Circulating levels of pregnancy proteins in early and late pregnancy in relation to placental tissue concentration.

The concentrations of human chorionic gonadotrophin (hCG), human placental lactogen (hPL), pregnancy specific beta 1 glycoprotein (SP1), ferritin (PP2) and placental protein 5 (PP5) were examined in maternal serum and placental tissue in early and late pregnancy. The circulating concentration of hPL, SP1, and PP5 were higher during late pregnancy than early pregnancy, that of hCG lower, and ferritin (PP2) levels showed no difference. Placental tissue levels of hPL and SP1 were higher in late pregnancy, hCG levels lower, and ferritin (PP2) and PP5 showed no change. The ratio of the concentration in maternal serum to that in placental tissue increased during pregnancy for all proteins with the exception of ferritin. It is proposed that the mechanism of secretion of trophoblast specific proteins varies widely and that this should be taken into account in the clinical interpretation of circulating levels in the mother.     

Stimulatory effects of extracellular calcium on chorionic gonadotrophin and placental lactogen release by human placental explants

The effects of sudden modifications of extracellular calcium ion concentration on human chorionic gonadotrophin (hCG) and human placental lactogen (hPL) release were investigated using term placental explants incubated in Krebs Ringer solution. The hCG and hPL releases were both stimulated when the extracellular Ca2+ concentration was increased. The hCG and hPL secretions elicited by the addition of extracellular Ca2+ were larger when placental explants were preincubated in alpha-calcium (no added calcium + EGTA) than in normo-calcium (1.5 mM). Removal of extracellular Ca2+ from the medium also elicited an increase in hCG and hPL release. However, this stimulatory effect of Ca2+ omission was partly suppressed by washing the explants prior to incubation in the alpha-calcium medium, and was completely abolished when alpha-calcium medium was supplemented with 1 mM cobalt. Our results indicate that changes in Ca2+ modify hCG and hPL release from term placental explants in a manner concordant with the 'stimulus-secretion coupling' concept.     

The concentrations of SP1 beta and SP1 alpha in retroplacental and peripheral blood at term

The concentrations of the two components of Schwangerschaftsprotein 1 (SP1 beta and SP1 alpha), human chorionic gonadotrophin (hCG) and human placental lactogen (hPL), were measured in peripheral venous blood and in retroplacental blood at term delivery in 22 women. Like hCG and hPL, the values of SP1 alpha were higher in the retroplacental than in the peripheral blood, as might be expected with placental secretory products. On the other hand, the concentration of SP1 beta showed a reverse gradient, being higher in the peripheral than in retroplacental blood.     

Effect of leptin on the regulation of placental hormone secretion in cultured human placental cells.

Placenta is an important source of leptin during pregnancy that contributes to the high plasma leptin levels in pregnant women. Leptin and its functional receptors are synthesized in trophoblast cells that, in turn, secrete gestational hormones supporting a paracrine or autocrine role for leptin in the endocrine activity of the placenta. In the present study we examined the effect of leptin on in vitro release of gestational hormones (human chorionic gonadotropin (hCG), human placental lactogen (hPL), progesterone, estrogens and testosterone) by human term placental cells in culture. Placentas at term were obtained immediately after delivery from mothers with uncomplicated pregnancies. Progesterone, hCG, hPL, estradiol, estrone, estriol and testosterone levels were measured by different assays in culture media of cells maintained in monolayer culture after incubation for 12, 24, 48 or 72 h with leptin or placebo. Incubation with leptin did not modify hCG, hPL, progesterone, estriol and estrone secretion for any of the doses and times assayed. However, leptin led to a dose-dependent decrease in estradiol release. This effect was observed when treatment with recombinant human leptin spanned from 12 to 72 h. At this time an increase in testosterone levels was observed in leptin-treated cells versus placebo. These results indicate that leptin can be considered a gestational hormone implied in the endocrine function of the placenta, with an important role in control of the production of steroid reproductive hormones in placental cells in vitro.     

