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.     

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.     

Capacity for hormone production of cultured trophoblast cells obtained from placentae at term and in early pregnancy

Problem: There is an increased doubt about the identity of isolated cytotrophoblast cells at term. Therefore, we compared pregnancy serum levels of three hormones [human placental lactogen (hPL), human chorionic gonadotropin (hCG), and leptin] with the capacity for hormone production of early placentae [EP; 8–13 weeks of gestation (WG)] and term placentae (TP; 38–42 WG).Methods: Serum levels of these hormones were determined in 15 paired maternal (7–41 WG) and fetal (37–41 WG) samples. Cytotrophoblast cells were isolated from term (TP; 38–42 weeks) and early (EP; 8–13 weeks) placentae by enzymatic digestion and subsequent purification on a Percoll gradient. These cells were cultured for 6 days. The production of the hormones hPL, hCG, and leptin was determined as release during culture + cell content after culture – cell content before culture.Results: Serum levels (mean ± SD; n ± 15) at 7–12 and 37–41 WG were 89,652 ± 21,431 and 13,620 ± 5854 mIU/ml for hCG, 400 ± 182 and 7088 ± 2030 ng/ml for hPL, and 12,675 ± 4266 and 32,236 ± 10,961 pg/ml for leptin, respectively. For cultured cells from EP and TP, hCG and hPL showed different patterns of release during the first 2–3 days. While the release of these two hormones by EP cytotrophoblast cells continued during 6 days in culture, their concentrations reached a plateau for TP cytotrophoblasts between 4 and 6 days. Leptin was undetectable (<15 pg/ml) in TP cell cultured media, while for EP all three hormones showed the same release profiles. Production calculated for 30,000 TP trophoblast cells cultured for 6 days (n = 8) was 2–31 mIU for hCG and 0.5–2 ng for hPL. For EP (n = 11), it was 50–1070 mIU for hCG, 15–323 ng for hPL, and 137–580 pg for leptin. Net synthesis of hCG and hPL for TP was >10-fold and <1-fold, respectively. In contrast, the production of all three hormones for EP was at least 100 times the initial cell content.Conclusions: These results demonstrate that trophoblasts from early pregnancy show much higher production rates of hCG, hPL, and leptin than at term. However, the in vitro findings are difficult to be reconciled with the different serum concentrations of the two hormones hPL and leptin observed during the course of pregnancy.     

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.     

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.     

In vitro secretory patterns of human chorionic gonadotrophin, placental lactogen and pregnancy-specific beta 1-glycoprotein

The control of secretion of the placental hormones human chorionic gonadotrophin (hCG) and human placental lactogen (hPL), and the trophoblastic protein pregnancy-specific beta-glycoprotein (SP1), is not well understood. During pregnancy, the hCG concentrations peak in the first trimester then decrease, while hPL and SP1 increase steadily throughout gestation. In order to determine whether the discordance between hCG secretion and that of hPL and SP1 observed in vivo also occur in vitro, we cultured placental explants with and without dibutyryl cyclic AMP (dbcAMP) and theophylline. Between 5 and 12 explants were used for each treatment in each experiment. The concentration of the proteins secreted into the media each day was measured by specific radioimmunoassays. The quantities of hPL and SP1 secreted per day declined in a parallel fashion after 24 hours under both basal and dbcAMP-stimulated conditions. The hCG output progressively decreased in the unstimulated cultures until 48 hours, at which time an increase in hCG secretion was observed. The dbcAMP-stimulated placentae significantly increased their hCG output at both 48 and 72 hours. These data show that hCG secretion is regulated differently from that of hPL and SP1. The results do not negate the possibility that term placental tissue may contain an inhibitor of hCG release that is removed by experimental manipulation in vitro.     

Linkage of human chorionic gonadotrophin and placental lactogen biosynthesis to trophoblast differentiation and tumorigenesis

Normal trophoblast of the human placenta elaborates at least two major protein hormones, chorionic gonadotrophin (hCG) and placental lactogen (hPL). Molar and choriocarcinoma tissues characteristically synthesize large amounts of hCG and hPL. To examine the role of trophoblast differentiation in the expression of the hCG and hPL genes, we studied the cytological distribution of their mRNAs in tissue sections of human hydatidiform mole and choriocarcinoma by in situ hybridization. Histologically, these tissues are in different stages of cellular differentiation. In normal placenta, hCG alpha/beta mRNA can be localized to some cytotrophoblasts and primarily to the syncytium, whereas hPL mRNA appears only in the syncytial layer. In hydatidiform mole, which still retains placental villous morphology, the hPL gene and hCG alpha and beta genes are expressed but are poorly localized because of the admixture of cyto- and syncytiotrophoblasts. By contrast, choriocarcinoma, which is devoid of placental villous pattern but in which the cyto- and syncytiotrophoblast-like components are distinguishable, expresses hCG alpha and beta in the syncytial-like areas but little, if any, hPL. These results suggest that a certain level of trophoblast differentiation, such as villous formation, is associated with hPL expression, while the hCG alpha gene and the hCG beta gene can be expressed in more disorganized tissues which contain cytotrophoblastic elements.     

