Susceptibility of Genomic Imprinting to Embryo Culture

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Susceptibility of Genomic Imprinting to Embryo Culture

  1. 1. Susceptibility of Genomic Imprinting to ART Mellissa Mann Children’s Health Research Institute Department of Obstetrics & Gynecology, and Biochemistry University of Western Ontario
  2. 2. 35-70 million couples involuntarily infertile 1 in 6 people of reproductive age ART Infertility and Assisted Reproductive Technologies  1-2% children born by ART
  3. 3. Children conceived via ARTs are at increased risk <ul><li>Intrauterine growth retardation </li></ul><ul><li>Premature birth </li></ul><ul><li>Low birth weight </li></ul><ul><li>Possibly genetic disorders </li></ul>
  4. 4. ART-induced perturbations in the mouse <ul><li>Reduced viability </li></ul><ul><li>Intrauterine growth retardation </li></ul><ul><li>Developmental abnormalities </li></ul><ul><li>Deviation in behaviour </li></ul>
  5. 5. Developmental abnormalities in in vitro produced livestock Large Offspring Syndrome <ul><li>Higher perinatal mortality </li></ul><ul><li>Breathing difficulties </li></ul><ul><li>Reluctance to suckle </li></ul><ul><li>Skeletal anomalies </li></ul><ul><li>Large organs </li></ul><ul><li>Cerebellar dysplasia </li></ul><ul><li>Increased prenatal loss </li></ul><ul><li>Large placentas </li></ul><ul><li>Large fetuses </li></ul><ul><li>Polyhydramnios </li></ul><ul><li>Parturition problems </li></ul>
  6. 6. Epigenetics : Heritable alterations in gene activity without a change in DNA sequence Obese and Yellow Skinny and Brown Duke University Medical Center
  7. 7. Epigenetics Epigenetics: Mediator of Environment, Development, and Disease Estrogen Disruptors Herbicides Pesticides Nutrition Drugs Vitamins Pathology Cancer Cardiovascular Disorders Growth Disorders Imprinting Disorders Lupus Neurological Disorders Pediatric Disorders Reproductive Disorders
  8. 8. Paternal allele Maternal allele Genomic Imprinting The unequal expression of the maternal and paternal alleles of a gene Imprinted or marked with their gametic-origin Maternal allele Paternal allele
  9. 9. Mouse/Human Imprinted Domains Kcnq1 Kcnq1ot1 Cd81 Ascl2 Th Ins2 H19 Igf2 Nap1l4 Slc22a1l Tssc3 Cdkn1c Mouse Distal 7/ Human 11p15.5 Frat3 Magel2 Ndn Mouse Central 7/ Human 15q11-13 Ipw Ube3A Atp10c Mkrn3 Snurf Snrpn snoRNA Genes IC IC Antisense Beckwith-Wiedemann Syndrome Critical Region 1 IC IC Prader-Willi Syndrome Angelman Syndrome Paternal Maternal Biallelic Imprinting Center IC Non-coding RNA
  10. 10. Paternal Genome Maternal Genome Methylation changes during mouse preimplantation development Methylated imprinted allele Unmethylated imprinted allele
  11. 11. Are genomic imprints maintained in preimplantation embryos after in vitro culture?
  12. 12. CAST Chromosome 7 B6(CAST7) B6 B6 X CAST B6 B6(CAST7) mice for use in allelic analyses F 1 Peg3 Snrpn H19 Kcnq1ot1
  13. 13. Trophectoderm ICM Blastocyst Whitten’s KSOMaa 2-cell In vitro preimplantation culture regimes
  14. 14. Paternal Maternal Loss of H19 imprinted expression occurs in a subset of individual blastocysts after culture in Whitten’s medium Vivo KSOMaa 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Whitten’s 100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 Blastocyst 14% p=0.002 6% p=0.006 63%
  15. 15. Loss of imprinting occurred during preimplantation development in culture, indicating that mechanisms that operate to maintain imprinting were disrupted.
  16. 16. What are the long-term effects of preimplantation development in culture? Cultured Blastocysts Recipient Mothers
  17. 17. Postimplantation embryos recovered at 9.5 days of pregnancy after preimplantation culture in Whitten’s medium.
