Cytokines in implantation.

Implantation is a complex process which involves the 'invasion' of the maternal endometrium by the trophoblast surrounding the developing blastocyst. In response to this interaction there is a cellular reaction within the endometrium which has some features analogous to invasion by a tumour and some which are more characteristic of an inflammatory response. In addition, and also in common with cancer and inflammation, there is a release of biologically active molecules, including cytokines, at and around the implantation site. The information on cytokines is complex and often contradictory but it is recognised that they play an important role in the successful establishment of pregnancy. The evidence for this role is examined in this review.

Monoclonal antibodies to human chorionic gonadotropin: implications for antigenic mapping, immunoradiometric assays, and clinical applications.

Monoclonal antibodies to intact hCG and free beta-subunit of hCG permit the recognition of different individual antigenic sites on the hCG molecule. At least seven different epitopes may be recognized on the native molecule and a further two on the free beta-subunit. These antibodies were used in a mouse Leydig cell bioassay system to compare the degree of inhibition of hCG-induced testosterone production. Two antibodies were particularly potent at inhibiting hCG action, suggesting that they bind near to the receptor-recognition site on the hCG molecular. One antibody had little effect on biological action and was presumably binding distant from the biologically active site on the hCG. Combinations of monoclonal antibodies in immunoradiometric assays were used to develop highly sensitive and specific assays to intact hCG, free beta-subunit of hCG, and beta-subunit as part of intact hCG. Using these assays it was possible to detect 0.1 ng/ml hCG in the presence of high levels of LH. In 106 serum samples from pregnant woman free beta-subunit was considerably higher in samples with low concentrations of intact hCG, suggesting that free beta-subunit is not a limiting factor in placental production of intact hCG in early pregnancy. Comparison of urinary to serum ratios of hCG and free beta-subunit using specific immunoradiometric assays showed a good correlation for intact hCG but not for free beta subunit which was present in very high concentrations in urine.

Synthesis of placental protein 12 by decidua from early pregnancy.

The synthesis and secretion of placental protein 12 (PP12) by early pregnancy decidua and trophoblast were studied in vitro from tissues obtained by curettage during elective termination of pregnancy (weeks 8-14). The tissue explants were incubated in Ham's F-10 medium for a 27-h period, and the PP12 levels in media and tissue homogenates were measured by RIA. De novo synthesis of PP12 was assessed by measuring the incorporation of radioactivity into PP12 after 20 h of incubation of tissues with 20 microCi/ml [35S]methionine. PP12 from the culture medium was immunoprecipitated with anti-PP12(A) antiserum, and the immunoprecipitate was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The initial tissue content of PP12 was 10- to 72-fold higher in decidua than in trophoblast. The total amount of radioimmunoassayable PP12 released into medium by decidual explants during the 27-h incubation period together with that present in the tissues at the end of incubation exceeded the initial tissue content by 242.7 +/- 63.7% (mean +/- SE). Only small amounts of PP12 were detected in media from trophoblast cultures. During the first 7 h of incubation, inclusion of cycloheximide had no effect on PP12 release by decidual explants in three of four experiments. Between 7 and 27 h, the amount of PP12 released by cycloheximide-treated tissues was 20.0 +/- 7% of that released by control tissues (P less than 0.01). Cycloheximide had no effect on PP12 release by trophoblasts. Decidual explants incorporated [35S]methionine into PP12, but trophoblasts did not. In sodium dodecyl sulfate-gel electrophoresis, the newly synthesized PP12 comigrated with the major band of purified PP12 corresponding to mol wt 29,000. These data clearly confirm that PP12 is a protein of decidual rather than trophoblastic origin, and indicate that decidua from early pregnancy has the ability to synthesize it.

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.

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)

Role of the ovary in the synthesis of placental protein-14.

Women with absent ovarian function provide an opportunity to investigate the ovarian contribution to secretion of placental proteins (PP), such as PP14, in early pregnancy. We present data on serum PP14 levels in 12 women (median age, 30.5 yr; range, 26-37 yr) with premature ovarian failure (POF) who conceived after ovum donation and embryo transfer with exogenous sex steroid support using transdermal (n = 5) or oral (n = 7) estradiol and vaginal (n = 8) or im (n = 4) progesterone. The women were closely monitored throughout early pregnancy, with measurement of serum levels of estradiol (E2), progesterone (P4), and PP14. Levels of E2 and P4 were entirely normal. Levels of PP14 were significantly subnormal (P = 0.008) in all 12 agonadal women compared with levels of PP14 in a control group of women with normal ovarian function between 6-12 weeks gestation. Basal and peak levels for subjects with absent ovarian function were 40 and 124 micrograms/L, respectively. For each week between 6-12 weeks of pregnancy, the mean serum levels of PP14 for women with normal ovarian function were between 706-940 micrograms/L. These observations support the concept that PP14 arises from the ovary in early pregnancy or that factors under the control of the maternal ovary are involved in its production by the endometrium.

