Anti-apoptotic effect of insulin-like growth factor (IGF)-I and its receptor in porcine preimplantation embryos derived from in vitro fertilization and somatic cell nuclear transfer

Insulin-like growth factor (IGF)-I is a receptor-mediated autocrine and/or paracrine growth and/or survival factor for mammalian embryo development. It is known to promote the growth and development of mouse preimplantation embryos. The present study was designed to investigate the effects of IGF-I (50 ng/ml), anti-IGF-I receptor antibody (50 ng/ml) and their combination on porcine preimplantation embryo development. Furthermore, the mechanism underlying the embryotropic effects of IGF-I was evaluated by monitoring the incidence of apoptosis and expression of apoptosis-related genes. In both in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos, culturing with IGF-I increased the rate of blastocyst formation and this embryotrophic effect was neutralized by culturing with IGF-I along with anti-IGF-I receptor (IGF-IR) antibody. Culturing IVF and SCNT embryos with IGF-I significantly increased the number of total cells in blastocysts and decreased the number of apoptotic nuclei. These effects of IGF-I were also neutralized by culturing with IGF-I along with anti-IGF-IR antibody. Expression of the anti-apoptotic Bcl-2 gene was increased, while expression of the pro-apoptotic Bax was decreased in both IVF and SCNT embryos cultured with IGF-I. In both IVF and SCNT embryos, anti-IGF-IR antibody along with IGF-I neutralized the effect of IGF-I on expression of Bcl-2 and Bax genes. In conclusion, the present study demonstrated that IGF-I through its specific receptors improved the developmental competence of IVF and SCNT embryos by decreasing the incidence of apoptosis and regulating apoptosis-related genes in porcine preimplantation embryos.     

Aberrant expression of miR-29a/29b and methylation level of mouse embryos after in vitro fertilization and vitrification at two-cell stage

Proper epigenetic modifications during preimplantation embryo development are important for a successful pregnancy. We aim to investigate the putative influence of in vitro fertilization (IVF) and vitrification on DNA methylation in mouse preimplantation embryos. The study groups consisted of blastocyst-derived vitrified two-cell embryos, nonvitrified embryos, and a control group of in vivo derived blastocysts. We assessed developmental competence, global DNA methylation, relative expression levels of miR-29a/29b, and their target genes, Dnmt3a/3b. Vitrified embryos had a lower developmental rate as compared with nonvitrified embryos. There was no significant decrease in blastocyst cell numbers among studied groups, whereas there was a steady decline in DNA methylation after IVF and vitrification. The levels of miR-29a/29b upregulated in the experimental groups as compared with the control group. IVF and vitrification caused Dnmt3a/3b downregulations in blastocysts. The results of this study have suggested that a relationship exists between IVF and embryo vitrification with methylation interruptions in the blastocysts.     

Haploidy but not parthenogenetic activation leads to increased incidence of apoptosis in mouse embryos.

Aneuploidy underlies failed development and possibly apoptosis of some preimplantation embryos. We employed a haploid model in the mouse to study the effects of aneuploidy on apoptosis in preimplantation embryos. Mouse metaphase II oocytes that were activated with strontium formed haploid parthenogenetic embryos with 1 pronucleus, whereas activation of oocytes with strontium plus cytochalasin D produced diploid parthenogenetic embryo controls with 2 pronuclei. Strontium induced calcium transients that mimic sperm-induced calcium oscillations, and ploidy was confirmed by chromosomal analysis. Rates of development and apoptosis were compared between haploid and diploid parthenogenetic embryos (parthenotes) and control embryos derived from in vitro fertilization (IVF). Haploid mouse parthenotes cleaved at a slower rate, and most arrested before the blastocyst stage, in contrast to diploid parthenotes or IVF embryos. Developmentally retarded haploid parthenotes exhibited apoptosis at a significantly higher frequency than did diploid parthenotes or IVF embryos. However, diploid parthenotes exhibited rates of preimplantation development and apoptosis similar to those of IVF embryos, indicating that parthenogenetic activation itself does not initiate apoptosis during preimplantation development. These results suggest that haploidy can lead to an increased incidence of apoptosis. Moreover, the initiation of apoptosis during preimplantation development does not require the paternal genome.     

