Influences of treatment of early pregnant mares with the progestin altrenogest on embryonic development and gene expression in the endometrium and conceptus

A positive influence of altrenogest treatment on a retarded development of the conceptus around the beginning of placentation in mares older than 8 years could be recently demonstrated. In the present study, effects of altrenogest treatment in early-pregnant mares on conceptus development and expression of endometrial and embryonic genes were investigated. Genes were chosen according to a possible involvement in embryo-maternal interaction and embryonic development in the equine species. Mares were treated with altrenogest (0.044 mg/kg bodyweight) or sunflower oil (placebo) from day 5 to 11 after ovulation. Embryos (altrenogest n = 13, placebo n = 12) and biopsies were collected on day 11. Pregnancy rate and embryonic size were not influenced by treatment (embryonic diameter: altrenogest 7.0 ± 2.5, placebo 6.5 ± 1.7 mm, n.s.). The percentage of luminal epithelial cells, superficial glandular epithelial cells and interstitial cells with nuclei staining positively for the progesterone receptor was significantly lower (P < 0.05) in samples collected from altrenogest-treated than from placebo-treated mares (e.g., luminal epithelium: altrenogest 1.9 ± 1.7%, placebo 23.0 ± 10.5%, P < 0.05). Staining for COX2 (cyclooxygenase-2) was not affected by treatment. In the endometrium a slight but significant increase in the number of PMN (polymorph nuclear neutrophils) was seen in response to treatment (altrenogest 0.8 ± 0.5 PMN/field, placebo 0.3 ± 0.3 PMN/field; P < 0.05). No differences in the relative gene expression of COX2, the receptors for progesterone, estrogens and growth hormone as well as for IGF (insulin-like growth factor) 1 and 2 were detected. The relative gene expression of aquaporin 3 in relation to β-actin differed significantly (P < 0.05) between embryos from altrenogest (3.2 ± 0.8) and placebo-treated mares (1.3 ± 0.2), but no other genes were affected. The study demonstrates down-regulation of progesterone receptors in the endometrium of early pregnant mares by treatment with the progestin altrenogest. This increased expression of aquaporin 3 in the conceptus was not related to changes in embryonic size or development.     

Effect of conceptus secretions on HOXA10 and PTGS2 gene expression, and PGE2 release in co-cultured luminal epithelial and stromal cells of the porcine endometrium at the time of early implantation

Homeobox A10 (HOXA10) gene expression was demonstrated in the endometrium of adult porcine uteri, however there is little information concerning the role of this gene in the pig. Objectives of the present study were to examine: 1) the expression of HOXA10 in the endometrium of cyclic and early pregnant gilts; 2) the effect of estradiol (E₂) and progesterone (P₄) on HOXA10 expression in porcine luminal epithelial (LE) and stromal (ST) cells in vitro; 3) the effect of E₂ and conceptus-exposed medium (CEM) on HOXA10 and prostaglandin endoperoxide synthase (PTGS2) gene expression and prostaglandin (PG) E₂ secretion from LE and ST cells in a co-culture model. The abundance of HOXA10 mRNA was increased on day 15 of pregnancy in comparison to day 15 of the estrous cycle. Moreover, increased HOXA10 mRNA level was detected in ST cells after E₂ and P₄ treatment. E₂ stimulated the expression of HOXA10 in LE cells cultured on collagen and pre-treated with steroids, but not in LE on plastic surfaces. Addition of CEM to LE cells cultured in collagen-coated inserts of the co-culture system resulted in elevated HOXA10 and PTGS2 gene expression and PGE₂ secretion in these cells, but not in ST cells cultured in basal compartments. ST cells directly treated with E₂ or CEM showed higher levels of HOXA10 and PTGS2 expression. Blocking of estrogen receptors with ICI-182,780 did not influence the stimulatory effect of CEM. We conclude that HOXA10 expression in the porcine endometrium is closely related to the implantation process and stimulated by conceptus products. Moreover, the co-culture system of LE and ST cells is a promising model for the study of endometrial response to conceptus-derived factors.     

