Reproductive Sciences recent issues

In the Spotlight

Maduro, M. R. — 2008-06-25

{beta}-HCG/LH Receptor ({beta}-HCG/LH-R) Expression in Eutopic Endometrium and Endometriotic Implants: Evidence for {beta}-HCG Sensitivity of Endometriosis

2008-06-25

Background: Luteinizing hormone (LH) and human chorionic gonadotropin (HCG) target their receptor in gonadal and nongonadal cells to stimulate steroidogenesis and cell growth. The aim of the present study was to investigate the expression of HCG/LH-R in endometriosis to elucidate a possible impact of LH and HCG on this disease. Materials and methods: Analysis of HCG/LH-R protein expression in 23 paired samples of ectopic and eutopic tissue of cycling women with endometriosis and in endometrial samples from 22 healthy controls was conducted via immunofluorescence. HCG and HCG/LH-R gene expression in endometriotic lesions was confirmed by reverse-transcriptase polymerase chain reaction. Results: In endometriotic implants, epithelial HCG/LH-R was found in 12/23 samples. No significant differences in HCG/LH-R levels were observed when compared with glands of uterine endometrium from the same patients or healthy controls. Messenger RNA transcripts for HCG were detected in all 12 samples, whereas HCG/LH-R mRNAs were observed in 10 of the 12 endometriotic lesions investigated. Conclusions: Although HCG/LH-R was not found to be selectively upregulated in endometriosis, the mere presence of HCG/LH-R in endometriotic tissue may suggest sensitivity of endometriosis to HCG and LH that target HCG/LH-R.

Induction of Growth Inhibition and Apoptosis in Human Uterine Leiomyoma Cells by Isoliquiritigenin

2008-06-25

Isoliquiritigenin(ISL), a calchone flavonoid, has cancer-preventing properties and is often used in Chinese medicine. In the present study, the authors use ISL to determine its effect on cell proliferation and cell cycle progression in primary cultured human uterine leiomyoma cells. Cell viability and cell proliferation assays were conducted. Flow cytometry, annexin V apoptosis assay, and DNA fragmentation assay were performed to determine the effect of ISL on cell cycle and apoptosis. The expression of cell cycle regulatory—related proteins was evaluated by Western blot. The cell viability and proliferation of uterine leiomyoma cells were significantly reduced by ISL treatment in a dose-dependent manner. Flow cytometry results showed that ISL induced subG1 and G2/M arrest. DNA fragmentation assay and annexin V apoptosis assays revealed apoptosis induction. ISL-induced growth inhibition in uterine leiomyoma cells was associated with increased p21^Cip1/ Waf1 expression in a p53-dependent manner. Activation of caspase-3 and downregulation of Bcl-2, cdk 2/4, and E2F, with a concomitant increase in dephosphorylation of Rb and poly—ADP-ribose polymerase cleavage, were observed. This study demonstrates that ISL inhibits cell proliferation by initiating apoptosis in human uterine leiomyoma cells coupled with increased cell cycle arrest. These results indicate that ISL could prove to be a promising chemopreventive and therapeutic agent against human uterine leiomyoma.

Estradiol 17-{beta} and Progesterone Modulate Inducible Nitric Oxide Synthase and High Mobility Group Box 1 Expression in Human Endometrium

2008-06-25

The aim of the present study is to investigate the effects of ovarian sex steroid hormones on the expression and the release of several locally active substances by human endometrium. Specific objectives are (1) to ascertain if estradiol 17-β (E2) and progesterone modulate inducible nitric oxide synthase (iNOS) expression and nitric oxide release; (2) to determine whether human endometrium can express High Mobility Group Box 1 (HMGB1), a multifunctional cytokine, and whether sexual steroid hormones can modulate this expression; and (3) to evaluate whether nitric oxide can influence HMGB1 expression in this tissue. Endometrial tissue was obtained from 40 healthy premenopausal women who underwent hysteroscopy for suspected benign gynecological conditions. Endometrium was incubated with E2, progesterone, or sodium nitroprusside, a nitric oxide donor. Nitrite assay was used to quantify stable nitric oxide metabolites in culture medium, and Western blot analysis was used to detect iNOS and HMGB1. Incubation of endometrium with E2 results in an increase in iNOS expression and nitric oxide metabolite production. The opposite effect is obtained by incubating tissues with progesterone. HMGB1 is expressed by human endometrium, and its expression is increased by E2 and decreased by progesterone. Incubation with sodium nitroprusside results in a reduction in HMGB1 expression. Both E2 and progesterone modulate iNOS expression and nitric oxide production in human endometrium. HMGB1 is expressed in the human endometrium, and its expression is modulated by E2, progesterone, and nitric oxide.

Clinical Application of Pulse Transit Time and Correlation With Intrapartum Fetal Heart Rate Monitoring: A Preliminary Study of 18 Full-Term Infants

Kawagoe, Y., Sameshima, H., Ikenoue, T. — 2008-06-25

The authors show that pulse transit time and blood pressure are reciprocal in fetal goat models. They applied this technique in clinical settings to correlate changes in pulse transit time with fetal heart rate monitoring patterns and acid-base status. In 18 uncomplicated pregnancies, pulse transit time was obtained from electrocardiograms to pulse oximeter waveform and averaged during each baseline period, defined by the interpretation of fetal heart rate monitoring. According to a > 10% change from the control value, chronological changes were categorized into shortened, unchanged, and prolonged. Pulse transit time was available in 82% ± 11% of the recordings. In 15 fetuses, 2 (13%) showed prolonged, 7 (47%) showed shortened, and 6 (40%) showed unchanged conditions. Comparisons of the shortened and unchanged categories revealed that severe variable deceleration was significantly increased, and half or more fetuses showed hypoxemia in the shortened category. Shortening of pulse transit time, theoretically indicating a hypertensive condition, was more frequently associated with severe variable decelerations, suggesting that the pulse transit time may supplement the interpretation of fetal heart rate monitoring.

