N-glycans of human amniotic fluid transferrin stimulate progesterone production in human first trimester trophoblast cells in vitro

AIMS: During pregnancy, the placenta produces a variety of steroid hormones and proteins. Several of these substances have been shown to exert immunomodulatory effects. Progesterone is thought to mediate some of these effects by regulating uterine responsiveness. The aim of this study was to clarify the effect of amniotic fluid transferrin and its N-glycans on the release of progesterone by first trimester trophoblast cells in vitro. METHODS: Cytotrophoblast cells were prepared from human first trimester placentae by trypsin-DNAse dispersion of villous tissue followed by a percoll gradient centrifugation and depletion of CD45 positive cells by magnetic cell sorting. Trophoblasts were incubated with varying concentrations (50-300 microg/ml) of transferrin from human amniotic fluid and serum as well as with N-glycans obtained from amniotic fluid transferrin. Culture supernatants were assayed for progesterone by enzyme-immunometric methods. RESULTS: The release of progesterone increased in amniotic fluid transferrin- and N-glycan-treated trophoblast cell cultures compared to untreated trophoblast cells. There was no stimulating effect of serum transferrin on the progesterone production of trophoblast cells. CONCLUSIONS: The results suggest that amnion-transferrin and especially its N-glycans modulate the endocrine function of trophoblasts in culture by up regulating progesterone secretion.     

Human amniotic fluid transferrin stimulates progesterone production by human trophoblast cells in vitro

OBJECTIVE: During pregnancy transferrin plays a key role as an iron transport protein to serve the increased fetal demands of iron. Transferrin is also present in relatively high concentrations in amniotic fluid [6], showing a different glycosylation compared with serum transferrin. The biological function of human amniotic fluid transferrin (hAFT) is still unknown. In addition trophoblast cells also synthesise transferrin. Transferrin synthesised by the trophoblast shows a special glycosylation. We found identical carbohydrate structure of hAFT and trophoblast transferrin. We investigated the influence of hAFT on the progesterone-, cortisol- and hCG-release of trophoblasts in culture compared with the influence of human holo- and apo-serum transferrin on the release of these hormones. MATERIAL AND METHODS: Cytotrophoblast cells were prepared from human term placentae by standard trypsin-DNAse dispersion of villous tissue followed by a percoll gradient centrifugation step. When placed in culture, the trophoblasts were incubated with varying concentrations (50-300 micrograms/ml) of human amniotic fluid- and serum-transferrin. Unstimulated cells of each placenta used as controls. Culture supernatants were assayed for progesterone, hCG and cortisol by enzyme-immunometric methods. RESULTS: Our results show, that the release of progesterone increased in hAFT-treated cell cultures compared to untreated cell cultures. Holo- and apo-serumtransferrin did not show any effect on the progesterone release by trophoblast cells in vitro. Neither hAFT nor holo- and apo-serum transferrin had any effect on the cortisol- and hCG-release in vitro. CONCLUSIONS: Progesterone is a marker for differentiation of trophoblasts in syncytiotrophoblasts. Only hAFT stimulates the progesterone production. We suggest, that hAFT can modulate the endocrine function of trophoblast cells in culture by regulating progesterone production.     

Regulation of progesterone production in human term trophoblasts in vitro by CRH,ACTH and cortisol (prednisolone)

Background: In most mammals, onset of labor is accompanied with progesterone withdrawal. In humans, cortisol blockade of progesterone is a possible mechanism involved in the initiation of labor. Therefore, aim of the study was to clarify the effect of CRH, ACTH and cortisol (prednisolone) on the release of progesterone by term trophoblast cells in vitro. Methods: Cytotrophoblast cells were prepared from human term placentas by standard dispersion of villous tissue followed by a percoll gradient centrifugation step. Trophoblasts were incubated with CRH, ACTH as well as with prednisolone Results: The release of progesterone is decreased in CRH- and ACTH-treated trophoblast cell cultures compared to untreated trophoblast cells. Addition of prednisolone in varying concentrations leads to an increase of trophoblast progesterone production. Conclusions: The results suggest that CRH and ACTH directly modulate the endocrine function of trophoblasts in culture by downregulating progesterone production. Prednisolone on the other hand showed a stimulating effect on progesterone production in term trophoblast cells in vitro. Because blockade of progesterone is a possible mechanism involved in initiation of labor, we may speculate that CRH and ACTH are directly involved in the auto- or paracrine regulation of this procedure.     

Human papillomavirus in amniotic fluid

Background

There is evidence to suggest that human papillomavirus (HPV) can cross the placenta resulting in in-utero transmission. The goal of this study was to determine if HPV can be detected in amniotic fluid from women with intact amniotic membranes.

