Preimplantation aneuploid embryos undergo selfcorrection in correlation with their developmental potential

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1 GENETICS Preimplantation aneuploid embryos undergo selfcorrection in correlation with their developmental potential Shiri Barbash-Hazan, B.Sc., a, *,# Tsvia Frumkin, M.Sc., a, * Mira Malcov, Ph.D., a Yuval Yaron, M.D., b Tania Cohen, M.Sc., a Foad Azem, M.D., a Ami Amit, M.D., a and Dalit Ben-Yosef, Ph.D. a a Racine IVF Unit and b Prenatal Diagnosis Unit, Genetic Institute, Lis Maternity Hospital, Tel-Aviv Sourasky Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Objective: To investigate the incidence of embryos self-correction during preimplantation development in terms of mosaicism and in correlation with its developmental stage. Design: Prospective study to compare the chromosome status of embryos on day 3 with that of day 5, in correlation with their developmental stage. Setting: In vitro fertilization unit of a university-affiliated hospital. Patient(s): Eighty-three aneuploid embryos. Intervention(s): Fluorescence in situ hybridization (FISH) reanalysis. Main Outcome Measure(s): Day 3 embryos classified as mosaic or chromosomally abnormal by preimplantation genetic screening (PGS) were reanalyzed on day 5. The results were evaluated in correlation with the embryos developmental stage. Result(s): Out of 83 day 3 aneuploid embryos, 15 (18.1%) were diagnosed with mosaicism. The FISH reanalysis on day 5 demonstrated that 27 embryos (32.6%) were partly or entirely normal disomic. Of these 83 aneuploid embryos, 8 (9.7%) underwent complete self-correction. The PGS results demonstrated that 26.5% of the embryos were trisomic, of which 41.0% underwent trisomic rescue by day 5. Self-correction was in correlation with the embryo s developmental stage, i.e., 38.1% of aneuploid embryos that developed to the blastocyst stage underwent self-correction compared with only 12.5% of embryos that only cleaved after biopsy. Conclusion(s): Our results demonstrate that self-correction of aneuploid and mosaic embryos occurs probably more significantly during development toward the blastocyst stage than in delayed embryos. In addition, trisomic embryos correct themselves more than other aneuploidies. These findings suggest that PGS results must be interpreted with caution. (Fertil Steril Ò 2009;92: Ó2009 by American Society for Reproductive Medicine.) Key Words: Preimplantation embryos, aneuploidy, mosaicism, self-correction, PGS Received May 19, 2008; revised July 10, 2008; accepted July 15, 2008; published online October 1, * The first two authors contributed equally to this work. S.B.-H. has nothing to disclose. T. F. has nothing to disclose. M.M. has nothing to disclose. Y.Y. has nothing to disclose. T.C. has nothing to disclose. F.A. has nothing to disclose. A.A. has nothing to disclose. D.B.-Y. has nothing to disclose. # Submitted by S. Barbash-Hazan as M.D. thesis, Faculty of Medicine, Ben Gurion University, Be er Sheba, Israel. Reprint requests: Dalit Ben-Yosef, Racine IVF Unit, Lis Maternity hospital, Tel-Aviv Sourasky Medical Center, Tel-Aviv, Israel. 6 Weizmann St., Tel-Aviv 64239, Israel (FAX: ; dalitb@tasmc. health.gov.il). Various embryo assessment procedures have been developed to select top-quality embryos with the best implantation potential for transfer during in vitro fertilization (IVF). Most of them are based on the morphologic appearance of the preimplantation embryo in each developmental stage. These methodologies include pronuclei scoring and the timing of first mitotic cleavage during the first day after fertilization (1 3), cleavage rate and pattern, compaction, degree of fragmentation, and mononuclearity of cells in the cleaving embryo (4). It is not clear, however, how many of these apparently normally developing good-quality embryos are chromosomally normal (euploid). The most practical technique for analyzing the chromosomal constitution of viable human preimplantation embryos is fluorescence in situ hybridization (FISH) analysis, which has been performed on human embryos obtained from infertile couples with repeated IVF failures as well as from fertile couples who are carriers of genetic diseases and who undergo preimplantation genetic diagnosis (PGD) (5 10). The results of these studies demonstrate that only 50% of IVF preimplantation embryos are euploid. The euploidy rate is only slightly higher in embryos of young fertile patients (55% 60%). It should be borne in mind that embryos that were available for FISH analysis had been obtained from individuals who had undergone hormonal stimulation for either 890 Fertility and Sterility â Vol. 92, No. 3, September /09/$36.00 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert

2 PGD or egg donation for research, including those who were fertile (3, 11, 12). Selection for only good-quality embryos, based on morphologic criteria, increased the proportion of euploid embryos to 60% 70% (12). Importantly, embryos with a normal cleavage rate have the best chance to be chromosomally normal (13), whereas either lagging or rapidly cleaving embryos have higher rates of chromosomal aberrations (12). Another strategy for selecting euploid embryos with the best implantation potential is based on the embryo s competence to reach the blastocyst stage at day 5 after fertilization (3, 14, 15). However, although culture to blastocyst allows self-selection of most viable embryos, still only 65% of them are chromosomally normal (3). Therefore, although the selection of top-quality embryos by means of morphologic parameters may increase the ability to choose chromosomally normal embryos, nonetheless 30% 40% of them will still be aneuploid. Preimplantation genetic screening (PGS) has been adopted as a method to select chromosomally normal embryos for transfer, aimed at increasing the pregnancy rate in repeated IVF failures with reasonable numbers of good-quality embryos. Preimplantation genetic screening (PGS) is based on the hypothesis that chromosomal errors could represent an underlying cause of the infertility, and that transfer of aneuploid embryos may explain some of the IVF failures. Recently however, two randomized controlled trials failed to show any benefit for performing PGS, using live birth rates as the measure of success (3, 16, 17). The debate on the usefulness of PGS is ongoing, and the only effective way to resolve this issue is to perform additional well designed and well executed randomized clinical trials (18). Aneuploid embryos are a consequence of either fertilization that involves an aneuploid oocyte or sperm which resulted from meiotic errors, or a mitotic error that occurred de novo after fertilization. Meiotic errors commonly lead to a uniformly aneuploid embryo, whereas mitotic errors usually lead to mosaicism. Preimplantation genetic screening identifies meiotic and post-zygotic chromosomal abnormalities. However, because it is based on FISH results obtained from only one or two biopsied blastomeres, it is not yet clear whether chromosomal screening of single blastomeres on day 3 truly represents the chromosomal constitution of the embryo that will eventually implant 3 4 days later. This uncertainty led the PGD Consortium to recommend that embryos that had been designated as aneuploid by PGS on day 3 undergo FISH reanalysis on day 5 (19). Subsequent studies that summarized FISH reanalyses of aneuploid embryos on day 5 demonstrated that the chromosomal constitution of a preimplantation embryo may change during early cleavages (20, 21). Indeed, it was suggested that some of them are capable of developing into normal euploid embryos or that there could be an increase in the normal diploid cells that comprise the blastocyst. Other reports, however, suggested that these embryos may accumulate additional chromosomal anomalies (20 24). TABLE 1 Embryo development profile of the 83 studied embryos. Fertilization Mean ± SD/cycle Oocytes PN Fertilization (2PN/MII) (%) 75.1% 18.3% Day 3 embryos Blastomeres/embryo High-quality a embryos 78.3% (% of total) Embryo biopsy n (% of total) Embryos with 1 cell biopsy 17 (20%) Embryos with 2 cell biopsy 66 (80%) Day 5 embryos n (% of total) Blastocysts 21 (25.3%) Compacted morula 33 (39.7%) Further cleavage 16 (19.3%) Cleavage arrest 13 (15.6%) Note: MII ¼ metaphase II; 2PN ¼ two pronuclei. a High-quality embryos indicate the presence of six to eight cells at grade I II or compacted morula. Because all of these studies were based on reanalysis of aneuploid embryos diagnosed by PGS that had been performed solely on one blastomere, they bear potential limitations of false negative and false positive results when mosaic embryos are involved. In the present study, we investigated the incidence of an embryo s self-correction during preimplantation development in terms of mosaicism and in correlation with its developmental stage to supplement our current knowledge on this subject. TABLE 2 FISH results of day 3 embryos a. Chromosomal constitution No. (%) Trisomy 22 (26.5%) Monosomy 23 (27.7%) Multiple aberration 14 (16.9%) Mosaic between blastomeres 15 (18%) normal/abnormal 7 (8.4%) abnormal/abnormal 8 (9.6%) Haploidy 1 (1.2%) Polyploidy 6 (7.3%) Undetermined 2 (2.4%) Note: FISH ¼ fluorescent in situ hybridiization. a Total of 83 aneuploid embryos. Fertility and Sterility â 891

