CIC Edizioni Internazionali. Preimplantation genetic screening: definition, role in IVF, evolution and future perspectives. Summary.

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1 Mini-review Preimplantation genetic screening: definition, role in IVF, evolution and future perspectives Antonio Capalbo 1 Cristina Poggiana 2 Cristina Patassini 2 Anna Checchele 2 Emiliano Scepi 2 Danilo Cimadomo 1,2 Filippo Maria Ubaldi 1,2 Laura Rienzi 1,2 1 GENERA, Center for Reproductive Medicine, Clinica Valle Giulia, Rome, Italy 2 GENETYX, Molecular Genetics Laboratories, Marostica (VI), Italy Address for correspondence: Antonio Capalbo, MD, PhD GENERA, Center for Reproductive Medicine Clinica Valle Giulia Via G. De Notaris 2b Rome, Italy capalbo@generaroma.it Summary Prenatal Genetic Screening (PGS) is a diagnostic technique used in assisted reproductive technologies (ART) in order to detect chromosomal aneuploidies in embryos obtained through in vitro fertilization (IVF). Aneuploidy is the main cause of preimplantation failure and pregnancy loss in women. The main goal of PGS strategy is to identify euploid embryos and prevent the abnormal ones from being transferred, thus increasing the efficiency of IVF treatment, in terms of higher implantation rate and lower miscarriage rate per transfer. PGS at blastocyst stage offers different advantages over PGS on polar body (PB) or single blastomere: a higher amount of starting material and a more robust genetic analysis, a minor impact of mosaicism, a lower impact of the biopsy on embryo vitality, development and implantation potential as well as optimization of laboratory workload and procedural costs. At present, three randomized controlled studies have been published showing a consistently higher implantation rate and lower miscarriage rate when euploid blastocysts were transferred compared to untested embryos. Accordingly, blastocyst stage PGS can be considered a validated method to enhance embryo selection in IVF. However, even when transferring a euploid embryo with excellent morphology, still 40-50% of these embryos fail to implant or produce a delivery. Blastocyst morphology and developmental timings, as well as morphokinetic do not seem to be good indicator to further enhance selection between euploid blastocysts. Thus, the attention must be turned to other criteria, in order to identify additional biomarkers of reproductive potential of euploid blastocysts. The purpose of this review is to give a full explanation of PGS technique, its advantages over conventional embryo selection and to define the next steps towards for the discovery of additional criteria to further enhance embryo selection that could be used together with aneuploidy screening. KEY WORDS: PGS, comprehensive chromosome screening, blastocyst stage biopsy. PGS: role in ART PGS is a diagnostic technique applied in IVF to identify chromosomally normal embryos for transfer. In recent years, the development of comprehensive chromosome screening (CCS) technologies, made of PGS a fundamental technique in ART. It is well known that, in preimplantation period, aneuploidies do not represent a strong selective negative barrier for human embryo development as in the post transfer period. In fact, while in newborn population their incidence is relatively low (approximately 0.3%) and mostly represented by trisomies for chromosomes 13, 18 and 21 and sex chromosome impairments, tracking backwards through the developmental stages their incidence greatly increases, involving all chromosomes and reaching an incidence of up to 60% in preimplantation embryos 56 Current Trends in Clinical Embriology 2015; 2 (2): 56-64

2 Preimplantation genetic screening: definition, role in IVF, evolution and future perspectives of women aged 40 years. A natural selection against aneuploid embryos from the preimplantation period onward prevents them from resulting in a live birth (1). Indeed aneuploidies are the cause of a significant number of spontaneous abortions (more than 60% of products of conception follow chromosomal abnormalities) (2) (Figure 1). Accordingly, the risk undergoing an IVF cycle without analyzing all embryo s chromosomes is to transfer aneuploid embryos that either fail to implant after transfer or cause a miscarriage early along gestation or in the worst case the establishment of a chromosomally abnormal pregnancy. The primary aim of PGS strategy is thus to obtain the same efficacy as conventional IVF, that is the same live birth rate per cycle, while significantly increasing the overall efficiency of an IVF treatment, that is minimizing related efforts and risks (Figure 2). The main historical indications for PGS analysis were: (i) advanced reproductive maternal age (usually defined as > 35 years