Embryo morphology, developmental rates, and maternal age are correlated with chromosome abnormalities*

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1 FERTILITY AND STERILITY Copyright {j 1995 American Society for Reproductive Medicine Printed on acid-free paper in U. S. A. Embryo morphology, developmental rates, and maternal age are correlated with chromosome abnormalities* Santiago Munne, Ph.D. t+ Mina Alikani, M.S.:j: Giles Tomkin:j: Jamie Grifo, M.D., Ph.D. Jacques Cohen, Ph.D.:j: The New York Hospital-Cornell University Medical College, New York, New York Objective: To determine some of the unresolved questions related to chromosome anomalies in early human embryos, such are the detection of any advanced maternal age effect; the complete assessment of mosaicism, which requires analysis of all cells; and the relationship with embryonic dysmorphism. Fluorescence in situ hybridization has been used in this study to answer these issues. Design: Fluorescence in situ hybridization analysis of human embryos using simultaneously probes for three or five chromosomes. Five hundred twenty-four cleavage-stage human embryos obtained by IVF were analyzed by fluorescence in situ hybridization. Embryos were allocated into three groups according to morphological and developmental characteristics (arrested; slow and/or fragmented; morphologically and developmentally normal). The embryos also were analyzed according to maternal age. Results: Dysmorphic embryos had higher rates of polyploidy and diploid mosaicism. Aneuploidy increased with maternal age in nonarrested embryos. Preimplantation genetic diagnosis successfully detected these abnormalities. Conclusion: This study demonstrates that, in morphologically and developmentally normal human embryos, cleavage-stage aneuploidy significantly increases with maternal age. The results suggest that implantation failure in older women largely could be due to aneuploidy. Fertil Steril 1995;64: Key Words: In vitro fertilization, 18-chromosome, 13-chromosome, 21-chromosome, trisomy, polyploidy, mosaicism, aneuploidy, preimplantation genetic diagnosis The purpose of this study was to analyze human preimplantation embryos for possible relationships between maternal age, development, and numerical chromosome abnormalities. Although a significant number of clinically recognized pregnancies miscarry because of chromosomal abnormalities, many embryos are unaccounted for, being lost before or Received February 2, 1995; revised and accepted April 3, * Presented at the 50th Annual Meeting of The American Fertility Society, San Antonio, Texas, November 4 to 9,1994, where it was awarded the prize paper ofthe Society for Assisted Reproductive Technology. t Reprint requests and present address: Santiago Munne, Ph.D., The Institute for Reproductive Medicine and Science, Saint Barnabas Medical Center, 101 Old Short Hills Road, Suite 501, West Orange, NJ 07052, (FAX: ). :j: Present address: The Institute for Reproductive Medicine and Science, Saint Barnabas Medical Center, West Orange, New Jersey. 382 Munne et at. Embryonic chromosome abnormalities shortly after implantation (1). Only 5% to 30% of them implant after IVF. The lost embryos may have high rates of chromosomal and morphological abnormalities (dysmorphism) (2-5). Reduced implantation may be caused by certain dysmorphisms but also may be affected by maternal age. Women over 35 years have the highest incidence of trisomic deliveries (6, 7), so a link may exist between lower implantation, higher aneuploidy, and maternal age. However, as far as the authors are aware, no clear connection between maternal age and aneuploidy has been shown for human oocytes or embryos; perhaps in part because of the scarcity of material and in part because of the inherent inefficiency of karyotyping procedures. Certain requirements must be met for a successful study of numerical chromosome abnormalities in preimplantation embryos. For example, cell division no longer occurs in arrested embryos, so their analy- Fertility and Sterility

2 sis must be done at the interphase stage. Individual chromosomes also must be analyzed to determine differences in aneuploidy rates among different chromosomes. Likewise, all or most cells of anyone embryo should be analyzed to distinguish mosaicism from other abnormalities. Many chromosome studies of pre implantation embryos have been performed by classical cytogenetic techniques. Actually, cytogenetic methods do not permit the analysis of cleavage-arrested blastomeres and, in others that have not arrested, only a proportion of blastomeres show metaphases. Also, optimal banding and specific identification of metaphase chromosomes in blastomeres are difficult to obtain. For both reasons, only 25% to 30% of embryos show clear results; and of those with clear results, <25% can have all their cells