REVIEW: The role of hCG in reproductive medicine
Human chorionic gonadotrophin (hCG) has an essential role in early pregnancy and is of fundamental importance in obstetrics and gynaecology. hCG measurement is used for early pregnancy testing and monitoring, biochemical prenatal screening and the assessment of gestational trophoblastic disease. hCG is used therapeutically to induce final oocyte maturation prior to oocyte retrieval for IVF/ICSI and recombinant hCG has recently been developed (Table 1). This review will relate to early pregnancy complications and reproductive medicine practice and will not deal in detail with its measurement in prenatal trisomy screening. Before considering the pathophysiological conditions that may lead to abnormalities of hCG synthesis and secretion, the expression and structure of both hCG and the hCG/LH receptor will be described.
Table 1. hCG in reproductive medicine.
|Therapeutic use||Ovulation induction||Trigger ovulation|
|Assisted conception||Oocyte maturation|
|In vitro maturation||Priming|
| ||Component of culture system|
|Serum assays||Monitoring|| |
|Early pregnancy||Diagnose viability|
| ||Diagnose ectopic pregnancy|
|Trophoblastic disease||Response to surgical evacuation|
| ||Response to chemotherapy|
|Prenatal testing||Aneuploidy screening|
| ||Component of double or triple test|
|Germ cell tumour marker||Response to treatment|
hCG is a member of the glycoprotein hormone family also comprising the pituitary derived follicle stimulating hormone (FSH), luteinising hormone (LH) and thyroid stimulating hormone (TSH).
Each hormone consists of a non-covalently bound α- and β-subunits where within a species the α-subunit is identical and hormone specificity is determined by the unique β-subunit.
The shared α-subunit is transcribed from a single gene on chromosome 6.1 It is expressed in the pituitary and the placenta. The β-subunit of hCG is encoded by multiple genes on chromosome 19 adjacent to the structurally related LHβ subunit gene.2,3 Both hCGα and hCGβ mRNA levels are increased by epidermal growth factor (EGF)4 whose receptors are expressed at high levels in the placenta.5 For hCGα this increase in mRNA has been shown to be via a cAMP response element (CRE) located in the hCGα promoter. EGF appears to phosphorylate CRE binding protein through the protein kinase C pathway in trophoblast cells.6 The hLHβ and hCGβ subunits are 85% homologous in amino acids. hCG has a C-terminal extension unique among the glycoprotein hormones; it is heavily glycosylated and is believed to have a role in extending its in vivo half life compared with LH which is secreted in a pulsatile fashion throughout the menstrual cycle. There are six separate hCGβ genes, all of which have been shown by RT-PCR to be transcribed, albeit with different efficiency.7
The glycoprotein hormone family is believed to share similar structural features and the structure of hCG has been elucidated.8,9 The α- and β-subunits each have remarkably similar folds. When the heterodimer is formed, the single loop of one subunit lies adjacent to the double loops of the other subunit. The β-subunit of hCG has six disulphide bonds and the α-subunit has five. The crystal structure revealed that the previously defined disulphide bonds could not all be made.10,11 These disulphide bonds appear to play an integral role in maintaining the heterodimeric structure. These structural features, together with the observation that the individual subunits are inactive, have implied that the quaternary structure provided by the assembly of the α- and β-subunits is important for hCG function. However, work with genetically fused α- and β-subunits have shown that the heterodimeric configuration is more important for assembly and secretion than for biological activity.12–14
hCG is heavily glycosylated with the carbohydrate content accounting for 30% of its molecular weight. There are two asparagine linked carbohydrate chains on each subunit and the carboxyl terminus of hCG has four O-linked glycosylation sites. The O-linked oligosaccharides extend the life of the hormone in the circulation. If the unique C-terminal extension of hCGβ bearing these oligosaccharides is added to FSHβ, then the in vivo half life of the hormone is increased.15 The Asn-linked carbohydrates may also be of importance in the folding and correct disulphide bond arrangement of hCG.16 The glycosylation status of the free α-subunit and hCG varies throughout pregnancy, with hCGα becoming more highly branched and both hCGα and hCG more fucosylated as gestation progresses.17 These carbohydrates prevent association of the α- and β-subunits, and the free α-subunits are linked to prolactin secretion.18,19
LH and hCG are thought to share a common receptor, transcribed from a single gene on chromosome 2,20 although there remains ongoing uncertainty about whether a separate hCG receptor could exist. The observation of a man with Leydig cell hyperplasia type II with high endogenous LH levels but low testosterone concentrations and delayed pubertal development suggested despite high LH levels the Leydig cells were not activated. Administration of exogenous hCG resulted in testosterone synthesis and subsequently, spermatogenesis. Genomic analysis revealed deletion of exon 10 of the LH receptor, which is identical to the normal male marmoset which also lacks exon 10. It is plausible that exon 10 is responsible for discriminating between LH and hCG action,21 suggesting a potential dual mechanism for hormone binding and signal transduction.
