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Susceptibility of Genomic Imprinting to ART Mellissa Mann Children’s Health Research Institute Department of Obstetrics & Gynecology, and Biochemistry University of Western Ontario Infertility and Assisted Reproductive Technologies 35-70 million couples involuntarily infertile 1 in 6 people of reproductive age ART 1-2% children born by ART Children conceived via ARTs are at increased risk Intrauterine growth retardation Premature birth Low birth weight Possibly genetic disorders ART-induced perturbations in the mouse Reduced viability Intrauterine growth retardation Developmental abnormalities Deviation in behaviour Developmental abnormalities in in vitro produced livestock Large Offspring Syndrome •Increased prenatal loss •Large placentas •Large fetuses •Polyhydramnios •Parturition problems • • • • • • Higher perinatal mortality Breathing difficulties Reluctance to suckle Skeletal anomalies Large organs Cerebellar dysplasia Epigenetics: Heritable alterations in gene activity without a change in DNA sequence Duke University Medical Center Skinny and Brown Obese and Yellow Epigenetics: Mediator of Environment, Development, and Disease Nutrition Vitamins Drugs Estrogen Disruptors Herbicides Pesticides Epigenetics Reproductive Disorders Cardiovascular Disorders Neurological Disorders Pathology Growth Disorders Pediatric Disorders Lupus Imprinting Disorders Cancer Genomic Imprinting The unequal expression of the maternal and paternal alleles of a gene Maternal allele Paternal allele Paternal allele Maternal allele Imprinted or marked with their gametic-origin Mouse/Human Imprinted Domains Mouse Distal 7/ Human 11p15.5 Slc22a1l Kcnq1 Cd81 IC Nap1l4 Tssc3 Cdkn1c IC Ascl2 Th Kcnq1ot1 Ins2 Igf2 H19 Beckwith-Wiedemann Syndrome Critical Region 1 Mouse Central 7/ Human 15q11-13 Magel2 Ndn Snurf Snrpn Ipw snoRNA Genes Ube3A Atp10c IC Frat3 Mkrn3 IC Antisense Prader-Willi Syndrome IC Angelman Syndrome Non-coding RNA Methylation changes during mouse preimplantation development Methylated imprinted allele Paternal Genome Unmethylated imprinted allele Maternal Genome Are genomic imprints maintained in preimplantation embryos after in vitro culture? B6(CAST7) mice for use in allelic analyses B6(CAST7) Peg3 Chromosome 7 CAST Snrpn F1 X CAST B6 B6 H19 B6 Kcnq1ot1 In vitro preimplantation culture regimes Trophectoderm ICM Whitten’s KSOMaa 2-cell Blastocyst Loss of H19 imprinted expression occurs in a subset of individual blastocysts after culture in Whitten’s medium 100 100 100 808080 60 6060 40 4040 20 20200 00 6% p=0.006 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 100 100 100 80 80 80 60 60 60 40 40 40 20 20 20 0 00 14% p=0.002 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 100 80 60 1 00 80 60 63% 40 20 40 20 0 0 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 Blastocyst 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. 100% 81% 80% 60% 40% 20% 32% 30% 8% 10% 11% 3% 3% 14% 5% 0% Empt y Severely Abnormal Abnormal Controls Whitt en's Delayed Normal Loss of imprinted expression occurs primarily in day 9.5 placentas after preimplantation culture 100 100 80 60 H19 80 60 40 20 0 40 20 0 100 100 80 60 60 Ascl2 80 40 40 20 20 0 0 100 Snrpn 100 80 80 60 60 40 40 20 20 0 0 100 100 80 Peg3 80 60 60 40 20 40 20 0 0 Vivo KSOMaa Whitten’s Embryo Vivo KSOMaa Whitten’s Placenta Loss of imprinted expression occurs primarily in day 9.5 placentas after preimplantation culture 100 13 H19 80 75 60 100 100 92 100 87 40 20 25 8 0 100 Ascl2 80 75 60 92 100 40 20 25 8 0 100 8 12 Snrpn 80 45 60 100 100 40 92 100 88 55 20 0 100 16 37 Peg3 80 27 60 100 100 40 100 83 63 73 20 0 V K Embryo W V K W Placenta Perturbations in imprinting persist long after embryos have been removed from culture. Loss of imprinting occurs more frequently in extraembryonic than embryonic lineages Preimplantation Culture Placenta Trophectoderm ICM Proximity to culture? ICM vs TE? Less redundancy? Blastocyst Embryo Day 9.5 ART children diagnosed with imprinting disorders Angelman Syndrome OR X Loss of Normal maternal methylation OR X Biparental origin Beckwith-Wiedemann Syndrome X OR X Biparental origin OR Loss of maternal methylation Sporadic imprinting defects may arise during ART procedures Assisted Reproductive Technologies PGC Oocyte maturation Humans Superovulation +/- GnRH treatment Oocyte retrieval IVF/ICSI In vitro culture Mice Superovulation In vitro culture Future Studies Assisted Reproductive Technologies PGC Oocyte maturation Mice Superovulation 1 In vitro culture 2,3 4,5 1. Do imprinting defects result from superovulation? 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. 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 Preantral Early antral Preovulatory Extraovarian Sensitivity Imprint Acquisition Spontaneous vs Induced Ovulation 2. Determine when imprint maintenance is lost during preimplantation development in culture. In vitro culture Examine imprinting of H19, Snrpn, Kcnq1ot1, Peg3 3. Does loss of imprinting occur in mouse embryos cultured in media used in human assisted reproduction? Trophectoderm ICM Global HTF H19 Snrpn Peg3 Kncq1ot1 One Step Two Step 2-cell G1.2/G2.2 H19 Snrpn Peg3 Kncq1ot1 P-1/Bl +SSS Blastocyst 4. Determine how disruptions lead to the selective loss of imprinting in the placenta Left horn Right horn In Vivo derived Whitten’s Right horn Day 5.5 Day 6.5 Day 7.5 Day 8.5 Examine imprinting of H19, Snrpn, Kcnq1ot1, Peg3 5. Determine the long-term affects of preimplantation culture on genomic imprinting and development Left horn Whitten’s Ultrasound biomicroscopy Day 9.5, Day 13.5, Day 17.5 Right horn In Vivo derived Ultrasound biomicroscopy at Day 9.5-10.5 of gestation Growth and Viability U U Crown-rump length E D D P Resorption U U D NT NT P E D Variable embryonic growth Calcium hydroxyapatite deposits Ultrasound biomicroscopy at Day 13.5 of gestation Beckwith-Wiedemann Syndrome Macrosomia Visceromegaly (liver, heart) Macroglossia ( tongue) Liver Limb Abdominal wall defects Polydactyly (limb) Cardiac defects (heart) Adrenal defects Lens defects (eye) Vertebral defects Hemihypertrophy Heart Polyhydramnios Placentomegaly/Placental hydrops Tongue Eye Acknowledgements University of Pennsylvania Marisa Bartolomei Richard Schultz QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. Michael Golding Anne Pin Liyue Zhang Sarah Lalone Julia Foster Brenna Market Lauren Magri Michelle Gabriel Morgan McWilliam QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture.