Pharmacogenomics of 17-alpha hydroxyprogesterone caproate for recurrent preterm birth: a case-control study.

OBJECTIVE: To compare maternal genotypes between women with and without significant prolongation of pregnancy in the setting of 17-alpha hydroxyprogesterone caproate (17-P) administration for the prevention of recurrent preterm birth (PTB). DESIGN: Case-control. SETTING: Three tertiary-care centres across the USA. POPULATION: Women (n = 99) with ≥ 1 prior singleton spontaneous PTB, receiving 17-P. METHODS: Women were classified as having successful prolongation of pregnancy during the 17-P treated pregnancy, in two ways: (1) Definition A: success/non-success based on difference in gestational age at delivery between 17-P-treated and untreated pregnancies (success: delivered ≥ 3 weeks later with 17-P) and (2) Definition B: success/non-success based on reaching term (success: delivered at term with 17-P). MAIN OUTCOME MEASURES: To assess genetic variation, all women underwent whole exome sequencing. Between-group sequence variation was analysed with the Variant Annotation, Analysis, and Search Tool (VAAST). Genes scored by VAAST with P < 0.05 were then analysed with two online tools: (1) Protein ANalysis THrough Evolutionary Relationships (PANTHER) and (2) Database for Annotation, Visualization, and Integrated Discovery (DAVID). RESULTS: Using Definition A, there were 70 women with successful prolongation and 29 without; 1375 genes scored by VAAST had P < 0.05. Using Definition B, 47 women had successful prolongation and 52 did not; 1039 genes scored by VAAST had P < 0.05. PANTHER revealed key differences in gene ontology pathways. Many genes from definition A were classified as prematurity genes (P = 0.026), and those from definition B as pharmacogenetic genes (P = 0.0018); (P, non-significant after Bonferroni correction). CONCLUSION: A novel analytic approach revealed several genetic differences among women delivering early vs later with 17-P. TWEETABLE ABSTRACT: Several key genetic differences are present in women with recurrent preterm birth despite 17-P treatment.

Epidermal growth factor increases lung liquid clearance in rat lungs.

Epidermal growth factor (EGF) has been reported to stimulate the proliferation of epithelial cells and increase Na+ flux and Na+-K+-ATPase function in alveolar epithelial cell monolayers. Increases in Na+-K+-ATPase in alveolar type II cells (AT2) have been associated with increased active Na+ transport and lung edema clearance across the rat alveolar epithelium in a model of proliferative lung injury. Thus we tested whether administration of aerosolized EGF to rat lungs would increase active Na+ transport and lung liquid clearance. Sixteen adult Sprague-Dawley male rats were randomized to three groups. To a group of six rats, an aerosol generated from 20 microgram of EGF in saline was delivered to the lungs, to a second group of five rats only aerosolized saline was delivered, and a third group of five rats without treatment served as the control. Forty-eight hours postaerosolization of rat lungs with EGF there was an approximately 40% increase in active Na+ transport and lung liquid clearance compared with control rats, in the absence of changes in 22Na+, [3H]mannitol, and albumin permeabilities. The Na+-K+-ATPase activity in AT2 cells harvested from these lungs was increased in rats that received aerosolized EGF compared with AT2 cells from both control rats and rats receiving aerosolized saline. These results support the hypothesis that in vivo delivery of EGF aerosols upregulates alveolar epithelial Na+-K+-ATPase and increases lung liquid clearance in rats.

Expression of the ErbB family of receptors in ovarian cancer.