  18. 18. Embryo Placenta H19 Ascl2 Snrpn Peg3 100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 Paternal Maternal Whitten’s KSOMaa Whitten’s KSOMaa Vivo Vivo Loss of imprinted expression occurs primarily in day 9.5 placentas after preimplantation culture
  19. 19. Loss of imprinted expression occurs primarily in day 9.5 placentas after preimplantation culture Embryo Placenta H19 Ascl2 Snrpn Peg3 W K W K V V Paternal Maternal Biallelic
  20. 20. Perturbations in imprinting persist long after embryos have been removed from culture.
  21. 21. Loss of imprinting occurs more frequently in extraembryonic than embryonic lineages Proximity to culture? ICM vs TE? Less redundancy? Placenta Embryo Day 9.5 Trophectoderm ICM Blastocyst Loss of imprinting Maintains/restores imprinting Preimplantation Culture
  22. 22. Sporadic imprinting defects may arise during ART procedures Beckwith-Wiedemann Syndrome Biparental origin ART children diagnosed with imprinting disorders Normal Biparental origin X X OR OR Loss of maternal methylation Loss of maternal methylation OR OR Angelman Syndrome X X
  23. 23. PGC Oocyte maturation Humans Superovulation +/- GnRH treatment Oocyte retrieval IVF/ICSI In vitro culture Superovulation In vitro culture Mice Assisted Reproductive Technologies
  24. 24. PGC Oocyte maturation Superovulation In vitro culture Mice Future Studies Assisted Reproductive Technologies 1 1. Do imprinting defects result from superovulation? 2,3 2. When is imprinting lost during preimplantation development in culture? 3. Does loss of imprinting occur in mouse embryos cultured in media used in human assisted reproduction? 4,5 4. Is loss of imprinting tissue-specific? 5. What are the long-term affects of preimplantation culture on genomic imprinting and development?
  25. 25. 1. Determine whether superovulation contributes to loss of imprinting Spontaneous vs Induced Ovulation Imprint Acquisition Extraovarian Sensitivity Preantral Early antral Preovulatory
  26. 26. 2. Determine when imprint maintenance is lost during preimplantation development in culture. Examine imprinting of H19, Snrpn, Kcnq1ot1, Peg3 In vitro culture
  27. 27. 3. Does loss of imprinting occur in mouse embryos cultured in media used in human assisted reproduction? Trophectoderm Blastocyst Global G1.2/G2.2 2-cell HTF P-1/Bl +SSS One Step Two Step H19 Snrpn Peg3 Kncq1ot1 H19 Snrpn Peg3 Kncq1ot1 ICM
  28. 28. Day 5.5 Day 6.5 Day 7.5 Day 8.5 Right horn 4. Determine how disruptions lead to the selective loss of imprinting in the placenta In Vivo derived Whitten’s Left horn Right horn Examine imprinting of H19, Snrpn, Kcnq1ot1, Peg3
  29. 29. Ultrasound biomicroscopy Day 9.5, Day 13.5, Day 17.5 In Vivo derived Whitten’s Left horn Right horn 5. Determine the long-term affects of preimplantation culture on genomic imprinting and development
  30. 30. Ultrasound biomicroscopy at Day 9.5-10.5 of gestation Growth and Viability Crown-rump length Variable embryonic growth Calcium hydroxyapatite deposits Resorption U D E P U D U D E P U D NT NT
  31. 31. Ultrasound biomicroscopy at Day 13.5 of gestation Placentomegaly/Placental hydrops Polyhydramnios Hemihypertrophy Vertebral defects Lens defects (eye) Adrenal defects Cardiac defects (heart) Polydactyly (limb) Abdominal wall defects Macroglossia ( tongue) Visceromegaly (liver, heart) Macrosomia Beckwith-Wiedemann Syndrome Limb Heart Eye Tongue Liver
  32. 32. Michael Golding Anne Pin Liyue Zhang Sarah Lalone Julia Foster Brenna Market Lauren Magri Michelle Gabriel Morgan McWilliam Acknowledgements University of Pennsylvania Marisa Bartolomei Richard Schultz

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