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Trypsin has been identified as a pancreatic protease comprising three isoenzymes, trypsin-1,-2, and-3. However, the gene for trypsinogen-3, PRSS3, also gives rise to additional variants, trypsinogen-4A and B, which differ from trypsinogen-3 only with respect to the leader-peptide part, and when activated are identical to trypsin-3. The unique overlapping leader peptides of trypsinogen-4A and B allowed us to develop a specific sandwich-type immunofluorometric assay that detects both these isoforms, but not trypsinogen-3 or activated trypsinogen-4. We measured the concentrations of trypsinogen-4 in various cell line lysates and bile of primary sclerosing cholangitis patients. Lysates of cell lines MDA-MB-231 and PC-3, and astrocytes contained trypsinogen-4, while the conditioned media from these cells did not, suggesting that trypsinogen-4, lacking a classical signal sequence, is not secreted from the cells. Interestingly, 5.7% of the 212 bile samples analyzed contained measurable (>2.4 mu g/l) trypsinogen-4. In conclusion, we have established a specific assay for trypsinogen-4 and demonstrated that trypsinogen-4 can be found in biological samples. However, the clinical utility of the assay remains to be established.

The concentrations of several diagnostic markers have been found to increase dramatically in critically ill patients with a severe disturbance of normal physiological homeostasis, without indication of the diseases they are normally associated with. To prevent false diagnoses and inappropriate treatments of critically ill patients, it is important that the markers aiding the selection of second-line treatments are evaluated in such patients and not only in the healthy population and patients with diseases the markers are associated with. The levels of trypsinogen isoenzymes, the trypsin inhibitor serine peptidase inhibitor Kazal type 1 (SPINK1), hCG and hCG beta, which are used as pancreatitis and cancer markers, were analyzed by immunoassays from serum samples of 17 adult patients who have undergone surgery of the ascending aorta during hypothermic circulatory arrest (HCA) with optional selective cerebral perfusion. Highly elevated levels of trypsinogen-1, -2 and -3, SPINK1 and hCG beta were observed in patients after HCA. This was accompanied by increased concentrations of S100 beta and NSE. In conclusion, this study highlights the importance of critically evaluating the markers used for aiding selection of second line of treatments in critically ill patients.

Glycodelin is a glycoprotein mainly expressed in well-differentiated epithelial cells in reproductive tissues. In normal secretory endometrium, the expression of glycodelin is abundant and regulated by progesterone. In hormone-related cancers glycodelin expression is associated with well-differentiated tumors. We have previously found that glycodelin drives epithelial differentiation of HEC-1B endometrial adenocarcinoma cells, resulting in reduced tumor growth in a preclinical mouse model. Here we show that glycodelin-transfected HEC-1B cells have repressed protein kinase C delta (PKC delta) activation, likely due to downregulation of PDKI, and are resistant to phenotypic change and enhanced migration induced by phorbo112-myristate 13-acetate (PMA). In control cells, which do not express glycodelin, the effects of PMA were abolished by using PKC6 and PDKI inhibitors, and knockdown of PKC delta, MEK1 and 2, or ERK1 and 2 by siRNAs. Similarly, transforming growth factor beta (TGF beta)-induced phenotypic change was only seen in control cells, not in glycodelin-producing cells, and it was mediated by PKC delta. Taken together, these results strongly suggest that PKC delta, via MAPI( pathway, is involved in the glycodelin-driven cell differentiation rendering the cells resistant to stimulation by PMA and TGF beta. (C) 2016 Elsevier Inc. All rights reserved.

OBJECTIVE Gestational diabetes mellitus (GDM) is a common metabolic disorder of pregnancy. Patients with GDM are at risk for high fetal mortality and gestational complications associated with reduced immune tolerance and abnormal carbohydrate metabolism. Glycodelin-A (GdA) is an abundant decidual glycoprotein with glycosylation-dependent immunomodulatory activities. We hypothesized that aberrant carbohydrate metabolism in GDM was associated with changes in glycosylation of GdA, leading to defective immunomodulatory activities. RESEARCH DESIGN AND METHODS GdA in the amniotic fluid from women with normal (NGdA) and GDM (DGdA) pregnancies was purified by affinity chromatography. Structural analysis of protein glycosylation was preformed by lectin-binding assay and mass spectrometry. Cytotoxicity, cell death, cytokine secretion, and GdA binding of the GdA-treated lymphocytes and natural killer (NK) cells were determined. The sialidase activity in the placental tissue from normal and GDM patients was measured. RESULTS GDM affected the glycosylation but not the protein core of GdA. Specifically, DGdA had a lower abundance of α2-6–sialylated and high-mannose glycans and a higher abundance of glycans with Sda (NeuAcα2-3[GalNAcβ1-4]Gal) epitopes compared with NGdA. DGdA had reduced immuosuppressive activities in terms of cytotoxicity on lymphocytes, inhibitory activities on interleukin (IL)-2 secretion by lymphocytes, stimulatory activities on IL-6 secretion by NK cells, and binding to these cells. Desialylation abolished the immunomodulation and binding of NGdA. Placental sialidase activity was increased in GDM patients, which may account for the reduced sialic acid content of DGdA. CONCLUSIONS Taken together, this study provides the first direct evidence for altered enzymatic glycosylation and impaired bioactivity of GdA in GDM patients.

background: The maternal –fetal interface has a unique immunological response towards the implanting placenta. It is generally accepted that a T-helper type-2 (Th-2) cytokine prevailing environment is important in pregnancy. The proportion of Th-2 cells in the peripheral blood and decidua is significantly higher in pregnant women in the first trimester than in non-pregnant women. Glycodelin-A (GdA) is a major endocrine-regulated decidual glycoprotein thought to be related to fetomaternal defence. Yet the relationship between its immunoregulatory activities and the shift towards Th-2 cytokine profile during pregnancy is unclear. methods: GdA was immunoaffinity purified from human amniotic fluid. T-helper, T-helper type-1 (Th-1) and Th-2 cells were isolated from the peripheral blood. The viability of these cells was studied by XTT assay. Immunophenotyping of CD4/CD294, cell death and GdA-binding were determined by flow cytometry. The mRNA expression, surface expression and secretion of Fas/Fas ligand (FasL) were determined by quantitative polymerase chain reaction, flow cytometry and ELISA, respectively. The activities of caspase-3, -8 and -9 were measured. The phosphorylation of extracellular signal-regulated kinases (ERK), p38 and, c-Jun N-terminal kinase was determined by western blotting. results: Although GdA bound to both Th-1 and Th-2 cells, it had differential actions on the two cell-types. GdA induced cell death of the Th-1 cells but not the Th-2 cells. The cell death was mediated through activation of caspase -3, -8 and -9 activities. GdA upregulated the expression of Fas and inhibited ERK activation in the Th-1 cells, which might enhance the vulnerability of the cells to cell death caused by a trophoblast-derived FasL. conclusions: The data suggest that GdA could be an endometrial factor that contributes to the Th-2/Th-1 shift during

Background The study was conducted to assess the effects of levonorgestrel (LNG) on hormonal behavior and on the secretory pattern of intrauterine glycodelin at the midcycle of ovulatory women. Study Design Thirty healthy sterilized women with normal ovarian function were studied during one control untreated cycle and one LNG-treated cycle. In the treated cycle, each woman received two doses of 0.75 mg of LNG 12 h apart during the preovulatory phase approximately 2 days before the LH surge. Daily follicle development recordings were performed until follicle rupture was observed, and serum glycodelin, LH, estradiol, estrone and progesterone were measured as well. In addition, glycodelin concentrations were assayed in uterine flushing obtained on Days LH+1 and LH+12. Results LNG did not modify follicle rupture in 20 of 30 women. In spite of ovulatory progesterone and the occurrence of follicle rupture in these women, luteal phase length was significantly decreased, as well as the serum concentrations of LH, estradiol and estrone in the periovulatory phase. Glycodelin in serum and uterine flushings was significantly elevated in the periovulatory phase when compared to control cycles. Conclusions LNG taken at the dose used in emergency contraception before the LH surge increased prematurely serum and intrauterine concentrations of glycodelin at the time of ovulation. Since there are well established glycodelin inhibitory effects upon fertilization, these results may represent an additional action of LNG in situations where the intervention did not interfere with ovulation.