Immunochemical analysis of the human chorionic gonadotrophin-like material secreted by 'normal' and neoplastic urothelial cells.

Material with the immunochemical characteristics of human chorionic gonadotrophin (hCG) is produced by bladder tumour cells in vitro and in vivo. In order to characterize this material further, media were collected from 17 cell cultures (three choriocarcinomas, seven bladder carcinomas and seven 'normal' urothelium). The hCG-like material was compared with pregnancy hCG and purified alpha-and beta-subunits by specific radioimmunoassays. Media were also submitted to affinity chromatography and the fractions further analysed by SDS-PAGE and Western blotting. It was shown that both the neoplastic and normal urothelium produced only free beta-subunit-like material. This urothelial 'beta-hCG' has the same molecular weight and electrophoretic mobility as that present in the intact hCG of pregnancy.

Amplification or rearrangement of the beta-human chorionic gonadotrophin (beta-hCG)--human LH gene cluster is not responsible for the ectopic production of beta-hCG by bladder tumour cells.

The beta-subunit of human chorionic gonadotrophin (hCG) is coded on chromosome 19 by the beta-hCG-hLH gene cluster. Genomic DNA has been isolated from bladder tumour cell lines which ectopically express beta-hCG. The beta-hCG-hLH gene cluster was probed for possible rearrangement or amplification and cells karyotyped for chromosome 19 abnormalities. No rearrangement or amplification of the gene cluster and no consistent abnormalities of chromosome 19 were found. The expression of beta-hCG by bladder tumours is therefore likely to be the result of altered gene regulation and not a rearrangement or amplification of this gene cluster.

The beta-subunit of human chorionic gonadotrophin exists as a homodimer.

The free beta-subunit of human chorionic gonadotrophin (hCGbeta) is well recognised as a product of many epithelial tumours. Recently, it has been shown that this ectopic production may have a functional relationship to tumour growth. The growth-promoting activity of hCGbeta may be explained by its structural similarity to a family of growth factors which all contain the same distinct topological fold known as the cystine-knot motif. Since the other members of this family all exhibit their activities as homo- and heterodimers, it is possible that the same may be true for hCGbeta. Using size-exclusion chromatography, low stringency SDS-PAGE and matrix assisted laser desorption/ionisation (MALDI) time-of-flight (TOF) mass spectrometry (MS) we have shown that pure preparations of hCGbeta contain hCGbetabeta homodimers. Size-exclusion chromatography revealed asymmetric elution profiles with a forward peak corresponding to the size-exclusion characteristic of a globular protein with an approximate mass of 44-54 kDa and a late shoulder centered around an elution position expected for a globular protein of approximately 29 kDa. Two immunoreactive hCGbeta species, of approximately 32 and 64 kDa, were clearly resolved by SDS-PAGE and Western blotting. When analysed by MALDI-TOF MS a |mf23 kDa monomer and a |mf46 kDa dimer were identified. Formation of hCGbetabeta homodimers is consistent with the behaviour of other cystine-knot growth factors and strengthens the inclusion of the glycoprotein hormones within this superfamily. It has yet to be determined whether it is this dimeric molecular species that is responsible for growth-promoting activity of hCGbeta preparations in tumours.

Molecular heterogeneity of pregnancy specific beta 1 glycoprotein: the effect on measurement by radioimmunoassay and electroimmunoassay.

A comparison of methods for the quantification of circulating pregnancy specific beta 1 glycoprotein in late pregnancy was performed to assess the influence of the presence of a high molecular weight glycoprotein with alpha 2 electrophoretic mobility (SP1 alpha) which is immunochemically identifiable with pregnancy-specific PBETA 1 glycoprotein (SP1 beta). Serum samples from 47 volunteers in the 3rd trimester of pregnancy were subjected to measurements of pregnancy specific beta 1 glycoprotein in rocket immunoelectrophoresis, quantitative crossed-immunoelectrophoresis and radioimmunoassay. Rocket immunoelectrophoresis gives a result which reflects the total SP1 content, i.e. SP1 alpha and SP1 beta while quantitative crossed-immunoelectrophoresis permits differentiation between the two molecules. Radioimmunoassay predominantly measures authenic SP1, i.e. SP1 beta in the presence of physiological amounts of SP1 alpha.

