Re: Questions about the accuracy of polar body analysis for preimplantation genetic screening

Sir, We thank Drs Fragouli and Wells and Drs Christopikou and Handyside for their interest in our paper (Capalbo et al., 2013). We welcome the opportunity to provide clarification and further stimulate discussion on this important topic. We acknowledge the possibility that biological variation due to mosaicism may produce a low error rate during trophectoderm (TE) based prediction of inner cell mass (ICM) chromosome complement. However, this aspect is considered to have a very low impact on the evaluation of polar bodies (PB) chromosomal screening accuracy according to the existing literature, including the papers mentioned by Fragouli and Wells in their letter. A very high rate of consistency between the comprehensive chromosome screening (CCS) of different TE samples from the same blastocyst and between TE and ICM was reported when a single method for chromosome screening was applied (Johnson et al., 2010; Northrop et al., 2010). In the Johnson paper all CCS results obtained from different TE biopsies derived from the same blastocysts were concordant. Furthermore, ICM and their relative TE samples gave the same results in 96.1% of cases. In Northrop et al., considering only embryos producing interpretable results in at least three out of four embryonic sections, 46 of 47 (98%) embryos had at least three concordant results in the four embryonic sections analysed. It is also interesting to note that Fragouli herself (Fragouli et al., 2008) found 100% consistency during chromosomal screening of 10 ICMs and their relative TE samples by CGH analysis. The conclusion of the authors in their paper was that ‘data obtained from TE biopsies can generally be considered diagnostic of ICM aneuploidy’. In addition, we have also observed 97.1% consistency between ICM and TE from 70 good quality blastocysts reanalysed by 9-chromosome FISH (Capalbo et al., 2011b). The higher estimation of blastocyst TE chromosomal mosaicism observed by Fragouli et al. in 2011 (Fragouli et al., 2011) may have been biased due to the methodology of the study. In fact, the intrinsic differences in the performance of the three different chromosomal screening methods used in that study may have contributed to the accuracy of the assessment of biological mosaicism prevalence. Finally, the clinical predictive value of TE-based CCS was shown to be very high in a recent prospective double blind study (Scott et al., 2012). In addition, it is worth to remember that aneuploidies predicted by PB testing leads to constitutive aneuploidies in the embryo ruling out the mosaicism issue related to TE-based PB accuracy assessment. This is further supported by the results of our study where the analysis of one blastomere sampled on day 3 of embryo development was also included. Indeed, most PB falseand -negative assignments were based on two consecutive confirmation analyses at day 3 and 5 of embryo development. In this regard, due to legal restrictions a study design as suggested by Christopikou and Handyside that included the analysis of multiple samples from TE and ICM would not be possible in our country, however we are confident that such approach would not add relevant additional information to the assessment of PB aneuploidy screening accuracy. Christopikou and Handyside also suggested that we give a misleading impression that polar body analysis is not very accurate. However, this consideration is based on their incorrect interpretation of our data. In their letter, they wrongly suggested that 94% (73 of 78) of PB predictions were confirmed as correct in the single blastomeres analysed in our study. On the contrary, in our study 16 out of 78 (20.5%) abnormal meiotic segregations had copy number gains or losses in either one or both PBs resulting in normal chromosome copy number in the corresponding embryos (Table I describes chromosomal segregation in PB1/PB2/blastomere and not PB1/PB2 and TE). It is also worth noting that Handyside himself found a very similar proportion (48/227; 21.1%) of chromosome segregation errors detected as copy number changes in the polar bodies that did not result in the predicted outcome in the corresponding zygote (Handyside et al., 2012). Finally, Christopikou herself (Christopikou et al., 2013) found 17% (17/100) of false-positive PB results plus 14 addition reciprocal aneuploidies in PB with normal copy number in the embryo that were considered as correct predictions without any prospective validation performed so far on PB1 log2 ratio accuracy to distinguish between whole chromosome and single chromatid aneuploidies (see comments below on this issue). However, in their upcoming paper, Christopikou et al. present their data focusing on the fact that 93% (65 of 70) of aneuploidies detected in the cleavage stage embryos were associated with copy number changes in the polar bodies giving the misleading impression that PB analysis is very accurate. It seems thus that all these data further and consistently demonstrate a low specificity of PB approach in predicting the normal chromosome copy number configuration in the embryo. Furthermore, in our study approximately half of the embryos had aneuploidies other than those arising from female meiosis. At the cleavage stage of embryo development, 25 new aneuploidies were recorded, 20 of which were confirmed at the blastocyst stage. These new aneuploidies can be equally the result of false-negative PB predictions or male and mitotic derived errors. Including these 25 de novo aneuploidies, the actual prediction of embryonic chromosomal complement based on PB testing decrease to 62 out of 87 aneuploidies recorded at the cleavage stage (71%) showing low sensitivity of PB testing in predicting cleavage stage embryo aneuploidies. Christopikou et al. (2013) reported 7% of aneuploidies detected only in the embryo with normal segregation pattern in PB. However, in the study by Christopikou et al., the actual incidence of post-zygotic