Glycodelin (encoded by PAEP gene) is a secreted lipocalin protein mainly expressed in reproductive tissues, but also in several tumour types. In the breast, glycodelin is expressed both in normal epithelial and cancerous tissue. To investigate the association of glycodelin with clinicopathological features of breast cancer and outcome of patients we evaluated the protein expression of glycodelin in a large series of breast tumours. Immunohistochemical analysis of tissue microarrays was used to study glycodelin expression on 399 sporadic and 436 familial non-BRCA1/2 tumours with strong family history. Gene expression analysis was used to define genes co-expressed with PAEP in sporadic and familial non-BRCA1/2 breast tumours. In the sporadic series, the glycodelin expression associated with low proliferation rate (P < 0.001), with a tendency towards well-differentiated tumours (grades 1 and 2, P = 0.012) and high cyclin D1 (P = 0.034) expression. However, in familial non-BRCA1/2 cases with strong family history glycodelin expression associated with a less favourable phenotype, i.e. positive lymph node status (P = 0.003) and HER2-positive tumours (P = 0.009). Moreover, the patients with glycodelin-positive tumours had an increased risk for distant metastases (P = 0.001) and in multivariate analysis glycodelin expression was an independent predictor of metastasis (hazard ratio (HR) = 2.22, 95% confidence interval (95% CI) = 1.22–4.03, P = 0.009) in familial non-BRCA1/2 breast cancer. Gene expression analysis further revealed different gene expression profiles correlating with the PAEP expression in the sporadic and familial non-BRCA1/2 breast cancers. Our findings suggest differential progression pathways in the sporadic and familial non-BRCA1/2 breast tumours expressing glycodelin.

Glycodelin is an endocrine-regulated glycoprotein that has significant effects on immune cells, apoptosis, reproduction, cell adhesion, differentiation and cancer. In reproduction, glycodelin contributes to capacitation and immunoprotection of spermatozoa, and it modulates sperm–oocyte binding, acrosome reaction and implantation. In endocrine-related cancer, the differentiation inducing effects of glycodelin are accompanied by growth restriction of malignant cells, decreased expression of oncogenes, increased expression of tumour suppressor genes and morphological reversion of the malignant phenotype. This review features these properties and clinical connections, highlighting the role of glycosylation in biological actions.

Glycodelin is a lipocalin family glycoprotein expressed mainly in reproductive tissues. It is involved in cell recognition, and its relationship with epithelial differentiation is well established. Glycodelin actually appears to drive epithelial differentiation. The evidence comes from studies employing endometrial and breast cancer cell lines. First, transfection of glycodelin cDNA into glycodelin-negative carcinoma cells results in reduced expression of oncogenes, increased expression of tumor suppressor genes, increased cell differentiation, and reduced carcinoma cell growth. Second, histone deacetylase inhibitors (HDACIs) induce glycodelin synthesis in endometrial cancer cells concomitantly with cell differentiation. This effect is blocked by specific down-regulation of glycodelin by RNA interference, suggesting that the effects of HDACIs are mediated by glycodelin. We recently found that glycodelin not only reduces carcinoma cell growth in vitro, but glycodelin cDNA transfection to MCF-7 breast carcinoma cells also reduces growth of these cells in vivo, demonstrated by xenograft tumor growth in mouse mammary fat pads. These results strongly suggest that glycodelin acts as a tumor suppressor in breast cancer. The findings are compatible with the observations that certain types of glycodelin-expressing ovarian and breast cancers have a more favorable prognosis compared to glycodelin non-expressing tumors. This research has therefore introduced a novel mechanism to control cancer cell growth. In this communication we review the differentiation-related effects of glycodelin.

Glycodelin is an example of a glycoprotein whose complex-type glycans mediate biological actions in human reproduction and immune reactions. Being attached to an identical protein backbone, glycodelin oligosaccharides vary significantly from one reproductive tissue to another and have an effect on its own secretion and role in cell communication. For instance, uterine glycodelin-A inhibits sperm-oocyte interaction by binding on the sperm head. This is a glycosylation-dependent phenomenon, in which fucosyltransferase-5 plays a key role. Glycodelin-S from seminal plasma binds evenly around the sperm head and maintains an uncapacitated state in the spermatozoa, until the isoform is detached during sperm passage through the cervix. Glycodelin-F from follicular fluid and Fallopian tube binds to the acrosomal region of the sperm head, thereby inhibiting both the sperm-oocyte binding and premature progesterone-induced acrosome reaction. The cumulus cells surrounding the oocyte can capture glycodelin-A and -F from the surrounding environment and convert these isoforms to a cumulus cell isoform, glycodelin-C. It differs by glycosylation from the other isoforms, and it too attaches on the sperm head, with the highest density in the equatorial region. Glycodelin-C is capable of detaching the sperm-bound inhibitory isoforms so that the sperm-oocyte binding is enhanced. Glycodelin-A also has immunosuppressive actions directed to cellular, humoral and innate immunity. Although these actions depend mainly on the protein backbone, glycosylation also plays a part. Glycosylated glycodelin may be involved in the protection of spermatozoa against maternal immune reactions, and glycodelin also has apoptogenic activity. Some glycosylation patterns of glycodelin may mask its apoptogenic domain. This review updates the recent research and clinical associations of glycodelin, highlighting the role of glycosylation.

Fertilization depends on successful binding of the spermatozoa to the zona pellucida of the oocyte. Glycodelin-A inhibits spermatozoa-zona pellucida binding. Previous data showed that glycodelin-A receptor(s) and zona pellucida protein receptor(s) on human spermatozoa are closely related. Using a chemical cross-linking approach, the glycodelin-A-sperm receptor complex was isolated. The receptor was identified to be fucosyltransferase-5 (FUT5) by mass spectrometry and confirmed with the use of anti-FUT5 antibodies. Sperm FUT5 was an externally oriented integral membrane protein in the acrosomal region of human spermatozoa. Biologically active FUT5 was purified from spermatozoa. Co-immunoprecipitation confirmed the interaction between glycodelin-A and sperm FUT5. Solubilized zona pellucida reduced the binding of glycodelin-A to sperm FUT5. An anti-FUT5 antibody and FUT5 acceptor blocked the binding of glycodelin-A to spermatozoa and the zona binding inhibitory activity of glycodelin-A. Sperm FUT5 bound strongly to intact and solubilized human zona pellucida. The equilibrium dissociation constant of sperm FUT5 binding to solubilized zona pellucida was 42.82 pmol/ml. These observations suggest that human sperm FUT5 is a receptor of glycodelin-A and zona pellucida proteins, and that glycodelin-A inhibits spermatozoa-zona binding by blocking the binding of sperm FUT5 to the zona pellucida.

Objective. Extracellular matrix (ECM) components regulate growth and differentiation of epithelial cells, and impaired cell–ECM interaction may lead to increased cell proliferation and tumorigenesis. Whereas ECM has been shown to alter cellular morphology and reduce proliferation of HEC-1B endometrial adenocarcinoma cells, little is known about the underlying changes in gene expression. The purpose of this study was to investigate these changes. Methods. We studied by cDNA array the effects of ECM components, as present in Matrigel™ basement membrane cell culture matrix, on gene expression in HEC-1B endometrial adenocarcinoma cells in respect of the same cells cultured on conventional plastic surface. Some of the changes were confirmed by protein analyses. Results. As expected, several growth-promoting genes were downregulated, while many genes associated with growth restriction were upregulated in Matrigel-grown carcinoma cells. Also, the expression of many 20S proteasome components was downregulated. The observed changes point to a less malignant phenotype of Matrigel-grown tumor cells, supported by reduced growth characteristics and morphology. Conclusion. The study provides further insight into the mechanisms whereby ECM components may participate in the regulation of cell growth—by reducing expression of growth-promoting genes and increasing expression of the genes associated with growth restriction.