ECG and Heart Rate Variability Changes in Preterm and Near-Term Fetal Lamb Following LPS Exposure

Blad, S., Welin, A.-K., Kjellmer, I., Rosen, K.G., Mallard, C. — 2008-06-25

The aim of this study is to evaluate the myocardial response in the preterm and near-term fetal lamb with infection. Chronically instrumented fetal lambs were exposed to lipopolysaccharide (LPS), and the fetal electrocardiogram (FECG) ST waveform was examined using STAN. Fetal heart rate variability (FHRV) was automatically analyzed by adapting a polynomial function to the RR sequence in the FECG. Preterm fetuses exposed to >90 ng/kg LPS died within 8 hours of LPS administration, a response not seen in near-term fetuses. In both surviving and nonsurviving preterm fetuses, cardiovascular responses were characterized by decreased arterial pressure, negative T waves, and tachycardia accompanied by an increase in FHRV. Similar changes were not observed in the near-term fetuses after LPS. The study shows that preterm lambs are more sensitive to LPS in terms of myocardial/cardiovascular response than the more mature fetuses are. High FHRV and negative ST waveform seem to characterize the LPS-induced stress response in preterm fetuses.

The Increase of Blood Flow in the Fetal Middle Cerebral Artery Correlates With the Onset of Labor at Term

2008-06-25

The objective of this cohort study is to evaluate the relationship between blood flow resistance in the fetal middle cerebral artery (MCA) and the beginning of spontaneous labor. A group of consecutive women (n = 664) with a singleton pregnancy was submitted to Doppler examination of fetal MCA between 24 and 41 weeks' gestation (median, 35 weeks). The study population was later divided into 2 groups according to the MCA pulsatility index (PI), expressed in multiples of the median (MoM) for each gestation week. The median time elapsed between the Doppler examination and the onset of spontaneous labor (which eventually occurred in 302 women, of whom 291 were at term) was significantly shorter in the group with MCA PI <0.74 MoM (5.5 days; interquartile interval, 2-10 days) than in the group with MCA PI ≥0.74 MoM (22.5 days; interquartile interval, 5-37.5 days; P < .001). Survival analysis and Cox regression confirmed that the MCA PI was independently associated with the number of days elapsed from Doppler to spontaneous labor (P < .001; Exp[B], 2.77; 95% confidence interval, 1.95-3.90) after correction for birth weight and umbilical artery PI. The present data suggest that, at term of pregnancy, a gestation-independent decrease of fetal cerebral vascular resistance precedes the onset of spontaneous labor.

Detection and Identification of Novel Metabolomic Biomarkers in Preeclampsia

2008-06-25

In a previous study, the ability of gas chromatography time-of-flight mass spectrometry to detect potential metabolic biomarkers in preeclampsia was demonstrated. In this study, the authors sought to validate their preliminary findings in an entirely different patient cohort using a complementary, novel, and powerful combination of analytical tools (ultra performance liquid chromatography and LTQ Orbitrap mass spectrometry system). Eight metabolites that appeared in the authors' previous patient cohort were identified as being statistically significant (P < .01) as discriminatory biomarkers. The chemical identities of these 8 metabolites were established using authentic chemical standards. They included uric acid, 2-oxoglutarate, glutamate, and alanine. This is the first study to identify, in an unbiased manner, a series of small-molecular-weight metabolites that effectively detect preeclampsia in plasma. The identity of these metabolites provides new insights into the pathology of this condition and raises the possibility of the development of a predictive test.

Thyroid Hormone Regulates Renocortical COX-2 and PGE2 Expression in the Late Gestation Fetal Sheep

Carey, L. C., Valego, N. K., Kai Chen, , Rose, J. C. — 2008-06-25

Cyclooxygenase 2 (COX-2) is important for development of the fetal kidney. Precisely how renal COX-2 expression is regulated in fetal life is unclear. The hypothesis that thyroid hormone positively regulates COX-2 and PGE₂ levels in the late gestation fetal kidney cortex was tested. Sham, thyroidectomized (TX), and TX + thyroid hormone replacement (R) fetal sheep were studied. TX was performed at 120 days gestational age (dGA). TX + R fetuses were continuously infused with thyroxine from 3 days after surgery until study completion. Fetal kidney cortex was obtained at 137 dGA for measurement of renal cyclooxygenase type-2 (COX-2) protein and PGE₂ metabolites. Renocortical COX-2 and PGE₂ levels were significantly lower in TX compared with sham and TX + R fetuses. There were no differences between sham and TX + R fetuses. These findings demonstrate that thyroid hormone positively regulates renal COX-2 and PGE₂ expression in the late gestation fetal sheep kidney.

The Relation Between Venous Reserve Capacity and Low Plasma Volume

2008-06-25

Objective: Prepregnant low plasma volume (LPV) is associated with subsequent gestational hypertensive disease. It is unknown to what extent an LPV affects the venous reserve capacity (VRC). We tested the hypothesis that LPV reduces the VRC, as indicated by presyncope or altered cardiovascular changes in response to head-up tilt. Study design: In 52 nonpregnant women with a history of preeclampsia or recurrent miscarriage, the authors assessed plasma volume, stroke volume, and cardiac output and determined blood pressure, heart rate, and autonomic responses to stepwise inflicted head-up tilt. Results: 12 participants had LPV, which related to presyncope when compared with subjects with normal plasma volume (NPV). Women with LPV without presyncope demonstrated a circulatory response comparable to NPV women at the expense of consistently higher heart rate. Conclusion: LPV decreases the capacity to cope with head-up tilt without affecting the response pattern, suggesting reduced VRC.

Reduction of Brain Injury in Neonatal Hypoxic--Ischemic Rats by Intracerebroventricular Injection of Neural Stem/Progenitor Cells Together With Chondroitinase ABC

2008-06-25

Perinatal hypoxia—ischemia (HI) remains a critical issue. Cell transplantation therapy could be a potent treatment for many neurodegenerative diseases, but limited works on this kind of therapy have been reported for perinatal HI. In this study, the therapeutic effect of transplantation with neural stem/ progenitor cells (NSPCs) and chondrotinase ABC (ChABC) in a neonatal HI rat model is evaluated. Histological studies showed that the unaffected area of the brain in animals treated with NSPCs together with ChABC was significantly larger than that in the animals treated with vehicle or NSPCs alone. The wet weight of the brain that received the combined treatment was also significantly higher than those of the vehicle and their individual treatments. These results indicate that intracerebroventricular injection of NSPCs with ChABC reduces brain injury in a rat neonatal HI model.

