Analysis Of Chromosome In Embryos With Multinucleation Or Vacuoles

Background and ObjectiveSince the world’s first IVF-ET (in vitro fertilization-embryo transfer IVF-ET) baby was born in1978, IVF-ET pregnancy rate and live birth rate were greatly improved with the development and maturity of technology.In pursuit of the ideal pregnancy rate, most domestic reproductive medical centers tend to increase the number of embryo transfer, so that significantly increased the multiple pregnancy rate, reachinged25to50percent, but directly harmed the health of mothers and children. ASRM suggested that women have an adequate response on the ovarian hyperstimulation transferred no more than two embryos once in order to induce the multiple pregnancy rates. Therefore, selecting embryos with high developmen potential and reducing the number of embryo transfer become a important proposition for reproductive workers with long-term efforts.Currently, the quality assessment tools of embryo in the pre-implantation including morphology score, metabolite analysis, preimplantation genetic diagnosis (preimplantation genetic diagnosis, PGD). Embryonic metabolite determination is still in the experimental stage, while morphology score is the most convenient and commonly used assessment tools.According to the number of cells, the degree of fragmentation and the uniformity of blastomeres on early embryo, laboratory workers can choose the high scores embryo to transplant. The number of cells reflects the development speed and vitality of the embryos. The amount and distribution of debris impact the potential of embryo implantation and the ability of developed to the blastocyst. But the Morphological assessment methods limits obviously, and can not avoid the transfer of genetic diseases, the embryo implantation rate reach only40to50percent, that means most of embryos ported to the womb can not continue to develop. Therefore, conditional center using FISH, PCR, CGH or gene chip technology for genetic analysis of some single gene disorders or chromosomal diseases in the gametes or the preimplantation embryo, and choosing healthy embryos for transplanting. The difficulties of PGD are cell biopsy, cells fixing and single-cell genetic diagnosis.Chromosomal abnormalities are one of the main reasons of low potential of embryonic development, implantation rate decreased, birth defects and perinatal death. Chromosome abnormalities of preimplantation embryo are mainly from the egg meiosis error, especially the first meiotic division, Minority from the sperm and other factors. The majority appeared the abnormal number of chromosomal (such as aneuploidy, haploid, polyploid chimera, etc.), and structural abnormalities is minority. At present, the only studies show the chromosomal abnormality rate of good morphological embryo can reach up to30percent (32.3to44.4percent).In view of the data between Embryo morphology and Chromosomal abnormalities is few, we intends to use fluorescence in situ hybridization (fluorescence in situ hybridization, FISH) to analysis the chromosome of single blastomere in three different forms of the embryos respectively, that are high morphological scores embryos, multinucleated embryos and vacuoles embryos, in order to provide the experimental basis for exploring more efficient and accurate embryo morphology evaluation criteria.Design and Methods1. Selection and grouping of the specimens(1) Embryos with high morphological scores.There were52embryos with high morphological scores obtained from conventional IVF-FET cycles during February2008to January2011in the reproductive medicine research center of Guangzhou general hospital of Guangzhou military command. The blastomeres in embryo remain ratio of<50%and≥2nuclear after thawing recovery. These embryos were divided into two groups according to women age (The group of<35years and the group of>35years).(2) Embryos with multinucleated cellsThere were30embryos obtained from conventional IVF-ET cycles during December2009to October2011in the reproductive medicine research center of Guangzhou general hospital of Guangzhou military command. These embryos were divided into two groups:group A (Embryos with multinucleated cells detected on day2and not multinucleated on day3) and group B (Embryos with multinucleation observed on days2presented two nuclei).(3) Embryos with vacuolesA total of36scrap embryos were collected and divided into three groups according to the appearance and diameter of vacuoles at Day1:group A with No vacuoles, group B with vacuoles<14um, group C with vacuoles>14um.2. Blastomeres detection by FISHA single blastomeres extracted, low permeability fixed, tablets baked, Chromosome abnormalities were detected using fluorescent in-situ hybridization (FISH) with X, Y,18and13/21chromosome specific probes, and the fluorescence signals are counted. 3. Statistical analysisThe comparion of two-sample rate was performed by x2, the chi-square test was used when n≥40and all T≥5; the continuous calibration chi-square test was used when n≥40and1<T<5; the R×C table chi-square test was used between the rate of multiple samples, and the Fisher’s exact test calculate the probability directly when n<40or1<T<5. Statistical software is SPSS17.0.Results and Discussion1.Analysis of chromosome in high morphological score embryos:There are16embryos with chromosomally abnormal in113florescence Nucleus and the abnormal rate is30.77%(16/52). The proportion of chromosome abnormalities in the group of<35years was26.47%, the group of>35years was38.89%. No significant difference between the two groups (P>0.05). The abnormal ratio is lower than the results report, showing that embryo morphology evaluation criteria of Cleavage stage stage D3is Reasonable.2. Analysis of chromosome in multinucleated cells embryos:14of19embryos are chromosomally abnormal in group A and the abnormal rate is73.68%;15of21embryos are chromosomally abnormal in group B and the abnormal rate is71.43%. Both of the two rates have significantly difference compared with30.77%(P<0.05). The results indicate that chromosomal abnormalities were significantly increased as long as the D2appeared multinucleated characteristics, no matter what type of D3and whether multinucleation. The two nuclear phenomena in D2may not normal in mitosis. Therefore, multinucleation is recommended in morphological evaluation syetem, and such embryos are not suggested to transfer.3. Analysis of chromosome in vacuoles embryos:13of36embryos are chromosomally abnormal and the abnormal rate is36.11%. The rate is not significantly different compared with30.77%(P>0.05).4of17embryos are abnormal in group A and the abnormal rate is23.53%;5of13embryos are abnormal in group B and the abnormal rate is38.46%;4of6embryos are abnormal in group C and the abnormal rate is66.67%. The Chromosomal abnormality rate between them have not significantly difference (P>0.05). For type B vacuoles (vacuoles can be seen in one blastomere or few small vacuoles can be seen in sevreal blastomeres), we can still choose to transplant when the other morphological indicators are good. But type C vacuoles (large vacuoles in more blastomeres) are not recommended to transplant.ConclusionIt is limited for embryo morphology assessment in cleavage stage and can not pick up chromosomally normal embryos completely. However, rational morphological indicators can do help to reduce the abnormal rate of chosen embryos. There are high abnormal rate in multinucleated embryos such as D2multinucleated and D3no multinucleation, or D2nuclear number is2, which are not fit to transplant. We can choose to transplant vacuoles embryos such as type B. In short, the important index of multinucleation and vacuolization are recommended to morphological evaluation syetem.