analyzed (4, 8). In contrast, fluorescence in situ hybridization can supply accurate information for >90% of blastomeres from an embryo (5, 9-11); but it will only supply information for chromosomes with specific DNA probes. Nevertheless, by using multiple-color and multiple-probe fluorescence in situ hybridization techniques simultaneously, it is now possible to differentiate between polyploidy, mosaicism, and aneuploidy. Mosaicism can be differentiated from fluorescence in situ hybridization failure and the mechanism of formation deduced when all or most cells of an embryo are analyzed (10-12). The present study shows for the first time that aneuploidy in morphologically normal cleavage-stage embryos markedly increases with maternal age. It also shows that embryonic dysmorphisms are correlated with polyploidy and mosaicism. MATERIALS AND METHODS Embryo Source and Classification Only monospermic embryos, embryos developing from dipronucleated zygotes, were used for this study. These embryos were obtained from patients undergoing IVF treatment for infertility or preimplantation genetic diagnosis at Center for Reproductive Medicine and Infertility, The New York Hospital-Cornell University Medical College. Embryos that had developed to the 5- to 12-cell stage by day 3, however, were replaced into patients after selective assisted hatching (13). Embryos that had not developed beyond the eight-cell stage on the 3rd day and had not cleaved during a 24-hour period ("arrested") were not replaced and could be used for this study. Dysmorphic nonarrested embryos also were used when enough embryos were available for replacement. The morphologically and developmentally normal embryos used in this study were obtained from three sources: [1] embryos deemed at high risk of carrying a genetic disease after preimplantation genetic diagnosis, [2] from patients with a maternal age over 42 years-after freezing spare embryos from these patients, pregnancies are obtained so very rarely that these embryos are usually available for research; and [3] patients with a maternal age over 41 years requesting preimplantation genetic diagnosis for trisomies at risk of arriving to term (XY, 18, 13, 21). Research on such embryos is permitted in accordance with two protocols endowed by the Human Investigation Committee of the New York Hospital Cornell University Medical College (protocol no for dysmorphic embryos and no for embryos rejected after preimplantation genetic diagnosis, and no for preimplantation genetic diagnosis of aneuploidy). Monospermic embryos were classified in three main groups, "arrested," "slow and/or fragmented," and "good," according to developmental fitness. Arrested embryos were considered those that did not develop beyond the eight-cell stage on their 4th day and had not cleaved for 2:24 hours by days 3 and 4. Slow and/or fragmented embryos were those that had not reached the eight-cell stage on their 4th day of development but had cleaved during day 3 or 4 (slow) or that cleaved normally, but had> 15% extracellular fragmentation (fragmented). Good embryos were those at the eight-cell stage by day 3 with < 15% fragmentation. Biopsy, Fixation and Fluorescence In Situ Hybridization Analysis Embryos were biopsied on day 4. A hole was drilled through the zona pellucida with acidified Tyrode's solution (ph = 2.4), and blastomeres were biopsied from each embryo by micromanipulation (14). Blastomeres were fixed individually on glass slides as described previously (11), dehydrated (70%,85%, 95% ethanol, 2 minutes each) and then stored at -20 C until analysis. Allor most fixed blastomeres from each embryo were analyzed by fluorescence in situ hybridization using simultaneously X-, Y-, and 18-chromosome-specific probes following Munne et al (5) without modification or by using simultaneously X-, Y-, 18-, and 13/21-chromosome-specific probes following Munne et al (11), also without modification. The hybridization targets for the X, 18, , and Y probes were a-satellite repeat clusters in the centromeric region and satellite-iii DNA at Yqh, respectively. The scoring and fluorescence in situ hybridization failure criteria from Munne et al (5) also were followed to distinguish fluorescence in situ hybridization failure from mosaicism when all or most cells of a particular embryo were analyzed. Munne et al. Embryonic chromosome abnormalities 383

