Molecular Human Reproduction, Vol. 6, No. 1, 5-10,
January 2000
© 2000 European Society of Human Reproduction and Embryology
Genetic diagnosis |
HCG concentration and receptor gene expression in placental tissue from trisomy 18 and 21
1 Academic Departments of Obstetrics and Gynaecology, Royal Free and University College Medical School, London, UK, 2 Laboratory of Molecular Biology and Medicine, Department of Obstetrics and Gynaecology, University of Louisville, Louisville, USA, and 3 The Research Laboratory on Reproduction, Free University of Brussels School of Medicine, Brussels, Belgium
Abstract
Trisomy 21 is associated with high maternal serum concentrations of intact human chorionic gonadotrophin 
(HCG) and free ß-HCG whereas these concentrations are markedly decreased in trisomy 18. In this study, we investigated the effect of trisomy 21 and 18 on endogenous HCG concentrations and luteinizing hormone (LH)/HCG receptor expression in placental villous tissue in eight trisomy 21, six trisomy 18 and 42 chromosomally normal samples, collected at 12–16 weeks gestation. The tissue concentrations of intact HCG, free -HCG and free ß-HCG subunits were measured using solid-phase two-site immunoradiometric assay. LH/HCG receptor expression was evaluated with immunohistochemistry and in-situ hybridization. Villous tissue in trisomy 21 contained higher ß-HCG concentrations than the controls (P < 0.05). In trisomy 18 cases, the ß-HCG concentration was lower than in the control group (P < 0.01). Both immunocytochemistry and in-situ hybridization demonstrated a more intense staining of the trophoblast in cases of trisomy 21 and 18, compared with controls with the strongest signal in cases of trisomy 18 (P < 0.01). We concluded that in trisomy 21 the high tissue HCG concentration and expression of LH/HCG receptor in the trophoblast may reflect the relative immaturity of the trophoblastic tissue whereas in trisomy 18, the very low concentration of endogenous HCG, associated with an over-expression of LH/HCG receptor in the trophoblast, is probably secondary to the poor differentiation of the cytotrophoblast.
HCG/placenta/pregnancy/receptors/trisomy
Introduction
Human chorionic gonadotrophin (HCG) is a glycoprotein hormone produced by the placental villous trophoblast. It consists of two subunits, and ß, which are synthesized separately and subsequently joined non-covalently (Lapthorn et al., 1994
). The gene encoding for -HCG is located on chromosome 6 (Fiddes and Goodman, 1979) and ß-HCG is encoded by a cluster of at least six genes located on chromosome 19 (Talmadge et al., 1983). Control of secretion of ß-subunits is thought to be the rate-limiting step to the production of the dimer (Nagy et al., 1994). HCG biosynthesis is inextricably linked to differentiation of the cytotrophoblast which integrates input from many different hormonal signalling pathways, e.g. cyclic AMP, insulin, calcium, interleukins, growth factors, placental-derived gonadotrophin-releasing hormone (GnRH) and HCG (Jameson and Hollenberg, 1993). Luteinizing hormone (LH)/HCG receptors have also been identified in human placental tissue, i.e. amnion, chorion, decidua and umbilical cord (Reshef et al., 1990, Licht et al., 1993a; Tao et al., 1995). Endogenous HCG appears to regulate subunit mRNA concentrations and HCG secretion and its effect is probably receptor-mediated in a paracrine and autocrine mechanism (Licht et al., 1998).
