Extraembryonic Coelomic Fluid Research Articles

Vertical transmission of African-lineage Zika virus through the fetal membranes in a rhesus macaque (Macaca mulatta) model.

Zika virus (ZIKV) can be transmitted vertically from mother to fetus during pregnancy, resulting in a range of outcomes including severe birth defects and fetal/infant death. Potential pathways of vertical transmission in utero have been proposed but remain undefined. Identifying the timing and routes of vertical transmission of ZIKV may help us identify when interventions would be most effective. Furthermore, understanding what barriers ZIKV overcomes to effect vertical transmission may help improve models for evaluating infection by other pathogens during pregnancy. To determine the pathways of vertical transmission, we inoculated 12 pregnant rhesus macaques with an African-lineage ZIKV at gestational day 30 (term is 165 days). Eight pregnancies were surgically terminated at either seven or 14 days post-maternal infection. Maternal-fetal interface and fetal tissues and fluids were collected and evaluated for ZIKV using RT-qPCR, in situ hybridization, immunohistochemistry, and plaque assays. Four additional pregnant macaques were inoculated and terminally perfused with 4% paraformaldehyde at three, six, nine, or ten days post-maternal inoculation. For these four cases, the entire fixed pregnant uterus was evaluated with in situ hybridization for ZIKV RNA. We determined that ZIKV can reach the MFI by six days after infection and infect the fetus by ten days. Infection of the chorionic membrane and the extraembryonic coelomic fluid preceded infection of the fetus and the mesenchymal tissue of the placental villi. We did not find evidence to support a transplacental route of ZIKV vertical transmission via infection of syncytiotrophoblasts or villous cytotrophoblasts. The pattern of infection observed in the maternal-fetal interface provides evidence of paraplacental vertical ZIKV transmission through the chorionic membrane, the outer layer of the fetal membranes.

Human chorionic gonadotropin (hCG) in the male reproductive tract

Normal hypothalamic-pituitary testicular and prostatic functions are essential for maintenance of male fertility, whereby glycoprotein hormones (GPH) as well as androgens are major endocrine and local regulators. We have investigated whether the GPH human chorionic gonadotropin (hCG) and the free alpha and beta subunits thereof are produced in the target organs themselves and potentially act as auto/paracrine modulators of fertility. Immunofluorometric assays (IFMAs) based on our panel of highly selective monoclonal antibodies, immunohistochemistry (IHC), confocal laser scanning microscopy (CLSM) and 1- and 2D gel electrophoreses with subsequent western blotting have been utilized for the detection of hCGα, hCGβ and its metabolite hCGβ core fragment (cf) in human testis, prostate and seminal plasma. Both organs synthesize hCGα and hCGβ, which are subsequently detectable at high concentrations in seminal plasma of healthy probands (n=17): hCGα 2630±520ng/mL (mean±S.E.M.), hCGβ 2±0.28ng/mL, hCGβcf and hCG 0.19±0.039ng/mL. These parameters significantly exceed physiological values, e.g. ten thousand-fold in the case of hCGα, in serum of young men (n=20): hCGα 0.142±0.054ng/mL (mean±S.E.M.), hCGβ 0.05ng/mL and hCG 0.004±0.003ng/mL. Levels of these markers were not correlated with sperm counts. Of all body fluids including those of pregnant women seminal plasma is the richest physiological source for genuine free i.e. non-dissociated GPHα (Mr,app 23k) which may even appear as di- or tetramers. Its concentration is similar to that observed in maternal serum (weeks 10–12 of gestation) and in extra-embryonic coelomic fluid. In contrast to those fluids where ratios of free subunits to hCG are in the range of 1:100 highly inverse ratios in the range of 10.000:1.000:1 were observed for hCGα:hCGβ:hCG in seminal plasma. hCGα is not derived from heterodimeric GPH suggesting hCG-independent functions of hCGα and hCGβ in male and female fertility.

Growth-promoting effects of different fractions of extra-embryonic coelomic fluid on embryonic development.