3 Determination of Specific Chromosome Aneuploidy Frequencies The incidence oftrisomy and monosomy 13 and 21 were calculated as the number of affected embryos divided by the number of embryos analyzed with the 13/21 probe. The incidences of all other abnormalities were calculated as the number of affected embryos divided by the total number of embryos, regardless of probe used. This is because aneuploidy for chromosomes X, Y, and 18; haploidy; polyploidy; and almost all mosaics could be detected using only three probes. Total or subtotal percentages of chromosome abnormalities also were calculated in this fashion. Definition of Limited and Extensive Mosaicism Diploid mosaicism possibly detrimental to embryonic development was defined as the occurrence of more than ~ths ofthe cells in a diploid embryo being chromosomally abnormal. This kind of mosaicism will be called "extensive" mosaicism, whereas "limited" mosaicism will refer to the rest of diploid mosaic embryos. The ~ths cutoff is not completely arbitrary. For example, assuming that abnormal cells will have impaired development, the embryo may arrest. This figure of ~ths is derived from our center's data on freezing and thawing of embryos, in which embryos that have lost up to 3 of 8 cells are able to develop normally to term, and embryo biopsy experiments (15). The distinction between extensive and limited mosaicism is not made for haploid and polyploid mosaic embryos because, in these, all cells are abnormal by definition. Scoring of Multinucleated Blastomeres Multinucleated blastomeres usually are arrested cells (16) so any embryos containing multinucleated blastomeres are expected to be developmentally incompetent. Although mosaicism and multinucleation are not exactly the same phenomenon, multinucleated blastomeres may be treated like abnormal cells, and embryos with multinucleated blastomeres as mosaic. Consequently, if an embryo has ~ths or more chromosomally abnormal cells and/or multinucleated blastomeres it is classified as extensive mosaicism. Statistical Analysis The X 2 test was used to compare morphological groups for abnormalities detected by the same probe. The Mantel-Haenszel X2 test was used to compare heterogeneous percentages, that is, those obtained after the addition of the percentage of abnormal em- 384 Munne et al. Embryonic chromosome abnormalities bryos found using X, Y, and 18 probes and those found using X, Y, 18, 13/21 probes. RESULTS Maternal Age and Embryonic Morphology Groups For this study a total of 524 monospermic embryos were included, 283 analyzed with X, Y, 18, 13/21 probes and the remainder with X, Y, and 18 probes. They were classified in three maternal age groups: 20 to 34, 35 to 39, and 40 to 47 years old. To determine the proportions for these groups within the total population of embryos in our IVF unit, we used a database prototype developed by Giles Tompkin to review 5,751 monospermic embryos obtained from 1,388 women. It showed that 36.1% were 20 to 34 years old, 41.1% were 35 to 39 years old, and 24.1% were 40 to 47 years old. The embryos were classified according to their morphology and developmental competence into three groups: arrested, slow and/or fragmented, and good embryos. The frequency of these groups among the IVF-obtained embryos included in the above database were 13.8%, 33.0% and 53.2%, respectively. Biopsy, Fixation, and Fluorescence In Situ Hybridization Efficiencies A total of 3,629 blastomeres from 524 monospermic embryos were biopsied on day 4 of development. The percentage of blastomeres lost after biopsy and fixation were 2.2% (8113,629) and 2.4% (86/3,629), respectively. A proportion of fixed blastomeres were used for other studies (n = 197). Of the remaining 3,265 fixed blastomeres, 2,867 were found to have a nucleus under phase-contrast observation, whereas the rest (398) were anuclear cells or large cytoplasmic fragments. Anuclear blastomeres (or large fragments) are a common event in embryonic development and not caused by technique failure. Mter fluorescence in situ hybridization analysis, 2,796 (97.5%) showed hybridization signals, 1,504 of which were analyzed with probes for X, Y, 18, and 13/21 and the rest with probes for X, Y, and 18. Seventyone cells (2.5%) were damaged (n = 13), covered with debris or cytoplasm (n = 5), without clear signals (n = 31), too condensed for clear analysis (n = 10), or lost (n = 12). In addition, 121 cells had fluorescence in situ hybridization errors: 74 with the 13/21 probe (4.9%, 74/1,504). Of these, 68 were false-negatives and 6 were false-positives. Thirty-two involved the 18 probe (0.9%, 27/2,867) and consisted of 24 falsenegatives and 8 false-positives. Fifteen involved the X or Y probes (0.4%, 11/2,867) and consisted of 14 false-negatives (all missing an X) and 1 false-posi- Fertility and Sterility

