USA hCG REFERENCE SERVICE

THE USA hCG REFERENCE SERVICE HAS RE-OPENED

THE USA hCG REFERENCE SERVICE IS NO LONGER AFFILIATED WITH THE UNIVERSITY OF NEW MEXICO, IT IS NOW A DIVISION OF COLE CHORIOGONADOTROPIN CONSULTING LLC

REFERRAL WITH USA hCG REFERENCE SERVICE
       To refer a patient to the USA hCG Reference service click here

Read Dr. Cole's extensive articles about the USA hCG Reference Service experience, hCG biology, and hCG, hyperglycosylated hCG and free ß-subunit levels in different conditions             Dr. Cole's Articles 

…..a simple explanation of why some men and women are positive in hCG pregnancy tests, of the role of hCG forms in cancer and gestational trophoblastic diseases, about important new gestational trophoblastic disease diagnoses, and an explanation of how the USA hCG Reference Service can optimally assist you.

Chapter 1. Introduction to hCG
Chapter 2. hCG production in women outside of pregnancy
Chapter 3. hCG production in men
Chapter 4. Gestation trophoblastic diseases in women
Chapter 5. The USA hCG Reference Service Protocol

Chapter 1. Introduction to hCG

        hCG stands for human chorionic gonadotropin. This is a category of molecules unto itself. A single molecule called hCG was discovered by Bernhard Aschner in Vienna in 1912. It was hormone that maintained progesterone production in early weeks of pregnancy. Today we see hCG as a group of structurally similar molecules, all detected by a serum total hCG pregnancy test as run by a laboratory, a clinic point-of-care pregnancy test, or a home over-the-counter urine pregnancy test. This group of molecules comprises hCG the hormone, hyperglycosylated hCG an autocrine (a molecule that acts on the same cells as produce it), free ß-subunit (an autocrine) and pituitary hCG a hormone (Table 1).

Table 1. The hCG group of molecules

        hCG the hormone performs multiple functions during pregnancy (Table 1)These include promotion of progesterone production by ovarian corpus luteal cells (3-8), promotion of adequate blood supply to the placenta during pregnancy (9-16), and promotion of growth and differentiation of the placenta and fetus during pregnancy (17-44). Hyperglycosylated hCG drives pregnancy implantation and placental growth during pregnancy (1,2,45,46). It also drives invasion, growth and malignancy by malignant gestational trophoblastic diseases, invasive mole, gestational trophoblastic neoplasm and choriocarcinoma (1,2,45,46). Free ß-subunit is produced by all advanced cancer primaries in humans, whether bladder cancer, lung cancer, a brain-related malignancy, or any other malignancy in the body. It promotes cancer cell growth, invasion and malignancy (47-55). Pituitary hCG during the menstrual cycle assists luteinizing hormone (LH) function (56-64), although concentration are miniscule and may not be detected by a pregnancy tests. Postmenopause and perimenopause higher levels of hCG are observed, mare like those detected by pregnancy test, but tis post-menopause hCG  has no known function (56-64).

          The USA hCG Reference Service measures uniquely (only service in world) all these forms of hCG, hCG, hyperglycosylated hCG and free ß-subunit, plus pituitary follicle stimulating hormone (FSH) to first determine the source of hCG-related molecules, placenta, cancer cell, gestational trophoblastic malignancy, and pituitary gland. One other hCG source exists, that is false positive hCG due to heterophilic antibodies in the blood. The USA hCG Reference Service identifies this source first by showing positive hCG in serum and negative hCG in urine. They also use Scantibodies HBR, an antibody to heterophilic antibodies too show false positive hCG. Finally, they measure hCG ß-core fragment in serum as a test for false positive hCG. hCG ß-core fragment is naturally only made in the kidney and deposited into urine. Its detection in serum demonstrates a possible false positive hCG.  Having demonstrated the source of hCG (Placenta, pituitary, cancer, gestational trophoblastic malignancy, false positive hCG) the USA hCG Reference Service then investigates (further blood tests) the reason for hCG production.

          The USA hCG Reference Service specializes in assisting men and women with positive hCG test results. Physicians refer case to the USA hCG Reference Service to discover the origin of hCG production outside of pregnancy in men and woman. The USA hCG Reference Service also aids woman with gestational trophoblastic diseases and neoplasms with correct diagnoses, with diagnosis of quiescent gestational trophoblastic disease, diagnosis of minimally aggressive or chemorefractory gestational trophoblastic neoplasm and in affirming diagnosis of aggressive gestational trophoblastic  neoplasm or choriocarcinoma. We make the differential diagnosis of choriocarcinoma or gestational trophoblastic neoplasm and placental site trophoblastic disease (PSTT). The USA hCG Reference Service is the gestational trophoblastic disease assistance center.

Chapter 2. hCG Production in Women Outside of Pregnancy

          All too often today, women are found positive in an hCG test or pregnancy test, who are truly not pregnant. These are women who are sure they are not pregnant because they are in menopause. There are also women who could be pregnant, but ultrasound shows no fetal sac of pregnancy. We say “all too often,” because today pregnancy hCG testing is very common, it is unknowingly a normal part of the work up prior to any surgery at any hospital, and prior to any invasive procedure, regardless of a woman’s age. The USA hCG Reference Service knows of 6 clear reasons why a person may be strangely positive in an hCG pregnancy test, a. Pituitary hCG; b. Quiescent gestational trophoblastic disease; c. Cancer; d. False positive hCG; e. Gestational trophoblastic neoplasm or choriocarcinoma; f. Familial hCG syndrome. There is no other know cause for somebody to be erroneously positive in and hCG pregnancy test, other than the 6 causes listed here. The USA hCG Reference Service is pleased to announce that most cases of hCG production in woman outside of pregnancy are due to pituitary hCG or quiescent gestational trophoblastic disease, or due to harmless or benign conditions. Cancer cases, gestational trophoblastic neoplasm/choriocarcinoma cases, false positive cases and familial hCG cases are observed rarely.

a. Pituitary hCG

          Pituitary hCG is normally and naturally produced at extremely low levels during the menstrual cycle. As reported, pituitary hCG levels are around 1/120th of the sister pituitary hormone, LH (63,64), or <2 mIU/ml of hCG, a levels barely detectable by any automated laboratory serum hCG test. hCG is strictly produced incidentally during menstrual years (56-64). On chromosome 19 is eight hCG ß-subunit genes and one LH ß-subunit genes all back to back (Figure 1). The hypothalamus of the brain signals the pituitary to make LH, this is done by the hypothalamus releasing gonadotropin releasing hormone (GnRH). Upon signaling LH ß-subunit to be produced it is hard for GnRH to not signal the neighboring hCG ß-subunit to be made. They both share a common α-subunit. Thus a small amount of hCG is normally made by the pituitary, this supplements LH in its normal menstrual activity. Pituitary hCG is 40 times more potent than LH (61), so that while the average person makes a tiny amount of hCG (about 1/120^th of the LH concentration (63,64)) it seemingly significantly enhances LH.

Figure 1. Mechanism of production of hCG by pituitary gonadotrope cells. There are 8 hCGß genes back to back with 1 LHß gene on chromosome 19. The GnRH signal from the hypothalamus is intended to stimulate the LHß gene, but cannot help also stimulating a small amount of hCGß genes. There is an excess of the common α-subunit for the ß-subunit to combine with.

LH activity promotes growth of ovarian follicle, meiosis, stigma formation, ovulation, formation of corpus luteum and progesterone production following ovulation. It is assumed that the pituitary hCG bunds the common LH/hCG receptor doing exactly the same, significantly enhacing LH menstrual cycle actions.

          When a woman enters menopause she stops ovarian functions and stops producing estrogen and progesterone or enter amenorrhea. In peri-menopause or the start of menopause, stoppage is partial leading to oligomenorrhea. Estrogen and progesterone normally attenuate the hypothalamus GnRH. When estrogen and progesterone are starting to halt (peri-menopause) or completely halt (menopause), GnRH pulses rise significantly, promoting pituitary LH levels and FSH levels higher than ever, and incidentally promoting pituitary hCG production higher than ever.

          In peri-menopause and menopause hCG may range from 1-39 mIU/ml or be very detectable by all hCG pregnancy tests. Pituitary LH levels rise to 40-140 mIU/ml and pituitary FSH is always >30 mIU/ml. As found, elevated hCG levels may be observed normally by somebody in menopause, perimenopause, or by somebody with history of having her ovaries removed, oophorectomy or bilateral salpingo-oophorectomy (BSO) (ovaries and fallopian tubes removed) (56-64). These elevated levels of hCG produced by the pituitary in menopausal states are completely natural, completely normal and totally harmless. When somebody is in menopause or peri-menopause, however, it is important for the USA hCG Reference Service after demonstrating hCG of pituitary origin, to exclude any possible cause for hCG production, cancer as the source of the hCG, or gestational trophoblastic disease as the source of the hCG.

          The normal USA hCG Reference Service tests performed on somebody with pituitary hCG, are hCG, LH and FSH. hCG Free ß-subunit is used to show cancer origin hCG and hyperglycosylated hCG to show gestational trophoblastic disease origin hCG. In the USA hCG Reference Service experience, in perimenopause hCG levels normally range from <1 to 22 mIU/ml and in menopause from <1 to 39 mIU/ml. The FSH test (>30 mIU/ml) is an accurate indicator of peri-menopause, or the possibility of detection of pituitary hCG (80).

b. Quiescent gestational trophoblastic disease

          Quiescent gestational trophoblastic disease is another common source of elevated hCG in the non-pregnant woman (65-67). Woman commonly have a miscarriage of pregnancy, which unknown to them is a hydatidiform mole a common source of miscarriages. Residual hydatidiform mole tissue remaining in the uterus mostly dies leaving a microdot of highly differentiated placenta tissue or syncytiotrophoblast cells remaining. This microdot produced regular hCG only, and no invasion promoter or hyperglycosylated hCG. This very benign microdot of tissue is harmless. It produces, however, regular hCG, leading to positive hCG pregnancy tests. In most cases, the microdot disperses or disappears in 6 months. In approximately 10% of cases, however, this microdot grows start to produce hyperglycosylated hCG with the appearance of cytotrophoblast cells and becomes malignant, with rising hCG levels, and needs chemotherapy (invasive gestational trophoblastic neoplasm).

The USA hCG Reference Service checks out everything in a woman found to have quiescent gestational trophoblastic disease. Quiescent disease is demonstrated by the finding of total hCG (2 mIU/ml to 210 mIU/ml) in a person, in the presence of zero hyperglycosylated hCG. Hyperglycosylated hCG is used to exclude the possibility of a gestational trophoblastic neoplasm, invasive mole or choriocarcinoma, FSH to exclude pituitary hCG. A free ß-subunit test is performed to exclude any possibility of non-trophoblastic (nothing to do with the placenta) cancer as an explanation for the hCG and positive pregnancy test. 

 c. Cancer

          As published (56-64), a cancer of any primary, when advanced, can produce hCG-like material. This is most commonly the free ß-subunit of hCG, as detected in serum samples, and its degradation product, ß-core fragment as detected in urine samples (56-64). Research now shows that advanced cancers not only produce free ß-subunit, but use the free ß-subunit to promote cancer cell growth and metastases. New vaccines are undoing clinical trial throughout the world. These generate an antibody to the free ß-subunit which removes free ß-subunit and stops it from promoting cancer growth. Hopefully these vaccines will be available by prescription in the near future. Cancer is a simple explanation for a positive hCG or pregnancy test, with no obvious pregnancy. The USA hCG Reference Service identifies cancer cases by the finding that >60% of the total hCG reactivity in serum is free ß-subunit, and by the supportive finding that >60% of the total hCG in urine is free ß-subunit or hCG ß-core fragment. If cancer is found a full malignancy work up, MRI of the head and pelvis and CT scan of the chest, and cancer blood work (blood cell checked for lymphoma and leukemia) is strongly indicated to find the cancer primary (can be anywhere in body).

d. False positive hCG

          False positive hCG results occur because of heterophilic antibodies in the serum. Heterophilic means cross-species. A heterophilic antibody is one that recognizes both human and animal antibodies. In an hCG test, hCG links together animal antibodies in the test tube, a cross-species antibody can bind and link together the animal antibodies causing a false positive result. Humans with history of mononucleosis or immunoglobulin A deficiency syndrome are at higher risk for false positive test results (68). Every hCG assay uses a different combination of anti-animal antibodies. Normally somebody that is falsely positive in test 1, may not be false positive in test 2 and so on.

          In the USA hCG Reference Service experience, a false positive hCG result can be between 2 mIU/ml and 1000 mIU/ml. A false positive case is identified by the USA hCG Reference Service by a mixture of results. Firstly, that the USA hCG Reference Service total hCG test result is 2-fold or greater different from the referring centers total hCG result. Secondly, we repeat the total hCG test using Scantibodies HBT blocking agent, an antibody to heterophilic antibodies. This repeat can give very different results indicating heterophilic antibodies and false positive hCG. Thirdly, the USA hCG Reference Service always runs its total hCG test multiple times and at multiple dilution. If the diluted assay result is not parallel (result x dilution), then a false positive case is indicated. The most absolute test for false positive hCG is urine hCG. Interfering heterophilic antibodies do not enter the urine. As such, somebody with false positive serum hCG results may be negative for hCG in urine. The finding of positive hCG in serum and negative hCG in urine is an absolute indicator of false positive hCG. The USA hCG Reference Service performs a urine creatinine test to make sure that urine is not negative due to very dilute urine. The USA hCG Reference Service also checks serum samples for false positive hCG by measuring hCG ß-core fragment in serum, a degradation product of hCG only normally present in urine. Its detection in serum would indicate false positive hCG.

e. Gestational trophoblastic neoplasm or choriocarcinoma

          Gestational trophoblastic neoplasm (choriocarcinoma without histology confirmation) and choriocarcinoma are malignancies that derive from normal pregnancies, and from hydatidiform moles of pregnancy (pregnancies comprising only placenta tissue, these derive from sperm fertilizing other sperm inside an egg, leading to a genetic disorder of pregnancy). Remaining deeply imbedded cytotrophoblast cells (placenta root cells) following delivery of a pregnancy and the afterbirth placenta, or surgical removal of a hydatidiform mole, become transformed so that they cannot fuse and become benign syncytiotrophoblast cells. Cells then produce hyperglycosylated hCG, which drives invasion, as in implantation of pregnancy. In implantation of pregnancy the cytotrophoblast fuse and loose the hyperglycosylated hCG or the invasion signal, and thus controlling implantation. The transformed trophoblast cell do not fuse so that they first invade the width of the uterus and then invade the body leading to pelvic, then lung and then brain metastases. Gestational trophoblastic neoplasm/choriocarcinoma is the most invasive malignant disease known to humans. It also responds exceedingly well to chemotherapy, so has a very high 5 years survival rate of 80-90% of cases.

          Gestational trophoblastic neoplasm/choriocarcinoma is marked by high production of hCG. Since the malignancy is mostly cytotrophoblast cells, gestational trophoblastic neoplasm/choriocarcinoma are marked by producing mostly or only hyperglycosylated hCG. It is by testing for hyperglycosylated hCG that the USA hCG Reference Service identifies gestational trophoblastic neoplasm or choriocarcinoma cases. Gestational trophoblastic neoplasm/choriocarcinoma cases urgently need MRI and CT scans to show the stage of disease. Cases are urgently given chemotherapy. 

f. Familial hCG syndrome

        Familial hCG syndrome is the most recently discovered cause for positive total hCG pregnancy tests, outside of pregnancy. As found in 5 cases, men and women can inherit elevated hCG production (69). Serum hCG levels of <1 to 153 mIU/ml have been observed, with hCG present in multiple men and women in a family, commonly in brothers and sisters and in one parent. Commonly, we hear that all have been erroneously diagnosed as pregnant prior to surgery, with surgeries being cancelled due to suspicion of pregnancy or cancer. In all cases, the patient are not producing hCG, pituitary hCG or hyperglycosylated hCG, excluding cancer, gestational trophoblastic neoplasm/choriocarcinoma, pituitary origin hCG and false positive hCG. These 5 cases were all producing a degraded form of hCG free ß-subunit, free ß-subunit missing the C-terminal peptide. This is a biologically inactive form of hCG, so that it does not interfere with ovulation or the menstrual cycle in woman or sperm production in men.

