A physiological, rather than a superovulated, post-implantation environment can attenuate the compromising effect of assisted reproductive techniques on gene expression in developing mice embryos.

Assisted reproductive techniques (ARTs) may perturb the pre-/peri-conception microenvironments, which subsequently threaten the health of offspring. This study aimed to investigate the effects of superovulation, vitrification, in vitro culture, and embryo transfer on the expression of epigenetic modulators, imprinted genes, and pluripotency markers in expanded blastocysts and Day-9.5 (D9.5) concepti. Results revealed that 53.4% (8/15) and 86.7% (13/15) of genes in the fetus and placenta, respectively, have similar patterns of transcription in all D9.5 concepti, despite the perturbed mRNA expression observed at the blastocyst stage for each embryo-production technique. These observations indicate a counterbalancing of the abnormal expression pattern analyzed at the blastocyst stage during post-implantation development, particularly when the uterus of a naturally synchronized foster mother is employed. Superovulation resulted in the most abnormal expression patterns compared to other treatment groups, although these same blastocysts were able to develop in a synchronized uterus. Thus, superovulation creates a hormonal environment that negatively affected gene expression and impairs fetal growth more adversely during post-implantation development than other ART protocols, such as in vitro culture, vitrification, or embryo transfer-although each did contribute negatively to the implantation and development process. Together, these results may have implications for treating infertility in humans.

Developmental competence of ovine oocytes after vitrification: differential effects of vitrification steps, embryo production methods, and parental origin of pronuclei.

Despite many advances in the field of oocyte cryopreservation, the adverse effects of cryopreservation on oocyte competence are still an open challenge in most mammalian species. Using ovine in vitro-matured oocytes, the differential effects of vitrification steps, embryo production methods, and parental origin of pronuclei were systemically investigated to unravel (1) the most critical stage (if any) of oocyte vitrification, (2) the most suitable method (if any) of embryo production for a vitrified oocyte, and (3) differential contributions of male or female pronuclear formation to the poor quality of vitrified oocyte. Although cryoprotectants used during vitrification had some inevitable adverse effects on oocyte competence, the damages caused by low temperature per se (chilling injury) were the main cause of poor quality of vitrified oocytes. When vitrified oocytes underwent either IVF or intracytoplasmic sperm injection (ICSI), embryo development rates were substantially lower than those of fresh ones. In contrast, when vitrified oocytes underwent either parthenogenetic activation (PA) or SCNT, embryo development rates were very similar to those of fresh ones. Evaluation of nuclear morphology after IVF, ICSI, PA, and SCNT oocytes revealed that vitrification had no apparent effect on the female (IVF, ICSI, and PA) and somatic (SCNT) pronuclear formation rates but significantly reduced male pronuclear formation after either IVF or ICSI compared with fresh counterparts. Quantitative analysis of transcripts revealed comparable mRNA abundances of CNX43, HSP90, GMNN, NPM, and OCT4 between vitrified and fresh oocytes, whereas CCNB, ATP1A1, and PAP transcripts were significantly lower in vitrified versus fresh oocytes. Although underlying mechanisms of poor quality of vitrified oocytes are multifactorial, the ability to obtain equivalent development after PA and SCNT, but not IVF and ICSI, in vitrified versus fresh oocytes may argue that the cytoplasm of vitrified oocyte has the necessary components to support in vitro embryonic development of the maternal, even adult somatic cell, chromosomes but fails to do so with sperm chromosomes.

Effect of epigenetic modification with trichostatin A and S-adenosylhomocysteine on developmental competence and POU5F1-EGFP expression of interspecies cloned embryos in dog.

Adult canine fibroblasts stably transfected with either cytomegalovirus (CMV) or POU5F1 promoter-driven enhanced green fluorescent protein (EGFP) were used to investigate if pre-treatment of these donor cells with two epigenetic drugs [trichostatin A (TSA), or S-adenosylhomocysteine (SAH)] can improve the efficiency of interspecies somatic cell nuclear transfer (iSCNT). Fluorescence-activated cell sorting (FACS), analyses revealed that TSA, but not SAH, treatment of both transgenic and non-transgenic fibroblasts significantly increased acetylation levels compared with untreated relatives. The expression levels of Bcl2 and P53 were significantly affected in TSA-treated cells compared with untreated cells, whereas SAH treatment had no significant effect on cell apoptosis. Irrespective of epigenetic modification, dog/bovine iSCNT embryos had overall similar rates of cleavage and development to 8-16-cell and morula stages in non-transgenic groups. For transgenic reconstructed embryos, however, TSA and SAH could significantly improve development to 8-16-cell and morula stages compared with control. Even though, irrespective of cell transgenesis and epigenetic modification, none of the iSCNT embryos developed to the blastocyst stage. The iSCNT embryos carrying CMV-EGFP expressed EGFP at all developmental stages (2-cell, 4-cell, 8-16-cell, and morula) without mosaicism, while no POU5F1-EGFP signal was observed in any stage of developing iSCNT embryos irrespective of TSA/SAH epigenetic modifications. These results indicated that bovine oocytes partially remodel canine fibroblasts and that TSA and SAH have marginal beneficial effects on this process.

