64 Impaired Post-Implantation Development Following Blastomere Biopsy is Associated with Placental Hypomethylation
Authors: Zacchini F. , Ogluska M. , Arena R. , Ptak G. E. 
Published: November 2018

The removal of one cell from cleaving embryos (blastomere biopsy, BB) is the main component of pre-implantation genetic diagnosis (PGD), a diagnostic test aimed to select only healthy embryos before transfer in utero. Studies on animals have suggested BB as risk factor for impaired development during both pre- and postnatal life. However, we still have incomplete knowledge on the real side effects of BB and the mechanisms underlying them. The present study was designed to evaluate whether BB or miscellaneous factors (in vitro culture, IVC; and embryo transfer, ET) may affect developmental potential of conceptuses obtained following BB (i.e. pregnancy rate/loss, fetal/placental weight, feto:placenta ratio) and placental epigenetic programming. To this aim, 3-month-old C57 females, naturally mated without hormonal stimulation, were used as donors. At 2.5 days post-coitum (dpc), 8-cell stage embryos were collected and subjected to BB in PBS (without Ca2+/Mg2+) supplemented with 0.4% BSA and antibiotics. Embryos were cultured in KSOM medium in an incubator at 37.5°C and 5% CO2. Two in vitro control groups were used: (1) IVC, embryos cultured in vitro, not subjected to biopsy; (2) ET, blastocysts collected at 3.5 dpc and directly transferred into recipient females. At 3.5 dpc, 10 to 12 blastocysts were transferred into pseudo pregnant females (n ≥ 7/group). An additional control group consisted of naturally conceived pregnancy (n = 8, in vivo control, CTR). At 18.5 dpc, conceptuses were collected and subjected to gross morphological evaluations (n > 20 conceptuses/group). Placentae (n ≥ 4/group) were analysed for 5-methylation and hydroxymethylation content by ELISA assay. Decimal variables were analysed using the Mann-Whitney test, and percentages were analysed with the Fisher exact test. Assessment of development to term revealed reduced survival rate in BB, IVC, and ET v. CTR (45.23, 47.82, 60, and 98.15% respectively; P < 0.05) and increased number of stillborn/congenital anomalies in BB v. CTR fetuses (9.5 v. 1.5%; P < 0.05). Gross morphological observations displayed increased placental weight in BB, IVC, ET v. CTR (0.15, 0.17, 0.16 and 0.11 g, respectively; P < 0.0001) and reduced fetal weight in BB v. IVC, ET, and CTR groups (0.88, 1.09, 1.16, and 1.15 g, respectively; P < 0.05). Also, a significant reduction of feto:placenta ratio was detected in BB, IVC, ET v. CTR (5.9, 6.3, 8.1 v. 10.4; P < 0.05). The ELISA assays on placentae revealed significant reduction of 5-methylcytosine content in BB v. CTR (22.24 v. 33.55%; P < 0.05) but no differences in hydroxymethylcytosine. Altogether, our results showed that BB is associated with impaired development to term (reduced fetal weight, increased risk of perinatal mortality, and congenital anomalies) and hypomethylation in full-term placentae. Thus, our preliminary study suggests that BB, rather than IVC and ET, to be a factor of risk for proper development in utero through perturbation of developmental epigenetic programming. Further analysis will be necessary to better characterise the epigenetic profile in both fetuses and placentae obtained following BB.