Reproductive Sciences current issue

Review: Luminescence as a Tool to Assess Pelvic Endometriosis Development in Murine Models

Defrere, S., Colette, S., Lousse, J.-C., Donnez, J., Van Langendonckt, A. — Mon, 16 Nov 2009 19:08:49 PST

Classic murine endometriosis models may be insufficient to evaluate the effect of therapeutic agents on endometriosis development, because the process of identification and measurement of induced lesions is often impeded, as implants are small and embedded in murine tissue. In this context, as summarized in the current review, luminescence techniques have proved useful for identifying and visualizing or quantifying endometriotic transplants. They are also a valuable tool for endometrial cell tracking in live animals, yielding further information by adding spatial and temporal dimensions to biological processes in vivo. Such approaches involve transplanting luminescently labeled murine or human endometrium into animals. Two main strategies are applied to label endometrium before injection: use of genetically modified tissue or tissue labeled with a fluorescent dye. Each model has its advantages and disadvantages, the choice of model depends on the study objectives/design (long- or short-term studies, homologous or heterologous model).

Elafin (SKALP/Trappin-2/proteinase inhibitor-3) Is Produced by the Cervix in Pregnancy and Cervicovaginal Levels Are Diminished in Bacterial Vaginosis

Mon, 16 Nov 2009 19:08:50 PST

Objectives. To examine cervicovaginal elafin production in pregnancy and determine its relationship in bacterial vaginosis. Study Design. Samples of cervicovaginal secretions were collected from women with uncomplicated singleton pregnancies (n = 112) below 20 weeks gestation. Bacterial flora was assessed using Nugent’s criteria, and levels of elafin were measured by enzyme-linked immunosorbent serologic assay (ELISA). Elafin expression in the cervix was also examined by immunohistochemistry. In vitro expression of elafin was examined using cervix and vaginal cell lines. Results. Elafin is expressed in the cervical glandular epithelium. Elafin was found in all 112 samples of cervicovaginal secretions and levels were diminished in women with bacterial vaginosis (P < .05). Interleukin 1β (IL-1β) stimulated elafin expression in cells derived from the endocervix, but not in those derived from the vaginal epithelium. Conclusions. Elafin is a component of cervicovaginal secretions in pregnancy, and levels are diminished in bacterial vaginosis. It may be an important component of innate immunity in the lower genital tract.

Possible Gene-Gene Interaction of KIR2DL4 With its Cognate Ligand HLA-G in Modulating Risk for Preeclampsia

Mon, 16 Nov 2009 19:08:50 PST

Preeclampsia (PE) is a leading cause of maternal and fetal mortality and morbidity that occurs only during pregnancy. Pregnancy is the only physiological situation where killer-cell immunoglobulin-like receptors (KIRs) may meet cognate nonself variants of human leukocyte antigen (HLA) allotypes. We previously reported that presence of fetal HLA-G*0106 was significantly associated with risk for PE in multigravid pregnancies. We have now tested the KIR2DL4 receptor gene for association with PE, as well as for its interaction with HLA-G in modulating disease risk, in a case-control study of 83 PE and 240 normotensive pregnancies. No significant association was observed between alleles of KIR2DL4 and PE in both maternal and fetal groups, either among primigravid or multigravid pregnancies. Alleles of KIR2DL4 and HLA-G were then analyzed together to determine whether particular variant ligand—receptor combinations were associated with an increased risk for PE. Gene-gene interaction analyses suggest that the presence of fetal HLA-G*0106 in combination with maternal KIR2DL4*006 is significantly associated with PE risk in multigravid pregnancies (P < .001). These data provide the first preliminary evidence suggesting that although KIR2DL4 itself is not associated with PE, it may modulate the effect of HLA-G*0106 on risk for PE.

Aberrant Processing of Plasma Vitronectin and High-Molecular-Weight Kininogen Precedes the Onset of Preeclampsia

Blumenstein, M., Prakash, R., Cooper, G. J. S., North, R. A., SCOPE Consortium — Mon, 16 Nov 2009 19:08:50 PST

To date, there is no reliable test to identify women in early pregnancy at risk of developing preeclampsia. Difference gel electrophoresis (DIGE) identified the plasma proteins vitronectin (VN) and high-molecular-weight kininogen (HK) in association with preeclampsia. In a longitudinal proteomics study, the plasma of preeclamptic patients (n = 6) was compared to healthy control participants (n = 6) before the onset of preeclampsia (week 20) and at the time of presentation with clinical disease (weeks 33-36). The 75-kd single-chain VN molecule increased 1.6- to 1.9-fold in preeclampsia, whereas the 65-kd moiety of the 2-chain VN molecule decreased 1.5- to 1.7-fold compared to healthy controls (P < .05). Immunoblots revealed differences in proteolytic processing of VN and/or HK in women who develop preeclampsia or preeclampsia further complicated by small-for-gestational-age. Vitronectin and HK may prove to be useful as early markers of fibrinolytic activity and neutrophil activation, which are known to be associated with preeclampsia.