Chapter 3. hCG Production in Men

        It is very strange for a man to have a positive pregnancy hCG test, but it is found and it happens. In such cases pregnancy, of course, is not a possibility. We ask you to read the section above on woman and a. Pituitary hCG. Elevated pituitary hCG can occur in a man with a non-functioning testis (no sperm, no testosterone). The lack of testicular estrogen and testosterone feedback to the hypothalamus can lead to excessive GnRH production, and to elevated GnRH-promoted pituitary hCG, LH and FSH production. We also ask you to look above at section c. Cancer, cancer is a possible explanation for positive hCG in a man. Another explanation that can be found in men and woman is False Positive hCG, see above section d. False Positive hCG. Finally, familial hCG syndrome occurs in both men and woman. Consider above section f. Familial hCG syndrome. Germ cell testicular cancer occur rarely in men, particularly young men in their teens and twenties, see below.

a.    Germ cell testicular malignancy

          Testicular germ cell malignancies, cancers common in young men, as they advance take on the morphology of female choriocarcinomas, or in many ways become female placenta tissue-like choriocarcinoma. These testicular germ cell malignancies include testicular choriocarcinomas, testicular yolk sac malignancies, testicular teratoma and testicular embyonal carcinoma. These carcinomas produce hyperglycosylated hCG, like choriocarcinoma (see above), which drives invasion and malignancies. The malignancies are identified by the detection of hyperglycosylated hCG in men and high total hCG levels like occur in choriocarcinoma in women. They are confirmed by MRI or ultrasound of the testicle. If testicular germ cell malignancy is identified chemotherapy is needed urgently. Like choriocarcinoma, the malignancy responds very well to chemotherapy with a high survival rate.

Chapter 4. Gestational Trophoblastic Diseases in Women

          Gestational trophoblastic disease are genetic oddments and malignancies associated with pregnancy. There are multiple categories of gestational trophoblastic diseases. All can be separately diagnosed by the USA hCG Reference Service.

a.         a. The origin of gestational trophoblastic diseases

          Complete and partial hydatidiform mole are the root gestational trophoblastic diseases. In these pregnancy disorders the genetic half set of chromosomes  (haploid set) in a sperm combines with the genetic half set in a second sperm, rather than with the genetic half set in an ACTIVE egg to form a zygote (70-72). The zygote grows, becoming 2 cells, then 4 cell and 8 cells and then in the absence of the appropriate female X chromosome signals fails to differentiate and become just placenta tissue (in partial moles, partial differentiation occurs leading to the presence of some fetal elements in the molar mass). The placenta cell of hydatidiform mole implant and then become a pregnancy comprising just placenta tissue (complete mole) or placenta tissue with limited fetal tissue (partial mole). The hCG levels in a hydatidiform mole pregnancy are extremely high due to the p[resence of primarily placenta tissue, between 150,000 and 1,500,000 mIU/ml at around 8 weeks of pregnancy.

          As illustrated in Figure 2. In normal pregnancy, a half set of chromosomes or haploid set (23X) comes from the maternal egg, and a half set of chromosomes or haploid set come from the sperm (23X or 23Y). If the combination after fertilization is 46XX a female results and if it is 46XY a male results. As show in Figure 2, a complete mole results from an empty egg, one lacking a chromosome half set. This most commonly occurs in woman partially through puberty or in peri-menopause.  A sperm penetrates this empty egg giving it a half set. A second sperm or duplicated spermal half set fertilizes it, making a 46XX and 46XY male and female complete mole, a pregnancy that is just placenta or a non-viable pregnancy.

          In a partial mole (Figure 2), an egg has an immature half set (haploid set) of chromosomes, that has not completed the maturation process or second stage of meiosis. This also most commonly happens when puberty is not complete or a woman is in peri-menopause. A sperm penetrates this egg leaving an active half set from the sperm in the egg. A second sperm or duplicated spermal half set then fertilizes this egg. A partial mole classically has triploid genetics in that it contains 3 sets of chromosomes, 2 male-origin sets and one inactive female set. A partial mole has an inactive female X chromosome. This partially signals the generation of some fetal elements. A partial mole comprises mostly placental tissue with some fetal elements or possible amniotic fluid, it is, however,  a completely non-viable pregnancy.

          Complete moles commonly lead to invasive disease or invasive mole (see below), partial moles rarely lead to invasive disease. Moles occur and have to be removed by dilation and curettage. The occurrence of moles is 1 in 900 pregnancies in the USA. The occurrence may be much higher (1 in 300 pregnancies) because most hydatidiform moles spontaneously abort in the first trimester of pregnancy, particularly partial moles. Hydatidiform mole is particularly common in third world countries where tribal groups are predominant. In tribal groups it is common to marry woman at 13 or 14 years, before they have completed puberty. Hydatidiform moles are more common in young pregnancies and pregnancies of woman close to menopause. The occurrence of moles may be as common as 1 in 50 pregnancies in tribal nations. Complete hydatidiform moles are commonly the precursor to invasive gestational trophoblastic diseases: gestational trophoblastic neoplasm, choriocarcinoma and placenta site trophoblastic tumor, thus are the root gestational trophoblastic diseases. 

Figure 2. Mechanism of chromosome set combination in normal pregnancy, complete hydatidiform mole and partial hydatidiform mole.

    b.    Hydatidiform mole 

          As described in the previous section, hydatidiform mole is the root gestational trophoblastic disease, occurring at a rate of 1 in 900 pregnancies in the United States and Western Europe. The real rate of hydatidiform mole is much higher, about 1 in 300 pregnancies when considering molar miscarriages of pregnancy. Hydatidiform moles occur with incidences as high as 1 in 50 pregnancies in some tribal nations like the Philippines, Indonesia and certain African countries.

          In a pregnancy which is a hydatidiform mole, women appear to have abdomens larger than expected for their date of pregnancy. This is largely due to the absence of amniotic fluid as a cushion to the pregnancy. Hydatidiform mole may be diagnosed by the finding of unduly high hCG total levels due to the sheer mass of placental tissue. While total serum hCG levels at 8 weeks of pregnancy maybe 20,000 mIU/ml to 180,000 mIU/ml for a normal term pregnancy, it may be 150,000 to 1,500,000 mIU/ml for a partial or complete hydatidiform mole. Unduly high hCG levels may be the first symptom of hydatidiform mole. Ultrasound can show a normal pregnancy from 6 weeks of gestation (weeks since start of last menstrual period) onwards (Figure 3), it can show a complete hydatidiform mole as a classical snowflake pattern, formed by placenta hygromatous cysts, and a partial hydatidiform mole as a mixture of hygromatous cysts, fetal elements and possibly amniotic fluid (Figure 3). The nature of a hydatidiform mole needs to always be definitively determined by cytogenetics. If it is diploid (46 chromosomes) then it is a complete mole, and if it triploid (69 choromosomes) then it is a partial mole. 

Figure 3. Ultrasound scan

A. Complete hydatidiform mole

B. Partial hydatidiform mole

c. Quiescent gestational trophoblastic disease   

     Quiescent gestational trophoblastic disease is an inactive form of invasive mole, gestational trophoblastic neoplasm or choriocarcinoma. All of the diseases are characterized by the presence of hyperglycosylated hCG, which drives invasion, tumor growth and malignancy, Hyperglycosylated hCG is produced by cytotrophoblast cells. When a hydatidiform mole is removed by dilation an curettage the hCG level may head to zero (see Figure 4), but serum total hCG level off around 100 mIU/ml (2-210 mIU/ml), and remains at around 100 (2-210 mIU/ml) for 6 months, then disappears. This is quiescent gestational trophoblastic disease (65,67). Only non-invasive regular hCG is produced by a pinpoint of differentiated syncytiotrophoblast cells. Quiescent gestational trophoblastic disease may follow evacuation of a hydatidiform mole, miscarriage of a pregnancy (actually a molar pregnancy), chemotherapy for invasive mole, chemotherapy for gestational trophoblastic neoplasm and chemotherapy for choriocarcinoma. The presence of quiescent disease is demonstrated by the USA hCG Reference Service by showing the absence of hyperglycosylated hCG (65,67).