Objective Glycodelin is a major secretory glycoprotein of differentiated endometrial epithelium, rarely expressed in proliferative endometrium or endometrial cancer. We aimed to elucidate its role in growth and gene expression of endometrial adenocarcinoma cells, and hypothesized that glycodelin affects cell growth and tumor-associated gene expression. Study design Endometrial adenocarcinoma HEC-1B cells were transfected with glycodelin cDNA in both antisense and sense orientations. Cellular morphology, cell proliferation, and gene expression were compared between native and transfected cells. Results Compared with native and antisense-transfected carcinoma cells, sense-transfected, glycodelin-producing carcinoma cells showed reduced proliferation, morphologic changes, and altered expression of cancer-related genes. Notably, anti-apoptotic Bcl-X L and MUC1 genes were down-regulated. Conclusion Reduction by glycodelin transfection of carcinoma cell proliferation and expression of MUC1 and Bcl-X L is significant because these genes are often overexpressed in human cancers—MUC1 is linked to invasive growth and metastases, and both confer resistance to chemotherapy. These results suggest a novel mechanism whereby malignant growth of endometrial adenocarcinoma cells is regulated.

Two binding proteins, SHBG and IGF-binding protein-1 (IGFBP-1), are both down-regulated by insulin and therefore could serve as potential indicators of the metabolic syndrome and hyperinsulinemia-related cardiovascular risk. We compared serum SHBG and IGFBP-1 as potential markers of abnormal glucose tolerance, the metabolic syndrome, diabetes mellitus, cardiovascular risk factors, and total, cardiovascular, and coronary heart disease mortality in elderly men. Of the original cohort of 1711 men, 524 were alive on January 1, 1989, and 413 participated in the 30-yr examination, of whom 335 men, aged 70-89 yr, formed the study group for the present analysis. Low SHBG and IGFBP-1 were both associated with an increased prevalence of abnormal glucose tolerance and the metabolic syndrome, but only SHBG was associated with diabetes mellitus. SHBG was less influenced by body mass index than IGFBP-1. Low SHBG indicated increased cardiovascular and coronary disease mortality; the association remained after adjustment for abnormal glucose tolerance, but not after adjustment for prevalent cardiovascular disease. IGFBP-1 had no association with mortality. It is concluded that low SHBG is a better indicator of increased cardiovascular mortality than low or high IGFBP-1.

The relation of maternal cytokine levels to retinopathy progression during diabetic pregnancy is a less studied subject. Therefore, we investigated levels of systemic proinflammatory markers, C-reactive peptide (CRP), interleukin-6 (IL-6) and circulating vascular cell adhesion molecule-1 (VCAM-1) during pregnancy and postpartum in relation to the progression of diabetic retinopathy (DR).A prospective follow-up study of 39 pregnant women with Type I diabetes and eight nondiabetic pregnant women was performed. DR was graded from fundus photographs. Plasma levels of systemic proinflammatory markers were measured by immunofluorometric assay (CRP) and by enzyme-linked immunosorbent assay (IL-6 and VCAM-1) in the first, second (diabetics only), third trimester of pregnancy, and 3 and 6 months postpartum (diabetics only).Our diabetic women had good glycaemic control (HbA1c 6.9 +/- 0.8). The levels of IL-6, VCAM-1, and CRP did not differ between diabetic and nondiabetic women throughout pregnancy and postpartum (repeated measures ANOVA between the groups). An association between CRP and progression of retinopathy was observed in diabetic women (P = 0.037). Additional evidence of inter-relationship could be revealed as CRP was higher in those diabetic women with worse glycaemic control (HbA1c) (P = 0.038).During pregnancy and postpartum, levels of proinflammatory factors (IL-6, CRP, VCAM-1) seem to be generally similar in Type I diabetic women compared to nondiabetic controls. However, CRP levels were higher in those diabetic women with progression of retinopathy and in those with worse glycaemic control.

Aims To evaluate the role of systemic angiopoietic factors in the progression of diabetic retinopathy during pregnancy. Methods In a prospective study of 26 pregnant women with diabetes and eight non-diabetic pregnant women, retinopathy was graded from fundus photographs. Plasma levels of angiopoietin-1, angiopoietin-2, human vascular endothelial growth factor A (hVEGF-A), and total soluble receptor of vascular endothelial growth factor (sVEGF) receptor-1 were measured during the first and third trimester and 3 months postpartum. Results In diabetic women, levels of angiopoietin-2 were 26.5 ng/ml (12.1–47.7) (median and range) during the first trimester, 2.9 ng/ml (0.6–3.5) during the third trimester, and 0.5 ng/ml (0.3–0.7) 3 months postpartum, compared with 44.3 (38.3–61.9), 5.7 (3.1–8.4) and 0.9 (0.6–4.9) ng/ml, respectively, in non-diabetic women (P = 0.002 between groups). Levels of angiopoietin-1 and sVEGF receptor-1 did not differ between the groups. Postpartum hVEGF-A levels were lowest in women with progression of retinopathy. In logistic regression analyses, progression of retinopathy during pregnancy was not explained by the levels of the angiopoietic factors. Conclusions The circulating levels of angiopoietic factors in pregnant diabetic women were either lower than (Ang-2) or similar to (Ang-1, hVEGF-A, VEGFR-1) those levels observed in non-diabetic pregnant women. The levels of angiopoietic factors measured here appear not to be connected with the progression of retinopathy during pregnancy.

The polycystic ovary syndrome (PCOS) is associated with an increased rate of early pregnancy loss (EPL). Hyperinsulinemia is an independent risk factor for EPL and has been found to decrease levels of glycodelin and IGF binding protein-1 (IGFBP-1), two major endometrial proteins. We hypothesized that serum glycodelin IGFBP-1 concentrations would be reduced in women with PCOS during the first trimester of pregnancy. Fasting serum insulin, glycodelin, and IGFBP-1 were measured, and oral glucose tolerance tests were performed in 72 women with PCOS and 62 normal women. Each woman was seen once and assigned to one of three gestational groups: wk 3-5, 6-8, and 9-11. The insulin sensitivity index during oral glucose tolerance test was lower in women with PCOS compared with normal women throughout the first trimester (P0.0001). Both serum glycodelin and IGFBP-1 were markedly lower in women with PCOS (for glycodelin: wk 3-5, P0.0001; wk 6-8, P = 0.03; wk 9-11, P = 0.19; and for IGFBP-1: wk 3-5 and 6-8, P0.0001; wk 9-11, P = 0.0003). Comparing women with PCOS who experienced EPL with those who did not, serum glycodelin was significantly lower during wk 3-8 (P0.02) and serum IGFBP-1 during wk 9-11 (P = 0.003). During the first trimester, serum glycodelin and IGFBP-1 concentrations are markedly decreased in PCOS, implicating endometrial epithelial and stromal dysfunction during periimplantation and early pregnancy as a possible mechanism for EPL in PCOS. These decreases are likely to be secondary to hyperinsulinemia and reduced insulin sensitivity.

