Choriocarcinoma-like human chorionic gonadotrophin (HCG) and HCG bioactivity during the first trimester of pregnancy

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(1)Human Reproduction vol.15 no.10 pp.2209–2214, 2000. Choriocarcinoma-like human chorionic gonadotrophin (HCG) and HCG bioactivity during the first trimester of pregnancy. Pascal Mock1,3, Galina Kovalevskaya2, John F.O’Connor2 and Aldo Campana1 1Department. of Obstetrics and Gynaecology, University Hospital, Geneva, Switzerland and 2Irving Center for Clinical Research, Columbia University College of Physicians and Surgeons, 630 W 168th Street, New York, NY 10032, USA 3To. whom correspondence should be addressed at: Department of Obstetrics and Gynaecology, Clinic of Infertility and Gynaecological Endocrinology, WHO Collaborating Center in Human Reproduction, University Hospital of Geneva, 32 Blvd de la Cluse, 1211 Geneva 14, Switzerland. E-mail: Pascal.Mock@hcuge.ch. The objective of this study was to evaluate the distribution of choriocarcinoma-like human chorionic gonadotrophin (HCG) isoforms during first trimester pregnancy and their relationship with in-vitro HCG bioactivity. This was done by means of a retrospective analysis of patients’ sera with first trimester normal intrauterine and abnormal (ectopic) pregnancies. Serum samples were obtained from 38 women with an amenorrhoea of <10 weeks. From these, 19 had a normal intrauterine pregnancy (IUP) and 19 an ectopic pregnancy (EP). Total immunoreactive HCG (HCGi), free β-HCGi and oestradiol were measured by enzyme immunoassays and bioactive HCG by the mouse Leydig cell bioassay. The alterations in HCG isoform content were measured by the combination of two immunometric assays, B152 for choriocarcinoma-like HCG and B109 for intact HCG detection and expressed as the B152/B109 ratio. Choriocarcinoma-like HCG isoforms ratio measured by B152 and B109 assays was significantly higher in the low subgroups of free β-HCGi and gestational age (P ⍧ 0.0111 and 0.0036 respectively). Whereas bioactive to immunoreactive HCG ratios (b/i ratio) were significantly higher when free βHCGi concentrations were low (P ⍧ 0.0010), no correlation was found between the variation of bioactivity (b/i ratio) and the proportion of choriocarcinoma-like HCG isoforms (B159/B108). It is concluded that in first trimester pregnancies (i) the modulation of HCG in-vitro bioactivity is not related to the variation of choriocarcinoma-like HCG isoforms secretion and (ii) the amount of choriocarcinomalike HCG isoforms secreted by the early trophoblast is predominant and may be the result of an early developmental regulation of glycosylation enzyme. Key words: bioactivity/choriocarcinoma/HCG/monoclonal antibodies/pregnancy. Introduction Human chorionic gonadotrophin (HCG), a member of the glycoprotein hormone family which includes pituitary hor© European Society of Human Reproduction and Embryology. mones such as LH, FSH and thyroid stimulating hormone (TSH), is composed of two distinct non-covalently associated subunits, namely the α- and β-subunits. Intact HCG, i.e. combined α- and β-subunits and its free subunits are secreted by trophoblast cells in particular by the syncytiotrophoblast during normal and abnormal pregnancies (Bousfield et al., 1994). It is generally accepted that the dimer HCG (α- and βsubunits) 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 β-subunit loop 2) was considered by previous reports as more prevalent after 8–10 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 choriocarcinoma2209.

