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Gonadotropin-releasing hormone effects on placental hormones during gestation: I. Alpha-human chorionic gonadotropin, human chorionic gonadotropin and human chorionic somatomammotropin.

The release of alpha-human chorionic gonadotropin (alpha hCG), gonadotropin human chorionic gonadotropin (hCG) and human chorionic somatomammotropin (hCS) in vitro from placentas of different gestational ages was studied. In addition, the effect of gonadotropin-releasing hormone (GnRH) on these hormonal releases, as related to the gestational age of the placenta cultured and the dose of GnRH, was determined. The basal release of alpha hCG and hCG was greatest at 9-13 wk of gestation (1000-1500 ng/mg and 250-350 ng/mg, respectively). Lowest release rates were at term (28 ng/mg and 20 ng/mg, respectively). Hormonal release declined with extended culture, except from the cultures of 13- and 15-wk placentas, in which the initially high release continued throughout the 8 days of culture. The initial release of hCS was low at 6 wk, increased to maximum rates by 15 wk, and was similar to the initial rate of release at term. Gonadotropin-releasing hormone stimulated the release of alpha hCG and hCG most dramatically in cultures of 16-wk and 17-wk placentas, where as much as a 400- and 250-fold increase, respectively, on Day 6 of culture was observed (p less than 0.0001). In term placenta cultures after 6 days in vitro, a 20-fold stimulation of alpha hCG and a 10-fold increase of hCG was effected by GnRH (p less than 0.001). The largest responses of alpha hCG and hCG to GnRH were observed when estrogen levels were low. Dose-related responses were observed in some placentas, yet in some instances, maximal effects were attained with all doses utilized in these studies (0.2 to 50 micrograms/ml). These data demonstrate that human placentas of different gestational ages have varying hormonogenic capabilities in vitro. The data also establish that synthetic GnRH is capable of stimulating alpha hCG and hCG production, but the degree and pattern of response to GnRH stimulation are related to the gestational age of the placental tissue and its time in culture. The most responsive period to exogenous GnRH stimulation of alpha hCG and hCG release was on Days 5 and 6 of culture, when basal estrogen release was very low. These data support the hypothesis that hCG release might be controlled by a chorionic GnRH stimulation and suggest that local steroid levels may modulate the hCG response to GnRH stimulation.

Chorionic Gonadotropin

Is deglycosylated human chorionic gonadotropin an antagonist to human chorionic gonadotropin? Characterization of deglycosylated human chorionic gonadotropin action in two testicular interstitial cell fractions.

In order to determine the significance of carbohydrate residues of human chorionic gonadotropin (hCG) in receptor interaction and signal transduction leading to steroidogenesis, the effect of deglycosylated hCG (DG-hCG) was studied in vitro with two different hCG-responsive purified testicular interstitial cell fractions. Fraction I light cells, previously found to bind 125I-labeled hCG with high affinity without producing testosterone, also bound 125I-labeled DG-hCG with high affinity (Kd 7.2.10(-10) M) without stimulating testosterone production. Fraction IV heavier cells, which produced testosterone in response to hCG without detectable high-affinity hCG-binding sites, neither bound DG-hCG nor sufficiently produced cAMP and testosterone in response. With the addition of intact hCG, DG-hCG inhibited cAMP levels, although not sufficiently to inhibit testosterone production. This observation was contrary to previous studies in which DG-hCG was shown to be an antagonist to hCG action. We conclude that: (a) DG-hCG retains its binding activity in light cells and this high-affinity binding is unrelated to steroidogenesis; (b) DG-hCG does not bind to heavier cells with high affinity and loses its biological activity as result of deglycosylation; (c) DG-hCG actions in this study strengthen the concept of two different hCG-responsive cells in the rat interstitium which, if not separated, will yield misleading data supporting the coexistence of hCG high-affinity binding and biological response in the same cell; and (d) DG-hCG partially antagonizes the activation of adenylate cyclase but does not block testosterone production, thus questioning the usefulness of this analogue in antagonizing the action of native hCG in rat testis.

Animals

Medical Research Council European trial of chorion villus sampling. MRC working party on the evaluation pf chorion villus sampling.