Immunocytochemical localization of placental lactogen and chorionic gonadotropin in the normal placenta and trophoblastic tumors, with emphasis on intermediate trophoblast and the placental site trophoblastic tumor

This report presents preliminary observations on the immunocytochemical localization of human chorionic gonadotropin (hCG) and human placental lactogen (hPL) in placental site trophoblastic tumors, hydatidiform moles, and choriocarcinomas and compares the findings with those of a similar immunocytochemical analysis of the placenta at various stages of development. In addition to cytotrophoblast (CT) and syncytiotrophoblast (ST), a third form of trophoblast designated "intermediate trophoblast" (IT) is present during normal pregnancy and in trophoblastic disease. Intermediate trophoblastic cells are mononucleate, larger than CT, and contain more abundant eosinophilic cytoplasm, resulting in a partial resemblance to ST. Intermediate trophoblast has distinctive immunocytochemical features that distinguish it from CT and ST. The localization of hPL and hCG in both IT and ST varies with the age of the placenta, with the type of trophoblastic neoplasm, and from one specimen to another within each category of tumor. Syncytiotrophoblast may contain both hormones in large amounts, whereas IT contains hPL predominantly and hCG focally. Cytotrophoblast is devoid of hCG and hPL except in choriocarcinoma, which may show focal weak staining for hCG. Immunocytochemical identification of hCG and hPL has proved helpful in clarifying the histogenesis of trophoblastic neoplasms and may also be of value in establishing their diagnosis and in determining their prognosis.     

Human choriogonadotropin (hCG) and placental lactogen (hPL) inhibit interleukin-2 (IL-2) and increase interleukin-1 beta (IL-1 beta), -6 (IL-6) and tumor necrosis factor (TNF-alpha) expression in monocyte cell cultures.

The effect of the human trophoblast hormones chorionic gonadotropin (hCG) and placental lactogen (hPL) on the expression of cytokines in peripheral blood mononuclear cell cultures was followed under a variety of culture conditions, (a) phytohemagglutinin stimulated cells (PHA-MSC), (b) allogenic mixed cells (AMC) and (c) spontaneously proliferating cells (SPC). A dose dependent enhanced release of IL-6, IL-1 beta and TNF-alpha by hCG and hPL was observed under all culture conditions. However, an inhibitory effect on the IL-2 release was seen in PHA-MSC by hPL and in AMC by hPL and hCG. The role of the suppression of IL-2 production/release on cytotoxicity towards trophoblast is discussed. These results suggest a sensitive, dose dependent hormonal control of the modulation of the immune response during pregnancy and strengthen the concept of a distinct regulation of monocytes and lymphocyte subpopulation by trophoblast hormones.     

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.     

Characterization of placental lactogen release from perifused human trophoblast cells

The dynamics of the release of human placental lactogen (hPL) under basal conditions and response to various secretogogues has been studied in perifused enriched hPL-producing cells from term placentae prepared by the isopycnic centrifugation of collagenase/hyaluronidase-dispersed placental cells on Percoll gradients. Under basal conditions, the perifused cells released hPL at a relatively constant rate for up to 24 h in culture. The mean rates of hPL release from cells (5 x 10(6) cells) from 18 normal full-term placentae varied from 1.8 to 20.2 ng/5 min (mean 7.7 ng/5 min). The cells from term placentae, however, did not release detectable amounts of chorionic gonadotrophin or the cytosolic enzymes lactic dehydrogenase and alkaline phosphatase. The amounts of hPL released by the perifused cells were inversely related to cell density with mean rates of hPL release by 2, 5, and 10 x 10(6) cells of 15.8, 8.6, and 5.7 ng/10(6) cells/0.5 h. The perifused cells responded to provocative stimuli (high-density lipoproteins (HDL), apolipoproteins AI, AII, and CI, partially purified hPL-releasing factor, phorbol esters, sn-1,2-diacylglycerol, and cAMP) in a manner qualitatively similar to enriched trophoblast cells and placental explants in static culture. Release of hPL in response to HDL, apoproteins AI, AII, and CI, and partially purified hPL-releasing factor was dose-dependent and occurred within 5 min of exposure. Basal and stimulated hPL release by perifused trophoblast cells that had been previously frozen at -70 degrees C for four weeks was identical to that of freshly dispersed cells from the same placenta. These experiments indicate that perifused trophoblast cells may be used as a model system to examine the dynamics of hPL release under basal conditions and in response to provocative stimuli.     

