1 Department of Obstetrics and Gynaecology, University Hospital, Geneva, Switzerland and 2 Irving Center for Clinical Research, Columbia University College of Physicians and Surgeons, 630 W 168th Street, New York, NY 10032, USA
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Abstract |
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Key words: bioactivity/choriocarcinoma/HCG/monoclonal antibodies/pregnancy
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Introduction |
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It is generally accepted that the dimer HCG (- and ß-subunits) structure changes during pregnancy with a higher molecular weight (Fein et al., 1980
) or sialic acid content (Wide and Hobson, 1987
) during the first trimester compared to late pregnancy. A change in the nature of several HCG isoforms seems to occur at around the 13th week of gestation with the appearance of less acidic HCG molecules later in pregnancy (Wide et al., 1994
). Whereas nicked HCG (HCGn, peptide bond cleavages within its ß-subunit loop 2) was considered by previous reports as more prevalent after 810 weeks of pregnancy (Cole et al., 1993
; O'Connor et al., 1994
), it appears recently that HCGn is not produced in significant quantities during normal pregnancy (Kovalevskaya et al., 1999a
).
Several studies have demonstrated for HCG as well as for the other gonadotrophins that the biological activity of the molecule may be influenced by its sialic acid content (Wide, 1962; Wide and Hobson 1987
; Burgon et al., 1996
, 1997
; Stanton et al., 1996
; Ronco et al., 1998
) or glycans (Matzuck et al., 1989
; Nemansky et al., 1995; Heikoop et al., 1998
) or even by the presence of peptide cleavage as in the nicked HCG (Cole et al., 1991
).
While most studies reported a difference in HCG bioactivity between the first and the last trimester of pregnancy which seems to be related to the above-mentioned HCG structural change, it was reported recently that a modulation of HCG bioactivity exists already during the early phase of placental development (amenorrhoea <10 weeks) in normal and abnormal pregnancies (Mock et al., 1998).
Interestingly, a recent study including IVF patients demonstrated that there were similar progressive changes of HCG isoforms from 1 to 4 weeks post-embryo transfer and progressively afterwards (Kovalevskaya et al., 1999b). These authors used a new immunoradiometric assay (IRMA) based on a monoclonal antibody which detect choriocarcinoma-like HCG. This assay appears to be particularly sensitive to changes in carbohydrate structure and to detect HCG isoforms present during early gestation (O'Connor et al., 1998
).
Since the importance of oligosaccharides for the biological activity of HCG and other gonadotrophins is well established (Thotakura and Blithe, 1995), it is postulated that HCG isoforms secreted in early pregnancy such as choriocarcinoma-like HCG known to have highly branched oligosaccharides (Elliott et al., 1997
) may be related to our recent observation of HCG bioactivity modifications. The aim of this study, therefore, was to analyse the relationship between both in-vitro bioactivity and these specific HCG isoforms.
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Materials and methods |
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Serum samples were obtained (one sample per patient) at the time of echographic diagnosis in normal IUP or at the day of laparoscopy in the EP. Immunoreactive free ß-HCG was measured by radioimmunoassay (free ß-HCG; CISbio International, Saclay, France) and total immunoreactive HCG by enzyme immunoassay (total HCG IMX; Abbott Abbott Park, USA) at a sensitivity of 0.1 ng/ml and 1 mIU/ml respectively. Oestradiol was measured by an automated enzyme immunoassay (Vidas; Biomérieux, Marcy-l'Etoile, France) at a sensitivity of 5 pg/ml.
Bioactive HCG (HCGb) was measured in triplicate by a mouse Leydig cell bioassay (Robertson and Binden, 1990) as recently described (Mock et al., 1998
).
Immunoradiometric assays B109-B108* and B152-B207* were used to measure intact HCG as previously described (O'Connor et al., 1988, 1998
).
