The relationship between blastocyst morphology, chromosomal abnormality, and embryo gender

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1 IN VITRO FERTILIZATION The relationship between blastocyst morphology, chromosomal abnormality, and embryo gender Samer Alfarawati, M.S. a,b Elpida Fragouli, Ph.D., a,b Pere Colls, Ph.D., c John Stevens, M.S., d Cristina Gutierrez-Mateo, Ph.D., c William B. Schoolcraft, M.D., d Mandy G. Katz-Jaffe, Ph.D., d and Dagan Wells, Ph.D., F.R.C.Path. a,b a Nuffield Department of Obstetrics and Gynaecology, John Radcliffe Hospital, Oxford, United Kingdom; b Reprogenetics U.K., Institute of Reproductive Sciences, Oxford Business Park North, Oxford, United Kingdom; c Reprogenetics, 3 Regent Street, Suite 301, Livingston, New Jersey; and d Colorado Center for Reproductive Medicine, Lone Tree, Colorado Objective: To assess correlation between blastocyst morphology and chromosomal status. Design: Observational research study. Setting: An IVF clinic and a specialist preimplanation genetic diagnosis (PGD) laboratory. Patient(s): Ninety-three couples undergoing IVF treatment in combination with chromosome screening of embryos. Intervention(s): Five hundred blastocysts underwent trophectoderm biopsy and comprehensive chromosome screening using comparative genomic hybridization (CGH). The morphology of the embryos was evaluated using standard methods. Main outcome measure(s): Association of aneuploidy and morphologic score. Result(s): A total of 56.7% of blastocysts were aneuploid. One-half of the grade 5/6 blastocysts were euploid, compared with only 37.5% of embryos graded 1/2, suggesting an effect of aneuploidy on blastocyst development. Aneuploidy also had a negative effect on inner cell mass and trophectoderm grades. Morphologically poor blastocysts had a higher incidence of monosomy and abnormalities affecting several chromosomes. The gender ratio was significantly skewed in relation to morphology. A total of 72% of blastocysts attaining the highest morphologic scores (5AA and 6AA) were found to be male, compared with only 40% of grade 3 embryos. Conclusion(s): Morphology and aneuploidy are linked at the blastocyst stage. However, the association is weak, and consequently, morphologic analysis cannot be relied on to ensure transfer of chromosomally normal embryos. A significant proportion of aneuploid embryos are capable of achieving the highest morphologic scores, and some euploid embryos are of poor morphology. Gender was associated with blastocyst grading, male embryos developing at a significantly faster rate than females. (Fertil Steril Ò 2011;95: Ó2011 by American Society for Reproductive Medicine.) Key Words: Morphology, chromosome, aneuploidy, blastocyst, preimplantation genetic screening, single embryo transfer, implantation, in vitro fertilization Received October 11, 2009; revised April 1, 2010; accepted April 6, 2010; published online May 26, S.A. has nothing to disclose. E.F. has nothing to disclose. P.C. has nothing to disclose. J.S. has nothing to disclose. C.G.-M. has nothing to disclose. W.B.S. has nothing to disclose. M.K.-J. has nothing to disclose. D.W. has nothing to disclose. Supported by the U.K. National Institute for Health Research Biomedical Research Centre Programme (D.W.). Reprint requests: Dr. Dagan Wells, Ph.D., F.R.C.Path., University of Oxford, Nuffield Department of Obstetrics and Gynaecology, Level 3, Women s Centre John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom (FAX: ; dagan.wells@obs-gyn.ox.ac.uk). To maximize the success rates of in vitro fertilization (IVF) treatments, a reliable means of recognizing the embryo with the greatest potential for producing a pregnancy is required. Currently, most methods of embryo evaluation involve the assessment of morphologic criteria, such as cell size and number, multinucleation, and the percentage of the embryo volume occupied by cellular fragments (1 4). However, it is acknowledged that such characteristics are only weakly correlated with IVF outcome, and consequently morphologic grading provides only a limited guide to embryo viability. Embryos achieving the best morphologic scores often fail to achieve implantation or do not produce a live birth, and those with poor scores sometimes succeed in producing a child. In many cases, the underlying cause of embryo arrest, implantation failure, or spontaneous pregnancy loss is the presence of numerical chromosomal abnormalities (aneuploidy). The high prevalence of aneuploidy in human oocytes produced during IVF treatments has been recognized for more than a quarter-century (5) and numerous studies have confirmed that human oocytes and preimplantation embryos are frequently affected by chromosome abnormalities (6 12). Indeed, for women >40 years old, it is not uncommon for more than one-half of the oocytes retrieved to be aneuploid (13). In the vast majority of cases chromosome imbalance of this type is lethal to the developing embryo. Many studies have searched for a link between aneuploidy and altered embryo morphology (14 25). Some associations have 520 Fertility and Sterility â Vol. 95, No. 2, February /$36.00 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert

2 been identified, e.g., aneuploidy rates are increased in embryos exhibiting cellular fragmentation, unevenly sized cells, multinucleation, or atypical cell numbers. However, in most cases, these correlations are weak. It is possible that the failure to find a reliable morphologic indicator of aneuploidy in previous studies has been a consequence of technical limitations. Virtually all research and clinical investigations have assessed only a limited number of chromosomes in each embryo, and it is therefore inevitable that some of the embryos categorized as euploid were in fact abnormal, with aneuploidies affecting chromosomes that were not tested. The present study used comparative genomic hybridization (CGH), a molecular cytogenetic method that allows simultaneous enumeration of all chromosomes (26). To date, there has been no attempt to correlate embryo morphology with the more detailed chromosome screening results provided by methods such as CGH. A reevaluation of a potential link between morphology and aneuploidy using data from comprehensive chromosome screening is therefore warranted. MATERIALS AND METHODS Patient Details, Embryo Culture, and Biopsy The data presented were derived from 93 patients undergoing assisted reproductive treatment at the Colorado (U.S.) Center for Reproductive Medicine. The study was conducted after obtaining Institutional Review Board approval. Patients were provided with counseling regarding the study, and signed consents were obtained. The average maternal age of patients was 38.5 years (range years). Most of the patients offered chromosome screening were of advanced reproductive age (>35 y), others had a history of unsuccessful IVF attempts (R2) or miscarriages (R2). Embryos that reached the blastocyst stage were biopsied and then vitrified to allow time for CGH analysis. Euploid embryos were warmed and transfered in a subsequent cycle. CGH results were available for 500 embryos. Ovarian stimulation, embryo culture, and biopsy were performed as previously reported (27). Embryo Scoring Embryos reaching the blastocyst stage were graded by using the system of Gardner and Schoolcraft (28). Blastocysts were given a number based on the degree of expansion and hatching status (from 1 to 6): 1 ¼ early blastocyst: the blastocele accounts for less than one-half of the volume of the embryo; 2 ¼ blastocyst: the blastocoel occupies more than one-half of the volume of the embryo; 3 ¼ full blastocyst: the blastocoel fills the embryo completely; 4 ¼ expanded blastocyst: the blastocoel is now larger than the early embryo, and the zona pellucida has begun to thin; 5 ¼ hatching blastocyst: trophectoderm (TE) cells have begun to herniate through the zona pellucida; and 6 ¼ hatched blastocyst: the blastocyst has completely escaped the zona pellucida. For blastocysts regarded to be full blastocysts and onward (grades 3 6), a second scoring step was performed under an inverted microscope to assess the inner cell mass (ICM) and the TE. For the ICM, the following descriptions are used: A ¼ tightly packed with many cells; B ¼ loosely grouped with several cells; and C ¼ very few cells. For the TE, the following grading is used: A ¼ many cells forming a cohesive epithelium; B ¼ few cells forming a loose epithelium; and C ¼ very few large cells. Comparative Genomic Hybridization The protocol used for processing biopsied cells and the validated CGH method were performed as described by Wells et al. (29), with modifications (30, 31). Briefly, biopsied cells were lysed and their DNA amplified using degenerate oligonucleotide primed polymerase chain reaction. The resulting DNA was subjected to nick translation, allowing nucleotides labeled with a green fluorochrome to be incorporated. DNA from a chromosomally normal individual was similarly amplified and labeled with a red fluorochrome. The two DNAs were mixed and then applied to normal chromosomes on a microscope slide. Red and green DNA fragments hybridized to each chromosome, producing fluorescence (i.e., green:red ratio) proportional