old; AMA); (ii) recurrent implantation failure (more than three failed IVF attempts; RIF) and (iii) recurrent pregnancy loss (more than three miscarriages; RPL). Data collected by the ESHRE PGD consortium IX showed that more than 60% of PGD cycles was actually PGS for AMA, RIF or RPL patients and that there is a constant increase in the number of the PGD cycles approached exclusively for euploid embryo selection (3). However, when looking at data form a recent study where embryo biopsies were analysed across the board of female age it is evident that all infertile patients seeking an IVF treatment are at high risk, ranging from 25 to 90%, of producing and transferring aneuploid embryos (4). Origin and mechanisms of aneuploidies in preimplantation embryos The origin of human aneuploidy is a multi-step process caused by errors at several distinct stages of oogenesis and enhanced by a lack of efficient checkpoints: the long prophase arrest in females contributes to aneuploidy because of age-dependent decay of components of the meiotic machinery; events occurring in the fetal ovary that influence the prophase interactions between homologous chromosomes have an important role; environmental effects may act at several different stages of oogenesis to influence the likelihood of mistakes (1). Capalbo et al. (5) provided a comprehensive description of the chromosomal segregation events taking place from female meiosis to the Figure 1 - Incidence of aneuploidies in preimplantation and prenatal period. A natural selection against aneuploid embryos from the preimplantation period onward results in the gap in terms of incidence of embryonic aneuploidies, when compared to maternal age, revealed by PGS versus prenatal diagnosis (PND). Implantation failure, but especially miscarriage events reside in this gap, and the only manageable tool to minimize these risks during IVF cycles is to perform CCS-based PGS on TE biopsies. Current Trends in Clinical Embriology 2015; 2 (2):

3 A. Capalbo et al. Figure 2 - The theory of PGS. PGS should obtain same efficacy, namely the same number of newborns per started cycle, and improved efficiency, namely less time, effort and risks to achieve this outcome, compared to standard IVF. By preventing aneuploid embryos from being transferred, PGS should ensure a lower miscarriage rate, a lower number of failed attempts and no newborns affected from chromosomal syndromes. blastocyst stage of preimplantation embryo development. In their study they accurately described the origin of female-derived meiotic aneuploidies based on the confirmation of PB data in both blastomere and trophectoderm (TE) samples. Their observations in AMA patients revealed a pattern of multiple meiotic errors, typically caused by chromatid miss-segregation and arising predominantly at meiosis II as a cause of chromatid nondisjunction. Most of the first meiotic aneuploidies arose as a consequence of premature sister chromatid predivision, leading to balancing events in about half the case during the second meiosis. Notably, all cases of MI errors balanced at MII had a normal mitotic chromosomal segregation until the blastocyst stage, suggesting no downstream effect of premature sister chromatid errors during preimplantation embryo development. The prevalence of aneuploidies in sperm is much less significant compared to oocytes, as it is never higher than 3-4% (3), and their exclusive incidence on embryo chromosomal component has been estimated to be 6,2% (6). Another source of aneuploidies lies in post-zygotic derived chromosomal segregation error that could arise during the first cleavage divisions of preimplantation embryos. These errors generate in preimplantation embryos the wellknown phenomenon of chromosomal mosaicism, which consists in the presence of more than one cell line with a different set of chromosomes. Mitotic chromosome errors are the main cause of this phenomenon and could be induced mainly by three mechanisms: anaphase lagging, non-disjunction and structural events of DNA damage of chromatid/chromosome breakage leading to structural rearrangements (e.g. duplications, translocations) (7, 8). An impressive influence of mosaicism up to 70% in preimplantation embryos has been reported in some previous studies (9-12), although some of them should be evaluated critically, especially if based on single cell analysis, since a different diagnosis in cells from the same embryo could be due to a technical error rather than real biological mosaicism. More recent studies suggest a prevalence of euploid/aneuploid mosaicism at the blastocyst stage lower than 5% (13, 14). What is the best time to perform biopsy? One of the main issues for the outcome of PGS programs concerns the type of cell to be biopsied and screened. The biopsied sample should be representative of the embryos chromosomal constitution and viability after transfer. There are different 58 Current Trends in Clinical Embriology 2015; 2 (2): 56-64

