Stimulation of Kaposi's sarcoma cell growth by urine from women in early pregnancy, the current source for clinical-grade human chorionic gonadotropin preparations.

Clinical-grade preparations of human chorionic gonadotropin (hCG) have been shown to be toxic to Kaposi's sarcoma (KS) cells. However, the results of clinical studies using commercial hCG preparations KS remain highly contradictory. More particularly, some hCG preparations could have a paradoxical growth effect on KS. Such discrepant results may be explained by the fact that the anti-KS activity is not associated with hCG itself but with one or more factors that are co-purified with the hormone. We found here that crude urine from first trimester pregnant women, the current source for commercial hCG, had a growth stimulatory effect on KS cells. By contrast, urine from last trimester pregnant women, from non-pregnant young women, from menopausal women and from men exhibited neither a growth stimulatory nor a growth inhibitory effect on KS cells. The amplitude of this pregnancy urine-associated pro-KS activity/hCG unit was higher than that achieved with clinical-grade hCG preparations. Partial co-purification of pregnancy-associated factors during the extraction procedure of commercial hCG from urine may explain the pro-KS activity achieved with some hCG preparations. We, therefore, suggest a cautious use of hCG purified from pregnancy urine for the treatment of KS.

Treatment of Kaposi's sarcoma with human chorionic gonadotropin.

Clinical-grade preparations of human chorionic gonadotropin (hCG) have been shown to be toxic to Kaposi's sarcoma (KS) cells. However, the mechanism of the anti-KS activity achieved with these preparations remains unclear. The results of clinical studies using commercial hCG preparations in human KS are also highly contradictory. The apparent controversy between different studies may be due to the fact that pro- and anti-KS components are present in varying proportions in different hCG preparations. As certain hCG preparations could not only lack the ability to control KS but also contain some contaminant KS growth factor(s), we suggest a cautious use of crude hCG for the treatment of KS.

HCG concentration and receptor gene expression in placental tissue from trisomy 18 and 21.

Trisomy 21 is associated with high maternal serum concentrations of intact human chorionic gonadotrophin alpha(HCG) and free beta-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 alpha-HCG and free beta-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 beta-HCG concentrations than the controls (P < 0.05). In trisomy 18 cases, the beta-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.

Epitope analysis and molecular modeling reveal the topography of the C-terminal peptide of the beta-subunit of human chorionic gonadotropin.

Human chorionic gonadotropin (hCG) belongs to a family of heterodimeric glycoprotein hormones that share a common alpha-subunit and a hormone-specific beta-subunit. Among the gonadotropin beta-subunits, greater than 85% homology exists between lutropin (hLH)beta and hCGbeta in their first 114 amino acid residues. However, unlike hLHbeta, hCGbeta contains a 31-amino acid hydrophilic stretch at its carboxyl end (CTPbeta: C-terminal peptide). Although the crystal structure of deglycosylated hCG has been solved, the topography of CTPbeta remains unknown. In this study, we have attempted to define the topology of CTPbeta using mAb probes. We investigated three epitopes on hCGalpha, which are hidden in the hCGalphabeta dimer. However, these epitopes are not hidden in hLH, which has a similar subunit interface to that of hCG, but lacks CTPbeta. This suggested that these epitopes are not masked at the subunit interface of hLH or hCG. Hence, we hypothesized that, in the case of hCG, these epitopes are masked by the CTPbeta. Consistent with this view, several treatments of hCG that removed CTPbeta unmasked these epitopes and enhanced their reactivity with the corresponding mAbs. In order to localise the position of CTPbeta on the alpha-subunit, we used an epitope-mapping strategy [N. Venkatesh & G. S. Murthy (1997) J. Immunol. Methods 202, 173-182] based on differential susceptibility of epitopes to covalent modifications. This enabled us to predict the possible topography of CTPbeta. Further, we were also able to build a model of CTPbeta, completely independently of the epitope-mapping studies, using a homology-based modeling approach [S. Krishnaswamy, I. Lakshminarayanan & S. Bhattacharya (1995) Protein Sci. 4 (Suppl. 2), 86-97]. Results obtained from these two different approaches (epitope analysis and homology modeling) agree with each other and indicate that portions of CTPbeta are in contact with hCGalpha in the native hCG dimer.

