Lipoprotein-associated PAF-acetylhydrolase activity in subjects with the metabolic syndrome

Plasma- and lipoprotein-associated activity of the platelet activating factor acetylhydrolase (PAF-acetylhydrolase, PAF-AH) plays an important role in inflammation and in atherosclerotic process, which are present in the metabolic syndrome (MS). Paraoxonase 1 (PON1) is an esterase associated with high-density lipoprotein (HDL) which contributes to the anti-atherogenic effects of this lipoprotein. We investigated the activities of both enzymes in 60 patients with MS and 110 age- and sex-matched subjects without it (non-MS group). Plasma PAF-AH activity was higher in the MS compared to the non-MS group, while HDL-PAF-AH and serum PON1 activities were lower in the MS compared to the non-MS group. Univariate regression analysis in the MS group showed that plasma PAF-AH activity was positively associated with systolic blood pressure, whereas HDL-PAF-AH activity was inversely associated with the homeostasis model assessments (HOMA) index. Both associations remained significant in the multivariate regression analysis, suggesting that insulin resistance and systolic hypertension are major determinants for the alterations in plasma and HDL-associated PAF-AH activity among those observed in MS patients.     

Pregnancy outcomes in women with repeated implantation failures after intracytoplasmic morphologically selected sperm injection (IMSI)

Background

The role of serum anti-Müllerian hormone (AMH) as predictor of in-vitro fertilization outcomes has been much debated. The aim of the present study is to investigate the practicability of combining serum AMH level with biological age as a simple screening method for counseling IVF candidates of advanced reproductive age with potential poor outcomes prior to treatment initiation.

Methods

A total of 1,538 reference patients and 116 infertile patients aged greater than or equal to 40 years enrolled in IVF/ICSI cycles were recruited in this retrospective analysis. A reference chart of the age-related distribution of serum AMH level for Asian population was first created. IVF/ICSI patients aged greater than or equal to 40 years were then divided into three groups according to the low, middle and high tertiles the serum AMH tertiles derived from the reference population of matching age. The cycle outcomes were analyzed and compared among each individual group.

Results

For reference subjects aged greater than or equal to 40 years, the serum AMH of the low, middle and high tertiles were equal or lesser than 0.48, 0.49-1.22 and equal or greater than 1.23 ng/mL respectively. IVF/ICSI patients aged greater than or equal to 40 years with AMH levels in the low tertile had the highest cycle cancellation rate (47.6%) with zero clinical pregnancy. The nadir AMH level that has achieved live birth was 0.56 ng/mL, which was equivalent to the 36.4th percentile of AMH level from the age-matched reference group. The optimum cut-off levels of AMH for the prediction of nonpregnancy and cycle cancellation were 1.05 and 0.68 ng/mL, respectively.

Conclusions

Two criteria: (1) age greater than or equal to 40 years and (2) serum AMH level in the lowest tertile (equal or lesser than 33.3rd percentile) of the matching age group, may be used as markers of futility for counseling IVF/ICSI candidates.     

Tetrapod phylogeny inferred from 18S and 28S ribosomal RNA sequences and a review of the evidence for amniote relationships [published erratum appears in Mol Biol Evol 1991 May;8(3):398]

The 18S ribosomal RNAs of 21 tetrapods were sequenced and aligned with five published tetrapod sequences. When the coelacanth was used as an outgroup, Lissamphibia (living amphibians) and Amniota (amniotes) were found to be statistically significant monophyletic groups. Although little resolution was obtained among the lissamphibian taxa, the amniote sequences support a sister-group relationship between birds and mammals. Portions of the 28S ribosomal RNA (rRNA) molecule in 11 tetrapods also were sequenced, although the phylogenetic results were inconclusive. In contrast to previous studies, deletion or down- weighting of base-paired sites were found to have little effect on phylogenetic relationships. Molecular evidence for amniote relationships is reviewed, showing that three genes (beta-hemoglobin, myoglobin, and 18S rRNA) unambiguously support a bird-mammal relationship, compared with one gene (histone H2B) that favors a bird- crocodilian clade. Separate analyses of four other genes (alpha- crystallin A, alpha-hemoglobin, insulin, and 28S rRNA) and a combined analysis of all sequence data are inconclusive, in that different groups are defined in different analyses and none are strongly supported. It is suggested that until sequences become available from a broader array of taxa, the molecular evidence is best evaluated at the level of individual genes, with emphasis placed on those studies with the greatest number of taxa and sites. When this is done, a bird-mammal relationship is most strongly supported. When regarded in combination with the morphological evidence for this association, it must be considered at least as plausible as a bird-crocodilian relationship.      