Methods

Residual amniotic fluid and cultured cell pellets from amniocentesis performed for prenatal diagnosis were used. PGMY09/11 L1 consensus primers and GP5+/GP6+ primers were used in a nested polymerase chain reaction assay for HPV.

Results

There were 146 paired samples from 142 women representing 139 singleton pregnancies, 2 twin pregnancies, and 1 triplet pregnancy. The women were 78% Caucasian, 5% African American, 14% Asian, and 2% Hispanic. The average age was 35.2 years with a range of 23–55 years. All samples were β-globin positive. HPV was not detected in any of the paired samples.

Conclusion

Given the age range, race, and ethnicity of the study population, one would anticipate some evidence of HPV if it could easily cross the placenta, but there was none.     

Proliferation of group B streptococci in human amniotic fluid in vitro

The group B Streptococcus is one of the most virulent organisms causing perinatal infection. Human amniotic fluid from the second and third trimesters was pooled and analyzed for electrolytes, protein, albumin, zinc, inorganic phosphorus, ferritin, lysozyme, and immunoglobulins. We inoculated replicates of specimens with known virulent strains of group B streptococci (893, 891, and 878) and Escherichia coli (C5) with Todd-Hewitt broth and normal saline solution used as controls. Group B streptococci strains 893 and 891 proliferated rapidly at rates similar to their rates in Todd-Hewitt Broth. Strain 878 grew at a rate slower than that of strains 893 and 891. The amniotic fluid specimens were similar with respect to factors reported as inhibitory to bacterial proliferation. Second- and third-trimester amniotic fluid supports the growth of group B streptococci as well as a culture medium optimized for bacterial growth. Strain-specific variance in group B streptococci growth rates in amniotic fluid may have clinical significance for those at risk for group B streptococci infection.     

Immunoreactive oestrogens and progesterone in amniotic fluid in twin pregnancies

Amniotic fluid concentrations of immunoreactive oestrogens and progesterone were measured at the time of caesarean section in 32 twin pregnancies; 25 women had an elective section and seven were in labour at the time of operation. No significant differences between concentrations in the amniotic fluid of the first and second twin were found in respect of conjugated and unconjugated oestrone, oestradiol, oestriol, oestetrol and unconjugated progesterone either before or during labour. It is unlikely that changes in oestrogens or progesterone in the amniotic fluid are responsible for the selective changes seen in prostaglandins and fetal adrenal steroid during labour in the first twin.     

Immunoreactive oestrogens and progesterone in amniotic fluid in twin pregnancies

Summary. Amniotic fluid concentrations of immunoreactive oestrogens and progesterone were measured at the time of caesarean section in 32 twin pregnancies; 25 women had an elective section and seven were in labour at the time of operation. No significant differences between concentrations in the amniotic fluid of the first and second twin were found in respect of conjugated and unconjugated oestrone, oestradiol, oestriol, oestetrol and unconjugated progesterone either before or during labour. It is unlikely that changes in oestrogens or progesterone in the amniotic fluid are responsible for the selective changes seen in prostaglandins and fetal adrenal steroid during labour in the first twin.     

Human fetal respiration. IV. Failure of severe distress to stimulate aspiration of amniotic fluid by the immature human fetus

Reported herein are studies on fetal breathing, specifically the inhalation of labeled amniotic fluid, by immature human fetuses so severely distressed that they subsequently died in utero. Decreased rather than increased amounts of the amniotic fluid label were found in the lungs of the distressed fetuses compared to the amounts in fetuses of comparable weight that were not distressed. We conclude from this study of the immature human fetus that severe distress depresses rather than stimulates aspiration of amniotic fluid.     

Neurogenic cells in human amniotic fluid

OBJECTIVE: The purpose of this study was to determine whether human amniotic fluid contains cells that harbor the potential to differentiate into neurogenic cells. STUDY DESIGN: Amniotic fluid cells (uncultivated or cultivated in standard or in neurogenic differentiation medium) were analyzed for morphologic neurogenic differentiation and for expression of cluster of differentiation 133 (marker for neuronal stem cells), nestin (neuronal progenitor cells), neurofilament (neurons), the p75 common neurotrophin receptor, the brain-derived neurotrophic factor and neurotrophin-3 and cyclic nucleotide phosphodiesterase (oligodendrocytes). RESULTS: The appearance of neurogenic cells was not detected in uncultivated cells, was sporadic after cultivation in standard medium but strongly increased in neurogenic differentiation medium, and was accompanied by the induction of the expression of the analyzed marker genes. CONCLUSION: For the first time, this study provides evidence that human amniotic fluid contains cells that express markers for neuronal stem and progenitor cells, which harbor the potential to differentiate into neurogenic cells.     