OBJECTIVE: To characterize the relative levels of the ErbB family of receptors and their relationship to one another in ovarian cancer. METHODS: A total of 17 serous cystadenocarcinomas were analyzed for epidermal growth factor receptor (EGF-R or ErbB-1) and ErbB-2, ErbB-3, and ErbB-4 receptor expression by Western blot analysis. Receptor levels were quantified by densitometry and expressed as relative densitometry units normalized to the level of alpha-tubulin. Linear regression analysis was used to analyze receptor group differences. A value of P < or = .05 was considered statistically significant. RESULTS: All 17 tumors expressed detectable levels of EGF-R, ErbB-2, and ErbB-3, but ErbB-4 expression was not detected. EGF-R levels correlated with ErbB-2 (r = .70, P < .01) and ErbB-3 (r = .52, P < .05) levels. The highest correlation was obtained between the levels of ErbB-2 and ErbB-3 (r = 0.81, P < .001). CONCLUSION: This study indicates an association between the levels of ErbB receptor family members in ovarian cancer. This association suggests that one or more coordinated regulatory mechanisms may be involved in determining their relative expression levels to one another.

Expression of epidermal growth factor and androgen receptors in ovarian cancer.

Ovarian cancer is the second most common malignancy of the female reproductive tract. Approximately 50% of ovarian cancers have elevated levels of epidermal growth factor receptor (EGFR). This overexpression is correlated with a poor prognosis for patient survival. Ovarian cancers also express a number of sex steroid receptors. The androgen receptor (AR) is the predominant sex steroid receptor and is expressed in over 80% of ovarian cancers. We investigated whether a relationship exists between EGFR and AR in ovarian cancer. Sixty serous cystadenocarcinomas were analyzed for their relative levels of EGFR and AR by Western blot analysis. Data were analyzed by Student's t test and linear regression analysis for statistical significance. More than 98% of the tumors expressed detectable levels of EGFR, while 65% of the tumors expressed detectable levels of AR. The levels of EGFR (mean +/- SEM) were found to be significantly (P < 0.01) higher in AR+ (516 +/- 15) than in AR- (304 +/- 57) tumors. EGFR levels significantly correlated to AR levels (r = 0.49, P < 0.001). These results demonstrate an association between EGFR and AR levels in ovarian cancer. Whether this association represents a causal or a casual relationship remains to be determined.

Expression of a truncated epidermal growth factor receptor-like protein (TEGFR) in ovarian cancer.

The epidermal growth factor receptor (EGFR) system has been implicated in the etiology of numerous cancers, including that of ovarian cancer. Elevated levels of EGFR are associated with poor patient prognosis. Moreover, a significant number of ovarian cancers express both the receptor and one of its ligands, suggesting an autocrine mechanism for autonomous tumor growth. Because of the implicated role of the EGFR system in neoplasia, a greater understanding of the factors involved in this system is necessary. We have recently characterized a truncated EGFR-like protein (TEGFR) in human placenta, and we now extend this investigation to ovarian cancer. We report that TEGFR is expressed in ovarian cancer and its level correlates to that of EGFR. Moreover, the level of TEGFR is reduced in metastatic compared to primary tumors. These results suggest that TEGFR may play a role in the EGFR system.

Possible role of variant RNA transcripts in the regulation of epidermal growth factor receptor expression in human placenta.

Epidermal growth factor receptor (EGFR) plays an important role in growth and differentiation. The human placenta expresses high levels of the receptor. In the placenta, as in many other human tissues, EGFR is encoded by two RNA transcripts of 5.8 kb and 10.5 kb. The placenta also expresses a putative truncated EGFR transcript of 1.8 kb, which encodes only the ligand binding domain of the receptor. The etiology and role of these variant EGFR transcripts is unknown. Using the human placenta as a model to study this area, we report 1) the relationships among these transcripts suggest that the induction of alternate pathways of EGFR RNA processing is involved in their etiologies; 2) the 10.5 kb transcript may be the principal transcript involved in determining the level of the protein receptor; and 3) the isolation of a soluble protein with characteristics consistent with a translational product corresponding to the 1.8 kb transcript, which may act in regulating the activity of EGFR. Together these results suggest that alternate processing of EGFR RNA into variant transcripts may represent a novel mechanism involved in the regulation of the receptor protein.

Effect of lipoxygenase products on choriogonadotropin secretion by cultured human choriocarcinoma JEG-3 cells pre- and post-stimulation with epidermal growth factor and a phorbol ester.

