Summary of 328 samples from 59 patients received for preimplantation genetic screening by array CGH: hints at early stage embryo development

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1 Summary of 328 samples from 59 patients received for preimplantation genetic screening by array CGH: hints at early stage embryo development Teresa A. Smolarek, PhD Division of Human Genetics, Department of Pediatrics, Cincinnati Children s Hospital Medical Center

2 Why do preimplantation genetic screening? Aneuploidy is frequent and has a negative effect on preimplantation development Need for testing the embryos to reduce number implanted Improve pregnancy rate by implanting normal embryos

3 Sample Preparation Cells are selected from the trophectoderm (becomes part of the placenta) Inner mass: part that will become the fetus; remains untouched

Pictures of the blastocysts http://www.fssc.com.

4 Pictures of the blastocysts 6DA301520E37C9991E09869E54FD5FAF8265&selectedIndex=137&ccid=LuXLwnF2&simid= &thid=OIP.M2ee5cbc d2d378fdfbab2d3bo0&ajaxhist=0

5 Helpful information on biopsy worksheet Number of cells Cell stage Cells visualized Additional comments

6 General information CGH assay using 24sure (Illumina); needed 24hr TAT Dye swap assay using Cy3 and Cy5 Approximately 3000 clones at 1Mb intervals Male and female references are run each time Looking for 10Mb or larger imbalances Receive 3-8 cells from each biopsy at day 5 or day 6 Average 5 samples per patient (range 1-14)

Workflow: 24sure Microarray chips 24 hour TAT: required for fresh embryo transfer Prepare Biopsied Cells Agarose Gel Electrophoresis Prepare Amplification Reaction

7 Workflow: 24sure Microarray chips 24 hour TAT: required for fresh embryo transfer Prepare Biopsied Cells Agarose Gel Electrophoresis Prepare Amplification Reaction controls Cell lysis/extraction Pre-amplification Amplification Labeling: Cy3, Cy5 Combination and Volume Reduction Hybridization (10-13hrs) Wash slides Scan slides Data Analysis

8 Hybridization 3-16 hrs (we use 10-13hrs) Standard dye swap assay: sample in one color reference in opposite color

9 Analyzing Samples Use BlueFuse software Add in deletions and/or duplications that are a min of 5 consecutive probes at the telomeres and a min of 10 consecutive probes interstitial to suggest an abnormality Samples are reviewed by 2 technologists and 1 Director

10 BlueFuse Software Sample vs Male Reference Sample vs Female Reference Fused Chart (Combined)

11 No amplification: data are very messy and difficult to interpret

12 Case 24: 47,XX,+21

13 Case 24: sample 7: monosomy 16

14 Complex: Case 24: sample 6

15 Complex with mosaicism Case 24: Sample 2

16 Case 10: male with mosaic terminal deletion of 22q Alternate normal 11; normal 22 Translocated chr Adjacent 1: del 11q, gain distal 22q gain 11q, del distal 22q Adjacent 2: tri 11, except ter, del prox 22q one 11, except ter 11q, tris prox 22q Gardner, Sutherland and Shaffer, Fourth Edition 3:1: Several combinations: common tri 11q and prox 22q

17 Case 10: sample 4: Alternate chromosome 11 chromosome 22

18 Case 10: sample 1 deletion 11q and gain of 22q: adjacent 1 chromosome 11 chromosome 22 p arm q arm 11q q11.21 q arm

19 Case 10, sample 3: distal tri11q; distal del 22q: Adj-1 chromosome 11 chromosome 22

20 Case 10, sample 2: mono most 11, except 11qter; prox tri 22q: Adj-2 chromosome 11 chromosome 22 These few markers are gains

21 Case 10, sample 5: tri most 11, distal 11q del; prox 22q del; distal tri 22q: 3:1, meiosis II error chromosome 11 chromosome 22

22 Case 10 summary Alternate: 2/9 Adjacent 1: 2/9 Adjacent 2: 2/9 3:1 and meiosis II nondisjn: 1/9 Unrelated abnormalities: 2/9 (loss 3p and complex)

23 Case year old with history of multiple miscarriages Mosaic monosomy 9

24 Case 39:Telltale sign of parental carrier of balanced translocation: loss/gain Gain of 6q Loss of 11p

25 Case 39: Gain of 23.2Mb from 6q and Loss of 22.2Mb from 11p Chr. Start End Start Cyto End Cyto Type Size (bp) Included 6 147,740, ,921,603 6q24.3 6q27 GAIN 23,180, ,848 22,527,270 11p p14.3 LOSS 22,294,423 23