In the early stages of embryonic development, many growth-promoting molecules must be provided by the maternal system. These factors may be supplied locally to the embryo, by the decidua, the placenta, or the yolk sac. In this study the growth-promoting potential of extra-embryonic coelomic fluid (EECF) and its fractions was investigated. The embryonic requirement of growth-promoting molecules may be studied by reducing the growth-supporting capacity of serum. Thus, ultrafiltration of rat serum was carried out for 8 h using Millipore filters with a molecular weight exclusion of 30 kDa. Rat embryos at 9.5 days of age were cultured for 8 days for anembryonic yolk sacs, and then EECF was collected and divided into three different molecular weight fractions by ultrafiltration. Rat embryos were cultured for 48 h in whole rat serum and the serum retenate (which has low growth-supporting capacity) in the presence and absence of EECF, its fractions, or in EECF only. Embryos grown in retenate showed severe growth retardation, and the addition of EECF significantly improved embryonic growth. The fraction which contained the molecules with molecular weight between 10 and 30 kDa had significantly more effect on embryonic development than the other fractions. This fraction of EECF was analysed by gel electrophoresis. Three of the four protein bands observed in this fraction were identified by amino-terminal sequencing as alpha-fetoprotein precursor (22 kDa), apolipoprotein A1 precursor (24 kDa) and fetal haemoglobin Y2 chain (14 kDa), none of which are likely to be responsible for the growth-promoting activity. To further investigate growth-promoting proteins, EECF was Western-blotted to nitrocellulose membranes and probed with antisera against rat prolactin, epidermal growth factor, insulin-like growth factors I and II and human placental lactogen. No immunoreactive bands were detected in the EECF, suggesting that either these proteins are not present or are present at levels too low to be detected. Although the growth-promoting effect of the EECF was demonstrated in this study, the molecules responsible remain uncharacterized.

Matrix metalloproteinases -2 and -9 and their endogenous tissue inhibitors in fetal membrane repair following fetoscopy in a rabbit model.

The cellular mechanisms underlying fetal membrane repair are poorly understood. Matrix metalloproteinases (MMP) and the endogenous tissue inhibitors of metalloproteinases (TIMP) play a key role in the control of turnover of extracellular matrix in fetal membranes at normal parturition and preterm prelabour rupture of the fetal membranes (PPROM). The time course of secretion of MMP-2 (72 kDa, gelatinase A) and MMP-9 (92 kDa, gelatinase B) and TIMP into extra-embryonic coelomic, allantoic and amniotic fluids in a rabbit model was examined. Furthermore, to evaluate their role in fetal membrane repair, the changes induced by fetoscopy at mid-gestation (23 days; gestation length is 32 days) were investigated. Zymography showed predominantly secretion of latent MMP-2 at 18, 23 and 30 days of gestation in all gestational compartments. Reverse zymography detected a broad range of TIMP activity with molecular weights of 27-30 kDa (TIMP-1, glycosylated TIMP-3 and TIMP-4), 24 kDa (unglycosylated TIMP-3) and 21 kDa (TIMP-2). Following fetoscopy, both MMP-2 and TIMP increased significantly in amniotic fluid and extra-embryonic coelomic fluid, but not in allantoic fluid, as demonstrated by densitometric analyses. These findings indicate a modulating role for MMP and TIMP in the repair processes following a surgically induced fetal membrane defect.

Secretion of matrix metalloproteinase-2, matrix metalloproteinase-9 and tissue inhibitor of metalloproteinases into the intrauterine compartments during early pregnancy.

Matrix metalloproteinases (MMPs) are important enzymes in tissue remodelling, a key event for the development of the fetal membranes and placenta and establishing the feto-maternal interface during early pregnancy. This study has examined the secretion of the gelatinases, MMP-2 (72 kDa) and MMP-9 (92 kDa), and the endogenous tissue inhibitors of metalloproteinases (TIMPs) into extra-embryonic coelomic and amniotic fluids, the two principal intra-uterine compartments of the first trimester, and compared them to amniotic fluid collected later in gestation. In extra-embryonic coelomic fluid, gelatin zymography demonstrated that MMP-2 (72 kDa) was the predominant gelatinase, with some MMP-9 present. A broad range of TIMPs corresponding to TIMP-1 and TIMP-2, glycosylated and unglycosylated TIMP-3 and TIMP-4 was detected in this compartment by reverse zymography and immunoblot analyses. There was little gelatinase or TIMP activity in amniotic fluid in the first trimester. In amniotic fluid from the second trimester after fusion of the membranes obliterating the extra-embryonic coelom, and at term elective caesarean section, MMP-2 is the predominant gelatinase present, with a broad spectrum of TIMPs. These findings demonstrate that predominantly MMP-2 and also MMP-9, regulated by a range of TIMPs, are involved in intra-uterine tissue remodelling during the establishment of pregnancy.