Transforming Growth Factor {beta}3 Regulates the Versican Variants in the Extracellular Matrix-Rich Uterine Leiomyomas

Mon, 16 Nov 2009 19:08:50 PST

Uterine leiomyoma are common, benign tumors that are enriched in extracellular matrix. The tumors are characterized by a disoriented and loosely packed collagen fibril structure similar to other diseases with disrupted Transforming growth factor β (TGF-β) signaling. Here we characterized TGF-β3 signaling and the expression patterns of the critical extracellular matrix component versican in leiomyoma and myometrial tissue and cell culture. We also demonstrate the regulation of the versican variants by TGF-β3. Using leiomyoma and matched myometrium from 15 patients, messenger RNA (mRNA) from leiomyoma and myometrium was analyzed by semiquantitative real time reverse transcription—polymerase chain reaction (RT-PCR), while protein analysis was done by western blot. Transforming growth factor β3 transcripts were increased 4-fold in leiomyoma versus matched myometrium. Phosphorylated-TGF-β RII and phosphorylated-Smad 2/3 complex were greater in leiomyoma as documented by Western blot. The inhibitor Smad7 transcripts were decreased 0.44-fold. The glycosaminoglycan (GAG)-rich versican variants were elevated in leiomyoma versus myometrial tissue: specifically V0 (4.27 ± 1.12) and V1 (2.01 ± 0.27). Treatment of leiomyoma and myometrial cells with TGF-β3 increased GAG-rich versican variant expression 7 to 12 fold. Neutralizing TGF-β3 antibody decreased the expression of the GAG-rich versican variants 2 to 8 fold in leiomyoma cells. Taken together, the aberrant production of excessive and disorganized extracellular matrix that defines the leiomyoma phenotype involves the activation of the TGF-β signaling pathway and excessive production of GAG-rich versican variants.

Gonadotropin Stimulation Increases the Expression of Angiotensin-(1--7) and Mas Receptor in the Rat Ovary

Mon, 16 Nov 2009 19:08:50 PST

We have previously shown the presence of immunoreactive angiotensin-(1—7) [Ang-(1—7)] in rat ovary homogenate and its stimulatory effect on estradiol and progesterone production in vitro. In the current study, we investigated the presence and cellular distribution of Ang-(1—7) and the Mas receptor, the expression of Mas and angiotensin-converting enzyme 2 (ACE2) messenger RNA (mRNA), and the enzymatic activity in the rat ovary following gonadotropin stimulation in vivo. Immature female Wistar rats (25 days old) were injected subcutaneously (SC) with equine chorionic gonadotropin (eCG, 20 IU in 0.2 mL) or vehicle 48 hours before euthanasia. Tissue distributions of Ang-(1—7), Mas receptor, and ACE2 were evaluated by immunohistochemistry, along with angiotensin II (Ang II) localization, while the mRNA expression levels of Mas receptor and ACE2 were evaluated by real-time polymerase chain reaction (PCR). In addition, we determined the activity of neutral endopeptidase (NEP), prolyl endopeptidase (PEP), and ACE by fluorometric assays. After eCG treatment, we found strong immunoreactivity for Ang-(1—7) and Mas primarily in the theca-interstitial cells, while Ang II appeared in the granulosa but not in the thecal layer. Equine chorionic gonadotropin treatment increased Mas and ACE2 mRNA expression compared with control animals (3.3- and 2.1-fold increase, respectively; P < .05). Angiotensin-converting enzyme and NEP activities were lower, while PEP activity was higher in the eCG-treated rats (P < .05). These data show gonadotropin-induced changes in the ovarian expression of Ang-(1—7), Mas receptor, and ACE2. These findings suggest that the renin-angiotensin system (RAS) branch formed by ACE2/Ang-(1—7)/Mas, fully expressed in the rat ovary and regulated by gonadotropic hormones, could play a role in the ovarian physiology.

Regulation of Human Umbilical Artery Contractility By Different Serotonin and Histamine Receptors

Santos-Silva, A. J., Cairrao, E., Marques, B., Verde, I. — Mon, 16 Nov 2009 19:08:50 PST

We studied the role of several serotonin (5-HT) and histamine receptors in the regulation of human umbilical artery (HUA) contractility. Among the 5-HT agonists used, only the 5-HT_2A and 5HT_1B/D agonists contracts HUA. The 5-HT-induced contractions were fully inhibited by ketanserin (5-HT_2A antagonist). The 5-HT₇-activation also relaxes and increases intracellular cyclic adenosine monophosphate (cAMP). Among the histamine receptor agonists, only betahistine (H₁ agonist) induced significant contractile effect. Histamine-induced contraction was partially relaxed by pyrilamine (H₁ antagonist). Betahistine-induced contraction was partially blocked by dimaprit (H₂ agonist) and by the H₃ agonist when a low concentration of forskolin is present. Both, H₂ and H₃ agonists increased the cAMP intracellular levels in HUA smooth muscle. These findings show that in HUA, 5-HT_2A- and 5-HT_1B/1D-activation lead to vasoconstriction and 5-HT₇-activation induces vasorelaxation. Concerning histamine receptors, H₁-activation induces contraction and H₂- and H₃-activation lead to vasorelaxation.