3 MATERIALS AND METHODS Study Material A total of 83 embryos classified by PGS as chromosomally abnormal were reanalyzed. Institutional Review Board approval for this study was obtained (295/03, 216/06), and written consent was provided by each couple. The aneuploid embryos were the result of 22 PGS cycles (19 women). Indications for PGS were repeated implantation failure (16 women), with an average (SD) of previous failed IVF cycles per subject, and/or recurrent miscarriages (6 women), with an average of previous pregnancy losses per subject. The mean maternal age was years (Table 1). The mean number of two-pronuclei (2PN) zygotes was per cycle (Table 2). Fertilization and Embryo Culture Ovarian stimulation, ovulation induction, and oocyte retrieval were performed as described previously (25). Intracytoplasmic sperm injection (ICSI) was performed to avoid contamination of the biopsied blastomere with sperm DNA or maternal DNA of cumulus cell origin. Metaphase II oocytes were meticulously denuded of cumulus cells using hyaluronic acid in combination with mechanical pipetting. Normal fertilization was determined by the presence of 2PN with two distinct polar bodies at h after ICSI using an inverted microscope at 400 magnification (TE; Nikon, Tokyo, Japan). Each embryo was incubated in a separate drop of medium to allow individual assessment and documentation at different time points during preimplantation development until the moment of intrauterine transfer. Embryo morphology was assessed on the mornings of days 2 and 3 following oocyte retrieval, and the number of blastomeres, degree of fragmentation cytoplasm uniformity, and the presence of early compaction were recorded. Embryo biopsy was performed on day 3 of embryo development at the 6 10-cell stage, as described previously (25). One blastomere was removed from embryos with <7 cells and two blastomeres were removed from embryos with R7 cells. Selected blastomeres were subjected to FISH analysis and classified as either normal or aneuploid. On day 5, chromosomally normal embryos were transfered back to the uterus and aneuploid embryos underwent FISH reanalysis. FISH Analysis The FISH analysis was performed at two time points during embryo development: 1) on day 3, on individually biopsied blastomeres as part of the PGS procedure; and 2) on day 5, when the entire developed embryo, including all blastomeres comprising it, was spread onto a glass slide and subjected to FISH analysis. Blastomeres and aneuploid embryos were spread onto a Superfrost Plus glass slide (Kindler, Freiburg, Germany) using 0.01 N HCl/0.1% Tween 20 solution (26). The spreading solution was used to dissolve the zona pellucida and cytoplasm and to expose the nuclei for FISH analysis. The slides were left to air dry, washed in phosphate-buffered saline for 1 min, and dehydrated by transfering through an ethanol series (70%, 85%, and 100%). All embryonic nuclei were scanned under a phase-contrast microscope. Fluorescence in situ hybridization was carried out according to the manufacturer s recommendations and as described previously (27). Two rounds of FISH procedures were carried out using Multivision PB Probe Panel (Vysis, Downers Grove, IL) for chromosomes 13, 16, 18, 21, and 22 in the first round and X, Y, and 15 (CEP X a-satellite, Xp11.1-q11.1, CEP Y a-satellite, Yp11.1-q11.1, and CEP 15 satellite III, 15q11.2; Vysis) in the second round. The FISH images were captured using a computerized system (FISHView; Applied Spectral Imaging, Migdal HaEmek, Israel), and the results were interpreted by two observers. The criterion for signal scoring was that signals had to be a minimum of a signal s width apart to be scored as two separate signals (28). Embryos were classified as normal when both nuclei of two analyzed blastomeres showed two signals for each chromosome investigated, aneuploid when both nuclei demonstrated the same chromosomal abnormality, and mosaic when the two nuclei demonstrated different results (one normal and the other abnormal, or two different abnormalities). If only one blastomere was