Immunolocalization of beclin 1, a bcl-2-binding,autophagy-related protein,in the human ovary: possible relation to life span of corpus luteum

Ovarian tissue homeostasis is maintained by highly regulated cyclic phases of cell proliferation/differentiation and programmed cell death. Compelling evidence indicates that both apoptotic and autophagic types of programmed cell death are involved in the regression of the corpus luteum (CL) in primate species. Beclin 1 is an autophagy-related protein that is involved in the inter-relationships between apoptosis and autophagy, through interaction with the anti-apoptotic protein bcl-2. We studied the presence and expression pattern of beclin 1 in the adult human ovary. In ovarian follicles, beclin 1 immunostaining was found in the theca layer, whereas granulosa cells were negative. After ovulation, beclin 1 immunostaining was present in both theca-lutein and granulosa-lutein areas. The expression of beclin 1 in granulosa-lutein cells was related to the functional and structural status of the CL, being strong at the early and mid luteal phases, barely detectable at the late luteal phase, and absent in granulosa-lutein cells in subsequent cycles. Our results indicated that beclin 1 expression was related to luteal cell survival rather than to cell death. Accordingly, persistent beclin 1 expression was found in granulosa-lutein cells under either physiological (i.e., CL of pregnancy) or pathological (irregularly regressing CL in climacteric women) conditions involving prolonged CL life span. Strong beclin 1 immunostaining was also found in ovarian androgen-producing cells (i.e., secondary interstitial and hilus cells). Our data thus suggest that beclin 1 plays important roles in the regulation of the life span of human CL and ovarian androgen-secreting cells, by maintaining autophagy at levels promoting cell survival rather than cell death. This study was subsidized by grant BFU 2005-01443 from the DGICYT (Spain).     

Effect of oestrus synchronization methods on oestrus behaviour, timing of ovulation and pregnancy rate during the breeding and low breeding seasons in Nili-Ravi buffaloes

The objective of this study was to determine the effect of oestrous synchronization methods on oestrous behaviour, timing of ovulation and pregnancy rate during the breeding and low breeding seasons in Nili-Ravi buffaloes. In Experiment 1, oestrous behaviour and timing of ovulation were determined from (n=34) oestruses. The mean (+/- S.E.M.) time of ovulation after the onset of standing oestrus was greater (P<0.05) in PGF(2alpha)-induced luteolysis (30.6+/-1.5h) compared to Ovsynch buffaloes (15.0+/-0.8h). In Experiment 2, pregnancy rates were compared between two methods of synchronization (detected oestrus and Ovsynch protocol) during the breeding and low breeding seasons. Pregnancy rates of buffaloes bred at detected oestrus (62.5%) or by the Ovsynch protocol (36.3%) during the breeding season did not differ significantly (P>0.05) from those which were inseminated during the low breeding season (55.5%) and (30.4%), respectively. This study demonstrates clearly that (1) timing of ovulation in Nili-Ravi buffalo is about 30h after the onset of standing oestrus and (2) buffaloes can be successfully synchronized with optimum fertility using either PGF(2alpha) alone (detected oestrus) or using (Ovsynch protocol) during low breeding season, to calve during the period when milk availability is short.     

NADPH- and iron-dependent lipid peroxidation inhibit aromatase activity in human placental microsomes

During pregnancy placenta is the most significant source of lipid hydroperoxides and other reactive oxygen species (ROS). The increased production of lipid peroxides and other ROS is often linked to pre-eclampsia. It is already proved that placental endoplasmic reticulum may be an important place of lipid peroxides and superoxide radical production. In the present study we revealed that NADPH- and iron-dependent lipid peroxidation in human placental microsomes (HPM) inhibit placental aromatase--a key enzyme of estrogen biosynthesis in human placenta. We showed that significant inhibition of this enzyme is caused by small lipid peroxidation (TBARS (thiobarbituric acid-reactive substances)<4nmol/mg microsomal protein (m.p.)). More intensive lipid peroxidation (TBARS>9nmol/mg microsomal protein) diminishes aromatase activity to value being less than 5% of initial value. NADPH- and iron-dependent lipid peroxidation also causes disappearance of cytochrome P450 parallel to observed aromatase activity inhibition. EDTA, alpha-tocopherol, MgCl(2) and superoxide dismutase (SOD) prevent aromatase activity inhibition and cytochrome P450(AROM) degradation. Mannitol and catalase have not effect on TBARS synthesis, aromatase activity and cytochrome P450 degradation. In view of the above we postulate that the inhibition of aromatase activity observed is mainly a consequence of cytochrome P450(AROM) degradation induced by lipid radicals. The role of hydroxyl radical in cytochrome P450 degradation is negligible in our experimental conditions. The results presented here also suggest that the inhibition of aromatase activity can also take place in placenta at in vivo conditions.     