Figure 4, Hydatidiform mole evacuation leading to quiescent gestational trophoblastic disease

        Quiescent gestational trophoblastic disease, lacking hyperglycosylated hCG does not grow, expand or invade, so is never responsive to continuing chemotherapy. It is recommended that all chemotherapy be halted when quiescent gestational trophoblastic disease is discovered. In about 10% of cases of quiescent gestational trophoblastic disease following hydatidiform mole or miscarriage of pregnancy, persistent disease, see below, invasive hydatidiform mole, may follow. When it follows, hCG instead of going down significantly and continuously rises, chemotherapy will be needed to destroy this malignancy. In about 25% of cases of quiescent gestational trophoblastic disease following treatment of choriocarcinoma, or following treatment of gestational trophoblastic neoplasm (choriocarcinoma, not proven by pathology), persistent disease follows.

          When a person has quiescent gestational trophoblastic disease it is very important for them to take a contraceptive pill to avoid getting pregnant at all costs. If someone achieved pregnancy, the invasive signal hyperglycosylated hCG produced during pregnancy implantation, could transform the quiescent gestational trophoblastic disease into recurrent invasive disease. Having a recurrent invasive disease at the same time as pregnancy is a total disaster. The recurrent disease may destroy the pregnancy. Abortion is recommended when this happens.  

          The USA hCG Reference Service Reference Service has consulted on well over one hundred cases of quiescent gestational trophoblastic disease (65, 67). You may call them the world experts on quiescent gestational trophoblastic disease. These have seen quiescent cases with total hCG ranging from 2.3 to 210 mIU/ml. These are considered the limits for quiescent gestational trophoblastic disease (65, 67).   

d. Invasive mole

          Invasive mole most commonly occurs in woman with history of complete hydatidiform mole. It rarely occurs after partial hydatidiform mole. Once a complete mole is evacuated by dilation and curettage, cytotrophoblast cells, which produce the invasion signal hyperglycosylated hCG might remain deeply implanted in the uterus. Sometimes as the hCG levels decline they form a shoulder or start to rise indicating invasive mole disease (Figure 5). Alternatively, hCG levels may reach zero,

then one week to six months later hCG levels start to incline indicating an invasive mole (Figure 5). In invasive mole cases, classically hyperglycosylated hCG accounts for 40 to 80% of the total hCG. Hyperglycosylated hCG produced by cytotrophoblasts cells, root placental cells, is the invasion signal in invasive mole.

          An invasive mole first invades the thickness of the uterus, or myometrial muscle (Stage 1). It then invades the pelvis, particularly the cervix and vagina (Stage 2). In then invades the liver and lungs (stage 3), and finally invaded the brain (stage 4). Sometimes invasive mole develops into choriocarcinoma-like disease, see below under e. Aggressive disease. The difference between an invasive mole and a gestational trophoblastic neoplasm/choriocarcinoma cases is that tissue maintains villous structure, like the villi that transfer nutrients during pregnancy. In gestational trophoblastic neoplasm/choriocarcinoma the tissue has lost all villous stracture.

          The Bagshawe protocol of chemotherapy agent is given in invasive mole cases. This is a regiment of agents that is proven to be affective in this disease (73). The Bagshawe protocol is-

1.     Methotrexate chemotherapy, daily intra-muscular injections for 5 days, repeat every 2 weeks

2.     Actinomycin D chemotherapy, daily intra-venous injections for 5 days, repeat every 2 weeks

3.     EMA-CO, weekly oscillating mixture of Etoposide, methotrexate and actinomycin D, and cyclophosphamide and onvocin (vincristine). Up to 7 bi-weekly regimens.

4.     EMA-EP, weekly oscillating mixture of Etoposide, methotrexate and actinomycin D, and etoposide and cisplatinin. Up to 7 bi-weekly regimens.

 It is extremely rare for major complication or death to occur in invasive mole, the survival rate is close to 100%. Occasionally invasive mole cases can become resistant to chemotherapy, see below under f. Minimally Aggressive disease.

 Figure 5. Declining hCG levels after hydatidiform mole evacuation and invasive mole. The upper panel show hCG rise 3 weeks after hCG levels have become undetectable. The lower panel shown an elbow in hCG decline following dilation and curettage (D&C).

e. Aggressive gestational trophoblastic neoplasm/choriocarcinoma

          Aggressive gestational trophoblastic neoplasm (no histology) or choriocarcinoma (histology confirmed) occurs when root cytotrophoblast cell transform, losing the ability to fuse and become non-invasive syncytiotrophoblast cells. Gestational trophoblastic neoplasm or choriocarcinoma is like a cancer of cytotrophoblast cells, driven by hyperglycosylated hCG, it can be one of the most invasive malignancies known to mankind, most commonly appearing in stage 3 and stage 4 with metastases to the lung, liver or brain. Even in stage 3 and 4 it is a very curable disease (80-95% complete survival rate), if recognized appropriately and treated rapidly with appropriate chemotherapy. In the USA hCG Reference Service experience aggressive cases of gestational trophoblastic neoplasm or choriocarcinoma mostly produce a high proportion of hyperglycosylated hCG, the invasion signal. As shown below in Table 2, the 33 cases of early (<2,000 mIU/ml) aggressive gestational trophoblastic neoplasm (choriocarcinoma, gestational trophoblastic neoplasm and invasive mole) referred to the USA hCG Reference Service. Thirty two of 33 cases were successfully treated with chemotherapy (methotrexate, actinomycin D or EMA-CO). The median hCG was 330 mIU/ml (IU/L) and the mean hyperglycosylated hCG was 79 ± 22%. The USA hCG Reference Service also saw 15 cases with advanced aggressive gestational trophoblastic neoplasm (Table 3). These patients all responded appropriately to chemotherapy except for case 198, which was advanced with 932,000 mIU/ml, with brain and lung metastases, did not respond to Xeloda, a very strange chemotherapy to give to someone with extremely advanced choriocarcinoma, she died (given by an General Oncologist, not a specialist, a Gynecologic Oncologist). The median total hCG in these advances case was 50,053 IU/L and the mean hyperglycosylated hCG was 91%. It is normal in cases with aggressive gestational trophoblastic neoplasm to use the proven Bagshawe chemotherapy protocol- The Bagshawe protocol of chemotherapy agent is given in all gestational trophoblastic neoplasm/choriocarcinoma cases. This is a regiment of agents that is proven to be most effective in these diseasee (73). The Bagshawe protocol is-

1.     Methotrexate chemotherapy, daily intra-muscular injections for 5 days, repeat every 2 weeks

2.     Actinomycin D chemotherapy, daily intra-venous injections for 5 days, repeat every 2 weeks

3.     EMA-CO, weekly oscillating mixture of Etoposide, methotrexate and actinomycin D, and cyclophosphamide and onvocin (vincristine). Up to 7 bi-weekly regimens.

4.     EMA-EP, weekly oscillating mixture of Etoposide, methotrexate and actinomycin D, and etoposide and cisplatinin. Up to 7 bi-weekly regimens.

          The USA hCG Reference Service can uniquely determine the grade or aggressiveness of gestational trophoblastic neoplasm or choriocarcinoma by measuring the extent to which hyperglycosylated hCG is the form of hCG produced. Unfortunately the hyperglycosylated hCG tests for gestational trophoblastic disease is not available anywhere else in the world. The USA hCG Reference Service can also detect chemorefractory cases with gestational trophoblastic neoplasm or choriocarcinoma, in that they characteristically have low hyperglycosylated hCG levels (low hCG doubling rate), or are slow growing malignancies. Gestational trophoblastic neoplasm or choriocarcinoma most commonly follows pregnancy (1 in 30,000 pregnancies) It also can follow a complete hydatidiform mole (1 in 100 hydatidiform moles). It is also important to double check the forms of hCG produced in a first occurrence or recurrence of gestational trophoblastic neoplasm or choriocarcinoma, could it be pituitary hCG generated in perimenopause, could it be false positive hCG, or is there any chance that it is coming from a malignancy.