In the Spotlight

Maduro, M. R. — 2008-06-25

Macrophages and Pregnancy

Mor, G., Koga, K. — 2008-06-25

Programming of Human Monocytes by the Uteroplacental Environment

McIntire, R. H., Ganacias, K. G., Hunt, J. S. — 2008-06-25

During human pregnancy, monocytes recruited to the uterus (decidua) are modified to promote immune defense and semiallogeneic pregnancy. The purpose of this study was to identify decidual factors involved in programming of monocytes into decidual macrophages by comparing the surface and secretory phenotypes of resting and interferon- (IFN-)—activated monocytes, unfractionated decidual cells, purified term decidual macrophages, and monocyte-derived macrophages. Surface markers for antigen presentation (HLA-DR, CD86), a membrane-bound cytokine interleukin (IL)—15, leukocyte immunoglobulin-like receptors (LILRB1, LILRB2), and secreted anti-inflammatory cytokines (transforming growth factor [TGF]—β1 and IL-10) were assessed. The results demonstrate that differentiated, activated monocytes closely resemble but are not identical to decidual macrophages. In addition to differential IFN- responsiveness, decidual macrophages were smaller than monocyte-derived macrophages and produced IL-10, which monocyte-derived macrophages did not. Only the unfractionated decidual cells secreted TGF-β1. These results suggest that activation, differentiation, and decidual signals cooperate to program monocytes into the decidual macrophage phenotype.

The Effect of Maternal Body Condition Score Before and During Pregnancy on the Glucose Tolerance of Adult Sheep Offspring

2008-06-25

This study investigates the effects of diet-induced changes in maternal body condition on glucose tolerance in sheep. Welsh Mountain ewes were established, by dietary manipulation, at a body condition score of 2 (lower body condition [LBCS], n = 17) or >3 (higher body condition [HBCS], n = 19) prior to and during pregnancy. Birth weight and postnatal growth were similar in LBCS and HBCS offspring. In young adulthood, LBCS offspring had increased fasting glucose levels (3.8 ± 0.07 vs 3.6 ± 0.05 mM, P < .05), poorer glucose tolerance (2274 ± 22.6 vs 2161 ± 33 min/mM, P < .01), and reduced insulin secretion (0.58 ± 0.05 vs 0.71 ± 0.07 nM/min, P = .07). Increased fasting glycemia, mild glucose intolerance, and impaired initial insulin secretory response, as observed in LBCS offspring, are indictors of increased diabetes risk in humans. These findings suggest that altered maternal body composition and an imbalance between the fetal and postnatal environment influence offspring glucose tolerance.

Identification of Proteomic Biomarkers of Preeclampsia in Amniotic Fluid Using SELDI-TOF Mass Spectrometry

2008-06-25

Objective: To identify proteomic biomarkers in amniotic fluid (AF) that can distinguish preeclampsia (PE) from chronic hypertension (CHTN) and normotensive controls (CTR). Methods: AF from women with PE, CHTN, and CTR were subjected to proteomic analysis by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. Results: Proteomic profiling of AF identified 2 biomarkers: peak X (17399.11 Da), which distinguished PE from CTR, and peak Y (28023.34 Da), which distinguished PE and CHTN from CTR. High performance liquid chromatography fractions containing the biomarkers were subjected to sodium dodecyl sulfate—polyacrylamide gel electrophoresis and in-gel tryptic digestion. The biomarkers were matched to proapolipoprotein A-I (peak Y) and a functionally obscure peptide, SBBI42 (peak X). Western blot analysis confirmed that AF from PE and CHTN had higher proapolipoprotein A-I levels than CTR. Conclusion: Proteomic analysis of AF can distinguish PE from CHTN and CTR. The discriminatory proteins were identified as proapolipoprotein A-I and SBBI42.

Hypoxia Modulation of Caveolin-1 and Vascular Endothelial Growth Factor in Ovine Fetal Membranes

Cheung, C. Y., Brace, R. A. — 2008-06-25

During normal pregnancy, amniotic fluid is absorbed from the amniotic compartment into fetal blood through the intramembranous blood vessels in the fetal membranes. It has been hypothesized that this transport process is mediated by transcytosis of caveolae-like vesicles. Because fetal hypoxia increases intramembranous absorption, the authors explore the effects of hypoxia on the gene expression of caveolin-1, a structural protein of caveolae, in ovine fetal membranes and cultured amnion cells. Near-term ovine fetuses were rendered hypoxic for 4 days. Caveolin-1 mRNA and protein levels were significantly reduced in the amnion and chorion but not in the placenta. In cultured ovine amnion cells incubated in 2% oxygen for 24 hours, hypoxia did not significantly alter caveolin-1 mRNA or protein expression. vascular endothelial growth factor mRNA levels were increased in response to hypoxia in the fetal membranes as well as in cultured amnion cells. The results indicate that hypoxia does not augment but instead down-regulates or has no effect on caveolin-1 gene expression in the amnion and chorion, suggesting that caveolin-1 may play a role as a negative regulator of amnion transport function under hypoxic conditions.

Corticotrophin-Releasing Hormone in Lipopolysaccharide-Stimulated Term Fetal Membranes and Amniotic Fluid From Term and Preterm Birth in African Americans and Caucasians

Menon, R., Arora, C. P., Hobel, C. J., Fortunato, S. J. — 2008-06-25

The objective of this study is to document differences in corticotrophin-releasing hormone (CRH), CRH receptor 1 (CRHR1), and CRH binding protein (CRHBP) gene expression in fetal membranes derived from African Americans and Caucasians in vitro in response to lipopolysaccharide (LPS) stimulation and to assess racial disparity in CRH concentrations in the amniotic fluid (AF) of women with spontaneous preterm birth (PTB). Fetal membranes (African American, n = 8; Caucasian, n = 8) at term, placed in an organ explant system, were stimulated with LPS. Microarray analysis documented differences in the mRNA expression pattern of CRH, CRHBP, and CRHR1 between races. CRH was measured in AF (a case [PTB]—control [term] study) and culture media. Between races, LPS significantly increased CRH and CRHR1 expression in African Americans and CRHBP in Caucasians, with no differences in controls. CRH was detectable only in LPS-stimulated African American membranes. AF CRH concentrations were higher in PTB compared with controls (P < .001), and no difference was noticed between races (P = .1). AF analysis did not document racial disparity in CRH concentrations in PTB. In fetal membranes, African Americans showed a higher expression and production of CRH in response to an in vitro stimulus.