小鼠体内外受精植入前胚胎全基因组甲基化模式比较

为考察体外受精、操作及培养环境对体外受精的小鼠植入前胚胎全基因组DNA甲基化模式的影响,本研究以体内受精的植入前胚胎作为对照,采用间接免疫荧光法检测小鼠体内外受精植入前胚胎基因组DNA甲基化模式.实验结果表明,体外受精各期植入前胚胎呈现出与之相应时期的体内受精植入前胚胎不同的DNA甲基化模式和水平,原核期甲基化水平较高,2-4-、8-细胞期明显降低,而桑葚胚和囊胚期又略有升高.各期体外受精植入前胚胎的基因组DNA甲基化水平都比同时期体内受精胚胎的甲基化水平低.本实验结果部分显示了体外受精、操作及培养环境可能对正常的DNA甲基化模式产生影响,造成体外受精植入前胚胎甲基化模式异常.     

Glucose and pyruvate metabolism of preimplantation goat blastocysts following in vitro fertilization and parthenogenetic activation

The energy metabolism of preimplantation embryos can be used to predict viability and postimplantation development. Although preimplantation development and mean blastocyst cell numbers of goat in vitro-fertilized (IVF) embryos and chemically activated parthenogenotes are comparable, mammalian parthenogenotes are not viable, with most dying shortly after implantation. The objective of this study was to compare glucose and pyruvate metabolism of IVF goat blastocysts with that of parthenogenetic blastocysts developing from chemically activated oocytes. Embryos derived from IVF and parthenogenotes produced by exposing oocytes to either ionomycin or ethanol followed by 6-dimethylaminopurine (6-DMAP) were cultured in G1.2/G2.2 sequential culture media. Metabolism was determined for individual blastocysts using [5-3H]glucose and [2-14C]pyruvate to determine glycolytic and Kreb's cycle activity, respectively. Data were analyzed by ANOVA. A significantly higher percentage of activated oocytes underwent cleavage and developed to the blastocyst stage compared to IVF oocytes (p < 0.05). There was no significant difference in glucose or pyruvate metabolism between IVF and parthenogenetically activated blastocysts. Mean glucose metabolism through glycolysis was 154.9 +/- 29.1, 130.3 +/- 17.1, and 129 +/- 16.5 pmol/embryo/3 h for IVF, ethanol-activated, and ionomycin-activated blastocysts, respectively. Mean pyruvate metabolism through the Kreb's cycle was 28.1 +/- 8.0, 15.8 +/- 4.2, and 24.4 +/- 4.4 in pmol/embryo/3 h for IVF, ethanol-activated, and ionomycin-activated blastocysts, respectively. Our results suggest that known differences in postimplantation development observed in IVF versus parthenogenetic embryos cannot be attributed to differences in pyruvate or glucose metabolism in the preimplantation blastocysts. Thus, these activation protocols result in embryos capable of appropriate regulation of key metabolic enzymes.     

Hypotaurine requirement for in vitro development of golden hamster one-cell embryos into morulae and blastocysts, and production of term offspring from in vitro-fertilized ova.

Almost 30 years after the first successful in vitro fertilization (IVF) in golden hamsters (Mesocricetus auratus), we report that IVF hamster embryos can develop in a chemically defined, protein-free culture medium into morulae and blastocysts, and produce normal offspring after transfer to recipients. When examined 96 h post-insemination, 82% (160/200) of IVF ova had cleaved to at least 2 cells, 55% (97/200) had developed beyond the 4-cell stage, and 22% (38/200) had developed into morulae/blastocysts. In vitro development of IVF embryos to greater than or equal to 8 cells was absolutely dependent on hypotaurine. Twenty living offspring were produced from transfer of IVF embryos to recipients, with an overall success rate of 5% and 17% for oviductal (2-cell) and uterine (8-cell/morulae) transfers, respectively. In vivo-fertilized pronucleate embryos collected 3 h after egg activation were less able to develop in vitro than embryos collected only 6 h later, revealing a critical influence of the oviduct within the first hours of embryo development. Hypotaurine partly compensated for the decreased oviductal exposure of early 1-cell embryos. Establishment of a key role for hypotaurine in hamster embryo development, support of IVF embryos to morula/blastocyst stages in vitro, and production of living offspring after IVF embryo transfer are significant steps towards the goal of obtaining comparative data on preimplantation embryogenesis.     

