Elevated levels of decidual insulin-like growth factor binding protein-1 in cervical fluid in early and mid-pregnancy are associated with an increased risk of spontaneous preterm delivery.

OBJECTIVE: To study whether elevated levels of decidual insulin-like growth factor binding protein-1 (IGFBP-1) in the cervical fluid of unselected asymptomatic women in early or mid-pregnancy are associated with spontaneous preterm delivery (PTD). DESIGN: Prospective population-based cohort study. SETTING: Maternity Clinics, University Central Hospital, Helsinki, Finland. POPULATION: A total of 5180 unselected pregnant women. METHODS: Cervical swab samples were collected during the first and second trimester ultrasound screening. The concentration of IGFBP-1 was measured by immunoenzymometric assay, which detects the decidual phosphoisoforms of IGFBP-1 (phIGFBP-1). Concentrations of 10 micrograms/l or more were considered to be elevated. MAIN OUTCOME MEASURE: Spontaneous PTD. Results In the first trimester, 24.5% of women, and in the mid-second trimester, 20.2% of women, had an elevated cervical fluid phIGFBP-1 level. The rates of spontaneous PTD before 32 and before 37 weeks of gestation were higher in women with an elevated cervical fluid phIGFBP-1 level, compared with women who had cervical phIGFBP-1 of <10 micrograms/l (1.1% versus 0.3% and 5.7% versus 3.2%, respectively). An elevated phIGFBP-1 level in the first trimester was an independent predictor for PTD before 32 and before 37 weeks of gestation, with odds ratios of 3.0 (95% CI 1.3-7.0) and 1.6 (95% CI 1.2-2.3), respectively. Cervical phIGFBP-1 levels of 10 micrograms/l or more in the first trimester predicted PTD before 32 and before 37 weeks of gestation, with sensitivities of 53.8% and 37.0%, respectively. The negative predictive values were 99.7% and 96.8%. CONCLUSIONS: Elevated cervical fluid phIGFBP-1 levels in the first trimester were associated with an increased risk of spontaneous PTD.

Factors affecting decidual IGFBP-1 levels in the vagina and cervix in the first and mid-second trimester of pregnancy.

OBJECTIVE: The aim of this study was to determine the concentrations of and factors associated with decidual insulin-like growth factor-binding protein-1 (IGFBP-1) in the lower genital tract in early- and mid-gestation in singleton pregnancies. DESIGN: Prospective population-based cohort study. SETTING: Maternity Clinic, Department of Obstetrics and Gynaecology, University Central Hospital, Helsinki, Finland. POPULATION: A total of 1702 unselected pregnant women undergoing the first- and the second-trimester ultrasound screening between April 2005 and December 2006. METHODS: The vaginal and cervical swab samples for assay of decidual IGFBP-1 and vaginal pH measurement were taken before transvaginal ultrasonography in the first trimester and in the mid-second trimester. Use of antibiotics, history of vaginal bleeding, and the history of sexual intercourse were questioned on both occasions. The concentration of IGFBP-1 was measured by a quantitative immunoenzymometric assay, which detects the decidual phosphoisoforms of IGFBP-1 (phIGFBP-1). The concentration of 10 micrograms/l was used as a cutoff when factors influencing phIGFBP-1 levels were analysed. MAIN OUTCOME MEASURES: The phIGFBP-1 concentrations in the vagina and the cervix and associations between the levels of > or =10 micrograms/l and selected factors. RESULTS: In the first trimester, the median (range) concentrations of phIGFBP-1 in vaginal and cervical samples were <0.3 micrograms/l (<0.3-176 micrograms/l) and 4.8 micrograms/l (<0.3-174 micrograms/l), respectively. During the second trimester, the corresponding values were <0.3 micrograms/l (<0.3-55 micrograms/l) in the vagina and 3.6 micrograms/l (<0.3-126 micrograms/l) in the cervix. In the vaginal samples, the frequency of phIGFBP-1 concentrations > or =10 micrograms/l was 5.8% in the first trimester and 1.5% in the second trimester (P < 0.001). In the cervical samples, the corresponding rates were 34.3 and 28.4%, respectively (P < 0.001). Of the factors studied, nulliparity (P < 0.001) and history of vaginal bleeding (P < 0.001) were independently associated with cervical phIGFBP-1 concentrations > or =10 micrograms/l during both trimesters. In addition, short cervical length (<30 mm) was associated with phIGFBP-1 concentration > or =10 micrograms/l in both vaginal and cervical samples in the second trimester in multivariate analysis. CONCLUSIONS: The rate of phIGFBP-1 concentrations > or =10 micrograms/l, both in the vagina and in the cervix, was significantly lower during the second trimester compared with the first trimester. The low rate of levels > or =10 micrograms/l in vaginal samples compared with cervical samples during both trimesters indicates that the exact site of sampling is important when phIGFBP-1 is used as a decidual marker. Nulliparity and history of vaginal bleeding were independently associated with phIGFBP-1 concentrations > or =10 micrograms/l in cervical samples during both trimesters.