Surface-active material in human amniotic fluid

Surface-active fractions similar to the biochemical constituents of the pulmonary surfactant system can be isolated from human amniotic fluid and lung tissue from week 10 of gestation through term. Amniotic fluid yielded 10 mg. per 100 ml. surface-active material at 10 to 28 weeks' gestation and 330 mg. per 100 ml. at term. Fetal lung at 10 weeks of gestation gave 1,233 mg. per 100 ml. and 1,670 mg. per 100 ml. at 4 days after birth. The constituent PC of these surface-active fractions from amniotic fluid and lung contained in excess of 60 per cent palmitoyl residues. PC associated with the surface-active fraction accounted for no more than 15 per cent of the total amniotic fluid concentration of this compound. Minimum surface tensions obtained with these preparations from amniotic fluid ranged from 2.5 dynes per centimeter at 33 weeks to 8.3 dynes per centimeter at 40 weeks. At 14 to 28 weeks, the surface-active fraction from lung gave a minimum surface tension of 18.1 dynes per centimeter and 7.0 dynes per centimeter by 4 days after birth. These findings are consistent with the observation that an increase in the activity or concentration of the fetal lung surfactant system takes place in the last 4 weeks of gestation. Some constituents of this system, or their derivatives which have possibly been altered as a result of biochemical changes, are found in amniotic fluid. We infer that the majority of PC in amniotic fluid is not directly associated with the biochemical constituents of the fetal lung surfactant system.     

Rapid in vitro replication of group B streptococcus in term human amniotic fluid

4-hour in vitro growth curves of a type III group B streptococcus (GBS) and Escherichia coli were examined in sterile term human amniotic fluid specimens. Both bacterial strains proliferated despite ratios of phosphate to elemental zinc in the range reportedly inhibitory for E. coli. After 4 h of incubation, despite comparable inocula, GBS numbers exceeded those of E. coli by 10-fold. The strikingly rapid growth of some GBS strains in amniotic fluid may represent yet another factor responsible for perinatal GBS infection of the human neonate.     

Phosphoenolpyruvate carboxykinase activity in human amniotic fluid

Amniotic fluid samples obtained from normal pregnancies during gestation were used to quantitate the levels of phosphoenolpyruvate carboxykinase (PEPCK) activity. It is concluded that: (1) PEPCK activity was highest in the amniotic fluid cells of midtrimester pregnancies as compared to those of pregnancies close to term; (2) the activity of PEPCK seen in the amniotic fluid supernatant was about one fourth to one half of that seen in the amniotic cells; (3) the amniotic fluid supernatant PEPCK activity fluctuated very little during the entire gestation.     

Fetal intestinal microvilli in human amniotic fluid

The intestinal microvilli of fetal origin in human amniotic fluid were purified by Ca2+ precipitation of contaminating organelles followed by differential centrifugation of microvillar membranes. In the purified preparation, the specific activity of the microvillar marker-enzymes maltase and sucrase increased about 77-fold over that in cell-free amniotic fluid. Significant contamination of the purified preparation by endoplasmic reticulum (microsomes) and lysosomes was ruled out on the basis of a low content of the marker enzymes glucose-6-phosphatase (microsomes) and acid phosphatase (lysosomes). Amniotic fluid microvilli contain typical enzymes of the fetal intestine including maltase, sucrase, trehalase, alkaline phosphatase and gamma-glutamyltransferase, and their morphology by electron microscopy resembles that of vesiculated intestinal microvilli. Prenatal detection of genetic diseases due to a deficiency of a protein expressed in these membranes or associated to abnormal microvilli seems feasible.     

Histocompatibility antigens, transferrin receptors and extra-embryonic markers of human amniotic epithelial cells in vitro

This study has shown that fresh amniotic epithelial cells which are negative by indirect immunofluorescence for HLA and Trf receptors become positive for both of these markers following prolonged culture. In addition, the amniotic antigens which are characteristic of fresh amniotic epithelial cells are lost and replaced by trophoblast antigens subsequent to maintenance of the cells in vitro. These observations suggest that, although the expression of HLA and Trf receptors are apparently negative in vivo, such gene products become activated and manifest in plasma membranes following exposure of the cells to culture conditions. In addition, the normal manifestation of the amnion antigens is lost following prolonged periods of culture. These findings indicate that the coding and expression of normally accepted transplantation antigens undergo a significant perturbation in vitro, and that markers of normal extra-embryonic epithelium in vivo are lost after the cells have been cultured in vitro.