Table 2. USA hCG Reference Service early (<2000 IU/L) malignant gestational trophoblastic disease cases (choriocarcinoma, gestational trophoblastic neoplasm and invasive mole). These case are divided into A. aggressive (hyperglycosylated hCG >40%), 33 cases, and B. minimally aggressive cases (hyperglycosylated hCG <40%), 36 cases . hCG-H is hyperglycosylated hCG, Mtx is methotrexate, and ActD is actinomycin D, Ifos is Ifosamide, TAH is trans-abdominal hysterectomy, TVH is trans-vaginal hysterectomy, BEP is bleomycin-etoposite-cisplatin, ICE is ifos-carpolplatin-etoposide, EMA-CO is weekly cycles of etoposide-methotrexate-actinomycin D with cyclophosphamide-vincristine, EMA-EP is weekly cycles of etoposide-methotrexate-actinomycin D with etoposide-cisplatin.

Table Table 3. Cases with choriocarcinoma and gestational trophoblastic neoplasm with advanced aggressive disease (>2,000 IU/L).

f.  Minimally aggressive gestational trophoblastic neoplasm/choriocarcinoma/ invasive mole

          Not all gestational trophoblastic neoplasm or choriocarcinoma cases are aggressive. Some cases can be minimally aggressive or slow growing, or chemorefractory or poorly responding to chemotherapy. All this information can be extracted by the USA hCG Reference Service from the proportion of hyperglycosylated hCG. We have consulted on cases with just 1% hyperglycosylated hCG that are extremely slow growing (the total hCG doubles every 2-3 months) or that poorly respond to all chemotherapy because they are slow growing. We call these cases minimally aggressive gestational trophoblastic neoplasm (no histology) or choriocarcinoma (has histology).

          Table 2 shows 33 early (<2000 IU/L) aggressive cases, and Table 3 show 15 advanced aggressive cases (>2,000 IU/L) of gestational trophoblastic neoplasm, choriocarcinoma and invasive mole. These are all fast growing cases with high hyperglycosylated hCG (mean 79% and 91%). As shown, 32 of 33 early aggressive cases, and 14 of 15 advanced aggressive cases appropriately responded to all chemotherapy. This is 96% positive response to chemotherapy. As shown in    Table 2, we also saw 36 cases with minimally aggressive disease (gestational trophoblastic neoplasm, choriocarcinoma and invasive mole). These patient all had slowly growing tumors, with hCG doubling rates of greater than two weeks. These patients also had very low cancer driving force or low hyperglycosylated hCG (Mean 13 ± 11%). As discovered, all 36 of 36 case were chemorefractory or reacted poorly to any chemotherapy. Some patients went through as many as 8 chemotherapy protocols (case 301, 447 and 271) with resistance to every chemotherapy regimen. In some respects, this is no surprise with chemotherapy not working with slow growing disease, since the theory of chemotherapy is to kill fast growing cells.

          Three case with chemorefractory minimally aggressive disease, having undergone numerous chemotherapy regimens halted further chemotherapy, could not stand thing any more, and turned to their faith and God (cases 198, 402 and 459). All three women agreed to supply the USA hCG Reference Service with blood samples for testing their disease and to revert to their physicians if disease became out of control (Figure 6).

Figure 6. The three cases (cases 198, 402 and 459) refusing further chemotherapy after not responding to low hyperglycosylated hCG and chemorefractory disease.

          As shown in Figure 6, in all 3 cases, disease advanced greatly over 4 to 8 months. All 3 women sought help once lung and brain metastases showed symptoms. Two were treated by their gynecologic oncologists with EMA-CO at this time (Bagshawe regimen) and survived (case 402 - - - - - - - - -, case 459 _______).  One of the 3 patients (case 198 ……………), with a total hCG of 964,000 was treated by a general oncologist with Xeloda, a strange non Bagshawe regimen for extreme advanced disease and died. It was clear from the three cases that as total hCG rises, so does percent hyperglycosylated hCG and aggressiveness of disease (Figure 6). This finding led to the observation that allowing total hCG to rise, probably will permit percent hyperglycosylated hCG to rise and would permit appropriate response to chemotherapy regimen.
          The USA hCG Reference Service was seemingly being referred an unusually high number of minimally aggressive gestational trophoblastic neoplasm case. Seemingly, many of these had become hopeless cases after 2 years of failure to respond to any chemotherapy. The USA hCG Reference Service, learning from the experience with the three women that refused further chemotherapy (Figure 6), proposed to test a new protocol. Under this protocol, women with chemorefractory minimally aggressive disease, would be withdrawn from any chemotherapy for approximately 4 months, or until total hCG had risen to >3,000 IU/L. At that time they would then be given EMA-CO chemotherapy from the Bagshawe regimen, even if EMA-CO had failed to work previously (failed to work because person had minimally aggressive disease, not due to resistance). This was somewhat of a risky protocol in that women can develop lung nodules when the hCG rises above 1500 mIU/ml. These can have potential complications. Amazingly, this protocol worked in 30 of 35 cases, and work very appropriately, with the women being cured (65). In the remaining 5 cases patient withdrew from the protocol because of the development of lung nodules. The USA hCG Reference Service now recommends this protocol in minimally aggressive cases. We have tried to share this procedure with other gestational trophoblastic disease treatment centers. There has been some resistance, however, because other centers do not have the critical hyperglycosylated hCG test and are unable to investigate it themselves. We are proposing some nationwide clinical trials (G.O.G. trials) to show this protocols worth in which we will measure hyperglycosylated hCG for all participants.
          We finish this section by warning patients with minimally aggressive or chemorefractory gestational trophoblastic disease to be careful. People die from these diseases, die primarily from complications of chemotherapy. Chemotherapy kills, we know of 3 cases that died from complications of bleomycin and etoposide.

 g. Placental Site Trophoblast Tumor (PSTT)

          Placental site trophoblastic tumor (PSTT) is a rare form of trophoblastic tumor involving trophoblastic cells that do not produce hyperglycosylated hCG. In that respect it is more like a non-trophoblastic malignancy, driven by cancer cytokines. PSTT may not have quite as good survival statistics as choriocarcinoma, gestational trophoblastic neoplasm and invasive mole. PSTT is best treated with surgical hysterectomy and by a separate chemotherapy protocol to the Bagshawe regimen. This biggest complication of PSTT is that to the pathologist it looks very similar to choriocarcinoma, and all too often wrong histological diagnoses are made. The USA hCG Reference Service has found a test that distinctly separates choriocarcinoma and PSTT. This is the free ß-subunit test. We show that most total hCG made in PSTT cases is free ß-subunit (74, 75). The USA hCG Reference Service uses the free ß-subunit test successfully to differentially diagnose PSTT, choriocarcinoma and gestational trophoblastic neoplasm.

Chapter 5. The USA hCG Reference Service Protocol

          The following protocol is used by the USA hCG Reference Service to monitor men and women with positive pregnancy tests, and to manage women with gestational trophoblastic diseases. After running these test protocols measuring total hCG, C-terminal peptide total hCG, intact hCG, free ß-subunit, ß-core fragment, hyperglycosylated hCG and nicked hCG tests, and FSH and creatinine tests, the USA hCG Reference Service prepares a formal report describing the test results and the inferred diagnosis. The report is prepared by Laurence A. Cole PhD, medical director of the USA hCG Reference Service. This is the USA hCG Reference Service. The high fees charged pay for approximately one week work involved.

a.    Total hCG testing

          The first testing is always total hCG testing. We chose the Siemens Immulite hCG test for this purpose, this test is unusual in that it uses two antibodies to the core of the ß-subunit. As such it detects hCG and all hCG-related molecules and their degradation products, hCG, hyperglycosylated hCG, nicked hCG, nicked hCG missing the ß C-terminal peptide, free ß-subunit, hyperglycosylated free ß-subunit, nicked free ß-subunit, nicked free ß-subunit missing the ß C-terminal peptide and ß-core fragment (76,77). We first measure serum total hCG in duplicate, and then ½, 1/5 and 1/10 (an if needed 1/100 and 1/1000) dilutions in duplicate. We also measure duplicate serum hCG with Scantibodies HBR added, an antibody to heterophilic antibodies to block interference. Then we test total hCG in urine in duplicate. The Siemens Immulite test has been shown to work equally well in serum and urine samples (78). We also test urine at 1/2, 1/5 and 1/10 dilution.
          From this combination of results we know what dilution to run all our test at. From the compilation of data, mean serum hCG, mean urine hCG, serum hCG with HBR blocking agent, and urine hCG we can determine if the serum hCG result is valid, or whether heterophilic antibodies are falsifying results. Firstly, heterophilic antibodies do not enter urine, so if patient is positive in serum and negative in urine it suggests heterophilic antibody interference or a false positive hCG (74). Many people have very dilute urine due to major liquid intake. We measure the concentration of the urine by a creatinine urine test. Secondly, if the USA hCG Reference Service total hCG results is two or more fold different from the referring centers total hCG results it suggests heterophilic antibody interference or a false positive hCG. Thirdly, if the diluted samples give results non-parallel to the undiluted samples (dilution x result) ¹ undiluted sample it suggests a heterophilic antibody or false positive result.