First-trimester chorion villus sampling has the advantage over second-trimester amniocentesis of allowing earlier prenatal diagnosis of various genetic and cytogenetic disorders in the fetus (and therefore earlier termination in affected pregnancies) but the relative safety and diagnostic accuracy remain unclear. Between 1985 and 1989, 3248 women seeking prenatal diagnosis, principally because of their age, were recruited to an international, multicentre, randomised comparison of the safety and diagnostic accuracy of the two techniques--5% of women allocated chorion villus sampling and 8% of those allocated amniocentesis were not tested, usually because of spontaneous miscarriage. 6% and 2% were retested, in most because of sampling failure. The endpoint of a liveborn infant who survived was achieved by 86% of women allocated chorion villus sampling and 91% of those allocated amniocentesis; statistical analysis, after appropriate weighting for a centre's contribution, showed that the typical difference between the groups was 4.6% (95% confidence interval 1.6-7.5%; p less than 0.01). This difference reflected more spontaneous fetal deaths before 28 weeks' gestation (2.9% [0.6-5.3%]); more terminations of pregnancy for chromosomal anomalies (1.0% [0.0-2.1%]); and more neonatal deaths (0.3% [-0.1 to 0.7%]). The difference in neonatal deaths was due to a preponderance of very immature liveborn infants in the chorion villus sampling group, and this factor also explained that group's longer mean stay in hospital. More abnormal diagnoses followed chorion villus than amniotic fluid analyses (5.6% vs 3.9%). This difference was largely due to diagnoses of trisomy 18 and of (usually mosaic) abnormalities known to be confined to the placenta. 3 terminated pregnancies were false positives, 1 tested by chorion villus sampling and 2 by amniocentesis, and 2 other mosaic cases diagnosed by chorion villus sampling may have been false positives. There was 1 false-negative result in the chorion villus sampling group. The possibility of earlier exclusion or diagnosis of some fetal disorders afforded by first-trimester chorion villus sampling must be set against its clinical risks.

Abortion, Induced

Fetomaternal transfusion depends on amount of chorionic villi aspirated but not on method of chorionic villus sampling.

Transcervical and transabdominal chorionic villus sampling are believed, on the basis of indirect evidence, to result in fetomaternal transfusion. We sought to measure this phenomenon by devising a simple method that would allow us to identify variables that influence fetomaternal transfusion. We investigated patients undergoing transcervical-chorionic villus sampling (n = 15) and transabdominal-chorionic villus sampling (n = 15), restricting the sample to subjects who required only a single catheter passage or needle insertion to obtain villi. Maternal serum alpha-fetoprotein was measured before and after the procedure along with alpha-fetoprotein concentration of the transport medium into which the villi had been aspirated. We first confirmed that the change in maternal serum alpha-fetoprotein levels after chorionic villus sampling, an indirect measure of fetomaternal transfusion, was indeed correlated with the alpha-fetoprotein concentration of transport medium into which the villi were aspirated (p = 0.0350). Fetomaternal transfusion next proved to be correlated with the amount of villi obtained (p = 0.0279). However, when adjusted for the amount of villi obtained, no significant difference was observed between transcervical and transabdominal-chorionic villus sampling with respect to the change in maternal serum alpha-fetoprotein levels after chorionic villus sampling (p = 0.8512). These data suggest that the magnitude of fetomaternal transfusion depends on the amount of villi obtained but not on the chorionic villus sampling method used.

Adult

A randomized comparison of transcervical and transabdominal chorionic-villus sampling. The U.S. National Institute of Child Health and Human Development Chorionic-Villus Sampling and Amniocentesis Study Group.