Expression of leptin ligand and receptor and effect of exogenous leptin supplement on in vitro development of porcine embryos

The present study investigated the expression of ligand and receptor for leptin, and the effect of leptin supplementation on preimplantation development of porcine in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos. The IVF embryos were produced using frozen boar semen and SCNT embryos were obtained by nuclear transfer of fetal fibroblasts into enucleated oocytes. The protein expression of leptin ligand and receptor was investigated in in vitro matured oocytes, 2-, 4- and 8-cell embryos, morulae and blastocysts derived from IVF and SCNT using immunofluorescence. Both the ligand and receptor were detected in in vitro matured oocytes and all stage of IVF and SCNT embryos. The IVF and SCNT embryos were cultured in modified North Carolina State University (mNCSU)-23 medium supplemented with various concentrations (0, 1, 10, 100 or 1000 ng/mL) of leptin. The rates of cleavage at day 2 and blastocyst formation at day 7, and cell number of blastocysts were monitored as experimental parameters. In SCNT embryos, supplementing with 1000 ng/mL leptin significantly (P<0.05) increased the rate of blastocysts formation (20.2% versus 12.9%) and total cell number (54.6+/-17.4 versus 45.1+/-15.2) compared to the control group. In IVF embryos, leptin supplementation did not affect preimplantation embryo development and cell number in blastocysts. In conclusion, the present study demonstrated the expression of leptin ligand and receptor and the embryotropic effect of leptin in SCNT embryos.     

Comparison of histone modifications in in vivo and in vitro fertilization mouse embryos

Histone modifications are thought to play important roles in various cellular functions. In this article, the distribution patterns of acetylation on histone H4, methylation on histone H3 lysine 9, and phosphorylation on histone H3 serine 10 were examined in in vivo and in vitro fertilization (IVF) preimplantation mouse embryos by using indirect immunofluorescence and scanning confocal microscopy. We desired to know whether the IVF, which has been widely used as a routine assisted reproductive technology in animal and human, was safe at the epigenetic level. As results, we found that there was no difference in these histone modification patterns in in vivo and IVF mouse embryos from zygote to blastocyst stage. Moreover, these histone modifications had different distributions at all examined stages, but they were consistent with the mouse embryo developmental stages.     

Vitrification of murine mature metaphase II oocytes perturbs DNA methylation reprogramming during preimplantation embryo development

Accurate reprogramming of DNA methylation occurring in preimplantation embryos is critical for normal development of both fetus and placenta. Environmental stresses imposed on oocytes usually cause the abnormal DNA methylation reprogramming of early embryos. However, whether oocyte vitrification alters the reprogramming of DNA methylation (5 mC) and its derivatives in mouse preimplantation embryo development remains largely unknown. Here, we found that the rate of cleavage and blastocyst formation of embryos produced by IVF of vitrified matured oocytes was significantly lower than that in control counterparts, but the quality of blastocysts was not impaired by oocyte vitrification. Additionally, although vitrification neither altered the dynamic changes of 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5 fC) before 4-cell stage nor affected the levels of 5 mC and 5-carboxylcytosine (5caC) throughout the preimplantation development, vitrification significantly reduced the levels of 5hmC and 5 fC from 8-cell stage onwards. Correspondingly, vitrification did not alter the expression patterns of Tet3 in preimplantation embryos but apparently reduced the expression levels of Tet1 in 4-cell and 8-cell embryos and increased the expression levels of Tet2 at morula stage. Taken together, these results demonstrate that oocyte vitrification perturbs DNA methylation reprogramming in mouse preimplantation embryo development.     