4 Preimplantation genetic screening: definition, role in IVF, evolution and future perspectives possible sources of genetic material that can be tested in the preimplantation period in patients undergoing an IVF cycle: (i) the first and second PBs (PBs approach); (ii) one or two blastomeres biopsied from 5- to 10-cell cleavage-stage embryos on Day 3 and (3) several trophoblast cells (usually 5-10) sampled from the blastocyst. Each of these stages offers specific diagnostic advantages as well as critical limitations. PBs-based PGS is the only practice ethically acceptable in some countries, and it is compatible with fresh ET after molecular diagnosis. However, this approach shows important limitations that could be source of errors: the inability to assess MI errors balanced at MII, the influence of male and mitotic-derived aneuploidies and the paucity of available material (around 10% of the oocytes tested remain without a conclusive diagnosis because of amplification failure in one or both PBs). Capalbo et al. (5) demonstrated that the accuracy of PBs approach was significantly lower compared to single blastomere analysis when results were correlated with blastocyst karyotype. These findings can be mainly explained by (i) the high false-positive rate obtained by PBs approach because of the inability to consistently identify the MI errors balanced at MII; (ii) the considerable proportion of female meiotic aneuploidy correction at the blastocyst stage and (iii) the high rate of male and/or mitotic derived aneuploidies observed in the embryo. These intrinsic limitations of PBs analysis may lead on one side to discard potentially healthy embryos and on the other side to the transfer of abnormal ones. At last, from an economic and logistic perspective, 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 (15). Single blastomere analysis is also affected by all the limitations that concern single cell diagnostics. From a technical point of view several artifacts can be introduced potentially causing false positive and false negative results. In particular, these artifacts can turn out in erroneous copy number assessments, since few loci or whole chromosomes could be under- or over-amplified (16), and can be listed as follows: i) Allele drop-out (ADO), that is random loss of alleles; ii) Preferential allocation (PA), namely over-amplification of specific genomic region or even a whole chromosome; iii) Allele drop-in (ADI), which is an artifact of whole genome amplification substituting an allele with another one; iv) Chimerical DNA molecules formation; v) failure of DNA amplification occurring more often. Moreover, none of the current methods for single-cell analysis can distinguish between a cell in G1-, S- or G2/M-phase of the cell cycle. This could determine biological false negative/ positive results, in case a cell during replication phase is retrieved for the analysis (17). Another biological issue regarding PGS analysis on a single blastomere at the cleavage stage resides in chromosomal mosaicism phenomenon. It is known that mosaicism phenomenon reaches its highest level at cleavage stage when the cell cycle control is carried out by maternal transcripts still present in the ooplasm and some checkpoint mechanisms lack an appropriate control until embryonic genome activation (18). Furthermore it is demonstrated that mosaic euploid embryos undergo a self-correcting mechanism at blastocyst stage (19) thus leading to an increasing risk of false positive diagnosis by cleavage stage PGS. Finally, Scott et al. (20) showed that even a single blastomere removal is sufficient to compromise embryo implantation potential, thus highlighting another noteworthy issue of performing PGS at the cleavage stage, that is embryo impairment. Blastocyst stage TE biopsy, instead, ensures a more robust evaluation of aneuploidies than previous strategies, since between five to ten cells are retrieved and analysed from the embryo. This translates in a significant reduction of the incidence of all the misdiagnosis risks derived from a single cell analysis. In fact the available material is more abundant, hence the risk of not obtaining a conclusive diagnosis is lower than in previous cases. Again an important aspect to consider in PGS analysis on randomly selected TE cells is the impact of mosaicism, and in particular the possibility of a non-random allocation of chromosomally abnormal cells exclusively to TE. Capalbo et al. (13) demonstrated that CCS of cells sampled from TE is unlikely to be confounded by mosaicism and accurately predicts the chromosome complement of the inner cell mass (ICM). The study design involved a preliminary acgh (array comparative genomic hybridization) analysis on a TE biopsy during blastocyst-stage PGS clinical cycles, followed by FISH (fluorescence in situ hybridization) re-analysis of three further fragments of TE and of the ICM from those blastocysts diagnosed as single and double aneuploid as well as euploid for chromosome copy number (unbalanced diploid embryos). The results of this research revealed that at the blastocyst stage of development, 79.1% of the aneuploidies was consti- Current Trends in Clinical Embriology 2015; 2 (2):

