Management of Abnormally Fertilized Zygotes? InVitro Correction of 3PN

In this newly developed protocol, and idea, is to manage those abnormally developed zygotes from different ART procedures. The investigators developed the plan and requirements needed to select the target extra nucleus or pronuclei to be extruded from fertilized egg in order to maintain developing healthy normal embryo.

As approved by Dyban and Baranov et al, about 15-18% of abortions caused by triploidy fertilization. One of sources for maternally triploidy is failure in the first meiotic division (Jacobs et al., 1978). One of Digynic triploidy is developed by fertilized giant oocyte (Dyban and Baranov, 1987) {nuclear but no cytoplasmic division in an oogonium or cytoplasmic fusion of two oogonia (Austin, 1960)}. Giant oocyte characterized with bigger diameter and will distinguished polar bodies at metaphase II. B. Rosenbusch et. al. 2002, cytogenetic study showed that extra haploid maternal copy associated with MII (46,XX/ 2N ) giant oocytes as well as triploidy with fertilized giant oocytes (3N with 69,XXX or 69,XXY). First Mitotic division plane with polar axes studies by Scott, 2001 , shows that Pn developed closer to 2nd polar body is the maternal origin PN. Giant oocytes were collected from different IVF cycles, to be injected with normal sperm using Intracytoplasmic sperm injection (ICSI). 18 hours post ICSI arranged for fertilization evaluation and PN removal for fertilized oocyte before syngamy starts. Video attached shows process of zygote manipulation by the way avoiding the division axis and focusing the extra maternal PN to be aspirated. Pronuclear transfer in human embryos for mitochondrial DNA correction started the methodology of pronuclear manipulation, for that possibility of utilizing of 3PNs developed embryos research tools can be started. We arranged to study available received giant oocytes during IVF cycles. Accordingly we arranged for pronuclear removal followed by FISH evaluation in order to targeting Normal males embryos that insure proper extra maternal pronucleus removal. Successful trials of maternal PN removal for giant oocyte collected from different cases summarized in table 1. All blastocyst developed arranged for FISH, so all embryos were utilized for cytogenetic evaluation. Recommendations: Further evaluations using STRs (Short tandem repeat ) should be used for maternal-paternal genome differentiation. NGS study is under evaluation for developed embryos for full CCS reporting and more genetic integrity. Epigenetic evaluation study recommended for triploidy corrected embryos for genetic expressions and early embryo developments as well as differentiation between paternal and maternal genomic activity.

Abnormal zygote fertilization, 3PNs correction, Giant oocyte management, triploidy fertilization, Maternal nucleus removal

abnormally produced oocyte after ART or Controlled ovarian stimulation. Correction of abnormally fertilized oocytes using nucleus removal.

Abnormally developed embryos these produced 3PNs. Correction of abnormally fertilized oocytes using nucleus removal.

At 18 hours after ICSi nucleus to be removed in-order to make genetic correction for abnormally developed embryos. Final result is normal embryos produced with 2PNs evaluated by FSIH study for 5 chromosomes. This indicated the genetic correction process was successful process, and applicable to produce normal growing embryos.

Availability of successful nucleus removal from zygote to be developed to active dividing embryo available for cytogenetic study. That indicated normal embryogenesis processing

chromosomes 13, 18, 21, X and Y to be screened using fluorescence insitu hybridization (FISH). In order to check primary euploid developed with male (XY) or female (XX) embryos.

availability of embryos to develop for day 5 giving chance for better cells evaluation and more genetic and cytogenetic studies.

Inclusion Criteria: Giant oocytes 3 PNs developed embryos at day 1 post ICSI. Exclusion Criteria: Patient refused to involving their abnormal oocytes at our study.

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