Glycodelin is a glycoprotein produced in many glands, particularly those of reproductive tissues. It appears as different glycoforms in amniotic fluid (glycodelin-A) and seminal plasma (glycodelin-S), but only glycodelin-A inhibits gamete adhesion. In the present study, glycodelin from secretory-phase endometrium, first-trimester pregnancy decidua, and midtrimester amniotic fluid was studied with respect to physicochemical properties, including glycosylation patterns and inhibitory activity of sperm-egg binding. Purified glycodelins from all these sources were similar in isoelectric focusing and in lectin immunoassays using lectins from Wisteria floribunda and Sambucus nigra. Likewise, the glycodelins inhibited sperm-egg binding in a dose-dependent manner, as measured by hemizona-binding assay. However, subtle quantitative physicochemical and biological differences were found between glycodelins from different sources as well as within the same tissue/fluid between different individuals. Differences were most pronounced between endometrial glycodelins from nonpregnancy and first-trimester pregnancy. The glycan structures studied by fast-atom bombardment mass spectrometry of individual amniotic fluid glycodelin-A samples also showed interindividual quantitative differences. In conclusion, glycodelins from different female reproductive tract tissues and amniotic fluid share substantial similarity, allowing all of them to be called glycodelin-A. However, these glycodelins exhibit quantitative physicochemical and functional differences between different sources and individuals.

Glycodelin is a glycoprotein released by secretory/decidualized endometrial glands. Its synthesis increases during pregnancy. Hormonal factors whose levels have been shown to change in diabetes (vascular endothelial growth factor, relaxin) may mediate the actions or regulate the synthesis of glycodelin. Cord serum glycodelin levels have not been studied in pregnancies complicated by diabetes. Cord serum glycodelin concentrations were measured at birth by an immunofluorometric assay in 62 normal pregnancies, in 67 pregnancies complicated by type 1 diabetes, and in 28 pregnancies complicated by insulin-treated gestational diabetes. The mean glycodelin concentration in cord serum was 2.7 ng/ml (standard error of the mean 0.6) in normal pregnancies. The concentration was not altered in pregnancies complicated by diabetes. Cord serum glycodelin concentrations were also unaltered in diabetic pregnancies with hypertensive disorders (chronic hypertension, pregnancy-induced hypertension or pre-eclampsia) or fetal macrosomia. There was a negative borderline correlation between cord serum glycodelin concentrations and the birth weight in pregnancies complicated by diabetes (r=−0.21, p=0.049). Decidual function, as assessed by cord serum glycodelin levels, is not markedly altered in diabetic pregnancies. The negative correlation between cord serum glycodelin and the birth weight of the newborns in diabetic pregnancies may be due to the decline in glycodelin levels with advancing pregnancy in the third trimester.

Background. The aim of the study was to evaluate whether the circulating levels of insulin-like growth factor-I (IGF-I) and its major circulating binding protein, IGFBP-3, are affected in premature rupture of membranes (PROM) and preterm delivery. Methods. The levels of IGF-I and IGFBP-3 were measured in 32 pregnant women with PROM and in 27 healthy gestational age-matched pregnant women. Statistical analyzes were performed by analysis of variance. Results. All the patients with PROM had preterm delivery, at a gestational age of 31.9 ± 0.4 weeks (mean ± SEM). In the control subjects, pregnancy proceeded to term. In the PROM patients, the serum IGF-I and IGFBP-3 levels (289 ± 21 ng/ml and 8248 ± 407 ng/ml, respectively) were not statistically different from those in the control subjects (275 ± 22 ng/ml and 7579 ± 488 ng/ml). Seventeen patients with PROM showed a rise in serum C-reactive protein, indicating subclinical intrauterine infection. Also in this subgroup of patients the levels of serum IGF-I (281 ...

Glycodelin is a glycoprotein that belongs to the lipocalin superfamily. Depending on glycosylation, glycodelin appears in various isoforms. In the uterus, glycodelin-A is the major progesterone-regulated glycoprotein secreted into uterine luminal cavity by secretory/decidualized endometrial glands. The other tissues expressing glycodelin include fallopian tubes, ovary, breast, seminal vesicle, bone marrow, and eccrine glands. Glycodelin-A potently and dose-dependently inhibits human sperm-egg binding, whereas differently glycosylated glycodelin-S from seminal plasma has no such effect. Absence of contraceptive glycodelin-A in the uterus during periovulatory midcycle is consistent with an open "fertile window." Glycodelin induced by local or systemic administration of progestogens may potentially reduce the fertilizing capacity of sperm in any phase of the menstrual cycle. Glycodelin also has immunosuppressive activity. Its high concentration at the fetomaternal interface may contribute to protection of the embryonic semiallograft. Besides being an epithelial differentiation marker, glycodelin appears to play a role in glandular morphogenesis, as transfection of glycodelin cDNA into a glycodelin-negative breast cancer cells resulted in formation of gland-like structures, restricted proliferation, and induction of other epithelial markers. These various properties, as well as the chemistry, biology, and clinical aspects of glycodelin, continue to be areas of active investigation reviewed in this communication.

Semenogelin plays an important role in sperm clotting and is degraded into smaller fragments by prostate-specific antigen (PSA) during clot liquefaction. Semenogelin and its fragments inhibit sperm motility in vitro. We studied the expression of semenogelin I mRNA and its localization in various tissues of the male genital tract. We also studied semenogelin concentrations with respect to sperm parameters and the outcome of in vitro fertilization. Semenogelin protein was detected by immunohistochemical staining and semenogelin I mRNA was detected by Northern blot analysis in the seminal vesicles and ampullary part of the vas deferens, whereas specimens from the prostate, epididymis, testis, and the female genital tract were negative. Using monoclonal antibodies against semenogelin, an immunofluorometric assay was developed to measure semenogelin levels in seminal plasma and to evaluate possible correlations with sperm parameters and fertilization in vitro. No correlation was found between the semenogelin concentration and the volume of the ejaculate, sperm concentration, sperm motility, or in vitro fertilization rate. Semenogelin levels were positively correlated with the total protein concentration in seminal plasma, and there was an inverse correlation between the concentration of semenogelin and that of PSA. The levels of semenogelin appear to bear no relationship to the in vitro fertilization capacity of the spermatozoa.

BACKGROUND Proteolysis of insulin-like growth factor-binding proteins (IGFBPs) may increase IGF-mediated growth stimulation and development of cancer in the organs producing large amounts of proteases, such as the prostate. METHODS We studied proteolysis of IGFBP-3 by three prostate-derived proteases, namely prostate specific antigen (PSA), human kallikrein 2 (hK2), and trypsin, and also by native seminal plasma. Cleavage of 125I-IGFBP-3 was studied by SDS–PAGE and autoradiography. We also used two different sandwich-type IGFBP-3 immunoassays, called “intact” and “total” IGFBP-3 assays. These assays differ in their capacity to recognize proteolytically degraded IGFBP-3. RESULTS HK2, PSA, and trypsin all cleaved IGFBP-3 at the concentrations normally present in seminal plasma. The IGFBP-3 cleavage by seminal plasma was inhibited by ZnCl2, which strongly inhibits hK2 and PSA, but not by a specific trypsin inhibitor. The IGFBP-3 fragments resulting from proteolytic cleavage by PSA, hK2, or trypsin were undetectable in the “intact IGFBP-3 assay,” whereas the “total IGFBP-3 assay” also detected the proteolytic fragments. No increased fragmentation of IGFBP-3 was found in serum of 659 men with elevated PSA concentrations, of whom 178 had a proven prostate cancer. Furthermore, the IGFBP-3 levels were not associated with the PSA concentrations. CONCLUSIONS These results show that, while seminal plasma and prostate-derived proteases can cleave IGFBP-3, in patients with prostate cancer the circulating IGFBP-3 is not significantly proteolyzed by either PSA, hK2, or trypsin. Prostate 50: 112–118, 2002. © 2002 Wiley-Liss, Inc.

