[Show abstract][Hide abstract] ABSTRACT: Vitrification is now the dominant approach for cryopreservation of human oocytes and embryos; however, serious disagreement persists, particularly about biosafety issues. Techniques are categorized as either ‘open’ or ‘closed’ according to occurrence of direct contact between the medium and liquid nitrogen during cryopreservation. Advocates of closed systems emphasize the potential danger of disease transmission mediated through liquid nitrogen, and praise the safety of their approach; those who use the open systems refer to the lack of evidence of disease transmission and regard their systems as more consistent and efficient. The purpose of this review is to clarify whether open and closed systems are really open and closed; if closed systems are safe and free of any danger of contamination; if closed systems are equally efficient as open ones for cryopreservation of human embryos and oocytes by considering overall outcome; and finally, if ethical and legal concerns are sound when risks and benefits are considered in a broader sense. On the basis of these answers, implementation of rational measures to lower the theoretical danger of disease transmission are proposed while maintaining the achievements in cryopreservation that have contributed substantially to the advancement in assisted reproduction techniques during the past decade.
[Show abstract][Hide abstract] ABSTRACT: Next generation sequencing (NGS) is now being used for detecting chromosomal abnormalities in blastocyst trophectoderm (TE) cells from in vitro fertilized embryos. However, few data are available regarding the clinical outcome, which provides vital reference for further application of the methodology. Here, we present a clinical evaluation of NGS-based preimplantation genetic diagnosis/screening (PGD/PGS) compared with single nucleotide polymorphism (SNP) array-based PGD/PGS as a control.
A total of 395 couples participated. They were carriers of either translocation or inversion mutations, or were patients with recurrent miscarriage and/or advanced maternal age. A total of 1,512 blastocysts were biopsied on D5 after fertilization, with 1,058 blastocysts set aside for SNP array testing and 454 blastocysts for NGS testing. In the NGS cycles group, the implantation, clinical pregnancy and miscarriage rates were 52.6% (60/114), 61.3% (49/80) and 14.3% (7/49), respectively. In the SNP array cycles group, the implantation, clinical pregnancy and miscarriage rates were 47.6% (139/292), 56.7% (115/203) and 14.8% (17/115), respectively. The outcome measures of both the NGS and SNP array cycles were the same with insignificant differences. There were 150 blastocysts that underwent both NGS and SNP array analysis, of which seven blastocysts were found with inconsistent signals. All other signals obtained from NGS analysis were confirmed to be accurate by validation with qPCR. The relative copy number of mitochondrial DNA (mtDNA) for each blastocyst that underwent NGS testing was evaluated, and a significant difference was found between the copy number of mtDNA for the euploid and the chromosomally abnormal blastocysts. So far, out of 42 ongoing pregnancies, 24 babies were born in NGS cycles; all of these babies are healthy and free of any developmental problems.
This study provides the first evaluation of the clinical outcomes of NGS-based pre-implantation genetic diagnosis/screening, and shows the reliability of this method in a clinical and array-based laboratory setting. NGS provides an accurate approach to detect embryonic imbalanced segmental rearrangements, to avoid the potential risks of false signals from SNP array in this study.
[Show abstract][Hide abstract] ABSTRACT: Recent studies involving a limited number of patients have indicated a correlation between aneuploidy and various morphokinetic parameters during preimplantation development. The results among different groups, however, have been inconsistent in identifying the parameters that are able to predict chromosomal abnormalities. The aim of this study was to investigate whether aneuploidy of human blastocysts was detectable by specific morphokinetic parameters in patients at increased risk of aneuploidy because of advanced maternal age, history of unsuccessful IVF treatments, or both. A longitudinal cohort study was conducted using 455 blastocysts from 138 patients. Morphokinetic features of preimplantation development were detected in a timelapse incubator. Blastocysts were subjected to trophectodermal biopsy and comprehensive chromosomal screening. Analyses were conducted by means of logistic mixed-effects models, with a subject-specific intercept. No statistical correlation between 16 commonly detected morphokinetic characteristics of in-vitro embryo development and aneuploidy was found. Results suggest that morphokinetic characteristics cannot be used to select euploid blastocysts in poor-prognosis patients regarded as candidates for pre-implantation genetic screening.