The LH/hCG receptor belongs to the structural superfamily of receptors which are coupled to G-proteins.22 There is a homology between the receptors of the glycoprotein hormones.23 They all have a large extracellular domain, seven transmembrane domains and a short C-terminal domain. The large extracellular domain (340 amino acids out of 674 for the LH receptor) is an unusual feature for this receptor family as most members have a small (30–50 amino acids) extracellular domain and bind small ligands, in contrast to the large glycoprotein hormones. Multiple species of mRNA transcripts for the LH receptor have been observed,22 however, on gonadal cells only the full-length protein has been detected.24
Expression of LH/hCG receptors
Receptors for LH/hCG are expressed in a variety of tissues in the reproductive system. Amnion and chorion express hCG receptor mRNA transcripts which are translated into receptor protein that can bind hCG and up-regulate COX-1 gene expression.25 Human cytotrophoblasts and syncytiotrophoblasts contain functional hCG receptors, suggesting a possible role for hCG in trophoblast invasion early in pregnancy.26 Moreover, smooth muscle and endothelial cells of umbilical vessels express hCG/LH receptors transcripts and the receptor protein, suggesting that hCG in cord blood and amniotic fluid may directly regulate vascular tone.27 Other reproductive tissues expressing functional hCG receptors include the endometrium,28 myometrium,29 fallopian tube,30 uterine cervix31 and granulosa cells. Additionally, lymphocytes from pregnant women express the hCG receptor gene,32 suggesting they may mediate hCG's immunoregulatory actions during pregnancy. It has to be acknowledged that interspecies differences do exist. Down-regulation of LH receptors during the luteal phase occurs in the rat, whereas in women LH receptor expression is maintained, and hence, hCG administration in the luteal phase would give differing responses (a rise in progesterone in women with n effect in the rat).
hCG receptors are also present in the brain,33 skin, including hair follicles, sebaceous and sweat glands.34 Moreover, the hCG receptor gene is expressed in benign prostatic hyperplasia and prostate carcinoma, suggesting that higher LH levels may play a role in these conditions.
hCG and pregnancy recognition
hCG is luteotropic in the early stages of pregnancy, maintaining progesterone production and endometrial support. hCG may be detected in maternal serum eight days following ovulation35 and in blastocysts as early as seven days after fertilisation.36 Compared with the α-subunit gene expression, which is sometimes detected in the cytotrophoblast cells, the β-hCG transcripts are restricted to the villous syncytiotrophoblast.
Levels of hCG in the maternal blood increase progressively in early pregnancy until peak levels are reached at seven to nine weeks.37 Thereafter they decline, until around 20 weeks when plasma levels remain comparatively low and constant until term. In the first trimester, there is episodic fluctuation in maternal hCG levels, representing pulsatile secretion, with a nadir at 1900 hours and peak levels at 0700 hours. The daily variation in maternal serum hCG concentrations can be up to 20%. There is no correlation between hCG levels early in pregnancy and fetal sex or birthweight. In late pregnancy, however, higher hCG levels are associated with female fetuses.38,39 In addition to the maternal circulation, hCG is detectable in extra-embryonic coelomic fluid early in pregnancy and in fetal blood, the latter deriving from the placenta and/or fetal kidney and adrenal.40 Regulation of hCG synthesis and secretion during pregnancy is not fully understood but the stage of trophoblast differentiation appears relevant as syncytia formation is linked to hCG production.41 However, the formation of a syncytium is not an absolute prerequisite for hCG expression, as trophectoderm produces hCG before implantation. A number of stimulating and inhibiting factors have been implicated in hCG secretion. Substances that increase hCG production, in vitro, include the cytokines interleukin-1 and interleukin-6, and GnRH, EGF and CSF-1 that stimulate adenylate cyclase and hence increase transcription hCG subunit genes via cAMP.42 hCG activates trophoblast adenylate cyclase leading to increased cAMP production.43
Furthermore, LH/hCG receptors have been demonstrated in trophoblastic cells therefore hCG could feedback positively acting in an autocrine manner. Glucocorticoids increase trophoblast hCG secretion by modifying the response to cAMP. Inhibin and activin, which are also produced by the placenta, modulate the release of GnRH and hCG; activin incites GnRH stimulation of hCG secretion and inhibin blocks it. Progesterone, like inhibin, has been shown to reduce hCG secretion in vitro.
