Preimplantation embryo biopsy: detection of trisomy in a single cell biopsied from a four-cell mouse embryo.

This paper describes an efficient technique for the production of metaphase spreads from single blastomeres biopsied from four-cell preimplantation mouse embryos. The karyotype obtained by chromosomal analysis of single biopsied cells is shown to be fully predictive of subsequent fetal karyotype. The data in this study also demonstrate that the entire process of embryo biopsy and karyotypic analysis of biopsied blastomeres does not adversely affect the ability of biopsied embryos to form fetuses after transfer into pseudopregnant recipients. This study has potential clinical relevance in that it demonstrates that chromosomally defective embryos can be accurately identified before implantation. In addition, the techniques developed in this study may facilitate more efficient procedures for the genesis of animal models for human disorders such as Down syndrome and Alzheimers disease.     

Birth of correctly genotyped calves after multiplex marker detection from bovine embryo microblade biopsies

We report a method for multiplex genotyping of bovine embryo microblade biopsies. We have tested the reliability of the method and the viability of the embryos in vitro and in vivo. Two polymorphic gene markers (GHR F279Y and PRLR S18N) associated with milk production traits and one marker for sex diagnosis (ZFX/ZFY) were genotyped simultaneously with a method that combines nested PCR and allelic discrimination. To test the accuracy of genotyping, in the first experiment the genotypes of 134 biopsies from in vitro produced embryos were compared to genotypes determined from the corresponding embryos after biopsy. The method proved to be highly accurate as only in three cases (two for PRLR S18N and one for GHR F279Y) out of 395 genotypes the genotype was in disagreement between the two samples. The viability of similarly biopsied embryos was tested in parallel: after 24-hr culture 94.6% of embryos recovered in vitro. In the second experiment, a total of 150 in vivo-produced embryos were biopsied on Day 7 and genotyped. After the genotyping results were obtained on Day 8, female embryos were selected for transfer. From a total of 57 selected embryos 43 were transferred individually and 14 as pairs. After single embryo transfers, 19 recipients became pregnant and after embryo transfers in pairs one became pregnant. The success of genotyping was tested with the genotypes of donors and bulls and also from the hair samples of born calves. All calves were females and of the same genotypes determined from the biopsy.     

Setting up equine embryo gender determination by preimplantation genetic diagnosis in a commercial embryo transfer program

Preimplantation genetic diagnosis (PGD) allows identifying genetic traits in early embryos. Because in some equine breeds, like Polo Argentino, females are preferred to males for competition, PGD can be used to determine the gender of the embryo before transfer and thus allow the production of only female pregnancies. This procedure could have a great impact on commercial embryo production programs. The present study was conducted to adapt gender selection by PGD to a large-scale equine embryo transfer program. To achieve this, we studied (i) the effect on pregnancy rates of holding biopsied embryos for 7 to 10 hours in holding medium at 32 °C before transfer, (ii) the effect on pregnancy rates of using embryos of different sizes for biopsy, and (iii) the efficiency of amplification by heating biopsies before polymerase chain reaction. Equine embryos were classified by size (≤300, 300–1000, and >1000 μm), biopsied, and transferred 1 to 2 or 7 to 10 hours after flushing. Some of the biopsy samples obtained were incubated for 10 minutes at 95 °C and the rest remained untreated. Pregnancy rates were recorded at 25 days of gestation; fetal gender was determined using ultrasonography and compared with PGD results. Holding biopsied embryos for 7 to 10 hours before transfer produced pregnancy rates similar to those for biopsied embryos transferred within 2 hours (63% and 57%, respectively). These results did not differ from pregnancy rates of nonbiopsied embryos undergoing the same holding times (50% for 7–10 hours and 63% for 1–2 hours). Pregnancy rates for biopsied and nonbiopsied embryos did not differ between size groups or between biopsied and nonbiopsied embryos within the same size group (P > 0.05). Incubating biopsy samples for 10 minutes at 95 °C before polymerase chain reaction significantly increased the diagnosis rate (78.5% vs. 45.5% for treated and nontreated biopsy samples respectively). Gender determination using incubated biopsy samples matched the results obtained using ultrasonography in all pregnancies assessed (11/11, 100%); untreated biopsy samples were correctly diagnosed in 36 of 41 assessed pregnancies (87.8%), although the difference between treated and untreated biopsy samples was not significant. Our results demonstrated that biopsied embryos can remain in holding medium before being transferred, until gender diagnosis by PGD is complete (7–10 hours), without affecting pregnancy rates. This simplifies the management of an embryo transfer program willing to incorporate PGD for gender selection, by transferring only embryos of the desired sex. Embryo biopsy can be performed in a clinical setting on embryos of different sizes, without affecting their viability. Additionally, we showed that pretreating biopsy samples with a short incubation at 95 °C improved the overall efficiency of embryo sex determination.     