Pregnancy and fetal characteristics after transfer of vitrified in vivo and cloned bovine embryos

This study was conducted to examine pregnancy progression and fetal characteristics following transfer of vitrified bovine nuclear transfer versus in vivo-derived embryos. Nuclear transfer (NT) was conducted using cumulus cells collected from an elite Holstein-Friesian dairy cow. Expanding and hatching blastocysts on Day 7 were vitrified using liquid nitrogen surface vitrification. Day 7 in vivo embryos, produced using standard superovulation procedures applied to Holstein-Friesian heifers (n=6), were vitrified in the same way. Following warming, embryos were transferred to synchronized recipients (NT: n=65 recipients; Vivo: n=20 recipients). Pregnancies were monitored by ultrasound scanning on Days 25, 45 and 75 and a sample of animals were slaughtered at each time point to recover the fetus/placenta for further analyses. Significantly more animals remained pregnant after transfer of in vivo-derived embryos than NT embryos at all time points: Day 25 (95.0 versus 67.7%, P<0.05), Day 45 (92.8 versus 49.1%, P<0.01) and Day 75 (70.0 versus 20.8%, P<0.0). There was no significant difference (P=0.10) in the weight of the conceptus on Day 25 from NT transfers (1.14+/-0.23 g, n=8) versus in vivo transfers (0.75+/-0.19 g, n=8). On Day 45, there was no significant difference in the weight of either fetus (P=0.393) or membranes (P=0.167) between NT embryos (fetus: 2.76+/-0.40, n=12; membranes: 59.0+/-10.0, n=11) or in vivo-derived embryos (fetus: 2.60+/-0.15, n=6; membranes: 41.8+/-5.2, n=4). However, on Day 75 the weight of the fetus and several of the major organs were heavier from NT embryos. These data suggest that morphological abnormalities involving the fetus and the placenta of cloned pregnancies are manifested after Day 45.     

Paternal influence on the time of first embryonic cleavage post insemination and the implications for subsequent bovine embryo development in vitro and fertility in vivo

The objectives of this study were: (1) to evaluate the effect of sire on the time from insemination to first cleavage following insemination in vitro and the relationship of this parameter to field fertility and (2) to establish the relationship between the kinetics of cleavage in vitro and oocyte developmental competence for bulls of known field fertility. Frozen semen from six bulls with 150-day non-return rates ranging from 57-78% was used. In experiment 1, after insemination with semen from one of the six bulls, presumptive zygotes were transferred to IVC in droplets of synthetic oviduct fluid. Droplets were examined at 24, 27, 30, 33, 36, 42, and 48 hr after insemination and the number of cleaved oocytes was recorded. Blastocyst yield was recorded on Days 6-, 7-, and 8-post insemination. In experiment 2, culture droplets were examined at 30, 36, and 48 hr after insemination. At each time point, the number of cleaved embryos was recorded and these embryos were transferred into new droplets and were cultured separately for the duration of the experiment. The proportion of embryos developing to the blastocyst stage was recorded for each of the groups for each bull. The best predictor of field fertility was a model containing 33-hpi-cleavage percentage only (r = 0.689, P < 0.0001). There was also a significant correlation between blastocyst yield and non-return rate, with Day 7 blastocyst yield having the highest correlation (r = 0.356), although this was relatively low in comparison. In experiment 2, irrespective of sire, a significantly higher proportion of those early-cleaving oocytes (before 30 hpi) developed to blastocysts than those cleaving later. In most cases, a higher proportion of blastocysts derived from early-cleaving oocytes hatched from the zona pellucida suggesting that such blastocysts are of superior quality to those derived from late-cleaving oocytes. In conclusion these data confirm our earlier observations that earliest cleaving zygotes are more competent in terms of development to the blastocyst stage than those that cleave later. This phenomenon is independent of the sire used. However, we have demonstrated that the kinetics of early embryonic development as measured by the timing of the first cleavage division post insemination vary between different bulls and that these differences can be used to discriminate between bulls of high and low bull field fertility.     