(2) P.Mock et al.. 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 invitro bioactivity and these specific HCG isoforms. Materials and methods Only women (n ⫽ 38) with amenorrhoea of ⬍10 weeks were included in this study. Among these, 19 patients had a normal intrauterine pregnancy (IUP), 19 women had an ectopic pregnancy (EP) of similar gestational age (mean: 43.6 and 46.6 days respectively). EP were diagnosed by laparoscopy and were all tubal pregnancies. IUP were confirmed by first trimester transvaginal echography and all cases of IUP had a normal term delivery. 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 β-HCG was measured by radioimmunoassay (free β-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; Biome´ rieux, 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 β-HCG (free β-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.. Results Figure 1 compares the ratio of HCG isoforms measured by B152 and B109 assays (B152/B109) and HCG b/i ratios between low and high subgroups of free β-HCGi for all pregnancies irrespective of their site of implantation. B152/ B109 and HCG b/i ratios were different between both subgroups (P ⫽ 0.0111 and P ⫽ 0.0010 respectively). Both the 2210. Figure 1. Comparison of human chorionic gonadotrophin (HCG) isoforms ratios measured by the B152-B207* and B109-B108* assays and HCG bioactive/immunoreactive (b/i) ratios in serum of first trimester pregnancies (n ⫽ 38) between low and high subgroups of free β-HCGi irrespective of the implantation site.. proportion of choriocarcinoma-like HCG isoforms (B152/ B109) and HCG bioactivity were significantly higher in the low free β-HCGi subgroup. However, there was no correlation between B152/B109 and HCG b/i ratios (result not shown). Furthermore, whereas the b/i ratio decreased with increasing immunoreative free β-HCGi (r2 ⫽ 0.38, P ⬍ 0.0001), B152/ B109 tended only to be inversely correlated without reaching statistical significance (r2 ⫽ 0.10, P ⫽ 0.0574). B152/B109 was not correlated to HCGi nor HCGb or serum oestradiol. The proportion of HCG isoforms measured by B152 and B109 assays (B152/B109) was evaluated according to the concentrations of free β-HCGi, total HCGi, oestradiol and gestational age in all first-trimester pregnancies. B152/B109 ratios were stratified in two subgroups (low or high) according to: (i) immunoreactive free β-HCG (free β-HCGi) ⬍10 ng/ml (low; mean 2.9, range 0.8–8.2) and ⬎10 ng/ml (high; mean 49.8, range 10–105), (ii) total HCG immunoreactivity ⬍5000 mIU/ml (low; mean 1580, range 175–3849) and 艌5000 mIU/ ml (high; mean 49 689, range 8110–127 820), (iii) serum oestradiol ⬍300 ng/ml (low; mean 155, range 38–298) and 艌300 ng/ml (high; mean 562, range 307-1123) and finally (iv) gestational age ⬍40 days (low; mean 36, range 32–39) and 艌40 days (high; mean 48, range 40–66). As shown in Figure 2, B152/B109 ratios showed only a trend to be lower in high subgroups of total HCGi and serum oestradiol, whereas there was a strong significant difference between both subgroups for gestational age (P ⫽ 0.0036) and free β-HCGi. Interestingly, GA was closely correlated to oestradiol and free β-HCGi in IUP only (r2 ⫽ 0.33, P ⫽ 0.0153 and r2 ⫽ 0.41, P ⫽ 0.0059). However, while there was no correlation between b/i ratio and gestational age, there was a strong negative correlation between B152/B109 ratios and gestational age for cases between 32 and 50 days of last menstrual period irrespective of the implantation site as shown in Figure 3. A similar comparison of parameters in both IUP and EP is shown in Table I. Using an analysis of variance with Scheffe´ ’s test, there was no difference in the gestational age between the different subgroups. Stratifying the data according to the.