Apoptosis in human term placenta is not increased during labor but can be massively induced in vitro.

Apoptosis in human placental villi is reported to increase until close to delivery. However, the involvement of the apoptotic process in the initiation of labor, and more particularly in relation to the decrease in placental perfusion during uterine contractions, remains unknown. The purpose of the study was to examine the reactivity of the apoptotic machinery in term placentae obtained before or after the onset of labor and after in vitro incubations. The incidence of apoptotic nuclei (< 1%) as evidenced by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method, and the histological distribution of immunoreactive Bcl-2, Bax, and Bcl-x proteins, were similar in placentae collected after delivery and before the onset of labor and in placental explants maintained overnight at 4 degrees C in a minimal salt-Hepes medium. By contrast, 28% of nuclei contained fragmented DNA when placental explants were incubated overnight at 37 degrees C. This marked increase was associated with a decrease in the intensity of the Bcl-2 immunostaining and an increase in the intensity of Bax and Bcl-x immunostaining. In conclusion, the present study clearly evidences the presence of an active apoptotic machinery in term placental cells that is not involved in normal parturition.

The contribution of maternal serum markers in the early prenatal diagnosis of molar pregnancies.

The aim of this study was to evaluate the usefulness of maternal serum markers in the early prenatal diagnosis of molar pregnancies. The ultrasound features, cytogenetic and histopathological findings of 10 cases of molar pregnancy diagnosed at 11-13 weeks of gestation were compared retrospectively with the maternal serum concentrations of human chorionic gonadotrophin (HCG), alpha fetoprotein (AFP), pregnancy-associated plasma protein A (PAPP-A) and pregnancy-specific beta1-glycoprotein (SP1). Free beta-HCG and intact HCG concentrations were very high [> or = 2.5 multiples of the median (MoM)] in all cases. AFP concentrations were extremely low in all cases of singleton complete moles (< or = 0.5 MoM) and were high in one case of twin complete mole, in one case of triploid partial mole and two cases of euploid partial mole (> or = 2.5 MoM). Serum PAPP-A and SP1 were high in complete moles. The combined use of ultrasound features, maternal serum proteins and fetal cytogenetic findings should enable the early differential diagnosis in utero and perinatal management of those molar pregnancies presenting with an anatomically normal fetus.

The thyrotrophic role of human chorionic gonadotrophin (hCG) in the early stages of twin (versus single) pregnancies.

OBJECTIVE: Human chorionic gonadotrophin (hCG) is known to possess thyroid-stimulating activity. The aim of the present study was to assess the role of hCG in stimulating the maternal thyroid gland in the early stages of normal gestation. STUDY DESIGN: Thirty euthyroid healthy women were investigated prospectively. In each, conception had been assisted by in vitro fertilization techniques, which allowed for the precise determination of gestational age. Women were subdivided into single (n = 17) and twin (n = 13) pregnancies. Serum intact hCG and its free alpha and beta subunits, TSH and free T4 concentrations were measured at 6, 8, 9, 10, 11, 15, 19, 22 and 32 weeks. RESULTS: In twin pregnancies compared with single pregnancies, peak hCG concentrations (9-11 weeks) were significantly higher (mean +/- SE 171,000 +/- 12,500 vs 65,500 +/- 7600 U/l; P < 0.001), and also much more prolonged. Human CG concentrations above 75,000 U/l lasted for less than 1 week in single, compared with up to 6 weeks in twin pregnancies. Free beta-hCG subunit concentrations paralleled those of intact hCG in both groups. The ratios of free beta-hCG subunit/total hCG were similar in single and twin pregnancies, and did not vary with gestation time. Concerning thyroid function, twin pregnancy was more frequently associated with a lowering of TSH, which was also more profound than in single pregnancies. Furthermore, while free T4 levels remained normal in single pregnancies, they were transiently supranormal (up to 52 pmol/l) in four twin pregnancies. CONCLUSION: In twin pregnancies the placenta produces larger amounts of hCG for a prolonged period of time than in single pregnancies. Both the amplitude and duration of hCG production (i.e. the global exposure of the thyroid gland to hCG) are responsible for increased thyroidal stimulation, leading more frequently to increased free T4 and suppressed TSH levels. The results emphasize the role of hCG in stimulating maternal thyroid function in the first trimester of pregnancy. Even though the production of a variant hCG molecule with potent thyrotrophic activity cannot be excluded, this hypothesis is not required to explain the data. Clinicians should be aware of the frequent occurrence of significant but transient biochemical hyperthyroidism associated with hCG stimulation in the early stages of gestation, particularly in twin pregnancies.