26 Case 39: Dad: 46,XY Normal 6q and 11p

27 Case 39: 46,XX,t(6;11)(q24.3;p14.3) Completed FISH confirmation using subtelomere probes

28 Most common reason for referral: multiple miscarriages (50.5%) Total Total Family balance: 70/325 (21.5%) multiple miscarriages 164 family balance 70 history of chromosome abnormality 33 adv mat age 25 pt wants embr tested 14 diminishing ovarian reserve 7 Hyster, using gest carrier 7 adv mat age, multiple miscarriages 4 Male factor 1 Grand Total 325

29 Overall: 50.9% normal; 24.7% single abn, 9.1% two abn; 6.7% complex Normal 167 single 81 two 30 complex 22 No amplification 20 Inconclusive 8 Grand Total 328

30 Gender: 55% female; 45% male Total Female Male Total Female 164 Male 134 Grand Total 298

31 Monosomy 22: most common single chromosome loss

32 +22: most common single chromosome gain

33 Whole chromosome loss/gain most common type of abnormality (57.6%) whole 76 arm 32 whole/mosaic 8 whole/arm 8 whole/arm/mosaic 6 whole/mosaic/arm 1 arm/mosaic 1 Grand Total 132 Arms: 24.2%

34 Reason for referral: family balancing Normal= 56.4% Total Normal single complex two Family balance Total Family balance 62 Normal 35 single 15 complex 9 two 3 Grand Total 62

35 multiple miscarriages 152 Normal 82 single 48 two 16 complex 6 Grand Total 152 Multiple miscarriages: 53.9% normal Total Normal single two complex multiple miscarriages Total

36 History of chromosome abn: 50% normal Total Normal two single complex history of chromosome abnormality Total history of chromosome abnormality 30 Normal 15 two 6 single 6 complex 3 Grand Total 30

37 Normal single two complex Normal single two complex single Normal two complex Age distribution and result Total Row Labels Count of abnormal summary < Normal 97 single 41 two 14 complex Normal 54 single 22 two 10 complex 8 >40 43 single 18 Normal 16 two 6 complex 3 Grand Total 300 Total < >40 Normal results most common in younger groups

38 Age vs result: Patients older than 40 with increase in single abnormalities Result < >40 Total Normal 97 (59.5%) 54 (57.4%) 16 (37.2%) 167 Age Single 41 (25.3%) 22 (23.4%) 18 (41.9%) 81 Two 14 (8.6%) 10 (10.6%) 6 (14%) 30 Complex 11 (6.8%) 8 (8.5%) 3 (7%) 22 Total

39 Conclusions Array analyses can be completed with few cells in 24 hrs Overall, approximately 50% of day 5-6 samples were normal Whole chromosome loss/gain was the most common type of abnormality Patients older than 40 had a higher percentage of embryos with single abnormalities There didn t appear to be a difference in the normal rate for family balancing (56.4%) vs. multiple miscarriages (53.9%) Analyses may help to predict segregation patterns for translocations or may help uncover apparently balanced translocation carriers

40 Acknowledgements Institute for Reproductive Health Erica Behnke, PhD Sherif Awadalla, MD, FACOG Michael Scheiber, MD, MPH, FACOG Dr. Thomas Burwinkel, MD, FACOG Illumina Gary Harton, BS, TS(ABB) BlueGnome; IVF Business Development Jim Zorzi BlueGnome; Territory Account Manager IVF Dan Saul Lead Technical Support Maila Crist Sr. Territory Account Manager GIVF Wayne S. Stanley, PhD, FACMG Harvey J. Stern, MD, PhD, FACMG CCHMC Lisa Dyer, PhD Stephanie Monzon Margie Hayes Brittany Jones Emily Liston Jenny Coffman Danielle Freppon Ambryn Laubenstein John White Emily Boyers Brenna Hott Shawn Mikulich Jennifer Glass, LGC Abby Masunga, LGC Jana Basil, LGC

Diagnosis of parental balanced reciprocal translocations by trophectoderm biopsy and comprehensive chromosomal screening

Diagnosis of parental balanced reciprocal translocations by trophectoderm biopsy and comprehensive chromosomal screening Lian Liu, MD Co-Authors: L. W. Sundheimer1, L. Liu2, R. P. Buyalos1,3, G. Hubert1,3,