NORMAL CONCEPTION AND IMPLANTATION
Conception occurs in the uterine tube, usually near the fimbrial end. The zygote transits through the tube in 3 to 5 days and moves to the site of implantation in another 1 to 2 days. During this time, the conceptus is dividing. At the time of implantation it has formed a blastocyst (a single layer of cells surrounding the central cavity). One area of the blastocyst's wall is 3 to 4 cells thick. This area is the embryonic pole, which soon becomes recognizable as an embryo. Trophoblast cells proliferate from the surface of the blastocyst invading and penetrating the endometrium so that the blastocyst burrows into the central layer of endometrium. This process begins on day 5 and 6 and is completed by day 9 or 10.
The trophoblast is the component of placenta arising from trophoectoderm. The invasive nature of trophoblast is responsible for attachment of blastocyst to the uterine wall. By day 10 syncytial (syncytiotrophoblast and cytotrophoblast) cells are identifiable. Beginning about this time, syncytial cells start secreting chorionic gonadotrophin which can be demonstrated using fluorescent staining. Trophoblast is of two types:
Cytotrophoblast, is the germinal cell, and is the cellular progenitor of the syncytiotrophoblast.
Syncytiotrophoblast, which is derived from cytotrophoblast only and lacks definite cell borders as compared to cytotrophoblast, which has got single, distinct nucleus. Lack of definite borders of syncytium obliges transfer across this structure.
According to the review done for processes of human implantation, that after apposition and adherence of the trophoectoderm of the blastocyst to the endometrial epithelial cells, implantation commences by intrusion of cytotrophoblasts between endometrial epithelial cells.1 This process of trophoblast invasion is facilitated by degradation of the extracellular matrix of the endometrium/ decidua, catalyzed by urokinase-type plasminogen activator, urokinase plasminogen activator receptor, and metalloproteinase's that are produced by selected cytotrophoblasts at various stages of implantation/ placentation. These functions of cytotrophoblasts invading the myometrium are indistinguishable from those of metastasizing malignant cells.
The capillary network of the most superficial portion of the endometrium is invaded by cytotrophoblasts. Subsequently, the arterioles and then the spiral arteries are invaded, and the walls of these vessels are destroyed.
During implantation, the spiral arteries acquire a lining of cells within the endothelium that is derived from the invading cytotrophoblasts. The vascular invasion leads to degenerative changes of arterial wall, affecting all layers of these vessels. The most striking change involves the vascular smooth muscle, which becomes non recognizable. The cytotrophoblasts that invade the spiral arteries can pass several centimeters along the vessel lumen indeed.
Normal Trophoblast Ultrastructure
Chorionic villi can be distinguished in the human placenta on about the 12th day after fertilization. With deeper blastocyst invasion into the decidua, the extra villous cytotrophoblast gives rise to the solid primary villi composed of cytotrophoblast core covered by syncytium. On the syncytial trophoblast there are prominent microvilli corresponding to “brush border” and associated pinocytotic vacuoles and vesicles are related to the absorbtive and secretary placental functions. Secondary villi are formed from cytotrophoblast which invades the solid trophoblast. After angiogenesis occurs from the mesenchymal core in situ, the resulting villi are termed tertiary.
Abnormal Trophoblast Ultrastructure
Some villi, in which failure o f angiogenesis results in a lack of circulation, become distended with fluid and form vesicles. A striking exaggeration of this process is characteristic of the development of hydatidiform mole. Proliferation of cellular cytotrophoblast at the tips of the villi produce the trophoblastic cell columns, which are not invaded by fetal mesenchyme but are anchored to the decidua at the basal plate. The villi are much larger than in a normal gestation, averaging 1.5 cm in diameter and there is usually very abundant tissue. It has to be differentiated from hydropic degeneration of an aborted conceptus in which excessive fluid or liquefaction of placental villous stroma is present but without undue trophoblastic hyperplasia.
It represents a spectrum of conditions ranging from hydatidiform mole to choriocarcinoma. Persistent trophoblastic disease (PTD) may follow either a molar pregnancy (complete or partial hydatidiform mole) or a non-molar event, for example an ectopic or normal pregnancy.
A significant proportion of cases of hydatidiform mole (HM) require chemotherapy for persistent gestational trophoblastic disease (PGTD) with the reported frequency of this complication being 8-36 percent. Various factors, including differing criteria for the diagnosis of PGTD and variations in the diagnosis and registration of HM, account for the wide range in reported frequency of this complication. The true frequency is probably 10-15 percent. The majority of cases of HM that are complicated by PGTD are complete moles (CHM), although PGTD has been described following partial moles (PHM) also.