p16INK4a-induced senescence is disabled by melanoma-associated mutations

The p16(INK4a)-Rb tumour suppressor pathway is required for the initiation and maintenance of cellular senescence, a state of permanent growth arrest that acts as a natural barrier against cancer progression. Senescence can be overcome if the pathway is not fully engaged, and this may occur when p16(INK4a) is inactivated. p16(INK4a) is frequently altered in human cancer and germline mutations affecting p16(INK4a) have been linked to melanoma susceptibility. To characterize the functions of melanoma-associated p16(INK4a) mutations, in terms of promoting proliferative arrest and initiating senescence, we utilized an inducible expression system in a melanoma cell model. We show that wild-type p16(INK4a) promotes rapid cell cycle arrest that leads to a senescence programme characterized by the appearance of chromatin foci, activation of acidic beta-galactosidase activity, p53 independence and Rb dependence. Accumulation of wild-type p16(INK4a) also promoted cell enlargement and extensive vacuolization independent of Rb status. In contrast, the highly penetrant p16(INK4a) variants, R24P and A36P failed to arrest cell proliferation and did not initiate senescence. We also show that overexpression of CDK4, or its homologue CDK6, but not the downstream kinase, CDK2, inhibited the ability of wild-type p16(INK4a) to promote cell cycle arrest and senescence. Our data provide the first evidence that p16(INK4a) can initiate a CDK4/6-dependent autonomous senescence programme that is disabled by inherited melanoma-associated mutations.     

Amino acid metabolism of bovine blastocysts: a biomarker of sex and viability

The ratio of male/female embryos may be modified by environmental factors such as maternal diet in vivo and the composition of embryo culture media in vitro. We have used amino acid profiling, a noninvasive marker of developmental potential to compare the effect of sex on the metabolism of bovine blastocysts conceived in vivo and in vitro. Blastocysts were incubated individually for 24 hr in a close‐to‐physiological mixture of amino acids and the depletion or appearance of 18 amino acids measured using HPLC. Blastocysts were then sexed by PCR. Amino acid depletion by in vitro‐produced blastocysts and expanded blastocysts was higher than in embryos conceived in vivo (P = 0.02). When cultured in vitro, female embryos exhibited increased depletion of arginine, glutamate, and methionine and appearance of glycine, while male embryos displayed increased depletion of phenylalanine, tyrosine, and valine. Overall, in vitro‐produced blastocysts exhibited sex‐specific differences in metabolic profiles of 7 out of 18 amino acids; in vivo‐produced, in 2 out of 18. These differences had disappeared by the expanded blastocyst stages. We have also shown that amino acid metabolism can predict the ability of bovine zygotes to develop to the blastocyst stage, providing “proof of principle” for the use of this technology in clinical IVF to select single embryos for transfer and thereby avoid the problem of multiple births. Mol. Reprod. Dev. 77: 285–296, 2010. © 2010 Wiley‐Liss, Inc.     

