Association of placental inflammation with fetomaternal hemorrhage and loss of placental mucin-1.

BACKGROUND: Fetomaternal hemorrhage (FMH) poses an immediate risk to the fetus and, in case of Rhesus-immunization, to future pregnancies. Given that altered endothelial permeability is part of the pathophysiology of inflammation, in this study we investigated whether placental inflammatory processes like chorioamnionitis (ChoA) or preeclampsia (PE) lead to increased rates of FMH compared to the established risk factor of placenta previa (PP). Putative accompanying markers of trophoblastic damage were also explored. METHODS: 40 patients (14 PE; 6 ChoA; 9 PP; 11 normal controls) were evaluated for FMH using a flowcytometric test kit, which is able to quantify FMH of 0.06% fetal cells. Placental tissue samples were immunostained for human placental lactogen (hPL), human chorionic gonadotropin (hCG), and mucin-1 (MUC1). MUC1 was evaluated as a potential serum marker of FMH. RESULTS: Patients with ChoA had a mean calculated FMH volume of 29 ml, compared to 4 ml in PE and 1 ml in PP and controls. MUC1 staining was reduced in PE and ChoA placenta samples, while elevated MUC1 serum concentration correlated positively with FMH. CONCLUSION: Diseases of placental inflammation are associated with FMH. Placental MUC1 staining is reduced and serum concentrations are increased in cases of FMH.

Severe spontaneous acute tumor lysis syndrome and hypoglycemia in patient with germ cell tumor.

Tumor lysis syndrome has been observed in patients with bulky, treatment-sensitive tumors, in particular hematological malignancies, especially after medical treatment (chemotherapy, corticosteroids, radiation, hormonal agents, and biological response modifiers). Tumor lysis syndrome has been observed also in solid malignancies and it very rarely occurs spontaneously. Tumor lysis syndrome-associated metabolic abnormalities include hyperuricemia, hyperphosphatemia, hyperkalemia, hypocalcemia and uremia. Severe hypoglycemia is another rare metabolic disorder, uncommonly associated with solid malignancies. The case described here is peculiar for the abrupt onset of these two rare conditions in a patient with a metastatic germ cell tumor.

Specificity and sensitivity of differentiation antigens in superficial soft tissue tumors: comparison of SMA, calponin, H-caldesmon, C-kit, PLAP and HPL.

We examined the expression pattern of smooth muscle actin (SMA), h-caldesmon (HCD), calponin (CALP), placental alkaline phosphatase (PLAP) and human placental lactogen (HPL) in benign and malignant spindle cell superficial soft tissue tumors in order to determine the role of these markers in differential diagnosis. Archival tissue from 38 patients with superficial smooth muscle cell and so-called fibrohistiocytic tumors (8 benign fibrous histiocytomas (BFHs), 6 dermatofibrosarcoma protuberans (DFPT), 9 malignant fibrous histiocytomas (MFHs), 9 leiomyomas (LMs) and 6 leiomyosarcomas (LMSs)) were immunostained with antibodies against SMA, HCD, CALP, PLAP and HPL. smooth muscle cell (SMC) tumors showed significantly high immunopositivity for HCD than that of so-called fibrohistiocytic tumors (p is less than or equal to 0.05) but 1/3 of DFPT and MFH cases and half of BFH cases also showed HCD immunopositivity; thus, this difference is debatable and not highly discriminative as expected. All tumor groups showed 100% immunopositivity for CALP. SMC tumors displayed significantly stronger and more widespread immunostaining pattern for PLAP than so-called fibrohistiocytic tumors (p < 0.05). Superficial soft tissue tumors did not express c-kit. In conclusion, HCD and PLAP can be used as ancillary immunomarkers in differential diagnosis of SMC tumors (Tab. 2, Fig. 7, Ref. 37).

Regulation of human placental development by oxygen tension.

Cytotrophoblasts, specialized placental cells, proliferate early in pregnancy and then differentiate into tumor-like cells that establish blood flow to the placenta by invading the uterus and its vasculature. In this study, cytotrophoblasts cultured under hypoxic conditions (2 percent oxygen), mimicking the environment near the uterine surface before 10 weeks of gestation, continued proliferating and differentiated poorly. When cultured in 20 percent oxygen, mimicking the environment near uterine arterioles, the cells stopped proliferating and differentiated normally. Thus, oxygen tension determines whether cytotrophoblasts proliferate or invade, thereby regulating placental growth and cellular architecture.