We hypothesized that hyperinsulinemia contributes to early pregnancy loss in the polycystic ovary syndrome by adversely affecting endometrial function and environment. Serum glycodelin, a putative biomarker of endometrial function, is decreased in women with early pregnancy loss. Insulin-like growth factor-binding protein-1 may also play an important role in pregnancy by facilitating adhesion processes at the feto-maternal interface. We studied 48 women with polycystic ovary syndrome before and after 4 weeks of administration of 500 mg metformin (n = 26) or placebo (n = 22) 3 times daily. Oral glucose tolerance tests were performed, and serum glycodelin and insulin-like growth factor-binding protein-1 were measured during the follicular and clomiphene-induced luteal phases of menses. In the metformin group, the mean (+/-SE) area under the serum insulin curve after glucose administration decreased from 62 +/- 6 to 19 +/- 2 nmol/L.min (P < 0.001). Follicular phase serum glycodelin concentrations increased 20-fold from 150 +/- 46 to 2813 +/- 1192 pmol/L (P < 0.001), and serum insulin-like-growth factor-binding protein-1 concentrations increased from 936 +/- 152 to 2396 +/- 300 pmol/L (P < 0.001). Similarly, luteal phase serum glycodelin concentrations increased 3-fold from 3434 +/- 1299 to 10624 +/- 1803 pmol/L (P < 0.001), and serum insulin-like growth factor-binding protein-1 concentrations increased from 1220 +/- 136 to 4916 +/- 596 pmol/L (P < 0.001). Uterine vascular penetration also increased in the metformin group, as did blood flow of spiral arteries, as demonstrated by a 20% decrease in the resistance index from 0.71 +/- 0.02 to 0.57 +/- 0.03 (P < 0.001). These variables did not change in the placebo group. We conclude that insulin reduction with metformin increases follicular and luteal phase serum glycodelin and insulin-like growth factor-binding protein-1 concentrations and enhances luteal phase uterine vascularity and blood flow in the polycystic ovary syndrome. These changes may reflect an improved endometrial milieu for the establishment and maintenance of pregnancy.

Glycodelin-A increased the secretion of interleukin-6, interleukin-13, and granulocyte-macrophage colony-stimulating factor from natural killer cells in the peripheral blood but does not affect their viability, cell death, and cytotoxicity. These data suggest that glycodelin-A contributes to the cytokine shift in early pregnancy.

We have produced human recombinant glycodelin in human kidney 293 cells and in Chinese hamster ovary (CHO) cells. Structural analyses by lectin immunoassays and fast atom bombardment mass spectrometry showed that recombinant human glycodelin produced in CHO cells contains only typical CHO-type glycans and is devoid of any of the N, N'-diacetyllactosediamine (lacdiNAc)-based chains previously identified in glycodelin-A (GdA). By contrast, human kidney 293 cells produced recombinant glycodelin with the same type of carbohydrate structures as GdA. The presence of a beta1-->4-N-acetylgalactosaminyltransferase functioning in the synthesis of lacdiNAc-based glycans in human kidney 293 cells is concluded to be the cause of the occurrence of lacdiNAc-based glycans on glycodelin produced in these cells. Furthermore, human kidney 293 cells were found to be particularly suited for the production of recombinant glycodelin when they were cultured in high glucose media. Lowering the glucose concentration and the addition of glucosamine resulted in higher relative amounts of oligomannosidic-type glycans and complex glycans with truncated antennae. Human glycodelin is an attractive candidate for the development of a contraceptive agent, and this study gives valuable information for selecting the proper expression system and cell culture conditions for the production of a correctly glycosylated recombinant form.

Serum insulin-like growth factor-binding protein-1 (IGFBP-1) is regulated by insulin and has been suggested to be a marker of the insulin resistance syndrome. The aim of this study was to investigate whether serum IGFBP-1 is associated with cardiovascular risk factors. Serum insulin-like growth factor-I and IGFBP-3 were also determined. 331 men aged 70-89 years were examined from the Finnish cohort of the Seven Countries Study. Serum IGFBP-1 concentrations were measured by immunofluorometric assay, and the results were analysed with respect to markers of cardiovascular risk, insulin resistance, and insulin secretion that were determined by using the homeostasis model assessment. Fasting serum IGFBP-1 correlated inversely with fasting serum insulin, serum insulin 2 h after oral glucose challenge test, body mass index, and serum triglycerides, and it correlated positively with age and serum high-density lipoprotein (HDL) cholesterol. After adjustment for age, fasting serum IGFBP-1 correlated positively with HDL cholesterol and inversely with fasting serum insulin and triglycerides. Serum IGFBP-1 correlated positively with insulin sensitivity and negatively with beta-cell function. In conclusion, low serum IGFBP-1 levels in elderly men are associated with a number of cardiovascular risk factors.

Glycodelin is a 28 kDa glycoprotein with structural homology to b-lactoglobulins, particularly expressed in steroidresponsive tissues of the female reproductive tract. We previously found that transfection of glycodelin cDNA into MCF-7 breast cancer cells induces differentiation into organized acinar epithelium and up-regulation of epithelial markers. In this study, we used immunohistochemistry, Northern blotting and reverse transcription-polymerase chain reaction (RT-PCR) analyses to study glycodelin expression in normal and in malignant breast tissues. The results were compared with the expression of estrogen (ER) and progesterone receptors (PR) and p53 tumor suppressor protein. Glycodelin was found in ductal and lobular epithelium of 6/6 normal breast tissues, 27/29 morphologically normal breast tissues from breast cancer patients, 6/6 benign lactating adenomas, 21/35 ductal carcinomas, 9/9 tubular carcinomas, 9/9 mucinous carcinomas, 3/3 mixed ductal/tubular carcinomas and 7/11 lobular carcinomas. In the latter, of particular interest was the presence of glycodelin in paranucleolar vacuoles of carcinoma cells. Northern blot analysis of fresh frozen tissues revealed the normal full length 0.9 kb mRNA of glycodelin in ductal breast carcinoma. Using RT-PCR analysis, glycodelin messenger ribonucleic acid was found in 13/13 ductal and in 3/3 tubular tumor tissues. We also detected a splicing variant lacking exon 4, which includes the nucleotide sequence encoding the potential N-glycosylation site at Asn-85. Our results demonstrate the synthesis of glycodelin in normal breast and breast cancer. In addition, we show that the paranuclear vacuole, characteristically present in lobular breast cancer cells, contains abundant amounts of glycodelin. Int. J. Cancer 83:738‐742, 1999. r 1999 Wiley-Liss, Inc.

Objective To study the effects of E2 on insulin-like growth factor binding protein-1 (IGFBP-1) and sex hormone-binding globulin (SHBG) production with cultured human HepG2 hepatoma cells. Design Experimental cell culture. Setting Academic medical center. Patient(s) None. Intervention(s) Addition of E2 to cell culture medium. Main outcome measure(s) Intracellular and released concentrations of IGFBP-1 and SHBG. Result(s) Estradiol did not affect the intracellular or extracellular IGFBP-1 concentration, whereas the intracellular SHBG concentration increased significantly in response to 0.5-2.5 microM of E2. Conclusion(s) Whereas the two binding proteins share a number of regulatory factors, their regulation by E2 is dissimilar in human hepatoma cells. Estradiol does not affect the intracellular or secreted IGFBP-1 concentration, but it does increase the production of SHBG.

Human glycodelin, a lipocalin with a high amino acid similarity to beta-lactoglobulins, appears as various glycoforms with different biological activities in endometrium (glycodelin-A) and seminal plasma (glycodelin-S). We found that the structures of these glycodelins and beta-lactoglobulin are similar. Despite this structural similarity, unlike beta-lactoglobulin, glycodelin-A binds neither retinoic acid nor retinol. It was impossible to detect any endogenous retinoids or steroids in any of the two purified glycodelins. Both their glycoforms share similar thermodynamic parameters of reversible denaturation suggesting that native folding of glycodelin-A and glycodelin-S is not influenced by the differences in glycosylation or by ligand binding.