恶性滋养细胞肿瘤细胞中胎盘激素的测定

应用免疫组化方法，采用抗绒毛膜促性腺激素（ｈＣＧ）、胎盘泌乳素（ｈＰＬ）、妊娠特异性ｐ１糖蛋白（ＳＰ１）抗体，检测９１份恶性滋养细胞肿瘤瘤细胞中胎盘激素的含量，其中侵蚀性葡萄胎２９份、转移性侵蚀性葡萄胎１２份，绒毛膜癌２９份、转移性绒毛膜癌２１份。结果：侵蚀性葡萄胎肿瘤细胞中ｈＣＧ的含量较绒毛膜癌明显减少（Ｐ＜０．００５），ｈＰＬ和ＳＰ，的含量较绒毛膜癌明显增多（Ｐ＜０．００５）；转移性侵蚀性葡萄胎肿瘤细胞中ｈＰＬ、ＳＰ＿１的含量较原发瘤为低（Ｐ＜０．０２５），转移性绒毛膜癌的ｈＣＧ及ｈＰＬ含量较原发瘤增多（Ｐ＜０．１，Ｐ＜０．０５）。提示：ｈＣＧ、ｈＰＬ和ＳＰ＿１在恶性滋养细胞肿瘤瘤细胞中含量的测定，对该瘤的诊断和分型有参考价值。     

In vitro effect of dopamine and pimozide on human chorionic gonadotropin secretion.

In human placental explants cultured in vitro, dopamine inhibited human chorionic gonadotropin (hCG) secretion into the culture media. In the control flasks, the level of hCG secretion was 751 +/- 215 mIU/gm of tissue. When 1 mM of dopamine was added, hCG levels decreased to 321 +/- 57.6 mIU/gm of tissue (n = 6, P less than 0.1)--5 and 10 mM of dopamine significantly inhibited hCG secretion. In contrast, 1 mM of pimozide enhanced hCG secretion by 1.8-fold compared to control levels (1,707 +/- 343 versus 3,117 +/- 0.005). This in vitro effect on hCG is similar to the effect of dopamine and pimozide on hCS secretion by placental explants.     

The relation between the concentration of placental specific proteins in retroplacental and peripheral blood.

Concentrations of four placental proteins: human placental lactogen (hPL), placental protein 5 (PP5), pregnancy specific beta 1 glycoprotein (SP1) and human chorionic gonadotrophin (hCG), and a normal serum component, alpha 2 macroglobulin, were measured in the peripheral circulation and in blood obtained from the retroplacental space in 20 women at term delivery. Levels of hPL and PP5 were higher in the retroplacental blood than in the peripheral circulation in all patients. By contrast, levels of SP1 and hCG were consistently lower in retroplacental blood than in the peripheral circulation. Similarly, levels of alpha 2 macroglobulin were lower in the retroplacental blood. It is suggested that this 'reverse' gradient is a technical arterfact. These findings are discussed in relation to synthesis of placental proteins in a site distal to the retroplacental space, and the introduction of a technical artefact in the collection of samples.     

Interrelationships of human chorionic gonadotropin, human placental lactogen, and pregnancy-specific beta 1-glycoprotein throughout normal human gestation

Human chorionic gonadotropin (hCG), human placental lactogen (hPL), and pregnancy-specific beta 1-glycoprotein (PSBG) were measured by radioimmunoassay in 270 samples of serum from women with uncomplicated pregnancies. All three proteins were significantly correlated with each other in individual samples of serum and with the estimated trophoblastic mass during the first trimester. No significant correlation could be demonstrated between the concentrations of hCG and PSBG in maternal serum during the second or third trimesters or between the concentrations of hCG and hPL during the second trimester. Levels of PSBG and hPL in serum were significantly correlated throughout all three trimesters. These findings suggest that the secretion of hCG, hPL, and PSBG may be regulated by similar control mechanisms during the first trimester of pregnancy. However, after this period, the factors that modulate the production of hCG differ from those that regulate the production of hPL and PSBG.     