[Show abstract][Hide abstract] ABSTRACT: Recent advances in embryology and related research offer considerable possibilities to accelerate genetic improvement in cattle breeding. Such progress includes optimization and standardization of laboratory embryo production (in vitro fertilization; IVF), introduction of a highly efficient method for cryopreservation (vitrification), and dramatic improvement in the efficiency of somatic cell nuclear transfer (cloning) in terms of required effort, cost and overall outcome. Handmade cloning (HMC), a simplified version of somatic cell nuclear transfer, offers the potential for relatively easy and low cost production of clones. A potentially modified method of vitrification used at a centrally located laboratory facility could result in cloned offspring that are economically competitive with elite animals produced by more traditional means. Apart from routine legal and intellectual property issues, the main obstacle that hampers rapid uptake of these technologies by the beef cattle industry is a lack of confidence from scientific and commercial sources. Once stakeholder support is increased, the combined application of these methods makes a rapid advance toward desirable traits (rapid growth, high quality beef, optimized reproductive performance) a realistic goal. The potential impact of these technologies on genetic advancement in beef cattle herds in which improvement of stock is sought, such as in northern Australia, is hard to overestimate.
[Show abstract][Hide abstract] ABSTRACT: Recent advancement in both human embryology and genomics has created a completely new situation for practical and widespread application of preimplantation genetic diagnosis and screening with a dramatic effect on assisted reproduction. The mapping of the first human genome and the advancement in sequencing technology and bioinformatics has led to the discovery of the exact genetic background of exponentially increasing number of diseases. In parallel, methods for culturing human embryos have also radically improved, enabling the late transfer, and the procedure of vitrification the safe cryopreservation. In consequence, refined genetic analyses have become available from blastocyst biopsy followed by the application of novel genomic methods. Furthermore, some studies suggest that by the selection of aneuploid embryos the pregnancy- and birth-rates can be increased. The amount and the depth of information obtainable from the embryos raise several technical and ethical questions that can be answered by further prospective randomized trials. Orv. Hetil., 2014, 155(35), 1375-1382.
Orvosi Hetilap 08/2014; 155(35):1375-1382. DOI:10.1556/OH.2014.29964
[Show abstract][Hide abstract] ABSTRACT: In somatic cell nuclear transfer (SCNT), donor cell reprogramming is considered as a biologically important and vulnerable event. Various donor cell pre-treatments with Xenopus egg extracts can promote reprogramming. Here we investigated if the reprogramming effect of one treatment with Xenopus egg extract on donor cells was maintained for several cell passages. The extract treatment resulted in increased cell-colony formation from early passages in treated porcine fibroblasts (ExTES), and increased development of cloned embryos. Partial dedifferentiation was observed in ExTES cells, shown as a tendency towards upregulation of NANOG, c-MYC and KLF-4 and downregulation of DESMIM compared with ExTES at Passage 2. Compared with our routine SCNT, continuously increased development of cloned embryos was observed in the ExTES group, and ExTES cloned blastocysts displayed hypermethylated DNA patterns and hypermethylation of H3K4me3 and H3K27me3 in ICM compared with TE. All seven recipients became pregnant after transferral of ExTES cloned embryos and gave birth to 7-22 piglets per litter (average 12). In conclusion, our results demonstrate that one treatment of porcine fibroblasts with Xenopus egg extract can result in long-term increased ability of the cells to promote their in vitro function in subsequent SCNT. Finally these cells can also result in successful development of cloned embryos to term.
Reproduction Fertility and Development 08/2014; 26(7):1017-31. DOI:10.1071/RD13147 · 2.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Handmade cloning (HMC) has been used to generate transgenic pigs for biomedical research. Recently, we found that parthenogenetic activation (PA) of porcine oocytes and improved HMC efficiency could be achieved by treatment with sublethal high hydrostatic pressure (HHP). However, the molecular mechanism underlying the effects of HHP treatment on embryonic development is poorly understood and so was investigated in the present study. Thus, in the present study, we undertook genome-wide gene expression analysis in HHP-treated and untreated oocytes, as well as in 4-cell and blastocyst stage embryos derived by PA or HMC. Hierarchical clustering depicted stage-specific genomic expression profiling. At the 4-cell and blastocyst stages, 103 and 163 transcripts were differentially expressed between the HMC and PA embryos, respectively (P<0.05). These transcripts are predominantly involved in regulating cellular differentiation, gene expression and cell-to-cell signalling. We found that 44 transcripts were altered by HHP treatment, with most exhibiting lower expression in HHP-treated oocytes. Genes involved in embryonic development were prominent among the transcripts affected by HHP. Two of these genes (INHBB and ME3) were further validated by quantitative reverse transcription-polymerase chain reaction. We also observed that HHP treatment activated expression of the imprinting gene DLX5 in 4-cell PA embryos. In conclusion, our genomic expression profiling data suggest that HHP alters the RNA constitution in porcine oocytes and affects the expression of imprinting genes during embryonic development.