BACKGROUND: Chorionic-villus sampling is done in early pregnancy to obtain fetal cells for the prenatal diagnosis of genetic and chromosomal defects. Transcervical chorionic-villus sampling has been shown to be safe and effective in national trials. Recently, an alternative transabdominal technique has been suggested as potentially easier and safer. METHODS: From April 1987 through September 1989, we prospectively compared transcervical and transabdominal chorionic-villus sampling in 3999 women with singleton pregnancies in whom the risk of a genetically abnormal fetus was increased. Women between 7 and 12 weeks of gestation underwent ultrasonographic evaluation of placental and uterine position. Those with active vaginal infections, active bleeding, or cervical polyps were excluded. If the obstetrician thought either sampling procedure was acceptable, the woman was asked to consent to random assignment to one of the two procedures. Both groups were followed to determine the outcome of pregnancy and the rate of spontaneous fetal loss after chorionic-villus sampling. RESULTS: Among the 3999 women who entered the study, sampling was attempted in 3873 (97 percent), 1944 of whom had been assigned to undergo transcervical sampling and 1929 to undergo transabdominal sampling. Of these 3873 women, sampling was eventually successful in 3863. Sampling was successful after a single insertion of the sampling instrument in 94 percent of the transabdominal procedures and 90 percent of the transcervical procedures. Among the women with cytogenetically normal pregnancies who had sampling because of maternal age, the rate of spontaneous fetal loss through 28 weeks of pregnancy was 2.5 percent in the transcervical-sampling group and 2.3 percent in the transabdominal-sampling group (difference, 0.26 percent; 95 percent confidence interval, -0.5 to 1.0 percent). CONCLUSIONS: Transabdominal and transcervical chorionic-villus sampling appear to be equally safe procedures for first-trimester diagnosis of fetal abnormalities.

Abdomen

Quantitation of whole molecule human chorionic gonadotropin and free beta-human chorionic gonadotropin subunit in the Abbott IMx analyser.

An assay for the quantitation of the serum levels of free and whole molecule-associated beta-subunit of human chorionic gonadotropin on the Abbott IMx analyzer is described. The assay detects human beta-chorionic gonadotropin with a sensitivity of approximately 0.5 IU/l while showing no cross-reactivity with follitropin (1000 IU/l) or thyrotropin (2.0 IU/l), and 0.02% cross-reactivity with lutropin (1000 IU/l). Haemoglobin (7.50 milligrams), bilirubin (0.50 milligrams), and triacylglycerols (10.6 milligrams) did not interfere with the assay. Pooled within-run, between-run, and total assay CVs were less than or equal to 5.2%, and less than or equal to 2.7%, and less than or equal to 6.3%, respectively. Values obtained with this assay correlated well (r = 0.98, n = 228) with those values obtained using the Hybritech TandemR-R HCG (Total Beta-HCG) IRMA. The normal range of the assay was found to be less than or equal to 5 IU/l (n = 311). The assay protocol provides results for up to 23 serum samples in approximately 47.5 minutes with the ability to report the human beta-chorionic gonadotropin concentrations of 5 specimens in approximately 15.5 minutes. We conclude that this is an acceptable assay for monitoring human beta-chorionic gonadotropin levels associated with normal pregnancy, reproductive pathology, and reproductive technology such as in vitro fertilization or embryo transfer.

Chorionic Gonadotropin

Amplification of the X-linked Drosophila chorion gene cluster requires a region upstream from the s38 chorion gene.

Genomic sequences controlling follicle cell-specific amplification of the X-linked Drosophila chorion gene cluster were mapped by P element-mediated transformation. Several DNA fragments containing the s38 gene and flanking sequences induced tissue-specific amplification, although replication levels were subject to position effects. Deletion analysis identified a 467-bp region upstream from the s38 transcription start site that contained sequences essential in cis for amplification. The essential region shared 32 bp of imperfect sequence homology with a previously identified region necessary for third chromosome chorion gene cluster amplification. This homologous segment contained a repetitive motif consisting of perfect and imperfect AATAC repeats; it was localized near the boundary of the essential domain since most, but not all, the repeats could be deleted without eliminating transposon-induced amplification. The repetitive region was not required for developmentally regulated s38 transcription, therefore our results identified at least one element required for amplification but not for chorion gene transcription. The homologous repetitive sequences within the amplification-essential regions may constitute part of the replication origins used to differentially replicate the two chorion domains during oogenesis.

Animals

[Prenatal cytogenetic diagnosis using chromosomal preparations of the native biopsy samples of chorionic villi, cultured chorionic cells and amniocytes].