Receptors for epidermal growth factor and insulin-like growth factor I and their relation to steroid receptors in human breast cancer.

Levels of epidermal growth factor receptor (EGF-R) and insulin-like growth factor receptor (IGF-R) in breast cancer tissue were evaluated. The binding of growth factors was compared to the content of estrogen receptors (ER) and progesterone receptors (PgR). EGF-R correlated negatively to the ER and PgR (Kendall correlation, P less than 0.001), whereas the IGF-R correlated positively to ER and PgR (analysis of variance, P less than 0.001). In contrast, no correlation was found between EGF-R and IGF-R. IGF-R binding was higher in tumor tissues than in adjacent normal tissues (Wilcoxon rank test, P less than 0.001), whereas the EGF-R binding in normal tissue did not differ from that in cancer tissue. The degree of differentiation in ductal breast cancer correlated to EGF-R (chi 2 test, P = 0.018), but not to IGF-R. The bindings of both growth factors were the same in metastases and primary breast tumors. Our results show that EGF-R and IGF-R are present in normal breast tissue and breast cancer tissue. The growth factor receptors are related to steroid receptor content and their presence is associated with malignant transformation of breast cells and dedifferentiation of breast cancer.

Expression of c-kit ligand messenger ribonucleic acids in human ovaries and regulation of their steady state levels by gonadotropins in cultured granulosa-luteal cells.

The c-kit ligand (KL), a ligand for the c-kit protooncogene receptor tyrosine kinase, is an important regulator of germ cell development in rodent gonads. However, no information about the role of KL in the ovaries of women or higher primates has been available. We studied the expression of KL messenger RNA (mRNA) in human ovaries and the effect of purified hCG and recombinant human FSH (rhFSH) on KL mRNA steady state levels in cultures of human granulosa-luteal (GL) cells obtained at oocyte harvest for in vitro fertilization. KL complementary DNA was generated by reverse transcription-polymerase chain reaction from human ovarian tissue RNA. Two alternatively spliced KL transcripts encoding 248-amino acid (aa) and 220-aa membrane-associated KL proteins were observed in GL cells and ovarian tissue. In Northern blot analysis of human ovarian and GL cell RNA, a major transcript of approximately 6.0 kilobases was detected. Specific mRNA transcripts for KL were detected in dot blot filter hybridization analyses, and the steady state levels of these mRNAs were lowered in cultured GL cells by both gonadotropins in a distinct time- and concentration-dependent manner. The KL mRNA levels of untreated and hCG- or rhFSH-stimulated GL cells were determined at 2- to 3-day intervals between days 2-10 of culture. An 8-h treatment with hCG was shown to decrease KL mRNA levels on days 2, 3, 5, and 7 of culture, whereas rhFSH decreased KL mRNA levels on days 5 and 7 of culture. Time-course and concentration-dependence studies were performed on days 2-7 of culture. Both gonadotropins decreased KL mRNA levels as early as 2 h after treatment. The maximal response to hCG and rhFSH treatment was observed at 7-24 h. Concentration-dependence studies performed 8 or 24 h after treatment indicated that the maximal inhibition occurred with 10-100 ng/ml hCG and 100-300 ng/ml rhFSH. We conclude that 1) the KL transcripts encoding 248- and 220-aa transmembrane proteins are expressed in vivo in the human ovary and in cultured human GL cells; and 2) KL transcript levels are rapidly decreased by gonadotropins in a time- and concentration-dependent manner in cultured GL cells. Thus, KL expression is hormonally regulated in human granulosa cells, and this growth factor may control the function of the ovarian follicle during the human menstrual cycle.

Characterization of functional type I insulin-like growth factor receptors from human choriocarcinoma cells.