HB-EGF but Not Amphiregulin or Their Receptors HER1 and HER4 Is Altered in Endometrium of Women With Unexplained Infertility

Aghajanova, L., Bjuresten, K., Altmae, S., Landgren, B.-M., Stavreus-Evers, A. — 2008-06-25

Heparin-binding epidermal growth factor—like growth factor (HB-EGF) and its receptors (HER1 and HER4) play a role in the human implantation process. Amphiregulin is a member of the EGF family but with unknown function in human fertility. It has been suggested that some women with unexplained infertility have defective endometrial development. The aim of this study is to determine the presence of amphiregulin and the receptors HER1 and HER4 in normal human endometrium throughout the menstrual cycle. In addition, the present study aims to compare endometrium from women with unexplained infertility with endometrium from women with male factor infertility and healthy fertile controls. Immunohistochemistry and real-time polymerase chain reaction were used to determine the expression of HB-EGF, HER1, HER4, and amphiregulin. The stromal staining of HER1 and the epithelial staining of HER4 were most intense in the mid- and late-secretory-phase endometrium. Amphiregulin did not vary during the menstrual cycle. In the mid-secretory phase, the protein expression of HB-EGF was lower in endometrium from women with unexplained infertility versus normal endometrium and endometrium from women with male factor infertility. HB-EGF and HER4 mRNA expression in mid-secretory endometrium of women with unexplained and male factor infertility were increased compared with normal controls. Impaired endometrial expression of certain members of the EGF family may contribute to infertility in some women with unexplained infertility.

DNA-Binding Ability of NF-{kappa}B is Affected Differently by ER{alpha} and ER{beta} and Its Activation Results in Inhibition of Estrogen Responsiveness

Guzeloglu-Kayisli, O., Halis, G., Taskiran, S., Kayisli, U. A., Arici, A. — 2008-06-25

Estrogenic effects involve interactions between estrogen receptors (ERs), response elements, and nuclear proteins. It is hypothesized that interaction between ER and NF- B may affect the regulation of responsive genes. Electrophoretic mobility shift assay (EMSA) was performed to assess if the interaction of ERs and NF- B affect their respective DNA-binding activities, and alkaline phosphatase assay was done to evaluate estrogenic activity. EMSA revealed that ERs inhibit DNA-binding of p50 and p65, whereas p50 did not impair ER binding. Stimulation with estradiol inhibited DNA binding of NF-B in ER-transfected endometrial stromal cells (ESCs). Moreover, activation of NF-B significantly decreased estrogen responsiveness of Ishikawa cells and ER-transfected ESC. Our results suggest that ERs downregulate NF-B-dependent gene activation by directly preventing DNA binding. However, NF-B-mediated inhibition of ER-dependent gene activation may be carried out indirectly rather than through a direct inhibition of ER-DNA binding. These findings offer new insight into the specific role of ER and could eventually help in developing therapeutics for endometriosis.

Regional Expression of Aquaporin 1, 4, and 9 in the Brain During Pregnancy

2008-06-25

Pregnancy is a state of physiologic adaptation, with significant changes in cardiovascular, renal, and hemodynamic systems. Aquaporins (AQPs) may play a role in facilitating these changes. While AQP expression has been assessed in several organs during pregnancy, little is known about its expression in the brain during pregnancy. Therefore, this study assesses the regional expression of AQP1, 4, and 9 during pregnancy and the postpartum period using real-time quantitative polymerase chain reaction. The authors show that AQP1, 4, and 9 are expressed in the anterior and posterior cerebrum, cerebellum, and brainstem of nonpregnant, midpregnant, late pregnant, and postpartum rats. The regional distribution pattern of AQP4 and 9 remained similar during gestation, whereas this pattern changed for AQP1. The expression levels of AQP1, 4, and 9 in the brainstem did not change with gestation, whereas changes were found in the anterior cerebrum for AQP4 and in the posterior cerebrum and cerebellum for all AQPs.

Expression and Distribution of Glucocorticoid Receptors in the Ovine Fetal Adrenal Cortex: Effect of Long-term Hypoxia

Root, B., Abrassart, J., Myers, D. A., Monau, T., Ducsay, C. A. — 2008-06-25

This study was designed to determine if long-term hypoxia (LTH) alters adrenal glucocorticoid receptor (GR) expression in the ovine fetal adrenal cortex. Ewes were maintained at 3820 m from ~30 to 138 to 140 days' gestation, and fetal adrenals were collected. Western analysis revealed two ~94-kDa GR- isoforms and a lower molecular weight (45 kDa) form. A decreasing trend in the ratio of 94-kDa/45-kDa bands following LTH suggested an increase in GR turnover. Immunohistochemistry demonstrated dense GR staining in the zona glomerulosa with minimal staining in the zona fasciculata in the control group, while dense staining was observed throughout the cortex in LTH. Western analysis and reverse transcription polymerase chain reaction confirmed that the GR- β isoform is not present or expressed at extremely low levels in the fetal adrenal, hypothalamus, pituitary, and placenta. These data indicate that LTH alters GR- function in the fetal adrenal cortex and suggest that GR-β is not expressed in sheep.

Gene Expression Profiling Reveals Putative HOXA10 Downstream Targets in the Periimplantation Mouse Uterus

Vitiello, D., Pinard, R., Taylor, H. S. — 2008-06-25

HOXA10 encodes a transcription factor required for endometrial receptivity and embryo implantation. The objective of this study was to identify and to characterize those molecular markers regulated by HOXA10 expression. The authors have identified putative HOXA10 target genes identified by microarray analysis employing a murine model of transient HOXA10 expression during the anticipated implantation window. Microarray analysis identified 40 statistically significant genes regulated by HOXA10 overexpression of which 31 genes were downregulated greater than 2-fold over control and 9 genes were upregulated. Cellular ontogenies of differentially expressed genes include cell adhesion molecules, signal transduction factors, and metabolic regulators. Semiquantitative real-time reverse transcriptase polymerase chain reaction confirmed regulation of selected candidate genes. Examples included clusterin (Clu), phoshoglycerate 3-dehydrogenase (3-Pgdh), and tumor-associated calcium signal transducer 2 (Tacstd2). Elucidation of these pathways will allow further characterization of the molecular mechanisms governing endometrial development, which also may function to enhance uterine receptivity.