(3) Choriocarcinoma-like HCG and HCG bioactivity in pregnancy. Figure 2. Comparison of B152/B109 ratios between high and low subgroups according to free β-HCGi, total HCGi, oestradiol and gestational age in first-trimester pregnancies (n ⫽ 38). NS ⫽ not significant.. negatively correlated to oestradiol (r2 ⫽ 0.363, P ⬍ 0.0001), B152/B109 was not correlated to oestradiol (results not shown).. Figure 3. Ratios of HCG isoforms measured by the B152-B207* and B109-B108* assays and HCG bioactivity to immunoreactivity (b/i) in first trimester pregnancies (n ⫽ 23) between 32 and 50 days of last menstrual period.. site of implantation, it was observed that in IUP and EP, the b/i ratio decreased significantly with increasing free β-HCGi, whereas the B152/B109 ratio showed only a trend to decrease (not significant, Table I). There was no difference in either HCG b/i nor B152/B109 ratios between matched IUP and EP subgroups. Values of free β-HCGi were not different when comparing matched IUP and EP subgroups. However, oestradiol concentrations were significantly different between the different free β-HCGi subgroups for both IUP and EP (P ⬍ 0.0001). Moreover, EP had significantly (P ⫽ 0.0026) lower oestradiol concentrations than IUP except when the lower subgroups were compared. Whereas b/i ratios appeared to be. Discussion The results of the present study indicate that the secretion profile of choriocarcinoma-like HCG isoforms, using B152 and B109 IRMA (B152/B109), changes in the maternal circulation during spontaneous first-trimester pregnancy. The distribution of these isoforms follows throughout early pregnancy a similar pattern to HCG bioactivity and is independent of the implantation site. However, no correlation was found between these choriocarcinoma-like HCG (B152/B109) and b/i ratios and serum oestradiol. It appears that these parallel modifications in HCG structure and bioactivity have never been reported in the literature in early spontaneous pregnancy. 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 α 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). 2211.

(4) P.Mock et al.. Table I. Gestational age, immunoreactive free β-HCG, oestradiol, HCG B152/B109 and bioactive/immunoreactive (b/i) ratios in both normal intrauterine pregnancies (IUP) and ectopic pregnancies (EP) according to the free β-HCG subgroups Implantation site. I. IUP All Low High II. EP All Low High. n. Gestational age in days (range). Free β-HCG ⫾SE. B152/B109 ratio ⫾SE. HCG b/i ratio ⫾SE. Oestradiol (pg/ml). 19 9 10. 42 (32–66) 40 (32–48) 44 (35–66). 27.2 ⫾ 7.9 3.9 ⫾ 0.7 48.1 ⫾ 11.7. 3.0 ⫾ 0.3 3.5 ⫾ 0.5 2.5 ⫾ 0.3. 0.9 ⫾ 0.1 1.3 ⫾ 0.1 0.6 ⫾ 0.2. 424.4 ⫾ 65.1 235.2 ⫾ 37.5 594.6 ⫾ 90.5. 19 11 8. 46 (35–61) 46 (35–61) 47 (41–58). 23.0 ⫾ 7.7 2.0 ⫾ 0.4 51.9 ⫾ 12.3. 2.6 ⫾ 0.3 3.1 ⫾ 0.5 1.8 ⫾ 0.2. 1.3 ⫾ 0.2 1.6 ⫾ 0.2 0.9 ⫾ 0.3. 208.1 ⫾ 38.0 111.1 ⫾ 27.3 341.4 ⫾ 54.4. NS ⫽ not significant. Statistics for b/i: in IUP: low versus high, P ⫽ 0.0141; in EP: low versus high, P ⫽ 0.0043. Statistics for gestational age: IUP low versus EP low P ⫽ NS, IUP high versus EP high P ⫽ NS. Statistics for B152/B109: in IUP: low versus high, P ⫽ NS; in EP: low versus high, P ⫽ NS.. 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 β-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 β-HCG immunoreactivity and gestational age. This finding strongly suggests that these specific isoforms are related to the differentiation process of trophoblastic tissue. Indeed, 2212. 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 β-HCG and HCG dimer immunoreactivity follow a similar pattern of secretion with a serum peak occurring between 8–10 weeks (Ozturk et al., 1988). Therefore, in early gestation when a low amount of free β-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 Nglycosylation 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 choriocarcinomalike 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), choriocarcinomalike 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.

(5) Choriocarcinoma-like HCG and HCG bioactivity in pregnancy. molecular forms of HCG and therefore less choriocarcinomalike 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 α-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 β-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. Acknowledgements We wish to thank Dr Paul Bischof, Dr Didier Chardonnens and Dr Jean-Michel Bidart for the helpful discussions and Lydia Delajoux for her excellent technical assistance. This work was supported in part by grant ESO7589 from the National Institute of Environmental Health Sciences and the Office of Research on Women’s Health, both at National Institutes of Health, USA.. References Birken, S., Krichevsky, A., O’Connor, J. et al. (1999) Development and characterization of antibodies to a nicked and hyperglycosylated form of HCG from choriocarcinoma patient. 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