4 Table 1 Chromosome Abnormalities Found in Arrested Embryos Maternal age groups 20 to 34 y 35 to 39 y 40to47y Total Embryos analyzed with X,Y,18, 13/21 probes Embryos analyzed with X,Y,18, probes Chromosome Abnormalities Monosomy YO and mosaic (66% multinucleated) Monosomy YO and polyploid (YY4[18]8[13/21]) Monosomy XO and diploid mosaic (60%)* Monosomy 18 Monosomy 18 and diploid mosaic (60%)* Monosomy 18 and polyploid (2X2Y3[18]8[13/21]) Trisomy 18 and mosaic (50% multinucleated) Trisomy 18 and polyploid (4X6[18]8[13/21]) Trisomy 13/21 Monosomy 13/21 and monosomy 18 Limited diploid mosaics Extensive diploid mosaics Polyploid with all the cells with same ploidy Polyploid mosaics Haploid mosaics Normal embryos Subtotalst A Aneuploidy for X,Y,18 B Aneuploidy for 13/2lt C Total aneuploidy (A + B) D Extensive diploid mosaicism E Total diploid mosaicism F Total polyploidy G Total haploid H Aneuploidy-polyploidy repeats I Aneuploidy-limited mosaic repeats J Aneuploidy-extensive mosaic repeats Total Including limited mosaics (C + E + F + G - H - I - J) Excluding limited mosaics (C + D + F + G - H - J) * Mosaic embroys were also considered aneuploid when the average of their cells, corrected for polyploidy and or haploidy, showed to be aneuploid for a specific chromosome type. t Values in parentheses are percentages (5.5) 2 (2.7) 4 (7.5) 9 (4.9) 0(0.0) 1 (2.9) 1 (2.9) 2 (2.2) 3 (5.5) 3 (5.6) 5 (l0.4) 11 (7.1) 4 (7.3) 26 (35.1) 8 (15.1) 38 (20.9) 11 (20.0) 29 (39.2) 10 (18.9) 50 (27.5) 25 (45.5) 30 (40.5) 26 (49.1) 81 (44.5) 0(0.0) 3 (4.1) 2 (3.8) 5 (2.7) 1 (1.8) 1 (1.4) 1 (1.9) 3 (1.6) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 1 (1.8) 1 (1.4) 2 (3.8) 4 (2.2) 37 (67.3) 63 (86.6) 40 (76.4) 140 (78.0) 30 (54.5) 60 (82.6) 38 (72.7) 128 (71.4) t Percentage of aneuploidy 13/21 = monosomic and trisomic embryos for 13/21 divided by the fraction of embryos analyzed with X,Y,18,13/21 probes. Percentage calculated according to the formulas indicated at the end of the category titles: (A + B), (C + E + F + G - H - I - J), or (C + D + F + G - H - J). tive (with an extra Y). The failure of the technique can be assessed as the number of nucleated blastomeres lost before fluorescence in situ hybridization (4.6%) plus the number of nucleated blastomeres not analyzable (2.5%) or with false results after fluorescence in situ hybridization (6.2%). This estimation is 13.3%. Anuclear blastomeres occurred more often in morphologically abnormal than in normal embryos: 24.8% ( cells) in arrested embryos, 11.0% (12711,156 cells) in slow and/or fragmented embryos, and 4.4% (72/1,641 cells) in good embryos. These differences are highly significant (P < 0.001). Comparison of Developmental Groups Tables 1, 2, and 3 describe the results obtained after analyzing 182 arrested, 154 slow and/or frag- mented, and 188 good embryos, respectively. Some (n = 131) of the embryos in the arrested and slow or fragmented groups have been described in previous work (6). Table 4 shows an estimate ofthe total chromosome abnormalities for each morphological group of embryos and shows that polyploidy is the main chromosomal abnormality in arrested embryos. In addition, polyploidy decreases with increasing embryonic competence, from 44.5% in arrested, to 2.1% in good embryos (P < 0.001). Maternal age did not affect polyploidy rates for any of the three morphological groups. Mter excluding polyploid and haploid embryos, extensive diploid mosaicism was found in 39.6% (381 96) of arrested embryos, 25.2% (33/131) of slow and/ or fragmented group, and 14.1% (25/177) of diploid good embryos, indicating a highly significant Munne et al. Embryonic chromosome abnormalities 385

5 Table 2 Chromosome Abnormalities in Slow and/or Fragmented Embryos Maternal age groups 20 to 34 y 35 to 39 y 40 to 47 y Total Embroys analyzed with X,Y,18, 13/21 probes Embryos analyzed with X,Y,18 probes Chromosome abnormalities Monosomy XO, monosomy 18, mosaic (50% multinucleated blastomeres) Monosomy YO and diploid mosaic (66% abnormal)* Monosomy 18 Monosomy 18 and diploid mosaic (57% abnormal)* Monosomy 18 and diploid mosaic (20% abnormal)* Trisomy 18 Monosomy 13/21 Double monosomy 13/21 Double monosomy 13/21 & mosaic (100% abnormal)* Monosomy 13/21 and diploid mosaic (13% abnormal)* Trisomy 13/21 Limited diploid mosaics Extensive diploid mosaics Polyploid with all the cells with same ploidy Polyploid mosaics Haploid mosaics Normal embryos Subtotalst A Aneuploidy for X,Y,18 B Aneuploidy for 13/21=1= C Total aneuploidy (A + B) D Extensive diploid mosaicism E Total diploid mosaicism F Total polyploidy G Total haploid H Aneuploidy-polyploidy repeats I Aneuploidy-limited mosaic repeats J Aneuploidy-extensive mosaic repeats Total Including limited mosaics (C + E + F + G - H - I - J) Excluding limited mosaics (C + D + F + G - H - J) (2.2) 5 (7.0) 3 (8.1) 9 (5.8) 1 (3.8) 3 (8.6) 6 (21.4) 10 (11.2) 2 (6.0) 805.6) 9 (29.5) ) 13 (28.3) 15 (21.1) 5 (13.5) 33 (21.4) 25 (54.3) 33 (46.5) 15 (40.5) 73 (47.4) 4 (8.7) 11 (15.5) 5 (13.5) 20 (13.0) 0(0.0) 2 (2.8) 1 (2.7) 3 (1.9) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 10.4) 1 (2.7) 2 (1.3) 1 (2.1) 2 (2.8) 1 (2.7) 4 (2.6) 30 (66.9) 51 (76.2) 28 (80.9) 109 (75.5) 18 (40.8) 34 (52.2) 19 (56.6) 71 (50.8) * Mosaic embryos were also considered aneuploid when the average of their cells, corrected for polyploidy