Specific receptors for insulin-like growth factor I (IGF-I) on cultured human choriocarcinoma cells (JEG-3 and BeWo) were characterized. The binding of 125I-labeled recombinant (Thr59)IGF-I to the cells was reversible and time, temperature, and pH dependent. Steady state of binding occurred after 16 h at 4 C, pH 7.4. Natural human IGF-I (hIGF-I), hIGF-II, recombinant (N-Met)IGF-I, rat multiplication-stimulating activity, and insulin were 200%, 37%, 37%, 1.6%, and 0.1% as potent as (Thr59)IGF-I in inhibiting the binding of [125I]iodo-(Thr59)IGF-I to JEG-3 cells, respectively. Epidermal growth factor was ineffective. The half-maximal displacement of [125I]iodo-(Thr59)IGF-I by unlabeled (Thr59)IGF-I occurred at 11 +/- 2 ng/ml (mean +/- SEM) in both JEG-3 and BeWo cells. Scatchard analysis of the competitive binding data revealed linear plots indicating a single species of binding sites with an association constant of 0.8 X 10(9) M-1 for the binding of [125I]iodo-(Thr59)IGF-I to both cell lines. The binding capacity was 30,000 and 20,000 sites/cell for JEG-3 and BeWo cells, respectively. Chemical cross-linking of [125I]iodo-(Thr59)IGF-I to JEG-3 cells revealed two receptor complexes of 130K and 260K. Their formation was completely inhibited by an excess of unlabeled (Thr59)IGF-I or hIGF-II. Increasing amounts of insulin affected both labeled bands equally, suggesting that the 130K and 260K bands represent the monomer and dimer forms, respectively, of the ligand-binding alpha-subunit of type I IGF receptor. (Thr59)IGF-I, in a dose-dependent manner, stimulated uptake of nonmetabolizable alpha-[3H]aminoisobutyric acid by JEG-3 cells, showing that the receptor is biologically active. Our results demonstrate that choriocarcinoma cells possess functional high affinity type I IGF receptors and suggest that IGF-I is involved in the growth-regulating processes of JEG-3 and BeWo cells. These cells may provide a useful model to study the role of IGFs in trophoblast physiology.

Insulin-like growth factor (IGF) binding protein from human decidua inhibits the binding and biological action of IGF-I in cultured choriocarcinoma cells.

The placenta expresses genes for insulin-like growth factors (IGFs) and possesses IGF-receptors, suggesting that placental growth is regulated by IGFs in an autocrine manner. We have previously shown that human decidua, but not placenta, synthesizes and secretes a 34 K IGF-binding protein (34 K IGF-BP) called placental protein 12. We now used human choriocarcinoma JEG-3 cell monolayer cultures and recombinant (Thr59)IGF-I as a model to study whether the decidual 34 K IGF-BP is able to modulate the receptor binding and biological activity of IGFs in trophoblasts. JEG-3 cells, which possess type I IGF receptors, were unable to produce IGF-BPs. Purified 34 K IGF-BP specifically bound [125I]iodo-(Thr59)IGF-I. Multiplication-stimulating activity had 2.5% the potency of (Thr59)IGF-I, and insulin had no effect on the binding of [125I] iodo-(Thr59)IGF-I. 34 K IGF-BP inhibited the binding of [125I] iodo-(Thr59)IGF-I to JEG-3 monolayers in a concentration-dependent manner by forming with the tracer a soluble complex that could not bind to the cell surface as demonstrated by competitive binding and cross-linking experiments. After incubating the cell monolayers with [125I]iodo-(Thr59)IGF-I in the presence of purified binding protein, followed by cross-linking, no affinity labeled bands were seen on autoradiography. In contrast, an intensely labeled band at 40 K was detected when the incubation medium was analyzed, suggesting that (Thr59)IGF-I and 34 K IGF-BP formed a complex in a 1:1 molar ratio. Also, 34 K IGF-BP inhibited both basal and IGF-I-stimulated uptake of alpha-[3H]aminoisobutyric acid in JEG-3 cells. RNA analysis revealed that IGF-II is expressed in JEG-3 cells. We conclude that decidual 34 K IGF-BP inhibits the cellular binding and biological action of IGFs in JEG-3 cells. Our data show that JEG-3 cells represent a cell type that can produce IGF, but not IGF-BPs. These cells may thus provide a useful model system for a better understanding of autocrine growth regulation mediated by the IGFs.

Placental protein 12 is a decidual protein that binds somatomedin and has an identical N-terminal amino acid sequence with somatomedin-binding protein from human amniotic fluid.