to the number of copies of the chromosome in the embryo cell sample. Chromosomes with a green:red ratio of R1.2 were considered to be present in excess (e.g., a trisomy), and those with a ratio of %0.8 were considered to have been lost (e.g., monosomy or nullisomy). RESULTS Of the blastocyst-stage embryos tested, 56.7% were found to be chromosomally abnormal (283 out of 500). A wide variety of aneuploidies were detected. Indeed, it was found that any chromosome can be affected by aneuploidy at the blastocyst stage. A total of 464 chromosomal abnormalities were detected in the aneuploid embryos, with monosomies and trisomies represented almost equally (225 and 239, respectively). Thirty of these errors were a consequence of chromosome breakage, resulting in partial losses or gains of chromosomal material, most of which affected the larger chromosomes (chromosomes 1 11 and chromosome X; Fig. 1). As expected, a strong association between advancing maternal age and aneuploidy was observed, but interestingly individual chromosomes were not equally affected by this phenomenon. Results from the embryos of patients (mean 34.6) years old were compared with those from patients (mean 41) years old. Chromosome 21 showed the greatest increase in the risk of aneuploidy (10-fold increase). Chromosomes 12, 14, and 18 each displayed a 5 6-fold increase, and for chromosomes 1, 2, 11, 15, 17, 20, and 22 the increase was 2-fold. All other chromosomes were associated with little or no change in aneuploid frequency with advancing age. Developmental Rate and Aneuploidy Regarding the growth rate of the embryos, there was a nonsignificant trend toward aneuploid embryos showing slower progression to the most advanced blastocyst stages. Whereas one-half (49.2%, 30 out of 61) of all grade 5 6 embryos were euploid, only 37.5% (18 out of 48) of grade 1 2 embryos were chromosomally normal. Interestingly, fast-growing (grade 5 or 6) aneuploid embryos were more often affected by trisomy than monosomy (ratio of monosomies to trisomies 1:1.7), whereas slower-growing (grade 3) embryos exhibited an equal frequency of monosomies and trisomies (Fig. 2). Complex chromosome abnormalities (abnormalities FIGURE 1 Number of whole (dark bars) and partial (light bars) aneuploidies affecting each chromosome. Fertility and Sterility â 521

3 FIGURE 2 Blastocyst morphologic grading and the proportion of euploid and aneuploid (divided by type) embryos. out of 57) female (Fig. 3). Furthermore, 60% (124 out of 207) of the slowest-growing embryos (grade %3) were female, compared with just 40% (83 out of 207) male. The gender-related difference in developmental stage is highly significant (Fisher exact test: P¼.001). Male embryos were 2.6 times more likely to produce a grade 5 or 6 blastocyst compared with female embryos. Nonsignificant differences between the genders in terms of ICM and TE morphology also were observed: 53% of male embryos achieved an A grade for the ICM compared with 47% of female embryos; 57% of male embryos achieved an A grade for the TE compared with 42% female embryos. Any gender-related differences in TE and ICM are probably a secondary effect of the more rapid developmental progress of the male embryos rather than any direct affect of gender on ICM/TE development. affecting more than two chromosomes) were twice as common among embryos displaying slow growth (grade %3) compared with fast developing embryos (grade 5 or 6). Developmental Rate and Embryo Gender Four hundred fifty-four of the embryos tested had a normal number of sex chromosomes (XX or XY), of which 229 (51%) were female (XX) and 225 (49%) were male (XY). Of the female embryos, 124 (54.2%) had autosomal aneuploidy, compared with 113 (50.2%) of the male embryos. These results demonstrate a 1:1 gender ratio for actively growing day-5 embryos and confirm that the aneuploidy rate is not influenced by the gender of the embryo. Interestingly, the most developmentally advanced blastocysts showed a substantial skew in the gender ratio: 72% (41 out of 57) of embryos scored as grade 5 or 6 were male versus only 28% (16 FIGURE 3 Proportion of male (dark bars) and female (light bars) embryos in each blastocyst development grade. Inner Cell Mass Development Related to Aneuploidy Morphologic evaluation of the ICM revealed a significant impact of aneuploidy (chi square: P<.004). A tightly packed ICM with many cells (grade A) was observed in 62% (121 out of 194) of normal embryos compared with 49% (116 out of 238) of aneuploid embryos. Embryos with a grade A ICM also displayed significant differences from those with a grade B or C ICM