Effect of estradiol and progesterone on human chorionic gonadotropin secretion in vitro

Many of the substances known to control the secretion of pituitary gonadotropins also modulate the secretion of human chorionic gonadotropin (hCG) by the placenta. In order to study the effect of estrogens and progestins on hCG secretion, term placental explants were cultured in culture media for 144 hours. During the culture period, hCG secretion increased after 48 hours, and a fortyfold increase was observed after 144 hours (p less than 0.001). Compared to concentrations of hCG in control cultures, secretion of hCG was markedly suppressed in the presence of progesterone 2.25 X 10(-5)M (p less than 0.001), a concentration similar to that found in term placental tissue (1.7 +/- 0.2 micrograms/gm of tissue). Suppression of hCG by progesterone occurred in a dose-response manner (r = -0.9100, p less than 0.01). Estradiol, an important steroid modulator of pituitary gonadotropins, did not significantly suppress the secretion of hCG, except in pharmacologic concentrations (10(-4)M), and physiologic concentrations of estradiol had no effect on the suppression of hCG by progesterone. These results suggest that the mechanism by which progesterone suppresses the secretion of hCG differs from the manner in which steroids modulate the secretion of pituitary gonadotropins.     

Glycodelin A and differentiation of first trimester trophoblast cells in vitro

Aim The glycoprotein, glycodelin A (GdA) is a main product of the maternal decidua in the first trimester of pregnancy and is secreted into the amniotic fluid. The purpose of this study was to investigate the effect of GdA on secretion and surface markers of isolated first trimester trophoblasts in vitro.Methods Cytotrophoblasts were prepared from human first trimester placentae and incubated with varying concentrations of GdA or transfected separately with the expression plasmid of GdA. Supernatants were assayed for human chorionic gonadotropin (hCG) protein concentrations. Expression of human placental lactogen (hPL), mucin 1 (MUC1) and the Thomsen–Friedenreich (TF) epitope was analysed in stimulated trophoblast cells and in unstimulated controls by immunocytochemistry.Results Glycodelin A induced a reduced expression of hPL compared with unstimulated controls. Expression of MUC1 was not affected by GdA. Freshly isolated trophoblast cells showed no TF expression but became positive for this antigen after 96 h of cultivation. GdA-stimulated trophoblast cells inhibited TF expression after 96 h of cultivation. GdA plasmids induced a significantly higher hCG production in transfected cells than in cells transfected with the empty plasmid.Conclusions The results obtained in this study suggest that GdA is involved in the differentiation of trophoblast cells. The treatment of GdA plasmid transfected trophoblast cells stimulated hCG production in isolated trophoblast cells and inhibited hPL and TF expression, suggesting a functional link between hCG and GdA.     

Immunohistochemical localization of hCG alpha, hCG beta CTP, hPL and SP1 on villous and extravillous trophoblasts in normal human pregnancy

Trophoblasts are divided into villous trophoblasts and extravillous trophoblasts, depending on whether they constitute a villous structure, and cell columns intervene between them. We conducted an immunohistochemical localization of human chorionic gonadotropin (hCG) alpha, hCG beta C-terminal peptide (CTP), human placental lactogen (hPL) and pregnancy-specific beta 1-glycoprotein (SP1) on the trophoblasts of normal human pregnancy, using forty-one hysterectomized pregnant uteri (6-22 weeks). In villous trophoblasts, the capacity to synthesize hCG alpha, hCG beta CTP, hPL and SP1 seemed to develop according to the morphological change from mononuclear cells to multinuclear cells. In contrast, the synthetic capacity of these proteins seemed not to correspond with the morphological change in extravillous trophoblasts: The location of hCG alpha, hCG beta CTP and SP1 was restricted to the mononuclear trophoblasts in the superficial decidua, while hPL was present extensively in extravillous trophoblasts, including multinuclear trophoblasts in the deciduomuscular junction. Therefore, it may be reasonably said that extravillous trophoblasts have many biological features distinct from villous trophoblasts and differentiate in an independent manner. Mononuclear trophoblasts in the cell column were negative for these proteins, which, together with morphological observations, strongly suggest the germinative nature of these cells.     