Increased incidence of pregnancy complications in women who later develop scleroderma: a case control study

Introduction  

Studies have shown that fetal progenitor cells persist in maternal blood or bone marrow for more than 30 years after delivery. Increased trafficking of fetal cells occurs during pregnancy complications, such as hypertension, preeclampsia, miscarriage and intra-uterine growth restriction (IUGR). Women with these pregnancy complications are significantly more often HLA-class II compatible with their spouses. Women who later develop scleroderma also give birth to an HLA-class II child more often. From these prior studies we hypothesized that preeclampsia and other pregnancy complications could be associated with increased levels of fetal cell trafficking, and later be involved in the development of scleroderma.     

Analysis of CCR5 and CX3CR1 gene polymorphisms in association with unexplained recurrent miscarriages among north Indian women

Context

Recurrent miscarriage (RM), defined as three or more consecutive losses before the 20th week of gestation, affects 0.5–2% of pregnant women. In over 80% of cases, RM remains unexplained after investigations, suggesting the involvement of genetic factors.

Objectives

The present study investigates the common polymorphisms of chemokine receptors CCR5 (NG_012637.1:g.5303A>G) and CX₃CR1 (NG_016362.1:g.21065C>T, Thr280Met and NG_016362.1:g.20971G>A, Val249Ile) and their association with recurrent miscarriages (RM) among north Indian women.

Participants and Methods

In a retrospective case-control study 200 well characterized patients with unexplained RM and 300 controls were genotyped for three polymorphic markers of CCR5 and CX₃CR1 by restriction digestion of PCR amplified fragments.

Results

Alleles and genotypes of CX₃CR1 Val249Ile revealed statistically significant associations with RM cases when compared with the controls. The homozygous variant genotype Ile/Ile was found to be significantly higher among patients (p = 0.0002) when compared with the homozygous wild type Val/Val genotype. The haplotype of CX₃CR1 that carried major alleles of Thr280Met and Val249Ile (T-V) showed statistically significant protective association (p < 0.0001, OR = 0.41, 95% CI = 0.31–0.54). The haplotype A-T-V (all wild type alleles) revealed a statistically significant protective association (p < 0.0001, OR = 0.41, 95% CI = 0.34–0.62), whereas the haplotypes G-T-I, A-T-I and A-M-V modified the risk of RM 1.9-fold, 5.5-fold and 5.1-fold respectively.

Conclusions

A common polymorphism of CX₃CR1 gene, Val240Ile is associated with the risk of RM in north Indian women. Risk of RM may also be modified by the presence of haplotypes T-I, M-V, G-T-I, A-T-I and A-M-V.     

Vitamin E sensitive genes in the developing rat fetal brain: a high-density oligonucleotide microarray analysis

Vitamin E (tocopherols and tocotrienols) is essential for normal neurological function. Recently we have reported that the neuroprotective properties of tocotrienols are much more potent than that of the widely studied tocopherols (Sen, C.K., Khanna, S., Roy, S. and Parker, L. (2000) J. Biol. Chem. 275, 13049–13055). The objective of this study was to evaluate whether (i) oral supplementation of tocotrienols during pregnancy is bioavailable to fetal and mother brains; (ii) short-term change in dietary vitamin E levels of pregnant rats influences gene expression profile of developing fetal brains. We report that dietary tocotrienol is bioavailable to both mother and fetal brains. The enrichment is more in fetal brain tissue. Using a GeneChip microarray expression profiling approach we have identified a specific set of vitamin E sensitive genes in the developing rat fetal brain.     

Meta-analysis of diagnostic test accuracy assessment studies with varying number of thresholds

Current meta-analytic methods for diagnostic test accuracy are generally applicable to a selection of studies reporting only estimates of sensitivity and specificity, or at most, to studies whose results are reported using an equal number of ordered categories. In this article, we propose a new meta-analytic method to evaluate test accuracy and arrive at a summary receiver operating characteristic (ROC) curve for a collection of studies evaluating diagnostic tests, even when test results are reported in an unequal number of nonnested ordered categories. We discuss both non-Bayesian and Bayesian formulations of the approach. In the Bayesian setting, we propose several ways to construct summary ROC curves and their credible bands. We illustrate our approach with data from a recently published meta-analysis evaluating a single serum progesterone test for diagnosing pregnancy failure.