Frequency of sex chromosomal mosaicism in bovine embryos and its effects on sexing using a single blastomere by PCR

Assessment of nuclear status is important when a biopsied single blastomere is used for embryo sexing. In this study we investigated the nuclear status of blastomeres derived from 8- to 16-cell stage in vitro fertilised bovine embryos to determine the representativeness of a single blastomere for embryo sexing. In 24 embryos analysed, the agreement in sex determination between a biopsied single blastomere and a matched blastocyst by polymerase chain reaction (PCR) was 83.3%. To clarify the discrepancies, karyotypes of blastomeres in 8- to 16-cell stage bovine embryos were analysed. We applied vinblastine sulfate at various concentrations and for different exposure times for metaphase plate induction in 8- to 16-cell stage bovine embryos. The 1.0 mg/ml vinblastine sulfate treatment for 15 h was selected as the most effective condition for induction of a metaphase plate (> 45%). Among 22 embryos under these conditions, only 8 of 10 that had a normal diploid chromosome complement showed a sex chromosomal composition of XX or XY (36.4%) and 2 diploid embryos showed mosaicism of the opposite sex of XX and XY in blastomeres of the embryo (9.1%). One haploid embryo contained only one X-chromosome (4.5%). Four of another 11 embryos with a mixoploid chromosomal complement contained a haploid blastomere with a wrong sex chromosome (18.2%). In conclusion, assessment of nuclear status of 8- to 16-cell stage bovine embryos revealed that morphologically normal embryos had a considerable proportion of mixoploid blastomeres and sex chromosomal mosaicism; these could be the cause of discrepancies in the sex between biopsied single blastomeres and matched blastocysts by PCR.     

Effect of ICSI and embryo biopsy on embryo development and apoptosis according to oocyte diameter in prepubertal goats

ICSI and embryo biopsy are routine methods used for assisted reproduction. However, their impact on embryo quality is still poor studied. Moreover, oocyte size is also a crucial factor for blastocyst production. In this study effect of oocyte size, ICSI and embryo biopsy was assessed in terms of incidence of apoptosis and blastocyst development. IVM-oocytes from prepubertal goats were fertilized by ICSI or IVF. Embryos obtained were divided depending on oocyte size, biopsied at day-4 post-insemination/injection and cultured for additional 4-5 days. Apoptotic cell number was assessed by TUNEL staining in day-4 embryos and blastocysts obtained. In each diameter group, ICSI did not affect embryo development, blastocyst cell number and embryo apoptotic grade in comparison to IVF. Embryo biopsy did not affect blastocyst rate and apoptotic cell number, but decreased blastocyst cell number (P=0.0018). Moreover, there was a negative relationship between blastocyst cell number and apoptotic grade (P<0.05). In conclusion, ICSI and embryo biopsy do not have negative effect on embryo quality and development. However, oocyte size has a positive relationship on blastocyst yield and quality.     

Polyethylene glycol-induced fusion of two-cell mouse embryo blastomeres

Polyethylene glycol (PEG) was found to be an effective fusion-inducing agent for early mouse embryo blastomeres. A brief exposure of zona-intact 2-cell embryos to 40% PEG induced fusion of blastomeres in > 80% of embryos, and the treatment did not interfere with subsequent development of embryos to the blastocyst stage.     

