DESIGN:We have developed a sequencing-based PGSmethod that allowsfor the detection of gross chromosomal abnormalities.

MATERIALS AND METHODS: The sequencing library preparation isbased on a novel transposase-based tagging protocol. The sequencing li-braries were sequenced with multiplexed, 36 cycle single read sequencingruns on an Illumina� MiSeq. Up to 12 samples were combined persequencing run. After alignment, only perfectly and uniquely aligning readswere kept for tag counting. The reference genome was divided into variablesized bins depending on genomic context and the number of reads that map toeach bin was counted. The read count per bin is subsequently correlated withcopy number. Turnaround time from cell biopsy to analyzed data was lessthan 12 hours.

RESULTS: In order to determine the limit of resolution, single cell, twocell and three cell aliquots of tissue culture cell lines with known aberrationswere assayed. 12Mb events were readily detectable even at the single cellinput level. The protocol was subsequently applied to single cells of cleav-age-stage embryos that had previously been characterized by FISH. Mono-somies of chromosome 13 and 14 were confirmed in one embryo andtrisomy of chromosome 13 was confirmed in another. Additional aberrationswere also detected that were previously unknown due to the non-comprehen-sive nature of FISH.

CONCLUSION: PGS by MPS is a rapid, comprehensive screeningmethod that allows for the detection of subchromosomal abnormalities in sin-gle cell biopsies.

Supported by: The authors (FK, TR, and BK) are employees of Illumina,Inc.

P-173 Tuesday, October 15, 2013

DEVELOPMENT AND VALIDATION OF A PACGENOMICS-AGI-LENT MICROARRAY SYSTEM FOR PREIMPLANTATION GE-NETIC SCREENING (PGS) IN PATIENTS UNDERGOING INVITRO FERTILIZATION (IVF). M. Li, H. Jin, R. Snyder, J. Zhou,L. Liu. PacGenomics, Thousand Oaks, CA.

OBJECTIVE: Agilent CGH array is the most commonly used platform forpostnatal CNV. However, it has not been used for aneuploidy PGS in IVFbecause of less reliability in gender confirmation due to sample-on-control(of one gender) co-hybridization and data analysis$ costly duel-color hybrid-ization$ time-consuming multiple manual data processing steps, which leadto potential human errors The aim of the study was to develop and validate anAgilent PGS pipeline to overcome such weaknesses.

DESIGN: Given the limitations of Agilent’s cohybridization and data anal-ysis methods, PacGenomics has developed 1) a novel hybridization method,which utilizes sample-on-sample and control-on-control co-hybridizationwith control DNA of both genders. 2) In-house developed data analysis soft-ware, which generates 2 log2 ratios for each sample relative to control DNAof both genders. 3) a fully automated scanning-data analysis-log2 ratio re-porting pipeline.Results were compared with NimbleGen, BlueGnome andCoriell cell lines.

MATERIALS AND METHODS: We used 42 embryonic samples in thestudy. Of those, we validated 15 (13 aneuploid, 2 euploid) samples with Blue-Gnome, 27 (10 aneuploid, 17 euploid) samples with NimbleGen. We alsovalidated 8 Coriell cell lines with known karyotype (1 aneuploid, 7 euploid).PicoPlex (Rubicon) was used for single cell amplification. Agilent 60k, Blue-Gnome 24sure BAC and NimbleGen 270k arrays were used. All labeling fol-lowed manufacturers’ protocols.

RESULTS: The ploidy detection results derived from the PacGenomics-Agilent pipeline had a 100% concordance with BlueGnome, NimbleGen re-sults, and Coriell cell line reports respectively (100% accuracy, sensitivityand specificity). P-values for aneuploidy calls were <0.05. The turn-aroundtime is 13 hours, which is the same as BlueGnome. The PacGenomics’ Agi-lent PGS pipeline allows us to

$ increase reliability of gender confirmation$ reduce costs$ minimize human errors.CONCLUSION: PacGenomics’s Agilent aneuploidy PGS platform is an

fast and accurate tool for PGS.

P-174 Tuesday, October 15, 2013

PREIMPLANTATION GENETIC SCREENING (PGS) ON DAY-5BLASTOCYSTS WITH A DAY-6 EMBRYO TRANSFER RESULTSIN A CLINICAL PREGNANCY RATE OF 71%. K. J. Tobler,a

R. Kaufmann,b R. Ross,b R. P. Dickey,c A. T. Benner,d W. G. Kearns.a,d

S198 ASRM Abstracts

aDepartment of Gynecology and Obstetrics, Division of Reproductive Endo-crinology and Infertility, Johns Hopkins Medical Institutions, Baltimore,MD; bFort Worth Fertility, Fort Worth, TX; cThe Fertility Institute of NewOrleans, Mandeville, LA; dCenter for Preimplantation Genetics, LabCorp,Rockville, MD.