Epitope mapping on intact, heated and reduced molecular variants of human chorionic gonadotrophin.

Monoclonal antibodies (MAbs) raised against purified human chorionic gonadotrophin (hCG) (n = 30) and synthetic peptides derived from hCG (n = 3) were able to recognize by Western blotting several hCG dimers (57-47 and 42 kDa), free beta-subunits (35-32, 26 and 16 kDa) and free alpha-subunit (21 kDa) which coexist in commercially available hCG preparations. According to differences in the immunoreactivity of hCG-related molecular forms observed under native or denaturing conditions such as boiling or reducing hCG samples before or after gel electrophoresis, nine classes of MAbs able to recognize different immunoreactive domains were determined. Three domains corresponded to continuous epitopes recognized by MAbs raised against hCG-related peptides. The six remaining domains, recognized by the other MAbs, contained discontinuous epitopes from which three were surface-oriented and three disguised in the holo-hormone. This solid-phase approach, combining native and denaturing conditions, represents a simple and powerful tool to screen the specificity of MAbs from varying sources and to investigate molecular variants of proteic hormone.

Comparative evaluation of a high lipase pancreatic enzyme preparation and a standard pancreatic supplement for treating exocrine pancreatic insufficiency in chronic pancreatitis.

OBJECTIVE: To compare the efficacy and safety of two enzyme-containing preparations, Pancrease HL (Cilag) containing 25,000 units of lipase per capsule and Creon (Triosol) with 8000 units of lipase per capsule, in patients with chronic pancreatitis and exocrine insufficiency. DESIGN: The study is a monocentric open crossover prospective study including 25 patients entered from March 1993 to May 1994. PATIENTS: Chronic pancreatitis was alcohol-related in 23 patients, previous surgery was performed in 9, 16 had diabetes and all had steatorrhoea (fat balance > 10g/24h). METHODS: Patients were investigated during four periods of 2 weeks, each one corresponding to a new treatment regimen: Pancrease HL, 3 capsules/day or Creon, 9 capsules/day, with or without omeprazole 20 mg/day. Stools were collected on the last 3 days at the end of each period when the patients were on a standard diet with a fixed daily intake of 100 g fat/day. RESULTS: Faecal fat, protein and energy excretion did not differ when both preparations were compared at roughly pharmaceutically equivalent doses. No significant improvement in fat and protein absorption was observed when omeprazole was taken with the pancreatic enzymes. However, omeprazole treatment was associated with a marked decrease in the fat-protein content ratio, suggesting an improvement in the fat digestive process but a decrease in the efficiency of protein digestion. Drug safety was comparable in the four groups of treatment. CONCLUSION: Pancrease HL with high lipase activity provides effective pancreatic enzyme replacement therapy in patients with chronic pancreatitis at an appreciably lower number of capsules per day than with standard preparations.

Total amount of circulating human chorionic gonadotrophin alpha and beta subunits in first trimester trisomies 21 and 18.