The first antibody was the capture antibody, the second with an asterisk, was a radioiodinated detection antibody. The antibody, B152, was developed by using as immunogen a hyperglycosylated and 100% nicked form of HCG produced by a single individual with choriocarcinoma (Birken et al., 1999). The B152 assay appears to be sensitive to changes in carbohydrate structure and can measure very early isoforms of HCG (O'Connor et al., 1998
), whereas the B109 assay detects isoforms of HCG which predominate later in pregnancy (Kovalevskaya et al., 1999b
). Since the B152 assay detects with greatly enhanced sensitivity HCG isoforms which appear earlier in pregnancy than isoforms measured by the B109 assay, the alteration of the B152/B109 ratio, as used in the present study, was assumed to reflect HCG isoforms change. Intra-assay variation was 6% for both assays, inter-assay variation was 12% for B109-B108* and 13% for B152-B207* assays. Sensitivity (least detectable dose) defined as +2 SD from the zero calibrator, was 1.0 fmol/ml for the B109-B108* assay and 2.2 fmol/ml for the B152-B207* assay.
Results were stratified in two subgroups (low and high) according to immunoreactive free ß-HCG (free ß-HCGi), total HCG immunoreactivity, serum oestradiol and gestational age.
Statistical analyses were performed on a Power Macintosh computer using the Statview programme. Data were compared by analysis of variance (ANOVA) or unpaired Student's t-test when appropriate.
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Results |
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Discussion |
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While most studies were focused on a difference of HCG bioactivity between the first and the last trimester, the present study confirms the previous finding that HCG bioactivity is altered already in first-trimester pregnancies (Mock et al., 1998). It was postulated that such a modification may be causally related to HCG isoform changes. Carbohydrate moieties such as glycans (oligosaccharides) and sialic acid are directly implicated in various molecular forms of HCG or other gonadotrophins and they appear to be related to the bioactivity (for reviews, see Thotakura et al., 1990
; Thotakura and Blithe, 1995
). Indeed, N-linked oligosaccharides on the
chain (Asn 52) of HCG seem to play an important role in signal transduction, resulting in a stimulation of steroidogenesis and cAMP production (Matzuck et al., 1989
). A linear correlation has been demonstrated for human FSH between sialic acid content, radioreceptor activity and in-vitro bioactivity (Stanton et al., 1996
).
HCG isoforms secreted by choriocarcinoma have highly branched oligosaccharides (Elliot et al., 1997). Recently, similar HCG isoforms have been detected in early pregnancy by an immunoradiometric assay B152 (O'Connor et al., 1998; Kovalevskaya et al., 1999b
). Indeed, it has been demonstrated that the distribution of the choriocarcinoma-like HCG isoforms synthesized by the JAR choriocarcinoma cell lines and measured by B152/B109 assays ratio was similar to that observed in early pregnancy. However, the structure of B152 recognized isoforms from early pregnancy has not yet been characterized and it is not clear whether these isoforms may be considered as having highly branched oligosaccharides residues and/or as being acidic isoforms with a high sialic acid content.
Interestingly, a recent report studied the different HCG isoforms during early human embryo development in an IVF programme. It was demonstrated that early blastocyst (day 11 after fertilization) produced more acidic HCG isoforms than the more advanced embryos (day 14). These results suggest that the bioactivity of HCG secreted by the blastocysts may change with time and with the differentiation of the trophectoderm (Lopata et al., 1997).
The observation in the current study that there is a similar decrease of secreted choriocarcinoma-like HCG isoforms and HCG bioactivity with increasing amount of free ß-HCG immunoreactivity may well correspond to the in-vitro HCG isoform pattern found for cultured blastocyst. However, because no correlation was found between B152/B109 isoforms ratio and HCG bioactivity, the direct implication of choriocarcinoma-like HCG on HCG in-vitro bioactivity can be ruled out. Because monoclonal antibody B152 preferentially binds to hyperglycosylated HCG isoforms, but not to those with sialic acid differences (Birken et al., 1999), it is postulated that the observed alteration of B152/B109 ratio during early pregnancy is related to a modulation of other HCG molecular isoforms such as sialic acid content which are prone to influence HCG bioactivity.