Radioreceptor assay for prolactin and other lactogenic hormones.

A radioreceptor assay with a sensitivity of 5 nanograms per milliliter has been developed for mammalian and avian pituitary prolactin, placental lactogenic hormones, and humnan growth hormone, using a membrane receptor preparation isolated from rabbit mammary glands. Prolactin preparations inhibited the binding of [(125)I]prolactin to receptors in direct proportion to the biological potency of these preparations. Thus, the radioreceptor assay provides a convenient and simnple assay for hormones which have lactogenic activity.

Quantitative analysis of pregnancy-associated plasma proteins in human placenta.

By immunochemical methods and simultaneous measurements of several normal plasma proteins, human placenta was shown to contain elevated quantities of four pregnancy-associated plasma proteins (PAPP's). In the order of increasing amounts, PAPP-A, PAPP-C, PAPP-B, and human chorionic somatomammotropin (PAPP-D) all were present in placenta extracts in quantities greater than could be expected on the basis of their content in maternal blood. In sharp contrast, the placental content of pregnancy zone protein could be entirely accounted for by the maternal plasma present in the placenta. All of the PAPP's appeared to be readily extractible from placental tissue with buffered saline, the large bulk of them being solubilized in the first extraction procedure. However, absorption studies indicated that appreciable quantities of the PAPP's were still present in the insoluble placental residue after 12 sequential extractions with saline. The chorioamniotic membranes were not significantly enriched in any of the PAPP's. Immunochemical analysis of unwashed placental tissue extracts for the PAPP's IgA, and IgM (maternal blood derived), as well as albumin and transferrin (maternal and fetal blood derived), permitted calculations to be made of the amount of blood and PAPP's in placenta. On the basis of these data, it was roughly estimated that a 400-g placenta (wet weight) would occupy 312 ml in volume, and would contain 144 ml of blood. Of this blood, 36 ml would be derived from the mother.

Studies of the secretion of monkey placental lactogen.

A radioimmunoassay for monkey placental lactogen (MPL) was developed to study the factors controlling the secretion of MPL. The sensitivity of the assay was 1 ng MPL per ml. Human and monkey growth hormone, and human placental lactogen (HPL) showed minimal cross-reactions with MPL. Maternal MPL concentrations as measured in 40 rhesus monkeys increased progressively throughout pregnancy to a mean of 5000 ng/ml at term while umbilical vein MPL was less than 50 ng/ml. After term delivery maternal MPL concentrations decreased rapidly with a t(1/2) of 20 min.After fetectomy but with retention of the placenta, MPL concentrations decreased by 25% reaching a plateau over a 6 hr period. Experimental abruption of the secondary placenta within 1 hr produced a 50% decrease in MPL concentration. After ligation of the fetal vessels supplying the secondary placental disc, MPL increased transiently and then decreased to levels significantly below those of the control period. These studies suggest MPL secretion is not directly controlled by the fetus but is sensitive to changes in placental blood flow. The pregnant rhesus monkey serves as a useful model for investigating factors which may regulate HPL secretion because of the close similarity between MPL and HPL secretion.

In vitro differentiation of villous trophoblasts from pregnancies complicated by intrauterine growth restriction with and without pre-eclampsia.

Highly purified (>99.99%) primary villous cytotrophoblasts from uncomplicated pregnancies and pregnancies complicated with intrauterine growth restriction (IUGR) alone, IUGR with pre-eclampsia (IUGR-PE) and PE alone were cultured for 5days and the extent of differentiation into syncytiotrophoblasts measured in terms of syncytialisation and secretion of chorionic gonadotropin (hCG) and placental lactogen (hPL). Three separate phenotypes were observed: (1) normal and IUGR-PE cells showed low syncytialisation and secretion of hCG and hPL, (2) IUGR cells showed the highest levels of syncytialisation and secretion and (3) PE cells showed high syncytialisation but low secretion. These results strongly suggest IUGR, IUGR-PE and PE to be distinct conditions in which villous cytotrophoblasts are either exposed to different environments or are genetically different.