The aim of this study was to evaluate the variations in the balance between total (free and combined) circulating alpha and beta subunits of human chorionic gonadotrophin (hCG) in trisomy 21 and 18. Maternal serum samples were collected at 10 and 11 weeks of gestation from 22 singleton pregnancies with trisomy 21 (n = 17) and trisomy 18 (n = 5) and 66 chromosomally normal controls, matched for gestational age. The hCG and free alpha and beta subunits circulating levels were measured using specific immunoradiometric assays and converted in a common unit system obtained using calibration of the assays with intact and thermally dissociated purified hCG preparation. In trisomy 21, the only significant difference from controls was in the free beta hCG level which was increased. In trisomy 18, intact hCG, free beta hCG as well as total alpha hCG and total beta hCG levels were significantly lower whereas the free alpha hCG level was significantly higher than in controls. The decrease in total beta hCG was more pronounced than the decrease in total alpha hCG resulting in a significant increase in the total alpha- to beta hCG subunit ratio in trisomy 18. These findings suggest some modifications in the biosynthesis and/or release rates of the hCG subunits in these trisomies.

Temporal relationship between the human chorionic gonadotrophin peak and the establishment of intervillous blood flow in early pregnancy.

The purpose of this study was to investigate the temporal relationship between the early pregnancy peak of circulating human chorionic gonadotrophin (HCG) concentration and the establishment of maternal blood flow in the placental intervillous space. The presence of blood flow echoes within intervillous space was determined by colour Doppler imaging from 44 women with clinically uncomplicated pregnancy between 6 and 18 weeks gestation. Circulating HCG, free alpha- and beta HCG subunits, oestradiol and progesterone concentrations were immunoassayed in blood samples collected at the time of Doppler examination. A continuous intervillous blood flow was detected in all cases with a gestational age > or = 11.7 weeks (n = 18) but never before this time. Circulating concentrations of free alpha HCG, oestradiol and progesterone were linearly or exponentially correlated with gestational age (r = 0.860, 0.903 and 0.538 respectively, all with P < 0.001), indicating steady increase of these hormones with advancing gestation. However, the best fitted lines were found to be parabolic for HCG (r = 0.771, P < 0.001) and beta HCG (r = 0.695, P < 0.001), their highest points corresponding to 11.24 and 10.74 weeks gestational age respectively. The close temporal relationship between the Doppler advent of intervillous maternal blood flow and the HCG peak suggests that the establishment of the intervillous blood flow is associated with the decline in circulating HCG concentrations.

The role of relaxin in the development of the uteroplacental circulation in early pregnancy.

OBJECTIVE: To evaluate the relation between the development of the uteroplacental circulation as assessed by Doppler velocimetry and the maternal blood relaxin concentration. METHODS: Transvaginal color Doppler investigation of the uteroplacental circulation was performed in 42 healthy women at 6-15 weeks' gestation before termination of pregnancy for psychosocial reasons. The resistance index (RI), pulsatility index (PI), and maximum peak velocity were recorded at the level of the main uterine artery, and the presence of intervillous flow was noted. Relaxin, hCG, 17 beta-estradiol (E2), and progesterone levels were measured in maternal venous blood. RESULTS: Limited intervillous flow was noted from 10 weeks' gestation and continuous intervillous flow from 12 weeks. An inverse relation was observed between the circulating levels of both E2 and progesterone and uterine artery RI and PI, whereas the relaxin level correlated positively with uterine RI and PI. Estradiol and progesterone levels also correlated positively with uterine peak systolic velocity and intervillous blood flow. Multiple linear regression analysis indicated that both hormones contributed to the decrease in downstream resistance to uterine blood flow with advancing gestational age, as assessed by uterine RI. In addition, relaxin contributed to the uterine RI and PI and to the intervillous blood flow. CONCLUSION: These data suggest that relaxin, E2, and progesterone may influence the changes in uterine blood flow that occur in early pregnancy. The role played by E2 and progesterone in the development of the uteroplacental circulation may be modulated by relaxin, constituting a novel function for this ovarian peptide.