Physiology of hCG
The physiological role of hCG throughout human pregnancy is not fully defined. What is clear is that the primary role of hCG in early pregnancy is to extend the functional life of the corpus luteum for a number of weeks rather than the 14 days in a non-pregnant menstrual cycle. Elegant studies utilising corpus luteum ‘rescue’ whereby the functional regression of the corpus luteum may be delayed by the administration of increasing doses of hCG prior to enucleation at open operation, simulates the changes that occur in early pregnancy.44 This model permitted the in vitro quantification of angiogenesis in response to hCG and both increased endothelial cell proliferation and vessel stabilisation were observed. This is in contrast to the late-luteal phase of a non-pregnant cycle where luteolysis is characterised by small vessel degradation.45 New vessel formation in the corpus luteum during early pregnancy is required to support both the synthesis of progesterone (through the delivery of precursors) and the secretion of progesterone into the circulation. That hCG plays an essential role is supported by the observation in primates that administration of hCG antisera leads to termination of pregnancy.46
Through its receptors on the endometrium, hCG may play a role in the implantation of the embryo. In myometrium and myometrial blood vessels, which express LH/hCG receptors, hCG may promote smooth muscle relaxation and myometrial vasodilation. Furthermore, via its receptors on trophoblast cells, hCG can stimulate adenylate cyclase and progesterone production. Therefore, hCG may have an autocrine–paracrine role in the placenta. hCG induces relaxin secretion by the corpus luteum during the luteal phase and in early pregnancy. Both relaxin and progesterone play an important role in the maintenance of early pregnancy. hCG stimulates cytotrophoblast VEGF secretion in vitro indicating a role in early placental angiogenesis.47
Its role in the maintenance of pregnancy at later gestations is not known but the observation that hCG reduces COX-2 expression, and hence prostaglandin E2, in culture of endocervical cells suggests a possible role in preventing cervical ripening. Furthermore, administration of hCG to mice may delay preterm delivery.48 The detection of hCG in cervicovaginal secretions from women in threatened preterm labour (24–34 weeks of gestation) was associated with a significant increase in preterm delivery. This preliminary report requires further evaluation but the authors suggest it has potential as a rapid inexpensive bedside test for women in threatened preterm labour.49
hCG also binds to TSH receptors and contributes to the increased maternal thyroxine levels usually observed in the first trimester of pregnancy. hCG may play a role in hyperemesis, which may be associated with an elevated free T4 level and suppressed TSH during the acute phase. Free thyroid hormones return to normal by about 20 weeks. Based on the interaction of both crude and purified hCG with receptors in human thyroid membranes, the thyrotropic activity of 1 IU hCG is equivalent to 0.5–0.8 μIU TSH.50
Fetal renal hCG production40 induces the secretion of testosterone by the fetal testes before the onset of pituitary LH secretion. The highest fetal serum testosterone levels coincide with peak maternal hCG concentrations. However, given that the onset of testosterone biosynthesis occurs at about 9 weeks and that hCG/LH receptors appear on the fetal Leydig cells at 12 weeks, the initial secretion of testosterone may be independent of hCG and LH. The role of hCG, if any, in ovarian development and differentiation is less clear. In addition, hCG stimulates dehydroepiandrosterone (DHEAS) production by the fetal adrenal gland.51In vitro studies show the free α-subunit of hCG stimulates decidual prolactin secretion.52 hCG promotes steroidogenesis in the placenta by stimulating the conversion of cholesterol to pregnenolone and progesterone, and promotes placental aromatisation. In addition, hCG promotes hydroxylation of placental steroids, although the precise mechanism remains uncertain.
