鼠胚大脑皮层子宫内电穿孔法基因转移体系的建立

体内电穿孔法转基因技术作为一种新型有效的转基因方法,被广泛应用于发育生物学的研究,特别是中枢神经系统发育。本研究旨在通过子宫内电穿孔法,建立鼠胚大脑皮层体内基因转移体系:将带有GFP的表达载体pCAGGS-IRES-EGFP显微注入胚胎期14.5d的鼠胚侧脑室中,电极沿与脑中缝平行的方向夹持鼠脑,在电压30V,脉冲时间50ms,脉冲数5的条件下方波电击,将外源基因转移到室管膜层细胞。胚胎被重新放回母体内,继续发育3d。分离被转染的鼠胚脑组织,沿冠状面冰冻切片,在荧光显微镜下观察到外源蛋白GFP在鼠胚大脑皮层中的瞬时表达。鼠胚大脑皮层子宫内电穿孔法基因转移体系的建立将为进一步研究皮层发育、神经元迁移、轴突导向等以及神经发生过程中相关基因功能奠定基础。     

The combination of polar body and embryo biopsy does not affect embryo viability

BACKGROUND: The biopsy of both polar bodies and a blastomere from the same embryo was investigated as an approach aimed at increasing the quantity of DNA available for genetic analysis in preimplantation embryos. METHODS: In 113 cycles, preimplantation genetic diagnosis (PGD) was performed for aneuploidy: 19 cycles underwent polar body biopsy, 32 cycles had both polar body and blastomere biopsy done, and the remaining 62 cycles underwent blastomere biopsy. The chromosomal analysis was performed in a two-round fluorescence in situ hybridization (FISH) protocol with probes specific for the chromosomes X, Y, 13, 15, 16, 18, 21 and 22. RESULTS: The morphological evaluation of the analysed embryos demonstrated similar rates of development irrespective of the biopsy procedure. Accordingly, the implantation rate did not differ significantly in the three biopsy groups and was 15% after polar body biopsy, 26% after the combined biopsy procedures of polar bodies and blastomeres, and 25% after blastomere biopsy. CONCLUSIONS: The removal of a blastomere subsequent to polar body biopsy does not seem to have negative effects on embryo viability. This approach could be especially valuable for a combined diagnosis of aneuploidy and single-gene disorders in preimplantation embryos generated by couples at high reproductive risk.     

Diagnostic efficiency, embryonic development and clinical outcome after the biopsy of one or two blastomeres for preimplantation genetic diagnosis

BACKGROUND: Preimplantation genetic diagnosis or screening (PGD, PGS) involves embryo biopsy on Day 3. Opting for one- or two-cell biopsy is a balance between the lowest risk for misdiagnosis on the one hand and the highest chance for a pregnancy on the other hand. METHODS: A prospective controlled trial was designed and 592 ICSI cycles were randomly assigned to the one-cell (group I) or the two-cell group (group II). Primary outcomes were diagnostic efficiency and embryonic development to delivery with live birth (analysed by cycle). The false-positive rate for the PCR cycles is presented as a secondary outcome (analysed by embryo). RESULTS: A strong significant correlation was observed between embryonic developmental stage on Day 3 and post-biopsy in vitro development on Day 5 (P < 0.0001). The influence of the intervention on Day 3 was less significant (P = 0.007): the biopsy of one cell is less invasive than the biopsy of two cells. PCR diagnostic efficiency was 88.6% in group I and 96.4% in group II (P = 0.008). For the fluorescence in situ hybridization (FISH) PGD cycles no significant difference in efficiency was obtained (98.2 and 97.5% in group I and II, respectively). Similar delivery rates with live birth per started cycle were obtained [58/287 or 20.2% in group I versus 52/303 or 17.2% in group II, P = 0.358; the absolute risk reduction = 3.05%; 95% confidence interval (CI): -3.24, 9.34]. Post-PGD PCR reanalysis showed six false positives in 97 embryos (6.2%) in group II and none in group I (91 embryos reanalysed). No false negatives were found. CONCLUSIONS: While removal of two blastomeres decreases the likelihood of blastocyst formation, compared with removal of one blastomere, Day 3 in vitro developmental stage is a stronger predictor for Day 5 developmental potential than the removal of one or two cells. The biopsy of only one cell significantly lowers the efficiency of a PCR-based diagnosis, whereas the efficiency of the FISH PGD procedure remains similar whether one or two cells are removed. Delivery rates with live birth per started cycle were not significantly different.     

The effects of cryopreservation and thawing on the development in vitro and in vivo of biopsied 8-cell mouse embryos.