In the current study, it appears that the distribution of choriocarcinoma-like HCG isoforms (B152/B109) is related to free ß-HCG immunoreactivity and gestational age. This finding strongly suggests that these specific isoforms are related to the differentiation process of trophoblastic tissue. Indeed, increased secretion of HCG is commonly used as an indicator of syncytiotrophoblast formation in in-vitro cytotrophoblast cultures (Kliman et al., 1986). During the first trimester of gestation, free ß-HCG and HCG dimer immunoreactivity follow a similar pattern of secretion with a serum peak occurring between 810 weeks (Ozturk et al., 1988
). Therefore, in early gestation when a low amount of free ß-HCG and HCG dimer are secreted by the trophoblast, trophoblast tissue may also be considered as less differentiated. It is postulated that in early gestational trophoblast as well as in choriocarcinoma, both considered as less differentiated tissues, some enzymatic alteration may lead to the biosynthesis of highly branched N-linked oligosaccharide. Thus, the higher proportion of choriocarcinoma-like HCG isoforms during early gestation, decreasing progressively with advancing gestational age, could be explained by incomplete glycosylation processing. Another explanation may be related to the highly proliferative pattern of trophoblast tissue as choriocarcinoma or villous cytotrophoblast cells. Indeed, it has been demonstrated in yeast that N-glycosylation genes, in particular the dolichol pathways ALG genes, play a central role in the cellular cycle (for review, see Kukurinska and Lennon, 1999). The expression of ALG 7, the first gene in the dolichol pathway of N-glycosylation protein synthesis, is down-regulated in cell-cycle arrest and may be involved in the down-regulation of cyclin expression (Lennon et al., 1997
). Thus, differentiation of trophoblast with advancing gestational age, which is accompanied by cell-cycle arrest (syncytium formation), may contribute to an alteration of HCG N-glycosylation. Taken together, the observations of the current study that there was a higher proportion of choriocarcinoma-like HCG isoforms when HCG secretion was low may be thus explained and/or related to the incomplete differentiation of trophoblast tissue as found during its early development. Since early cytotrophoblasts before the 6th week of gestation appear to secrete dimer HCG (Maruo et al., 1992
), choriocarcinoma-like HCG isoforms may be even related to the secretion of this area of villous trophoblast. Thereafter, as syncytialization of trophoblast is increasing during the first trimester and promoted by HCG (Shi et al., 1993
), differentiated syncytiotrophoblasts may thus secrete more well processed glycosylated molecular forms of HCG and therefore less choriocarcinoma-like HCG isoforms.
The physiological implication of this higher amount of choriocarcinoma-like HCG in early pregnancy is obviously not well understood. A recent study demonstrated that glycosylation of free -HCG subunit is different between early and late second trimester with increasing amounts of core fucosylation and of triantennary glycans being secreted. Interestingly, the type of triantennary branching observed on early pregnancy free ß-HCG was similar to glycan structures found on HCG associated with invasive mole and choriocarcinoma (Nemansky et al., 1998
). While it is known that choriocarcinoma-derived HCG isoforms have a highly branched oligosaccharides structure (Elliot et al., 1997), B152 isoforms in normal early pregnancy have not as yet been characterized. Therefore, it is not known if they are similar to choriocarcinoma-derived HCG isoforms or if they correspond to HCG isoforms with a higher content in sialic acid or both. However, because in early pregnancy trophoblast is more invasive, in particular the cytotrophoblastic cells, the finding that there is a modulation of choriocarcinoma-like HCG during early pregnancy may be related to trophoblast invasiveness.
In conclusion, this study shows that changes in in-vitro HCG bioactivity are not related to the presence of choriocarcinoma-like HCG isoforms in early pregnancy and that known changes in HCG bioactivity must thus be attributable to other HCG molecular forms. However, the in-vivo bioactivity, which is related to the biological half life of the molecule, remains to be determined, awaiting purification of the HCG isoform. Furthermore, the amount of secreted choriocarcinoma-like HCG isoforms is predominant during early gestation and may be the result of an early developmental regulation of glycosylation enzyme in trophoblast. Such modifications of HCG isoforms and bioactivity during the invasive period of the trophoblast may be in favour of an implication of HCG itself in the trophoblast invasiveness and thus in the implantation process.
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Acknowledgments |
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Notes |
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References |
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Submitted on February 21, 2000; accepted on July 6, 2000.