Placental production of human chorionic gonadotrophin alpha and beta subunits in early pregnancy as evidenced in fluid from the exocoelomic cavity.

Levels of human chorionic gonadotrophin (hCG) and of its free alpha and beta subunits were measured using specific immunoradiometric assays in exocoelomic fluid (ECF) and maternal serum (MS) collected from five pregnant women at 6.6-8 weeks of gestation. Mean levels of hCG and its free subunits were significantly (P < 0.001) higher in ECF than in MS: 3.5-fold for hCG, 600-fold for free alpha hCG and 38-fold for beta hCG. There was no correlation between either hCG levels or levels of its free subunits in ECF and MS. On a molar basis, the quantity of free alpha hCG subunit expressed as a percentage of the total (free+combined) amount was 83% in ECF and 2.7% in MS (P < 0.001). The amount of free beta hCG subunit as a percentage of the total was 22% in ECF and 3.5% in MS (P < 0.001). The ratio of the total amounts of alpha- and beta hCG subunits amounted to 4.6 in ECF and 0.99 in MS (P < 0.001). The heterogeneity of hCG was further investigated by polyacrylamide gel electrophoresis followed by immunoblotting. Several bands with molecular mass ranging from 42 to 57 kDa, corresponding to hCG dimers, were immunodetected in ECF and MS with anti-alpha hCG and anti-beta hCG monoclonal antibodies. A free 35 kDa beta hCG immunoreactive band was found in ECF and MS. A free alpha hCG immunoreactive band was observed at 23 kDa in ECF and at 21 kDa in MS. These findings suggest that the exocoelomic cavity is a reservoir where hCG and its subunits produced by trophoblast accumulate directly.(ABSTRACT TRUNCATED AT 250 WORDS)

Total amounts of circulating human chorionic gonadotrophin alpha and beta subunits can be assessed throughout human pregnancy using immunoradiometric assays calibrated with the unaltered and thermally dissociated heterodimer.

The aim of the present study was to determine the variations in the balance between total (free plus combined) circulating alpha and beta subunits of human chorionic gonadotrophin (hCG) throughout human pregnancy. The equivalence between the International Units (IU) of hCG (IRP 75/537) and those assigned to the alpha (IRP 75/569) and beta (IRP 75/551) free subunits was experimentally determined by using intact and thermally dissociated hCG. Heat exposure (2 min at 100 degrees C) of hCG preparations resulted in a complete dissociation of hCG into free, soluble and intact alpha and beta subunits. The hCG and alpha and beta subunit contents of unaltered and heated hCG preparations were assessed by specific immunoradiometric assays. The amount of immunoreactive subunits dissociated by heat from hCG could then be evaluated on a molar basis. Circulating hCG and its free alpha and beta subunits were immunoassayed in 836 blood samples collected from healthy pregnant women at different gestational ages. After conversion of hCG and its subunits into a common IU system, the gestational profiles of the total amounts (free plus combined) of alpha- and beta hCG subunits increased together and peaked at 9-10 weeks of gestation. Thereafter, total alpha and beta subunits decreased and subsequently remained stable until term. The decline in total alpha hCG subunit was less marked than that of total beta hCG subunit. The alpha- to beta hCG ratio was equimolar until 10 weeks of gestation when it increased almost fourfold until term (P < 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)

Serum levels of intact human chorionic gonadotropin (HCG) and its free alpha and beta subunits, in relation to maternal thyroid stimulation during normal pregnancy.