Effects of cyclic adenosine monophosphate on human chorionic gonadotropin and estradiol output by cultured human placental cells

We have previously shown that adenosine-3',5'-cyclic monophosphate (cAMP) inhibits basal estradiol output in human trophoblast cells. The objective of this study was to investigate further the effect of 8-bromo-cAMP on the conversion of C19 androgens to estradiol by placental cells. Trophoblast cells were prepared from human term placenta and maintained in monolayer culture. On days 3 and 4 of culture, these cells were treated with dehydroepiandrosterone sulfate, androstenedione, or testosterone with or without the concomitant presence of 8-Br-cAMP. 8-Br-cAMP markedly enhanced human chorionic gonadotropin secretion into the culture medium. On the other hand, the concomitant addition of 8-Br-cAMP with the androgen precursors led to an inhibition of estradiol output. The concentrations of androstenedione and dehydroepiandrosterone in the culture medium after treatment with dehydroepiandrosterone sulfate were elevated by the concomitant presence of 8-Br-cAMP. From these results we conclude that 8-Br-cAMP enhances human chorionic gonadotropin output in human term placental cells, whereas the presence of 8-Br-cAMP in cells given androgen precursors inhibits estradiol output, probably at the level of aromatization.     

Endocrinologic events in early pregnancy failure

Fourteen women experiencing early pregnancy failure have been studied during the time of conception and at frequent intervals until spontaneous abortion occurred. Serial measurements of serum estradiol, progesterone, 17α-hydroxyprogesterone, prolactin, human placental lactogen (hPL), and human chorionic gonadotropin (hCG) were determined; regular sonar scanning allowed the time of fetal death to be determined to within 7 days in six patients and a diagnosis of blighted ovum to be made in the remainder. In all patients serum progesterone and estradiol concentrations were within the normal range up to 7 weeks but appeared to decrease from about 8 weeks' gestation whether or not a living fetus was present. The placenta continued to produce hCG and hPL but, despite the continuing presence of hCG, the levels of 17α-hydroxyprogesterone declined to concentrations below those associated with normal pregnancy. These data suggest that the placenta may require a particular stimulus to take over production of progesterone and estradiol.     

Gestational age-related inhibition of placental hCG, alpha hCG and steroid hormone release in vitro by a GnRH antagonist

Human placental tissues have been shown to contain gonadotrophin-releasing hormone-(GnRH)-like activity. Thus, the effect of a potent GnRH antagonist (N-Ac-Pro1,D-p-Cl-Phe2,D-Nal(2)3,6-GnRH, obtained from Syntex Laboratories) on placental hormonal release was studied. Explant cultures of placentae of 6 to 15 weeks' gestation were studied. This GnRH antagonist did not inhibit the alpha human chorionic gonadotrophin (alpha hCG), human chorionic gonadotrophin (hCG), oestrone or oestradiol release from the six- and nine-week placental cultures, but greatly suppressed the release of these hormones in the placental cultures from 13- and 15-week gestations. Synthetic GnRH partially reversed the action of this antagonist on the hormonal releases in the 15-week placental cultures. These data demonstrate a gestational age-related action of this antagonist on placental hormonal release. Thus, a role for the endogenous GnRH-like activity of the placenta in the control of placental hormonogenesis is indicated.     

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。     

Immunocytochemical localization of chorionic gonadotropin, placental lactogen, and placental alkaline phosphatase in the diagnosis of complete and partial hydatidiform moles

Complete hydatidiform moles (CHMs) and partial hydatidiform moles (PHMs) represent different clinicopathologic entities with characteristic morphologic and cytogenetic findings. In the absence of cytogenetic data, the histologic distinction between these lesions and abortuses showing hydropic swelling (AHS) may be difficult. An immunocytochemical study analyzing the distribution of human chorionic gonadotropin (hCG), human placental lactogen (hPL), and placental alkaline phosphatase (PlAP) in CHMs, PHMs, and AHS was undertaken to determine whether the expression of these trophoblastic proteins might assist in the differential diagnosis. A total of 24 CHMs, 22 PHMs, and 13 AHS were selected on the basis of established morphologic criteria. Thirty-four specimens of abortuses without hydropic swelling and normal placentas, ranging from 6 to 24 weeks gestational age, were similarly analyzed. The immunocytochemical localization of the three trophoblastic proteins, predominantly in syncytiotrophoblast (ST), was scored using a semiquantitative scoring system. In CHMs hCG is widely distributed and PlAP is patchily distributed in ST regardless of the gestational age, whereas hPL tends to increase with increasing gestational age. In contrast, in PHMs hPL is more widely distributed in ST compared with CHMs regardless of gestational age, while PlAP increases with increasing gestational age; in PHMs the distribution of hCG is markedly less than in CHMs except early in the first trimester when the staining patterns are similar. The different patterns of distribution of hCG, hPL, and PlAP may reflect differences in the pathobiology of trophoblast in CHMs and PHMs and appear to be useful in the differential diagnosis of these conditions.     

