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Chromosomal aneuploid is very common in IVF-ET, which is defined by that the number of chromosomes is more,or less than 2.Trisomy is the most common type of aneuploid,especially in embryos derived from women with advanced maternal age. The formation mechanism of aneuploid is an important focus in medical genetic research field. It is considered that aneuploid is often caused by chromosomal nondisjunction during cell division stage, chromatid predisjunction during meiotic division stage, and loss of chromosome. The disequilibrium of genes located in aneuploidy chromosomes result in sponeous abortion, embryonic death, which account for nidation and pregnancy failure, abortion, embryo development block, malformation. So it is important to screen aneuploid of embryos in order to estimate utility value for transfer.For the moment during IVF-ET,the number of pronucleus is observed and scored routinely 16～18h after fertilization. Embryos choosed to transfer are usually from 2PN, which are considered as normal fertilization, while embryos are considered as abnormal fertilization without pronucleus,sometime with one or more than 2 pronucleuses, and those embryos are often discarded.Clinical pregnant rate is around 30%～40%, and it seems lower. It has been convinced that ,the lower rate is correlated with chromosomal aneuploid.Many people considered that, most of embyos from abnormal fertilization are aneuploidy and they are not suitable for transfer.However,it is shown that part of abnormally fertilized embryos, especially 1PN embryos, are convinced to be normally fertilized with disomy in some studies. Some scholars advised that these embryos should be choosed to transfer when there is no 2PN embryo. It was reported that a healthy child was bom after transferring embryo form 1PN. In most reproductive medicine center, embryos from 2PN are often choosed for transfer.When there is none of 2PN embryos, whether to choose abnormally fertilized embryos from 0PN, 1PN for transfer is controvertial. To observe the pronuclear number of embryos of IVF can’t show a genuine aneuploid outcome and it affects the evaluation of value for ET .Many scholars analyzed monosomy and trisomy of chromosome 13,18,21,X,Y of embryos by FISH.It is focused on aneuploid of embryos of women with advanced maternal age and repeated miscarriage ,factors on the formation and aneuploid of polyspermous fertilization. Several domestic scholars studied on aneuploid of embryos from 1PN, but there is no affirmative conclusion on whether to select these embryos to transefer .The aneuploid and value for transfer from embryos derived from 0PN ,and the factors such as fertilization ways,ages,oocytes counts,clinical protocols ,which lead to high risk of abnormality are rarely reported in domestic research field. In this research, chromosomal aneuploid is analyzed by FISH,which aims at providing theoritical evidence for abnormally fertilized embryos tansefer from 0PN,1PN in clinical practice,when there is no normal fertilized embryo for ET.ObjectivesTo explore factors on formation of aneuploid and value for embryo transfer by analyzing aneuploid of chromosome 21 of embryos derived from IVF-ET cycles by FISH, and to provide theoritical evidence for abnormally fertilized embryos transfer from 0PN,1PN in clinical practice,when there is no 2PN normally fertilized embryo.MethodsAbandoned embryos for research were recruited from patients who were performed with IVF/ICSI treatment in Reproductive Medical Center in the First Affiliated Hospital of Zhengzhou University from Apr 2006 to Jan 2007. The patient gave informed consent for FISH analysis on their abanded embryos.AH embryos were collected and divided into 0PN,1PN,2PN,≥3PN groups by number of pronucleus; <30,30-34,≥35groups by ages; <17,≥17 groups by average of oocyte counts; IUI+IVF protocol, GnRH-a short protocol, GnRH-a long protocol groups by clinical protocols.To use aspirate-pull method to biopsy embryos whose zone drilled by a non-contact diode laser system (OCTAX-Laser shot).To use Tween-20/HCl+ Methanol/ glacial acetic acid methods to fix blastomeres. To observe adequate signals after hybridization with probe LSI21.To compare aneuploid rate and differences of each group.All data were dealed with SPSS13.0 statistical package by t-test and x~2 test.α=0.05 is considered as test criterion for statistical analyse.Results1. Bioposy outcomes 292 embryos were bioposied and 289 blastomeres were recruited,of which 277 blastomeres were integrated with clear nucleuses, successful bioposy rate was 95.8%.2. Fix and hybridyzation outcomes250 of 277 blatomeres wer successfully fixed, and fluorescence signals can be deteced in 235 blatomeres.The fixing rate was 90%,while deteced signals rate was 94.0%. 