The Utility Of Mate Pair Library Sequencing For Reciprocal Translocation PGD

BackgroundBalanced chromosome translocation is the rearrangement of chromosome structure. It is about 1/625 individuals carries a balanced chromosome translocation, they always have normal phenotype, but they are at increased risk for recurrent abortion, fetal death, stillbirth, offspring with congenital abnormalities and mental retardation. Among the balanced translocation, reciprocal translocation and Robertsonian translocation are the main causes of recurrent abortion. In theoretical, the chance of producing normal gametes for the reciprocal tanslocation carrier is 1/18, and 1/6 for Robertsonian translocation carrier, so the translocation carriers are always suggested to receive the prenatal diagnosis. These couples, the wife or the husband either of them is the translocation carrier, and they are ready to undergo the assisted reproductive technology, they should choose PGD. The methods of PGD for translocation carriers include fluorescence in situ hybridization polymerase chain reaction-array comparative genomic hybridization and single nucleotide polymorphism. Most of Chinese IVF centers take aCGH and SNP array as main techniques. Next generation sequencing is the basis of copy number variation sequencing. Now CNV-seq has shown its advantages because it is high throughput、 high sensitivity and lowcost. It is difficult to distinguish the normal embryos and balanced embryos for translocation carrier through PGD, normal and balanced embryos have the same chance to be transferred. If the balanced embryos are transferred, and the woman get pregnant, the offspring will be another translocation carrier, face the same problems as his or her parents. So this study performed the PGD for tanslocation carriers with CNV-seq, validated the efficiency of this technique, combined mate pair library sequencing with PCR to differentiate the normal and balanced embryos, set up a new effective method for translocation PGD.Objective1.Validate the efficiency of CNV-Seq for translocation PGD;2. Establish and optimize the procedure of differentiate normal and balanced embryos with mate pair library sequencing and PCR;3. Test the accuracy of mate pair library sequencing in differentiating normal and balanced embryos;4. Improve the pregnant rate of translocation carriers through transferring the normal embryos which have been detected.Methods1. The women who were included in the study receiving the controlled ovarian hyperstimulation, oocytes retrivaling was performed when the follicles matured monitoring by the ultrasound. The husband retrieved sperm, ICSI, continue to incubate after fertilization, blastomeres or blastocysts were biopsied;2. The cell or cells were undergoing WGA, screening the diplody with normal copy numbers;3. Diploid embryo was transferred, calculated the implantation rate, ongoing pregnancy rate and live birth rate;4. When the patients enrolled the mate pair experiment, drawed blood from translocation carrier, sequencing and established mate pair library, found the breakpoint;5. Designed and tested the primer according to the breakpoint, detected if the diploid embryo had the same breakpoint as translocation carrier with PCR, if it had,it was balanced;6. Transferred the normal embryo, after 18-20weeks, got the amniotic fluid, performed chromosome karyotyping;7. Compared the PCR result with chromosome karyotyping, evaluated the diagnose efficiency of mate pair library sequencing.Results1.113 embryos were biopsied of 21 couples,98 (86.7%) embryos detected by CNV Seq; found out the diploid embryos, even detect out 0.8Mb copy number variance, in addition,20% mosaic was found;2. All embryos of 8 couples (38.1%) were abnormal, no embryos were available for transfer,13 couples had at least one diploid embryo,13 couples underwent frozen thawed embryo transfer, the implantation rate was 78.6%, the ongoing pregnancy rate was 70%, the live birth rate was 60%;3. Combined mate pair library sequencing with PCR to differentiate normal and balanced embryos, optimized the procedure, transferred 2 normal embryos and 2 balanced embryo for 3couples, ongoing pregnancy rate was 100%;4. Chromosome karyotyping was performed use the amniotic fluid, the results were as same as the results of PCR, turn out that the mate pair library sequencing was accuracy.Conclusions1. The CNV-seq which was at the basic of NGS, was available for translocation PGD, because of high sensitivity, get a higher implantation rate、ongoing pregnancy rate and live birth rate;2. Verified that translocation carriers were at high risk of producing aneuploid embryos;3. CNV-seq was high resolution, except for detecting the micro deletion and duplication, even can be used to find out mosaic;4. CNV-seq、mate pair library sequencing and PCR can be used to differentiate normal and balanced embryos for translocation PGD, and the methods were accurate.