The possible impact of cryopreservation on biopsied 8-cell mouse embryos was investigated. Biopsied and control 8-cell embryos were cryopreserved using a slow freezing and quick thawing protocol with 1,2-propanediol as a cryoprotectant. The cryopreservation process did not affect either the recovery or the survival of biopsied embryos, when compared with intact controls; however, sham controls survived significantly better than biopsied 8-cell embryos (88.6 versus 74.2%, P less than 0.001). When fully and partially intact surviving embryos were cultured in vitro to the blastocyst stage, there was no difference in the proportions of embryos which formed blastocysts (biopsy 97.2%, intact control 98.4% and sham control 93.7%). The developmental potential and fetal development in vivo following embryo transfer were not impaired when assessed on day 17 of pregnancy. Cryopreservation of biopsied 8-cell mouse embryos is therefore a feasible approach to storing embryos while analysis of the biopsied material is carried out.     

植入前遗传学诊断的安全性和可靠性探讨

植入前遗传学诊断(preimplantation genetic diagnosis,PGD)是在胚胎植入前对配子或体外受精胚胎进行遗传学分析的一项诊断技术,目的在于从源头预防遗传性疾病的发生,从而改善有遗传性疾病高风险夫妇的妊娠结局.近年来,随着分子生物学技术的进步,越来越多遗传性疾病的发生机制被阐明,PGD的诊断范围逐渐扩展,其应用周期数也日益增多.新技术的开展如比较基因组杂交及其微阵列提高了PGD诊断的准确性.然而,这项技术的安全性问题也引起了人们高度重视.作者从透明带开孔技术、不同时期胚胎活检、胚胎活检细胞数目以及遗传分析技术的可靠性等方面对PGD的安全性进行了探讨.

Abstract：

Preimplantation genetic diagnosis (PGD) refers to a procedure to genetically analyze embryos prior to implantation, in order to prevent the occurrence of specific inherited disorders before conception and improve the outcome of high-risk pregnancy with genetic disorders. In recent years, with the advance of molecular biology techniques, more and more genetic diseases have been elucidated, and PGD has been gradually expanding its scope and applications. New technologies, such as microarray comparative genomic hybridization (array CGH), are developed to improve the accuracy of diagnosis. However, the safety of this procedure has aroused great attention. In this article, authors will review the safety of zona opening procedures, different biopsy procedures at different stages, and removal of one or two cells from cleavage-stage embryos. The reliability of genetic analysis technologies will be discussed as well.     

Preimplantation diagnosis: Blood analysis of mice born following single-cell embryo biopsy

Single-cell embryo biopsy is an important technique in preimplantationdiagnosis. The development of the mouse embryo and fetus, andthe results of some analyses after birth following embryo biopsy,have been demonstrated to be normal. Histopathological analysisof mice born following single-cell embryo biopsy with a physicalmethod (zona puncture) also showed normal organ and cell structure,thus demonstrating the safety of embryo biopsy. This experimentanalysed the parameters of blood cells and the blood chemistryof 28 mice born following single-cell embryo biopsy. White cellcount, red cell count, haemoglobin level and platelet countof the blood, and plasma sodium, potassium, chloride, bicarbonate,calcium, glucose, albumin, protein, creatinine, urea, bilirubin,aspartate transaminase, creatine kinase and lactate dehydrogenaseof the blood were not significantly different between the micefrom the biopsied and the control embryo groups, which againdemonstrated the safety of single-cell embryo biopsy. The remainingtotipotent cells in the biopsied embryos would thus be expectedto develop to the correct cell counts and normal organic functionsaccording to the intact hereditary messages. Further studieson the safety of embryo biopsy (including long-term observationafter birth) and the improvement of the different biopsy techniquesand skills for preimplantation diagnosis are necessary.     

Decompaction and biopsy of late mouse morulae: assessment of in-vitro and in-vivo developmental potential.

The present study reports a biopsy technique on decompacted late mouse morulae. Zygotes were collected from hyperstimulated F1 hybrids (C57BL6j females x CBAca males) and cultured in modified Earle's balanced salt solution supplemented with 0.5% bovine serum albumin until the late morula stage (92 h post administration of human chorionic gonadotrophin). Decompaction was obtained by exposure of the embryos to Ca(2+)-Mg(2+)-free phosphate-buffered saline (PBS) or an aqueous solution of ethylene-diaminetetraacetic acid (EDTA) and glycine. Using micromanipulation, a single blastomere was aspirated without removal or softening of the zona. The impact of the different decompaction procedures and the biopsy technique was studied by vital staining, by in-vitro culture up to the early egg-cylinder staged and by the recovery of living mice after transfer to pseudopregnant foster mothers. Our investigations revealed no impact of decompaction and biopsy on immediate viability, as assessed by fluorescein diacetate staining. A significant reduction (P less than 0.05) in the number of mouse morulae that reached the early egg-cylinder stage in vitro was observed after the biopsy procedure. We observed that after this microbiopsy technique, successful pregnancies can be obtained but at a lower percentage compared to controls (P less than 0.01).     

Precise sex selected births of mice following single cell embryo biopsy and Y-linked testis-specific gene analysis

We report on the birth of 39 mice following single cell embryobiopsy and precise sex determination following in-vitro fertilization.Polymerase chain reaction was used to amplify fragments of themouse testis-specific gene sequence (pYMT2/B) on the Y chromosomeand the ovary-specific gene (ZP₃) sequence on chromosome 5 fromthe single biopsied cell. Embryo biopsy was not associated withany deleterious effects.     

