| Purpose: By using MALBAC for whole-genome amplificantion,we performed NGS method on culture medium used to culture human blastocysts and obtained the ploidy information of all chromosomes.Then we validated these results by comparing each with their corresponding whole donated embryo,so as to validated the sensitivity and specificity of noninvasive chromosome screening method.Method: The fertilized D2/D3 embryos achieved via ICSI had been donated for research voluntarily and signed a consent form.The embryos were placed into G-2PLUS droplets which were changed every 24 hours.We collected the culture media on D5/D6 and whole embryos after laser drilling.Then we performed MALBAC and NGS method on the samples to obtain the ploidy and more than 50 Mb microdeletion and microduplication information of all chromosomes,as well as the copy number of mitochondrial DNA.Results:1.Material source: 26 blastocysts and culture medium were included.15 blastocysts were fresh and others were thawed.8 were collected on Day 5 and 18 on Day 6.2.Successful rate: All blastocysts were sequenced successfully,the successful rate was 100 %(26/26).3 culture media samples failed,with the successful rate of 88.5 %(23/26).22 blastocysts were laser-drilled and the culture medium was sequenced successfully,with a successful rate of 100 %.3.The chromosome information of blastocysts:(1)The normal rate of 26 blastocysts was 57.7 %(15/26)and mosaic rate was 26.9 %(7/26).(2)The normal and mosaic rate of D5 blastocysts were 75.0 %(6/8)and 12.5 %(1/8),which were 50.0 %(9/18)and 33.3 %(6/18)in D6 blastocysts.(3)The normal and mosaic rate of 11 thawed blastocysts were 45.5 %(5/11)and36.4%(4/11),which were 66.7 %(10/15)and 20.0 %(3/15)in fresh blastocysts.4.Comparison of NICS and blastocyst:(1)The mosaic rate of NICS was 39.1 %(9/23)and the 9 samples were not included in the following comparative analysis.(2)Of the 11 normal or less variable blastocysts,8 were normal in NICS and 3 false positive results.The specificity and false positive rate were 72.7 %(8/11)and 27.3 %(3/11).(3)Of the 3 abnormal blastocysts,2 were abnormal in NICS and 1 false negative result.The sensitivity and false negative rate were 66.7 %(2/3)and 33.3 %(1/3).(4)The positive and negative predictive value were 40.0 %(2/5)and 88.9 %(8/9).5.Mitochondrial DNA:(1)The mitochondrial DNA copy number of all samples were sequenced successfully,the successful rate was 100 %.(2)There was no significant correlation between the copy number of mitochondrial DNA and the chromosome euploidy and so on.(3)There was significant correlation between the mitochondrial DNA copy number of embryos and their culture medium.Conclusion:1.Embryo release free DNA into culture medium which can be increased by laser drilling.2.The free DNA in culture medium can be detected by WGA and NGS method to predict embryo chromosome information and provide a noninvasive preimplantation genetic screening method.3.NICS can avoid invasiveness and reduce the safety problems caused by PGS.Its sensitivity and specificity still needs further experimental verification.4.Embryo release mitochondrial DNA into culture medium which was positively correlated with the copy number of embryo mitochondrial DNA.5.The blastocysts are also with high proportion of chromosome abnormalities. |
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