Human Chorionic Gonadotropin(hCG)
It is a glycoprotein which is biologically very similar to luteinizing hormone (LH) both of which act via plasma membrane LH/hCG receptor. hCG is produced almost exclusively in the placenta, but is synthesized in fetal kidney, and a number of fetal tissues produce the beta-subunit or intact hCG molecule.
hCG is the ideal tumor marker for trophoblastic disease/tumors. It is a placental hormone that is secreted by the syncytiotrophoblast, and serves to maintain corpus luteum function and preserve progesterone secretion during the early stages of gestation. In a normal pregnancy it can be detected about 5 days after conception and reaches its peak at 8-10 weeks of pregnancy.
Although syncytiotrophoblast is the physiological source of hCG, an hCG like substance has been detected in a wide variety of normal human tissues and low levels can be measured in normal human plasma.
In patients with clinically evident trophoblastic disease, pregnancy tests are usually positive and can be used as a quick, though not necessarily the most sensitive test for hCG.
The clinical utility of early methods of hCG detection were limited because of the difficulty of distinguishing hCG from LH. The development of assay with use of an antiserum to hCG beta unit made possible the quite sensitive detection of hCG production, even with LH present in the serum.2
Rise and Fall of Beta hCG
Detection in serum
8-9 days after conception
Peak of hCG (100,000 mIu/mL)
60-70 days after ovulation
Nadir of hCG
Fall after normal delivery
Fall after abortion
Fall after H. mole
Biochemistry of hCG
hCG is a glycoprotein hormone composed of two dissimilar subunits, alpha and beta, joined noncovalently.
In addition to regular hCG, at least 5 key variants are present in serum samples:
- hyperglycosylated hCG
- nicked hCG
- hCG missing the beta-subunit c-terminal peptide
- free beta-subunit
- nicked free beta subunit
- multiple combinations of these variations.
The same molecules plus beta-core fragments are detected in urine samples.3–11
Serum levels of biologically active hCG(non-nicked) rise exponentially in the first trimester of pregnancy, doubling every two days, rising to a peak at about 10 weeks of gestation. Levels decrease from the 10th to 20th week of gestation, reaching approximately 20 percent of peak levels. They remain at around this concentration until term.12 Regular hCG and hyperglycosylated hCG are non nicked. Hyper-glycosylated hCG is the predominant form of hCG produced in the 2-3 weeks following implantation of pregnancy but it decreases to 25 percent of total hCG by 4th week. In GTD the hyperglycosylated fraction and nicked hCG increases and ideally these should be assessed for proper follow-up.4
The percentage of variants of hCG are low, they can vary greatly between individuals. In a recent study of 176 first trimester pregnancy serum samples,0-59 percent nicking was detected.13 The extent of nicking of hCG is much greater and much more variable in trophoblastic disease patients serum and urine samples (0-100% of hCG levels).14–16
Detection of hCG
A very small number of laboratories continue to use the competitive radioimmunoassay (RIA) methods developed in 1950. Currently immunometric assay is used to detect hCG, where at least one antibody directed against the beta-subunit is used to differentiate between hCG and LH.
In gestational trophoblastic disease, hyperglycosylated hCG is commonly the principal form of hCG present. Failure to appropriately detect this hCG variant is a common cause of failure to detect active disease or a recurrence or persistence of trophoblastic disease.5–7,10
Tests using hCG, beta-subunit C-terminal antibodies do not detect hCG, missing beta-subunit and C-terminal and may yield misleading results or may miss persistence or recurrence of trophoblastic disease or other germ cell malignancies. Unfortunately, over one half of commercial laboratory serum hCG assays used today use an antibody against the beta-subunit C-terminal peptide. Only one test detects all the pertinent forms of hCG, the DPC immulite hCG.
Common Problems with hCG Determination in Trophoblastic Disease
- Phantom hCG refers to persistent mild elevation of beta hCG, leading physicians to consider treating patients for choriocarcinoma when in reality no true beta hCG or trophoblastic disease is present. False positive low levels (<100 mIu/ml) of hCG, free beta subunit, and beta core fragment are rarely detected in the serum of nonpregnant, reproductive women but may lead to a false diagnosis of choriocarcinoma. Human heterophilic antibodies in serum samples can cause false positive hCG results. There have been multiple publications on the issue of false positive hCG problem.5,11,13,17
- hCG immunoreactivity detected in urine but not serum. One may be measuring beta core fragment in urine. This is the final hCG related molecule to clear the body 3 to 6 weeks after evacuation of hydatidiform mole. Alternatively, the serum assay does not detect nicked hCG which may be the principle immunoreactivity remaining in the circulation.