5 A. Capalbo et al. tutional, while 20.9% of them was mosaic. However, only 4% of the blastocysts was found to be mosaic diploid/aneuploid, being at risk of misdiagnosis due to mosaicism when testing at the blastocyst stage. A more recent study performing a blinded comparison of different TE biopsies from the same blastocyst and using different aneuploidy screening methods, found a consistent chromosome copy number diagnosis in 99.4% (2561/ 2576; 95%CI ) of the chromosomes analysed (21). These data support the theory that the impact of mosaicism could be critical at day 3 of embryo development, but it has definitely less influence at the blastocyst stage. One consideration that had been argued is that postponing the embryo transfer in a period subsequent to cleavage stage could result in a decrement in pregnancy rate. This possibility has been excluded because Glujovsky et al. (22) comprehensively demonstrated by a Cochrane review entailing 12 randomized controlled trials (RCTs) that the extension of the culture to the latest stage of preimplantation development increases the number of live births per transfer during IVF cycles. Finally, another issue that has been raised against the biopsy at blastocyst stage is the possible damage that the removal of a small number of cells could cause to embryo viability and implantation rate. Scott et al. (20) performed a non-selection paired RCT to determine if cleavage or blastocyst stage embryo biopsy affect reproductive competence. After selecting two embryos for transfer, one was randomized to biopsy and the other to control. Both were transferred within shortly thereafter. The biopsy was submitted for microarray analysis and single-nucleotide polymorphism (SNP) profiling. Fetal DNA was obtained from maternal blood or buccal DNA from the neonate after delivery and compared with that of the embryonic DNA. A match confirmed that the biopsied embryo implanted and developed to term, whereas a nonmatching indicated that the control embryo had led to the delivery. This paired non-selection study clearly demonstrated that the stage of embryonic development when biopsy is performed significantly affects the safety of the procedure. TE biopsy at the blastocyst stage had no meaningful impact on the developmental competence of the embryo as measured by implantation and delivery rates, although blastomere biopsy at the cleavage stage produced a dramatic 39% relative reduction in the probability that an embryo would implant and progress to delivery. One possible explanation to the fact that biopsy at blastocyst stage is safer than at cleavage stage is that the technique involves removal of a smaller proportion of the embryo s total cellular content. Another explanation could be provided by the fact that only extra-embryonic (trophectoderm) cells are biopsied. In contrast, the lineage specific developmental fate of an individual blastomere is unpredictable by morphology (23). Finally, it is possible that blastocysts possess increased tolerance to manipulation compared with cleavage-stage embryos as a result of having already undergone embryonic genome activation (20). From an economic and logistic perspective, blastocyst stage PGS on TE biopsy, conversely to previous strategies and especially to PBs biopsy one, represents the less expensive approach and the easiest to implement. These is mainly due to the fact that only developmentally competent embryos would reach to this stage and be screened for aneuploidies, while incompetent ones will arrest at previous stages of development. This results also in PGS costs reduction with the considerable advantage of being able to increase the patient population that can benefit from this technology during their IVF cycle (Table 1). Is blastocyst morphology useful to select between euploid embryos? One outstanding issue is to implement embryo evaluation methods beyond aneuploidy screening to further enhance selection among euploid blastocysts. Capalbo et al. (24) performed a multicenter retrospective observational study to assess if conventional blastocyst morphological evaluation correlates with euploidy (as