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Table 3 Analysis results for immediate and persistent changes in DNA methylation after ED exposure of G1 prospermatogonia

From: Deleterious effects of endocrine disruptors are corrected in the mammalian germline by epigenome reprogramming

    Analysis level 1 Level 2 Level 3 Level 4
  Sample A Sample B Sample A to B Hits Common in G1R and G2R Hits Common in MGCs and sperm Hits TGI Hits
Group 1: CpG_G1R_MGC Oil BPA Test 1 112 Test A (test 1 v 7) 1 (1)     
  BPA Oil Test 2 72 Test B (2 v 8) 3 (3)     
  Oil VZ Test 3 129 Test C (3 v 9) 0 (0) Test Q (3 v 13) 1 (1) Test C v G 0 (0)
  VZ Oil Test 4 81 Test D (4 v 10) 0 (0) Test R (4 v 14) 2 (1) Test D v H 0 (0)
  Oil DEHP Test 5 130 Test E (5 v 11) 4 (1)     
  DEHP Oil Test 6 120 Test F (6 v 12) 6 (3)     
Group 2: CpG_G2R_MGC Oil BPA Test 7 61       
  BPA Oil Test 8 98       
  Oil VZ Test 9 59    Test S (9 v 15) 4 (2)   
  VZ Oil Test 10 135    Test T (10 v 16) 2 (1)   
  Oil DEHP Test 11 89       
  DEHP Oil Test 12 167       
Group 3: CpG_G1R_SPERM Oil VZ Test 13 76 Test G (13 v 15) 4 (4)     
  VZ Oil Test 14 76 Test H (14 v 16) 4 (3)     
Group 4:           
CpG_G2R_SPERM Oil VZ Test 15 196       
  VZ Oil Test 16 99       
Group 5: Imp_G1R_MGC Oil BPA Test 17 46 Test I (17 v 23) 0 (0)     
  BPA Oil Test 18 35 Test J (18 v 24) 0 (0)     
  Oil VZ Test 19 93 Test K (19 v 25) 1 (1) Test U (19 v 29) 1 (1) Test K v O 0 (0)
  VZ Oil Test 20 51 Test L (20 v 26) 1 (1) Test V (20 v 30) 0 (0) Test L v P 0 (0)
  Oil DEHP Test 21 83 Test M (21 v 27) 0 (0)     
  DEHP Oil Test 22 59 Test N (22 v 28) 1 (0)     
Group 6: Impr_G2R_MGC Oil BPA Test 23 30       
  BPA Oil Test 24 45       
  Oil VZ Test 25 29    Test X (25 v 31) 0 (0)   
  VZ Oil Test 26 120    Test Y (26 v 32) 4 (4)   
  Oil DEHP Test 27 27       
  DEHP Oil Test 28 111       
Group 7: Impr_G1R_SPERM Oil VZ Test 29 56 Test O (29 v 31) 3 (2)     
  VZ Oil Test 30 58 Test P (30 v 32) 1 (0)     
Group 8: Impr_G2R_SPERM Oil VZ Test 31 114       
  VZ Oil Test 32 93       
  1. After in utero exposure of G1 MGC to vehicle control (‘oil’) or one of the EDs, the level of DNA methylation was measured in reprogrammed G1R and G2R fetal MGCs and adult spermatozoa by MIRA-chip and Nimblegen microarrays. CpG-promoter arrays were used in groups 1 to 4 and custom imprinting arrays were used in groups 5 to 7. At the first level of analysis, we performed 32 tests comparing the log2 ratio MIRA versus input values. To detect a change between sample A and B, peaks were identified in the average value (n = 3 for MGC and n = 2 for sperm) of A samples first and were compared with the MIRA intensity of the average of sample B at the same locations. The number of hits where a change occurred with greater than ±5% and Fisher’s exact test P value (P <0.05) were tabulated. At level two, we compared the results of two level-1 tests (in parentheses) to find common changes between G1R and G2R in MGCs (Tests A-F and I-N) and in sperm (Tests G-H and O-P); the number of common hits is provided and those that changed in the same direction are given in parentheses. At level three, we compared the results of two level-1 tests to find common changes between MGC and sperm in the same generation, G1R (Test Q-R and U-V) or G2R (S-T and X-Y). At level four, we compared the results of four level-1 tests in search for true transgenerational epigenetic inheritance: immediate effect in G1R MGCs, maintained into G1R sperm, G2R prospermatogonia, and G2R sperm, thereby having the potential to affect G3 soma. Note the low number of hits in levels 1 to 3 and lack of hits at level 4, even at the low cutoff values applied.