and or haploidy, showed to be aneuploid for a specific chromosome type. t Values in parentheses are percentages. =1= Percentage of aneuploidy 13/21 = monosomic and trisomic embryos for 13/21 divided by the fraction of embryos analyzed with X,Y,18,13/21 probes. Percentage calculated according to the formulas indicated at the end of the category titles: (A + B), (C + E + F + G - H - I - J), or (C + D + F + G - H - J). (P < 0.001) increase of diploid mosaicism with increasing dysmorphism. Aneuploidy increased with embryonic competence, from 7.1% in arrested embryos, to 23.8% in good ones (P < 0.005). Haploidy was found to be represented equally in all three morphological groups. Excluding limited mosaicism, chromosome abnormalities increased with dysmorphism overall, from 71.4% in the arrested group to 41.3% of good embryos (P < 0.001). Comparison of Age Groups Tables 1 to 4 show the total of chromosome abnormalities divided in the three maternal age groups, and Table 5 shows aneuploidy results by chromosome and by maternal age groups. Aneuploidy was 386 Munne et ai. Embryonic chromosome abnormalities found to increase with maternal age from 5.4% in embryos from women 20 to 34 years old to 27.7% in women :=::40 years (P < 0.001). In good embryos, aneuploidy increased with maternal age, from 4.0% in embryos from women 20 to 34 years of age to 37.2% in embryos from 40 to 47 years old women (P < 0.005). Similarly, aneuploidy in slow embryos increases from 6% in embryos from women 20 to 34 years of age to 29.5% in embryos from 40- to 47- year-old women (P < 0.025). However, no significant increase of aneuploidy was detected in arrested embryos with maternal age (Table 4). The increase in aneuploidy mostly was due to aneuploidy of chromosomes 13/21 (P < 0.001) because the increase for gonosomes and chromosome 18 was not statistically significant. Whereas the increase in 13/21 Fertility and Sterility

6 Table 3 Chromosome Abnormalities in Good Embryos Maternal age groups 20 to 34 y 35 to 39 y 40to47y Total Embryos analyzed with X,Y,18, 13/21 probes Embryos analyzed with X,Y,18 probes Chromosome abnormalities MonosomyXO TrisomyXXY Monosomy 18 Monosomy 18 and mosaic (30% abnormal)* Trisomy 18 Monosomy 13/21 Monosomy 13/21 and mosaic (33% abnormal)* Monosomy 13/21 and trisomy 18 Monosomy 13/21 and monosomy 18 Trisomy 13/21 Trisomy 13/21 and trisomy 18 Trisomy 13/21 and monosomy XO Trisomy 13/21 and monosomy YO Trisomy 13/21 and trisomy XXX Trisomy 13/21 and mosaic (26% to 37% abnormal)* Trisomy 13/21 and mosaic (38% to 60% abnormal)* Limited diploid mosaics Extensive diploid mosaics Polyploid with all the cells with same ploidy Polyploid mosaics Haploid mosaics Normal embryos Subtotalst A Aneuploidy for X,Y,18 B Aneuploidy for 13/21:j: C Total aneuploidy (A + B) D Extensive diploid mosaicism E Total diploid mosaicism F Total polyploidy G Total haploid H Aneuploidy-polyploidy repeats I Aneuploidy-limited mosaic repeats J Aneuploidy-extensive mosaic repeats Total Including limited mosaics (C + E + F + G - H - I - J) Excluding limited mosaics (C + D + F + G - H - J) * Mosaic embryos were also considered aneuploid when the average of their cells, corrected for polyploidy and or haploidy, showed to be aneuploid for a specific chromosome type. t Values in parentheses are percentages (4.0) 2 (4.2) 6 (6.7) 10 (5.3) 0(0.0) 1 (5.3) 18 (30.5) 19 (18.4) 2 (4.0) 3 (9.4) 24 (37.2) 29 (23.8) 3 (6.0) 9 (18.8) 13 (14.4) 25 (13.3) 20 (40.0) 22 (45.8) 33 (36.7) 75 (39.9) 2 (4.0) 1 (2.1) 1 (1.1) 4 (2.1) 1 (2.0) 3 (6.3) 3 (3.3) 7 (3.7) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 4 (4.4) 4 (2.1) 0(0.0) 0(0.0) 3 (3.3) 3 (1.6) 25 (50.0) 29 (63.6) 54 (70.5) 108 (65.8) 8 (16.0) 16 (36.5) 38 (52.7) 62 (41.3) :j: Percentage of aneuploidy 13/21 = monosomic and trisomic embryos for 13/21 divided by the fraction of embryos analyzed with X,Y,18,13/21 probes. Percentage calculated according to the formulas indicated at the end of the category titles: (A + B), (C + E + F + G - H - I - J), or (C + D + F + G - H - J). aneuploidy started after 35 years of age, the increase for gonosomes and chromosome 18 started only after 40 years of age. The latter trends were, however, not significant. Extensive mosaicism was found more often in the 35- to 39-year-old group of embryos (P < 0.005). Examples of mosaic and polyploid embryos have been described previously by us (5, 12, 17), and no new types of mosaic or polyploid embryos have been found since then. Multinucleation In addition to chromosome abnormalities, 13.3 (367/2,765) multinucleated blastomeres were found. As explained above, those cells were considered abnormal, and embryos containing them were mosaic. A significant decrease (P < per embryo and P < per cell) of multinucleated blastomeres with increasing developmental competence was found: 47.2% arrested embryos had at least one multinucleated blastomere and 23.9% of all the cells from arrested embryos were multinucleated blastomeres; 45.6% slow and/or fragmented embryos had at least one multinucleated blastomere and 12.0% of all the cells from slow and/or fragmented embryos were multinucleated blastomeres; 34.4% good embryos had at least one multinucleated blastomere; and Munne et ai. Embryonic chromosome abnormalities 387