Circulating cell-free fetal messenger RNA levels after fetoscopic interventions of complicated pregnancies.

OBJECTIVE: The aim of this study was to examine fetal gene expression in maternal plasma after fetoscopic intervention for twin-twin transfusion syndrome or congenital diaphragmatic hernia. STUDY DESIGN: Twelve women with pregnancies that were complicated by twin-twin transfusion syndrome and 10 women carrying fetuses with congenital diaphragmatic hernia were sampled before and sequentially after treatment. Levels of glyceraldehyde-3-phosphate dehydrogenase, human placental lactogen, and gamma globin messenger RNA were measured by real-time reverse transcriptase polymerase chain reaction amplification. RESULTS: At all time points, glyceraldehyde-3-phosphate dehydrogenase messenger RNA levels were higher in the congenital diaphragmatic hernia cases than in the twin-twin transfusion syndrome cases (P < .05), but during the immediate postoperative observation period, there were no significant changes in glyceraldehyde-3-phosphate dehydrogenase, human placental lactogen, or gamma globin messenger RNA levels in individual patients or patients who were grouped by procedure. CONCLUSION: Fetoscopic intervention of complicated pregnancies does not affect circulating fetal messenger RNA levels, which is in contrast to earlier observations that circulating fetal DNA levels increase after laser ablation for twin-twin transfusion syndrome. Plasma glyceraldehyde-3-phosphate dehydrogenase messenger RNA levels could be a potential novel biomarker for fetal trauma.

Immunocytochemical evaluation of human esophageal neoplasms and preneoplastic lesions for beta-chorionic gonadotropin, placental lactogen, alpha-fetoprotein, carcinoembryonic antigen, and nonspecific cross-reacting antigen.

Human esophageal neoplasms were studied in comparison to normal, uninvolved, and preneoplastic human esophageal epithelium for the presence of human chorionic gonadotropin (HCG), human placental lactogen (HPL), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), and nonspecific cross-reacting antigen (NCA) using the unlabeled antibody peroxidase-antiperoxidase technique. HCG immunoreactivity was identified in 10 of 33 squamous cell carcinomas (33%), in 1 of 6 adenocarcinomas (17%), and 1 of 6 preneoplastic esophageal lesions (17%); while 9 of 33 squamous cell carcinomas (33%) and 1 of 6 adenocarcinomas (17%) contained immunoreactive AFP. Immunoreactive HPL was detected in 6 of 33 squamous cell carcinomas (20%), but in none of the adenocarcinomas. Neither AFP nor HPL immunoreactivity was identified in the 6 hyperplastic lesions which were studied. When stained with an antiserum that was able to detect both CEA and NCA, 27 of 33 squamous cell tumors (82%) and 6 of 6 adenocarcinomas (100%) showed positive immunostaining reactions. Of these, 8 squamous cell carcinomas and 1 adenocarcinoma were subsequently shown to contain only NCA immunoreactivity, while 19 squamous cell carcinomas and 5 adenocarcinomas contained both NCA and CEA immunoreactivity. NCA immunoreactivity alone was identified in 3 of 6 preneoplastic lesions and NCA and CEA immunoreactivity in 1 of 6 preneoplastic lesions. None of the markers was detected in 8 specimens of normal esophageal epithelium which were studied as controls, nor in 6 specimens of uninvolved esophageal epithelium obtained from patients with esophageal cancer. Most tumors expressed 2 or 3 markers, and some tumors were identified which expressed up to 4 of the 5 markers investigated. Only 3 tumors failed to express any of the markers studied. No association was found between the degree of tumor differentiation and presence or absence of HCG immunoreactivity. However, HPL immunoreactivity was more common in poorly differentiated squamous cell carcinomas. In contrast, immunoreactive AFP was more common in well-differentiated squamous cell carcinomas than in other tumor types. Similarly, both CEA and NCA were more frequently expressed in well-differentiated squamous cell carcinomas, adenosquamous carcinomas, and adenocarcinomas than in less differentiated tumors. Our results suggest that HCG, HPL, AFP, CEA, and NCA are tumor-associated antigens in esophageal cancer. Therefore, they could be of value in screening tests for esophageal neoplasms and could be useful in subclassification of esophageal neoplasms.