OBJECTIVE: To determine if a day-5 blastocyst biopsy and a fresh day-6embryo transfer following PGS using comparative genomic hybridizationmicroarrays (aCGH) is an alternative to embryo freezing / PGS and a futurefrozen embryo transfer (FET).DESIGN: Retrospective.MATERIALS AND METHODS: 241 embryos were obtained from 59

couples undergoing 59 in vitro fertilization (IVF) and clinically indicatedPGS. The mean maternal age was 35.7 years (28-43). All embryos werebiopsied at the blastocyst stage of development and the trophectodermcell(s) underwent lysis, DNA extraction and amplification using a randompriming protocol. This was followed by 23-chromosome pair aCGH micro-array using the BlueGnome 24sure chip and BlueFuse software analysis.Euploid embryos were transferred the morning of day-6 of embryo devel-opment.RESULTS: A total of 236 molecular karyotypes were obtained from 241

biopsied embryos. Five embryos showed no evidence of DNA amplification.All women had euploid embryos available for transfer. The overall clinicalpregnancy rate per IVF cycle in this cohort undergoing aCGH microarrayPGS was 71% (42/59). Two spontaneous miscarriages occurred: the cytoge-netic tissue analysis of the first one was 46, XY and the second one had nofetal tissue to analyze.CONCLUSION: This preliminary study suggests that PGS using 23-chro-

mosome aCGHmicroarrays on day-5 trophectoderm cells with a day-6 trans-fer is a promising strategy to consider for patients undergoing IVF and PGS.This technology offers IVF/PGS patients an opportunity to undergo a freshembryo transfer and reduce the requirements for a future FET.

P-175 Tuesday, October 15, 2013

KARYOMAPPING(KM)ASAUNIVERSALPREIMPLANTATIONGE-NETIC DIAGNOSIS (PGD) FOR BETA-GLOBIN GENE (HBB) MUTA-TIONS COMBINED WITH HUMAN LEUKOCYTE ANTIGEN (HLA)GENOTYPING: A PROOF-OF-PRINCIPLE STUDY. S. Coskun,a

W. Qubbaj,a S. AlHassan,a S. Natesan,b K. Awratani,a A. Handyside.b aKingFaisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia; bBlueG-nome an Illumina Company, Cambridge, United Kingdom.

OBJECTIVE: Diseases caused by over 700 mutations in HBB are preva-lent in the Mediterranean countries. PGD is one of the effective methodsused in prevention of transmission to the offspring. However, classicalPGD requires extensive validation for each mutation/patient. KM has beensuggested to be a universal method of PGD. The reliability of KM withoutany prior test development was tested following standard PGD with haplo-typing (PGH) in 4 patients with 3 different HBB mutations and HLA geno-typing.DESIGN: Proof-of-principle.MATERIALS AND METHODS: Four patients underwent PGD for HBB

mutations. The diagnosis was made by sequencing and PGH. All patientsbecame pregnant and delivered healthy babies. Following the delivery, ablinded KM was performed from the remaining MDA products. Karyomapsof each embryo from multiple displacement amplification (MDA) productswere constructed to predict the presence of any HBBmutations and HLA ge-notype. Results were compared to the sequencing and PGH analysis of theoriginal cycles.RESULTS: Amplified DNA following MDA was obtained from 30 em-

bryos biopsied on day 3. Seven unaffected embryos identified bysequencing and PGH were transferred and four unaffected children wereborn. KM was performed on the leftover MDA products and DNA fromchildren born following PGD. The average call rates and AB call ratesfor DNA were 96 and 30%, and for MDA products were 72 and 15%,respectively. The results of KM and PGH were 100% concordant. Therewas discrepancy between the results of sequencing, and PGH/KM in 3 em-bryos (10%) which stem from allele drop out of during sequencing. Oneembryo although concordant among the techniques as normal, KM/PGHshowed only maternal allele at the HBB locus suggesting monosomy forthe chromosome. Moreover, KM provided the euploidy status of all chro-mosomes.CONCLUSION: KM can be reliably used in PGD of HBB mutation

without prior test development or knowledge of the specific mutations andmight prevent the transfer of aneuploidy embryos.

Vol. 100, No. 3, Supplement, September 2013