Reproduction Fertility and Development 03/2014; 26(3):469-84. DOI:10.1071/RD13037 · 2.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: During the past decade, cryopreservation of oocytes and embryos has become one of the most exciting areas in human-assisted reproduction. Improvement of the old techniques and introduction of alternative procedures have radically increased the overall efficiency and extended applicability. In parallel, the development of other laboratory techniques (embryo culture and preimplantation diagnostics) as well as medical, social, legal and cultural demands required the rapid and widespread exploitation of the new possibilities. This review summarizes the main features of the technical development, including both achievements and controversies, and outlines the emerging assisted reproductive technique strategies based partially or predominantly on cryopreservation. Application of these new achievements may substantially modify our approach in many fields and increase the overall efficiency of infertility or fertility preservation treatments.
Expert Review of Obstetrics & Gynecology 01/2014; 8(2). DOI:10.1586/eog.12.80
[Show abstract][Hide abstract] ABSTRACT: Minipigs have become important biomedical models for human ailments due to similarities in organ anatomy, physiology, and circadian rhythms relative to humans. The homeostasis of circadian rhythms in both central and peripheral tissues is pivotal for numerous biological processes. Hence, biological rhythm disorders may contribute to the onset of cancers and metabolic disorders including obesity and type II diabetes, amongst others. A tight regulation of circadian clock effectors ensures a rhythmic expression profile of output genes which, depending on cell type, constitute about 3-20% of the transcribed mammalian genome. Central to this system is the negative regulator protein Cryptochrome 1 (CRY1) of which the dysfunction or absence has been linked to the pathogenesis of rhythm disorders. In this study, we generated transgenic Bama-minipigs featuring expression of the Cys414-Ala antimorphic human Cryptochrome 1 mutant (hCRY1(AP)). Using transgenic donor fibroblasts as nuclear donors, the method of handmade cloning (HMC) was used to produce reconstructed embryos, subsequently transferred to surrogate sows. A total of 23 viable piglets were delivered. All were transgenic and seemingly healthy. However, two pigs with high transgene expression succumbed during the first two months. Molecular analyzes in epidermal fibroblasts demonstrated disturbances to the expression profile of core circadian clock genes and elevated expression of the proinflammatory cytokines IL-6 and TNF-α, known to be risk factors in cancer and metabolic disorders.
PLoS ONE 10/2013; 8(10):e76098. DOI:10.1371/journal.pone.0076098 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to compare the survival and in vitro development of sheep oocytes after open pulled straw vitrification and different final concentrations of permeable cryoprotectants. In 5 identical replicates of two experiments, in vitro matured (IVM) oocytes were vitrified by the Open Pulled Straw (OPS) method, then warmed, and the surviving ones were subjected to parthenogenetic activation. In Experiment 1, survival rate of oocytes after vitrification in 33% ethylene glycol was higher than in 33% DMSO or a mixture of 17.5% ethylene glycol and 17.5% DMSO (87.64 vs. 77.43 vs. 69.39%, respectively). The cleavage and blastocyst rates were higher after vitrification in mixture group than in ethylene glycol and DMSO (46.81 and 15.5 vs. 37.55 and 9.12 vs. 29.51 and 6.40%, for cleavage and blastocyst rates in different groups, respectively). In Experiment 2, elevated concentrations of vitrification solutions were used. The survival rate was higher after vitrification in 40% ethylene glycol and in the mixture of 20% ethylene glycol and 20% DMSO than in 40% DMSO (90.22 vs. 87.56 vs. 75.34%, respectively). Cleavage and blastocyst rates were also higher in the ethylene glycol and ethylene glycol – DMSO mixture group than in DMSO alone group (50.67 and17.60 vs. 49.13 and 14.45 vs. 33.86 and 9.81% for cleavage and blastocyst rates in different groups, respectively). The survival rates between the two experimental groups was higher in 40% ethylene glycol group, 40% mixture group and 33% ethylene glycol group than in 40% DMSO group, 33% mixture group and 33% DMSO group. Cleavage and blastocyst rates were higher in 40% ethylene glycol group, 40% mixture group and 33% mixture group than in 40% DMSO group, 33% ethylene glycol group and 33% DMSO group. All cleavage and blastocyst rates in both the experiments were lower than those of the non-vitrified control group (87.00 and 45.00, respectively). In conclusion, although ethylene glycol group and ethylene glycol – DMSO mixture group gave better survival and cleavage – blastocyst rates than DMSO group, the survival rates were lower than the control group and hence the technique could be further improved to get better results after OPS vitrification of IVM sheep oocytes.