Previous studies using arachidonic acid and preferential inhibitors of the arachidonic acid pathway have implicated the lipoxygenase system in choriogonadotropin (hCG) secretion by JEG-3 cells. Presently, JEG-3 cells are used in order to examine the effect of lipoxygenase products on hCG secretion. Results show that 30 microM 15-hydroxyeicosatetraenoic acid (15-HETE) induces an approximately 3-fold increase in basal hCG secretion, while 5-HETE, 12-HETE, and leukotriene LTA4 have no significant effect. In addition, 15-HETE potentiates the stimulation of hCG secretion induced by 3 nM epidermal growth factor (EGF), but has no significant effect on the stimulation of hCG induced by 22 nM tetradecanoylphorbol acetate (TPA). The present study further implicates the arachidonic acid pathway in the control of hCG secretion and documents that the effect of EGF can be rate-limited by a product of the lipoxygenase system.

Epidermal growth factor receptor numbers in male and female mouse primary hepatocyte cultures.

Epidermal growth factor receptors (EGF-R) were measured in adult male and female mouse primary hepatocyte cultures. On culture day 1, female hepatocytes had significantly fewer EGF-R than male hepatocytes (1.3 x 10(4) versus 6.2 x 10(5) per cell). Over the next three days, morphological changes consistent with progressive heptocyte dedifferentiation were observed. During this period, EGF-R numbers progressively increased in female cultures and decreased in male cultures, and by day 4 the sexual difference in EGF-R numbers was obliterated. These results indicate that a relationship exists between the degree of differentiation in hepatocyte cultures and the expression of EGF-R on the cell surface.

Competitive studies with dehydroepiandrosterone sulfate and 16 alpha-hydroxydehydroepiandrosterone sulfate in cultured human choriocarcinoma JEG-3 cells: effect on estrone, 17 beta-estradiol, and estriol secretion.

The estradiol (E2) to estriol (E3) ratio during human pregnancy depends on fetal liver hydroxylation of fetal adrenal dehydroepiandrosterone sulfate (DHEAS) and conversion by the trophoblast of DHEAS and 16 alpha-hydroxy-DHEAS (16 OH-DHEAS) to estrone (E1), estradiol (E2), and estriol (E3), respectively. It is not known whether the conversion of DHEAS into E1 and E2 influence the conversion of 16OH-DHEAS into E3 and vice versa. To examine this question, we studied these interactions in human choriocarcinoma JEG-3 cells. In serum-free medium (Dulbecco's Modified Eagle's Medium), JEG-3 cells secreted hCG [27 +/- 3 (+/- SEM) ng/mg cellular protein X 24 h] and progesterone (22 +/- 2.5), but neither C-19 nor C-18 steroids. The addition of DHEAS resulted in secretion of E1 and E2; at a concentration of 500 ng DHEAS/ml, the secretion of E1 (1 +/- 0.16) and E2 (11 +/- 3.1) was maximal, while E3 remained undetectable. The addition of 1000 ng 16 OH-DHEAS/ml resulted in maximum E3 secretion (13 +/- 1.8), while E1 and E2 remained undetectable. The addition of increasing concentrations of DHEAS to cultures exposed to 1000 ng 16OH-DHEAS/ml caused a decrease in E3 secretion and increased secretion of E1 and E2. Conversely, addition of increasing concentrations of 16OH-DHEAS in cultures exposed to 500 ng DHEAS/ml resulted in inhibition of E1 and E2 secretion and increased E3 secretion. A concentration of 16OH-DHEAS that inhibited the conversion of DHEAS into E1 and E2 neither altered the intracellular to extracellular steroid ratios (approximately 0.1) nor reduced the secretion of DHEA, androstenedione, and testosterone. The inhibitory effect of 16OH-DHEAS was minimal at low DHEAS concentrations (favoring the secretion of E1 and E2) and was greatly enhanced at concentrations of DHEAS that induced maximum E1 and E2 secretions. The results indicate that in trophoblastic cells, the metabolism of DHEAS can modulate E3 secretion, and the metabolism of 16OH-DHEAS can modulate the secretion of E1 and E2; and this regulatory mechanism appears to take place at the level of the aromatase system.