Comparison of two manipulation methods to produce in vitro fertilized, biopsied and vitrified bovine embryos

The objective of this study was to compare the overall efficiency, measured by in vitro embryonic survival, and practical value of bovine in vitro embryo production, biopsy, vitrification, and direct transfer technology using 2 different manipulation methods for biopsy. Slaughterhouse-derived oocytes were matured in vitro, fertilized (Day 0) with frozen-thawed, Percoll-separated spermatozoa and cultured on a granulosa cell monolayer. In Experiment 1, one or two blastomeres were expelled from Day 4 embryos by mechanical force through a hole made by partial zona dissection. Using a darning needle hole system for individual culture of biopsied embryos from Day 4 to Day 7.5, the blastocyst per oocyte rate was 50%, and 76% of the blastocysts survived subsequent vitrification and direct in-straw rehydration. Attempts to increase the cell number of the biopsies by further in vitro culture were unsuccessful. In Experiment 2, Day 7 and Day 8 embryos were manually biopsied before or after vitrification. When biopsy was performed before vitrification, 98% of the embryos survived manipulation, and 86% of these re-expanded after vitrification and in-straw dilution. Biopsy after vitrification was less efficient, since only 69% of the embryos survived both processes. The cumulative efficiency of embryo production, Day 7.5 biopsy and vitrification--in-straw direct rehydration was lower (P < 0.001) than that of Day 4 biopsy and Day 7.5 vitrification (29 vs 38%, respectively). However, a Day 7.5 biopsy may have the more practical application since the size of the biopsy is larger and the process is not as time-consuming as the long-term individual culture of the biopsied embryos.     

Biopsy of preimplantation mouse embryos: development of micromanipulated embryos and proliferation of single blastomeres in vitro

We have developed a technique to sample the preimplantation embryo, which may, in the future, be applied to prenatal diagnosis of genetic disease. Using micromanipulation, we aspirated a single blastomere from 4-cell mouse embryos. This procedure had no effect on in vitro development; 98% of control and 94% of biopsied embryos reached the blastocyst stage after 48 h in culture. Furthermore, after transfer to pseudopregnant recipient mice, the rate of fetal development of biopsied embryos was not significantly different from control embryos, although implantation rate was significantly reduced (mean +/- SD: biopsied 53.1 +/- 4.0, control 81.8 +/- 8.4, p less than 0.001). For the first time we have produced monolayer cell cultures derived from single preimplantation blastomeres. Individual biopsied blastomeres were cultured in vitro on different extracellular matrix components. Significantly greater cell proliferation was obtained in wells coated with fibronectin (FN), laminin (LN), and a complex of laminin and nidogen (LNC) than in a less specific matrix of swine skin gelatin (SSG). Mean (+/- SE) cell nuclei number per well after 6 days in culture was 6.4 +/- 2.1, 11.9 +/- 1.5, 19.8 +/- 2.9, and 20.9 +/- 2.6 in wells coated with SSG, LN, FN, and LNC respectively.     

Use of zona drilling for safe and effective biopsy of murine oocytes and embryos

With the mouse as a model, we have used zona drilling to devise procedures for safe removal of the first polar body or one or more blastomeres from cleaving embryos. These methods require minimal disruption of the zona pellucida and little or no direct contact between microtools and the materials to be biopsied. Of 175 eggs subjected to the polar body biopsy procedure, 1 was killed, and 165/174 survivors were fertilized (94.8%). For blastomere biopsy, embryos from the 2- to 16-cell stage were incubated in a chelating medium containing 100 mM sucrose for at least 30 min. The zonae were then drilled, and one or more blastomeres were "pushed" out through the hole by pressure exerted against the zona at some distance from the drilling site. In all 85 embryos biopsied, one or more additional intact blastomeres were successfully removed. Moreover, 83/84 biposied embryos that were subsequently cultured developed into blastocysts (98.8%). Although acid Tyrode's solution was used in this study, mechanical methods of zona opening were also effective. The data indicate that oocyte and embryo biopsy assisted by zona drilling is safe and does not appear to affect fertilization or development, and as such, it is applicable to genetic diagnostic procedures.     