Aneuploid pregnancies are associated with changes in maternal serum concentrations of intact HCG and its subunits and these variables are currently used between 11–17 weeks gestation as markers for the clinical screening of trisomy 21 and 18 (Brizot et al., 1995a, Extermann et al., 1998). In most cases of trisomy 21, maternal serum concentrations of intact and free ß-HCG are increased, whereas little variation is found in maternal serum-free -HCG (Spencer, 1993; Jauniaux et al., 1996) and for fetal serum total HCG (Abbas et al., 1995). The placental mRNA expression was not altered in trisomy 21 suggesting that the increase in maternal serum-free ß-HCG found in the majority of these cases is due to changes in the post-transcriptional mechanism of HCG protein biosynthesis (Brizot et al., 1995b). Immaturity of the feto–placental function or a change in the balance of placental versus fetal protein secretion have been the two main hypotheses proposed to explain these endocrinological changes associated with trisomy 21 (Chard, 1991). In trisomy 18 pregnancies, maternal serum-intact and serum-free ß-HCG and fetal serum total HCG are markedly decreased (Abbas et al., 1995; Jauniaux et al., 1996) and serum-free -HCG subunit is raised only slightly (Brizot et al., 1995a). In these pregnancies, there is a decrease in placental expression of ß-HCG but -HCG mRNA is not different from normal. This probably results from an impairment in the transcription of the corresponding gene which affects the ß to a greater extent than the subunit and has been linked to a global trophoblastic dysfunction (Brizot et al., 1996). This study was undertaken to evaluate the effect of trisomy 21 and 18 on HCG endogenous concentrations and LH/HCG receptor expression in placental villous tissue.
Materials and methods
Tissue sampling
Samples of placental villous tissue were collected from pregnancies presenting at 12–16 weeks of gestation with fetal chromosomal abnormalities (trisomy 21, n = 8; trisomy 18, n = 6) and pregnancies with chromosomally normal fetuses (n = 42) undergoing termination for psychosocial reasons. In all cases, gestational age was calculated from the last menstrual period and confirmed by ultrasound measurements of crown–rump length (CRL). Written informed consent was obtained in each case and each woman had a hypan dilator (Dilapan®; Gynotech, Middlesex, NJ, USA) inserted into the cervix 12h pre-operatively for cervical preparation. The study was approved by the University College London Hospitals Committee on the Ethics of Human Research.
Pure samples of placental tissue were obtained during the surgical procedure from the definitive placenta by transcervical biopsy under abdominal ultrasound guidance. The samples were immediately fixed in Bouin's solution and subsequently embedded in paraffin and small pieces of villous tissue were snap-frozen in liquid nitrogen. These samples were kept frozen at –70°C until assayed.
HCG assay
Samples of villous tissue were homogenized individually in 3 volumes of Tris–HCl buffer (0.1 mol/l, pH 7.6). The homogenate was centrifuged at 10 000 g for 20 min at 4°C and the supernatant was used for the assay. All samples were assayed in duplicate for intact HCG, free -HCG and free ß-HCG subunits using solid-phase two-site immunoradiometric assay (IRMA) kits from BioMerieux (Mercy-l'Etoile, France). These assays, using monoclonal antibodies, were calibrated against the First International Reference Preparations 75/537 for HCG-dimer, 75/569 for free -HCG subunits and 75/551 for free ß-HCG subunits. Sensitivities were 1 mIU/ml for HCG and 0.03 mIU/ml for both free subunits. Intra- and inter-assay coefficients of variation were <7 and <10% respectively, for each assay.
Immunohistochemistry
This procedure was performed by an avidin–biotin immunoperoxidase method, as previously described (Reshef et al., 1990; Licht et al., 1993a). Briefly, the 4 µm thick sections cut from the placental tissue blocks were deparaffinized, rehydrated amd treated with 0.3% hydrogen peroxide (H2O2) in methanol to block endogenous peroxidase activity. After three washes with phosphate-buffered saline (PBS), non-specific binding sites were blocked and then incubated overnight at 4°C with a 1:500 dilution of a polyclonal LH/HCG receptor antibody raised against a synthetic N-terminus amino acid sequence of 15–38. The receptor antibody also recognizes the truncated receptor protein. The next day, the sections were washed, incubated with biotinylated secondary antibody and avidin–biotin–immunoperoxidase complex and then exposed to diaminobenzidine and H2O2. For the procedural controls, the LH/HCG receptor antibody was replaced with normal rabbit serum. The same slides were used for trisomy cases and controls to allow direct comparison.