Placental protein 12 (PP12) was originally isolated from term human placenta and adjacent membranes. Recently we found that the site of PP12 synthesis is decidua but not placenta. In this work, the purity of PP12 was first tested by sodium dodecyl sulfate polyacrylamide slab-gel electrophoresis and by reverse phase HPLC, and the N-terminal amino acid sequence of 15 residues was determined by a liquid-phase sequencer. A single amino acid sequence of Ala-Pro-Trp-Gln-Cys-Ala-Pro-Cys-Ser-Ala-Asp-Glu-Leu-Ala-Leu was obtained showing identity to the known N-terminal amino acid sequence of somatomedin-binding protein from human amniotic fluid. Like the latter, PP12 bound somatomedin (insulin-like growth factor I) as demonstrated in gel chromatography by a shift in the elution pattern of [125I]iodo-insulin-like growth factor I after incubation with PP12. These data show that PP12 is a somatomedin-binding protein and extend through previous literature on PP12 the existing knowledge on the physiology and pathophysiology of somatomedin-binding protein(s) in human reproduction and cancer.

Synthesis of placental protein 12 by human decidua.

The synthesis and secretion of placental protein 12 (PP12) were studied in tissue culture using explants of decidua, amnion, chorion, and placenta from seven full term pregnancies. The total amounts of PP12 in media and tissues were measured by RIA, and new protein synthesis and secretion by decidual explants were demonstrated by the incorporation of [35S]methionine into PP12 after 20 h of incubation with 12.5 microCi/ml [35S]methionine. Cycloheximide was used to study the effect of a protein synthesis inhibitor on the secretion of PP12 by decidua. Significantly more PP12 (P less than 0.001) was released into the medium from decidual explants than from chorion and amnion explants throughout the experimental period of 24 h. When incubated under identical conditions, placental explants released no detectable PP12. In decidual tissues and their culture media, the total amount of PP12 was 127.4% higher after incubation than before incubation (P less than 0.001). No increase was found when chorion and amnion were cultured. The addition of cycloheximide into cultures decreased the total amount of PP12 in the decidua and in its culture medium by more than 50%, indicating that one part of PP12 in decidua was performed and another part was newly synthesized. Decidual explants incorporated [35S]methionine into immunoprecipitable PP12 indicating new PP12 synthesis. In gel filtration, 77% of decidual [35S] PP12 eluted in the same position as purified PP12. In sodium dodecyl sulfate polyacrylamide gel electrophoresis, the migration mobility of [35S]PP12 was identical with that of purified PP12. Our results clearly demonstrate that PP12 is a decidual rather than a placental protein.

Secretory endometrium synthesizes placental protein 14.

Saline extracts of human nonpregnant endometrium were found to contain placental protein 14 (PP14). The tissue PP14 content was highest in the late secretory phase (median, 7.7 mg/g protein; n = 14), whereas proliferative endometrium (n = 8) was either PP14 negative or showed a low PP14 content (median, 0.15 mg/g protein). By immunoperoxidase staining, PP14 was localized in the glandular epithelial cells of endometrium. In tissue culture, secretory endometrium released more PP14 than proliferative endometrium, and cycloheximide markedly decreased this release. Synthesis of PP14 by secretory endometrium was demonstrated by incorporation of [35S]methionine into immunoprecipitable PP14. These results show that PP14 is synthesized and secreted by the nonpregnant endometrium.

Synthesis of placental protein 12 by human endometrium.

We have previously shown that placental protein 12 (PP12) is synthesized and secreted by human term pregnancy decidua in vitro. In the present study, fragments of proliferative and secretory phase endometrium were cultured in media in the presence and absence of progesterone (P) and 17 beta-estradiol (E2) for 96 h. The PP12 concentrations in the media and tissues were measured by RIA, and de novo synthesis was investigated by measuring the incorporation of [35S]methionine into PP12. Before culture, PP12 could not be detected in any proliferative endometria, whereas all secretory endometria contained PP12. All secretory endometria released PP12 into the medium in the presence and absence of added P and E2. Secretory endometria released significantly more PP12 than proliferative endometria. Three of seven proliferative endometria did not release PP12 in the absence of P, but all did so after P had been added. The addition of P to culture medium caused a 2.4-to over 71-fold increase in PP12 secretion over control values in proliferative endometria and up to a 3.5-fold increase in secretory endometrium. E2 had no significant effect. Cycloheximide totally inhibited the PP12 release induced by P from proliferative endometrium, and in secretory endometrium, it either totally blocked PP12 release or inhibited the stimulation due to P. [35S]Methionine was incorporated into immunoprecipitable PP12 in cultures of secretory and P-treated proliferative phase endometria. These results demonstrate de novo synthesis of PP12 by nonpregnant endometrium in tissue culture and suggest that the biosynthesis and secretion of PP12 by nonpregnant endometrium are regulated by P.

