IVF and embryo transfer (ET) affect murine placenta and embryo development

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loci, as evidenced by animal models and an increased incidence of imprinting disorders in children conceived via ART. We have previously presented preliminary evidence demonstrating that ART conditions may also result in global alterations of genomic methylation in murine blastocysts. We now present microarray data to further test this hypothesis. DESIGN: Mouse model, DNA microarray analysis MATERIALS AND METHODS: C57/BL6 female mice were crossed with C3H male mice to obtain F1 hybrid embryos. For ART conditions, female mice were injected with 5U of PMSG followed 48 hours later with 5U of hCG. Mice were then mated and the resulting embryos were harvested on E0.5 and cultured in HTF media until E3.5. For the control group, female mice were mated in estrus without superovulation and the resulting embryos were flushed from the oviducts at E3.5. Genomic DNA was prepared from morphologically normal blastocysts by standard methods for 4 ART embryos and 4 control embryos. DNA was digested with a methylation sensitive restriction enzyme and then ligated to double-stranded linkers. PCR with a biotinylated primer was then performed. Amplified DNA fragments were purified via streptavidin paramagnetic particles and then re-amplified using multiple strand displacement whole genome amplification. The microarray contained 2 to 3 oligonucleotides for each expected DNA sequence from the original digest of chromosome 7. Eight hybridizations were performed with color reversal. The first 4 experiments compared control embryos to control embryos; the second 4 experiments compared control to ART embryos. RESULTS: Genomic DNA from ART blastocysts exhibited a similar degree of genomic methylation as controls. CONCLUSIONS: These results indicate that the aberrant DNA methylation observed under ART conditions are not a global phenomenon but may be present only a certain, highly sensitive loci. Supported by: University of Vermont.

O-87 Monday, October 19, 2009 5:45 PM IVF AND EMBRYO TRANSFER (ET) AFFECT MURINE PLACENTA AND EMBRYO DEVELOPMENT. L. Delle Piane, W. Lin, A. Donjacour, X. Liu, A. Revelli, P. F. Rinaudo. Ob/Gyn, Reproductive Medicine Division, University of Turin, Torino, Italy; Obstetrics, Gynecology & Reproductive Sciences, University of California San Francisco, San Francisco, CA. OBJECTIVE: Abnormal placentation is a potential mechanism to explain the increased incidence of low birth weight observed after IVF. This study evaluates if the method of conception and ET affect placentation and fetal development DESIGN: Mouse model of IVF MATERIALS AND METHODS: IVF blastocysts, from CF1 female and B6D2F1/J male mice, were transferred to the uteri of recipients (IVF group). Two control groups were created: in vivo embryos fertilized and grown in vivo; flushed blastocysts (FB) embryos fertilized in vivo, flushed out of the uterus at the blastocyst stage and transferred to recipients. At 14 days post fertilization (DPF), implantation sites were collected, fixed in 4% paraformaldehyde and stained; embryos and placentas were weighed and developmental stage (DS) evaluated. Placenta areas were calculated using Image Processing toolkit (Photoshop). Parametric statistic was applied as appropriate RESULTS: Embryos and placentas after the ET procedure (FB and IVF groups) are smaller than in vivo embryos and show developmental delay; importantly, these differences persist after correcting for DS. In addition, IVF embryos and placentas are consistently smaller than FB embryos for all parameters tested.Gross morphology of the placenta and ratio labyrinth/ fetal area are equivalent in the 3 groups. Measurements at 14 DPF

Developmental stage Placenta (mg) Embryo (mg) Placenta/embryo Fetal area (mm) Labyrinth (mm) Labyrinth/fetal area

In vivo(n¼42)

FB(n¼57)

IVF(n¼49)

13.74a 74.40a 202.97a 0.44a 5.39a 3.71a 0.69a

12.80b 68.00a 98.74b 0.72b 7.50b 5.26b 0.70a

12.42c 58.10b 64.44c 0.95c 4.55c 3.14a 0.69a

Different superscripts if p
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