Isolation of prolactin from human amniotic fluid.

Amniotic fluid, obtained from women at term, was centrifuged and concentrated by ultrafiltration. The concentrated amniotic fluid was purified by gel filtration on Sephadex G-100 and ion-exchanged chromatography on DEAE-cellulose. The fractions obtained during purification procedures were assayed for prolactin and somatotropin activities by respective radioimmunoassays. The prolactin-rich fraction obtained from ion-exchange chromatography was further purified by isoelectric focusing. A yield of 0.21 mg of highly pruified prolactin containing 40 units/mg was obtained from 1 liter of the amniotic fluid. The prolactin was free of somatotropin and human placental lactogen.

Characterization of bovine placental lactogen as a glycoprotein with N-linked and O-linked carbohydrate side chains.

In a previous report we showed that purified bovine placental lactogen (bPL) exists in two isoforms in the 31,000-33,000 Mr range, each with at least five isoelectric variants differing in approximately 2 orders of magnitude in isoelectric points (pI) 4-6. The multiple isoelectric variants are unique to the bovine hormone. In an effort to determine the nature of these variants endo- and exoglycohydrolase digestions were conducted to determine if this hormone was glycosylated. Analysis of peptide/N-glycosidase F and endoglycosidase F digests of radioiodinated bPL on one-dimensional gel electrophoresis showed a Mr decrease from 31,000 to 24,000 and 33,000 to 26,000 for the two isoforms. Digestion with a mixture of neuraminidase plus mixed exoglycosidases resulted in a Mr decrease of 4,000. Digestion with neuraminidase resulted in a Mr decrease of 2,000. Further analysis of peptide/N-glycosidase F- and neuraminidase-treated bPL by two-dimensional gel electrophoresis showed the isoelectric variants shifted from pI 4.4-6.3 to 4.9-8.0. The sialic acid residues on the N-linkage are responsible for the pronounced acidic character of bPL, but do not account for the residual charge heterogeneity as the different isoelectric variants persist after sialic acid removal. The apparent Mr of the protein after removal of N-linked carbohydrate residues is similar to that of PRL and GH. These enzymatic digestion results demonstrate the presence of N-linked complex oligosaccharide residues attached to the beta-amide group of an asparagine residue. Analyses of the sugar content of the molecule were consistent with the presence of one biantennary N-linked and two O-linked carbohydrate chains.

The development and characterization of a homologous radioimmunoassay for mouse placental lactogen.

A highly specific and sensitive homologous radioimmunoassay (RIA) has been developed for mouse placental lactogen(mPL). Mouse pregnancy serum and placental extracts showed parallel dilution-response curves, and no significant cross-reaction was seen with either mouse growth hormone or mouse prolactin. The sensitivity of the assay ranged from 0.5 to 1.0 ng/ml, and the intra- and inter-assay coefficients of variation were 5 and 11%, respectively. By RIA, mPL levels were detectable on day 9 of pregnancy (1 ng/ml) and increased until day 14 (100-150 ng/ml) in both C3H and BALB/c strains. On days 14-18 of pregnancy, mPL levels were maintained (95-125 ng/ml) in C3H mice, while they continued to increase (greater than 250 ng/ml) in BALB/c mice. In both strains, a midpregnancy peak in prolactin-like activity was detected by a radioreceptor assay but not by the mPL RIA.

Identification of "big" human placental lactogen in placenta and serum.