regarding the types of aneuploidy detected. For embryos with a high-quality ICM, complex aneuploidy was much less common, detected in only 1.7% (4 out of 237) of embryos with ICM grade A, compared with 11.1% (21/ 190) with ICM grade B. Trophectoderm Development Related to Aneuploidy The distribution of TE grades differed significantly between euploid and aneuploid embryos (chi square: P¼.019). Assessment of embryos graded 1, 2, or 3 showed that 46.4% (90 out of 194) of those that were euploid had a grade A TE (many cells forming a cohesive epithelium) compared with 34.9% (83 out of 238) of those with an aneuploidy. Embryos with poor TE development (grade C) displayed a 2.5-fold increase in the probability of aneuploidy compared with those with grade A. As TE grade declines from A to B to C, there appears to be an increase in the incidence of aneuploidy involving more than one chromosome. The percentage of blastocysts affected by aneuploidy for two or more chromosomes with TE graded A, B, and C was 13% (23 out of 173), 23% (56 out of 242), and 29% (5 out of 17), respectively. However, this was not statistically significant with a sample of this size. Size of Chromosome Involved in Aneuploidy The impact of the size of the chromosome affected by aneuploidy on morphology was explored by comparing embryos with abnormalities affecting the largest chromosomes (chromosomes 1 5) with embryos carrying aneuploidies affecting the smallest chromosomes (21 and 22). For the purpose of this comparison, only embryos with aneuploidy affecting a single chromosome were considered. Embryos with imbalance affecting large chromosomes exhibited a strong trend toward slower development (Fig. 4) as well as poorerquality ICM and TE. Aneuploid embryos with an ICM or TE grade of B were equally likely to have an abnormality of a large chromosome or a small chromosome. Whereas only a minority (39%) of aneuploid embryos with an ICM or TE grade of A had an abnormality affecting a large chromosome. There were insufficient blastocysts with a grade C ICM or TE and a single chromosome abnormality to make a meaningful evaluation of embryos in that category. 522 Alfarawati et al. Aneuploidy and blastocyst morphology Vol. 95, No. 2, February 2011

4 FIGURE 4 Comparison of the growth rate for embryos with aneuploidy affecting chromosomes 1 5 (n ¼ 17; dark bars) and embryos with aneuploidy affecting chromosomes 21 or 22 (n ¼ 38; light bars). Embryos not graded for inner cell mass and trophectoderm had a morphologic score of <3. DISCUSSION Earlier studies have reported associations between aneuploidy and several distinct abnormal morphologies (14 25). However, in most cases the correlations have been weak, and no morphologic marker permitting accurate differentiation of euploid embryos from those with a lethal chromosome abnormality has yet been identified. This failure may simply demonstrate that cleavagestage morphology is a poor guide to aneuploidy. Alternatively, it may be a consequence of errors in the classification of embryos as normal or aneuploid, because previous studies using fluorescence in situ hybridization (FISH) only assessed between one-fifth and one-half of the chromosomes in each cell. The present study illustrates that aneuploidy can affect any chromosome (Fig. 1). It is therefore inevitable that some embryos from earlier studies, with abnormalities affecting untested chromosomes, were wrongly categorized as euploid. Additionally, much of the existing data concerning the effect of aneuploidy on morphology comes from embryos classified by analysis of a single cell after routine preimplantation genetic screening. The high frequency of chromosomal mosaicism at the cleavage stage, including occasional diploid:aneuploid mosaicism, makes it likely that some errors in assigning embryos to normal/abnormal categories were made. In the present study, CGH analysis was used, allowing all of the chromosomes to be accurately assessed. Furthermore, testing was based on the analysis of several cells rather than just one, an approach that theoretically reduces the risk of misclassification due to mosaicism (31). Finally, analysis was conducted at the blastocyst stage (day 5), whereas almost all earlier attempts to associate embryo morphology and aneuploidy focused on earlier stages of preimplantation development (up to day 3). We suggest that the approach used in the present investigation allows conclusions regarding aneuploidy and blastocyst morphology to be drawn with a high level of confidence. More than one-half (56.7%) of all blastocysts tested were chromosomally abnormal. A strong association