In-situ hybridization
This procedure was also performed on 4 µm thick sections of placental tissue following a previously described method (Angerer et al., 1987). The sections were deparaffinized, rehydrated, treated for 30 min at 37°C with 1.2 µg/ml proteinase K and dehydrated. Then the sections were prehybridized at 45°C for 4 h and hybridized overnight at 58°C in double-strength SSC (single-strength SSC = 150 mmol/l sodium chloride and 15 mmol/l sodium citrate, pH 7.0) containing 50% formaldehyde, 5-strength Denhardt's solution, 50 mmol/l sodium phosphate, 100 µg/ml of denaturated salmon sperm DNA, tRNA and 2x107 cpm/ml of [35S]-labelled antisense or sense riboprobe transcribed from full length LH/HCG receptor cDNA (developed within the laboratory of Molecular Biology and Medicine). After hybridization, the sections were washed twice at 65°C for 30 min each time with double-strength SSC containing 0.1% sodium dodecyl sulphate (SDS), then washed once with single-strength SSC/0.1% SDS, and finally washed once with 0.1 strength SSC/0.1% SDS. The washed sections were dehydrated and coated with emulsion. After 1 week's exposure, slides were developed and mounted with polymount.
Quantification of immunostaining and in-situ hybridization signals
The intensity of the signals were visually scored by three observers while examining the slides under a light microscope. The scale of 1+ to 4+ was used. One + being the lightest above background and 4+ being the darkest. The immunostaining in both cell types was taken into account during scoring.
Statistical analysis
The data were analysed using a biomedical processing statistics package (Statgraphics, Manugistics, Rockville, USA). Standardized kurtosis was used to determine whether the samples derived from a normal distribution. Because some distributions were skewed, data are presented as medians and interquartile ranges. Each trisomy case was matched with one (immunostaining and in-situ hybridization) or three controls (HCG assays) of same gestational age obtained from the same population during the same period of time. Differences in median of placental intact HCG, free -HCG and free ß-HCG concentrations between the trisomy groups and the corresponding controls were tested by the Mann–Whitney W rank test at the 95% confidence level. Differences in the immunostaining and the in-situ hybridization score were evaluated using Kruskal–Wallis rank test. P < 0.05 was considered to be statistically significant.
Results
Concentration of HCG proteins in villous tissue
Table I shows the data for -HCG, ß-HCG and intact HCG in trisomy 21 and 18 and for the corresponding control groups. There was a significantly higher free ß-HCG median concentration in cases of trisomy 21 than in the controls (W = 48; P < 0.05). In trisomy 18 cases, the free ß-HCG median concentration was significantly lower than in the control group (W = 94; P < 0.01). There were no significant differences between the groups for intact HCG and free -HCG.
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Distribution of LH/HCG receptors
LH/HCG receptors were expressed in all samples of placental villi from both trisomy and control groups. They were localized mainly to the syncytiotrophoblast. In all cases, there was a weaker staining of the cytotrophoblast, in comparison with syncytiotrophoblast, whereas Hofbauer and endothelial cells in the villous stromal tissue contained few or no receptors.
Immunohistochemistry demonstrated a more intense staining of the trophoblast in all cases of trisomy 18, compared with both cases of trisomy 21 and controls (Figure 1). In all cases of trisomy 21, the trophoblast staining was also more intense than that in the controls. All trisomy 18 and 21 placentas showed a significant difference (t = 15.4; P < 0.001) from normal placentas, and the median (interquartile range) immunostaining scores were 2.3 (2.0–2.7) for controls, 3.1 (2.8–3.3) for trisomy 21 and 3.2 (2.9–3.3) for trisomy 18 (Figure 2).
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In-situ hybridization showed a similar pattern for LH/HCG receptor mRNA distribution as immunocytochemistry with the strongest signal in cases of trisomy 18 (Figure 3
). The in-situ hybridization scores were significantly higher (t = 18.1; P < 0.001) for trisomy 21 and 18 and the median (interquartile range) scores were 1.3 (1.0–1.5) for controls, 2.4 (1.9–2.5) for trisomy 21 and 3.0 (2.3–3.0) for trisomy 18 (Figure 2
).