Because of increasing evidence for the heterogeneity of polypeptide hormones, studies of the molecular species of human placental lactogen (hPL) were initiated. When extracts of freshly delivered human placentas were passed over Sephadex G-100 in 0.05M ammonium carbonate, three immunoreactive peaks were detected. In addition to a peak corresponding to native hPL (Kav = 0.39) and one in the void volume, a consistent peak which eluted before hPL (Kav = 0.20) was present. The latter represented 2-25% of total hormonal activity and could be rerun without significant conversion to hPL. In 8M urea, the peak continued to behave as a large molecular weight form on both Sephadex chromatography and on polyacrylamide disc gel electrophoresis. Extraction procedures at both neutral and alkaline pH produced similar quantities of the larger material. [125I]iodo-hPL was not converted to the larger form by the conditions of extraction or analysis. These properties are consistent with a larger molecular weight, non-aggregated form of hPL. In comparison with the native hormone, the idsplacement curves for the larger form were parallel in radioimmunoassay studies. Sera obtained from pregnant women during various stages of gestation also showed consistent evidence for a large molecular weight form of the hormone. These observations provide direct evidence, both in placental tissue and in serum for "big" hPL.

Partial characterization of mouse placental lactogen.

Serum, placental, extracts, and placental incubation medium from 12- and 17-day-pregnant mice were fractionated by gel exclusion chromatography on Sephadex G-100. The eluates were monitored for activity by a radioreceptor assay (RRA) for lactogenic hormones that detects placental lactogen (PL) and PRL. RRA activity in serum and placental extracts and incubation medium from both days of gestation eluted in two peaks, the first emerging in the void volume (Vo) and the second with an elution volume (Ve) to Vo ratio of 1.96, which was similar to the Ve to Vo ratio of a human PL marker. Fractions in the second peak stimulated casein synthesis in mouse mammary gland explants. The RRA activity of the eluates was not due to the presence of PRL, as determined by RIA, and therefore the activity in the second peak was identified as monomeric PL. Electrophoresis of mouse placental extracts and incubation medium from both days 12 and 17 of gestation on 10% polyacrylamide gels showed only one band of RRA activity (Rf = 0.22). The present results show there are no differences between the elution profiles on Sephadex G-100 and the electrophoretic mobilities of PL from serum, placental extracts, and placental incubation media from days 12 and 17 of pregnancy in mice.

Immunohistochemical localization of placental lactogen in binucleate cells of bovine placentomes.

Bovine placental lactogen (bPL) has been isolated from bovine trophoblast and characterized as a 32 K mol wt protein which exists in three different forms which differ in their isoelectric point values and their amino acid compositions. Two of the three forms have been shown to have both bovine GH (bGH)- and bovine PRL (bPRL)-like activities equal on a molar basis to bGH and BPRL in radioreceptor assays. It has been postulated that, in sheep, PL is delivered to the maternal circulation by the migration of fetal binucleate cells from the trophoblast across the fetal-maternal boundary into the uterine epithelium. To determine whether an analogous situation exists in the cow, antibodies to bPL were used to localize bPL in bovine placentomes and to measure its concentration in fetal and maternal sera. For cytology, bPL was localized on sections of placentomes from midgestation and term bovine placentas using an indirect immunoperoxidase technique. Stained binucleate cells were demonstrated throughout the trophoblast, often in close association with the microvillous boundary which separates the trophoblast from the maternal epithelium. In cross-sections of fetal villi, binucleate cells with cytoplasmic processes extending into and through the uterine epithelium were immunostained as well as cells within the plane of the uterine epithelium in close approximation or apposition to the maternal basement membrane. RIA demonstrated bPL to be present in maternal sera in concentrations of 1-2 ng/ml and in fetal sera at 5-12 ng/ml. These data are consistent with the hypothesis that binucleate cell migration accomplishes the delivery of bPL to the maternal circulation.

Maternal and fetal concentrations of ovine placental lactogen measured by radioimmunoassay.