Decidual transformation of human extrauterine mesenchymal cells is associated with the appearance of insulin-like growth factor-binding protein-1.

Human extrauterine decidual cells were studied for the presence of insulin-like growth factor-binding protein-1 (IGFBP-1) by using an avidin-biotin complex immunoperoxidase method with a purified monoclonal antibody 6303 against IGFBP-1. It is well established that human decidua expresses mRNA for IGFBP-1 and synthesizes and secretes this protein. We now describe, for the first time, that cells morphologically similar to uterine decidual cells found beneath the peritoneal mesothelium in inguinal hernia and in the cervical mucosa during normal pregnancy and in the mesenchymal tissue of the Fallopian tubes and ovaries at term react with the monoclonal antibody for IGFBP-1 equally to endometrial decidual cells. These results imply that the extrauterine mesenchymal cells, which have undergone decidual transformation, are not only morphologically but also functionally, similar to their counterparts in the uterus. Secondly, the data suggest that IGFBP-1 expression is associated with a special type(s) of cells and a certain stage of differentiation rather than special organs in the body.

Soluble 34K binding protein inhibits the binding of insulin-like growth factor I to its cell receptors in human secretory phase endometrium: evidence for autocrine/paracrine regulation of growth factor action.

The human secretory phase endometrium synthesizes and secrets a 34K insulin-like growth factor (IGF)-binding protein designated placental protein 12. We now report that membrane preparations of human endometrium possess IGF receptors that complete with the soluble binding protein for binding to IGF-I. Multiplication-stimulating activity and insulin were 1% and 0.1% as potent as recombinant (Thr59)IGF-I in inhibiting the binding of [125I](Thr59)IGF-I to endometrial membranes. Scatchard plots for the IGF-I binding data were curvilinear, and the apparent affinities [Ka = 1.4 +/- 0.2 ( +/- SEM) X 10(9) M-1] for (Thr59)IGF-I (high affinity site) did not change during the menstrual cycle. Affinity cross-linking of [125I](Thr59)IGF-I to endometrial membranes revealed two major bands with mol wt of 130K and 260K on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. The 130K band is consistent with the alpha-subunit of the type I IGF receptor. The 260K band is either the type II IGF receptor or represents cross-linking (dimer) of two alpha-subunits of the type I receptor. A less intense band at about 40K was also seen in all membrane preparations. It comigrated with the cross-linked purified 34K binding protein. The band was more intensely labeled when the tracer was cross-linked to proteins in the cytosol fractions of late secretory phase endometria. By specific RIA, the 34K binding protein was detected in the cytosol of late secretory phase endometria only. Newly synthesized binding protein, which contaminated membrane preparations, caused an apparent increase in the binding of (Thr59)IGF-I to the membranes prepared from late secretory phase endometria when studied by competitive binding assay. In contrast, purified binding protein prevented the binding of [125I](Thr59)IGF-I to membrane receptors, as confirmed by affinity cross-linking. These results suggest that the 34K IGF-binding protein, secreted by the human endometrium in a cyclic fashion, has a significant role in inhibiting the receptor binding and, thus, the possible biological action of IGF-I in the endometrium in an autocrine/paracrine manner.

Different insulin-like growth factor binding species in human placenta and decidua.

Previous studies demonstrated that human decidua secretes a 34K insulin-like growth factor binding protein (34K IGF-BP) earlier designated placental protein 12, whereas placenta contains IGF receptors and IGF mRNA. We studied binding of [125I]IGF-I to paired placental and decidual tissues using competitive binding, gel filtration, RIA, and cross-linking techniques, and compared the binding characteristics of these two tissues which are located in close proximity in vivo. The effect of decidual cytosols on [125I]IGF-I binding to placental membranes also was studied. Consistent with previous data the dominating binding species in placental membranes were IGF receptors. In contrast, the binding of [125I]IGF-I to decidual membranes was mainly due to binding species with mol wts of 34K and 39K. The 34K IGF-BP was more readily detected in decidual cytosols than in decidual membranes and little was detected in placental cytosols. Purified 34K IGF-BP as well as decidual cytosols inhibited [125I]IGF-I binding to placental receptors. The inhibitory effect of decidual cytosol on IGF receptor binding was linearly correlated to the decidual content of 34K IGF-BP. The results suggest that the decidual 34K IGF-BP might act as a local modulator of the IGF action at the interface between the decidua and the placenta.