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Discussion
Previous immunohistochemistry studies have shown that -HCG mRNA is localized to the differentiating cytotrophoblast and syncytial regions, while ß-HCG mRNA is localized primarily to the syncytiotrophoblast (Hoshina et al., 1985; Hay, 1988). The rate of differentiation of cytotrophoblast into syncytiotrophoblast appears to be the main factor that leads to HCG synthesis in the syncytiotrophoblast (Hay, 1988). Newly synthesized HCG is rapidly secreted by the syncytiotrophoblast and thus only a limited storage of HCG molecules were found in granules of the very early placenta but not later in pregnancy (Morrish et al., 1987). LH/HCG receptors are found in the villous trophoblast and also in the extravillous trophoblast within the decidua (Tao et al., 1995). Since the latter type of trophoblastic cell does not secrete HCG, the HCG that activates receptors comes from the villous trophoblast to act in a paracrine manner. Endogenous HCG working via these receptors regulates the differentiation of the cytotrophoblast into the syncytiotrophoblast and also HCG secretion (Shi et al., 1993). The results of the present study indicate that the changes in intact HCG and subunit concentrations in placental tissue from fetuses presenting with trisomy 21 and 18 are similar to those found in maternal blood and are associated with an increase in LH/HCG receptor expression. These findings suggest that trisomy 21 and 18 are associated with modification in both HCG synthesis and paracrine regulation.
LH/HCG receptors have been localized in gonadal and non-gonadal tissues and their expression has been recently described as a function of implantation in humans (Rao, 1996). In the uterus, they are expressed in endometrial stromal cells, endometrial glands, myometrial smooth muscle and T lymphocytes. There is evidence that chronic exposure of endometrial stomal cells to moderate to high LH or HCG concentrations results in a decrease in receptor number by increasing the degradation of mRNA transcripts rather than decreasing the transcription rate of the gene (Rao and Sanfilippo, 1997). The and ß subunits of HCG have no effect, suggesting that the complete native hormone is required. In villous tissue, it has been found that the use of a receptor specific antibody increases cytotrophoblast differentiation and HCG secretion (Shi et al., 1993). Furthermore, concentrations of HCG that regulate its own synthesis can decrease the trophoblastic transcription of HCG receptor gene (Licht et al., 1993a). A similar but inverted mechanism may explain the increased expression in LH/HCG receptor that we found in trisomy 18. This chromosomal abnormality is almost always associated with marked trophoblastic hypoplasia (Jauniaux and Hustin, 1998) together with a depression of the placental tissue expression of ß-HCG and synthesis of HCG (Brizot et al., 1996; Jauniaux et al., 1996). Our data suggest that in trisomy 18, the chronically lower concentrations of endogenous HCG promotes the over-expression of LH/HCG receptors in the syncytiotrophoblast and the poor differentiation of the cytotrophoblast.
In normal pregnancies, intact HCG and free ßHCG subunit concentrations rise rapidly in maternal serum peaking at 9–10 weeks gestation, followed by a fall during the second trimester. In contrast, the -subunit concentrations rise continuously during the first and second trimesters (Nagy et al., 1994). The HCG profile can be explained by a physiological lack of self-regulation of HCG biosynthesis in early pregnancy. While at term, the concentration of HCG self-regulates its synthesis via LH/HCG receptors. In early pregnancy, the receptors are truncated and probably non-functional until 9 weeks gestation (Rao, 1996). This lack of self-regulation may allow HCG to rapidly increase to peak concentrations. Once the peak concentrations are reached, which may vary with the individual, the self-regulatory mechanism causes a rapid fall in HCG concentrations. In contrast with trisomy 18, trisomy 21 of <17 weeks is not associated with specific morphological changes of the trophoblast (Jauniaux and Hustin, 1998) and the trophoblastic expression of and ß subunits of HCG is not different from that found in normal pregnancies of similar gestational age (Brizot et al., 1995b). Thus the increase in tissue HCG concentrations and LH/HCG receptor expression, in cases of trisomy 21 could simply reflect the relative immaturity of the trophoblastic tissue, as previously suggested (Chard, 1991).