- Considering the heterogeneity of hCG, particularly with regards to gestational trophoblastic diseases and hCG producing malignancies (testicular cancer and germ cell tumors), it is essential to use an hCG test that measures all forms of hCG and its free subunits in managing all cases. The only test which fulfills this is DPC immulite hCG (equal detection of regular hCG, nicked hCG, hCG missing the β –subunit C-terminal peptide, hyperglycosylated hCG, and their free β –subunits and the β –core fragment). To avoid misdiagnoses or failure to detect persistence or recurrence, one should find out what test is being used by the laboratory before submitting samples from patients with gestational trophoblastic diseases.
- Cross-reactivity following multiple courses of combination chemotherapy, patients may experience ovarian dysfunction, with resulting increases in serum levels of luteinizing hormone(LH). Increased levels of LH may cross react with hCG and falsely give the impression of persistent low levels of hCG. In order to prevent confusion related to LH cross reactivity, it is prudent to place patients on oral contraceptives during intensive combination chemotherapy to suppress LH levels.
- Persistent low levels of hCG following a pregnancy or hydatidiform mole are commonly because of a non-invasive condition, quiescent gestational trophoblastic disease, or unexplained elevated hCG. These conditions do not appear to respond to chemotherapy. If hCG results begin to increase after becoming undetectable, this could either be persistent trophoblastic disease or quiescent gestational trophoblastic disease. These need to be differentiated. They can be differentiated using a hyperglycosylated hCG test or by observing whether hCG results consistently or sharply rise (i.e. 5, 10, 20, 100, 600 mIU/mL) indicative of persistent or invasive disease requiring chemotherapy, or whether they rise and plateau (i.e. 5, 10, 20, 22, 18, 25, 24 mIU/mL) consistent with quiescent gestational trophoblastic diseases, in which case, chemotherapy should probably be withheld.
To summarize, it has been emphasized that false positive levels of hCG has led to unnecessary treatment of the patients. Because of the pivotal role of serum concentration of hCG in the diagnosis and follow-up of these tumors, concurrent testing of urine for hCG should be done to show that serum hCG values are representative of true hCG concentrations.
Difference between Beta Subunits of Various hCG Molecules:3–10
PERSISTENT TROPHOBLASTIC DISEASE
The spectrum of persistent trophoblastic disease (PTD) extends from benign invasive mole to life threatening choriocarcinoma. It is defined as a state in which the trophoblast persists with continued elevation of serum or urine hCG levels, but exact diagnosis is undermined due to lack of histopathologic study. From clinical point of view it is not necessary to differentiate the two as treatment is same. This is the only disease where diagnosis can be made on clinical or biochemical parameters. Metastatic spread to distant organs can occur early, even in the absence of disease in the uterus or pelvis. Trophoblast can persist in the body after molar pregnancy, abortion, ectopic pregnancy or normal pregnancy. Classically PTD is present following molar pregnancy. H-mole precedes 50 percent of the patients of PTD. In 25 percent of patients it is preceded by normal pregnancy and in 25 percent abortion or ectopic pregnancy. It is a relatively uncommon condition and unless affiliated with a treatment center, most gynecologists never gain sufficient experience to develop the judgment required to individualize the therapy appropriately.
The Current FIGO Criteria to Diagnose Post Molar GTD Includes18
- Four values or more of hCG documenting a plateau (neither a decrease or an increase >10% of hCG value) over at least 3 weeks; days 1,7,14 and 21(using a normal hCG regression curve after evacuation of molar pregnancy permits the clinician to establish whether the given hCG value is still within the normal range).
- A rise of hCG of 10 percent or greater for 3 values or longer; over at least 2 weeks; days 1, 7 and 14.
- The presence of histologic choriocarcinoma.
- Persistence of hCG 6 months after mole evacuation.
Presentation of Patients
- Following molar pregnancy-vaginal bleeding
- Following abortion- vaginal bleeding, history of repeated D & C
- Following normal delivery bleeding off and on, not responding to any routine treatment.
Post Molar Persistent Trophoblastic Disease
In the majority of cases, approximately 70-90 percent have invasive mole and 10-30 percent have choriocarcinoma. The risk of having metastatic disease ranges from 6-25 percent.