Spatial arrangement of individual 4-cell stage blastomeres and the order in which they are generated correlate with blastocyst pattern in the mouse embryo

In the unperturbed development of the mouse embryo one of the 2-cell blastomeres tends to contribute its progeny predominantly to the embryonic and the other to the abembryonic part of the blastocyst. However, a significant minority of embryos (20-30%) do not show this correlation. In this study, we have used non-invasive lineage tracing to determine whether development of blastocyst pattern shows any correlation with the orientation and order of the second cleavage divisions that result in specific positioning of blastomeres at the 4-cell stage. Although the orientation and order of the second cleavages are not predetermined, in the great majority (80%) of embryos the spatial arrangement of 4-cell blastomeres is consistent with one of the second cleavages occurring meridionally and the other equatorially or obliquely with respect to the polar body. In such cleaving embryos, one of the 2-cell stage blastomeres tends to contribute to embryonic while the other contributes predominantly to abembryonic part of the blastocyst. Thus, in these embryos the outcome of the first cleavage tends to correlate with the orientation of the blastocyst embryonic-abembryonic axis. However, the order of blastomere divisions predicts a specific polarity for this axis only when the earlier 2-cell blastomere to divide does so meridionally. In contrast to the above two groups, in those embryos in which both second cleavage divisions occur in a similar orientation, either meridionally or equatorially, we do not observe any tendency for the 2-cell blastomeres to contribute to specific blastocyst parts. We find that all these groups of embryos develop to term with similar success, with the exception of those in which both second cleavage divisions occur equatorially whose development can be compromised. We conclude that the orientations and order of the second cleavages are not predetermined; they correlate with the development of blastocyst patterning; and that the majority, but not all, of these cleavage patterns allow equally successful development.     

Sexing and multiple genotype analysis from a single cell of bovine embryo

We described a procedure for multiple genotype analysis (determination of sex and of three genetic markers) from a single cell derived from bovine preimplantation embryo. It consists of primer extension preamplification-polymerase chain reaction (PEP-PCR) and subsequent single assay or multiplex PCR. A single blastomere that was isolated by microaspiration from bovine embryos at the 16- to 32-cell stage then was lysed and was subjected to the PEP-PCR. When testing 75 embryos, efficiency of genotyping by standard PCR for kappa-casein, growth hormone (GH) and prolactin (PRL) polymorphic alleles was 91, 88 and 89%, respectively. Sexing efficiency in the multiplex PCR was 91%, based on the amplification of Y-specific locus using kappa-casein internal standard. The microaspiration of a single blastomere was shown not to be invasive for the embryos. It did not alter their development potential in vitro (P > 0.05), as was seen by obtaining a similar percentage of embryos developing further into the blastocyst stage in the group subjected to biopsy (44/75, 59%) and in the control group of embryos (30/50, 60%).     

Splitting and biopsy for bovine embryo sexing under field conditions.

Improvements on embryo micromanipulation techniques led to the use of embryo bisection technology in commercial embryo transfer programs, and made possible the direct genetic analysis of preimplantation bovine embryos by biopsy. For example, aspiration and microsection, allow bovine embryos sexing by detection of male-specific Y-chromosome in a sample of embryonic cells. We report on the application of the methodologies of splitting and biopsy of bovine embryos in field conditions, and on the results of embryo sex determination by the polymerase chain reaction (PCR). Pregnancy rates achieved with fresh bisected or biopsied embryos (50 to 60%) were similar to the fresh intact embryos (55 to 61%). The PCR protocol used for embryo sexing showed 92% to 94% of efficiency and 90 to 100% of accuracy. These results demonstrate these procedures are suitable for use in field conditions.     

Effects of removal of necrotic blastomeres from mouse cryopreserved embryos on blastocyst formation and hatching

To evaluate whether the developmental potential of embryos that were partially damaged after freezing and thawing can be improved by removal of necrotic blastomeres. Eight-cell mouse embryos were cryopreserved using 1,2-propanediol and sucrose as cryoprotectant with slow cooling procedure. After thawing, blastocyst formation and hatching of fully intact embryos were compared between no treatment and with laser-assisted hatching. For partially intact embryos, the effects of removal of necrotic blastomeres with micromanipulation were evaluated. Laser-assisted hatching of mouse cryopreserved fully intact embryos significantly increased blastocyst hatching (63.4% versus 48.3%, P<0.05), but had little effect on blastocyst formation (72.0% versus 70.1%, P>0.05). The removal of necrotic blastomeres from partially damaged mouse cryopreserved embryos with micromanipulation significantly increased blastocyst formation (52.9% versus 32.0%, P<0.05) and blastocyst hatching (41.2% versus 22.0%, P>0.05) compared with the control group. The developmental potential of partially damaged cryopreserved embryos can be improved by removal of necrotic blastomeres with micromanipulation.     