There are a number of pathophysiological conditions that may lead to abnormal regulation of the hCG receptor and/or increased synthesis and secretion of hCG.
Abnormalities in the expression of hCG receptors
Young anovulatory women, with or without polycystic ovarian syndrome, with endometrial hyperplasia and carcinomas have increased expression of hCG receptors,53 implicating LH and LH/hCG receptor overexpression in human endometrial carcinomas.54 hCG receptor gene expression is significantly reduced in fibroids, compared with normal myometrium, it is possible this may be a factor in controlling fibroid growth.29
Following the abnormalities in the expression levels of hCG receptor, a number of activating and inhibiting mutations have been defined. Several mutations in this receptor gene lead to constitutive activation of Gs α-subunits and activation of adenylate cyclase, leading to an increase in cAMP in the absence of ligand. These constitutively activating mutations result in increased testosterone production in Leydig cells, leading to the clinical syndrome of ‘Testotoxicosis’. In this familial male-limited precocious puberty, inherited in an autosomal dominant pattern, affected individuals present prior to adrenarche with signs of rapid virilisation and growth acceleration, but final adult height is reduced because of premature epiphyseal closure.
Inactivating mutation of the hCG receptor have also been shown to occur. These mutant receptors bind to the ligand (LH, hCG) normally, but do not induce an increase in cAMP, which may be related to a defective interaction between the receptor and G proteins. The phenotype for these inactivating mutations differs between men and women. In men, there is primary testicular failure in utero and as a consequence a 46 XY male presents with female-appearing external genitalia (male pseudohermaphrodism). Homozygous females with inactivating hCG receptor mutations have primary amenorrhoea with ovarian resistance to LH.
‘Abnormal’ hCG synthesis and secretion
hCG levels are markedly elevated in multiple pregnancy and trophoblastic disease. Theca lutein ovarian cysts may arise during pregnancy, these are most frequently found in women with multiple pregnancy, diabetes mellitus, hydatidiform mole or Rhesus isoimmunisation, and usually indicate high hCG levels.
hCG levels in maternal blood are higher in pregnancies complicated by chromosomal abnormalities.55 Trisomy 21 is associated with high maternal serum concentrations of intact hCG and free β-hCG whereas these concentrations, compared with controls, are significantly decreased in trisomy 18, probably secondary to the poor differentiation of the cytotrophoblast.56 hCG has formed part of the biochemical triple test widely used for prenatal screening for trisomies.
hCG is also secreted by non-trophoblastic tissue. Elevated hCG concentrations are more likely to be found in patients with gynaecological (28.9%), breast (21%), gastrointestinal (18%), lung (10%) tumours, as well as melanoma, carcinoid, genitourinary, haematopoetic tumours.57 Elevated circulating hCG concentrations can also be found in benign conditions such as cirrhosis and inflammatory bowel disease. Patients are often asymptomatic although gynaecomastia may present in men. It is not entirely clear whether increased oestrogen is due to the gonadal response, to the tumour's ability to produce oestradiol or to the conversion of DHEAS to oestradiol.
The observation of regression of HIV-associated Kaposi's sarcoma during pregnancy in some women led to investigation of hCG as a potential mediator of this effect. Induction of apoptosis was demonstrated58 in Kaposi sarcoma cell lines, although it was not clear if this was solely due to hCG or other factors within the commercial urinary preparation. The discrepancies between clinical trials and in vitro studies support the notion that the hCG-associated factors are important.59 Furthermore, urinary hCG, β-hCG and interestingly to a lesser extent, recombinant hCG inhibited angiogenesis in a mouse model,60 suggesting that active hCG fragments or other components of urinary hCG contributed to the inhibitory effects on tumour growth and may mediate pregnancy-related regulation of cell death. hCG, in particular, the free β-subunit, may also have a role in promoting the development of epithelial tumours by reducing apoptosis.61 It is not clear whether these opposing effects are mediated by the same mechanisms but is an area that requires investigation.