assessed by CCS of TE biopsies) and implantation potential. The study included the data analysis of 956 blastocysts with conclusive CCS results obtained from 213 infertile AMA, RIF and RPL patients. Single frozen embryo transfer (FET) cycles of 215 euploid blastocysts were performed where it was possible to track the implantation outcome of each embryo transferred. Prior to TE biopsy for CCS, blastocyst morphology was assessed and categorized in four groups (excellent, good, average and poor quality). The developmental rate of each embryo reaching the expanded blastocyst stage was defined according to the day of biopsy post-fertilization (Day 5 and Day 6 biopsied blastocysts were defined as faster and slower growing embryos, respectively). Among the embryological variables assessed (morphology and developmental rate), only blastocyst morphology slightly correlated with CCS da- 60 Current Trends in Clinical Embriology 2015; 2 (2): 56-64

6 Preimplantation genetic screening: definition, role in IVF, evolution and future perspectives Table 1 - Advantages and disadvantages of chromosome testing on PBs, blastomeres and TE biopsies. POLAR BODIES BLASTOMERE TROPHECTODERM ADVANTAGES Only practice ethically acceptable in some countries Compatible with fresh ET Compatible with fresh ET Highest worldwide expertise Compatible with fresh ET Robust genetic diagnosis Lower impact of mosaicism No impact on the developmental competence of the embryo Less expensive and time consuming approach ta. In particular, euploidy rate was 56.4, 39.1, 42.8 and 25.5% in the excellent, good, average and poor blastocyst morphology groups, respectively. The implantation potential of euploid embryos was the same, despite different morphologies and developmental rates (Figure 3). This study provides knowledge for a better laboratory and clinical management of blastocyst stage PGS cycles suggesting that the commonly used parameters of blastocyst evaluation (morphology and developmental rate) are not good indicators to improve the selection among euploid embryos. Therefore, all poor morphology and slower growing expanded blastocysts should be biopsied and similarly considered for FET cycles. This knowledge will be of critical importance to achieve similar cumulative live birth rates in PGS programs compared with conventional IVF, avoiding the potential for exclusion of low quality, but viable embryos from the biopsy and transfer procedures. A prospective nonselection study is currently ongoing in our centers to corroborate these findings. Conclusions and future perspectives DISADVANTAGES Paucity of available material Unable to assess MI errors balanced at MII Unable to assess the influence of male and mitotic-derived aneuploidies Prone to technical errors of single cell analysis Time-consuming and wasteful technique Unknown effect on the implantation potential and developmental competence of the embryo Paucity of available material Prone to technical errors of single cell analysis Unable to distinguish between a cell in G1-, S- or G2/M-phase Highest prevalence of chromosomal mosaicism phenomenon along preimplantation development Impact on the implantation potential and developmental competence of the embryo Not compatible with fresh ET with most of the genetic screening platform used in PGS Need optimized embryo culture system and cryopreservation protocols The purpose of this review was to emphasize the key role that CCS based PGS, especially if performed at the blastocyst stage, plays in ART. When an effective PGS strategy is implemented in IVF programs, then many advantages can be expected, ranging from increased implantation rate, because euploid embryos implant at a higher rate compared to chromosomally abnormal embryos, and a significant decrease in abnormal pregnancies occurrence and in the abortion rate. Importantly, the implementation of PGS might lead to adopt a single ET policy also in poor prognosis patient avoiding any kind of obstetrical and neonatal complication associated with multiple pregnancies. Furthermore, it is expected that in PGS programs less time would be invested to achieve a pregnancy, since non-useful and potentially detrimental ETs will be avoided. Up to date, several RCTs have been published providing evidences of a higher efficiency when CCS-based PGS on TE biopsies is performed with respect to standard IVF, in different patient populations and adopting different methods of 24chromosome screening (qpcr, acgh or asnp) (25-27) (Table 2). However, we still lack RCTs providing evidences on a per cycle base, thus aiming at certifying that the efficacy of IVF, namely the number of baby born per started treatment, is not compromised by PGS. Although there is growing evidence that aneuploidy may be the single most important factor determining the ability of an em- Current Trends in Clinical Embriology 2015; 2 (2):