PTD can be classified clinically as follows:
- Non metastatic: No evidence of metastasis
No risk factors:
Short duration (< 4 months)
Pre therapy beta hCG < 40,000 mIU/ml
No brain and liver metastasis
No antecedent term pregnancy
No prior chemotherapy
Any risk factor:
Long duration (>4 months since last pregnancy)
Pre therapy beta hCG >40,000 mIU/ml
Brain and liver metastasis
Antecedent term pregnancy
It should by definition always follow a molar pregnancy. Invasive mole is the most common form of persistent GTT following a mole, and is probably 6 to 10 times more common than choriocarcinoma.
Histologically, invasive mole is a benign condition, but because access to the plentiful venous plexus of the myometrium and pelvis, trophoblastic embolization to vagina and lungs occurs in upto 25 percent of cases. The diagnosis of invasive mole requires histologic confirmation of myometrial invasion, and therefore is usually made in a hysterectomy specimen. With modern treatment, however, the diagnosis of invasive mole is rarely confirmed as hysterectomy is generally not necessary in patients with persistently elevated hCG titers after evacuation of a mole, nor is biopsy of metastatic foci needed prior to administration of cytotoxic chemotherapy.
Grossly, invasive moles appear as hemorrhagic, erosive masses invading myometrium. Perforation is possible when there is full-thickness involvement. Microscopically, as one would expect, the diagnostic feature is the presence of molar villi and trophoblast within the myometrial wall or at extrauterine sites. In extrauterine sites, the distinction from choriocarcinoma is based solely on the presence of villi, which may be sparse in number. In addition, deported invasive moles usually form masses within blood vessels, without significant invasion into surrounding tissue.
The differential diagnosis of invasive mole is relatively limited as few other lesions within the myometrium contain both chorionic villi and trophoblast. Placenta accreta also has villi and trophoblast invading myometrium, but in contrast to invasive mole the villi are the normal (i.e. not enlarged or hydropic) and the trophoblast has a normal proliferative pattern. From a clinical standpoint it is often difficult to distinguish between invasive mole and choriocarcinoma. Both result in elevated hCG levels and both can give rise to secondary lesions in lung and elsewhere. Fortunately, it is not necessary to distinguish between these two entities for treatment purposes and the clinical term persistent GTD is often used without attempting to distinguish between invasive mole and choriocarcinoma.
Over all 15 percent of patients with malignant GTN are found to have metastatic disease. The trophoblastic cells invade locally into the myometrium, gaining access into capillaries and small veins. Venous metastasis can occur into the subvaginal venous plexus, resulting in vaginal metastasis or into the main venous system with metastasis to the parametrium and lungs. While direct shunting into the systemic circulation rarely occurs, the majority of disseminated metastasis develop only after pulmonary metastasis have become established. From pulmonary nodules, hematogenous dissemination can occur via the systemic circulation. From left side of heart the metastasis can take place to any organ.
Sites of Metastasis
- Lungs (60-80%)
- Vagina (30%)
- Pelvis (20%)
- Brain (10%)
- Liver (10%)
- GI tract
- Lymph node
Pretreatment Staging Evaluation
- Full history
- Proper physical examination including pelvic examination
- Serum beta hCG
- Complete blood count
- Liver function tests
- Kidney function tests
- Clotting function studies
- Imaging investigations
- X-ray chest
- CT chestThere is a little controversy regarding routine use of CT chest in metastatic workup.Why do CT chest if X-ray chest can pick up the metastasis. It has been seen that 29-41 percent of patients of GTD treated as non- metastatic as per X-ray chest findings, have occult lesions in chest, which have been missed by X-ray.19 Hence, CT chest should be done for a patient of PTD if X-ray chest is negative. As has been already explained that once the metastasis occurs in the lungs the trophoblastic cells can embolize to the left side of the heart and eventually to any other organ. It would be a tragedy to miss the diagnosis of a high-risk metastasis in a patient on the basis of a negative chest X-ray and delay appropriate aggressive initial therapy.
- USG abdomen and pelvis
- CNS assessment if required.
CNS assessment has to be done in all high-risk patients: beta hCG >50,000 mIU/ml, patients with 5 or > metastasis visible on CT scan of thorax or with single nodule greater than 3 cms. For evaluating CNS, CT head and lumbar puncture is done provided there is no clinical evidence of raised intracranial pressure. An abnormal CSF-beta HCG is interpreted as one where the ratio with serum beta hCG is > 1:60.
Prognostic scoring of patient to be done as per revised FIGO scoring system.20