OBJECTIVE Body builders may use growth hormone (GH) and anabolic steroids to increase fitness and muscle mass. GH increases serum concentrations of IGF-I and IGFBP-3. The combined effects of GH and anabolic androgenic steroids on IGFBP-3 and IGF-I levels are not known under authentic substance-abusing conditions. The aim of this study was to investigate this in substance-abusing power athletes. DESIGN AND PATIENTS Nine healthy, non-obese and non-competing male power athletes, all aggressive substance abusers, used GH and/or anabolic steroids independently of this study. Blood samples were taken both during and between the drug intake. Sixteen substance non-abusing wrestlers served as controls. MEASUREMENTS Serum IGF-I concentration was measured by radioimmunoassay and the IGFBP-3 concentration was measured by two immunofluorometric assays, one detecting proteolytic fragments of IGFBP-3. The capacity of serum to proteolytically cleave IGFBP-3 was studied by the proteolysis assay. RESULTS While growth hormone increased the IGFBP-3 and IGF-I concentrations, anabolic steroids decreased the same. Concomitant use of growth hormone and anabolic steroids decreased the IGFBP-3 concentration in five out of six abuse periods in spite of the fact that the IGF-I concentration remained elevated in four of them. However, in two men who were on low calorie diet both the IGF-I and IGFBP-3 concentrations decreased during combined GH/anabolic steroid abuse. No proteolytic fragmentation of IGFBP-3 was observed. CONCLUSION Massive abuse of anabolic steroids decreases both the basal and GH-stimulated IGFBP-3 concentrations, whereas its effects on serum IGF-I concentration are variable and affected by low calorie diet. This study demonstrates that detection of GH doping by measuring the IGF-I and/or IGFBP-3 levels has notable confounding factors.

Intrauterine devices (IUDs) exert contraceptive action by interfering with sperm transport, ovum development, fertilization and implantation. Glycodelin A (GdA) is a uterine glycoprotein that has local contraceptive activity by inhibiting sperm-egg binding. GdA is normally absent from endometrium during the fertile midcycle and it is not expressed until the fifth postovulatory day. The phase of menstrual cycle addressed in this study covers the phase when conception is most likely to follow an unprotected intercourse and when GdA is normally absent. We present here evidence that levonorgestrel-releasing IUD (LNg-IUD) is accompanied by 'inappropriate' expression of GdA in endometrium between days 7 and 16 of the menstrual cycle (six out of six cases). The same was also found in copper-releasing IUD (Cu-IUD)-wearing women, but less frequently (four out of 11 cases, P0.0345, Fisher's exact test). In-situ hybridization localized GdA mRNA into endometrial glands in the midcycle endometrium, confirming the cellular site of synthesis. Based on the potent inhibitory activity of GdA on sperm-egg binding, the presence of GdA in uterine glands of IUD wearers may lead to prior exposure of sperm to contraceptive GdA, thus contributing to the contraceptive activity of the IUD.Glycodelin A (GdA), a uterine glycoprotein that exerts local contraceptive activity by inhibiting sperm-egg binding, is generally absent from the endometrium during the fertile midcycle and is not expressed until postovulatory day 5. In this study of endometrial specimens collected from 6 Finnish users of the levonorgestrel-releasing IUD, "inappropriate" expression of GdA between days 7 and 16 of the cycle was observed in every case. Endometrium samples showed epithelial atrophy and stromal decidualization regardless of the duration of IUD use. GdA was localized to endometrial glands, with only sporadic faint patches in the stroma. Unexpected expression of GdA was also detected in 4 of 11 endometria from women with copper-releasing IUDs. Based on the potent inhibitory activity of GdA on sperm-egg binding, the presence of GdA in the uterine glands of hormone-releasing IUD users may lead to prior exposure of sperm to contraceptive GdA, thus contributing to the fertility control activity of the device.

Objective: To examine the effects of the synthetic somatostatin-analogue octreotide and human recombinant insulin on the release of insulin-like growth factor binding protein-1 (IGFBP-1) and P from human granulosa-luteal cells. Design: Primary culture of human granulosa-luteal cells. Setting: Academic research laboratory. Patient(s): Women undergoing oocyte retrieval for IVF-ET because of tubal infertility. Intervention(s): Octreotide or insulin were added to the cultures; sampling of culture medium was performed after 48 hours. Main Outcome Measure(s): Insulin-like growth factor binding protein-1 and Result(s): Octreotide significantly inhibited IGFBP-1 (58.8% compared with controls) and P release (65.1% compared with controls). Insulin abolished IGFBP-1 release while stimulating P release (200.7% compared with controls). There was a significant and positive correlation between IGFBP-1 and P levels. Conclusion(s): Octreotide and insulin have a significant effect on human granulosa-luteal cell function in terms of IGFBP-1 and P release. Our results suggest a local ovarian mechanism for the recently observed effects of octreotide in the treatment of women with polycystic ovary syndrome.

Linear growth is often impaired after successful liver transplantation. The cause is multifactorial; poor graft function and long term glucocorticoid treatment are the main factors responsible. The efficacy and safety of recombinant human GH (rhGH) treatment were assessed in eight growth-retarded children (five boys and three girls) with liver transplants. Immunosuppression comprised azathioprine, cyclosporin, and methylprednisolone. rhGH was administered in a dose of 1 IU/kg x week, given by daily sc injections. The median age at the start of treatment was 9.7 yr (range, 5.9-14.9 yr). All but one of the patients remained prepubertal during treatment. The median growth rate increased from 3.2 to 7.l cm/yr (P = 0.025) and height SD score increased from -3.9 to -3.1 (P = 0.036) during the first year of rhGH treatment. Serum insulin-like growth factor I and insulin-like growth factor-binding protein-3 levels increased significantly during treatment. Graft function was normal in all except one patient, and no rejections or other serious side-effects were documented. In conclusion, rhGH treatment is effective in short, non-GH-deficient, liver-transplanted children receiving long term glucocorticoid treatment. Due to potential risk of allograft rejection, close monitoring of liver function and immunosuppression is required.

Objective To study whether the effect of cotreatment with human biosynthetic GH improves the outcome of poor IVF responders. Design A double-blind placebo-controlled study using a GnRH agonist (GnRH-a) and gonadotropin in a "boost" flare-up protocol for ovarian stimulation together with either placebo, 4, or 12 IU of human GH followed by oocyte retrieval and IVF-ET. Patients Twenty-two patients with previously demonstrated poor responses in at least two assisted reproductive technology cycles were recruited. Interventions Pretreatment and post-treatment blood samples and daily morning blood samples during ovarian stimulation were collected after an overnight fast. Human GH or placebo and GnRH-a were administered SC; gonadotropin was administered IM. Oocytes were collected by ultrasound-guided transvaginal aspiration of follicles. Embryos were cultured in vitro and transferred transcervically. Main Outcome Measures Serum E 2 , FSH, GH, insulin-like growth factor-I (IGF-I), IGF binding protein 1 (IGFBP-1), and IGFBP-3 concentrations. Number of FSH ampules, follicles, oocytes, embryos, and pregnancies. Results No improvement in cycle outcome was demonstrated with daily adjuvant human GH administration with either 4 or 12 IU. Serum IGF-I levels were highest in the 12 IU human GH group and lowest in the placebo group. Serum IGFBP-3 levels increased 2 days after IGF-I levels in the 12 IU human GH group only. Serum IGFBP-1 levels were unchanged in all groups. Conclusion Poor IVF responders do not benefit from cotreatment with human GH during their ovarian stimulation.

Linear growth is more often impaired after liver than after renal transplantation (Tx) in childhood. As similar triple immunosuppression was used in our liver and renal transplant recipients, we were able to compare growth and endocrine function between 19 prepubertal liver and 35 renal transplant recipients. There were no significant differences in median age, weight-for-height index, or height standard deviation score at Tx. Seventy-eight percent of the liver Tx patients, but only 7% of the renal Tx patients, were below the normal range for height 3 years after Tx. Graft function was good in both liver and renal transplant recipients 3 years after Tx. There was no significant difference in growth hormone secretion, serum insulin-like growth factor (IGF)-I, and IGF-binding protein-3 levels, or in methylprednisolone and cyclosporine doses. However, the blood cyclosporine levels were significantly higher in the liver transplant recipients (P = 0.001 1 year and P = 0.005 2 years after Tx). Cortisol production was significantly lower in the liver transplant recipients (P = 0.002 1 year and P = 0.049 2 years after Tx), which suggests greater steroid-mediated suppression of adrenal function. Growth inhibition is more often observed in liver than in renal transplant recipients on similar triple immunosuppression, and may not be related to deficient function of the growth hormone-IGF-I axis. Similar cyclosporine doses result in higher plasma levels of the drug and similar methylprednisolone doses result in more inhibited adrenal cortisol production in liver transplant recipients. In children with organ transplants, cyclosporine and methylprednisolone should be administered on an individual basis.