Aging is associated with decreased suppression of insulin-like growth factor binding protein-1 by insulin.

We determined whether aging influences circulating insulin-like growth factor-binding protein-1 (IGFBP-1) concentrations and, if so, whether this effect is explained by altered regulation of IGFBP-1 by insulin. Fasting levels of glucose, insulin, and IGFBP-1 were measured in 94 healthy volunteers, ages 24-93 yr. To determine the effect of aging on insulin-induced suppression of IGFBP-1, an oral glucose tolerance test (OGTT) was performed in 10 older (72-92 yr) and 10 younger (24-58 yr) nonobese subjects, matched for sex and body mass index. For all ages combined, the mean glucose level (+/- SE) averaged 4.9 +/- 0.1 mmol/L, and there was no significant change with aging. Fasting insulin and IGFBP-1 concentrations increased with advancing age (r = 0.37, P < 0.001 for age vs. insulin and r = 0.47, P < 0.001 for age vs. IGFBP-1). However, there was no correlation between insulin and IGFBP-1 concentrations. In multiple linear regression analysis, the age-related increase in IGFBP-1 was independent of body mass index. During the OGTT, the mean insulin concentration was significantly higher in the older group compared with the younger group (P < 0.001). Serum IGFBP-1 concentrations were higher in the fasting state as well as during the OGTT, and the mean percent decrease of IGFBP-1 below baseline was significantly smaller in the older compared to the younger subjects at 3 h (35 +/- 5% vs. 55 +/- 2%, P < 0.01). We conclude that aging is associated with decreased suppression of serum IGFBP-1 by insulin, as demonstrated by 1) lack of the inverse correlation between fasting insulin and IGFBP-1 seen in young adults; 2) concurrent elevation of fasting insulin and IGFBP-1 concentrations; and 3) a blunted decrease in serum IGFBP-1 during an OGTT.

Intrauterine progestin induces continuous insulin-like growth factor-binding protein-1 production in the human endometrium.

The effect of local intrauterine progestin on endometrial insulin-like growth factor-binding protein-1 (IGFBP-1) production was studied in 60 women using a levonorgestrel-releasing intrauterine device (IUD). Intrauterine progestin was a potent stimulator of stromal cell IGFBP-1 production, with 97% of endometrial specimens showing positive staining by immunohistological methods. After 5 yr or more of intrauterine progestin exposure, 100% (n = 20) of the tissues remained strongly positive for IGFBP-1. The IGFBP-1 content in endometrial tissue homogenates reached values as high as 10 micrograms/mg protein when measured by immunoradiometric assay. In contrast to the continuous endometrial IGFBP-1 production induced by local progestin, no such effect could be found in endometria from subjects with sc progestin-releasing implants or copper IUDs. Although the levonorgestrel-releasing IUD had a striking effect on local endometrial IGFBP-1 production, it had no effect on serum IGFBP-1 levels. By Western ligand blot analysis, the domainating IGF-binding species in endometria exposed to intrauterine progestin was of 28K mol wt, corresponding to IGFBP-1, whereas no IGFBP species of 31-43K, corresponding to IGFBP-2 or IGFBP-3, were detected.

Evidence of high circulating testosterone in women with prior preeclampsia.

Women with prior preeclampsia are characterized by hyperinsulinemia and a 2- to 3-fold excess risk of hypertension and ischemic heart disease in later life. We therefore studied whether these women present changes in pituitary, ovarian, and endothelial factors that could also affect the risk of vascular disorders. Twenty-two women with prior preeclampsia and 22 control women matched by age and body mass index were studied an average of 17 yr after delivery. Women with prior preeclampsia had elevated serum free testosterone levels (20.6 +/- 2.2 vs. 15.0 +/- 1.3 pmol/L, mean +/- SE, P = 0.03), an elevated free androgen index (3.2 +/- 0.5 vs. 1.9 +/- 0.2, P = 0.04), and an elevated free testosterone estradiol ratio (0.089 +/- 0.017 vs. 0.046 +/- 0.006, P = 0.02). The levels of insulin-like growth factor binding protein-1 decreased as expected during a 3-h oral glucose tolerance test without differences between the groups. Levels of FSH, LH, androstenedione, dehydroepiandrosterone sulfate, and endothelin-1, as well as urinary output of prostacyclin and thromboxane A2 metabolites, showed no difference between study groups. A history of preeclampsia an average of 17 yr earlier thus appears to be associated with elevated levels of testosterone, which may contribute to the increased risk of vascular morbidity in such women.