Analysis of the results of prenatal cytogenetic diagnosis carried out in the first and second pregnancy trimesters in more than 300 women permitted comparing the efficacies of two methodologic approaches, diagnostic amniocentesis and chorion sampling , with due consideration for the methodologic errors typical of these methods and of the tested biologic material. Up to 5% of the diagnoses are erroneous if the diagnosis is based on chorion sampling data, whereas in amniocentesis the share of diagnostic errors is lower by an order. The authors have given a theoretical rationale for and tried a methodologic approach, involving the employment of the 'direct' chromosomal preparations from villous chorion biopsy specimens and the so-called 'maintained' cell culture technique, that permits obtaining chromosomal preparations of higher quality and, consequently, helps improve the accuracy of chromosomal diagnosis.

Adult

Investigations of chorionic villi after chorionic villus sampling (CVS). Correlation of morphological with clinical and laboratory data.

This report documents the first 262 cases of chorionic villus sampling (CVS) performed in parallel with cytogenetic and morphological investigations. Histomorphological examination of these CVS specimens gave suitable results in about 96% (251 cases). Of the latter, 201 samples (80.1%) exhibited villi and 176 (70.1%), maternal tissue. Viability and maturation of the chorionic villi were determined light microscopically even in cases with few villus trees. Smooth avascular villi with poorly defined margins observed under an inverted microscope, less than 10 mitoses after short-term incubation, and reduced growth of cell cultures were significantly correlated with sampling at the chorion laeve by means of histomorphologic criteria. Villi from cases exhibiting cytogenetically proved chromosomal abnormalities were characterized by molar degeneration or stromal fibrosis, or both, in 4 out of 9 cases, including 3 mosaics. In early abortions (within 3 weeks after CVS), an unexpectedly high rate of pathohistological changes within maternal tissue was evident. These results need further confirmation by investigation of a greater number of samples with immunohistochemical and morphometric methods.

Adult

The possible evolutionary significance of repeat elements near and within an early chorion gene in the late chorion locus of Bombyx mori.

Three beta-type early chorion gene copies (6F6.1, 6F6.2, and 6F6.3) are dispersed in the late region of chorion locus Ch1-2. Detailed analysis of the 5'-flanking region and the intron of 6F6.1 shows that they contain sequences that are homologous to Bombyx mori Bm1 repeat elements. Interestingly, the Bm1-type segment of the intron is interrupted by the insertion of a sequence that shows significant similarities with part of an intron of B. mori and Bombyx mandarina fibroin genes, and with part of the 3'-flanking region of B. mori prothoracicotropic hormone and tRNA-Glu genes; this sequence may represent a new repetitive, possibly transposable, element of B. mori. Following the Bm1-homologous sequence of the 6F6.1 5'-flanking region and preceding the gene promoter region, a short DNA segment shows sequence motifs that are also present in the ErA.1 promoter region. The occurrence of these sequences near one end or within the Bm1 repeat element is suggestive of complex sequence transfer events. Comparative analysis of known B. mori chorion alpha-gene promoters and of Bm1 repeat elements suggests, with marginal statistical significance, that these two sets of sequences contain common elements.

Animals

Enhancing effects of human macrophage colony-stimulating factor on the secretion of human chorionic gonadotropin by human chorionic villous cells and tPA30-1 cells.

Human macrophage colony-stimulating factor (hM-CSF) concentration-dependently enhanced the secretion of human chorionic gonadotropin (hCG) by primary cultured human cytotrophoblastic cells and a human placental cell line, 3A-SubE (tPA30-1). Since this effect appeared 12 hours after the addition of hM-CSF and disappeared when protein synthesis was inhibited, it was surmised that hCG synthesis was enhanced by hM-CSF. When anti fms (hM-CSF receptor) antibody was added, hCG secretion by cultured human cytotrophoblasts in early pregnancy markedly decreased. These findings demonstrate that hM-CSF acts on the chorionic villous cells and promotes hCG synthesis by these cells.

Cell Line

Immunohistochemical localization of thrombomodulin in chorionic diseases of the uterus and choriocarcinoma of the stomach. A comparative study with the distribution of human chorionic gonadotropin.

