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ORIGINAL RESEARCH |
From the Facultad de Medicina, Escuela de Nutrición y Dietética, Universidad Central de Venezuela, Caracas; Departamento de Biología de Organismos, Universidad Simón Bolívar, Sartenejas, Baruta; Laboratorio de Bioenergética Celular, Instituto Venezolano de Investigaciones Científicas, Altos de Pipe; and Fundamatin, Laboratorio de Endocrinología, Maternidad Concepción Palacios, Caracas, Venezuela.
Address reprint requests to: Ysabel C. Casart, MD, Departamento de Ciencias Fisiológicas, Escuela de Nutrición y Dietética, Facultad de Medicina, Universidad Central de Venezuela, Ciudad Universitaria, Caracas, Venezuela; E-mail: casarty{at}hotmail.com.
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ABSTRACT |
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METHODS: Peripheral blood samples from women in the third trimester with preeclampsia (n = 30) or normal pregnancies (n = 30) were assayed for immunoactive and bioactive hCG (mouse Leydig cell testosterone production assay).
RESULTS: Serum bioactive hCG levels tended to be lower than normal, and immunoactive hCG levels tended to be higher in women with preeclampsia, but the differences were not statistically significant. However, the ratio of bioactive to immunoactive hCG was significantly lower than normal for preeclamptic women (0.71 ± 0.28 vs. 1.15 ± 0.35 for normotensive pregnant women [mean ± standard deviation], P < .001).
CONCLUSION: The ratio of bioactive to immunoreactive serum hCG is lower among preeclamptic than among normotensive pregnant women.
Preeclampsia is a relatively common syndrome, dangerous for mother and infant, unpredictable in its onset and progression, and untreatable except through termination of the pregnancy.1 Preeclampsia affects up to 7% of pregnant women and is considered a leading cause of fetal growth restriction and infant morbidity and mortality. Yet its cause remains poorly understood.
Several studies have reported an association between unexplained increases in maternal second-trimester serum hCG levels, determined by immunometric methods, and subsequent development of preeclampsia.2 However, hCG levels measured by immunometric methods are not always similar to those determined by the bioactivity technique,3,4 apparently because immunologic techniques do not evaluate factors, such as spatial conformation of the glycoprotein molecule, levels of glycosylation, and binding to receptors. In this work, we measured the levels of immunoactive hCG and bioactive hCG in serum from normotensive or preeclamptic women in the third trimester of pregnancy.
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MATERIALS AND METHODS |
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Only women with severe preeclampsia5 were considered for this study. Gestational age was calculated from the 1st day of the last menstrual period, unless ultrasonography before 16 weeks demonstrated a discrepancy of at least 10 days, in which case ultrasonographic dating of the pregnancy was used for calculations.6 Serum samples were collected (one sample per subject) at time of diagnosis of preeclampsia and during a routine examination of normotensive women. The study was approved by the Institutional Review Board of the Maternity Hospital "Concepción Palacios" and by the Bioethics Committee of the Instituto Venezolano de Investigaciones Científicas, and all women gave written informed consent.
Immunoreactivity of total hCG (nicked + non-nicked + free ß) was measured by enzyme immunoassays (Chiron Diagnostic, Emeryville, CA, USA) at a sensitivity of 2.0 mIU/mL and with a between-assays variation coefficient of 3.8%. Bioactive hCG levels were measured in triplicate by a mouse Leydig cell bioassay, using WHO LH 80/552 as standard.7 The samples were diluted in Dulbecco’s minimal essential medium—20 mM HEPES (N-[2-hydroxyethyl]piperazine-N'-2-ethanesulfonic acid) without bicarbonate or glutamine—supplemented with 1% bovine serum albumin (Sigma, St. Louis, MO; fraction V) and 4% calf serum. Leydig cells were obtained by mechanical dispersion of testes from mice 6–8 weeks old. After 4 hours of incubation of the cells with serum, the testosterone concentration in the medium was measured using a radioimmunoassay (DPC, Sunnyvale, CA). In all cases, the samples were diluted sufficiently, to keep data within the reference curve. The results are expressed as mean ± standard deviation (SD). The Mann-Whitney U test was used for statistical evaluation. P < .05 was considered significant.
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RESULTS |
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lists the average concentrations of hCG determined by immunoassay and by bioassay for the two groups. The immunoactive hCG concentration tended to be higher and the bioactive hCG concentration to be lower in women with preeclampsia, but in both cases the difference was not statistically different from those of the normotensive pregnant women. The bioactive to immunoactive hCG cutoff giving maximum sensitivity (80%) and specificity (70.1%) was 0.9. Figure 1 shows the ratios of bioactive to immunoactive hCG for each of the subjects of the two groups. Whereas most of the women with normal pregnancies showed ratios greater than 0.9, most of the preeclamptic women showed ratios less than 0.9 (mean of all of the values, normotensive and preeclamptic women). Consequently, the ratio of bioactive to immunoactive hCG was significantly lower than normal for preeclamptic women (0.71 ± 0.28 vs 1.15 ± 0.35 for normotensive women [mean ± SD], P < .001).