Portal insulin concentrations rather than insulin sensitivity regulate serum sex hormone-binding globulin and insulin-like growth factor binding protein 1 in vivo.

Serum sex hormone-binding globulin (SHBG) and insulin-like growth factor-binding protein 1 (IGFBP-1) concentrations have been suggested to be useful markers of insulin sensitivity. As the production of both proteins is inhibited by insulin in the liver, we postulated that their concentrations reflect whole body insulin sensitivity only when the latter parallels changes in endogenous insulin secretion. To test this hypothesis, we determined SHBG and IGFBP-1 concentrations, whole body insulin sensitivity (euglycemic insulin clamp; serum free insulin, approximately 400 pmol/L), and serum insulin and C peptide concentrations in 13 type 1 diabetic patients lacking endogenous insulin secretion and 34 matched normal subjects. Whole body insulin sensitivity was 50% lower in the type 1 diabetic patients (20 +/- 3 mumol/kg.min) than that in the normal subjects (40 +/- 3 mumol/kg.min; P < 0.001). Despite this, serum SHBG (45 +/- 4 vs. 29 +/- 2nmol/L; P < 0.002) and IGFBP-1 (14 +/- 3 vs. 2 +/- 1 micrograms/L; P < 0.002) concentrations were increased in the type 1 diabetic patients. In the normal subjects, SHBG (r = -0.49; P < 0.01) and IGFBP-1 (r = -0.49; P < 0.01) were inversely correlated with serum C peptide and positively correlated with whole body insulin sensitivity (r = 0.54; P < 0.005 and r = 0.54; P < 0.005, respectively). In the type 1 diabetic patients, SHBG and IGFBP-1 concentrations were disproportionately increased when related to insulin sensitivity, but appropriate when related to estimated portal insulin concentrations. Serum T4, free testosterone, and estradiol concentrations were similar in both groups. We conclude that SHBG and IGFBP-1 reflect hepatic insulinization and can only be used as markers of insulin sensitivity in individuals with intact insulin secretion.

Cellular localization of c-jun messenger ribonucleic acid and protein and their relation to the proliferation marker Ki-67 in the human endometrium.

We studied cellular c-jun messenger RNA (mRNA) expression in the human endometrium during the menstrual cycle (n = 47) and in human decidua during pregnancy (n = 8), by using digoxigenin-labeled RNA probes in in situ hybridization. The same tissue samples were also analyzed for c-Jun protein and the proliferation marker Ki-67. In the proliferative endometrium, strong expression of c-jun was detected in luminal and glandular epithelium as well as in fibroblast-like stromal cells. During the early luteal phase, strong hybridization signals were identified in both epithelial and stromal compartments, with the strongest hybridization in the stromal cells beneath the epithelium. c-jun mRNA was markedly diminished in luminal and glandular epithelia of mid- and late secretory phase endometria, but it remained unchanged in the stroma. Regardless of the phase of the menstrual cycle, significant hybridization was identified in endothelial cells in the endometrium and myometrium, and a low signal was detected in myometrial muscle cells as well. During early gestation, weak expression of c-jun mRNA was observed in glandular epithelial cells and in decidualized stromal cells. In term pregnancy decidua, only low-level hybridization was detected in a few decidual cells. Nuclear immunostaining of c-Jun was detected in luminal and glandular epithelia and in stroma throughout the menstrual cycle. The location of Ki-67 antigen was temporally related to the c-jun mRNA expression in cycling endometrium and pregnancy decidua. From our data we conclude that 1) c-jun mRNA is differentially expressed in endometrial epithelial and stromal cells; 2) c-jun mRNA is cyclically regulated in the human endometrial epithelium; 3) c-jun mRNA expression is temporally related to epithelial proliferation in the endometrium; and 4) c-Jun protein is present in the human endometrium throughout the menstrual cycle.