          If a false positive result is indicated further tests are run. First we run our specialized total hCG assay using a C-terminal peptide determinant and secondly our specialized intact hCG assay on the serum assay. These use different animal antibodies to the Siemens Immulite 1000 assay. If these tests give very different result to the Siemens Immulite test, then false positive hCG due to heterophilic antibodies is indicated. As a final test to confirm a case of false positive hCG, we use our urine hCG ß-core-fragment test. This assay measure the final breakdown product of hCG free ß-subunit which is only found in urine samples and never found in serum. If it is positive with a serum sample then heterophilic antibodies must be present which causes a false positive hCG (74,79).

b.    Free ß-subunit testing

          All serum samples contain some free ß-subunit, it is usually from the dissociation of hCG and hyperglycosylated hCG as occurs normally, but it never accounts for more than 20% of total hCG. We see serum free ß-subunit accounting for 20% to 80% of total hCG as a marker for placental site trophoblastic disease (PSTT) (74,75,79). Urine ß-core fragment is the urine terminal degradation product of hCG free ß-subunit. We measure urine free ß-subunit and urine ß-core fragment, if these are 20% to 80% of total urine hCG it confirms the diagnosis of PSTT. PSTT is finally confirmed by patient having a history of gestational trophoblastic disease. This data together leads to the PSTT diagnosis.

          Almost all advanced cancers of the body produce a free ß-subunit, whether bladder cancer, ovarian cancer, endometrial cancer, hepatic cancer, breast cancer, lung cancer or a brain based malignancy. The finding of >60% free ß-subunit in serum or urine of a person positive in a pregnancy tests is indicative of cancer. This is confirmed by measuring free ß-subunit and ß-core fragment in urine samples (are they >60% of total hCG). When we report this to physicians it can create many problems, where is the cancer. Normally we recommend that the physician arrange an MRI of the head and pelvis and CT of the chest. We also recommend appropriate blood work to exclude leukemia or a lymphoma.

c.    Hyperglycosylated hCG testing

          Hyperglycosylated hCG is the tumor promotion signal produced in invasive mole, gestational trophoblastic neoplasm and choriocarcinoma in woman. It is also the signal produced by testicular germ cell malignancies in men. In woman, the finding of undetectable hyperglycosylated hCG suggests quiescent gestational trophoblastic disease. This can follow the disappearance of hCG from a hydatidiform mole, and the disappearance of hCG from a miscarriage of pregnancy (pregnancy was a mole). In these cases, hCG plateaus as it drops below 210 IU/L. Quiescent gestational trophoblastic disease can follow chemotherapy for invasive mole, gestational trophoblastic neoplasm or choriocarcinoma. Quiescent gestational trophoblastic disease is an inactive disease that requires no chemotherapy (65-67).

          Hyperglycosylated hCG is an incredibly good test for assessing the grade of invasive mole, gestational trophoblastic neoplasm and choriocarcinoma (66). Is it a surprise that measurement of the molecule that drives invasion in gestational trophoblastic disease is invaluable to assess the diseases aggressiveness? As established by the USA hCG Reference Service, disease producing >40% hyperglycosylated hCG or total hCG can be classed as aggressive, and disease producing >80% hyperglycosylated hCG is classed as very aggressive. On the other hand, disease producing less that 40% hyperglycosylated hCG is considered minimally aggressive (65). As found, it is the minimally aggressive disease that is most commonly slow growing and chemorefractory or poor responding to chemotherapy. The USA hCG Reference Service recommend a new protocol for treating such cases, see section 3f minimally aggressive disease.

d.    Other hCG-related assays

          The USA hCG Reference Service runs multiple other assays as needed to reach a final diagnosis. We have a C-terminal peptide-dependent total hCG assay, that only detect hCG-forms containing the ß-subunit C-terminal peptide. This is useful in detecting degraded or inactive hCG forms such as that produced in familial hCG syndrome. We have an intact hCG test that measures hCG αß dimer only. We have used this test to identify men and women taking hCG supplements for dietary purposes and for testosterone-promotion muscle building purposes (having a very pure non-degraded form of hCG). During our 10 years of practice we have identified two case with Munchausen’s syndrome, individuals that took hCG to make themselves appear sick. We also have a specific test for nicked hCG that can differentiates active hCG and inactive or nicked hCG. This is useful for examining women with positive hCG tests having normal menstrual periods.

e.    FSH testing

          In women in perimenopause or postmenopause it is common to detect total hCG in the blood. This is coming naturally from the pituitary gland (56-64). If a woman has a history of oophorectomy or ovary removal it places her in amenorrhea or in menopause. We have observed menopausal woman with <1 to 39 mIU/ml of hCG. When a menopausal woman has a positive pregnancy test, we first exclude any possibility of false positive hCG, then exclude any chance of cancer (serum free ß-subunit and urine ß-core fragment tests), and finally exclude any possibility of gestational trophoblastic neoplasm or choriocarcinoma (serum hyperglycosylated hCG test). Having excluded false positive test, cancer and gestational trophoblastic neoplasm, we consider pituitary hCG. The critical issues is whether the woman is in perimenopause or menopause. We run an FSH test to make this discrimination. If the FSH is >30 IU/L then menopause or perimenopause is confirmed (80). Monopause or perimenopause combined with positive total hCG test indicates normal/natural pituitary hCG after excluding the possibilities of cancer and gestational trophoblastic neoplasm.

References

1.       Sasaki Y, Ladner DG, Cole LA: Hyperglycosylated hCG the source of pregnancy failures. Fertil Steril 2008; 89:1871-1786.

2.       Cole LA, Dai D, Butler SA, Leslie KK, Kohorn EI: Gestational trophoblastic diseases: 1. Pathophysiology of hyperglycosylated hCG-regulated neoplasia. Gynecol Oncol 2006;102:144-149

3.       Rao CV, Griffin LP, Carman FR Jr. Prostaglandin F2 alpha binding sites in human corpora lutea. J Clin Endocrinol Metab 1977;44:1032-1037

4.       Strott CA, Yoshimi T, Ross GT, Lipsett MB: Ovarian physiology: relationship between plasma LH and steroidogenesis by the follicle and corpus luteum; effect of HCG. J Clin Endocrinol Metab 1969;29:1157-1167

5.       Cedard L, Varangot J, Yannotti S: The metabolism of estrogens in human placentas artificially maintained in survival by perfusion in vitro. Comptes Rendus Hebdomadaires Seances de l'Academie des Sci 1962; 254: 1870-1871.

6.       Azuma K, Calderon I, Besanko M, MacLachlan V, Healy DL: Is the luteo-placental shift a myth? Analysis of low progesterone levels in successful art pregnancies. J Clin Endocrinol Metab 1993;77:195-198

7.       Mayerhofer A, Fritz S, Grunert R, Sanders SL, Duffy DM, Ojeda SR, Stouffer RL: D1-Receptor, DARPP-32, and PP-1 in the primate corpus luteum and luteinized granulosa cells: evidence for phosphorylation of DARPP-32 by dopamine and human chorionic gonadotropin. J Clin Endocrinol Metab 2000;85:4750-4757

8.       Pierce JG, Parsons TF: Glycoprotein hormones: structure and function. Ann Rev Biochem 1981;50:65-95.

9.       Rao CV: Differential properties of human chorionic gonadotropin  and human luteinizing hormone binding to plasma membranes of bovine corpora luteal. Acta Endocrinol 1979;90:696-710.