Chemical enhancement in embryo development and stem cell derivation from single blastomeres

Several chemicals targeting the mitogen-activated protein (MAP) kinase signaling pathway, which play an important role in regulating cell growth and differentiation, have shown enhancing effects on the development of the inner cell mass (ICM) and the derivation of ES cells. However, investigation of such chemicals on early embryonic development and the establishment of ES cell lines has not been elucidated. This study was aimed to determine if ACTH, MAP2K1 inhibitor [MAP2K1 (I)], and MAPK14 inhibitor [MAPK14 (I)] could enhance the development of the ICM in preimplantation mouse embryos and blastocyst outgrowths, and the establishment of ES cell lines from blastomeres of early embryos. We have demonstrated that both MAP2K1 (I) and MAPK14 (I) delay early embryo development and inhibit the development of embryos from early blastomeres. On the other hand, ACTH had a positive effect on embryos derived from early blastomeres. As a result, 17 ES cell lines were established. Among these ES cell lines, nine and five ES cell lines were established from single blastomeres of two-cell embryos with and without the supplement of ACTH, respectively. In addition to two-cell isolated blastomeres, three ES cell lines were established from blastomeres of four-cell embryos only with the supplement of ACTH. Our results suggest that ACTH can enhance the derivation of ES cells from single blastomere-derived embryos.     

Dual blastomere analysis improves reliability of preimplantation trembler mouse diagnosis

Dual blastomere biopsy and independent blastomere analysis dramatically improved preimplantation diagnostic reliability as confirmed by testing the remaining biopsied eight-cell mouse embryo. The autosomal dominant trembler mouse point mutation was selected as a model for human preimplantation diagnosis because: (1) single cell assay failure is predicted to be the highest when testing autosomal dominant mutations; (2) point mutations represent the most common of all mutation categories and the most demanding mutation to assay reliably; and (3) the trembler mouse point mutation in peripheral myelin protein 22 (Pmp22) is a model of human Charcot-Marie-Tooth type 1A disease. Mathematical models predict our experimental results assuming amplification of 80% of each target allele as well as trembler sperm DNA contamination in 1 of 44 normal biopsied single blastomeres. Single blastomere analysis correctly predicted the genotype in only 84% of embryos that would have been implanted as normal. In contrast, when independent tests of both biopsied blastomeres agreed, test results were confirmed in 20 of 21 (95.2%) of the remaining six-cell biopsied embryos designated as normal. Thus, biopsied six-cell embryo confirmation demonstrated that dual biopsied blastomere analysis improved test reliability remarkably. Received: 20 November 1996 / Accepted: 4 April 1997     

激光辅助制作小鼠嵌合体胚胎的研究

目的：探索激光辅助体外制作小鼠嵌合体胚胎的方法。方法：激光辅助去除不同发育阶段体外受精胚胎的透明带,随机组合卵裂球体外培养,观察胚胎融合情况。结果：没有透明带的卵裂球体外能够“自发”融合,并且融合胚胎在体外培养环境中,能够发育至囊胚期,显微镜观察其形态基本与二倍体胚胎无差别。结论：激光辅助方法获得裸露的卵裂球能够在体外培养环境制作嵌合体胚胎。     

Effect of donor embryo cell number and cell size on the efficiency of bovine embryo cloning