The main objective of the present study was to present additional evidence of the potentially important thyrotropic role of hCG to regulate the maternal thyroid gland during normal pregnancy. Sequential determinations (first and last trimesters) of intact hCG, free alpha and beta-hCG subunits concentrations (using monoclonal IRMAs), and assessment of parameters of thyroid function and thyroid volume were carried out in 62 pregnant women who exhibited during the first trimester of gestation low TSH levels (< or = 0.20 mU/L), and compared to 276 pregnant women with normal TSH levels. The prevalence of having low serum TSH represented 18% of all pregnancies, with almost one half of cases who transiently had undetectable TSH levels. Lowering of TSH was associated with high hCG levels, and occurred primarily during the first trimester. About 10% of women with low TSH presented transient gestational thyrotoxicosis, frequently associated with vomiting. In comparison to control subjects, women with a suppressed serum TSH had significantly and markedly higher intact hCG and free beta-hCG subunit concentrations. The results suggest that TSH reduction may result from a relative oversecretion of both intact hCG and free beta-hCG subunits, compatible with three hypotheses: a) transient overexpression of the beta-hCG gene, leading to enhanced production of hCG heterodimer; b) increased glycosylation of circulating hCG, with in turn a prolonged half life; c) larger syncytiotrophoblast mass with increased hCG production.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein and steroid levels in embryonic cavities in early human pregnancy.

Biochemical analysis including concentrations of urea, creatinine, human chorionic gonadotrophin (HCG), oestradiol, progesterone, and alpha-fetoprotein (AFP), two-dimensional gel electrophoresis, and the affinity of AFP for Concanavalin A (Con A)-Sepharose were performed on samples of exocoelomic and amniotic fluids retrieved by transvaginal puncture and maternal serum from 25 normal pregnancies between 5 and 12 weeks of gestation. Biochemical assays showed that during this period of gestation no differences in urea concentration were found between fluids from the three compartments, whereas creatinine concentration decreased significantly (P < 0.001) from maternal serum to amniotic fluid. The exocoelomic fluid contained significantly (P < 0.001) higher concentrations of oestradiol, progesterone and HCG than both maternal serum and amniotic fluid. AFP concentration was similar in amniotic and exocoelomic fluids and significantly (P < 0.001) lower in maternal serum. Between the second and the third months of gestation, urea concentration decreased significantly (P < 0.05) and oestradiol, HCG and AFP increased significantly in maternal serum (P < 0.05, P < 0.05, P < 0.001, respectively). During the same period of gestation, exocoelomic fluid concentrations of urea and HCG decreased significantly (P < 0.005, P < 0.001, respectively). Comparison of the two-dimensional gel patterns obtained from maternal serum with those from exocoelomic amniotic fluids revealed no significant qualitative differences, except for several small proteins. These results suggest that protein pathways across materno-embryonic membranes are not simply passive transfers.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of human chorionic gonadotrophin, oestradiol and progesterone on uteroplacental and corpus luteum blood flow in normal early pregnancy.

A transvaginal colour and pulsed Doppler study was performed on 44 women with normal pregnancies between 5 and 16 weeks of gestation. Maternal levels of human chorionic gonadotrophin (HCG), free alpha-HCG subunit, free beta-HCG subunit, 17 beta-oestradiol and progesterone were determined in sera obtained at the time of Doppler examination. Uterine peak systolic velocity (PSV) and alpha-HCG and 17 beta-oestradiol levels increased significantly (P < 0.001) from the second to the fourth month of gestation, whereas uterine and spiral resistance index (RI) decreased significantly (P < 0.005 and P < 0.001, respectively) with gestational age. Levels of HCG and beta-HCG peaked significantly (P < 0.01) during the third month of gestation. Corpus luteum PSV and RI and progesterone levels did not vary significantly with gestational age. Multiple regression analysis showed that gestational age was the only significant (P < 0.05) contributor to uterine PSV and spiral RI variability. In addition to gestational age, 17 beta-oestradiol had a significant (P < 0.001) influence on uterine RI. Both corpus luteum PSV and RI were significantly (P < 0.01 and P < 0.05, respectively) related to progesterone levels. Corpus luteum PSV was also significantly (P < 0.05) related to 17 beta-oestradiol levels and RI to HCG levels.(ABSTRACT TRUNCATED AT 250 WORDS)