Loss of imprinting occurred during preimplantation development in culture, indicating that mechanisms that operate to maintain imprinting were disrupted.
What are the long-term effects of preimplantation development in culture? Cultured Blastocysts Recipient Mothers
Postimplantation embryos recovered at 9.5 days of pregnancy after preimplantation culture in Whitten’s medium.
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
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
Perturbations in imprinting persist long after embryos have been removed from culture.
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
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
PGC Oocyte maturation Humans Superovulation +/- GnRH treatment Oocyte retrieval IVF/ICSI In vitro culture Superovulation In vitro culture Mice Assisted Reproductive Technologies
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?
1. Determine whether superovulation contributes to loss of imprinting Spontaneous vs Induced Ovulation Imprint Acquisition Extraovarian Sensitivity Preantral Early antral Preovulatory
2. Determine when imprint maintenance is lost during preimplantation development in culture. Examine imprinting of H19, Snrpn, Kcnq1ot1, Peg3 In vitro culture
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
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
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
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
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