To establish reliable criteria for the evaluation of nuclear donor embryos, we studied the effect of cell number and cell size of in vitro produced day 6 donor morulae on the rate of blastocyst formation following nuclear transfer to in vitro matured oocytes. In experiment 1, donor embryos were divided into three groups with low (25–34), intermediate (40–55), and high (60–81) blastomere numbers. Transfer of nuclei from day 6 morulae with intermediate and high cell numbers resulted in a significantly higher blastocyst rate (31% and 32%, respectively) than use of nuclei from day 6 morulae with low cell numbers (17%) or nuclei from day 7 morulae with 50–83 blastomeres (19%). This suggests that blastomeres from the developmentally advanced day 6 morulae are more viable than blastomeres from retarded embryos. In experiment 2, we evaluated the effect of blastomere size in day 6 donor morulae with intermediate (40–55) or high (60–81) cell numbers on the efficiency of nuclear transfer. In both classes of embryos, small blastomeres were better nuclear donors than large blastomeres. The rates of development to the blastocyst stage were 28% versus 15% (40–55 cells) and 41% versus 25% (60–81 cells), suggesting that small blastomeres include a higher proportion of totipotent cells than the polarized large blastomeres. Our results demonstrate that blastomere number and size markedly affect the efficiency of nuclear transfer and therefore are useful criteria for evaluating nuclear donor embryos. These parameters are easy to determine and may therefore be helpful to improve the efficiency of cattle cloning. © 1995 wiley-Liss, Inc.     

The effect of oxygen on the development of preimplantation mouse embryos in vitro

The optimal oxygen tension for development of preimplantation mouse embryos to the blastocyst stage in vitro was found to be between 2.5% and 5%. One- and two-cell embryos had a more sharply defined range of oxygen tension capable of supporting development than 8-cell and morula stages. At all stages of development, more embryos developed to the blastocyst stage under 5% O2 compared to the numbers of developing under higher oxygen tensions (20% and 40% O2). The blastocysts developing under 20% O2 had fewer blastomeres than those which developed under 5% O2. As the time required for development to the blastocyst stage in vitro increased, there were fewer blastomeres present at the blastocyst stage. These results indicate that the cleaving mouse embryo has an optimal oxygen requirement in vitro of about 5%. At higher oxygen tensions, fewer embryos develop to the blastocyst stage and in those which do develop, there are fewer cell divisions. If a gradient of oxygen tension exists across the blastomeres from the outside of the embryo to its centre, the blastomeres might be using this gradient to obtain imformation about their location within the embryo and respond accordingly. Thus blastomeres on the outside at a higher oxygen tension would divide at a slower rate and form trophectoderm whereas those on the inside at a lower oxygen tension would divide more rapidly and contribute to the inner cell mass.     

Generation of transgenic rabbits by the novel technique of chimeric somatic cell cloning

A novel technique of chimeric somatic cell cloning was applied to produce a transgenic rabbit (NT20). Karyoplasts of transgenic adult skin fibroblasts with Tg(Wap-GH1) gene construct as a marker were microsurgically transferred into one, previously enucleated, blastomere of 2-cell non-transgenic embryos, while the second one remained intact. The reconstructed embryos either were cultured in vitro up to the blastocyst stage (Experiment I) or were transferred into recipient-females immediately after the cloning procedure (Experiment II). In Experiment I, 25/102 (24.5%) embryos formed blastocysts from whole embryos and 46/102 (44.12%) embryos developed to the blastocyst stage from single non-operated blastomeres, while the reconstructed blastomeres were damaged and degenerated. Thirteen (12.7%) embryos did not exceed 3- to 4-cell stages and 18 (17.7%) embryos were inhibited at the initial 2-cell stage. Out of 14 blastocysts which were subjected to molecular analysis, the transgene was detected in the cells of 4 blastocysts. In Experiment II, 163/217 (75.0%) embryos were transferred into 9 pseudopregnant recipient-rabbits (an average of 18 embryos per recipient). Four recipient-females (44.4%) became pregnant and delivered a total of 24 (14.7%) pups. Molecular analysis confirmed that two pups (1.2%), one live and one stillborn, showed a positive transgene signal. Live transgenic rabbit NT20 appeared healthy and anatomically as well as physiologically normal. The results of our experiments showed that transgenic adult skin fibroblast cell nuclei, which have been introduced into the cytoplasmic microenvironment of single enucleated blastomeres from 2-cell stage rabbit embryos, are able to direct the development of chimeric embryos not only to the blastocyst stage but also up to term.