Automated microinjection of recombinant BCL-X into mouse zygotes enhances embryo development

Progression of fertilized mammalian oocytes through cleavage, blastocyst formation and implantation depends on successful implementation of the developmental program, which becomes established during oogenesis. The identification of ooplasmic factors, which are responsible for successful embryo development, is thus crucial in designing possible molecular therapies for infertility intervention. However, systematic evaluation of molecular targets has been hampered by the lack of techniques for efficient delivery of molecules into embryos. We have developed an automated robotic microinjection system for delivering cell impermeable compounds into preimplantation embryos with a high post-injection survival rate. In this paper, we report the performance of the system on microinjection of mouse embryos. Furthermore, using this system we provide the first evidence that recombinant BCL-XL (recBCL-XL) protein is effective in preventing early embryo arrest imposed by suboptimal culture environment. We demonstrate that microinjection of recBCL-XL protein into early-stage embryos repairs mitochondrial bioenergetics, prevents reactive oxygen species (ROS) accumulation, and enhances preimplantation embryo development. This approach may lead to a possible treatment option for patients with repeated in vitro fertilization (IVF) failure due to poor embryo quality.     

Effects of growth hormone on the ultrastructure of bovine preimplantation embryos

Growth hormone (GH) has recently been shown to promote the development of preimplantation embryos. The aim of our study was therefore to analyze the effects of GH on the morphology and ultrastructure of the cells of bovine preimplantation embryos produced by in vitro fertilization (IVF). In order to determine the physiologically optimal morphology of blastocysts, ex vivo embryos obtained by uterine flushing were also included in the study. As shown by transmission electron microscopy, treatment with GH induced the elimination of glycogen storage in cells of the inner cell mass of 7-day-old embryos. GH also stimulated the exocytosis of lipid vesicles in the inner cell mass and trophectoderm cells of these embryos. Quantitative analysis of micrographs demonstrated a higher volume density of embryonic mitochondria in 7-day-old embryos cultured with GH than in control embryos. Treatment with GH regularly resulted in an improvement of the ultrastructural features of embryos produced in vitro, thus resembling the morphology of ex vivo embryos. Scanning electron-microscopy studies demonstrated that GH altered the structure and the pore size of the zona pellucida of blastocysts. Our studies imply that GH can modulate carbohydrate, lipid, and energy metabolism and influence transportation processes in the early IVF embryo.This work was supported by the Deutsche Forschungsgemeinschaft (FOR 478/1)     

Preimplantation genetic analysis of translocations: case-specific probes for interphase cell analysis

Carriers of balanced translocations show an increased risk of infertility and spontaneous abortions, because of errors in gametogenesis, and constitute a significant fraction of patients seeking assisted reproduction. The objective of this study was to design approaches for preimplantation diagnosis of chromosome translocations and to apply such techniques to the selection of chromosomally normal or balanced embryos prior to their transfer to the mother’s womb. Three slightly different approaches were assessed by means of chromosome-specific, non-isotopically labeled DNA probes and an assay based on fluorescence in situ hybridization- to score and characterize chromosomes in single blastomeres biopsied from embryos on their third day of development. The three approaches were used for preimplantation genetic diagnosis involving four couples who had enrolled in our IVF program and in which one of the partners was a carrier of one of the following translocations: 46,XX,t(12;20)(p13.1;q13.3), 46,XY,t(3;4) (p24;p15), 45,XY,der(14;15)(10q;10q), and 46,XY,t(6;11) (p22.1;p15.3). A total of 33 embryos were analyzed, of which 25 (75.8%) were found to be either unbalanced or otherwise chromosomally abnormal. Only a single embryo could be transferred to patients A and D, whereas three embryos were transferred to patient B in a total of two IVF cycles. Transfer of two embryos to patient C resulted in an ongoing pregnancy. Re-analysis of non-transferred embryos with additional probes confirmed the initial results in 95% (20/21) of the cases. In conclusion, case-specific translocation tests can be applied to any translocation carrier for the selection of normal or chromosomally balanced embryos prior to embryo transfer. This is expected significantly to increase the success rates in IVF cycles of translocation carriers, while preventing the spontaneous abortion or birth of abnormal offspring. Received: 13 January 1998 / Accepted: 24 March 1998     