In the Spotlight

Maduro, M. R. — 2008-05-22

Tug of War Between Ovarian Stimulation and Endometrial Receptivity

Huang, S. J. — 2008-05-22

Review Article: Steroid Hormones and Uterine Vascular Adaptation to Pregnancy

Chang, K., Lubo Zhang, — 2008-05-22

Pregnancy is a physiological state that involves a significant decrease in uterine vascular tone and an increase in uterine blood flow, which is mediated in part by steroid hormones, including estrogen, progesterone, and cortisol. Previous studies have demonstrated the involvement of these hormones in the regulation of uterine artery contractility through signaling pathways specific to the endothelium and the vascular smooth muscle. Alterations in endothelial nitric oxide synthase expression and activity, nitric oxide production, and expression of enzymes involved in PGI₂ production contribute to the uterine artery endothelium—specific responses. Steroid hormones also have an effect on calcium-activated potassium channel activity, PKC signaling pathway and myogenic tone, and alterations in pharmacomechanical coupling in the uterine artery smooth muscle. This review addresses current understanding of the molecular mechanisms by which steroid hormones including estrogen, progesterone, and cortisol modulate uterine artery contractility to alter uterine blood flow during pregnancy with an emphasis on the pregnant ewe model.

Demonstration of the Essential Role of Protein Kinase C Isoforms in Hyperglycemia-Induced Embryonic Malformations

Zhiyong Zhao, , Wu, Y.-K., Reece, E. A. — 2008-05-22

To address the role of PKC isoforms in hyperglycemia-induced apoptosis and malformations in the embryos of diabetic pregnancies, expression of PKC, β1, β 2, , , , and was examined in the neural tube of rat embryos and showed to overlap with the regions of increased apoptosis. Levels of activated (phosphorylated) PKC , β2, and were increased in the embryos of diabetic dams whereas those of PKC and were decreased when compared with those in control groups. Cytosolic phospholipase A₂ (cPLA₂) was also activated. Blocking the activity of PKC , β2, and using isoform-specific inhibitors in embryos cultured in hyperglycemia (40 mM) reduced malformation rates when compared with those in untreated hyperglycemic and euglycemic (8.3 mM) groups. These observations demonstrate that PKC, β2, and play an essential role in diabetic embryopathy. Activation of cPLA₂ was also decreased, suggesting that PKCs mediate the hyperglycemic effects through the cPLA₂-phospholipid peroxidation pathway.

The Impact of Ovarian Stimulation With Recombinant FSH in Combination With GnRH Antagonist on the Endometrial Transcriptome in the Window of Implantation

2008-05-22

The aim of this prospective paired cohort study is to elucidate the impact of ovarian stimulation with recombinant follicle-stimulating hormone in combination with gonadotropin-releasing hormone antagonist on the endometrial transcriptome. Oocyte donors underwent endometrial biopsy during the implantation window of the nonstimulated cycle and following ovarian stimulation with recombinant follicle-stimulating hormone and gonadotropin-releasing hormone antagonist but no luteal progesterone supplementation (n = 4). Microarray analysis showed 142 genes to be significantly upregulated and 98 significantly downregulated. Significantly upregulated genes included those sequencing for the chemokine ligand CXCL 13, the Dickkopf homolog, steroidogenic acute regulatory protein, and homeobox C6. Also upregulated were genes inhibited by progesterone, such as insulin-like growth factor binding protein 5. In conclusion, ovarian stimulation with follicle-stimulating hormone and gonadotropin-releasing hormone antagonist dysregulates the expression of many genes involved in cell adhesion, T-cell receptor signaling, and regulation of signal transduction. These data suggest that dysregulation of the endometrial transcriptome in the stimulated cycle is not fully attributable to supraphysiological sex steroid levels at the folliculo-luteal transition.

Altered Endothelin Receptor Binding in Response to Nitric Oxide Synthase Inhibition in the Pregnant Rat

Neerhof, M. G., Jilling, T., Synowiec, S., Khan, S., Thaete, L. G. — 2008-05-22

The authors evaluate the expression of endothelin-1 (ET-1) and its receptors in the uterus and placenta during maternal nitric oxide synthase (NOS) inhibition. Timed-pregnant rats received L-NAME (2.5 mg/kg/h) or saline from day 14 to 21 of gestation. Uterine and placental tissues collected on day 21 were assayed for preproET-1, ET_A, and ET_B mRNA expression; localization and expression of ET-1 and receptor proteins; and receptor activity. NOS inhibition did not affect preproET-1 mRNA expression in the placenta or uterus. ET_A expression decreased in the uterine free wall, but no other changes in receptor mRNA expression were observed in the uterus or placenta. ET-1 and receptor proteins were unchanged. Placental ET_A and ET_B receptor binding decreased. Uterine ET_A receptor binding decreased in the placental bed. ET-1, a prominent mediator during NOS inhibition, is not of uterine or placental origin. Reduced receptor binding activity is the primary means by which these tissues regulate their response to ET-1 in the setting of NOS inhibition.

Evidence of Endothelial Dysfunction in Preeclampsia and Risk of Adverse Pregnancy Outcome

2008-05-22

The purpose of this study is to investigate whether endothelial dysfunction, as assessed by elevated cellular fibronectin (cFN), in women with preeclampsia is associated with an increased risk of preterm and/or small-for-gestational-age (SGA) births. Maternal plasma cFN was measured by enzyme-linked immunosorbent assay in samples collected at admission to delivery in 605 normotensive women, 171 women with transient hypertension, and 187 women with preeclampsia. Logistic regression was used to estimate the risk for preterm delivery, SGA, or both. Elevated cFN in women with preeclampsia was associated with an increased risk of both preterm and SGA births (odds ratio, 3.0; confidence interval [CI], 1.0-8.7) compared with women with preeclampsia without elevated cFN. The risk of preterm birth was 14.7-fold higher (CI, 8.1-26.7) and the risk of SGA was 6.8-fold higher (CI, 3.5-13.1) in women with preeclampsia, hyperuricemia, and elevated cFN compared with normotensive women. Elevated cFN is prevalent among women with preeclampsia and identifies women at increased risk of preterm delivery and SGA.