Simple, fast, and efficient method of manual oocyte enucleation using a pulled Pasteur pipette.

Cloning mammals by somatic cell nuclear transfer entails the replacement of oocyte chromosomes with the nucleus of a somatic cell. A major step in this technique is to efficiently produce large batches of enucleated oocytes, a process that requires considerable micromanipulation skills and expensive equipments. Here, a simple, fast, and efficient method of manual oocyte enucleation was introduced that can be adopted in every laboratory with the minimum equipments. Common laboratory glass pipettes were pulled on the flame of a burner and then used for manual bisection or enucleation of sheep and goat zona-free oocytes by passing them through the discontinuous cutting border of culture medium and mineral oil. The described techniques showed a certain efficiency to conveniently bisect or enucleate large batches of sheep, and goat oocytes being pre-treated with demecolcine. The method may be straightforward for simple manipulation of oocytes of other species and for development of automated cloning methods as well.

Nuclear transfer technique affects mRNA abundance, developmental competence and cell fate of the reconstituted sheep oocytes.

The effect of technical steps of somatic cell nuclear transfer (SCNT) on different aspects of cloned embryo development was investigated in sheep. In vitro-matured oocytes were enucleated in the presence or absence of zona and reconstituted by three different SCNT techniques: conventional zona-intact (ZI-NT), standard zona-free (ZF-NT) and intracytoplasmic nuclear injection (ICI-NT). Stepwise alterations in nuclear remodeling events and in mRNA abundances, throughput and efficiency of cloned embryo development and cell allocation of the resulted blastocysts were assessed. Early signs of nuclear remodeling were observed as soon as 2 h post-reconstitution (hpr) for fusion-based methods of nuclear transfer (ZI-NT and ZF-NT) but were not observable until 4 hpr with the ICI-NT method. The relative mRNA abundances of HSP90AA1 (HSP90), NPM2 and ATPase genes were not affected by i) presence or absence of zona, ii) oocyte enucleation method and iii) nuclear transfer method. After reconstitution, however, the relative mRNA contents of POU5F1 (OCT4) with the ZI-NT and ZF-NT methods and of PAPOLA (PAP) with ZF-NT were significantly lower than those for the ICI-NT method. Zona removal doubled the throughput of cloned blastocyst development for the ZF-NT technique compared with ZI-NT and ICI-NT. Cleavage rate was not affected by the SCNT protocol, whereas blastocyst yield rate in ICI-NT technique (17.0±1.0%) was significantly (P<0.05; ANOVA) higher than in ZF-NT (7.1±1.5%) but not in the ZI-NT group (11.2±3.3%). Despite the similarities in total cell number, SCNT protocol changed the distribution of cells in the blastocysts, as ZF-NT-cloned blastocysts had significantly smaller inner cell mass than ZI-NT. These results indicate that technical aspects of cloning may result in the variety of cloning phenotypes.

Cloned sheep blastocysts derived from oocytes enucleated manually using a pulled pasteur pipette.

The potential applications of a simplified method of somatic cell nuclear transfer (SCNT) that is improved in both efficiency and throughput is considerable. Technically, a major step of SCNT is to produce large pools of enucleated oocytes (cytoplasts) efficiently, a process that requires considerable micromanipulation skill and expensive equipment. Here, we have developed an efficient and high-throughput method of manual oocyte enucleation using a simple device, a pulled Pasteur pipette, that can be connected to standard zona-free method of embryo reconstitution. Common Pasteur pipettes were pulled on a flame to produce finely drawn pipettes with inner diameters approximately less than half the oocyte diameter (∼50-60 µm), and slightly larger than cytoplasmic protrusion (∼20-30 µm) that was induced after demecolcine treatment of MII-stage oocytes. Oocyte manipulation was performed under a stereomicroscope by either bisecting the oocyte into two approximately equal demioocytes (blind manual enucleation), or by positioning the oocytes so that the cytoplasmic extrusion that contains the MII chromosome mass is removed with the minimum amount of cytoplasm (oriented manual enucleation). The survival rate of the manually enucleated oocytes was 81.4-91.5%, comparable to standard zona-free method of oocyte enucleation (>95%). A total of 80-120 oocytes could be enucleated in 10 min, which was considerably higher than standard zona-free enucleation method. In vitro development rates of cloned embryos derived from manually enucleated cytoplasts with varying cytoplasmic volumes (50%, 95%, and 100%) was comparable, and embryonic developmental rates of the two latter groups were at least as good as standard zona-free method. The manual method of oocyte enucleation described here can be learned and mastered for simple, fast, and cheap production of cloned embryos with comparable efficiency to other available methods.