The effect of dehydroepiandrosterone sulfate on de novo and low-density lipoprotein-stimulated progesterone secretion by human choriocarcinoma JEG-3 cells.

It has been suggested that fetal adrenal steroids affect the secretion of progesterone by the human trophoblast and decrease the progesterone/estrogen secretory ratio at the time of parturition. In the present study, cultured human choriocarcinoma JEG-3 cells were used as an experimental model in order to examine the effect of dehydroepiandrosterone sulfate on both de novo and low-density lipoprotein-stimulated progesterone secretion. In serum-free and cholesterol-free Dulbecco's modified Eagle's medium, JEG-3 cell cultures demonstrated a significant secretion of both pregnenolone and progesterone. A 200% to 300% increase in pregnenolone and progesterone secretion was achieved by physiologic low-density lipoprotein concentrations added to Dulbecco's modified Eagle's medium while androgens and estrogens remained undetectable. Dehydroepiandrosterone sulfate added to Dulbecco's modified Eagle's medium was actively converted into C-19 and C-18 steroids but had no significant effect (up to 20 micrograms/ml) on basal (de novo) progesterone secretion. In contrast, the addition of dehydroepiandrosterone sulfate (1 to 5 micrograms/ml) to cultures grown in Dulbecco's modified Eagle's medium plus low-density lipoprotein induced a dose-related inhibition of progesterone and a return of that secretion to basal levels.

Effects of epidermal growth factor, phorbol myristate acetate, and arachidonic acid on choriogonadotropin secretion by cultured human choriocarcinoma cells.

Epidermal growth factor (EGF) binds specifically (Ka = 4 X 10(9) M-1; 1.3 X 10(11) receptors/mg cellular protein) to JEG-3 cells, which respond in the succeeding 24 h by a 400% increase in hCG secretion without a significant change in cell number. Since JEG-3 cells store less than 2% of the 24-h hCG secretion, a significant increase in hCG in the culture medium reflects increased hCG biosynthesis. It is observed that a tumor promoter, phorbol myristate acetate (PMA), binds specifically to the cells (Ka = 5 X 10(8) M-1; 3.9 X 10(11) receptors/mg), reduces (33%) the affinity but not the number of EGF receptors, and stimulates hCG to the same extent as does EGF. The relative potencies of PMA (100%), phorbol dibutyrate (25%), and nonesterified phorbol (no effect) to stimulate hCG parallel their known tumor-promoting activities. Since phospholipids and fatty acids have been implicated in the mechanism of action of EGF and PMA, the effects of arachidonic acid (AA) and inhibitors of its metabolism were studied. The addition of AA had no effect on basal or PMA-stimulated hCG secretion, but it potentiated the effect of EGF by 200%. Indomethacin (a cyclooxygenase inhibitor) had an effect similar to that of AA. Nordihydroguairetic acid (a cyclooxygenase and lipoxygenase inhibitor) reduced by 90% basal and EGF- or PMA-stimulated hCG secretion. These results indicate that the AA pathway can influence the effects of EGF and PMA on hCG secretion and suggest an intermediary role for the lipoxygenase system.

Characterization of an immune suppressor from transformed human trophoblastic JEG-3 cells.

Cultured human choriocarcinoma JEG-3 cells secrete an immunosuppressor that inhibits lymphocyte proliferation stimulated by either an antigen or a mitogen. In this study, the immunosuppressive factor was characterized by three methods: ion-exchange and exclusion chromatography, partition in organic solvents, and thin-layer chromatography on silicic acid. This JEG-3 cell factor appeared to be a protein complex of about 150,000-200,000 Da that contained an immunologically active polar lipid. The structural and functional characteristics of JEG-3 cell immunosuppressor are similar if not identical to those of SIF, a suppressor lymphokine derived from T cells. These secretions from transformed trophoblastic cells may correspond to normal placental products or represent a function of malignant cells.