available for diagnosis, the embryo could be classified as either normal or aneuploid but not mosaic. If a blastomere showed aneuploidy for two or more chromosomes, it was defined as having multiple aberrations (MAB). A FISH reanalysis on the entire embryos was performed on day 5 using the same probe panel and the same protocol described for day 3 blastomeres. RESULTS A total of 83 embryos were analyzed on day 3, of which 65 (78.3%) were defined as high-quality embryos, with an average of blastomeres per embryo. Two blastomers were biopsied from each of 66 embryos (80%) which had R7 cells and subjected to FISH analysis (Table 1). The FISH results demonstrated that 22 embryos (26.5%) were trisomic, 23 (27.7%) were monosomic, and 14 (16.9%) had MAB (aneuploidy of >1 chromosomes) (Table 2). Mosaic embryos could also be identified, because two blastomeres were biopsied from most of the embryos studied: 15 embryos (18%) were found to be mosaic, with seven (8.4%) displaying mosaicism of both normal and abnormal cells and eight (9.6%) displaying different aneuploidy between the two analyzed blastomeres. In addition, one embryo was haploid, six embryos (7.3%) were polyploid, and two (2.4%) were undetermined (Table 2). For the purpose of FISH reanalysis, aneuploid embryos were further incubated for another 2 days, and 70 out of 83 (84%) embryos continued to develop following blastomere biopsy: 21 (25.3%) underwent blastulation, 33 (39.7%) underwent compaction, and 16 (19.3%) further cleaved but with no signs of compaction (Table 1). The FISH reanalysis on day 5 was performed on whole embryos, regardless of the stage of development they had reached. The average 892 Barbash-Hazan et al. Self-correction of aneuploid embryos Vol. 92, No. 3, September 2009

4 TABLE 3 Day 5 reanalysis of the 83 aneuploid embryos studied. Day 5 embryos No. (%) Mean no. of cells analyzed per day 5 embryo Embryos with confirmation of 26 (31.3%) day 3 FISH results Embryos with partly or entirely 27 (32.6%) normal cells Embryos that acquired 30 (36.1%) additional aneuploidies FISH results Normal 8 (9.6%) Trisomy 8 (9.6%) Monosomy 7 (8.4%) Mosaic between blastomeres 54 (65%) Normal/abnormal 19 (23%) Abnormal/abnormal 35 (42%) Haploidy 1 (1.2%) Polyploidy 5 (6%) number of nuclei analyzed from each day 5 embryo was (1 36 nuclei/embryo, median 7) (Table 3). The FISH results of day 5 embryos confirmed those of day 3 in 26 embryos (31.3%), 30 (36.1%) had acquired additional aneuploidies, and 27 (32.6%) demonstrated normal cells (Table 3) and presumably had undergone self-correction. Of these 27 corrected embryos, eight (9.7%) underwent complete self-correction (presenting 100% normal cells) and were diagnosed as euploid on day 5, eleven (13.2%) had 51% 99% normal cells, and eight (9.7%) had %50% normal cells. Ten of the embryos that underwent major self-corrections (i.e., >50% normal cells) were either trisomic or mosaic on day 3 (Table 4). We further investigated trisomic rescue as one of the proposed mechanisms leading to self-correction, and found that 9 of the 22 trisomic embryos (41.0%) had >50% normal cells at day 5 (Tables 2 and 4). Our results demonstrate a linear correlation between preimplantation embryo development and self-correction rate (Fig 1). The embryos that reached the blastocyst stage by day 5 of development had the highest self-correction rate, 38.1% (8 out of 21), compared with a rate of 24.2% (8 out of 33) correction in compacted morula and only 12.5% (2 out of 16) for embryos that further cleaved but showed no signs of compacting (Fig. 1). DISCUSSION Not all morphologically normal IVF embryos are also chromosomally normal. Although day 3 embryo morphology has been recently shown to be a selection marker of euploidy among advanced maternal age subjects, it had a poor predictive value for euploidy in younger women who may have other factors responsible for embryo dysmorphism (4). Indeed, a high rate of aneuploidy and mosaicism is revealed even when only top-quality IVF embryos are analyzed by FISH. For obvious ethical reasons, comparative studies on chromosome abnormalities in in vivo derived and in vitro produced human embryos are not available. Studies on other mammalian species (mouse, cow, sheep), however, have demonstrated a higher rate of aneuploidy in IVF embryos compared with their in vivo counterparts (29). Collectively, these data indicate that human IVF embryos probably have a higher degree of aneuploidy compared with the naturally