Failure to conceive and pregnancy loss, in both natural conception and IVF, are mainly caused by chromosome aneuploidies, whose occurrence exponentially increases with advancing female age [20]. Molecular analyses performed after natural conception or spontaneous miscarriage highlighted that trisomies arise mainly due to an impaired female meiosis, in particular the first meiotic division [21]. Thus, fertility decrease with increasing maternal age is basically ascribable to aneuploidies’ increase due to an oocyte aging issue. This is mainly determined by the long-lasting arrest in the prophase of MI, which ranges from fetal life up to oocyte recruitment for final maturation, occurring between the menarche and the menopause. Unfortunately, despite the unique possibility to perform PGS without directly operating on the embryo, which makes of PB-based PGS the only practice ethically acceptable in some countries, and its compatibility with fresh ET after molecular diagnosis, this approach soon showed important limitations.

One study in particular shed light on the main drawbacks of PBs approach [17]. It was designed as a sequential biopsy associated with aCGH analysis of PBs, blastomere, and TE from the same embryo, which led to an elegant and comprehensive view on chromosomal segregation patterns from female meiosis and throughout preimplantation development up to the blastocysts stage. A unique possibility to infer the etiology of aneuploidies in AMA patient population as well as the accuracy of PB-based chromosome screening in predicting the chromosomal complement of resulting embryos was provided. The results reported in this study firstly confirm the inefficiency of PB1-only approach, because both PBs are needed since approximately half of female-derived aneuploidies in the embryos arose as a consequence of errors originating during the second meiotic division. With the inclusion of the PB2 data, more accurate information inferring oocyte chromosome copy number can be obtained. However, the inability to assess MI errors balanced at MII, the relevant proportion of meiotic errors selected against and corrected during preimplantation development, and the influence of male and mitotic-derived aneuploidies were all important source of errors described in the study and representing inescapable pitfalls of this approach which are sensibly compromising its reliability in predicting embryos’ chromosomal complement.

In this scenario, these intrinsic and technical limitations of PB analysis may result in one case in discarding viable embryos, while in the other in transferring abnormal ones. It is also worth highlighting that Handyside and colleagues found a consistent proportion (21.1 %) of chromosome segregation errors detected as copy number changes in the PBs not resulting in the expected outcome in the corresponding zygote [22], which is also similar to what reported by Capalbo and colleagues in the study previously described. Moreover, also in a recent paper published by Christopikou and colleagues, 17 % of false-positive PB results were observed based on the follow-up analysis performed on the resulting embryos [23]. As previously mentioned, one of the major concerns of PB-based PGS relates to the difficulties in detecting precocious sister chromatid errors balancing in MII that was shown to be one of the major mechanisms contributing to female-derived aneuploidies in embryos [17, 22]. In the matter of this, Forman and colleagues demonstrated in a good prognosis patient population that when reciprocal aneuploidy occurs from MI premature separation of sister chromatids and compensation in MII, the resulting embryo is usually normal for that chromosome [24]. The same authors also showed in a different paper that most of these embryos could result in a chromosomally normal child after ET [25]. Thus, future studies are required to assess whether these embryos should be reanalyzed or the signal intensity of the data is reliable enough to distinguish between chromatid and whole chromosome impairments. In particular, the threshold values should be prospectively set by reanalyzing cases in which PB reciprocal aneuploidies occurred and making blinded predictions of the chromosomal status of the embryo.

At present, all the data reported so far in the scientific literature consistently demonstrate a low accuracy of PB approach in predicting the actual copy number configuration in the embryo and that reciprocal aneuploidies in PBs inevitably require a follow-up analysis in the resulting embryo. Therefore, many concerns related to whether the accuracy achievable using PB screening is good enough to improve the IVF clinical outcome still remain [17, 26, 27]. Another major problem related to PB biopsy is the paucity of material that is available. In our hands as well as in the practice of other qualified centers, around 10 % of the oocytes tested remain without a conclusive diagnosis because of amplification failure in at least one of the two PBs [28]. If PGS aims at improving IVF outcomes, it is crucial that results are obtained from all embryos tested. Economic and logistic issues should then be also considered. In particular, PBs screening results as the most time-consuming and least cost-effective among PGS approaches and it is also independent from oocyte developmental potential, since part of the analyzed oocytes/zygote will never reach to the blastocyst stage and be transferred. In synthesis, all these evidences together resulted in the breakdown of PBs approach, while some investigators proposed to move the biopsy stage forward along preimplantation development. They suggested blastocyst stage TE biopsy, arguing that several advantages could be brought from this novel intriguing approach [29]. Several efforts have been invested then in order to highlight the concrete possibilities offered by this breakthrough and to uncover its technical and clinical opportunities.