Upregulation of PSCDBP, TLR2, TWIST1, FLJ35382, EDNRB, and RGS12 Gene Expression in Human Myometrium at Labor

O'Brien, M., Morrison, J. J., Smith, T. J. — 2008-05-22

The regulatory mechanisms underlying myometrial smooth muscle contractility during labor are poorly understood. The authors therefore investigated the transcriptional profile of the changes that occur in the human myometrium at term pregnancy when compared with that at labor. Microarray technology was used to identify differentially expressed genes in human myometrium at labor. Real-time fluorescence reversetranscriptase polymerase chain reaction (RT-PCR) was subsequently performed to verify the microarray data. Semiquantitative RT-PCR, Western blotting, and microscopy methodologies were also used. Certain novel genes were found to be upregulated in human myometrium at labor. Of these, PSCDBP, TLR2, TWIST1 , FLJ35382, andRGS12 have not been previously characterized or identified in human myometrium. EDNRB is the other novel labor-associated gene whose reported expression is also upregulated at labor. All 6 genes were expressed on human myometrial smooth muscle cells. These novel upregulated genes are involved in multiple pathways that may be associated with a variety of cellular processes including inflammation, transcriptional regulation, and intracellular signaling.

Alterations in Saliva Steroid Hormone Levels After Oral Mifepristone Administration in Women With Pregnancies of Greater Than 41 Weeks' Gestation

Fassett, M. J., Lachelin, G. C. L., McGarrigle, H. H. G., Wing, D. A. — 2008-05-22

The objective of this study is to describe the effects of oral mifepristone administration on saliva levels of estradiol, estriol, progesterone, and cortisol in women with postdates pregnancy. As an adjunct to a randomized controlled trial comparing 200 mg oral mifepristone to placebo for cervical ripening and labor induction in women with pregnancies greater than 41 weeks' gestation, saliva samples were obtained before drug administration and every 6 hours thereafter for 24 hours. Estradiol, estriol, progesterone, and cortisol levels were measured by radioimmunoassay. Ninety-seven participants received mifepristone, and 83 received placebo. Saliva steroid hormone data were available for 71 mifepristone-and 60 placebo-treated women. Mean baseline saliva estradiol, estriol, progesterone, and cortisol levels were similar between study groups. At 24 hours after study medication administration, saliva estradiol, estriol, progesterone, and cortisol levels in the mifepristone group were significantly elevated compared with baseline. There was no significant change in hormone levels in the placebo group. Oral mifepristone significantly increased saliva estradiol, estriol, progesterone, and cortisol compared with placebo. This may reflect mifepristone's antiglucocorticoid properties. These hormone elevations may contribute to the mechanism by which mifepristone causes cervical ripening and increases myometrial activity.

Loss of Cyclin G1 Expression in Human Uterine Leiomyoma Cells Induces Apoptosis

2008-05-22

Observations from the authors' laboratory suggest a physiological role for increased cyclin G1 protein levels in human uterine leiomyoma. The hypothesis of the present study is that the strategic modulation of cyclin G1 by antisense technology will inhibit the survival of in vitro—grown uterine leiomyoma cells. Cultured uterine leiomyoma cells were transfected with cyclin G1 ribbon-type antisense oligonucleotide (cyclin G1 RiAS) to effectively reduce cyclin G1 expression. Cell viability, in situ terminal deoxyuridine nick end-labeling (TUNEL) assay, flow cytometry, DNA fragmentation, and expression of cell cycle regulatory—related proteins were evaluated by Western blot. Antisense oligonucleotides compromised uterine leiomyoma cell viability and inducted apoptosis in a caspase-independent mechanism. In situ TUNEL and DNA fragmentation revealed apoptosis induction, and fluorescent-activated cell sorting analysis showed increased sub-G1-phase cells. Furthermore, abrogation of cyclin G1 enhanced p53 accumulation, phosphorylation of p53 at Ser-15 residue, and increased expression of cyclin-dependent kinase inhibitors p21 and p27. These data imply that cyclin G1 expression is associated with growth promotion and the potential utility and novelty of using ribbon-type antisense oligonucleotides as a gene therapy strategy to treat human uterine leiomyoma.

The Influence of Pregnancy and Gender on Perivascular Innervation of Rat Posterior Cerebral Arteries

Aukes, A. M., Bishop, N., Godfrey, J., Cipolla, M. J. — 2008-05-22

The authors investigated the influence of pregnancy and gender on the density of trigeminal and sympathetic perivascular nerves in posterior cerebral arteries (PCA) and the reactivity to norepinephrine and calcitonin gene-related peptide (CGRP). PCAs were isolated from nonpregnant, late-pregnant, postpartum, and male rats, mounted and pressurized on an arteriograph chamber to obtain concentration-response curves to norepinephrine and CGRP. Arteries were immunostained for CGRP-, tyrosine hydroxylase—, and protein gene product 9.5 (PGP 9.5)—containing perivascular nerves, and nerve density was determined morphologically. Pregnancy had a trophic effect on trigeminal perivascular innervation (P < .01 vs male); however, this was not accompanied by a change in reactivity to CGRP. Sympathetic and PGP 9.5 nerve densities were not altered by pregnancy or gender, and there were no differences in reactivity to norepinephrine. Together, these results suggest that the increase in trigeminal innervation during pregnancy is more related to nociception than in controlling resting cerebral blood flow.

Placental Matrix Metalloproteinase--1 Expression Is Increased in Labor

Vu, T.-D., Yun Feng, , Placido, J., Reznik, S. E. — 2008-05-22

Matrix metalloproteinases (MMPs) are now known to process a broad spectrum of cell surface molecules and to function in several important biological processes. Testing for differences in gene expression in human placental chorionic villi in the absence or presence of labor, using cDNA microarray analysis, revealed that labor was associated with increased expression of MMP-1 gene expression in 5 placentas collected after term normal spontaneous deliveries compared with 5 placentas collected after term nonlaboring cesarean deliveries. Fibronectin 1 and collagen XVII, 2 other proteins involved in the homeostasis of the extracellular matrix, were also found to be upregulated in labor. MMP-1 was further tested in individual samples and found to be consistently overexpressed in labor. While previous microarray analyses have focused on either uterine tissue or the fetal membranes, the data presented here indicate for the first time that placental chorionic villus genes are likely to affect the initiation of parturition through altered processing of cell surface molecules by MMP-1.