Follistatin and activin A in extra-embryonic coelomic and amniotic fluids and maternal serum in early pregnancy.

Follistatin is a specific binding protein which controls bioavailability of activins and inhibins which have an important role in fetal development. In the first trimester of pregnancy bioactive dimeric inhibins are found at high concentrations in the extra-embryonic coelomic fluid, but the distribution of follistatin and activins is not known. We have used recently developed immunoassays for follistatin, activin A and activin AB to determine their presence in the intrauterine compartments during early pregnancy. Follistatin was present in highest concentrations in the extra-embryonic coelomic fluid (11.72 +/- 1.70 ng/ml; median +/- SEM), with less in maternal serum (6.35 +/- 4.58) and lowest amounts in amniotic fluid (0.97 +/- 0.52). Follistatin concentrations in extra-embryonic coelomic fluid were highly correlated with both dimeric inhibin isoforms. Activin A was present in only barely detectable amounts in some samples of extra-embryonic coelomic fluid (41% of samples) and maternal serum (26%) and was undetectable in all amniotic fluid samples. Activin AB was undetectable in all compartments. The presence of follistatin in the amniotic and extra-embryonic coelomic fluids may regulate the availability of bioactive activins and inhibins which are released into the intrauterine compartments during the development of the fetus and placenta in early pregnancy.

Differential concentrations of monocyte chemotactic protein-1 and interleukin-8 within the fluid compartments present during the first trimester of pregnancy.

Monocyte chemotactic protein-1 (MCP-1) and interleukin-8 (IL-8) are important chemokines which effect the chemotaxis of monocytes and neutrophils, respectively. There is increasing evidence that such chemokines play an integral role in the control and maintenance of a normal pregnancy from implantation to parturition. However, little is known about the sites of secretion and function of MCP-1 and IL-8 in particular with respect to establishment of the placenta and membranes during first trimester. The aim of this study was therefore to investigate the concentrations and localization of MCP-1 and IL-8 in amniotic fluid and extra-embryonic coelomic fluid (EECF) collected by ultrasound-guided needle aspiration and maternal serum during the first trimester of pregnancy. Using specific enzyme-linked immunosorbent assays, MCP-1 was present at high concentrations in the EECF, significantly higher than those in amniotic fluid and maternal serum. IL-8 was also present predominantly in the EECF with concentrations being significantly higher than the low values detected in maternal serum and the very low amounts found in amniotic fluid. This strict compartmentalization of these cytokines in the fluid compartments of early pregnancy may be important for establishment and development of a viable pregnancy.

Activin A and inhibin B in extra-embryonic coelomic and amniotic fluids, and maternal serum in early pregnancy

Activin A and inhibin B levels were measured, using a two-site enzyme immunoassay, in extra-embryonic coelomic fluid, amniotic fluid and maternal serum samples retrieved from 23 healthy pregnant women, at 8 ( n=8), 9 ( n=8), and 10 ( n=7) weeks of gestation. Dimeric activin A and inhibin B were measurable in all samples. Median (± sem) activin A concentrations in coelomic fluid (0.98 ± 0.34 ng/ml) were significantly higher than in maternal serum (0.68 ± 0.05 ng/ml) and in amniotic fluid (0.09 ± 0.04 ng/ml) ( P<0.05). Maternal serum activin A levels were significantly higher than amniotic fluid concentrations. Median (± sem) inhibin B concentrations in coelomic fluid (24.32 ± 6.02 pg/ml) were significantly higher than in maternal serum (5.94 ± 0.97 pg/ml) and in amniotic fluid (6.31 ± 1.53 pg/ml) ( P<0.05), while no significant difference between maternal serum levels and amniotic fluid concentrations was found. No significant difference in activin A and inhibin B levels in extra-coelomic fluid, amniotic fluid, and maternal serum throughout the 3 weeks of pregnancy was found. The present study showed that coelomic fluid is an important reservoir of activin A and inhibin B, supporting the hypothesis that the extra-embryonic coelom may have a secretory role during the first 11 weeks of gestation.