Thrombomodulin (TM) is a newly described protein that functions as a potent natural anticoagulant by converting thrombin from a procoagulant protease to an anticoagulant. TM is found on endothelium of the blood and lymph vessels and on syncytiotrophoblast of the placenta in humans. In the current study, we localized TM by immunohistochemistry in chorionic diseases of the uterus and choriocarcinomas of the stomach. The syncytiotrophoblast of hydatidiform mole contained TM. Uterine choriocarcinoma also had TM in the neoplastic syncytiotrophoblastic cells, although some of the neoplastic syncytiotrophoblastic cells showed no TM expression. Gastric choriocarcinoma never showed TM expression in the neoplastic syncytiotrophoblastic cells. However, all of the syncytiotrophoblastic cells had human chorionic gonadotropin (hCG) in hydatidiform mole, uterine choriocarcinoma, and gastric choriocarcinoma.

Biomarkers, Tumor

Evolution of the autosomal chorion cluster in Drosophila. II. Chorion gene expression and sequence comparisons of the s16 and s19 genes in evolutionarily distant species.

We present a total of 13 kb of DNA sequences, encompassing autosomal chorion genes and their flanking DNA in four species of the genus Drosophila. Against a background of extensive divergence in introns and even in parts of the coding regions, islands of strong conservation are evident in the proximal 5' flanking and 5' untranslated sequences. An extragenic region of strong conservation is seen downstream of the last chorion gene in the autosomal cluster. The conserved DNA elements may be related to the conserved regulatory features of this cluster, including gene amplification and tissue- and temporally regulated transcription.

Amino Acid Sequence

Gene evolution and regulation in the chorion complex of Bombyx mori. Hybridization and sequence analysis of multiple developmentally middle A/B chorion gene pairs.

Twenty-two pairs of chorion genes belonging to the A and B multigene families have been characterized and mapped within two segments of a 320 kb (1 kb = 10(3) bases or base-pairs) chromosomal walk in the domesticated silkmoth Bombyx mori. Eighteen of the gene pairs belong to two groups that are typified by the previously characterized A/B.L12 and A/B.L11 chorion gene pairs, and are defined by two respective types of short (approx. 280 base-pairs) bidirectional promoter sequences. In the chromosome, the L12-like and L11-like pairs are interspersed with each other and with the remaining four gene pairs, which have unrelated promoter sequences. We have sequenced the promoter regions and adjacent small exons of all L12-like and L11-like A and B genes in the walk. The L12-like promoters are highly conserved, whereas L11-like promoters are somewhat more variable. Reconsideration of previous data on RNA accumulation and disappearance during choriogenesis, in the light of the sequences, indicates that L12-like genes are developmentally early-middle, while L11-like genes correspond to two developmental subgroups, middle I and middle II. Sequence comparisons of all these promoters, as well as the previously characterized promoters of the developmentally late HcA and HcB genes, identify short elements of possible regulatory significance. The sequences, as well as extensive cross-hybridization analysis with short probes derived from the reference A/B.L12 gene pair, under carefully controlled conditions of stringency, indicate the occurrence of sequence transfers among A or B genes. These sequence transfers, which could result from gene conversions or unequal crossovers, are less abundant than in the HcA and HcB families, but do result in a patchwork of similarities and differences in the A and B genes. The transfers appear to be least frequent between the moderately divergent A genes that belong to different temporal classes, while the L12-like and L11-like B genes appear to be extensively homogenized in sequence.

Animals

Chorionic gonadotropin, chorionic somatomammotropin, and prolactin in the uterine vein and peripheral plasma of pregnant rhesus monkeys.

Seven adult female rhesus monkeys were laparotomized at days 22, 42, and 157 of pregnancy and blood was collected from the uterine vein and peripheral circulation. Plasma samples were analyzed for monkey chorionic gonadotropin (mCG), monkey chorionic somatomammotropin (mCS), and prolactin by radioimmunoassay. Levels of mCG at day 22 of pregnancy were approximately 250 ng/ml; however, during the later stages of gestation mCG was either nondetectable or less than 0.7 ng/ml. There was no statistical difference in prolactin concentrations between days 22 and 42 of pregnancy, mean levels being between 176-424 ng/ml, but by day 157 prolactin levels of greater than 2,000 ng/ml were recorded. No statistical difference existed between peripheral and uterine vein concentrations of either mCG or prolactin at any of the stages of gestation examined. At day 22, mCS was not detectable; however, at day 42 of gestation mCS titers averaged 1.5 micrograms/ml and 2.3 micrograms/ml in the peripheral and uterine vein plasma, respectively. A statistically significant mCS increase occurred by day 157, levels in the periphery and uterine vein averaging 11.0 micrograms/ml and 16.3 micrograms/ml, respectively. Uterine vein titers of mCS were significantly higher than peripheral titers at both days 42 and 157. Thus, the highest levels of mCG were present during early pregnancy, whereas the highest levels of mCS and prolactin were present during late pregnancy.