between maternal age and aneuploidy was observed, increasing from 51% in embryos of patients aged (mean 34.6) years to 60.7% in the embryos of women aged (mean 41) years. Although most of the chromosomes displayed an increase in the probability of aneuploidy with advancing age, the effect was much more dramatic for certain chromosomes, hinting at the existence of different chromosome-specific mechanisms leading to aneuploidy. The chromosome most affected by age was 21, which displayed a 10-fold increase in aneuploidy rate in the older group of patients. This compared with a mean 2.3-fold increase in aneuploidy for all chromosomes. With few exceptions, abnormalities affecting the largest chromosomes, monosomies, and abnormalities affecting multiple chromosomes simultaneously are not observed during prenatal testing. This suggests that such severe anomalies are lethal before the end of the first trimester of pregnancy, although the precise point at which affected embryos succumb remains to be defined. The present study indicates that embryos carrying these types of abnormalities are often capable of forming blastocysts. However, there is evidence that the abnormalities have begun to have a negative effect on embryo development by this time, resulting in poorer ICM and TE development and having a significant impact on blastocyst growth rate. In contrast to the present data, one earlier study suggested that most monosomies are incapable of forming viable blastocysts (32). We speculate that the evolution of highly optimized blastocyst culture methods over the past decade, providing improved support for embryos, may explain the progression of monosomic embryos in the present study. Up until day 3 after fertilization, the survival of embryos with severe forms of aneuploidy is probably facilitated by persistence of proteins (and to a lesser extent, mrna) derived from the oocyte. However, after the onset of embryonic gene expression, at the 4 8-cell stage, the embryo must increasingly rely on the products of its own genome (33). Thus, aneuploidy is likely to have a progressively more dramatic effect as development advances beyond day 3. Many of the highly abnormal embryos detected during the present study had poor TE development and may have been incapable of implantation. Others, with poor ICM formation, may have had the capacity to implant but would probably have been incapable of producing organized fetal tissues, perhaps culminating in a blighted ovum or an early miscarriage. Regarding blastocyst morphology as an indicator of chromosome abnormality, we found increased aneuploidy among blastocysts with poor morphologic scores and a greater likelihood of euploidy for embryos with good scores. However, as with earlier attempts to link aneuploidy and morphology, the distinction was not clear. Fifty-two percent of embryos achieving the top two grades (5AA and 6AA) were euploid, but 48% were abnormal. Conversely, 63% of embryos graded <3 were abnormal, but 37% were euploid. In theory, for the patients examined in the present study, the risk of transfering an aneuploid embryo could have been reduced from 56% (if no morphologic analysis was performed) to 48% if only embryos of 5AA/6AA grades were transfered. However, achieving this modest reduction in aneuploidy risk depends on the availability of embryos with the highest morphologic grades. In reality, not every cycle produces such embryos. Interestingly, the most obvious association between chromosomes and morphology concerned embryo gender rather than aneuploidy. Overall, there was a 1:1 gender ratio for the blastocysts Fertility and Sterility â 523

5 studied. However, male embryos achieved significantly better morphologic scores than equivalent female embryos: 72% of grade 5 and 6 blastocysts were found to be male, whereas 60% of grade 3 blastocysts were found to be female. The disparity in embryo score between male and female is unlikely to reflect any difference in competence, but clearly demonstrates that male embryos tend to reach the final stages of blastocyst development at a faster rate than females. Similar observations have been reported in several mammalian species (34 36). The underlying biologic cause remains unproven at this time, but it may be associated with differences in gene dosage compensation related to X-chromosome inactivation during early preimplantation development, or with the presence of growth factor gene(s) on the Y-chromosome. In summary, aneuploidy affected more than one-half of the blastocysts in this study (mean maternal age 38.5 y). 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