Enhancement of ectopic beta-human chorionic gonadotrophin expression by interferon-alpha.

Treatment of three beta-human chorionic gonadotrophin (beta-hCG)-expressing bladder tumour cell lines with interferon-alpha (IFN-alpha) (5000 U/per 10(6) cells) enhanced the rate of beta-hCG secretion from 34.2 +/- 0.9 to 102.5 +/- 0.1 mIU/10(6) cells per 72 h in cell line 5637; 111.15 +/- 11.75 to 261.8 +/- 51.75 mIU/10(6) cells per 72 h in cell line RT112 and 503.25 +/- 28.55 to 1361.65 +/- 110.3 mIU/10(6) cells per 72 h in cell line SCaBER. IFN-gamma had no effect on the rate of beta-hCG secretion. Both interferons reduced the growth rate of the cells: incorporation of radiolabelled thymidine was reduced by 15-45% in the presence of IFN-alpha and by 20-53% with IFN-gamma. Enhancement of beta-hCG secretion by IFN-alpha was dose-dependent over the range 5-50,000 U/10(6) cells. Analysis of cell cycle profiles by flow cytometry showed no increase in the proportion of cells in the G0G1 phase in cultures treated with IFN-alpha. The conceptus of some species produces substances which are either luteotrophic or anti-luteolytic. In sheep, the corpus luteum is maintained by ovine trophoblast protein-I, which has been shown to have structural homology with human IFN-alpha. In primates and a few other higher mammals, early pregnancy is maintained by chorionic gonadotrophin. IFN-alpha is also an early product of the human conceptus. We have now shown that IFN-alpha enhances the ectopic production of the beta-subunit of hCG by bladder tumour cells. This study suggests a direct transcription/translational effect of this cytokine on the expression of a reproductive endocrine gene.

Chromatographic characterization of insulin-like growth factor-binding proteins in human pregnancy serum.

Insulin-like growth factor-binding proteins (IGFBPs) in maternal and umbilical cord sera have been analysed by combinations of gel filtration chromatography, affinity cross-linking and electrophoresis. On gel filtration chromatography, the majority of circulating IGF-I in non-pregnant and pregnant women was present in the large molecular mass (150 kDa) binding proteins (IGFBP-3). In umbilical cord serum, by contrast, most IGF-I was present in the 40 kDa binding proteins (consisting of IGFBP-1 and IGFBP-2). Western blots demonstrated an apparent progressive attenuation of IGFBP-3 and IGFBP-2 in serum from pregnant women with an increase in IGFBP-1. After prior cross-linking with disuccinimidyl suberate, the 150 kDa fractions (IGFBP-3) from non-pregnant and pregnant serum showed a similar pattern on SDS-PAGE (several bands at different molecular masses). However, IGFBP-2 (one of the components of the 40 kDa fractions) was undetectable, even after cross-linking, in serum from pregnant women later than 8 weeks of gestational age and in a mixture of maternal serum at term delivery and serum from non-pregnant women. This suggests that serum IGFBP-2 was degraded by specific proteases present in pregnancy serum. Following acid treatment, the 150 kDa fractions from pregnancy serum were split into smaller subunits or fragments while the 40 kDa fractions remained unchanged, suggesting that the 40 kDa binding proteins, are acid-stable. The present data demonstrate that IGFBP-3 is the principal IGFBP in pregnancy serum even though there is an apparent reduction in serum IGFBP-3 activity as revealed on Western blots. The absence of IGFBP-2 in serum from pregnant women may be due to degradation by proteases. In the fetal circulation IGFBP-1 and IGFBP-2 appear to be the major binding proteins for IGF-1.

Is ovarian relaxin a stimulus to placental protein 14 secretion in pregnancy?

The objective of this study was to investigate whether relaxin might be involved in placental protein 14 (PP14) secretion by measuring serum levels of PP14 during labour and post partum in normal women with a term pregnancy given vaginal human recombinant relaxin (rhRlx) gel for induction of labour. A randomized double-blind controlled trial was conducted on 11 women with a singleton pregnancy at term admitted for induction of labour. Comparison of serum PP14 and relaxin concentrations in the control and treated groups of subjects revealed that there was an increase in serum relaxin concentrations in women receiving 3 mg or 6 mg relaxin. There was no difference in serum PP14 levels between the control and treatment groups. These findings do not support the hypothesis that relaxin is involved in the control of PP14 secretion. However, the failure of any response might be a consequence of the very small increase in systemic levels of relaxin produced by topical vaginal administration of rhRlx. Furthermore, these measurements were made in late pregnancy and hence may not relate to the events in early pregnancy, when serum levels of PP14 are maximal.