Cryosurvival of in vitro produced embryos as affected by health status effect of oocyte donor cow.

In vitro embryo production and embryo vitrification of genetically superior cows that culled inevitably due to health problems can accelerate genetic progress. This study was carried out to investigate whether maternal age and health status effects of high genetic merit cows affect cryosurvival and developmental competence of IVP embryos. In this sense, the effects of ageing and four common culling causes of dairy cows [repeat breeding (RPB), udder problems (UPM), chronic endometritis (CRE), and lameness (LAM)] on in vitro embryo development, and in vivo developmental competence after embryo vitrification were evaluated. The mean number of oocytes obtained per cow did not vary significantly between donors indifferent groups. Cleavage rates in RPB (86.0+/-4.2%), SEN (81.3+/-2.5%) and CRE (77.6+/-6.3%) cows which were comparable to control (95.9+/-1.5%) but were significantly higher than the related rate of UPM donors (50.6+/-2.6%). Importantly, there was no significant difference between the blastocyst rates of different groups. Mean overall survival rate was not different between the groups and was not affected by the blastocyst production rate. There was no significant difference between pregnancy rates of different groups. The results of the present study indicated that in cattle, neither ageing, nor these four diseases affect ovarian potential in terms of the yield and quality of in vitro embryo development.

Vitrification of in vitro produced bovine embryos: effect of embryonic block and developmental kinetics.

In order to investigate whether the kinetics and stage of embryo development affect cryosurvival of in vitro produced bovine embryos, cleaved embryos were categorized in six groups based on their developmental kinetics regarding the stage of embryonic block in bovine (8-16 cell stage): I and II--early (day 2) and late (day 3) 5-8 cell, III and IV--early (day 3) and late (day 4) 8-16 cell, and V and VI--early (day 4) and late (day 5) morula. The cryosurvival and developmental competence of these embryos were compared with each other and also with the corresponding control groups. The potential of 5-8 cell stage embryos to survive vitrification and further develop towards blastocyst stage was significantly lower than vitrified and un-vitrified 8-16 cell and morula stage embryos. These results suggest that, the survival rate and potential of embryos to develop towards blastocyst stage might be affected by the kinetic of the embryo development. Moreover, the results of this study indicated that the optimal stages of early embryo vitrification are post-embryonic block.

Specific activation requirements of in vitro-matured sheep oocytes following vitrification-warming.

Oocyte vitrification and assisted oocyte activation have increasingly important roles in assisted reproductive technology. Yet, an important area of concern with matured oocyte cryobiology is that elements of oocytes intimately involved in metaphase-II arrest may be modified by cryopreservation. By comparing different cellular characteristics of unvitrified, vitrified-warmed, and unvitrified-activated oocytes, the present study investigated how vitrification-warming process may affect developmental competence of in vitro-matured sheep oocytes following parthenogenetic activation. Structural, ultrastructural, and molecular analyses indicated that the characteristics of vitrified-warmed oocytes vastly differed from fresh oocytes, instead resembling unvitrified-activated oocytes. For unvitrified oocytes, the highest blastocyst yield (41.8 ± 0.6%) was achieved using the maximum ionomycin concentration (5 µM), and importantly, the duration of ionomycin treatment was not of utmost importance at this concentration. In contrast, the maximum blastocyst yield of vitrified-warmed oocytes (28.4 ± 1.4%) was achieved with a minimal duration of ionomycin treatment (1 min), and further extending the duration dramatically reduced developmental potential of vitrified-warmed oocytes. These results suggested that vitrified-warmed oocytes may need an activation protocol different from unvitrified oocytes. In this respect, unvitrified oocytes were more sensitive to the concentration rather than the duration of ionomycin treatment when compared with vitrified oocytes, which were sensitive to the treatment duration. These results may provide a platform to improve the potential applications of vitrified oocytes in medicine and agriculture.

Potential applications of sheep oocytes as affected by vitrification and in vitro aging.