Development of Lactococcus lactis encoding fluorescent proteins,GFP, mCherry and iRFP regulated by the nisin-controlled gene expression system

Fluorescent proteins are useful reporter molecules for a variety of biological systems. We present an alternative strategy for cloning reporter genes that are regulated by the nisin-controlled gene expression (NICE) system. Lactoccocus lactis was genetically engineered to express green fluorescent protein (GFP), mCherry or near-infrared fluorescent protein (iRFP). The reporter gene sequences were optimized to be expressed by L. lactis using inducible promoter pNis within the pNZ8048 vector. Expression of constructions that carry mCherry or GFP was observed by fluorescence microscopy 2 h after induction with nisin. Expression of iRFP was evaluated at 700 nm using an infrared scanner; cultures induced for 6 h showed greater iRFP expression than non-induced cultures or those expressing GFP. We demonstrated that L. lactis can express efficiently GFP, mCherry and iRFP fluorescent proteins using an inducible expression system. These strains will be useful for live cell imaging studies in vitro or for imaging studies in vivo in the case of iRFP.     

Embryo survival and recipient pregnancy rates after transfer of fresh or vitrified,in vivo or in vitro produced ovine blastocysts

The aim of this study was to assess the effect of production system and of cryopreservation of ovine embryos on their viability when transferred to recipients. The experimental design was an unbalanced 2 x 2 factorial design of two embryo production systems (in vivo versus in vitro) and two embryo preservation conditions prior to transfer (transferred fresh versus transferred after vitrification/warming). For the production of blastocysts in vivo, crossbred donor ewes (n=30) were synchronised using a 13-day intravaginal progestagen pessary. Ewes received 1500 IU equine chorionic gonadotropin (eCG) 2 days before pessary withdrawal, and were mated 2 days after pessary withdrawal and embryos were recovered surgically (6 days after mating). Blastocysts were produced in vitro (IVP) using standard techniques. Recipients (n=95) were synchronised using a progestagen pessary and received 500 IU eCG at pessary removal and were randomly assigned to receive (two per recipient) in vivo fresh (n=10), in vivo vitrified (n=10), in vitro fresh (n=35) or in vitro vitrified (n=40) blastocysts. Recipients were slaughtered at day 42 of gestation and foetuses recovered. Pregnancy and embryo survival rates were recorded and analysed using CATMOD procedures. Foetal weights and crown-rump lengths were recorded and analysed using generalised linear model (GLM) procedures. There were no statistically significant interactions between the effects of embryo production system and preservation status at transfer on pregnancy rate and embryo survival. The pregnancy rate following transfer of fresh IVP blastocysts was lower (P<0.07) than that of in vivo embryos (54.3% versus 90.0%, respectively). Vitrification resulted in a decrease in pregnancy rate, the effect being more pronounced in the case of IVP embryos (54.3-5.0%, P<0.001) compared with in vivo embryos (90.0-50.0%), although the absolute change was similar (49.3% versus 40.0%). Transfer of fresh IVP blastocysts resulted in a higher proportion of single (78.9% versus 33.3%) and lower proportion of twin (21.1% versus 66.7%) pregnancies than those produced in vivo. This was reflected in a significant difference in embryo survival rate (fresh: 32.8% versus 75.0%, P<0.01; vitrified: 2.5% versus 35.0%, P<0.001, for IVP and in vivo blastocysts, respectively). Similarly, all pregnancies resulting from the transfer of vitrified/warmed IVP blastocysts were single pregnancies, while 40% of those from vitrified/warmed in vivo blastocysts were twin pregnancies; this was reflected in an embryo survival rate of 35.0% versus 75.0%, respectively. There was a significant effect (P=0.0184) of litter size on foetal weight but not on foetal length (P=0.3304). Foetuses derived from the fresh transfer of IVP blastocysts were heavier (6.4+/-0.2g versus 5.8+/-0.2g, respectively, P<0.05) and longer (5.2+/-0.1cm versus 4.8+/-0.1cm, respectively, P<0.01) than those derived from fresh in vivo blastocysts. There was no difference in these parameters as a consequence of vitrification of IVP embryos. However, in vivo blastocysts subjected to vitrification resulted in heavier (6.6+/-0.3g versus 5.8+/-0.2g, respectively, P=0.055) and longer (5.2+/-0.1cm versus 4.8+/-0.1cm, respectively, P<0.05) foetuses than their counterparts transferred fresh.