Ovarian hyperstimulation syndrome
Ovarian hyperstimulation syndrome is a serious iatrogenic complication of controlled ovarian hyperstimulation, exacerbated and perpetuated by the presence of circulating levels of hCG. The condition is characterised by ascites, ovarian enlargement and increased capillary permeability. The pathophysiology of ovarian hyperstimulation syndrome is poorly understood but much interest has focussed on vascular endothelial growth factor (VEGF) as a possible mediator of these changes.62 VEGF mRNA expression in luteinised granulosa cells increases with hCG63 and hCG induces VEGF production in ovarian hyperstimulation syndrome model rats.64 Furthermore, hCG up-regulated VEGF expression in luteinised granulosa cells from women who developed severe ovarian hyperstimulation syndrome but had no effect on VEGF expression in control patients,65 suggesting that hCG plays a significant role in the pathogenesis of ovarian hyperstimulation syndrome.
There is a bimodal incidence following IVF–ET with early onset relating to the follicle and oocyte number and later onset (7–10 days following embryo transfer) almost invariably relates to endogenous hCG synthesis from a successful treatment cycle66 and is exacerbated by multiple pregnancy.67 The choice of luteal support, in pituitary desensitised cycles, following IVF favours progesterone as hCG support is associated with a higher incidence of ovarian hyperstimulation syndrome.68 There are however other potential mechanisms for the development of ovarian hyperstimulation syndrome including renin–angiotensin, nitric oxide and the kinin–kallikrein system,69 but the strong association with pregnancy and ovarian hyperstimulation syndrome implicates hCG as a causative factor.
Around 50 years ago, measurement of hCG by crude bioassays made possible the diagnosis of pregnancy. Since then more sensitive and specific radio-immunoassays have replaced these bioassays. The antibodies used for these assays are raised against the β-subunit of hCG, and principally detect the intact hCG molecule, the major form of the hCG molecule circulating in the blood during pregnancy, rather then the free β-subunit. All commercially available hCG radio-immunoassay kits are calibrated against an agreed-upon standard. There are two standards for hCG assays: the 1st International Reference Preparation (IRP) of hCG for Immunoassay—1974, and the 2nd International Standard of hCG for Bioassay—1964. One microgram of the IRP is equivalent to 12 IU, and 1 μg of the second International Standard corresponds to 5.7 IU of the IRP. The most recent third IRP Preparation IS 75/537 has in vivo and in vitro biopotency of 13 IU per 1 μg (range 8.3–16.8 IU).70 The WHO guidelines suggest that when comparing potency of different hCG isomers that moles/L be used instead of IU. Therefore, the same serum sample assayed with kits utilising different standards may give different results. The importance of this observation is realised if quantitative measurements are required or if comparisons with other laboratories are necessary.
There are over a hundred immunoassays commercially available for quantifying hCG-related molecules in serum or urine. Each immunoassay measures non-nicked hCG (the hormone) and one of seven combinations of the other hCG-related molecules present in pregnancy serum and urine samples, which include nicked hCG, glycosylated hCG, hCG missing the C-terminal extension, free α-subunit, large free α-subunit, free β-subunit, nicked free β-subunit and β-core fragment.71 Variations between immunoassays may occur and are more important for abnormal pregnancy (miscarriage or ectopic) than for a normally progressing pregnancy. hCG assays are used clnically not only in the diagnosis and management of pregnancy, but also in other disorders.
The detection of hCG in serum or urine serves as the basis of the pregnancy test. The sensitivity of hCG radio-immunoassays makes possible the diagnosis of pregnancy before the first missed menstrual period. Serum hCG assays, which have a sensitivity of 25 IU/L, can detect pregnancy within 8–10 days postovulation, compared with urinary assays which detect hCG 14–18 days after ovulation in a fertile cycle.
The serum concentrations of hCG follow a characteristic pattern during pregnancy, as discussed earlier. Quantitative hCG assays have become an integral part of the management of early pregnancy complications. Sequential monitoring of maternal serum hCG concentrations are particularly helpful when transvaginal ultrasound shows no intrauterine pregnancy in a woman with a positive urinary test. In normal pregnancies, hCG titres in maternal serum double every 1.3 to 2.3 days. If serum hCG levels are ‘low’ and do not follow the normal pattern, it suggests that the pregnancy is not viable but cannot determine the site of implantation. Suboptimal rises in serum β-hCG may be due to either a failing intrauterine or ectopic pregnancy. A high index of suspicion should be maintained until hCG levels return to non-pregnant levels or ultrasound confirms a non-viable intrauterine pregnancy.