7 A. Capalbo et al. Figure 3 - Implantation rate of embryos of different morphology and developmental timing. a) Implantation rate in excellent, good, average and poor blastocyst morphology groups; b) Implantation potential of euploid embryos at different developmental rates. Table 2 - Randomized controlled trials published on the use of blastocyst stage comprehensive chromosome screening. *Single embryo transfer. **Double embryo transfer. ***Frozen embryo transfer. METHODS CONCLUSIONS REFERENCES Good prognosis patients (age <35; no previous miscarriages); acgh based CCS and fresh ET N103 Increased ongoing implantation rate of euploid blastocysts ( %) (17) Good prognosis patients (21-42 years); qpcr based CCS and fresh ET N155 Female age <43; qpcr based CCS and fresh or frozen ET N175 Increased ongoing implantation rate of euploid blastocysts ( %) Similar ongoing pregnancy rate of eset* of euploid blastocyst ( %) and lower multiple pregnancies compared with DET** of untested blastocysts (0 53.4%); Similar cumulative pregnancy rate (69 72% fresh + 1 FET ) of eset of euploid blastocysts compared with DET of untested blastocysts Better obstetrical and perinatal outcomes (lower risk for preterm delivery, low birth weight and Neonatal Intensive Care Unit admission because no multiple pregnancies were obtained in the PGS group) (18) (19) 62 Current Trends in Clinical Embriology 2015; 2 (2): 56-64

8 Preimplantation genetic screening: definition, role in IVF, evolution and future perspectives bryo to implant and form a viable pregnancy, in some cases the transfer of a chromosomally normal, morphologically perfect embryo does not guarantee an established pregnancy. Clearly, there are other, less well defined aspects of embryo biology that are also critical for successful development. Future research in IVF is required to identify additional biomarkers of reproductive potential and to further enhance selection among euploid blastocysts. The advent of next generation sequencing (NGS) technologies is opening a new avenue of research in IVF to study embryo biology on a multidimensional scale and to use new knowledge to develop non invasive biomarker for embryo assessment. It is now possible to study single cells genome, epigenome, transcriptomics and secretomics at high resolution. NGS strategies have the potential to simultaneously provide data on mtdna copy number and mutations together with chromosome aneuploidies. This may provide an extra dimension to embryo screening. The first challenge is finding markers that will remain valid in all sorts of genetic backgrounds, hormonal treatments and clinical conditions. The second challenge is related to create a robust process to assess the level of a given marker or a combination of them in a clinical environment. This would be the goal to achieve in the near future. References 1. Nagaoka SI, Hassold TJ, Hunt PA. Human aneuploidy: mechanisms and new insights into an age-old problem. Nat Rev Genet. 2012;18;13 (7): Heffner LJ. Advanced maternal age-how old is too old? N Engl J Med. 2004;4;351(19): Cimadomo D, Capalbo A, Rienzi L, Ubaldi FM. Preimplantation genetic screening and related issues. Maternal & prenatal testing. CLI. 2014;6. 4. Franasiak JM, Forman EJ, et al. 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9 A. Capalbo et al. 22. Glujovsky D, Blake D, Farquhar C, et al. Cleavage stage versus blastocyst stage embryo transfer in assisted reproductive technology. Cochrane Database Syst Rev. 2012;11;7:CD Hansis C, Edwards RG. Cell differentiation in the preimplantation human embryo. Reprod Biomed Online. 2003;6(2): Capalbo A, Rienzi L, Cimadomo D, et al. Correlation between standard blastocyst morphology, euploidy and implantation: an observational study in two centers involving 956 screened blastocysts. Hum Reprod. 2014; 0(0): Yang Z, Liu J, Collins GS, Salem SA, Liu X, Lyle SS, Peck AC, Sills ES, Salem RD. Selection of single blastocysts for fresh transfer via standard morphology assessment alone and with array CGH for good prognosis IVF patients: results from a randomized pilot study. Mol Cytogenet. 2012;5(1): Forman EJ, Hong KH, Ferry KM, Tao X, Taylor D, Levy B, Treff NR, Scott RT Jr. In vitro fertilization with single euploid blastocyst transfer: a randomized controlled trial. Fertil Steril. 2013;100(1): Scott RT Jr, Upham KM, Forman EJ, Hong KH, Scott KL, Taylor D, Tao X, Treff NR. Blastocyst biopsy with comprehensive chromosome screening and fresh embryo transfer significantly increases in vitro fertilization implantation and delivery rates: a randomized controlled trial. Fertil Steril. 2013;100(3): Current Trends in Clinical Embriology 2015; 2 (2): 56-64