Preimplantation diagnosis: Histopathological analysis of mice born following single cell embryo biopsy

Single cell embryo biopsy is a useful but invasive techniquefor preimplantation diagnosis. Biopsy may be performed by physical(direct zona puncture) or chemical methods (zona drilling withacid solution). This study has analysed the safety of a physicalmethod of embryo biopsy in the mouse. Six adult mice (male andfemale), three from biopsied embryos and three from a controlgroup (non-biopsied) were subjected to histopathological analyses.Macroscopically, the anatomy and morphology of the internalorgans in both groups were normal. Microscopic analyses of 15major organs, which included the brain, heart, lung, liver,kidney, stomach, intestine, voluntary muscle, spleen, pancreas,adrenal, thymus, skin, testis (male) and ovary (female), inboth groups were all normal. These results showed that carefulsingle cell embryo biopsy by direct zona puncture performedat the 8-cell embryo stage had no adverse influence on the macroscopicand microscopic structure of the organs. The remaining pluripotentialcells of biopsied embryos developed normal microstructure accordingto the hereditary messages. Ideally, the safety of embryo biopsyrequires observation of three stages after embryo biopsy, namelyembryonic and fetal development before birth, neonatal assessmentand long-term monitoring after birth.     

Proliferation of blastomeres from biopsied cleavage stage human embryos in vitro: an alternative to blastocyst biopsy for preimplantation diagnosis

Normally fertilized human embryos were biopsied at cleavagestages on the third day after in-vitro fertilization (IVF).One or two blastomeres at the 8-cell stage were removed andco-cultured with the biopsied embryos. Embryos and blastomereswere assessed daily for morphological development until day6, when the number of cells were counted by labelling the nuclei.In all, 53% of the biopsied embryos (25 out of 47) reached theblastocyst stage between day 5 and 6 and the proportion wasthe same irrespective of the number of cells removed. Therewas no significant difference between biopsied embryos fromwhich one or two blastomeres respectively had been removed withregard to total cell numbers at the blastocyst stage (56.2 ±3.0 and 64.7 ± 5.5), number of trophectoderm (45.4 ±3.5 and 44.0 ± 5.7) and inner cell mass cells (14.0 ±1.2 and 16.6 ± 1.8). Overall, 72% of the isolated blastomeresdivided at least once over 3 days in culture and 50% dividedmore than once. The mean overall cell number after 3 days inculture was 3.7 ± 0.48 per blastomere (range 1–8cells) if one cell was removed and 6.9 ± 1.0 if two cellswere removed. If the undivided blastomeres are excluded, themean cell number was 4.8 ± 0.51 and 8.3 ± 1.0respectively. Over this period, 55% of the blastomeres cavitated.Of the blastomeres taken from embryos that developed to theblastocyst stage, 92% divided and 76% cavitated. In those fromarrested embryos, 50% divided (P < 0.002) and 32% cavitated(P < 0.003). From the first group 8% of blastomeres and fromthe second group 41% of blastomeres neither divided nor cavitated(P < 0.008). We conclude that as a more consistent alternativeto blastocyst biopsy, cryopreserving biopsied cleavage stageembryos and culturing blastomeres would increase the numberof cells available for genetic analysis. This could facilitatepreimplantation diagnosis of inherited disease by improvingreliability and possibly allowing combined detection of chromosomaland single cell defects. Further studies will investigate thegenetics of proliferated blastomeres.     

Impact of blastomere biopsy and cryopreservation techniques on human embryo viability

The purpose of the present study was to evaluate the effect of cryopreservation on 55 embryos which had one blastomere biopsied for preimplantation genetic diagnosis of aneuploidy before freezing. The thawing outcome was compared to that obtained in 94 embryos which derived from our conventional freezing programme in patients with comparable characteristics who were treated in the same period. Their embryos were morphologically similar but the incidence of aneuploidy was 100% in the biopsy group and unknown in the controls. The percentage of embryos which survived intact after thawing was significantly lower in the biopsied group compared to the controls (9 versus 25% respectively; P < 0.025), whereas the rate of lysis was superior among biopsied embryos (34 versus 13% in the controls; P < 0.001). Similarly, the survival index was higher in the frozen-intact embryos than in the embryos which were frozen after biopsy (61 versus 38%; P < 0.001). No empty zonae resulted in the control group, while six were found after thawing biopsied embryos. In the second part of the study, blastomere biopsy was implemented on 102 thawed embryos generated by 16 patients. The chromosomal analyses revealed that 49 were normal, leading to the transfer of 2.5 +/- 0.8 embryos per patient. Only three clinical pregnancies were obtained, and are presently ongoing. In conclusion, the present findings discourage the use of conventional cryopreservation protocols in strategies involving preimplantation genetic diagnosis in human reproductive medicine. Adequate protocols are required for freezing and thawing embryos which have been subjected to biopsy procedures.