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DISCUSSION |
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Preeclampsia is associated with poor placentation and an incomplete physiologic adaptation of the spiral arteries, which prevents them from dilating to accommodate the increased uteroplacental blood flow of late gestation.9 Placental tissue produces cytokines, hormones, and growth factors, essential for regulating the fetomaternal unit. The production of these substances is regulated by a network of intrauterine interactions, through paracrine and/or autocrine mechanisms. Imbalances between local and external signals may produce or reflect a variety of gestational disorders.10
Decreased bioactivity of hCG can be the consequence of alterations in its structure. Human chorionic gonadotropin is a glycoproteic molecule, and any alteration in its proteic and/or glycolytic fractions may modify its biologic action without affecting its capacity to bind to its receptors.11 Studies performed in vitro have shown that once released, the hCG molecule might be degraded quickly through the activity of enzymes, released by macrophages related to the trophoblastic tissue. Fragmentation of the circulating hCG molecule can explain a diminished bioactivity.12 In this regard, it has been shown that removal of sialic acid from hCG molecules through treatment with neuroamidases decreases their bioactivity.13 The totally deglycosylated hCG does not lose its capacity to bind to receptors, but its capacity to stimulate production of cyclic adenosine monophosphate is strongly diminished.
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Footnotes |
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Received December 15, 2000. Received in revised form March 29, 2001. Accepted April 5, 2001.
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REFERENCES |
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2. Hsu CD, Chan DW, Iriye B, Johnson TRB, Hong S-F, Repke JT. Elevated serum human chorionic gonadotropin as evidence of secretory response in severe preeclampsia. Am J Obstet Gynecol 1994;170:1135–8.[Medline]
3. Dafau ML, Veldhuis J. Pathophysiological relationships between the biological and immunological activities of luteinizing hormone. Ballieres Clin Endocrinol Metab 1987;1:153–76.[Medline]
4. Jaakkola T, Ding Y-Q, Kellokumpu-Lehtinen P, Valavaara R, Martikainn H, Tapanainen J, et al. The ratios of serum bioactive/immunoactive luteinizing hormone and follicle stimulating hormone in various clinical conditions with increased and decreased gonadotropin secretion: Reevaluation by a highly sensitive immunometric assay. J Clin Endocrinol Metab 1990;70:1496–505.[Abstract]
5. Casart Y, Proverbio T, Marín R, Proverbio F. Comparative study of the calcium adenosine triphosphatase of basal membranes of human placental trophoblasts from normotensive and preeclamptic pregnant women. Gynecol Obstet Invest 2001;51:28–31.[Medline]
6. Ashour AMN, Lieberman ES, Wilkins LE, Repke JT. The value of elevated second-trimester ß-human chorionic gonadotropin in predicting the development of preeclampsia. Am J Obstet Gynecol 1997;176:438–42.[Medline]
7. Robertson WR, Binden SP. The in vitro bioassay of peptide hormones. In: Hutton JC, Siddle K, eds. Peptide hormones: A practical approach. New York: IRL Press, 1990:120–2.
8. Diaz-Cueto L, Mendez JP, Barrios de Tomasi J, Lee JY, Wide L, Veldhuis JD, et al. Amplitude regulation of episodic release, in vitro biological to immunological ratio, and median charge of human chorionic gonadotropin in pregnancy. J Endocrinol Metab 1994;78:890–7.[Abstract]
9. Muttukrishna S, Knight PG, Groome N, Redman CWG, Ledger WL. Activin A and inhibin A as possible endocrine markers for pre-eclampsia. Lancet 1997;349:1285–8.[Medline]
10. Petraglia F, Santuz M, Florio P, Simoncini T, Luisi S, Plaino L, et al. Paracrine regulation of human placenta: Control of hormonogenesis. J Reprod Immunol 1998;39: 221–33.[Medline]
11. Cole L, Kardana A. Discordant results in human chorionic gonadotropin assays. Clin Chem 1992;38:263–70.
12. Cole LA. Immunoassay of human chorionic gonadotropin, its free subunits, and metabolites. Clin Chem 1997;43: 2233–43.
13. Dunkel L, Jia X-CH, Nishimori K, Boime I, Hsueh AJW. Deglycosylated human chorionic gonadotropin (hCG) antagonizes hCG stimulation of 3',5'-cyclic adenosine monophosphate accumulation through a noncompetitive interaction with recombinant human luteinizing hormone receptors. Endocrinology 1993;132:763–9.[Abstract]
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