A specific and sensitive homologous RIA for ovine placental lactogen (oPL) has been developed. The assay is specific for oPL in that ovine pituitary PRL (oPRL), GH (oGH), and other pituitary hormones, as well as rat, caprine, bovine, monkey, and human PLs exhibit no cross-reaction in the assay. The lower limit of sensitivity of the assay is 1.0 ng/ml. In uterine vein samples oPL was measurable after day 40 of gestation and in peripheral sera after day 48 (3.5 +/- 1.9 ng/ml). Peripheral plasma oPL concentrations reached peak levels by day 131--141 (649 +/- 205--565 +/- 347 ng/ml), then began to decline approximately 5 days before parturition and disappeared rapidly after delivery. No significant circadian variation in serum oPL concentrations was found during a 4-day period of sampling from day 120--125 of gestation. Fetal serum oPL levels ranged from 24--150 ng/ml throughout pregnancy. oPL was measurable in fetal membranes as early as day 20--30 (0.21 +/- 0.06 microgram/g wet wt), whereas the concentration in maternal caruncles at this time was 0.1 +/- 0.06 microgram/g wet wt. In the placentomes, peak concentrations occurred around day 101--130. Peak concentrations of oPL in allantoic fluid were found between day 35--50, with detectable levels as early as day 18. In amniotic fluid, oPL was measurable between day 40--50. Concentrations of oPL in urine from the pregnant ewe never exceeded 5 ng/ml.

Immunohistochemical visualizations of prolactin, growth hormone, and a substance resembling placental lactogen in the in situ and ectopic pituitary in the hamster.

The effects of transplanting neonatal adenohypophyseal tissue into the hamster cheek pouch on the presence of intracellular materials related to PRL in the allografts were examined using immunohistochemistry. In situ pituitary and placental tissue were used as control. Cells with antirat PRL-reactive sites were scarce in the grafts, and radioimmunoassayable serum PRL was not detectable in hypophysectomized hosts with grafts. Antirat GH- and antihuman placental lactogen (hPL)-reactive sites were visualized in the grafts and in situ pituitary tissue. Intracellular material in hamster placental tissue was visualized with anti-hPL only. Results of various immunohistochemical procedures using in situ pituitary tissue and antirat GH and anti-hPL antisera indicated that three cell types could exist: 1) a cell type visualized with only antirat GH, 2) a cell type visualized with only anti-hPL, and 3) a cell type, the most frequently observed, visualized with both antisera. The physiological significance of this intracellular product, which resembles hPL and perhaps hamster PL immunologically, in hamster adenohypophyseal cells is unknown. Additionally, our data indicate that the ectopic site for pituitary transplantation in the hamster may influence the cell types present in the grafts.

The purification and characterization of ovine placental lactogen.

Ovine placental lactogen (oPL), has been purified approximately 1,000-fold from sheep cotyledons using conventional protein purification procedures. Radioreceptor assays using rabbit liver particulate fractions for growth hormone (RRA-GH) and using rabbit mammary gland particulate fractions for prolactin (RRA-PRL) were employed to monitor the hormonal activities. The molecular weight of oPL is approximately 22,000 as determined by gel filtration on Sephadex G-100, and its isoelectric point as determined by isoelectric focusing is 8.8. In the two RRA's, the displacement curve of oPL is parallel to bovine growth hormone (bGH) and ovine prolactin (oPRL) standards and the ratio of GH-activity to PRL-activity of oPL is 1:2. In a body weight gain assay using hypophysectomized rats, oPL has a growth-promoting potency of 1.3 U/mg. In rabbit mammary explants, oPL stimulates casein synthesis. In a receptor assay for growth hormone using human liver, oPL and hGH are equipotent in competing for receptor sites, suggesting that oPL and hGH have common structural features that are lacking in other non-primate hormones.

Mouse placental lactogen-I: RIA and gestational profile in maternal serum.

A RIA for mouse placental lactogen-I (mPL-I) was developed using recombinant mPL-I as the standard, radioligand, and antigen for antiserum production. Displacement curves for dilutions of serum and placental extracts from pregnant mice were parallel to the recombinant mPL-I standard curve. Serum from male and nonpregnant female mice and high concentrations of mouse PRL, GH, PL-II, proliferin, and proliferin-related protein did not cross-react in the assay. mPL-I appeared in maternal serum on day 6 of pregnancy. Its concentration remained low until day 8 and them increased to a very large peak on days 9-11 (maximum concentration, approximately 8 micrograms/ml). The mPL-I concentration declined after day 11, but the hormone could be detected at low concentration in maternal serum until the end of pregnancy. On day 10 of pregnancy, the mPL-I concentration of maternal serum was correlated with litter size. Fractionation of serum from 10-day pregnant mice by size exclusion chromatography indicated the absence of high mol wt forms of mPL-I in the circulation.