Immunoblot studies of the acid-labile subunit (ALS) in biological fluids, normal human serum and in children with GH deficiency and GH receptor deficiency before and after long-term therapy with GH or IGF-I respectively.

The aims of this investigation were (a) to study the presence of immunoreactive forms of the acid-labile subunit (ALS) in different human biological fluids, (b) to define the age dependence of serum ALS in normal children and adults and (c) to compare the regulation of ALS by GH or IGF-I in children with GH deficiency (GHD) and GH receptor deficiency (GHRD) before and after 1 year of therapy with GH or IGF-I, respectively. Selected human biological fluids from different consenting volunteers and serum from 68 normal children and 5 adults were analysed. Four children diagnosed as GHD and 7 children diagnosed as GHRD were treated with recombinant human (rh) GH at a dosage of 0.05 mg/kg/day s.c. or rhIGF-I at a dosage of 120 micrograms/kg twice daily s.c., respectively, for 12 months. Immunoreactive forms of ALS were studied by Western immunoblot using a specific rabbit antiserum derived against synthetic human ALS and quantified by laser densitometry analysis. Serum from children with GHD or GHRD were sampled before and at 6 and 12 months of therapy; serum from these patients had been also assayed at baseline for determination of IGF-I and IGF binding protein (IGFBP)-3 by radioimmunoassay and immunoradiometric assay, respectively. An immunoreactive 85 kDa doublet of ALS was detected in serum, plasma, follicular, peritoneal and synovial fluid, but not in urine, seminal plasma, amniotic or extra-embryonic coelomic fluids. Assessment of serum from newborns to adults revealed an age dependence; the ALS doublet was low, but detectable, in newborns, increased during adolescence and remained constant in adulthood. ALS levels were significantly lower in GHD (P = 0.02) and in GHRD children (P = 0.001) than in age-matched controls. Treatment with rhGH in GHD children produced a 2.7-fold increase in serum ALS concentrations at 6 months of therapy (P = 0.01), which was maintained after 1 year of treatment (P = 0.006), leading to normalization of ALS concentrations. In contrast, administration of rhIGF-I to GHRD children failed to increase and normalize serum ALS levels either at 6 or 12 months of therapy. Immunoreactive forms of acid-labile subunit are present in serum and plasma, as well as in follicular, peritoneal and synovial fluids, suggesting that acid-labile subunit can either cross the capillary barrier or be secreted locally. Acid-labile subunit concentrations are age-dependent with a sharp increase during adolescence, and are reduced in GH deficient and GH receptor deficient children. While treatment with rhGH is able to increase and normalize acid-labile subunit concentrations in GH deficient children, therapy with rhIGF-I fails to increase serum acid-labile subunit levels in GH receptor deficient patients. These data suggest that acid-labile subunit is directly GH-regulated, and that IGF-I cannot increase acid-labile subunit levels, as assessed by Western immunoblot.

Gestational age-dependent expression of insulin-like growth factor-binding protein-1 (IGFBP-1) phosphoisoforms in human extraembryonic cavities, maternal serum, and decidua suggests decidua as the primary source of IGFBP-1 in these fluids during early pregnancy.

The insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) are important regulators of fetal and maternal tissue development during pregnancy. Posttranslational modification of IGFBP-1 yields up to six IGFBP-1 phosphovariants and a nonphosphorylated form, which in vitro, have some different properties. Nonphospho IGFBP-1 has less affinity for IGFs than the phospho isoforms and also may have IGF-independent actions. Herein, we have investigated the complement of IGFBP-1 phosphoisoforms present in extraembryonic coelomic (EEC) fluid, amniotic fluid (AF), and maternal serum (MS) throughout human gestation. Also, to determine potential tissue source(s) of IGFBP-1 in these fluids, we have quantified IGFBP-1 and examined IGFBP-1 phosphoisoforms in conditioned media (CM) from maternal decidua, fetal liver, and fetal kidney explants throughout gestation. Western immunodetection revealed that IGFBP-1, present in EEC and AF in early pregnancy and in CM from early pregnancy decidua, is primarily in the nonphosphorylated form. MS in this period contains primarily the nonphospho form and, as in nonpregnant adults, the highly phosphorylated form of IGFBP-1. The phosphorylation profile of IGFBP-1 in AF, MS, and decidua CM changes as pregnancy progresses. All the IGFBP-1 phosphoisoforms ultimately are produced by decidua and are present in midgestation MS, and all but the most highly phosphorylated form are present in AF. In late gestation, MS contains primarily the highly phosphorylated form. In contrast, profiles in CM from explants of fetal liver and kidney at different gestational ages remain unchanged. Nonphosphorylated IGFBP-1 is the primary form in fetal kidney CM, whereas fetal liver CM contains all IGFBP-1 phosphoisoforms. Concentrations of IGFBP-1 in fetal liver and kidney CM are significantly lower (482 +/- 146 and 120 +/- 32 ng/mL x 100 mg wet wt tissue, respectively) than in decidua CM (11,417 +/- 2,358 ng/mL x 100 mg wet wt tissue). The data cumulatively suggest that maternal decidua is the primary source of IGFBP-1 in EEC, AF, and MS in early pregnancy and that fetal liver and kidney are not likely significant contributors. The presence of nonphospho IGFBP-1 in AF, EEC, and MS suggests an important role for this isoform during early gestation.

Maternal and fetal levels of methionine and homocysteine in early human pregnancy.

To investigate methionine metabolism during normal human embryonic development by measuring levels of methionine and total homocysteine in samples of maternal serum, extra-embryonic coelomic fluid, and amniotic fluid. Cross-sectional observational study. Collaboration between St Bartholomew's Hospital, London, and the University Hospital of Nijmegen in The Netherlands. Twenty-three women with uncomplicated pregnancies between 8 and 12 weeks of gestation before surgical termination of an ultrasonographically normal fetus. Maternal serum samples were collected prior to surgery. Samples of extra-embryonic fluid and amniotic fluids were obtained by transvaginal ultrasound-guided coelocentesis and amniocentesis. Methionine was measured using an amino acid analyser and total homocysteine by high performance liquid chromatography. Levels of methionine were four times higher in extra-embryonic coelomic fluid and twice as high in amniotic fluid compared with maternal serum. In contrast, the total homocysteine concentrations were much lower in both extra-embryonic coelomic fluid and amniotic fluid than in maternal serum. All differences were significant (P < or = 0.01). The comparatively high concentrations of methionine in extra-embryonic coelomic fluid and amniotic fluid, and the concomitant low levels of total homocysteine in these fluids, suggest a role for methionine metabolism during early human pregnancy.

Inhibin in extra-embryonic coelomic and amniotic fluids and maternal serum in early pregnancy.

Inhibins are present in maternal serum during pregnancy. However, the presence of inhibins in the compartments surrounding the fetus in early pregnancy is not well defined. Using novel specific enzyme-linked immunosorbent assays we have demonstrated that the bioactive dimeric inhibin forms, inhibin-A and inhibin-B, and the immunoreactive inhibin forms containing pro- and alpha C sequences are present in different amounts in the extra-embryonic coelomic and amniotic fluids and maternal serum between 8-11 weeks gestation. Of the bioactive dimeric inhibins, both inhibin. A (mean +/- SEM 236.0 +/- 24.8 pg/ml) and inhibin-B (62.0 +/- 8.6 pg/ml) are present in extra-embryonic coelom whereas no dimeric inhibin is present in the amniotic fluid and only inhibin-A (360.2 +/- 32.9 pg/ml) is present in maternal serum. Furthermore, pro-alpha C-related immunoreactivity is present at high concentrations in the extra-embryonic coelom (591.7 +/- 60.5 pg/ml), amniotic fluid (452.4 +/- 76.8 pg/ml) and maternal serum (539.4 +/- 39.5 pg/ml). These findings would indicate that at this stage of gestation inhibin-A, inhibin-B and immunoreactive pro- alpha C-containing inhibin production are likely to arise from different sources including the fetus, placenta and fetal membranes and maternal sources including the ovary. Inhibins may be important regulators of fetal and placental development and involved in the establishment of pregnancy.