A recent study of women presenting with pain or bleeding in early pregnancy has defined the slowest rise in serial serum hCG concentrations associated with viable intrauterine pregnancies.72 The slowest rise recorded with a viable pregnancy over 48 hours was 53%, which is lower than previously accepted and suggests that management should be more conservative to reflect this.
Spontaneous and missed miscarriage
Broadly speaking, there are two distinct patterns of hCG secretion seen in spontaneous miscarriage. In one pattern, trophoblastic activity appears to be normal and then decreases, suggesting that conception is associated with abnormal development of the fetus and subsequently miscarries as the hCG levels fall and progesterone synthesis declines. In the second, hCG levels are abnormally low from the start, because of poor trophoblastic development and conceptus failure.73
In addition, quantitative hCG measurements have been used to predict spontaneous miscarriage.74 Urinary hCG levels of less than 10,000 IU/L between 8 and 16 weeks of pregnancy are a poor prognostic indicator in patients with threatened miscarriage; levels over 20,000 IU/L are associated with a good pregnancy outcome. Furthermore, serum hCG, oestradiol and progesterone are also correlated with pregnancy outcome in women with threatened miscarriage, with hCG as the best predictor. If serum hCG levels are greater than 18,000 to 20,000 mIU/mL at eight weeks or more of gestation, miscarriage is unlikely to occur, compared with levels less than 10,000 mIU/mL.75
This remains an important cause of mortality in women accounting for one in eight deaths in the UK in the last Confidential Enquiry.76 The evolution of serum hCG assays, alongside high resolution ultrasound, has contributed to the earlier diagnosis of ectopics with opportunities for conservative, medical or laparoscopic management.77,78 In a suspected ectopic pregnancy, measurement of hCG will help to determine whether the patient is pregnant, and whether the titres of hCG are consistent with a normal intrauterine pregnancy or suggestive of an abnormal one, in which case hCG production is low for that gestational age. The agreed threshold for detecting intrauterine pregnancy on transvaginal ultrasound is controversial79 and ranges between 1000 and 2000 IU/L hCG. The detection of an adnexal mass or free fluid suggests an ectopic gestation although the woman's clinical condition and past history must be taken into account when deciding on her management.
Quantitative determination of serum hCG may permit a conservative approach in a small subgroup of women. In a study analysing the safety and efficacy of the conservative approach in the management of ectopic pregnancy, the initial hCG value (<1000 IU/L) and its following trend were found to be the most important prognostic factors.80 Serum progesterone concentrations may complement hCG levels in discriminating pregnancies that may resolve without intervention irrespective of site.81
In order to distinguish normal from abnormal gestations (ectopic pregnancies and spontaneous miscarriages) before six weeks, investigators have measured urinary β-core fragment, the urine degradation product of β-hCG.82 In early pregnancy (four to six weeks), β-core fragment correlated positively with gestational age which was not apparent in abnormal counterparts, suggesting that β-core fragment may be a promising marker to differentiate normal early pregnancies from abnormal gestations. Interestingly, however, a more recent report measured hCG isoforms and observed that free hCG β-subunit measurement in serum was as sensitive as total serum hCG but had a significantly higher specificity. This may form the basis of a single test for ectopic pregnancy in the future83 if larger studies assessing the use of hCG subunits in diagnosing early ectopic gestations confirm these preliminary findings.
Serum hCG monitoring following conservative laparoscopic surgery for ectopic pregnancy or following medical management with methotrexate is mandatory in order to detect persistent trophoblastic disease (approximately 5% following salpingotomy) or medical treatment failure.
Different molecular forms of hCG, in serum and urine, are elevated in pre-eclampsia.84 Measuring free β-subunit of hCG (serum)85,86 or hCG β-subunit core fragment (urine)87 in the second trimester as a screen for predicting pre-eclampsia has been studied. Serum free β-subunit of hCG did not predict pre-eclampsia,85 however, elevated levels of urine hCG β-subunit core fragment were associated with the subsequent development of pre-eclampsia.87 In a prospective randomised controlled study, a strong association between elevated maternal serum β-hCG levels in the third trimester and pregnancy-induced hypertension has been described.88 Furthermore, elevated β-hCG levels were more commonly seen in severe proteinuric pregnancy-induced hypertension.