Blastocyst stage PGS on TE biopsy ensued the sharp failure of cleavage stage PGS on blastomere biopsy and the betrayed promises of PBs biopsy ones. Understandably, the same issues concerning these previous strategies were moved against this different approach. Thus, in order to prove its efficiency, a fruitful series of evidences were produced and published in literature up to date and more studies are currently in the pipeline. Hereafter, a review of the main evidences reported up to date will be provided. At first it was solved the doubt about a possible impact of the biopsy on embryo developmental potential. A particular concern dealt with the risk of decreasing the pregnancy rate per started cycle by postponing the time of the transfer beyond the cleavage stage, that is, extending embryo culture to the blastocyst stage. This issue in particular arises from the risk of embryo developmental arrest either at the time of compaction or at the time of cavitation, which, especially in poor prognosis patients, can reduce the pool of blastocysts to be screened for aneuploidies and potentially transferred. However, in the scientific literature there is absolutely no evidence that transferring embryos at the cleavage stage can result in higher pregnancy rate per stimulation cycle in poor prognosis patients compared to the use of blastocyst transfer policy and, reasonably, extended culture has not been considered to lead to embryo waste. In this regard, Guerif and colleagues [30] reported on an RCT highlighting that, in a poor prognosis patient population, the pregnancy rates per stimulation cycle was similar after both fresh ETs and frozen ETs when using a cleavage or blastocyst stage ET policy. This suggested that the extension of the culture to the latest stage of preimplantation development does not reduce the number of live births after IVF.

Blastocyst biopsy can be performed in two different ways. In the first one described by Schoolcraft and colleagues in 2010, a zona opening is made at the cleavage stage to prompt TE cells herniation on day 5 or 6 and to facilitate the biopsy procedure [31]. The main drawbacks of this method relate to the extra manipulation of embryos at the cleavage stage, especially in the current trends of contemporary IVF culture that are exploiting closed culture systems from fertilization to the blastocyst stage, and to the risk of Inner Cell Mass (ICM) herniation that may require a second hole in the zona pellucida. A different method for TE biopsy has been then recently described by our group not requiring zona breaching and avoiding any potential stress at the cleavage stage, as well as allowing a more physiological growth of embryos to the blastocyst stage [32]. As far as the impact of biopsy on embryo development is concerned, Scott and colleagues reported, in the same elegant and powerful study previously mentioned in this chapter, no significant differences in implantation rate between untested biopsied and non-biopsied blastocysts [18]. It is not clear whether this is ascribable to a smaller proportion of total cells removed from the blastocyst, to a higher stress tolerance of the blastocyst with respect to other stages of preimplantation development, or to the preservation of the ICM counterpart which originates the fetus, but still this represents a further advantage of postponing the time of the biopsy.

A crucial point of discussion is the accuracy of the analysis and the information that can be obtained from a randomly selected TE sample biopsied at the blastocyst stage. Certainly, blastocyst stage TE biopsy ensures a more accurate assessment of meiotic aneuploidies than previous strategies, since between five and ten cells are retrieved and analyzed from the embryo compared to the analysis of a single cell that is commonly performed on blastomeres and PBs. This translates in a significant reduction of the incidence of all the misdiagnosis risks derived from a single-cell analysis. In fact, all confirmation studies reported so far based on FISH reanalysis of aneuploid blastocysts following TE biopsy and CCS found between 98 and almost 100 % of correct aneuploidies prediction of meiotic errors [33–35]. Also, in a recent study from our group, we provided the first assessment of the reliability of blastocyst stage aneuploidy screening by the analysis of multiple TE biopsies from the same blastocyst with the use of different CCS methods [36]. The analysis was based on the real-time quantitative Polymerase Chain Reaction (qPCR) blinded reanalysis of 120 s biopsies of aneuploid blastocysts previously screened by TE aCGH and showed a consistent chromosome copy number diagnosis in 99.4 % (2,561/2,576; 95 % CI 99.0–99.7) of the chromosomes analyzed. The remaining 0.6 % was due to either technical variation between CCS techniques or occasionally by biological variation due to the presence of chromosomal mosaicism.