Animals

Regulation of chorionic gonadotropin-alpha and chorionic somatomammotropin messenger ribonucleic acid expression by 8-bromo-adenosine 3',5'-monophosphate and dexamethasone in cultured rhesus monkey syncytiotrophoblasts.

We wished to establish an in vitro culture system to examine gene expression in the context of differentiated function with rhesus monkey syncytiotrophoblasts. Chorionic villous tissue from placentas obtained at cesarean section was dispersed with trypsin and DNase and fractionated on a 5-70% Percoll gradient. When placed in culture, cells from a mononuclear fraction demonstrated to be very highly enriched (95-97% pure) for cytotrophoblasts aggregated and began to form syncytia within 24 h in culture, reminiscent of placental syncytiotrophoblast formation. The migration and fusion of individual cytotrophoblasts to form multinuclear syncytia were documented with time-lapse video microscopy. Incorporation studies with tritiated thymidine supported the conclusion from videomicroscopy that syncytia form by the fusion of individual cells and the addition of mononuclear cells to existing syncytia, rather than by endomitosis. The syncytiotrophoblast marker pregnancy-specific beta 1-glycoprotein (SP1) was immunocytochemically identified in both intact placenta and cultured syncytiotrophoblast cells. With cells isolated from placentas obtained on day 28, 50, 70, or 140 of pregnancy, treatment with 8-bromo-cAMP increased both rhesus monkey CG alpha-subunit (mCG alpha) and chorionic somatomammotropin (mCS) mRNA levels by an average of 4-fold. Increases of up to 2.5-fold were seen with mCG alpha mRNA in as little as 2 h after treatment, with a statistically significant average response seen within 6 h. The response with mCS required at least 24 h before a significant effect was seen. Actin mRNA levels were generally unchanged or suppressed by this treatment, indicating that the effect of 8-bromo-cAMP is relatively specific for the hormone mRNAs. Treatment of syncytiotrophoblasts with dexamethasone, but not progesterone or androstenedione, resulted in an approximately 4-fold increase in mCG alpha mRNA levels within 6 h of treatment. Steroid treatment did not affect mCS mRNA levels. Treatment with 4.5-400 nM GnRH or 0.1 to 100 ng/ml basic fibroblast growth factor likewise had no effect on the level of either mRNA, suggesting that any actions of these factors on hormone secretion are not effected via changes in steady state mRNA. These results communicate that the expression of the mRNAs for rhesus monkey CG alpha and CS in syncytiotrophoblast are regulated by steroid hormone- and cAMP-mediated pathways.

8-Bromo Cyclic Adenosine Monophosphate

Evolution of the autosomal chorion cluster in Drosophila. III. Comparison of the s18 gene in evolutionarily distant species and heterospecific control of chorion gene amplification.

We present a total of 6.1 x 10(3) base-pairs of DNA sequences, encompassing the s18 gene and flanking regions within the autosomal chorion cluster of three Drosophila species. Against a background of extensive divergence in the intron and even in parts of the coding region, islands of strong sequence conservation are evident. These are particularly notable in the 5' flanking DNA where they extend to approximately -600 base-pairs from the transcription start site. The most conserved segment of the entire chorion cluster is 71 base-pairs in the s18 5' flanking DNA, which in D. melanogaster is part of a region defined functionally as containing amplification control elements (ACE3 region). Transformation analysis, using chimeric transposons of D. melanogaster and D. grimshawi DNA, revealed that amplification control elements of D. grimshawi can support amplification in D. melanogaster. The functionally defined ACE3 region of D. grimshawi includes the conserved 71 base-pair segment, but also non-conserved sequences further upstream, which apparently enhance amplification.

Amino Acid Sequence