7 Table 4 Comparison of Chromosome Abnormalities in Arrested, Slow and/or Fragmented, and Good Embryos by Maternal Age* Maternal age groups Differences between 20 to 34 y 35 to 39 y 40to47y maternal age groups Total aneuploidyt Arrested =1: NS Slow P < Good =1:11 lip < Differences between morphological groups NS NS =l:p < 0.05 Extensive diploid mosaicismt Arrested P < Slow NS Good NS Differences between morphological groups P < NS NS Total polyploidyt Arrested NS Slow NS Good NS Differences between morphological groups P < P < P < * Values are percentages. NS, not significant. t Aneuploidy and total abnormality significance were calculated using the Mantel-Hazel test to take into account that aneuploidy for 13/21 could only be detected in embryos analyzed with 13/21 probe. Mosaicism and polyploidy significance were calculated using a X 2 test. 9.9% of all the cells from good embryos were multinucleated blastomeres. The chromosomal constitution of individual multinucleated blastomeres has been described previously (5, 11, 18), and no new multinucleated blastomere types were found. Preimplantation Genetic Diagnosis of Trisomies at Risk of Arriving to Term Fluorescence in situ hybridization on single blastomeres was applied for the preimplantation genetic diagnosis of X, Y, 18, 13, and 21 aneuploidy in human embryos obtained from women 40 or older undergoing IVF. The analysis of all the cells in discarded embryos was used to estimate the overall fluorescence in situ hybridization failure rate for the test (5.4%). Preimplantation genetic diagnosis was performed in 11 women of 2:40 years (average 41.3 years). Ofthe 39 chromosomally abnormal embryos, 4 were nonetheless transferred because the risk of misdiagnosis was higher (6.5%) than the risk ofimplantation (1110,000) for such abnormality (monosomy 21). The risk estimate was based on results of 283 embryos analyzed with X, Y, 18, and 13/21 probes. In total 32 embryos were transferred, and one patient became pregnant and gave birth to a normal healthy baby. Of the nontransferred embryos, 37 were reanalyzed and gave the same result, Table 5 Aneuploidy Events for Gonosomes, 18, and 13/21 Chromosomes at Different Maternal Ages Maternal age groups Fraction analyzed with X,Y,18, 13/21 probes MonosomyXO MonosomyYO Trisomy XXY Trisomy XXX Total gonosomes Monosomy 18 Trisomy 18: Total chromosome 18 Monosomy 13/21 Double monosomy 13/21 Trisomy 13/21 Total chromosome 13/21 * NS, not significantly different. 20 to 34 y to 39 y 40to47y Total P NS* NS (0.08) t 2t t 33t <0.001 t Each double monosomy 13/21 is counted as two events in the totals. 388 Munne et al. Embryonic chromosome abnormalities Fertility and Sterility

8 Table 6 Preimplantation Diagnosis ofxy, 18, 13,21 Chromosome Aneuploidy: Clinical Results Embryos with no result (n = 12) 13/21 probe not clear (n = 10) Multinucleation (n = 2) Embryos diagnosed as normal (n = 34) Transferred (n = 28) Reanalyzed (n = 6) Confirmed (n = 5) Error (undetected trisomy 13/21) (n = 1) Embryos diagnosed as abnormal (n = 39) Transferred (monosomy 13/21) (n = 4) Not reanalyzed (n = 4) Reanalyzed (n = 31) Aneuploid (n = 19) Polyploid (n = 4) Haploid (n = 2) Mosaics (80% to 100% abnormal) (n = 5) Error (false trisomy 13/21) (n = 1) Error rate in reanalyzed embryos 5.4% (2137) Aneuploidy rate 33% (24/73) one, a trisomy 13/21, was misdiagnosed as normal, and another, a normal one, was misdiagnosed as trisomy (5.4% error rate). In total, 33% (24173) of the embryos were aneuploid, and 15% (11173) were mosaics, haploids, or polyploids. The results are shown in Table 6. DISCUSSION This study shows that aneuploidy is the most common abnormality in normally developing embryos after follicular stimulation and IVF. Aneuploidy also increases significantly with maternal age. This corroborates the hypothesis that oocytes of older women are more prone to nondisjunction caused by meiotic errors (19). Aneuploidy rates for specific chromosomes in human embryos have not been studied before because of the difficulty of identifying specific chromosomes when using karyotyping. In this study we were able to determine these rates for gonosomes, chromosome 18, and chromosomes 13 and 21, combined. Assuming that apparently normally developing embryos also have the highest chance of developing to term, a 23.8% rate of aneuploidy for only four chromosomes may seem to be high when compared with the rates in clinically recognized pregnancies. An