The coelomic cavity — A reservoir for metals

OBJECTIVE: The concentrations of metals in fluids surrounding the first trimester fetus were measured. STUDY DESIGN: Atomic absorption spectrometry was used to measure concentrations of metals in matched samples of amniotic and extraembryonic coelomic fluids in 17 women between 9 and 12 weeks of pregnancy. RESULTS: Concentrations of calcium, magnesium, iron, copper, and manganese (but not zinc, cadmium, or lead) were significantly higher in coelomic than in amniotic fluid. There was no significant difference between levels of iron, manganese, and lead to controls and amniotic fluid or between concentrations of manganese, cadmium, and lead in controls and coelomic fluid. There was no relationship between the concentrations of each metal in amniotic and coelomic fluid. CONCLUSION: The extraembryonic coelom is an important site of concentration of metals in early pregnancy. This might represent a store of metals essential for normal embryonic and fetal development or constitute a defense mechanism against environmental conditions adverse to the fetus.

Relaxin levels in amniotic fluid, extraembryonic coelomic fluid and maternal serum in early human pregnancy.

Separately identified samples of amniotic fluid and extraembryonic coelomic fluid together with maternal serum were collected from 22 women between 8 and 11 weeks of pregnancy and analysed for relaxin by immunoassay. Relaxin levels in maternal serum (median 1085 pg/ml; range 390-1259 pg/ml) were substantially higher than those in extraembryonic coelomic fluid (median 57.5 pg/ml; range 17-145 pg/ml; P < 0.0001; Mann-Whitney U-test). In turn, the levels of relaxin in coelomic fluid were higher than those in amniotic fluid (median 10 pg/ml; range 10-37 pg/ml; P < 0.0001; Mann-Whitney U-test). A linear correlation was found between relaxin levels in maternal serum and coelomic fluid (r = 0.68; P = 0.001) but there was no relation between levels in the other fluid compartments.

The role of glycosylation in regulating the glycoprotein hormone free alpha-subunit and free beta-subunit combination in the extraembryonic coelomic fluid of early pregnancy

The extraembryonic coelomic fluid (EECF) represents a major compartment in the fetal-placental unit during the first trimester of pregnancy. The compartment is composed of the fluid contained between the chorionic and amniotic membranes. The levels of glycoprotein hormone free alpha-subunit and free beta-subunit in the EECF far exceed those in the amniotic fluid or maternal serum. Furthermore, the level of free alpha in this compartment is twice that of intact hCG. We purified the glycoprotein hormone free alpha-subunit from a pool of EECF. This free alpha-subunit was found to be larger in size than the alpha-subunit of intact hCG. The size difference was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reduced and denatured conditions. The carbohydrate composition of the EECF free alpha-subunit indicated a higher degree of oligosaccharide branching, as evidenced by larger amounts of fucose, sialic acid, galactose, and N-acetylglucosamine than were present on combined hCG alpha. These differences in size and carbohydrate composition argue strongly against the concept that free alpha-subunits might originate from dissociation of intact hCG or "nicked" hCG. The free subunits of the EECF were evaluated for their ability to combine with the corresponding subunit obtained by dissociation of intact hCG. EECF free beta was able to combine with hCG alpha to form intact hCG. In contrast, EECF free alpha was unable to combine with hCG beta to form intact hCG. However, after removal of the asparagine-linked glycans by treatment with N-glycanase, most of the previously uncombinable free alpha-subunits were able to combine with hCG beta. These data demonstrate that the N-linked oligosaccharide(s) of EECF free alpha function to prevent the molecule from combining with the available and combinable free beta-sub-units that coexist in the same physiological compartment during early pregnancy. In view of the large amount of free alpha that is present in the EECF and the observation that, in vitro, free alpha can stimulate uterine decidual cell PRL secretion, together with the close apposition of free alpha-producing cells to decidual cells, it is likely that EECF free alpha has a function in early pregnancy. Carbohydrate modifications generated during the biosynthesis of EECF free alpha-subunit ensure that a population of free alpha molecules can exist in the presence of substantial quantities of free beta-subunits, and correspondingly, these same carbohydrate modifications function to permit the existence of free beta-subunits in the same gestational compartment with free alpha molecules.