Retrograde time-scale analysis of human placental lactogen, beta human chorionic gonadotropin, and unconjugated estriol levels in human maternal serum from the onset of spontaneous labor

We analyzed on a retrograde time scale which calculated in maternal serum, from the onset of spontaneous labor, human placental lactogen (hPL), beta human chorionic gonadotropin (beta-hCG), unconjugated estriol (E3) levels, and the ratios among these hormones in the normal late pregnancy. Maternal serum hPL, beta-hCG, and unconjugated E3 levels were measured simultaneously and serially in regular menstrual sera from 27 women in late pregnancy (total 155 samples) by radioimmunoassay. The peak level of hPL was found at 2 weeks before labor, and the peak of beta-hCG was found during 2-4 weeks before the onset of spontaneous labor. On the other hand, the mean level of E3 rose slightly with advancing gestational age. The hormonal ratios of hPL to E3 and beta-hCG to E3 decreased gradually toward the onset of labor, but the ratios of hPL to beta-hCG did not change. From these data, it is possible to conclude that the onset of spontaneous labor can be predicted by measuring the levels of hPL and unconjugated E3 in maternal peripheral serum.     

Immunocytochemical localization of human chorionic gonadotropin and placental lactogen in normal placentae

Preliminary observations on immunocytochemical localization of human chorionic gonadotropin (hCG) and human placental lactogen (hPL) were made in 35 placentae of normal pregnancy at various stages of development. By analysing the processing regularities of the two hormones and comparing the characteristics of immunocytochemical localization and hematoxylin-eosin stain on various trophoblasts in the normal placentae, the findings showed: (1) presence of the three types of trophoblasts, namely, cytotrophoblast (CT), intermediate trophoblast (IT), and syncytiotrophoblast (ST) was confirmed in normal placentae of first-trimester pregnancy. (2) IT has distinctive immunocytochemical features that distinguishes itself from CT and ST. In the first-trimester, ST contains a large amount of hCG which sharply diminishes thereafter, but hPL in ST increases with the fetal age. IT contains hPL all through the pregnancy period and the peak-value occurs in the second trimester. CT is devoid of hCG and hPL. The results indicated: (1) IT is more like ST but different from CT. (2) IT contains chiefly hPL and hCG only locally in early pregnancy which demonstrates that the processing of hPL is in the more well differentiated cells whereas hCG is in the less differentiated cells.     

Differentiation and secretory activities of cultured human placental cytotrophoblast

Ultrastructural, autoradiographic, immunofluorescent and biochemical techniques were used to characterize primary cultures of term placental cytotrophoblast in order to gain insight into the differentiation and secretory capacities of the cellular component of human trophoblast. Trypsin treatment of placental villi allowed isolation of a predominantly cytotrophoblast cell population that maintained viability up to 13 weeks in monolayer culture. Autoradiographic studies of tritiated thymidine incorporation identified a smaller diameter mononucleated cell population that was mitotically active and developed into larger diameter mononucleated cells and into multinucleated cells during culture. Ultrastructurally, cultured cells formed desmosomes, had an extensive network of cytoplasmic microfilaments and contained the organelles for hormone synthesis and secretion. These cells secreted steroid hormones, secreted Schwangerschafts protein I, actively incorporated tritiated glycoprotein precursors and expressed surface immunoreactivity for the beta-subunit of human chorionic gonadotrophin (hCG). However, medium concentrations of hCG and human placental lactogen dropped rapidly to undetectable levels after 14 days in primary culture. Cells grown beyond confluence differentiated into 1 to 2 mm structures with a villus-like histology. Our studies indicate that cytotrophoblast can secrete steroids, cytotrophoblast differentiation occurs in vitro in the absence of maternal tissues, hCG synthesis occurs in cultured cytotrophoblast and medium concentrations of placental protein hormones are not the best indicators of cell viability for cultures of cytotrophoblast.