At 17–23 weeks, trophoblast culture from trisomy 21 placentas show an increase in HCG secretion and subunit mRNA content (Eldar-Geva et al., 1995). This may be due to different regulatory mechanisms of HCG biosynthesis at this stage of gestation or to the loss of some regulatory mechanisms in vitro. Choriocarcinoma cells, which are mononuclear, are not induced to differentiate by either low or high concentrations of HCG (Licht et al., 1993b). As in early normal pregnancy, this lack of self-regulation of HCG biosynthesis is not due to the absence of receptors. In fact the receptors are present in abundance but they are truncated and primarily located in and around the nucleus of choriocarcinoma cells (Licht et al., 1993b). A similar mechanism may explain higher LH/HCG receptor concentrations and the high tissue and circulating HCG concentrations in the placenta of trisomy 21 pregnancies. The microheterogeneity of the HCG molecular structure has been highlighted in recent years by the finding of variations in its sialic acid content (Nagy et al., 1989). Although, no specific HCG molecular forms have been isolated in cases of trisomy 21, the absence or the excessive production of one or more forms not binding to the LH/HCG receptor could also explain the present findings.
Notes
4 To whom correspondence should be addressed at: Academic Department of Obstetrics and Gynaecology, University College London Medical School, 86–96 Chenies Mews, London WC1E 6HX, UK 
References
Abbas, A., Chard, T. and Nicolaides, K. (1995) Fetal and maternal hCG concentration in aneuploid pregnancies. Br. J. Obstet. Gynaecol., 102, 561–563.[ISI][Medline]
Angerer, L.M., Cox, K.H. and Angerer, R.C. (1987) Demonstration of tissue specific gene expression by in situ hybridization. Methods Enzymol., 152, 649–661.[ISI][Medline]
Brizot, M.L., Snijders, R.J.M., Butler, J. et al. (1995a) Maternal serum hCG and fetal nuchal translucency thickness for the prediction of fetal trisomies in the first trimester of pregnancy. Br. J. Obstet. Gynaecol., 102, 127–132.[ISI][Medline]
Brizot, M.L., Jauniaux, E., Mckie, A.T. et al. (1995b) Placental expression of and ß subunits of human chorionic gonadotrophin in early pregnancies with Down syndrome. Hum. Reprod., 10, 2506–2509.
Brizot, M.L., Jauniaux, E., Mckie, A.T. et al. (1996) Placental mRNA expression of and ß human chorionic gonadotrophin in early trisomy 18 pregnancies. Mol. Hum. Reprod., 2, 463–465.
Chard, T. (1991) Biochemistry and endocrinology of the Down's syndrome pregnancy. Ann. N.Y. Acad. Sci., 626, 580–596.[Medline]
Eldar-Geva, T., Hochberg, A., deGroot, N. and Weinstein, D. (1995) High maternal serum chorionic gonadotropin level in Downs' syndrome pregnancies is caused by elevation of both subunits messenger ribonucleic acid level in trophoblasts. J. Clin. Endocrinol. Metab., 80, 3528–3531.[Abstract]
Exterman, P., Bischof, P., Marguerat, P. and Mermillod, B. (1998) Second-trimester maternal serum screening for Down's syndrome: free ß-human chorionic gonadotrophin (HCG) and alphafetoprotein, with or without unconjugated oestriol, compared with total HCG, alphafetoprotein and unconjugated oestriol. Hum. Reprod., 13, 220–223.