The role of glycosylation in regulating the glycoprotein hormone free alpha-subunit and free beta-subunit combination in the extraembryonic coelomic fluid of early pregnancy.

The extraembryonic coelomic fluid (EECF) represents a major compartment in the fetal-placental unit during the first trimester of pregnancy. The compartment is composed of the fluid contained between the chorionic and amniotic membranes. The levels of glycoprotein hormone free alpha-subunit and free beta-subunit in the EECF far exceed those in the amniotic fluid or maternal serum. Furthermore, the level of free alpha in this compartment is twice that of intact hCG. We purified the glycoprotein hormone free alpha-subunit from a pool of EECF. This free alpha-subunit was found to be larger in size than the alpha-subunit of intact hCG. The size difference was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reduced and denatured conditions. The carbohydrate composition of the EECF free alpha-subunit indicated a higher degree of oligosaccharide branching, as evidenced by larger amounts of fucose, sialic acid, galactose, and N-acetylglucosamine than were present on combined hCG alpha. These differences in size and carbohydrate composition argue strongly against the concept that free alpha-subunits might originate from dissociation of intact hCG or "nicked" hCG. The free subunits of the EECF were evaluated for their ability to combine with the corresponding subunit obtained by dissociation of intact hCG. EECF free beta was able to combine with hCG alpha to form intact hCG. In contrast, EECF free alpha was unable to combine with hCG beta to form intact hCG. However, after removal of the asparagine-linked glycans by treatment with N-glycanase, most of the previously uncombinable free alpha-subunits were able to combine with hCG beta. These data demonstrate that the N-linked oligosaccharide(s) of EECF free alpha function to prevent the molecule from combining with the available and combinable free beta-sub-units that coexist in the same physiological compartment during early pregnancy. In view of the large amount of free alpha that is present in the EECF and the observation that, in vitro, free alpha can stimulate uterine decidual cell PRL secretion, together with the close apposition of free alpha-producing cells to decidual cells, it is likely that EECF free alpha has a function in early pregnancy. Carbohydrate modifications generated during the biosynthesis of EECF free alpha-subunit ensure that a population of free alpha molecules can exist in the presence of substantial quantities of free beta-subunits, and correspondingly, these same carbohydrate modifications function to permit the existence of free beta-subunits in the same gestational compartment with free alpha molecules.(ABSTRACT TRUNCATED AT 400 WORDS)

Insulin-like growth factors (IGFs), IGF-binding proteins (IGFBPs), and proteolyzed IGFBP-3 in embryonic cavities in early human pregnancy: their potential relevance to maternal-embryonic and fetal interactions.

Insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) are believed to be important in fetal growth and development. In the current study, the developmental changes in the IGF and IGFBP axis were examined in 23 paired samples of human amniotic fluid (AF), extraembryonic coelomic (EEC) fluid, and maternal serum (MS) between 9 and 12 weeks gestation. Levels of IGF-I were very low in AF (7 +/- 3 ng/mL) and EEC (10 +/- 3 ng/mL) compared to those in MS (237 +/- 42 ng/mL). In contrast, IGF-II concentrations were 210 +/- 36 and 174 +/- 22 ng/mL in AF and EEC, respectively, and were approximately 25% of MS serum levels (884 +/- 122 ng/mL). There was no dependence on gestational age for either peptide in AF or EEC during the period of gestation examined. IGFBP-1 levels in AF increased about 20-fold (1.6 +/- 0.3 to 33.0 +/- 0.1 ng/mL) between 9 and 12 weeks of pregnancy, and IGFBP-1 levels were nearly 2 orders of magnitude higher in EEC, increasing about 100-fold (365 +/- 119 to 3014 +/- 100.0 ng/mL) by the end of the first trimester. In contrast, IGFBP-1 levels were low in MS (24.9 +/- 3.5 ng/mL) and showed no gestational age dependence. Using RIA, high levels of IGFBP-3 were found in EEC (2062 +/- 177 ng/mL) and MS (6590 +/- 357 ng/mL) compared to those in AF (152 +/- 24 ng/mL). Levels of IGFBP-3 in MS and EEC did not change significantly with gestational age, whereas an increase in IGFBP-1 was observed in AF after the tenth week of pregnancy. In contrast to high levels of IGFBP-3 in MS and EEC, determined by RIA, the 37- to 43-kilodalton IGFBP-3 doublet was barely detectable by Western ligand blot analysis. This discrepancy suggested the presence of an IGFBP-3 protease in EEC, as has been found in MS, that decreases the affinity of this BP for IGF peptides and, therefore, renders it less readily detectable by Western ligand blot analysis. Using [125I]IGFBP-3 as substrate, lower levels of IGFBP-3 protease activity were detected in EEC compared to MS, and nearly undetectable levels were found in AF. By Western immunoblotting, a smaller (28-kilodalton) immunoreactive form of IGFBP-3 was detected only in MS and EEC, suggesting proteolyzed IGFBP-3 in MS and EEC, but not in AF, during this gestational period.(ABSTRACT TRUNCATED AT 400 WORDS)