developed embryos which are not accessible for research. Aneuploid pregnancies may result in miscarriage, stillbirth, or ultimately the birth of a child with an abnormality, such as trisomy 21 or monosomy X (Turner syndrome) (30). The incidence of chromosomal abnormalities in early spontaneous abortions (<7 weeks) is more than 60% compared with 5% in induced abortions at 10 weeks (31 33). This is TABLE 4 Correction rate of embryos through day 5. Chromosomal constitution at day 3 % Correction within 5 days No. of embryos (% of total) Trisomy or mosaic Other aberrations 100% 8 (9.7%) 5 T 1 M, 1 tetra, 1 mab <100% and >50% 11 (13.2%) 2 T þ 1 T*, 2 N/M 3 M,1 haploid, 2 mab %50% 8 (9.7%) 1 T*,1 M 4 mab, 1 poly, 1 haploid Total embryos with corrected cells 27 (32.6%) Note: haploid ¼ haploidy; M ¼ monosomy; mab ¼ multiabnormal; N/M ¼ mosaic for normal and monosomy; poly ¼ polyploidy; T ¼ trisomy; T* ¼ mosaic of two different trisomies; tetra ¼ tetraploidy. Fertility and Sterility â 893

5 FIGURE 1 Correction rate in relation to developmental stage at day 5. Correction rate * (%) Blastocyst 24.2 Compacted Morula 12.5 Further Cleavage probably an underestimation of the true rate of chromosome abnormalities in preimplantation embryos and early fetuses, owing to early natural selection of chromosomal aberrations. These data suggest that the incidence of chromosome abnormality at conception is high and that natural selection occurs before and after implantation. The FISH results of human IVF embryos demonstrated that around 70% of the best-quality embryos are chromosomally normal. This estimation, however, is based on the extrapolation of values to the entire embryo from single blastomeres sampled for PGS application (12, 34). Some of the aneuploid embryos, however, subsequently acquire chromosomal abberations at a later stage (21). Conversely, some of the aneuploid embryos undergo self-correction during cleavage and propagation toward the blastocyst stage (22 24). Munne et al. (24) compared PGS results with the chromosomal status of the derived blastocysts 2 days later and found that 65% of them comprised normal cells. Li et al. (23) examined whole blastocysts and classified 44% of the embryos as euploid. It was previously demonstrated that the process of culturing to the blastocyst itself allows self-selection of euploid embryos (3, 35). In addition, euploid day 3 embryos develop more blastocysts than aneuploid ones (35, 36). Indeed, when embryos from all developmental stages, including delayed embryos, were reanalyzed by FISH, only 20% of the aneuploid ones had undergone self-correction (20, 22). 7.6 Arrest *Correction rate ¼ embryos with >50% of normal cells in relation to developmental stage. We present new data on self-correction of aneuploid and mosaic embryos, and demonstrate its correlation to embryo development. For the analysis of self-correction, we combined both the >50% corrected embryos (i.e., partially corrected) and the completely corrected embryos. The rationale for doing this was based on the observation that when at least two-thirds of the cells comprising the mosaic preimplantation embryo are normal, it can develop into a normal blastocyst and even to a normal live birth (23). Therefore, although mosaicism seems to be a frequently occurring phenomenon during the preimplantation period, it can be naturally eliminated during further development. The present results are based on FISH analysis of an average of nine cells per day 5 embryo, thereby enabling us to illustrate a more accurate and representative picture of the embryos chromosomal status at this stage. Peura et al. (37) recently showed that embryonic stem cells derived from PGS-aneuploid embryos were mostly euploid, suggesting that not all aneuploidies diagnosed in IVF embryos are permanent. Two mechanisms have been suggested to explain self-correction: the high incidence of mosaicism within preimplantation embryos, and trisomic rescue. The most common mechanisms leading to aneuploidy mosaicism are postzygotic chromosome loss, chromosome gain, and mitotic nondisjunction (38). The incidence of mosaicism can be as high as 50% of the preimplantation embryos (39, 40). This high rate of mitotic errors is likely due to the deregulation of the mitotic processes and/or temporary relaxation of the centromere function during the several cell divisions that take place after fertilization (38). Additionally, it was speculated that in vitro culture conditions may increase