Human ovarian follicular fluid contains a number of insulin-like growth factor binding proteins (IGFBP) of which IGFBP-3 is the most abundant. IGFBP-3 synthesis is growth hormone-regulated. We studied the effect of prostaglandin F2 alpha (PGF2 alpha) on IGFBP-3 secretion by cultured human granulosa-luteal cells from follicular aspirates of women participating in an in-vitro fertilization programme. The IGFBP-3 concentration was measured using a specific monoclonal immunofluorimetric assay. Contrary to a previous report on unstimulated follicles, this study demonstrated a positive correlation between follicular fluid IGFBP-3 concentration and follicular size. PGF2 alpha was found to stimulate in a dose-dependent fashion the secretion of IGFBP-3. Significant (P < 0.05) effects were found at PGF2 alpha concentrations of 10(-8), 10(-7) and 10(-6) M. Because IGFBP-3 inhibits progesterone production stimulated by insulin-like growth factor (IGF)-I, the PGF2 alpha-induced stimulation of IGFBP-3 production may be one of the mechanisms whereby PGF2 alpha exerts its luteolytic effect via the IGF system.

BACKGROUND Trypsinogen 3 is a minor trypsinogen isoform in the pancreas. In contrast with trypsin 1 and 2, trypsin 3 degrades pancreatic secretory trypsin inhibitor, which may lead to an excess of active trypsin and acute pancreatitis (AP). We developed an immunoassay for trypsinogen 3 and studied whether an assay of serum trypsinogen 3 is of clinical utility in the diagnosis of AP. METHODS Monoclonal antibodies were generated using recombinant human trypsinogen 3 as the antigen and used to establish a sandwich-type immunoassay. We analyzed serum trypsinogen 3 concentrations in 82 patients with AP and 63 patients with upper abdominal pain (controls). The reference interval was determined using serum samples from 172 apparently healthy individuals. RESULTS The measuring range of the trypsinogen 3 assay was 1.0–250 μg/L. Intra- and interassay CVs were CONCLUSIONS We established for the first time a specific immunoassay for trypsinogen 3 using monoclonal antibodies. Patients with AP were found to have increased serum concentrations of trypsinogen 3. The availability of this assay will be useful for studies of the clinical utility of trypsinogen 3.

OBJECTIVE Gestational diabetes mellitus (GDM) is a common metabolic disorder of pregnancy. Patients with GDM are at risk for high fetal mortality and gestational complications associated with reduced immune tolerance and abnormal carbohydrate metabolism. Glycodelin-A (GdA) is an abundant decidual glycoprotein with glycosylation-dependent immunomodulatory activities. We hypothesized that aberrant carbohydrate metabolism in GDM was associated with changes in glycosylation of GdA, leading to defective immunomodulatory activities. RESEARCH DESIGN AND METHODS GdA in the amniotic fluid from women with normal (NGdA) and GDM (DGdA) pregnancies was purified by affinity chromatography. Structural analysis of protein glycosylation was preformed by lectin-binding assay and mass spectrometry. Cytotoxicity, cell death, cytokine secretion, and GdA binding of the GdA-treated lymphocytes and natural killer (NK) cells were determined. The sialidase activity in the placental tissue from normal and GDM patients was measured. RESULTS GDM affected the glycosylation but not the protein core of GdA. Specifically, DGdA had a lower abundance of α2-6–sialylated and high-mannose glycans and a higher abundance of glycans with Sda (NeuAcα2-3[GalNAcβ1-4]Gal) epitopes compared with NGdA. DGdA had reduced immuosuppressive activities in terms of cytotoxicity on lymphocytes, inhibitory activities on interleukin (IL)-2 secretion by lymphocytes, stimulatory activities on IL-6 secretion by NK cells, and binding to these cells. Desialylation abolished the immunomodulation and binding of NGdA. Placental sialidase activity was increased in GDM patients, which may account for the reduced sialic acid content of DGdA. CONCLUSIONS Taken together, this study provides the first direct evidence for altered enzymatic glycosylation and impaired bioactivity of GdA in GDM patients.

Summary OBJECTIVE We determined the relationship of short-term changes in circulating insulin concentrations, resulting from an oral glucose load, to those in both sex hormone binding globulin (SHBG) and insulin-like growth factor binding protein 1 (IGFBP-1) and assessed the effect of a short-term low calorie diet on the levels of SHBG and IGFBP-1 during an oral glucose tolerance test. DESIGN A within-group comparison of biochemical indices during an oral glucose tolerance test before and after calorie restriction. PATIENTS AND METHODS Six obese women with polycystic ovary syndrome with mean (SD) BMI 34 2 (3 4) kg/m2 were studied before and after one month on a very low calorie diet (350 kcal/day; Cambridge diet). Each subject was given a 75-g oral glucose load after an overnight fast and blood samples were taken every 30 minutes for 3 hours. These were analysed for glucose, insulin, SHBG, and IGFBP-1. RESULTS All the women lost weight (range 1.7–9.5 kg). The SHBG concentrations did not change significantly during the oral glucose tolerance test but there was a highly significant decline in IGFBP-1 levels both before (0 min, mean (SD) 27 3 (10 6); 180 min, 8.9 (4.2) μg/l) and after (0 min, 28 4 (12 1); 180 min, 6 2 (2 1) μg/l, P< 0 001) dieting. The sum of the SHBG concentrations during the test, however, was significantly lower prior (129 9 (405) nmol/l) to calorie restriction than after (164 3 (70 6) nmol/l), whereas there was no significant effect of dieting on the IGFBP-1 response to glucose. CONCLUSIONS The changes in insulin and SHBG concentrations found after dieting have been confirmed. SHBG levels, in contrast to IGFBP-1, do not change in response to a short-term increase in insulin or glucose concentrations. The difference in the response of the two binding proteins may be explained by differences in their half-lives in the circulation or the regulation of mRNA for the peptides by insulin. This study confirms that insulin regulates both SHBG and IGFBP-1 but that there is a difference in the time course of the response of the two proteins to insulin.

The aim was to study the levels of amniotic fluid and maternal serum insulin-like growth factor binding protein-1 levels at 15 to 16 weeks' gestation with regard to fetal growth in the third trimester.Amniotic fluid (n = 148) and maternal serum (n = 129) levels of insulin-like growth factor binding protein-1 were prospectively measured.Elevated amniotic fluid insulin-like growth factor binding protein-1 levels were related to fetal growth retardation. At a cutoff value of 3 multiples of the median a high-risk group comprising 16% of all pregnant women was identified. This group included 40% of all small-for-gestational age (birth weight10th percentile) and 55% of all very-small-for-gestational-age (birth weight5th percentile) infants. Maternal serum insulin-like growth factor binding protein-1 levels were not significantly elevated in these groups.In most cases of intrauterine growth retardation the disturbance may exist before the condition is detectable by ultrasonography. An elevated amniotic fluid insulin-like growth factor binding protein-1 level may be an early sign.