Thin-layer chromatography for separation of phospholipids in amniotic fluid.

A method using a 1-dimensional, thin-layer chromatographic technique for the separation of phospholipids in amniotic fluid is described. The phospholipids of major importance in evaluating fetal lung maturity were separated: sphingomyelin, lecithin, and, notably, phosphatidylglycerol. Preliminary clinical results correlate the increasing appearance of phosphatidylglycerol with increasing lecithin : sphingomyelin ratios.

Endometrial prostaglandin F content in women wearing nonmedicated or progestin-releasing intrauterine devices.

Prostaglandin F (PGF) was measured in endometrial samples from eight women wearing Lippes Loops and 14 women wearing Progestaserts after 6 months' use of the intrauterine devices (IUDs). In addition, in 37 women wearing dydrogesterone-T IUDs, endometrial PGF was measured after 1 month, 3 to 7 months, and 8 to 12 months of use. Endometrial samples were also obtained from 51 women without IUDs. The following means and standard errors of endometrial PGF, expressed in picograms per microgram of endometrial protein, were obtained in women without IUDs: early proliferative phase, 3.11 +/- 1.0; midproliferative phase, 4.1 +/- 0.7; late proliferative phase, 4.4 +/- 1.0; midluteal phase, 7.13 +/- 1.0; and late secretory phase, 6.08 +/- 1.0. The midproliferative phase mean was significantly different from the midsecretory phase mean (P is greater than 0.05). Except for one group, no differences were noted during the midproliferative and midsecretory phases among the groups of women wearing medicated and nonmedicated IUDs, when compared with values for women without IUDs. The only difference noted was during the midsecretory phase in the dydrogesterone group at 8 to 12 months. In these women, the mean endometrial PGF content was 2.8 +/- 0.8, significantly different (P is greater than 0.05) from that of women without IUDs (6.1 +/- 1.0).

The effect of intrauterine progesterone treatment on the endometrial prostaglandin F content in the rabbit.

The effect on rabbit endometrial prostaglandin F caused by progesterone delivered directly to the uterus was investigated. Four groups of animals were used in the experiment: (1) no treatment (control); (2) an empty Silastic capsule (as an intrauterine device [IUD]) was inserted in one horn and the other horn was sham-operated; (3) a Silastic capsule releasing 150 microng of progesterone/day was placed in one horn and the other horn was sham-operated; (4) a Silastic capsule releasing progesterone was placed in one horn and the opposite horn received an empty Silastic capsule. In group 1, which received no treatment, no difference was noted. In group 2, the prostaglandin content of the horn containing an empty IUD was significantly higher than that of the sham-operated horn. In group 3, the same significant difference was noted between the prostaglandin content of the IUD-containing, progesterone-treated horn and the sham-operated horn. In group 4, no significant difference was observed between the horn containing an inert IUD and that containing a progesterone-releasing device. The addition of progesterone to an IUD does not significantly affect the elevated prostaglandin content of the endometrium caused by an inert IUD.

PIP: The effect of intrauterine progesterone treatment on the endometrial prostaglandin F (PGF) content was investigated in the rabbit. The following groups of animals were employed: 1) no treatment, 2) an empty Silastic capsule as an IUD in 1 horm and the other horm sham-operated, 3) a Silastic capsule releasing 150 mcg of progesterone/day in 1 horn and the other sham operated, and 4) a Silastic capsule releasing progesterone in 1 horn and an empty Silastic capsule in the other horn. No difference was noted in Group 1. In Group 2 the PGF content of the horn containing an empty IUD was significantly higher (p less than .05) than that of the sham-operated horn. In Group 3, the progesterone-treated horns contained a significantly higher (p less than .05) PGF content than the sham-operated horn. In Group 4 no marked difference was observed between the horn containing an inert IUD and that containing a progesterone-releasing device. It is concluded that the addition of progesterone to an IUD does not markedly affect the elevated PGF content of the endometrium caused by an inert IUD.