3.FISH outcomes of blastomeres3.1 Choromosomal aneuploid outcomes of IVF cyclesIn IVF cycles, disomy rate of 0PN group and 1PN group were 53.3%,58.1% respectively, no significant statistical difference was observed (P>0.05) ,however both were less than 73.1% of 2PN group(P<0.05).Chromosomal multisomy of≥3PN group was 81.9%, which were significantly more than 0PN group, 1PN group,2PN group with 20.0%, 19.4%, 11.5% (P<0.05). Chromosomal disomy of≥3PN group was 9.1%, which were significantly less than 0PN group,1PN group,2PN group with 53.3%,58.1%,73.1% (P<0.05) .Embryonic chromosomal abnormality rate of 0PN and 1PN groups were 46.7%,41.9%,which was more than 26.9% Of 2PN group,while less than 36.9% of≥3PN group (P<0.05) .3.2 Choromosomal aneuploid outcomes of ICSI cyclesIn ICSI cycles, disomy rate of 0PN group and 1PN group were 45.2%%,31.0% respectively, no significant statistical difference was observed (P>0.05) ,however disomy rate of 2PN group was 70.9%, which was significantly more than other groups (P < 0.05 ) . In≥3PN group, chromosomal disomy rate was 9.1%, which was significantly less than 2PN group (P<0.05 ) ,multisomy rate was 67.8%, which was significantly more than other groups (P<0.05) .Embryonic chromosomal abnormality rate of 0PN and 1PN groups were 55.8%,69.0%,which was more than 29.1% Of 2PN group (P<0.05) .3.3 Fertilization methods and embryonic choromosomal abnormalityIn IVF group, choromosomal abnormality rate of 1PN group was 41.9%, significantly less than that of ICSI group with 69.0%. Choromosomal abnormality rate of≥3PN group was 90.9%, which was significantly more than that of ICSI group with 71.0% (P<0.05) .compared with ICSI group with54.8%,50.0%,20.8%,80.0%, no significant difference was found (P>0.05) . No significant statistical difference was observed between 0PN group and 2PN group (P>0.05) .3.4 Ages and embryonic choromosomal abnormalityCompared with <30 group,with choromosomal abnormality rate of 27.9%, 15.4%, 8.3%,37.5%, that of 0PN,1PN,2PN,≥3PN embryos in≥35 group were 66.7%, 64.5%,47.1%,90.3% respectively, which were significantly higher (P<0.05) ; that of 0PN,1PN,≥3PN embryos in 30～34 group were 59.0%,86.8%,84.0%, which were significantly higher, too (P<0.05) .But that of 2PN was 23.8% and no significant statistical difference was observed (P>0.05) . No significant statistical difference was observed between≥35 group and 30～34 group on choromosomal abnormality rate (P>0.05) .3.5 Oocytes counts and embryonic choromosomal abnormalityOocytes counts≥17group, choromosomal abnormality rate of 0PN,1PN,2PN,≥3PN groups were 55.9%,63.3%,20.7%,80.0%,compared with <17group with 48.1%, 45.2%, 38.1%, 82.4%, no significant statistical difference was observed (P>0.05) .3.6 Different clinical protocols and embryonic choromosomal abnormalityIn IUI+ IVF group, choromosomal abnormality rate of 0PN,1PN,2PN,≥3PN groups were 23.1%,25.0%,7.1%,35.7%, which were significantly less than that of GnRH-a short protocol group with 70.8%、75.0%、31.3%、96.2% (P<0.05) ;Among IUI+IVF group, choromosomal abnormality rate of 1PN,2PN,≥3PN groups were significantly less than that of GnRH-a long protocol group with 58.3%、40.0%、91.7% (P<0.05) , but no significant statistical difference was observed between IUI+ IVF group and GnRH-long protocol group in choromosomal abnormality rate of 0PN embryo (P>0.05) . No significant statistical difference was observed between GnRH-a short protocol group and GnRH-long protocol group on choromosomal abnormality rate (P>0.05) .Conclusion1. The disomy rate of embryos from 0PN and 1PN derived from IVF cycles are 45.2%～58.1%, which indicates normal fertilization. These embryos are avalible and suitable for transfer when there is no 2PN embryos. PGD and PND are necessary to screen chromosomal abnormalities. 1PN embryos derived from ICSI cycles are not suitable to transfer for its higher mulitisomy rate of 69.0%.2. The disomy rate of embryos from 2PN is 70.9%～73.1%.These embryos are the best choice to transfer.Choromosomal abnormality rate of 2PN embryos is 8.3%～11.5%, which is probable one of key factors on lower clinical pregnant rate.3.Embryos derived from≥3PN are not suitable for ET,because of higher mulitisomy rate of 67.8%～81.9%. 4.Advanced maternal age,especially >35; higer dose of Gonodotrophin in COH protocols may result in higer abnormality rate.5. In IVF cycles,choromosomal abnormality rate of 1PN embryo is more than that of ICSI cycles,while abnormality rate of≥3PN embryo is less.All aboved indicates that fertilization methods may lead to formation of chromosomal abnormality.But number of oocytes retrieved may have no effect on embryonic chromosomal abnormality.
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