In the Spotlight

Maduro, M. R. — 2008-04-17

Review Article: Expression and Function of Toll-Like Receptors at the Maternal--Fetal Interface

Koga, K., Mor, G. — 2008-04-17

Toll-like receptors (TLRs) form the major family of pattern recognition receptors that are involved in innate immunity. Innate immune responses against microorganisms at the maternal—fetal interface may have a significant impact on the success of pregnancy because intrauterine infections have been shown to be strongly associated with certain complications of pregnancy. At the maternal—fetal interface, TLRs are expressed not only in the immune cells but also in nonimmune cells such as trophoblasts and decidual cells. Moreover, their expression patterns vary according to the stage of pregnancy. Here we will describe potential functions of TLRs in these cells, their recognition and response to microorganisms, and their involvement in the innate immunity. The impact of TLR-mediated innate immune response will be discussed via animal model studies, as well as clinical observations.

Expression of Fox Head Protein 1 in Human Eutopic Endometrium and Endometriosis

Lulu Fu, , Girling, J. E., Rogers, P. A. W. — 2008-04-17

The objective of this study is to examine the localization and expression of FOXP1 in human endometrium during the menstrual cycle and in endometriotic lesions and endometrial adenocarcinoma. FOXP1 protein expression was analyzed by immunohistochemistry and Western blot. FOXP1 expression was significantly different between glandular epithelial and stromal nuclei and cytoplasm in both endometrial functionalis and basalis. FOXP1 immunostaining was significantly reduced in the early secretory stage in comparison to the mid proliferative stage in the functionalis and the early proliferative stage in the basalis. FOXP1 expression was found in endometriotic lesions but not in endometrial adenocarcinoma. Multiple protein bands of FOXP1 were identified, and their presence varied considerably among patients. Protein expression levels were significantly higher in the mid and late secretory stages in comparison to early proliferative and early secretory stages. FOXP1 protein is present in human endometrium with evidence of dependent changes in expression.

The Impact of ACTH Receptor Knockdown on Fetal and Adult Ovine Adrenocortical Cell Function

Yixin Su, , Rose, J. C. — 2008-04-17

Preparing the mammalian fetus for birth requires an increase in fetal plasma glucocorticoid levels. The mechanisms facilitating this increase are not fully known. It has been shown in sheep that the prepartum elevation in fetal plasma cortisol is accompanied by increases in adrenocorticotropin receptor (ACTH-R) expression in the fetal adrenal and in the adrenal responsiveness to stimulation. To determine the significance of the upregulation in ACTH-R expression on fetal adrenal function, the authors used small interfering RNA targeted to the ovine ACTH-R to reduce receptor expression and studied responses to stimulation in ovine adrenal cells. They studied fetal cells from late gestation after responsiveness had increased. They also studied adult cells to determine if maturation would influence the impact of receptor expression suppression on responsiveness. Fetal and adult cells were obtained, dispersed, transfected with receptor-targeted small interfering RNA or scrambled small interfering RNA, and subsequently stimulated with ACTH. Cells and media were harvested for measurements of gene and protein expression and cyclic adenosine monophosphate (cAMP) and cortisol levels. The ability of ACTH to upregulate its receptor or steroid acute regulatory protein was attenuated in fetal (P < .01) and adult cells (P < .01) by small interfering RNA treatment; the blockade was more pronounced in the adult cells (P < .01). The small interfering RNA treatment also blocked the cAMP response to ACTH in fetal (P < .001) and adult (P < .05) cells. This was accompanied by marked reductions in cortisol responses in both (P < .001 and P < .01, respectively). These data suggest that upregulation of the ACTH-R expression in late gestation is essential for the increase in adrenal steroidogenic capacity occurring then. The data also indicate that a reduction in the ACTH-R expression blocks the ability of the peptide to stimulate early steps in the steroidogenic pathway event after maturation is complete.

Shotgun Proteomic Analysis of Vaginal Fluid From Women in Late Pregnancy

Klein, L. L., Jonscher, K. R., Heerwagen, M. J., Gibbs, R. S., McManaman, J. L. — 2008-04-17

Liquid chromatography and tandem mass spectrometry without prior fractionation (shotgun proteomics) were used to analyze vaginal fluid from patients admitted for signs and symptoms of preterm labor. The patients had an average age of 26.3 ± 5.9 years, a gestational age of 30.5 ± 2.5 weeks, and a median cervical dilation of 1 cm (range, 0-6 cm). None of the patients exhibited signs of vaginal infection at the time of enrollment. Shotgun proteomics yielded reproducible identifications (R = 0.973) of more than 40 proteins in vaginal fluid samples, such as plasma proteins, epithelial structural proteins, and several immunoregulatory proteins, including some that were previously linked to intra-amniotic infection. This initial characterization of the vaginal fluid proteome using a nonbiased, high-throughput technique yields reproducible results in late pregnancy. The presence of host defense proteins in vaginal fluid suggests that this technique may be useful for future study of inflammation-related preterm birth.

Peripubertal Hyperinsulinemia Upregulates Phosphatidylinositol 3-Kinase/Akt Pathway in Rat Ovaries

Chakrabarty, S., Nagamani, M. — 2008-04-17

Objective: Polycystic ovary syndrome (PCOS) usually develops during peripuberty and is often associated with hyperinsulinemia. Paradoxically, hyperinsulinemic patients with PCOS develop peripheral insulin resistance but retain ovarian insulin sensitivity. We investigated the effect of peripubertal hyperinsulinemia on insulin signaling pathways in rat ovaries. Methods: Hyperinsulinemia was induced in peripubertal female rats by infusing insulin (0.14 IU/day) for 4 weeks. Control animals received normal saline. At autopsy, trunk blood was collected for insulin assay; ovaries were collected for examining the effect of hyperinsulinemia on phosphatidylinositol 3-kinase/Akt (PI3-K/Akt) and mitogen-activated protein kinase (MAPK/ERK1/2) pathways. Results: Compared with control, ovarian protein levels of total Akt, phospho-Akt, and phospho-glycogen synthase kinase-3β were upregulated and phospho-phosphatase and tensin homolog was downregulated in hyperinsulinemic animals. The MAPK/ERK1/2 pathway was unaffected. Conclusions: Peripubertal hyperinsulinemia upregulates the PI3-K/Akt pathway in rat ovaries. Ovarian insulin sensitivity in hyperinsulinemic patients with PCOS is potentially retained by this mechanism.