Gestational trophoblastic disease
Gestational trophoblastic disease/neoplasia encompasses a spectrum of proliferative trophoblast abnormalities. The neoplasm develops in the trophoblast portion of the blastocyst and retains placental characteristics. hCG measurements are important in diagnosing and monitoring gestational trophoblastic disease.89
While not all trophoblastic neoplasms secrete excessive amounts of hCG, serum hCG concentrations above 500,000 IU/L are strongly suggestive. Levels of hCG in molar pregnancies are typically much greater than in choriocarcinoma. In suspected cases of trophoblastic disease, findings may include a large for gestational age uterus, the passage of trophoblastic vesicles or exaggerated symptoms of pregnancy such as hyperemesis. Typical ultrasonic features of a complete mole are of a snow storm appearance but triploid partial moles may have a coexistent fetus and subtler placental abnormalities that are only detectable histologically.
Once a molar pregnancy is evacuated, there usually is a continuous fall in hCG titres, with most women achieving remission and no measurable hCG within 15 weeks.90 Furthermore, 87% of women whose regression curves deviate from normal during the postevacuation period can be identified by six weeks.90 Serial monitoring of hCG provides an indicator of response to chemotherapy for choriocarcinoma and cerebrospinal fluid levels can be used to detect and monitor CNS involvement.91 The co-ordination of follow up of women with gestational trophoblastic disease is through regional centres in the United Kingdom.
A series of women with persistently low hCG levels following pregnancy, with normal imaging (colour Doppler, ultrasound and MRI of the uterus and ovaries), were monitored for up to six years.92 Methotrexate administration had failed to reduce hCG concentrations. One patient developed a pulmonary placental site trophoblastic tumour, after two years, and it is therefore possible that trophoblastic cells may remain quiescent for years before manifesting as a trophoblastic tumour. Long term observation is therefore advisable if women have low levels of hCG (even when radiological imaging is negative) until levels become undetectable. Pituitary MRI should also be considered as a hCG secreting pituitary tumour has been recently reported.93
Finally, it is important to recognise the condition referred to as ‘phantom choriocarcinoma syndrome’/phantom hCG (pseudohypergonadotrophinaemia),94 where there is persistent mild elevations of serum hCG in patients with no history of trophoblastic disease. In addition, there is absence of hCG in the urine and no ‘dilutional parallelism’, that is, when serum is diluted, levels do not decrease parallel to the dilution. This is in contrast to the case series above92 wherein hCG was detectable in serum and urine. Awareness of this phenomenon should avoid the inappropriate use of cytotoxic chemotherapy to treat patients for presumed occult choriocarcinoma particularly as ‘real’ and phantom hCG can be distinguished biochemically.
hCG in non-trophoblastic tumours and normal cells
Around 70% of non-seminomatous germ cell tumours and 5–10% of seminomas are accompanied by elevated serum hCG levels.95,96 Furthermore, 20% of other non-trophoblastic cancers are associated with immunoreactive hCG in blood.57 Compared with trophoblastic hCG, ‘ectopic’ hCG is predominantly the α-subunit which exceeds the production of ectopic hCG. In addition, α-subunit produced by non-trophoblastic tissue is larger than the ‘normal’α-subunit with decreased ability to combine with hCG-β, suggesting abnormalities of the protein synthesised by the tumour with respect to the polypeptide backbone, carbohydrate content or production of a hCG-α precursor.97 hCG levels in cancer patients are usually only modestly raised (<500 IU/L).
The presence of hCG is not specific for cancer, given that immunoreactive hCG has been found in low concentrations in blood of normal men and women.98,99 Serum levels of hCG in normal men range from 0.02 to 0.8 IU/L, and in normal pre- and postmenopausal women between 0.02 to 0.2 and 0.02 to 2.8 IU/L, respectively. This immunoreactive hCG is probably produced by the pituitary gland and may be under gonadotrophin releasing hormone (GnRH) regulation.
Aberrant hCG assay results
False-positive pregnancy results or discordant quantitative hCG levels can occur as a result of a number of factors, including anti-hCG antibodies present in people who have been treated with hCG, those with hyperlipidaemia and elevated immunoglobulin levels. By performing assays on serial dilutions of the specimen, discordant results can often be identified. If values do not decline in relation to the dilutions, assay interference can be inferred.