The impact of mosaicism on the reliability of the diagnosis, and especially the possibility of a nonrandom allocation of chromosomally abnormal cells exclusively to TE in case of mosaicism [37–39], is in fact another important point to be considered when blastocyst biopsy is performed on randomly selected TE cells. To this end, high concordance between ICM and TE chromosomal complement has been reported in the most recent literature [11, 35, 40], suggesting no preferential allocation of abnormal cells in a mosaic blastocyst, and that the analysis of a TE sample can be considered diagnostic of the ICM. In order to properly perform this analysis, our group conceived and published a novel method of ICM biopsy, which led to the total absence of TE cells contamination [35]. In the same paper, a preliminary aCGH analysis on a TE biopsy during blastocyst stage PGS cycle was performed, which was followed by a FISH reanalysis of three further TE fragments and of the whole ICM from those embryos diagnosed as aneuploid. The ultimate aim of this study design was to define the real influence of mosaicism on the accuracy of the diagnosis. Constitutional aneuploidies were reported in 79.1 % of cases, while mosaic in 20.9 % of cases. However, the real risk of an uncertain diagnosis due to mosaicism when testing at the blastocyst stage accounts for only 4 % of aneuploid blastocysts that were detected to be mosaic diploid/aneuploid (embryos showing a mosaic error as the only aneuploidy).

A very interesting finding of this study was that all cases of high-grade diploid/aneuploid mosaicism where abnormal cells constituted more than 40 % of the blastocysts were diagnosed as aneuploid by the original blastocyst stage aneuploidy screening cycle. This data suggested that blastocyst stage PGS performed on a randomly selected TE sample is able to avoid also the transfer of mosaic embryos with a very high prevalence of abnormal cells that might have a poor clinical outcome on pregnancies (Fig. 7.1). Indeed it is well known that, when compatible with life, mosaicism can be associated with poor fetal outcomes and neonatal morbidity. Looking at mosaicism data in prenatal diagnosis, the overall incidence of mosaicism is very low and reported to range between 1.22 % and 1.32 % after spontaneous pregnancies and after IVF care, respectively, where true mosaicism accounted only for 0.3–0.44 % of pregnancies [41]. These data suggest that the incidence of mosaicism has been overestimated in preimplantation genetics but also that mosaic embryos can be subjected to a negative selective pressure following ET resulting in failure of implantation or early embryo loss, and at last highlight the potential benefit of blastocyst stage PGS to detect and avoid the transfer of mosaic embryos with a high prevalence of abnormal cells. However, even though all these preclinical studies were sufficient to assess no impairment of implantation potential following TE biopsy and the high diagnostic accuracy and reliability of a CCS approach for embryonic aneuploidy, they were not sufficient themselves to determine whether the test has a true clinical value.

Thus, in order to demonstrate whether a clinical benefit results from application of aneuploidy screening at the blastocyst stage and to determine the specific magnitude of this benefit, four RCTs were published up to date using different CCS methods and investigating the use of this approach in different patient populations [31, 42–44]. Taken together, all these RCTs consistently reported an increased sustained implantation and live birth rate (relative increase between 28 and 40 % with respect to the control group) following the transfer of euploid blastocyst compared to the transfer of untested embryo, suggesting that blastocyst stage aneuploidy screening can be considered today a validated technology to improve embryo selection and clinical outcome per transfer in IVF. What is still missing are RCTs evaluating live birth rate per stimulation cycle to assess whether blastocyst stage PGS can result also in similar efficacy compared to standard care according to an intention to treat analysis. A final interesting argument of discussion deals with the implementation of this PGS approach in clinical practice. Regarding this point from an economic and logistic perspective, blastocyst stage PGS on TE biopsy, conversely to previous strategies and especially to PBs biopsy one, represents the most convenient and easy to implement approach. This is mainly due to the fact that only developmentally competent embryos would reach to this stage, while incompetent ones will arrest at previous stages of development. Thus, only reproductively competent embryos will be screened for aneuploidies. This results in PGS cost reduction with the considerable advantage of being able to increase the patient population that can benefit from this technology during their IVF cycle. To summarize, blastocyst stage PGS approach has passed thorough several preclinical and clinical validation steps and fulfilled so far all the requirements that we may expect from an ideal PGS strategy.