Differential Gene Expression Profiling in HELLP Syndrome Placentas

Kang, B.-Y., Tsoi, S., Shan Zhu, , Shenghui Su, , Kay, H. H. — 2008-04-17

The aim of this study was to identify differentially expressed genes by suppression subtractive hybridization (SSH) in HELLP placentas. Two cDNA libraries were constructed; HSI (HELLP subtracted induced or upregulated) and HSS (HELLP subtracted suppressed or downregulated). Two hundred eighty-eight cDNA clones were sequenced; 37 were matched to GenBank entries and included genes in cell communication and organization, cellular processes, genetic information processing, and metabolic processes. A subgroup of 11 genes of interest was further selected for real-time quantitative polymerase chain reaction confirmation. Results showed no differences in expression of chosen upregulated genes between HELLP and non-HELLP placentas; 6 HELLP downregulated genes were significantly suppressed. Two genes related to production of secreted proteins, CTHRC1 and SERPINE2. SERPINE2 (PAI-1) is a soluble protease inhibitor and is a potential biomarker by Western blot analysis, and the protein is significantly decreased in HELLP placentas. SERPINE2 might be tested clinically in patients for early diagnosis of HELLP syndrome.

Jugular Lymphatic Maldevelopment in Turner Syndrome and Trisomy 21: Different Anomalies Leading to Nuchal Edema

2008-04-17

Increased nuchal translucency (NT), morphologically known as nuchal edema, is an ultrasound marker for aneuploidy. Turner syndrome presents with massive NT, called cystic hygroma. Conflicting data exist as to whether cystic hygroma and increased NT are different entities. Both are associated with jugular lymphatic distension. The authors investigated jugular lymphatics of trisomy 21, Turner syndrome, and normal karyotype fetuses. Fetuses were investigated using immunohistochemistry for blood vascular, lymphatic, and smooth muscle cell markers. Trisomy 21 fetuses showed nuchal cavities within the mesenchymal edema negative for endothelial markers. These were extremely large in Turner fetuses, showing similar characteristics. The skin showed numerous dilated lymphatics in the case of trisomy 21 and scanty small lymphatics in Turner fetuses. A jugular lymphatic sac was present in control and trisomy 21 fetuses and was enlarged in trisomy 21 cases. In Turner fetuses, no jugular lymphatic sac was observed. Nuchal edema in trisomy 21 and Turner syndrome appears to be a similar entity caused by different lymphatic abnormalities.

A Digenic Combination of Polymorphisms Within ESR1 and ESR2 Genes Are Associated With Age at Menarche in the Spanish Population

2008-04-17

In the present study, the authors look at an association of genetic variants within estrogen synthesis and signaling pathways and age at menarche (AAM) in Spanish women. They analyzed 9 polymorphisms in 6 different genes in 714 well-characterized postmenopausal women from Spain. They performed a quantitative trait locus study of these markers individually or in digenic combinations in relation to AAM. None of the studied markers, with the exception of the follicle-stimulating hormone receptor (P = .013), were significantly associated with AAM in the Spanish population, and no marker demonstrated an association of statistical significance after multiple testing corrections ( P > .0055). In contrast, linear regression analysis suggests epistatic interactions including ESR1 and ESR2 loci in relation to AAM in the series (P = .003). The results suggest that epistatic interactions of ESR1 and ESR2 alleles could be associated with advancing AAM among Spanish women.

Effect of Relaxin on TGF-{beta}1 Expression in Cultured Vaginal Fibroblasts From Women With Stress Urinary Incontinence

Yan Wen, , Zhao, Y.-Y., Polan, M. L., Chen, B. — 2008-04-17

The objective of this study is to compare relaxin's effect on transforming growth factor (TGF)— β1 and latent TGF-β1—binding protein (LTBP-1) in vaginal fibroblasts from women with stress urinary incontinence (SUI) to continent women (controls) in both phases of the menstrual cycle. Fibroblasts were treated with relaxin. TGF-β1 levels were measured by enzyme-linked immunosorbent assay. LTBP-1 expression was evaluated by Western blot. In the proliferative phase, total TGF-β1 level in the supernatant, cells, and extracellular matrix (ECM) of SUI fibroblasts decreased with increasing relaxin concentration (P < .05). Active TGF-β1 levels increased at a low concentration of relaxin (P < .05) in the supernatant but decreased in the ECM of SUI fibroblasts at high concentration (P < .05). In the secretory phase, total TGF-β1 levels decreased with relaxin treatment (P < .05) in the supernatant, cells and ECM of both women with SUI and controls. Relaxin decreased the levels of total and active TGF-β1 in the ECM isolated from SUI vaginal fibroblasts.

Lipoxygenase Pathway Receptor Expression in Ovarian Cancer

2008-04-17

Objective: To determine the expression of lipoxygenase (LOX) pathway receptors in ovarian cancer as a potential target for anti-LOX—based therapy. Study design: Paraffin-embedded tumor samples from epithelial ovarian cancer patients were used to construct tissue microarrays to stain for the proposed sites of inhibition of a LOX inhibitor (5-LOX, LTB4-BLT1, and LTB4-BLT2). Results: 245 samples were available for interpretation. Strong expression was demonstrated in 45%, 34%, and 6% of ovarian cancer for LTB4-BLT2, LTB4-BLT1, and 5-LOX, respectively. Expression of LTB4-BLT2 correlated with advanced stage III/IV disease (P = .05), suboptimal debulking (P = .07), and platinum resistance (P = .03). No correlation was seen with regard to disease-free survival. Conclusions: LOX pathway receptor expression was found in the majority of cancers evaluated. Additionally, LTB4-BLT2 expression portends worse clinical parameters for ovarian cancer. Thus, further investigation on the role of LOX pathway in ovarian cancer is warranted.