Circulating levels of IGF-I and IGF-binding protein-1 throughout pregnancy: relation to birthweight and maternal weight.

Serum levels of IGF-I and IGF-binding protein (IGFBP-1) have been determined in the maternal circulation between 11 and 42 weeks of gestation in women not in labour (n = 335) and in the maternal and fetal circulations at the time of delivery between 37 and 42 weeks (n = 55). Maternal serum (MS) IGF-I levels increased during pregnancy and showed a significant positive correlation with maternal weight (P = 0.0033) but no correlation with birthweight. The MS IGFBP-1 levels did not change during the second and third trimesters and showed a negative correlation with birthweight, maternal weight, placental weight and MS glucose (P = 0.0002, P < 0.0001, P = 0.047, P = 0.024 respectively). MS IGFBP-1 levels were higher in small-for-gestational age babies than in average-for-gestational weight babies (P = 0.026) and lower in the large-for-gestational weight group (P = 0.048). There was a significant rise in mean MS IGFBP-1 levels during labour (P = 0.0005). These findings suggest that IGFBP-1 may be an important factor in pathological growth retardation.

The concentration of insulin-like growth factor-I and insulin-like growth factor-binding protein-1 in human umbilical cord serum at delivery: relation to fetal weight.

Serum levels of insulin-like growth factor-I (IGF-I) and insulin-like growth factor-binding protein-1 (IGFBP-1) have been determined by radioimmunoassay in the maternal circulation (n = 91) and in the umbilical artery (n = 56) and vein (n = 90) of man. In both the umbilical artery and vein, the concentration of serum IGF-I showed an inverse correlation with birthweight (P less than 0.005 and P less than 0.001 respectively); the mean serum IGF-I levels in the small-for-gestational-age (SGA) group were significantly higher than those in average-for-gestational-age (AGA) neonates (P less than 0.01 and P less than 0.001 respectively). However, maternal serum IGF-I showed no association with birthweight and there was no significant difference between the SGA and AGA groups. These observations imply that the production of IGF-I in the maternal and fetal compartments is independent and that there is unlikely to be transfer of IGF-I across the placenta. Serum IGFBP-1 levels in both maternal and umbilical cord blood (artery and vein) showed an inverse relation to birthweight (P less than 0.001, P less than 0.005 and P less than 0.001 respectively). Increased IGFBP-1 levels in the umbilical artery and vein were observed in the SGA group. These findings suggest that IGFBP-1 might inhibit the action of IGF-I in both the maternal and the fetal compartments and that the rise in IGFBP-1 could be a primary factor in retardation of fetal growth. Alternatively, circulating IGF-I and IGFBP-1 levels may only be a secondary reflection of local tissue events involved in fetal growth.

Human chorionic gonadotrophin and subunit composition of maternal serum and coelomic and amniotic fluids in the first trimester of pregnancy.

Sixteen matched samples of first trimester amniotic fluid (AF), extraembryonic coelomic fluid (EECF) and maternal serum (MS) were assayed for intact human chorionic gonadotrophin (hCG) and free subunits. Total beta-hCG (free beta-subunit and intact hCG) levels in the EECF (median 410 kIU/l) were 61 times greater than levels in AF (median 6.73 kIU/l) and 2.8 times greater than in MS (median 141.5 kIU/l). Levels of intact hCG in the EECF (median 245 kIU/l) were 142 times greater than in AF (median 1.73 kIU/l) and 1.6 times greater than in MS (median 157 kIU/l). Free alpha-subunit levels in EECF (median 17.3 mg/l) were 66 times greater than in AF (median 0.262 mg/l) and 12 times greater than in MS (median 1.3 mg/l). Virtually all of the total beta-hCG immunoreactivity in MS can be attributed to intact hCG, but only 60% of total beta-hCG in the EECF and 20% of that in AF can be accounted for by the intact hormone. In both EECF and AF the free alpha-subunit was a major constituent; on a molar basis the ratio of free alpha:free beta:intact hCG was 1:1.2:0.3 in AF, 1:0.6:0.5 in EECF and 1:0:5 in MS. Chromatography of MS, EECF and AF on Sephadex G-100 confirmed the hCG and subunit composition of the fluids. On the basis of these findings it seems likely that previous studies showing very high levels of hCG in AF during the first trimester may have incorrectly sampled the EECF. In reality, the levels of total hCG (and free subunits) are low in the AF, and only 20% is intact hCG.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-like growth factor-binding protein-1, glucose tolerance and fetal growth in human pregnancy.