the occurrence of mitotic errors in IVF embryos (41). Mosaicism is detected in about 1% 2% of chorionic villous samples (CVS) during the first trimester of pregnancy (42). A systematic DNA polymorphism analysis of CVS, however, demonstrated that some cases of the confined placental mosaicism will undergo postzygotic aneuploid correction (42). With pregnancy progression through the second trimester, generalized mosaicism of both the placenta and the fetus is observed in only 0.1% 0.4% of viable pregnancies, as detected in amniotic fluid cell cultures (43). It was suggested that there is a preferential allocation of euploid cells to the inner cell mass during early cleavage stage, when the aneuploid cells are directed toward the extra-embryonic compartments (trophectoderm). Thus, a mosaic morula can develop within a chromosomal dichotomy between the inner cell mass and the trophectoderm (29, 40). Conversely, others have shown that the chromosomal abnormalities observed in human blastocysts are equally present in both compartments of the blastocyst (44). The second mechanism suggested for explaining self-correction is trisomic rescue, whose exact mechanism is not known: anaphase lagging or nondisjunction in early postzygotic cell divisions have been proposed (24). We report a 26.5% trisomy rate in day 3 embryos, with 41% of them (9 out of 22) having undergone significant self-correction (>50% of normal cells within a day 5 embryo), probably 894 Barbash-Hazan et al. Self-correction of aneuploid embryos Vol. 92, No. 3, September 2009

6 by a mechanism of trisomic rescue. In accordance, Rubio et al. (35) demonstrated that the trisomic embryos develop more blastocysts than other aneuploid ones. Mosaicism can be identified by PGS only when at least two blastomeres are biopsied, as was done in the current study. Earlier reports have debated between the need to sample two blastomeres for efficient and accurate FISH results and the potential damage incurred to the embryo. Goossens et al. (45) recently reported, in a prospective randomized study, on the diagnostic efficiency and clinical outcome after the biopsy of either one or two blastomeres for PGD, demonstrating that live birth rates were not significantly different in embryos from which two blastomeres were biopsied. When discussing discrepancy between day 3 FISH analysis and day 5 reanalysis, one also needs to bear in mind the possibility of an error in FISH analysis stemming from technical limitations of the procedure. The false-negative and false-positive rates are estimated to be 7% 15 % (28, 46), although the error rate is only 5%, when using probes for two different loci on the same chromosome (38). In conclusion, our current investigation s contribution is threefold. First, because two blastomeres were FISH analyzed in day 3 embryos, mosaicism and its correlation to self-correction prevalence by day 5 could be studied. Second, our study demonstrated that self-correction of aneuploid and mosaic embryos is more likely to occur during development toward the blastocyst stage than in delayed embryos. The fact that whole embryos were reanalyzed on day 5 of development independent of their developmental status or morphologic quality enables the evaluation of self-correction in correlation with the embryo s developmental stage. The results demonstrate that viable embryos developing with a normal cleavage rate and pattern are more capable of correcting their aneuploidy. Moreover, embryos with higher correction capability may catch up and reach normal cleavage and developmental rates. Finally, we showed that trisomic embryos correct themselves more than other aneuploidies, probably owing to self-correction governed by the mechanism of trisomic rescue. Our results further strengthen the accumulating data indicating that PGS on day 3 of embryo development is not always representative of the chromosomal constitution of the implanting embryo. However, PGS may have an important prognostic value in couples with normal karyotype who produce morphologically normal embryos but still experience recurrent IVF failures. In these cases, two blastomeres should be aspirated to increase the accuracy of the PGS results and take into account mosaicism where possible. Nevertheless, the presence of a high aneuploidy rate among IVF embryos as detected by PGS may guide the couple and their physician to choose another route of parenthood. REFERENCES 1. 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