Therapeutic use of hCG
In assisted conception, multifollicular development is usually achieved by daily administration of FSH injections using either purified urinary preparations or more recently developed recombinant FSH. Urinary purified LH is not available in high enough doses to induce oocyte maturation100 and hCG has been used as a surrogate for LH.
In ovulation induction programmes, hCG is usually given to induce ovulation as oocyte release is necessary for conception to occur as opposed to solely maturation in IVF/ICSI where follicular aspiration and physical retrieval of the oocyte is performed. The detection of LH/hCG receptors in the human cervix suggests that pre-ovulatory LH concentrations (or administered hCG), in addition to inducing ovulation, may directly influence endocervical epithelial cells to affect mucus secretion, and hence, sperm transport.31
Whether hCG should be used routinely in clomiphene ovulation induction has not been fully addressed however. In a study of clomiphene stimulation prior to IUI, patients were prospectively randomised to hCG or to await the onset of spontaneous ovulation.101 There was no difference in pregnancy rate between the two approaches but large probably multicentre trials would be required to answer this question. Ovulation has been observed to occur approximately 38 hours following 6000 IU hCG intramuscularly.102
Purified urinary preparations of hCG in a dose of 5000 or 10,000 IU have been used for maturation prior to IVF after an initial study found a significantly worse outcome with lower doses.103 The route of administration (intramuscular or subcutaneous) does not appear to affect clinical outcome and oocyte retrieval is scheduled for approximately 36 hours following hCG administration. The optimal size of the developing follicle with respect to retrieving an oocyte appears to be between 18 and 20 mm,104 however, the criteria for hCG administration vary between programmes. Acquisition of LH receptors by the pre-ovulatory follicle is important in natural cycles to respond to the LH surge and in stimulated cycles for hCG to be effective in promoting oocyte maturation and ovulation.
Empty follicle syndrome is a term used to describe the unexpected failure to retrieve oocytes from pre-ovulatory follicles. The incidence has been reported to be up to 0.5%105 and has been attributed to patient error in injecting the hCG, interbatch variation in the urinary purified form or rapid clearance of hCG from the patients circulation.
Recombinant hCG is available and appears from preliminary studies to be at least as effective as urinary derived hCG with fewer local injection site reactions and higher serum progesterone concentrations, however, there was a trend toward a higher incidence of ovarian hyperstimulation syndrome.106–108 Recombinant LH (15,000–30,000 IU) compared with urinary hCG (5000 IU) was as effective in final oocyte maturation prior to IVF–ET. However, a single injection of shorter acting rLH was associated with a significantly lower incidence of ovarian hyperstimulation syndrome, which is clearly beneficial.109
In vitro maturation of follicles (IVM) has many potential applications in reproductive medicine including future fertility preservation, eliminating ovarian hyperstimulation syndrome and reducing the financial costs by avoiding expensive gonadotrophin preparations. hCG has been used both for in vivo priming of immature follicles and as a component of the culture system for oocyte maturation. Initial IVM success rates were poor but the introduction of in vivo hCG priming (administered 36 hours prior to retrieval) accelerated the maturation process and resulted in satisfactory pregnancy rates.110 Interestingly, additional pretreatment with FSH for six days while increasing serum oestradiol did not improve the maturation, fertilisation or pregnancy rate compared with hCG priming alone.111
Culturing oocytes in vitro was equally successful in the presence of either recombinant hCG or recombinant LH,112 suggesting both hormones are effective in promoting oocyte maturation.
Optimal priming regimes and in vitro culture systems may be different for subgroups of patients (e.g. PCOS and non-PCOS) and further work on this may improve the overall success rates of IVM.
hCG has vital roles in inducing angiogenesis in embryo implantation and early trophoblastic invasion and in maintaining the stability of the corpus luteum. Furthermore, these potent angiogenic properties are relevant to the pathogenesis of ovarian hyperstimulation syndrome, and to tumour development and metastasis. The development of sensitive assays for the isoforms and subunits of hCG may find application in the rapid diagnosis of important early pregnancy problems such as ectopic or non-viable pregnancies.
Therapeutically, recombinant hCG appears at least as effective as urinary hCG for inducing ovulation and oocyte maturation prior to IVF although data on overweight women are currently awaited.