The present study was carried out to investigate how the interactions between aging, vitrification and post-warming interval affect the credibility of sheep MII-oocytes for in vitro fertilization (IVF), intracytoplasmic injection (ICSI), and parthenogenetic activation (PA). According to our results, aged oocytes had significantly higher rates of chromosome and spindle abnormalities compared to young oocytes. However after vitrification-warming, the total rates of these abnormalities were not significantly different between aged and young oocytes. Unvitrified-aged, and vitrified young and aged oocytes had comparable ultrastructural characteristics, whereas they were completely dissimilar in compared with unvitrified-young oocytes. Although mRNA abundance was reduced during vitrification-warming in both aged and young oocytes, the post-warming interval could improve the relative mRNA abundance. Aged oocytes had lower capacity for IVF and ICSI in compared with young oocytes, but had similar pattern for PA process. The vitrification process decreased developmental competence of both aged and young oocytes in compared with young ones, particularly when warmed oocytes were rested for 2 h before IVF, ICSI and PA. The results of the present study showed that in vitro aged oocytes had higher capacity to be used for parthenogenetic studies rather than IVF and ICSI. Furthermore, it was shown that vitrified oocytes had a time-dependent decline in quality and developmental potential. Notably, the speed of this decline was higher in vitrified-young oocytes, indicating that the vitrified oocytes do not require to be rested post warming. Conclusively, the results of this study can be useful in preserving in vitro aged oocytes to provide a valuable and easy access source of oocytes for research purposed studies.

Time dependent effect of post warming interval on microtubule organization, meiotic status, and parthenogenetic activation of vitrified in vitro matured sheep oocytes.

It is a common practice to rest vitrified-warmed matured oocytes for 1-3 h, as a treatment to recover spindle and cytoskeleton, before commencing a further treatment. Vitrified-warmed matured oocytes, however, are very sensitive and may resume meiosis spontaneously during this recommended rest time. Therefore, the aim of this study was to assess spindle and chromosome status as well as developmental competence of vitrified in vitro matured sheep oocytes activated parthenogenetically, either 0 h (immediately) or 2 h (delayed) after warming. There was no significant effect of post-warming interval on the proportion of degenerated oocytes. Evaluation of chromosomes and meiotic spindle configuration showed that 11.11% of oocytes in the immediate group and 8.82% of oocytes in the delayed group had normal chromosomal alignment on well-structured spindles, compared to non-vitrified group (79.41%). Meanwhile, majority of the chromosomal abnormalities in the immediate and delayed groups were categorized as absent (unobservable) (77.78%) and anaphase II (70.59%), respectively. Oocytes in immediately activated group showed significantly higher blastocyst rate (28.86%) compared to delayed activated group (16.47%). In conclusion, the results suggest that post-warming interval may have important consequence on meiotic progression and parthenogenetic activation of vitrified oocytes. In sheep, it appears that chemical activation without having to await microtubule reorganization improves embryonic development.

Antioxidant supplementation of culture medium during embryo development and/or after vitrification-warming; which is the most important?

PURPOSE: To determine the most optimal stage for antioxidant supplementation of culture medium to improve developmental competence, cryotolerance and DNA-fragmentation of bovine embryos. METHODS: Presumptive zygotes were first cultured in presence or absence of beta-mercaptoethanol (beta-ME), for 8 days. Subsequently, half of the expanded blastocysts developed in both groups were vitrified, warmed within 30 min and post-warming embryos along with their corresponding non-vitrified embryos were cultured for two further days in presence or absence of (100 microM) betaME. RESULTS: For vitrified and non-vitrified embryos, the best effect was found when betaME was added from day 1 of in vitro culture in continuation with post-warming culture period. Day 1-8 supplementation significantly increased the rates of cleavage, day 7 and day 8 blastocyst production. For non-vitrified embryos, betaME addition during day 1-8 and/or 9-10 of embryo culture improved both hatching rate and quality of hatched embryos. For vitrified embryos, however, the percentage of DNA-fragmentation (18.5%) was significantly higher (p < or = 0.05) than that of embryos developed in absence of betaME but supplemented with betaME during post-warming period (13.5%). CONCLUSIONS: Exogenous antioxidant increases the chance of embryos, even those of fair-quality, to develop to blastocyst. However, antioxidant inclusion during in vitro embryo development is not sufficient to maintain the redox state of these embryos during the critical period of post-warming embryo culture, and therefore, there should be a surplus source of exogenous antioxidant during post-warming embryo culture.

Study of bacterial infections among the patients with suspected cutaneous leishmaniasis.

The main objective of this study was to evaluate the prevalence of secondary bacterial infections in the patients with cutaneous lesions. The patients admitted to leishmaniasis laboratory of faculty of health, Tehran university of medical sciences from October 2004 to June 2005 were subjected in this study. Clinical samples were analyzed using standard bacteriological and parasitological methods. One hundred seventy three patients were subjected to this study and leishmania was found in 84 (48.5%) cases. According to bacteriological experiments, 47 cases (55.9%) had been also infected by bacterial infections. The most prevalent bacterial isolates included group D Streptococcus (19.1%), Enterococcus spp. (19.1%) and Staphylococcus aureus (12.7%). The findings of current study indicated that the bacterial infections are still an important problem in the patients with cutaneous leishmaniasis and should be considered in treating these patients.