Fiddes, J.C. and Goodman, H.M. (1979) Isolation, cloning and sequence analysis of the cDNA for the -subunit of the human chorionic gonadotropin. Nature, 281, 351–356.[Medline]
Hay, D.L. (1988) Placental histology and the production of human choriogonadotrophin and its subunits in pregnancy. Br. J. Obstet. Gynaecol., 95, 1268–1275.[ISI][Medline]
Hoshina, M., Boothby, M., Pattillo, R. et al. (1985) Linkage of human chorionic gonadotropin and placental lactogen biosynthesis to trophoblast differentiation and tumorigenesis. Placenta, 6, 163–172.[ISI][Medline]
Jameson, J.L. and Hollenberg, A.N. (1993) Regulation of chorionic gonadotropin gene expression. Endocr. Rev., 14, 203–221.[ISI][Medline]
Jauniaux, E. and Hustin, J. (1998) Chromosomally abnormal early ongoing pregnancies: Correlation of ultrasound and placental histological findings. Hum. Pathol., 29, 1195–1199.[ISI][Medline]
Jauniaux, E., Nicolaides, K.H., Nagy, A.-M. et al. (1996) Total amount of circulating human chorionic gonadotropin and ß subunits in first trimester trisomies 21 and 18. J. Endocrinol., 148, 27–31.[Abstract]
Lapthorn, A.J., Harris, D.C., Littlejohn, A. et al. (1994) Crystal structure of human chorionic gonadotropin. Nature, 369, 455–461.[Medline]
Licht, P., Cao, H., Lei, Z.M. et al. (1993a) Novel self-regulation of human chorionic gonadotropin biosynthesis in term pregnancy human placenta. Endocrinology, 133, 3014–3025.[Abstract]
Licht, P., Cao, H., Lei, Z.M. et al. (1993b) Lack of self-regulation of human chorionic gonadotropin biosynthesis in human choriocarcinoma cells. J. Clin. Endocrinol. Metab., 78, 1188–1194.[Abstract]
Licht, P., Losch, A., Dittrich, R. et al. (1998) Novel insights into human endometrial paracrinology and embryo–maternal communication by intrauterine microdialysis. Hum. Reprod. Update, 4, 532–538.
Morrish, D.W., Marusyk, H. and Siy, O. (1987) Demonstration of specific secretory granules for human chorionic gonadotropin in placenta. J. Histochem. Cytochem., 35, 93–101.[Abstract]
Nagy, A.M., Meuris, S. and Robyn, C. (1989) Inventory of the molecular heterogeneity of purified human chorionic gonadotrophin preparations as revealed by immunoelectrotransfer. Med. Sci. Res., 17, 771–773.
Nagy, A.-M., Glinoer, D., Delogne-Desnoeck, et al. (1994) Total amounts of circulating and ß subunits can be assessed throughout pregnancy using immunoradiometric assay calibrated with the unaltered and thermally dissociated heterodimer. J. Endocrinol., 140, 513–520.[Abstract]
Rao, Ch.V. (1996) The beginning of a new era in reproductive biology and medicine: Expression of low levels of functional luteinizing hormone/human chorionic gonadotropin receptors in nongonadal tissues. J. Physiol. Pharmacol., 47, 41–53.
Rao, Ch.V. and Sanfilippo, J.S. (1997) New understanding in the biochemistry of implantation: Potential direct roles of luteinizing hormone and human chorionic gonadotropin. Endocrinologist, 7, 107–111.
Reshef, E., Lei, Z.M., Rao, Ch.V. et al. (1990) The presence of gonadotropin receptors in non-pregnant human uterus, human placenta, fetal membranes and decidua. J. Clin. Endocrinol. Metab., 70, 421–429.[Abstract]
Shi, Q.J., Lei, Z.M., Rao, Ch.V. and Lin, J. (1993) Novel role of human chorionic gonadotropin in differentiation of human cytotrophoblast. Endocrinology, 132, 1387–1395.[Abstract]
Spencer, K. (1993) Free -subunit human chorionic gonadotropin in Down syndrome. Am. J. Obstet. Gynecol., 168, 132–135.[ISI][Medline]
Talmadge, K., Boorstein, W.R. and Fiddes, J.C. (1983) The human genome contains seven genes for the ß-subunit of chorionic gonadotropin but only one gene for the ß-subunit of luteinizing hormone. DNA, 2, 281–289.[ISI][Medline]
Tao, Y.X., Lei, Z.M., Hofmann, G.E. and Rao, Ch. V. (1995) Human intermediate trophoblasts express gonadotropin/luteinizing hormones receptor gene. Biol. Reprod., 53, 591–597.[Abstract]
Submitted on July 2, 1999; accepted on October 20, 1999.
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