Stimulation of hCG and inhibition of hPL in isolated human trophoblast cells in vitro by glycodelin A

The immunosuppressive protein glycodelin A (formerly named PP14) is produced by human decidua and secreted in the maternal circulation. Glycodelin A concentrations in serum have been used as indicators of endometrial function. The purpose of this study was to investigate the effect of glycodelin A on human chorionic gonadotropin (hCG) and human placental lactogen (hPL) release by freshly isolated cytotrophoblasts (in vitro). Cytotrophoblasts have been prepared from human term placenta by the three-step trypsin-DNase dispersion method of villous tissue followed by a percoll gradient centrifugation step. When placed in culture, the isolated mononuclear trophoblasts differentiated into syncytial counterparts within 12-72 h after plating. Trophoblasts were incubated with varying concentrations (60-300 microg/ml) of glycodelin A. Glycodelin A was isolated and purified by chromatographic methods from amnion fluid. Supernatants of the trophoblast cell cultures were assayed for hCG and hPL by immunological methods. The release of hCG is increased in glycodelin A-treated trophoblast cell cultures compared to untreated trophoblast cells. Glycodelin A inhibits the production of hPL in vitro. Differences in Glycodelin A stimulated cells and untreated controls are statistical significant. hCG and hPL are markers for the differentiation process of trophoblast cells to syncytial trophoblasts. The results imply that glycodelin A secreted by decidualised endometrium modulates endocrine function, as well as the differentiation of trophoblasts in culture.     

Progesterone and human placental lactogen inhibit leptin secretion on cultured trophoblast cells from human placentas at term.

The placenta is an important source of leptin production that contributes to the state of hyperleptinemia observed in pregnant women. Moreover, the synthesis of leptin and its receptors by syncytiotrophoblast cells suggests a potential paracrine or autocrine action of leptin in the placenta. In the present study we examined the effect of gestational hormones, human chorionic gonadotropin (hCG), human placental lactogen (hPL), progesterone and estradiol, on in vitro leptin release by human term trophoblast cells in culture. Placentas at term were obtained immediately after delivery from mothers with uncomplicated pregnancies. Leptin levels were measured by enzyme-linked immunosorbent assay in culture media of trophoblasts maintained in monolayer culture for 24, 48 and 72 h with different hormonal treatments or placebo. Treatment with hPL and progesterone led to a time- and dose-dependent decrease in leptin release that was statistically significant after 24 h, with a maximal effect after 72 h of incubation. In contrast, incubation with estradiol and hCG did not have exhibit any effect on leptin secretion at any of the doses and times assayed in this work. The results obtained in this study support that leptin can be considered a gestational hormone implied in the endocrine function of the placenta and that its secretion is at least partially regulated by steroid and peptidic reproductive hormones in trophoblast cells in vitro.     

The extent and variability of effects of culture conditions on the secretion of human chorionic gonadotrophin and interleukin-6 by human, term placental explants in culture

Culture of explants derived from third trimester human placenta is used in a range of contexts as an experimental model that retains tissue architecture. This study aimed to explore the variability between, and within, individuals of secretion by explants of human chorionic gonadotrophin (hCG) and interleukin-6 (IL-6). Standard culture medium contained hydrocortisone, insulin, retinoic acid and serum. Under these conditions explants displayed significant differences in the time-course and extent of hCG secretion. Peak hCG secretion varied between 1.19 and 242 mIU/mg protein/h (coefficient of variation (CV) = 111%) and could occur between days 4 and 7 of culture. hCG secretion was more variable if explant protein was < 400 microg. Unadjusted day 7 hCG secretion showed marked variation: intra-placental CV = 15%, inter-placental CV = 86%. When day 7 hCG secretion was standardised by day 6 secretion, intra-placental CV was 6.9%, inter-placental CV was 4.0%. When this standardisation was applied, hCG secretion during day 7 of culture was not affected by removal of hydrocortisone, insulin or serum from the medium or by the addition of tumour necrosis factor-alpha (TNF-alpha). The secretion of IL-6 during day 7 of culture (standardised by taking natural logarithms) was increased markedly by the addition of TNF-alpha but unaltered by removing hydrocortisone, insulin or serum. Thus, we have shown that although variable, secretion by placental explants can be used to investigate how placental tissue adapts to different culture conditions. However, explants of the same protein content may have markedly different secretory properties.     