Enhanced Expression of P2X4 and P2X7 Purinergic Receptors in the Myometrium of Pregnant Rats in Preterm Delivery Models

Urabe, S., Miyoshi, H., Fujiwara, H., Yamaoka, K., Kudo, Y. — Mon, 16 Nov 2009 19:08:50 PST

The expression levels of P2X purinergic receptors were determined in the myometrium of pregnant rats using the quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR). The messenger RNAs (mRNAs) of P2X4 and P2X7 were expressed most strongly. The expression levels of these receptors increased during the late stages of pregnancy; at the time of delivery, the mRNA levels of P2X4 and P2X7 had increased to 1.9 and 3.2 times the day 19 values, respectively. We also explored the roles of P2X receptors in hormone-induced and inflammation-induced preterm delivery models. In the former, mifepristone caused the P2X4 and P2X7 mRNA levels to increase to 2.1 and 4.1 times the control values, respectively. In the latter, lipopolysaccharide (LPS) caused the mRNA levels of P2X4 and P2X7 to increase dramatically to 7.4 and 18.6 times the control values, respectively. These findings suggest that increased P2X4 and P2X7 receptor expression in pregnant rats is related to uterine contraction leading to term and preterm delivery.

The Role of IGF-1 and Ghrelin in the Compensation of Intrauterine Growth Restriction

Mon, 16 Nov 2009 19:08:50 PST

The role of insulin-like growth factor 1 (IGF-1) and ghrelin in intrauterine growth restricted (IUGR) neonates in comparison to appropriate for gestational age (AGA) ones was investigated. Levels of IGF-1/insulin-like growth factor binding protein 3 (IGFBP3), ghrelin, insulin, and cortisol were determined in 20 singleton, full-term IUGR and 20 respective AGA neonates at birth (umbilical cord-UC), on days 1 (d1) and 4 (d4) postnatally. The ratio of IGF-1 to birth weight was higher in IUGR than in AGA in both UC (18.2 ± 1.2 vs14.4 ± 0.9, P = .05) and d1 (9.6 ± 0.5 vs 6.8 ± 0.3, P = .05). A significant positive correlation was found between IGF-1 and ghrelin levels and a negative one between IGFBP3 and ghrelin only in IUGR. In both groups, fetal IGF-1 levels negatively correlated with fetal cortisol levels. Intrauterine growth restricted neonates demonstrate a relative IGF-1 resistance in an attempt to drive energy toward survival on the expense of growth. The observed correlations between ghrelin and IGF-1/IGFBP3 postnatally indicate that ghrelin might play a role in the compensation of intrauterine undernutrition, promoting postnatal growth.

Do Alterations in Placental 11{beta}-Hydroxysteroid Dehydrogenase (11{beta}HSD) Activities Explain Differences in Fetal Hypothalamic-Pituitary-Adrenal (HPA) Function Following Periconceptional Undernutrition or Twinning in Sheep?

Mon, 16 Nov 2009 19:08:50 PST

Periconceptional undernutrition (UN) in sheep accelerates fetal hypothalamic-pituitary-adrenal (HPA) axis activation, resulting in preterm birth. In contrast, twin conception suppresses fetal HPA function and delays prepartum HPA activation. We hypothesized that these dissimilar effects on fetal HPA activity result from different influences of maternal glucocorticoid (GC) on maturation of the fetal HPA axis, mediated via different activities of placental 11β-hydroxysteroid dehydrogenase (11βHSD) isozymes. We examined the effects of twinning and maternal periconceptional UN from 60 days before until 30 days after mating on the ontogeny of placental 11βHSD-1 and -2 enzyme activities. At day 85 of gestation, placental 11βHSD-2 activity was lower in UN than in normally nourished (N) fetuses (P < .05) and was higher in twins than in singletons (P < .05). Furthermore, placental 11βHSD-1 activity was not different between nutritional groups but was higher in twins than in singletons (P = .01). At day 85, fetal plasma cortisol (P < .001) and cortisone (P < .001) concentrations were lower in UN than in N fetuses, but the cortisol to cortisone ratio was higher in UN than in N fetuses (P = .01). There was no effect of fetus number on plasma cortisol or cortisone concentrations or on the ratio of cortisol to cortisone at day 85. Therefore, periconceptional UN and twinning may result in the alterations of placental 11βHSD isozyme activities at particular times during gestation. Changes in these activities during critical periods of fetal development could affect transplacental transfer or placental generation of GCs that reach the fetus, potentially influencing the timing of activation of the fetal HPA axis, fetal maturation, and hence the development and health later in life.