10.     Berndt S, Blacher S, d'Hauterive PS, Thiry M, Tsampalas M, Cruz A, Pequeux C, Lorquet S, Munaut C, Noel A, Foidart JM:  Chorionic gonadotropin stimulation of angiogenesis and pericyte recruitment. J Clin Endocrinol Metab 2009;94:4567-4574

11.     Toth P, Li X, Rao CV, Lincoln SR, Sanfillipino JS, Spinnato JA, Yussman MA: Expression of functional human chorionic gonadotropin/human luteinizing hormone receptor gene in human uterine arteries. J Clin Endocrinol Metab 1994;79:307-315

12.     Lei ZM, Reshef E, Rao CV: The expression of human chorionic gonadotropin/luteinizing hormone  receptors in human endometrial and myometrial blood vessels. J Clin Endocrinol Metab 1992;75:651-659.

13.     Zygmunt M, Herr F, Keller-Schoenwetter S, Kunzi-Rapp K, Munstedt K, Rao CV, Lang U, Preissner KT: Characterization of human chorionic gonadotropin as a novel angiogenic factor. J Clin Endocrinol Metab 2002;87:290-5296.

14.     Herr F, Baal N, Reisinger K, Lorenz A, McKinnon T, Preissner KT, Zygmunt M: hCG in the regulation of placental angiogenesis. Results of an in vitro study. Placenta 2007;28(Suppl A):S85-93

15.     Zygmunt M, Herr F, Munstedt K, Lang U, Liang OD: Angiogenesis and vasculogenesis in pregnancy. Euro J Obstet Gynecol Reprod Biol 2003;110 (Suppl 1):S10-18.

16.     Toth P, Lukacs H, Gimes G, Sebestyen A, Pasztor N, Paulin F, Rao CV: Clinical importance of vascular hCG/LH receptors-A review. Reprod Biol 2001;1:5-11.

17.     Shi QJ, Lei ZM, Rao CV, Lin J: Novel role of human chorionic gonadotropin in differentiation of human cytotrophoblasts. Endocrinol 1993;132:387-395.

18.     Cronier L,  Bastide B, Herve JC, Deleze J, Malassine A: Gap junctional communication during human trophoblast differentiation: influence of human chorionic gonadotropin. Endocrinology 1994;135: 402-408.

19.     Akoum A, Metz CN, Morin M: Marked increase in macrophage migration inhibitory factor synthesis and secretion in human endometrial cells in response to human chorionic gonadotropin hormone. J Clin Endocrinol Metab  2005;90:2904-2910

20.     Matsuura T, Sugimura M, Iwaki T, Ohashi R, Kanayama N, Nishihira J: Anti-macrophage inhibitory factor antibody inhibits PMSG-hCG-induced follicular growth and ovulation in mice. J Assist Reprod Genet 2002;19:591-595.

21.     Wan H, Marjan A. Cheung VW,  Leenen PJM, Khan NA, Benner R, Kiekens RCM: Chorionic gonadotropin can enhance innate immunity by stimulating macrophage function. J Leukocyte Biol 2007;82:926-933.

22.     Kamada M, Ino H, Naka O, Irahara M, Daitoh T, Mori K, Maeda N, Maegawa M, Hirano K, Aono T: Immunosuppressive 30-kDa protein in urine of pregnant women and patients with trophoblastic diseases. Eur J Obstet Gynecol Reprod Biol 1993;50:219-225.

23.     Noonan FP, Halliday WJ, Morton H, Clunie GJA: Early pregnancy factor is immunosuppressive. Nature 1879;278:649-651.

24.     Majumdar S, Bapna BC, Mapa MK, Gupta AN, Devi PK, Subrahmanyam D: Pregnancy specific proteins: suppression of in vitro blastogenic response to mitogen by these proteins Int J Fertil 1982;27:66-69.Bottom of Form

25.     Reshef E, Lei ZM, Rao CV, Pridham DD, Chegini N, Luborsky JL: The presence of gonadotropin receptors in nonpregnant human uterus, human placenta, fetal membranes, and decidua. J Clin Endocrinol Metab 1990;70:421-430.

26.     Zuo J, Lei ZM, Rao CV: Human myometrial chorionic gonadotropin/luteinizing hormone receptors in preterm and term deliveries. J Clin Endocrinol Metab 1994;79:907-911 

27.     Eta E, Ambrus G, Rao V: Direct regulation of human myometrial contractions by human chorionic gonadotropin. J Clin Endocrinol Metab 1994;79:1582-1586,

28.     Doheny HC, Houlihan DD, Ravikumar N, Smith TJ, Morrison JJ: Human chorionic gonadotrophin relaxation of human pregnant myometrium and activation of the BKCa channel. J Clin Endocrinol Metab 2003;88:4310-4315

29.     Edelstam‌ G, Karlsson C‌, Westgren M‌, Löwbeer‌ C, Swahn ML: Human chorionic gonadatropin (hCG) during third trimester pregnancy. Scand J Clin Lab Invest 2007; 67: 519-525.

30.     Goldsmith PC, McGregor WG, Raymoure WJ, Kuhn RW, Jaffe RB: Cellular localization of chorionic gonadotropin in human fetal kidney and liver. J Clin Endocrinol Metab 1983; 57:54-61

31.     Abdallah MA, Lei ZM, Li X, Greenwold N, Nakajima ST, Jauniaux E, Rao CV: Human Fetal nongonadal tissues contain human chorionic gonadotropin/ luteinizing hormone receptors. J Clin Endocrinol Metab 2004;89:952-956.

32.     Rao CV: Chapter 11: Paradigm shift on the targets of hCG actions. In: Human chorionic gonadotropin (hCG) ed. Cole LA, Elsevier, Oxford UK, 2010, in press.

33.     Rao CV: Nongonadal actions of LH and hCG in reproductive biology and medicine. Sem Reprod Med 2001;19:1-119.

34      Rao, CV: An overview of the past, present and future of nongonadal hCG/LH actions in reproductive biology and medicine. Sem Reprod Endocrinol 2001;19:7-17.

35.     Rao CV, Lei ZM: The past, present and future of nongonadal hCG/LH actions in reproductive biology and medicine. Molec Cell Endocrinol 2007;269:2-8.

36.     Rao CV, Li X, Toth P, Lei ZM: Expression of epidermal growth factor, transforming growth factor-alpha and their common receptor genes in human umbilical cords. J Clin Endocrinol Metab 1995;80:1012-1020.

37.     Rao CV, Li X, Toth P, Lei ZM, Cook VD: Novel expression of functional human chorionic gonadotropin/luteinizing hormone receptor in human umbilical cords. J Clin Endocrinol Metab 1993;77:1706-1714.

38.     Wasowicz, Derecka K, Stepien A, Pelliniemi L, Doboszynska T, Gawronska B, Ziecik AJ: Evidence for the presence of luteinizing hormone–chorionic gonadotrophin receptors in the pig umbilical cord. J Reprod Fertil 1999;117:1-9.

39.     Ohlsson R, Larsson E, Nilsson O, Wahlstrom T, Sundstrom P: Blastocyst implantation precedes induction of insulin-like growth factor II gene expression in human trophoblasts. Developm 1989;106:555-55

41.     Licht P, Fluhr H, Neuwinger J, Wallwiener D, Wildt L: Is human chorionic gonadotropin directly involved in the regulation of human implantation? Molec Cellul Endocrinol 2007; 269: 85-92.

42.     Fluhr H, Bischof-Islami D, Krenzer S, Licht P, Bischof P, Zygmunt M: Human chorionic gonadotropin stimulates matrix metalloproteinases-2 and -9 in cytotrophoblastic cells and decreases tissue inhibitor of metalloproteinases-1, -2, and -3 in decidualized endometrial stromal cells. Fertil Steril 2008; 90:1390-1395.

43.     Reis FM, Cobellis L, Luisi S, Driul L, Florio P, Faletti A, Petraglia F: Paracrine/autocrine control of female reproduction. Gynecol Endocrinol  2000; 14: 464-475

44.     Ticconi C, Zicari A, Belmonte A, Realacci M, Rao ChV, Piccione E: Pregnancy-promoting actions of HCG in human myometrium and fetal membranes. Placenta 2009; 28: S137-143.