Serotonin localization and its functional significance during mouse preimplantation embryo development

Serotonin is a neurotransmitter functioning also as a hormone and growth factor. To further investigate the biological role of serotonin during embryo development, we analysed serotonin localization as well as the expression of specific serotonin 5-HT1D receptor mRNA in mouse oocytes and preimplantation embryos. The functional significance of serotonin during the preimplantation period was examined by studying the effects of serotonin on mouse embryo development. Embryo exposure to serotonin (1 microM) highly significantly reduced the mean cell number, whereas lower concentrations of serotonin (0.1 microM and 0.01 microM) had no significant effects on embryo cell numbers. In all serotonin-treated groups a significant increase in the number of embryos with apoptotic and secondary necrotic nuclei was observed. Expression of serotonin 5-HT1D receptor mRNA in mouse oocytes and preimplantation embryos was confirmed by in situ hybridization showing a clearly distinct punctate signal. Immunocytochemistry results revealed the localization of serotonin in oocytes and embryos to the blastocyst stage as diffuse punctate cytoplasmic labelling. It appears that endogenous and/or exogenous serotonin in preimplantation embryos could be involved in complex autocrine/paracrine regulations of embryo development and embryo-maternal interactions.     

Methods of embryo scoring in in vitro fertilization

It has been 25 years since the introduction of in vitro fertilization (IVF) for treatment of infertility. During this time very dynamic advances have taken place in all aspects of assisted reproductive technology (ART). The rapid improvement in embryological methods, especially these related to preimplantation embryo evaluation are of great importance. This article is a review of embryo classification systems utilized in ART programs. The most widely used scoring systems of zygotes and embryos (including blastocysts) are described. Additionally, the advantages of advanced embryo classifications in relation to ART success rates are presented.     

Increasing Live Birth Rate by Preimplantation Genetic Screening of Pooled Polar Bodies Using Array Comparative Genomic Hybridization

Meiotic errors during oocyte maturation are considered the major contributors to embryonic aneuploidy and failures in human IVF treatment. Various technologies have been developed to screen polar bodies, blastomeres and trophectoderm cells for chromosomal aberrations. Array-CGH analysis using bacterial artificial chromosome (BAC) arrays is widely applied for preimplantation genetic diagnosis (PGD) using single cells. Recently, an increase in the pregnancy rate has been demonstrated using array-CGH to evaluate trophectoderm cells. However, in some countries, the analysis of embryonic cells is restricted by law. Therefore, we used BAC array-CGH to assess the impact of polar body analysis on the live birth rate. A disadvantage of polar body aneuploidy screening is the necessity of the analysis of both the first and second polar bodies, resulting in increases in costs for the patient and complex data interpretation. Aneuploidy screening results may sometimes be ambiguous if the first and second polar bodies show reciprocal chromosomal aberrations. To overcome this disadvantage, we tested a strategy involving the pooling of DNA from both polar bodies before DNA amplification. We retrospectively studied 351 patients, of whom 111 underwent polar body array-CGH before embryo transfer. In the group receiving pooled polar body array-CGH (aCGH) analysis, 110 embryos were transferred, and 29 babies were born, corresponding to live birth rates of 26.4% per embryo and 35.7% per patient. In contrast, in the control group, the IVF treatment was performed without preimplantation genetic screening (PGS). For this group, 403 embryos were transferred, and 60 babies were born, resulting in live birth rates of 14.9% per embryo and 22.7% per patient. In conclusion, our data show that in the aCGH group, the use of aneuploidy screening resulted in a significantly higher live birth rate compared with the control group, supporting the benefit of PGS for IVF couples in addition to the suitability and effectiveness of our polar body pooling strategy.