Small Ruminant Research 05/2013; Volume 112(Issue 1):Pages 136-140. DOI:10.1016/j.smallrumres.2012.12.017 · 1.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Technology of somatic cell nuclear transfer (SCNT) has been adapted worldwide to generate transgenic animals, although the traditional procedure relies largely on instrumental micromanipulation. In this study, we used the modified handmade cloning (HMC) established in cattle and pig to produce transgenic sheep with elevated levels of omega-3 (n-3) fatty acids. Codon-optimized nematode mfat-1 was inserted into a eukaryotic expression vector and was transferred into the genome of primary ovine fibroblast cells from a male Chinese merino sheep. Reverse transcriptase PCR, gas chromatography, and chromosome analyses were performed to select nuclear donor cells capable of converting omega-6 (n-6) into n-3 fatty acids. Blastocysts developed after 7 days of in vitro culture were surgically transplanted into the uterus of female ovine recipients of a local sheep breed in Xinjiang. For the HMC, approximately 8.9% (n = 925) of reconstructed embryos developed to the blastocyst stage. Four recipients became pregnant after 53 blastocysts were transplanted into 29 naturally cycling females, and a total of 3 live transgenic lambs were produced. Detailed analyses on one of the transgenic lambs revealed a single integration of the modified nematode mfat-1 gene at sheep chromosome 5. The transgenic sheep expressed functional n-3 fatty acid desaturase, accompanied by more than 2-folds reduction of n-6/n-3 ratio in the muscle (p<0.01) and other major organs/tissues (p<0.05). To our knowledge, this is the first report of transgenic sheep produced by the HMC. Compared to the traditional SCNT method, HMC showed an equivalent efficiency but proved cheaper and easier in operation.
PLoS ONE 02/2013; 8(2):e55941. DOI:10.1371/journal.pone.0055941 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Preimplantation genetic diagnosis and screening are widely accepted for chromosomal abnormality identification to avoid transferring embryos with genetic defects. Massively parallel sequencing (MPS) is a rapidly developing approach for genome analysis with increasing application in clinical practice. The purpose of this study was to use MPS for identification of aneuploidies and unbalanced chromosomal rearrangements after blastocyst biopsy. Trophectoderm (TE) samples of 38 blastocysts from 16 IVF cycles were subjected to analysis. Low-coverage whole genome sequencing was performed using the Illumina HiSeq2000 platform with a novel algorithm purposely created for chromosomal analysis. The efficiency of this MPS approach was estimated by comparing results obtained by Affymetrix single nucleotide polymorphism (SNP) array. Whole Genome Amplification (WGA) products of TE cells were detected by MPS with an average of 0.07X depth and 5.5% coverage of the human genome. Twenty-six of embryos (68.4%) were detected as euploid, while 6 embryos (15.8%) contained uniform aneuploidies. Four of these (10.5%) were with solely unbalanced chromosomal rearrangements, whereas the remaining 2 embryos (5.3%) showed both aneuploidies and unbalanced rearrangements. Almost all these results were confirmed by the SNP array, with the exception of one sample, where different size of unbalanced rearrangements were detected possibly due to chromosomal GC bias in array analysis. Our study demonstrated MPS could be applied to accurate detect embryonic chromosomal abnormality with a flexible and cost-effective strategy and higher potential accuracy.
Biology of Reproduction 01/2013; 88(3). DOI:10.1095/biolreprod.112.106211 · 3.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lack of animal models with human-like size and pathology hampers translational research in atherosclerosis. Mouse models are missing central features of human atherosclerosis and are too small for intravascular procedures and imaging. Modeling the disease in minipigs may overcome these limitations, but it has proven difficult to induce rapid atherosclerosis in normal pigs by high-fat feeding alone, and genetically modified models similar to those created in mice are not available. D374Y gain-of-function mutations in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene cause severe autosomal dominant hypercholesterolemia and accelerates atherosclerosis in humans. Using Sleeping Beauty DNA transposition and cloning by somatic cell nuclear transfer, we created Yucatan minipigs with liver-specific expression of human D374Y-PCSK9. D374Y-PCSK9 transgenic pigs displayed reduced hepatic low-density lipoprotein (LDL) receptor levels, impaired LDL clearance, severe hypercholesterolemia, and spontaneous development of progressive atherosclerotic lesions that could be visualized by noninvasive imaging. This model should prove useful for several types of translational research in atherosclerosis.
Science translational medicine 01/2013; 5(166):166ra1. DOI:10.1126/scitranslmed.3004853 · 14.41 Impact Factor