explanation for such phenomenon is that many aneuploid embryos must be eliminated before a pregnancy is recognized clinically. For instance, the data in this study show that monosomy is as frequent as trisomy (except for gonosomal monosomy), whereas, with the exception of monosomy 21 (111,000 karyotyped abortions), the other autosomal monosomies normally undetected are in clinically recognized pregnancies. Because nondisjunction produces disomic and nullisomic gametes with the same fre- quency, monosomic embryos must be the ones eliminated during the first days or weeks of pregnancy. This has been described in the mouse (20). Trisomy 18, 13, and 21 are found in only 4% of chorionic villi samples performed in women 40 to 44 years old (6, 7). In contrast, 35% of good embryos were aneuploid for these chromosomes, suggesting that most trisomies are eliminated before clinical recognition. The fact that 37.2% of good embryos from women 40 or older were aneuploid for 4 chromosome pairs may suggest that for 23 pairs the number of aneuploid embryos may exceed 100%. However, other chromosomes may be involved less commonly in aneuploidy. The chromosomes analyzed in this study, together with chromosome 16, are the most common trisomies found in clinically recognized pregnancies. Similarly, the current results confirm preliminary findings after karyotyping of human preimplantation embryos (8). The other factor that may explain why aneuploidy in this age group of embryos will not reach 100% is the relationship between single and multiple aneuploidy. For example, taking all the morphological groups together, the number of double aneuploidies accounts for 19.1% (9/47) of all aneuploidies in embryos from women:2=: 40 years compared with only 4.5% (1/22) for women ::s; 39 years. The fact that most aneuploid embryos develop normally to the eight-cell stage is not in contradiction with the high mortality rate at later stages. The embryonic genome may not be activated until the four- to eight-cell stage (21) and, consequently, any detrimental effect caused by aneuploidy will not be detected until later. Similarly, because the genome is not activated, the aneuploid embryonic genome cannot be actively producing dysmorphisms. The decrease of aneuploidy with embryonic dysmorphism observed in this study remains puzzling. One explanation may be the difficulty of detecting aneuploid zygotes once these become polyploid mosaic. Because arrested aneuploid embryos have a 21% chance of being polyploid-mosaic, an aneuploid zygote is often very difficult to identify once it becomes a polyploid mosaic embryo. Although it is demonstrated here that maternal age is linked to aneuploidy in cleavage-stage human embryos, more research is needed to determine whether this phenomenon affects all chromosomes or only a few and whether this is an IVF phenomenon or if it also occurs in embryos from natural cycles. Polyploidy and multinucleation are the main chromosome abnormalities in arrested embryos. These phenomena normally decrease with embryonic competence. Polyspermic fertilization is unlikely to be the cause of polyploidy in arrested embryos because only two pronuclei are observed after insemination (5). Some of these embryos may have originated from Munne et al. Embryonic chromosome abnormalities 389

9 diploid gametes. It also can be argued that uniformly polyspermic embryos remain undetected when asynchronous pronuclear formation occurs. However, polyspermic embryos normally become mosaic as a result of an abnormal first division (12), whereas polyspermic embryos do not appear to arrest more often than monospermic ones, at least during the first 2 days of development. Furthermore, uniformly polyploid embryos have a very low chance of having originated from polyspermic zygotes. In some monospermic embryos DNA synthesis may continue, even though cellular division has stopped, producing polyploidy. In some of these cases, even karyokinesis may continue, producing multinucleation in almost half of the cells. Artley et al (22) demonstrated that DNA synthesis is not prevented by cleavage arrest. Even if karyokinesis and gene activation do not fail, impaired cytokinesis may cause arrest because there are insufficient cells to produce a functional inner cell mass (22). But in any case, because most polyploid embryos arrest around the four- to eight-cell stage, before the onset of genome activation (21), oocyte quality or embryo culture conditions cannot be discounted as the primary cause. Similarly, extensive diploid mosaicism (>3/8 cells abnormal) was found to significantly decrease with developmental competence. Again, assuming that the embryonic genome is not active, extensive mosaicism cannot produce dysmorphism. However, cytoplasmic impairment may produce both mosaicism and polyploidy, through cytoskeletal and spindle