Immunohistochemical localization of HLA antigens and placental proteins (alpha hCG, beta hCG CTP, hPL and SP1 in villous and extravillous trophoblast in normal human pregnancy: a distinctive pathway of differentiation of extravillous trophoblast

Immunohistochemical localization of HLA antigens and placental proteins (alpha hCG, beta hCG CTP, hPL and SP1) in villous and extravillous trophoblast at various stages of normal human gestation were studied, using hysterectomy specimens. In the chorionic villi, the capacity for synthesizing placental proteins seemed to develop in parallel with the morphological change from mononuclear cells to multinucleated syncytiotrophoblast and no villous trophoblast expressed HLA antigens. In contrast, extravillous trophoblast, including the multinucleated trophoblastic cells at the deciduomuscular junction, expressed HLA-A, -B, and -C, and their capacity for synthesizing placental proteins did not seem to correspond with the degree of morphological change: the location of alpha hCG, beta hCG CTP and SP1 was restricted to mononuclear trophoblast in the superficial decidua, while hPL was present extensively in extravillous trophoblast. These findings strongly suggest that extravillous trophoblast possesses many distinctive biological features and differentiates in an independent manner. Mononuclear trophoblast forming the cell columns was also positive for HLA-A, -B, and -C, and no placental protein was demonstrated in these cells; this, together with previous morphological observations, may indicate the germinative nature of these cells.     

Extremely low placental lactogen hormone (hPL) values in an otherwise uneventful pregnancy preceding delivery of a normal baby

A case of a normal pregnancy and delivery with extremely low placental lactogen hormone (hPL) values in maternal blood is presented. The low hPL-values were due to the fact that the placenta only produced about 1/25 of the normal estimated output, calculated on the basis of the hPL-concentration in the intervillous spaces. The concentrations of progesterone, the placenta-specific beta-glycoprotein (SP1) and total estriol in serum were normal, while prolactin and chorionic gonadotropin (hCG) were considerably elevated. Glucose levels were normal. At the ultrastructural level the actual placenta under study did not differ from a normal term placenta. In spite of the very low concentrations of hPL there was a good milk secretion, and the mother was still breast-feeding her baby 11 months after the delivery. Basal level of prolactin was at this time normal.     

Maternal serum levels of placental proteins after in vitro fertilisation and their implications for prenatal screening

OBJECTIVES: To determine whether the serum levels of pregnancy-associated plasma protein A (PAPP-A), pregnancy-specific beta(1)-glycoprotein (SP1), placental lactogen (hPL) and human chorionic gonadotrophin (hCG) are different in pregnancies obtained after in vitro fertilisation (IVF) and embryo transfer (ET) in comparison to spontaneous pregnancies. Assessment of the need to establish normal medians for biochemical trisomy screening in IVF pregnancies. METHODS: The population comprised 96 IVF-ET pregnancies, of which 79 came from fresh gonadotrophin-stimulated cycles and 17 from embryo transfers without gonadotrophin stimulation (natural cycle IVF, frozen embryo transfers), and 156 spontaneous pregnancies. A single blood sample was obtained between 7 + 0 and 16 + 3 weeks. PAPP-A, SP1, hPL and hCG were quantified and the levels compared between gonadotrophin-stimulated IVF, steroid-only- or non-stimulated IVF, and controls with respect to gestational age using non-parametric statistical analysis. RESULTS: PAPP-A and hPL levels were reduced after stimulated IVF in early gestation (before 10 pregnancy weeks); SP1 followed the same trend without reaching statistical significance. hCG tended to be increased after IVF treatment including non-gonadotrophin-stimulation cycles, and also beyond 10 pregnancy weeks. CONCLUSION: Reduced PAPP-A with increased hCG yields an increased risk in screening for foetal trisomy 21. We confirm recently published observations but do not recommend the establishment of normal medians for IVF pregnancies since the extent of the deviations is varying according to the different stimulation protocols and dosages of gonadotrophins used.     

In vitro perfusion study of the human placenta at term: preliminary results.