Pregnancy-specific beta 1-glycoprotein and chorionic gonadotropin-like immunoreactivity during the latter half of the cycle in women using intrauterine contraception.

In a cross-sectional study, the serum levels of pregnancy-specific beta 1-glycoprotein (PSBG), hCG, human LH, and progesterone were measured by RIAs during 94 mid or late luteal phases of 69 women using oral contraceptives. Subsequent spontaneous menstruation took place in every cycle. None of the women using oral contraceptives had any PSBG or hCG-like immunoreactivity in serum. In women with intrauterine devices, PSBG was found in six cycles (6.4%) and hCG-like immunoreactivity was demonstrated in one cycle only, where PSBG also was present. In two out of six PSBG-positive cycles, menstruation was delayed by 5 and 16 days. Although rare, the transient occurrence of trophoblastic antigens in women using intrauterine contraception is taken as evidence for an occult pregnancy in these apparently infertile cycles.

PIP: Serum levels of pregnancy-specific beta-1-glycoprotein (PSBG), human chorionic gonadotropin (hCG), human luteinizing hormone (LH), and progesterone were measured by radioimmunoassays during 94 mid- or late luteal phases of 69 women using IUDs and 34 women using oral contraceptives (OCs) in a cross-sectional study. hCG-like immunoreactivity was found in 1 of 94 cycles (1.1%) of women using IUD contraception, and in none of those taking OCs. The positive hCG reaction was found on Day 27 of the cycle. In the hCG-positive sample, hCG concentration was 19 mIU/ml. Cross-reaction by LH was unlikely since the LH concentration was 82 ng/ml, well below the cross-reacting level of the assay. PSBG was found in 6 of 94 cycles (6.4%) of IUD users, and in none of OC users. Levels varied from 11-21 ng/ml, and PSBG was found on Days 24-27 of the cycle. 1 cycle demonstrated both hCG and PSBG, and the PSBG concentration was 15 ng/ml. In trophoblastic antigen-positive IUD cycles, menstruation was delayed from the expected date by 3 or more days in 2 cases. In the case where both markers were found, menstruation was delayed by 16 days. Aside from menstrual delay in 2 subjects, no typical changes were noted in the bleeding pattern of those cycles where trophoblast-specific antigens were detected. Although rare, the transient occurrence of trophoblastic antigens in women using IUDs was taken as evidence for occult pregnancy in these apparently infertile cycles.

Relationship between carbohydrate metabolism and serum insulin-like growth factor system in postmenopausal women: comparison of endometrial cancer patients with healthy controls.

Insulin is a major regulator of circulating insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1), suppressing the hepatic production of IGFBP-1. Postmenopausal age, obesity, hypertension, and impaired glucose tolerance, which are known risk factors for endometrial cancer, are all associated with hyperinsulinemia and insulin resistance. In this study, we investigated the relationship among serum insulin, glucose, insulin-like growth factors (IGF-I and IGF-II), and IGFBP-, -2, and -3 in 32 nondiabetic postmenopausal women with endometrial cancer and in 18 healthy controls. The mean fasting levels of glucose and insulin were higher, whereas the mean basal IGF-I, IGF-II, and IGFBP-3 levels were lower in the endometrial cancer patients than in the healthy control subjects. The mean fasting IGFBP-1 and IGFBP-2 levels did not differ between the groups, and no correlation was found between fasting insulin and IGFBP-1 concentrations or between insulin and IGFBP-2 concentrations in either of the study groups. During an oral glucose tolerance test, the mean glucose levels at 1 and 3 h as well as the mean insulin level at 3 h were significantly higher in the endometrial cancer patients than in the controls, and the area under the glucose curve was larger in the first group. An oral glucose load resulted in a similar fall in serum IGFBP-1 levels in endometrial cancer patients and controls (51% and 55% at 3 h). When the cancer patients were divided into two subgroups according to the body mass index (kilograms per m2), the obese group had higher glucose and insulin indices than the nonobese group. No difference was found by the same measures in healthy controls. The fasting serum IGFBP-1 levels tended to be lower in the obese than in the normal weight subjects, but the difference did not reach statistical significance. In summary, these results provide preliminary evidence that the inverse relation between fasting insulin and IGFBP-1, well established in children and young adults, disappears in elderly women, although short term suppression by insulin still occurs. Further, our data indicate that in addition to carbohydrate metabolism, postmenopausal women with endometrial cancer have alterations in their circulating IGF system compared to controls.