The growth-regulating actions of IGFs are modulated by their binding proteins (IGFBPs). The serum concentration of IGFBP-1 is down-regulated by insulin, and in-vitro studies have demonstrated that IGFBP-1 secretion from various tissues and cells can be stimulated by theophylline, forskolin, oestrogen and progesterone. We have studied the effects and mechanisms of thyroid hormone action on IGFBP-1 gene expression and secretion by human hepatoma cells in vitro. Tri-iodothyronine dose-dependently enhanced IGFBP-1 secretion in serum-free HepG2 cell cultures after 24–48 h of exposure, as measured by a specific immunofluorometric assay. This was accompanied by an increase (+ 50%) in the amount of IGFBP-1 mRNA, which could be prevented by cycloheximide, a protein synthesis inhibitor. Cycloheximide transiently enhanced (+ 200%) the accumulation of IGFBP-1 mRNA at 3–12 h of incubation, when no effect of tri-iodothyronine was observed. It is concluded that thyroid hormone stimulates IGFBP-1 secretion slowly by enhancing IGFBP-1 gene expression by a protein mediator. The acute stimulation of IGFBP-1 gene transcription by cycloheximide associates this gene with a number of growth-related genes encoding growth- and tumour-associated peptides.

Background Glycodelin-A interacts with spermatozoa before fertilization, but its role in modulating sperm functions is not known. Zona pellucida-induced acrosome reaction is crucial to fertilization and its dysfunction is a cause of male infertility. We hypothesized that glycodelin-A, a glycoprotein found in the female reproductive tract, potentiates human spermatozoa for zona pellucida-induced acrosome reaction. Methods Glycodelin isoforms were immunoaffinity purified. The sperm intracellular cAMP concentration, protein kinase-A (PKA) and extracellular signal-regulated kinase (ERK) activities, and intracellular calcium were measured by ELISA, kinase activity assay kits and Fluo-4AM technique, respectively. The phosphorylation of inositol 1,4,5-trisphosphate type-1 receptor (IP3R1) mediated by ERK was determined by western blotting. Zona pellucida-induced acrosome reaction was detected by Pisum sativum staining. Results Pretreatment of spermatozoa with glycodelin-A significantly up-regulated adenylyl cyclase/PKA activity and down-regulated the activity of ERK and its phosphorylation of IP3R1, thereby enhancing zona pellucida-induced calcium influx and zona pellucida-induced acrosome reaction. Glycodelin-F or deglycosylated glycodelin-A did not have these actions. Treatment of spermatozoa with a protein kinase inhibitor abolished the priming activity of glycodelin-A, whilst ERK pathway inhibitors mimic the stimulatory effect of glycodelin-A on zona pellucida-induced acrosome reaction. Conclusions Glycodelin-A in the female reproductive tract sensitizes spermatozoa for zona pellucida-induced acrosome reaction in a glycosylation-specific manner through activation of the adenylyl cyclase/PKA pathway, suppression of extracellular signal-regulated kinase activation and up-regulation of zona pellucida-induced calcium influx. The action of glycodelin-A may be important in vivo to ensure full responsiveness of human spermatozoa to the zona pellucida.

Human trypsin isoenzymes share extensive sequence similarity, but certain differences in their activity and susceptibility to inhibitors have been observed. Using phage display technology, we identified seven different peptides that bind to and inhibit the activity of trypsin-3, a minor trypsin isoform expressed in pancreas and brain. All of the peptides contain at least two of the amino acids tryptophan, alanine and arginine, whereas proline was found closer to the N-terminus in all but one peptide. All peptides contain two or more cysteines, suggesting a cyclic structure. However, we were able to make synthetic linear variants of these peptides without losing bioactivity. Alanine replacement experiments for one of the peptides suggest that the IPXXWFR motif is important for activity. By molecular modeling the same amino acids were found to interact with trypsin-3. The peptides also inhibit trypsin-1, but only weakly, if at all, trypsin-2 and -C. As trypsin is a highly active enzyme which can activate protease-activated receptors and enzymes that participate in proteolytic cascades involved in tumor invasion and metastasis, these peptides might be useful lead molecules for the development of drugs for diseases associated with increased trypsin activity.

Expression of insulin-like growth factor binding protein-1 mRNA in human fetal kidney. Expression of insulin-like growth factor binding protein-1 (IGFBP-1) messenger RNA (mRNA) was studied in tissues of human fetuses from 15 to 23 weeks of gestation. Northern blot analysis revealed IGFBP-1 mRNA in the fetal liver and kidney but not in other fetal tissues, including the brain, heart, lung, skeletal muscle and spleen. Studies by in situ hybridization histochemistry showed that, in all fetal kidneys tested, the IGFBP-1 mRNA was localized preferentially to the epithelial cells of the collecting ducts, as well as to the cells of developing glomeruli and in the subcapsular nephrogenic mesenchyme. Less intense labeling for IGFBP-1 mRNA was seen in the connective tissue stroma of the medullary pyramids. A weak signal was detected in the mature glomeruli, and in the cells of the medullary mesenchyme and capsular connective tissue. IGFBP-1 protein was detected by immunoperoxidase staining mostly around small blood vessels but not in the respective endothelium. The protein was also present in many epithelial cells of the collecting ducts and in stromal connective tissue. These results show that the predominant sites of IGFBP-1 transcription in the developing kidney are those with most active differentiation.

Human endometrium is composed of three layers: stratum compactum, stratum spongiosum and stratum basale. Stratum compactum is the superficial layer made up of openings of the uterine glands and some stromal cells. Stratum spongiosum is the middle layer containing mainly dilated glands and little stroma. Stratum basale is the deepest layer adjoining the muscularis. It consists of primordial glands and compact stroma. Stratum compactum and stratum spongiosum form stratum functionale which is subject to cyclic changes and is removed during menstruation.

BACKGROUND Glycodelin-C is a glycodelin isoform isolated from the cumulus matrix. It stimulates spermatozoa-zona pellucida binding. Here, we report the isolation and characterization of a novel glycodelin interacting protein (GIP) from human cumulus matrix. METHODS GIP was purified by liquid chromatograph and identified by mass spectrometry. The interaction of GIP with glycodelin, matrix molecule and spermatozoa were investigated. RESULTS Mass spectrometry analysis suggested that GIP contained the N-terminal region of alpha2-macroglobulin, confirmed by western blot with anti-alpha2-macroglobulin antibody. GIP bound to native but not deglycosylated glycodelin-C in native gel electrophoresis, suggesting that the binding was glycosylation-dependent. GIP did not bind to capacitated and uncapacitated human spermatozoa. The cumulus cells could convert exogenous labeled alpha2-macroglobulin into GIP in vitro. GIP interacted with hyaluronic acid, a major component of the cumulus matrix. Glycodelin-C bound to hyaluronic acid-coated agarose beads in the presence of GIP. Human spermatozoa acquired the hyaluronic acid-GIP-bound glycodelin-C during incubation in vitro. CONCLUSION The hyaluronic acid-GIP complex formed in the cumulus matrix retains and concentrates glycodelin-C in the cumulus matrix for displacing sperm-bound glycodelin-A and -F and stimulating the zona binding activity of the spermatozoa traversing through the cumulus mass.

Human decidua of early pregnancy contains considerable numbers of CD3-CD56+ natural killer (NK) cells. In this study, two major protein products of the decidua, placental protein 14 (PP14) and placental protein 12 (PP12), were tested for the ability to regulate human NK cell activity. In vitro overnight exposure to PP14 of blood lymphocytes or purified large granular lymphocytes (LGL) resulted in suppression of cytotoxicity against K562 target cells in a 4-h 51Cr release assay. The NK inhibition was dependent on concentrations of PP14, being detectable at 5 micrograms/ml and reaching maximum at 50 micrograms/ml. Manifestation of PP14-induced NK suppression required 18-h contact with NK cells. The suppression of NK activity by PP14 was not abolished by indomethacin. In a target binding assay the number of PP14-treated LGL binding to K562 was comparable to that of untreated ones. By contrast with PP14, PP12 produced no effects on NK cells. These results indicate that PP14 suppresses the function of NK cells, which might be involved in prevention of maternal immune rejection of fetus at the fetomaternal interface.