Dual perfusion of the human placental lobule in vitro is a useful method for studying the transfer of molecules across the placenta, including the transfer of endogenous substances and xenobiotics. To establish the first model of in vitro placental dynamic study in our country, we used a dual recirculating perfusion system modified from that described by Miller et al. A placental lobule without tears or gross lesions was chosen. Both the fetal and maternal sides of the placenta were perfused with Medium 199 in addition to heparin, glucose, dextran and antibiotics. Perfusate samples were obtained periodically and analyzed for blood gas, glucose, lactate, human placental lactogen (hPL) and human chorionic gonadotropin (hCG). The stability of this human placental lobule preparation during 10 hours of perfusion was reflected by the stability of the arterial pressure and by the constant volume in the fetal compartment. A constant rate of oxygen was delivered by the maternal circulation system, and a steady rate of oxygen was gained by the fetal circulation system. Neither oxygen nor glucose consumption by the tissue was significantly reduced during the period of perfusion. The releasing rates of hCG and hPL did not change significantly during perfusion. The development of this dual perfusion system for the human placenta can provide for the study of placental hemodynamics and transplacental transport in perinatal medicine in our country.     

Morphological and immunohistochemical study on specimens of trophoblastic diseases in which the presence of a villous formation could not be established

The histopathological discrimination between malignant trophoblastic diseases and benign trophoblastic diseases depends on the presence or absence of a villous structure. However, molar extravillous trophoblasts and cells in some placental site trophoblastic tumors (PSTT) of a benign nature, lack a villous structure. We therefore observed the morphology of trophoblastic cells which do not constitute a villous structure, including choriocarcinoma cells, and analyzed the location of placental proteins in these cells immunohistochemically. The results were as follows: 1. Molar extravillous trophoblasts were composed of large mononuclear cells and multinuclear cells. Most of them were positive for hPL and negative for hCG and SP1. 2. Choriocarcinoma consisted of cytotrophoblast-like cells, syncytiotrophoblast-like cells, large mononuclear cells and multinuclear cells resembling large mononuclear cells. HCG was noted in syncytiotrophoblast-like cells and large mononuclear cells, while hPL and SP1 were found only in syncytiotrophoblast-like cells. 3. PSTT was made up of large mononuclear cells and multinuclear cells which contained abundant hPL and very little hCG and SP1 or none at all. Molar extravillous trophoblasts were clearly distinguishable from choriocarcinoma cells in terms of their morphology and the location of placental proteins. In contrast, it seemed difficult to distinguish cells of PSTT from molar extravillous trophoblasts on a cell level.     

Discordant secretion of pregnancy specific beta 1-glycoprotein and human chorionic gonadotropin by human pre-embryos cultured in vitro.

OBJECTIVE: To study and compare the secretion of pregnancy specific beta 1-glycoprotein (SP1) and human chorionic gonadotropin (hCG) by human pre-embryos, cultured in vitro, with their respective morphological development. DESIGN: Spare human pre-embryos from randomly selected women participating in a program of in vitro fertilization (IVF) were studied prospectively. SETTING: Pre-embryos were cultured, and hormone release was determined in academic research laboratories. PATIENTS, PARTICIPANTS: Pre-embryos (n = 108) cultured for 14 days after fertilization in Ham's F-10 medium (GIBCO Ltd., Paisley, Scotland) were observed, and hCG and SP1 were measured in the culture media at regular intervals. MAIN OUTCOME MEASURES: Discordant secretion of SP1 and hCG. RESULTS: Of the 98 bipronucleate pre-embryos, 53.6% formed blastocysts, 17.3% of which hatched. Human chorionic gonadotropin was detected from day 7 after fertilization concomitantly with blastocyst formation, thereafter showing a logarithmic increase (maximum 10,650 mIU) until the first signs of embryonic disintegration. Pregnancy-specific beta 1-glycoprotein release started 3 to 4 days after fertilization independently of the morphological development and the future production of hCG, thereafter displaying a nonlogarithmic increase (maximum 41 ng). CONCLUSIONS: Hormone secretion and morphological development are unique for each pre-embryo. Human chorionic gonadotropin and SP1 seem to have different biochemical and physiological regulation.