosylated hCG, dissociation half-time 10-16 days . A nicked or cleaved hCG is produced. Nicked hCG is most evident in serum and urine samples in the later trimesters of pregnancy. It may be the principal form of hCG detected in choriocarcinoma patients, and in germ cell and other cancer cases. nicked hCG is cleaved or cut in the ß-subunit peptide between residues 47 and 48. It can be cleaved between residues 43 and 44 or 44 and 45 in some choriocarcinoma cases Although cleaved, the molecules are held together by the intra-peptide disulfide linkages; no amino acids or sugar residues are missing . Nicking occurs by the action of proteolytic enzymes in the placental, mole or cancer tissue, or in the circulation.

http://www.hcglab.com/process1.gif Process 1

Nicked hCG is unstable (dissociation half-time 10-16 days at 37 deg. versus >40 days for non-nicked hCG)

http://www.hcglab.com/process3.gif Process 3

Nicked free ß-subunit is rapidly removed from the circulation. It may be degraded in the kidney to ß-core fragment, and excreted into urine .

ß-core fragment may be the terminal degradation product of hCG. The linked sugar moieties on ß-subunit core fragment are also degraded. While the molecular weight of hCG is 39,000 the molecular weight of ß-core fragment is only 10,000. From 8 week of gestation until term, ß-core fragment is the principal hCG ß-subunit-related molecule in urine samples. Urine ß-core fragment may be the only hCG-related molecule detectable (in either serum or urine) in certain germ cell cancers, placental-site trophoblastic tumors, in ovarian cancer, bladder cancer and certain other malignancies (FIGURE.. Degradation of nicked free ß-subunit to ß-core fragment in the kidney.

http://www.hcglab.com/process4.gif Process 4

PRINCIPAL OF HCG TEST AND OF DISCORDANT RESULTS

A. PRINCIPALS

Most hCG tests or pregnancy test used today, whether a home urine test, a physician's office urine or blood test, or a clinical laboratory blood test are "sandwich assays". Sandwich assays use at two or more animal antibodies raised against different sites on hCG. Usually a mouse monoclonal antibody against one site on the hCG molecule, and a mouse monoclonal, or a sheep, rabbit or a goat polyclonal antibody against a second distant site on the hCG molecule. One antibody, the capture antibody, is in a solid phase permanently attached to a tube, plate, membrane, or bead. The second antibody, the tracer antibody, is labeled with a dye, with radioactivity, as an identifier. This antibody is in the liquid phase (SEE FIGURE 1, BELOW). Blood (serum or plasma) or urine is added to the assay system. After a short incubation period the hCG binds both the solid phase and liquid phase antibodies linking them. In this way it forms a sandwich or bridge between the solid support or capture antibody and the tracer antibody with the attached label. In this way the label becomes immobilized (SEE FIGURE 2, BELOW). After washing away the serum or plasma, and the excess tracer antibody, the amount of label attached to the solid support is measured. This is directly proportional to the amount of hCG (SEE FIGURE 3, BELOW).

FIGURE 1. Device with solid phase capture antibody to one site on hCG, and liquid phase tracer antibody (label shown by red star) to second or distant site on hCG

FIGURE 2. Serum or urine containing hCG (shown as ab) added to device. The hCG forms a sandwich or bridge between capture and tracer antibody.

FIGURE 3. Excess tracer antibody is washed away. Amount of label or tracer (red star) is measured. This is proportional to amount of hCG.

In a home pregnancy test or physician's office test, you have a disposable plastic device. It has an absorbent stem which is placed in flow of urine, or an opening/window in the plastic into which drops of urine are placed. The urine moves through the plastic device. In part of the device shielded by plastic the hCG (if present) attaches to the tracer or liquid phase antibody with a blue or red or gold color

B. DISCORDANT RESULTS WITH THE hCG TEST

Over fifty different quantitative hCG assays (clinical laboratory blood tests) are sold in the United States. Over eight different antibody binding sites have been identified on different part of the hCG molecule. Each commercial test uses a different combination of capture and tracer antibodies which recognize any two of the eight different binding site. One problem with hCG assays is the heterogeneity of hCG, its different synthetic forms and degradation products. This problem is compounded by the wide variation in hCG concentrations observed in serum and urine samples throughout pregnancy. Assays that use different combinations of antibodies recognize different molecules. An assay, for instance, that uses an antibody against the hCG ab subunit interface as capture antibody and an antibody against the core of the b subunit as tracer antibody, will recognize normal pregnancy hCG (non-nicked hCG), but might not detect nicked or damaged hCG, an important component of hCG immunoreactivity in trophoblastic disease and cancer.