Two hundred Chinese primigravidae had 50 g 3-h oral glucose tolerance tests (OGTTs) twice in pregnancy; between 20 and 24 weeks and between 30 and 34 weeks of gestation. In 149 women, a single sample was taken for insulin-like growth factor-binding protein-1 (IGFBP-1) measurement 0, 1, 2 or 3 h after the glucose load at both visits; in 55 women IGFBP-1 levels were estimated in all four OGTT samples. Fetal growth was assessed by ultrasound performed at the first and second visit and, if possible, at term, and by anthropometry of the neonate. Cord serum IGFBP-1 was measured in 144 of the babies. Mothers who developed gestational diabetes were excluded. Maternal levels of IGFBP-1 were inversely related to glucose levels at 0, 1 and 2 h in the third trimester of pregnancy. IGFBP-1 measured at 1 h in an OGTT increased between the second and third trimester. There was an inverse correlation between maternal IGFBP-1 measured in the second trimester and all fetal measurements at that time, and with most neonatal measurements and birthweight. Levels of IGFBP-1 in the third trimester were inversely correlated to neonatal abdominal circumference, skinfold thickness and birthweight. Cord blood IGFBP-1 was inversely related to growth of abdominal circumference. The strongest inverse relationship was between IGFBP-1 and maternal weight. Fasting glucose in the second trimester was positively correlated to fetal subcutaneous fat and growth of abdominal circumference. In the third trimester it was related to fetal abdominal circumference, the growth of abdominal circumference, birthweight and neonatal skinfold thickness.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification and purification of a new protein from water-soluble extracts of human adrenal gland.

An homogenate of human foetal adrenal gland was subjected to negative immunoabsorption by column chromatography using anti-whole human serum coupled to Sepharose 4B. Two peaks were eluted and used to immunize rabbits. The antisera produced were absorbed and tested for specificity by double immunodiffusion. Two antigens, which appeared to be specific to the adrenal gland, were identified having molecular weights of 25 000 and 65 000 as determined by gel filtration. The lower molecular weight antigen was isolated by physicochemical methods and found to be a protein. The amino acid composition is reported.

Circulating levels of GH-releasing hormone and GH during human pregnancy.

To study the potential role of GH-releasing hormone (GHRH) in maintaining circulating levels of GH during pregnancy, 302 maternal plasma samples were collected from non-fasted subjects at various stages of pregnancy and assayed for GHRH using a 'two-site' immunoradiometric assay. The GH and placental lactogen levels were also determined. In addition, maternal plasma samples taken during labour, amniotic fluid and cord blood were also assayed for these hormones. Maternal plasma GHRH levels were similar to non-pregnant levels throughout gestation despite fluctuations in GH values which were always higher than non-pregnant levels. There was no significant difference between GHRH levels in maternal plasma and cord blood although high GH levels were observed in the latter. These findings suggest that peripheral GHRH levels do not play an important role in maintaining circulating GH levels during pregnancy.

Regulation of human villous trophoblast by insulin-like growth factors and insulin-like growth factor-binding protein-1.

Many studies have implicated the insulin-like growth factors (IGFs) and insulin-like growth factor-binding protein-1 (IGFBP-1) in the control of the feto-maternal interface of human pregnancy, but many of the data are from cell lines derived from primary trophoblast or from extravillous trophoblast. We have obtained highly enriched villous cytotrophoblast (VCT) from first trimester and term human placentae, and investigated the effects of IGF-I, IGF-II and phosphoisoforms of IGFBP-1. First trimester villous trophoblast cells were regulated by all these factors. IGF-II increased cell numbers 3.5-fold after 96 h in culture, and IGF-I had less effect (1.5-fold increase) (both P<0.05). IGF-II also had a greater effect on the levels of matrix metalloproteinase (MMP)-2 and MMP-9. Phosphorylated and non-phosphorylated iso-forms of IGFBP-1 added alone increased cell numbers and MMP levels (P<0.05). IGFBP-1 did not modify the effects of IGF-II on cell numbers or on MMP production. Term VCT numbers and MMP production in vitro were unaffected by IGFs (P>0.05). Cell numbers were increased only by 100 nM IGFBP-1 isoforms (P<0.05), whereas MMP levels released from term cells were optimally increased by 1-10 nM IGFBP-1. Overall, our data show that IGFs regulate only first trimester, but not term, VCT. IGFBP-1 regulates VCT from both gestations, but the effects are concentration and end-point specific. In particular, first trimester cell numbers are more affected by low levels of IGFBP-1, whereas high levels of IGFBP-1 are needed to increase MMP and the converse applies to term VCT; low levels of IGFBP-1 have more effect on MMP levels.