Pregnancy-associated plasma protein A levels in maternal serum, extraembryonic coelomic and amniotic fluids in the first trimester

First trimester maternal serum levels of pregnancy-associated plasma protein A(PAPP-P) are reduced in women with a Down's syndrome pregnancy. We have examined the concentration of this molecule in the amniotic (AF) and extraembryonic coelomic (EECF) fluids surrounding the developing fetus. Maternal serum levels of PAPP-A were elevated in all samples and steadily rose from a median of 480 mIU/1 at 8 weeks to a median of 6375 mIU/1 at 14 weeks gestation. Levels of PAPP-A were low in EECF and undetectable in the AF until 14 weeks gestation. This pattern of distribution is in contrast to that of most other trophoblast-associated antigens. This may reflect PAPP-A physiology and its specific production by the syncytiotrophoblast.

Levels of insulin-like growth factor-binding protein-2 and insulin-like growth factor-II in maternal serum, amniotic fluid and extraembryonic coelomic fluid at 9-20 weeks of pregnancy.

Insulin-like growth factor-II (IGF-II) and IGF-binding protein-2 (IGFBP-2) were measured in amniotic fluid, extraembryonic fluid and maternal serum from 20 women with apparently normal first trimester pregnancies prior to termination. A further 111 specimens of amniotic fluid were collected from women at 10-20 weeks of pregnancy. Levels of IGFBP-2 were similar in coelomic fluid and maternal serum. Levels in amniotic fluid were lower than those in serum and coelomic fluid (Mann-Whitney test; P = 0.0002 and P < 0.0001 respectively). The levels of IGF-II were much higher in maternal serum than in coelomic fluid, and higher in the latter than in amniotic fluid (Mann-Whitney test; P < 0.0001 for both situations). The levels of IGFBP-2 were relatively low at 10-11 weeks (medians 19.8 and 61.1 micrograms/l) but thereafter increased to 20 weeks (median 1400 micrograms/l). The levels of IGF-II showed a similar pattern. The findings suggest that the role of IGF-II and IGFBP-2 in the regulation of growth or differentiation of the fetus or of its surrounding membranes may change with advancing pregnancy.

Concentrations of vitamins A and E in amniotic fluid, extraembryonic coelomic fluid, and maternal serum in the first trimester of pregnancy.

Paired samples of amniotic fluid and coelomic fluid were obtained by transvaginal ultrasound guided amniocentesis from 15 women with an ultrasonographically normal pregnancy between 8 and 12 weeks' gestation. Vitamins A and E were measured in the two pregnancy fluids and in maternal serum by high performance liquid chromatography with detection by ultraviolet absorption and fluorimetry respectively. Concentrations of vitamins A and E were higher in maternal serum than in coelomic fluid and were generally undetectable in amniotic fluid. All differences in concentration were significant. The vitamin E/cholesterol ratio was similar in maternal serum and coelomic fluid. No correlation was shown between the vitamin concentrations in the three fluids. These findings suggest that the coelomic cavity plays a part in the maternofetal exchange of these vitamins.