45.     Hamade AL, Nakabayashi K, Sato A, Kiyoshi K, Takamatsu Y, Laoag-Fernandez JB, Ohara N, Maruo T: Transfection of antisense chorionic gonadotropin ß gene into choriocarcinoma cells suppresses the cell proliferation and induces apoptosis. J Clin Endocrinol Metab 2005;90:4873-4879.

46.     Kamijo T, Rajabi MR, Mizunuma H, Ibuki Y: Biochemical evidence for autocrine/paracrine regulation of apoptosis in cultured uterine epithelial cells during mouse embryo implantation in vitro. Molec Human Reprod 1998;4:990-8.

47.   Acevedo HF, Hartstock RJ: Metastatic phenotype correlates with high expression of membrane-associated complete ß-human chorionic gonadotropin in vivo. Cancer 1996; 78:2388-2399.

48.     Li D, Wen X, Ghali L, Al-Shalabi FM, Docherty SM, Purkis  P, Iles RK: hCGß expression by cervical squamous carcinoma - in vivo histological association with tumor invasion and apoptosis. Histopathology, 2008;53:147-155.

49.     Iles RK: Ectopic hCGß expression by epithelial cancer: Malignant behavior metastasis and inhibition of tumor cell apoptosis. Molec Cellul Endocrinol 2007;260:264-270

50.     Iles RK: Human chorionic gonadotrophin and its fragments as markers of prognosis in bladder cancer. Tum Mark Upd  1995; 7:161-166

51.     Cole LA: b-core fragment (b-core UGP or UGF). Tum Mark Upd 1994; 6:69-75

52.     Mountzouris G, Yannapoulis D, Barbatis C, Zaharof A, Theodorou C: Is ß human chorionic gonadotrophin production by transitional cell carcinoma of the bladder a marker of aggressive disease and resistance to radiotherapy? Br J Urol 1993;72:907-909

53.     Butler SA, Iles RK: Ectopic human chorionic gonadotrophin ß secretion by epithelial tumors and human chorionic gonadotrophin ß-induced apoptosis in Karposi’s sarcoma Is there a connection? Clin Cancer Res  2003; 9:4666-4673

54.     Bellet D, Lazar V, Bleche I, Paradis V, Giovangrandi Y, Paterliru P: Malignant transformation of nontrophoblastic cells in association with the expression of chorionic gonadotropin ß genes normally transcribed in trophoblastic cells. Cancer Res 1997; 57:516-523.

55.     Butler SA, Ikram MS, Mathieu S, Iles RK: The increase in bladder carcinoma cell population induced by the free beta subunit of hCG is a result of an anti-apoptosis effect and not cell proliferation. Brit J Cancer  2000; 82:1553-1556

56.        Matsuura S, Ohashi M, Chen HC, Shownkeen RC, Hartree AS, Reichert LE Jr, Stevens VC, Powell JE: Physicochemical and immunological characterization of an hCG-like substance from human pituitary glands. Nature 1980;286:740-1.

57.     Cole LA, Khanlian SA, Muller CY: Detection of hCG peri- or post-menopause an unnecessary source of alarm. Am J Obstet Gynecol 2008;198:275-9.

58.     Cole LA, Sasaki Y, Muller CY: Normal production of hcg in menopause: A medical management dilemma. N Eng J Med 2007;356:1184-6.

59.     Gronowski AM, Fantz CR, Parvin CA, Sokoll LJ, Wiley CL, Wener MH, Grenache D: Use of serum FSH to identify perimenopausal women with pituitary hCG. Clin Chem 2008; 54:652-6.

60.     Cole LA, Khanlian SA: Inappropriate management of women with persistent low hCG results. J Reprod Med 2004;49:423-32.

61.     Birken S, Maydelman Y, Gawinowicz MA, Pound A, Liu Y, Hartree AS: Isolation and characterization of human pituitary chorionic gonadotropin. Endocrinol 1996;137:1402-11.

62.     Hoermann R, Spoettl G, Moncayo R, Mann K: Evidence for the presence of human chorionic gonadotropin (hCG) and free beta-subunit of hCG in the human pituitary. J Clin Endocrinol Metab 1990;71:179-86.

63.     Odell WD, Griffin J: Pulsatile secretion of human chorionic gonadotropin in normal adults. N Engl J Med 1987;317:1688-91.

64.     Odell WD, Griffin J: Pulsatile secretion of chorionic gonadotropin during the normal menstrual cycle. J Clin Endocrinol Metab 1989;69:528-32.

65.     Cole L.A, Muller Y: hCG in the Management of Quiescent and Chemorefractory Gestational Trophoblastic Diseases, Gyn Oncol 2010;116:3-9.

66.     Cole LA: Use of hCG Tests for Evaluating Trophoblastic Diseases: Choosing an Appropriate hCG Assay, False Detection of hCG, Unexplained Elevated hCG, and Quiescent Trophoblastic Disease. In: Gestational Trophoblastic Disease, 2nd Edition (Eds: Hancock BW, Newland ES, Berkowitz RS, Cole LA), Chapman and Hall, London, pp 130-142, 2002

67.     Cole LA, Butler SA, Khanlian SA, Giddings A, Muller CY, Seckl MJ, Kohorn EI: Gestational trophoblastic diseases: 2. Hyperglycosylated hCG as a Reliable Marker of Active Neoplasia. Gyn Oncol 2006;102:150-158.

68.     Knight AK, Bingemann T, Cole L, Cunningham-Rundles C: Frequent false positive beta human chorionic gonadotropin in immunoglobulin A deficiency. Clin Exper Immunol 2005;141:333-337.

69.     Cole LA, Laidler LL: Inherited hCG. J Reprod Med, 2010;55:99-102.

70.     Ohama K, Kajii T, Okamoto E, Fukuda Y, Imaizumi K, Tsukahara M, Kobayashi K, Hagiwara: Dispermic origin of XY hydatidiform moles Nature. 1981;292:551 – 552.

71.     Pattillo RA, Sasaki S, Katayama, KP, Roesler M, Mattingly RF: Genesis of 46,XY Hydatidiform Mole Obstet Gynecol Surv 1982;37:1-5.

72.     Pattillo RA, Sasaki S, Katayama KP, Roesler M, Mattingly RF: Genesis of 46,XY hydatidiform mole. Am J Obstet Gynecol. 1981;141:104-5.

73.     Hancock BW, Newland ES, Berkowitz RS, Cole LA: Gestational Trophoblastic Disease, 2nd Edition (Eds:), Chapman and Hall, London, pp 1-440, 2002

74.     Cole LA, Khanlian SA, Muller CY, Giddings A, Kohorn EI, Berkowitz R. Gestational trophoblastic diseases: 3. Human Chorionic Gonadotropin Free ß-subunit a Reliable Marker of Placental Site Trophoblastic Tumors. Gyn Oncol 2006;102:159-163.

75.     Cole LA, Khanlian SA, Muller CY. Blood Test for Placental Site Trophoblastic Tumor (PSTT) and for Non-Trophoblastic Malignancy for Evaluating Patients with Low Positive hCG Results. J Reprod Med 2008;53:457-464.

76.     Cole LA, Shahabi S, Butler S, Mitchell H, Newlands ES, Behrman HR, Verrill HL. Utility of commonly used commercial hCG immunoassays in the diagnosis and management of trophoblastic diseases. Clin Chem 2001;47:308-315.

77.     Cole LA, Sutton JM, Higgins TN. Higgins, Cembrowski GS. Between-Method Variation in hCG Test Results, Clin Chem 2004;50:874-882.

78.     Cole LA. Background hCG in healthy non-pregnant women, 
Clin Chem 2005; 51:1765-66.

79.     Cole LA, Laidler L, Muller C. USA hCG Reference Service, 10 year 
report, Clin Biochem, 2010 in press.

80.     Gronowski AM, Fantz CR, Parvin CA, Sokoll LH, Wiley CL, Wener MH, Grenache DG: Use of Serum FSH to Identify Perimenopausal Women with Pituitary hCG Clin Chem 2008; 54: 652-656,

REFERRAL WITH USA hCG REFERENCE SERVICE

   To refer a patient to the USA hCG Reference service click here