malfunction, cellular division block, or other mechanisms. It has been demonstrated recently that the impairment of the sperm, by generating for instance abnormal zygote centrioles, also may produce chromosome abnormalities (23). Mosaicism in monospermic diploid embryos was caused mostly by mitotic nondisjunction or tetraploidization of one or more cells. Based on totipotency evaluations from embryo biopsy (15) and freeze-thaw data from our center, embryos that have lost up to three of eight cells are able to develop normally to term. In the event that the few abnormal cells in a limited diploid mosaic have suboptimal proliferation, that embryo still may develop normally. Consistent with this hypothesis is the fact that multinucleated blastomeress seem to arrest (16) whereas polyploid cells in mostly diploid blastocysts are found primarily in the trophectoderm (24), and, consequently, both types of cells probably do not participate in the formation of the inner cell mass. Preimplantation genetic diagnosis with fluorescence in situ hybridization has been used for the diagnosis of X-linked diseases and aneuploidy (9,11, 12). Two of the findings from the present work have direct implications in the new field of preimplanta- 390 Munne et al. Embryonic chromosome abnormalities tion genetic diagnosis. One is the high efficiency of fluorescence in situ hybridization when applied to single cells. The other is that mosaicism is so common that it could lead to misdiagnosis in certain cases of preimplantation genetic diagnosis. The efficiency of fluorescence in situ hybridization is obvious. It is already possible to screen for most aneuploid embryos at risk of producing a full-term trisomy. The failure rate may be reduced further by analyzing two blastomeres, or a blastomere and a polar body, or both polar bodies. As the maternal component is by far the most significant factor for aneuploidy increase with parental age, polar body analysis alone would be enough to prevent most fullterm trisomic pregnancies. However, if a mosaic embryo were biopsied, misdiagnosis may occur. The most common event would be that a mosaic embryo would be confused for a normal embryo and transferred or confused for an aneuploid or polyploid embryo and rejected. The risk that a mosaic embryo could lead to misdiagnosing an aneuploid embryo for a normal one is very slim, although possible. Mosaicism can lead to serious misdiagnosis when the preimplantation genetic diagnosis is performed by polymerase chain reaction. For instance, a cell with monosomy 7 from a mosaic embryo that is also compound heterozygote for cystic fibrosis could be diagnosed as a simple carrier for one of the mutations and transferred under the false assumption that it would not be affected. The reality of human reproduction may be that a large proportion of embryos is affected genetically. This generally is indicated by a high incidence of miscarriage as well as failed conception. Selective replacement of human embryos after IVF traditionally has been based on relatively subjective parameters such as cleavage and morphology. Routine assessment of the genetic function of all embryos should become mandatory, once noninvasive methods have been developed. The first few steps toward this practice are proposed here. Embryo selection based on development rate and morphology may be effective in preventing replacement of a proportion of abnormally polyploid and mosaic embryos. However, for aneuploidy there are no superficial criteria (11). Fertility declines with maternal age, becoming more apparent in women >30 years. Yet, infertile women >40 years successfully can carry pregnancies with eggs donated by younger women, indicating that the uterine factor is negligible (25). The problem in the older age group is that apparently morphologically normal embryos could be affected. Genetic diagnosis therefore could have two advantages to individual patients: to reduce the risk of trisomy and to increase the chance of pregnancy. We have applied aneuploidy assessment to 11 women Fertility and Sterility

10 undergoing IVF for infertility treatment. Preliminary results confirm that common trisomies potentially can be avoided after assisted conception. A larger group of patients should be evaluated to test the hypothesis that the pregnancy rate can be improved. In addition, this study probably should involve assessment of all chromosomes. In addition, preimplantation genetic diagnosis will provide reproductive biologists with tools to study abnormalities of fertilization, syngamy, and cleavage. Acknowledgments. The authors acknowledge the embryological skills of Alexis Adler, B.S.; Adrianne Reign, B.S.; Toni Ferrara, B.S.; Cindy Anderson, M.L.T.; Elena Kissin, M.S.; and Sasha Sadowy, B.A. We are grateful to J. Michael Bedford, D.V.M., Zev Rosenwaks, M.D., and William Ledger, M.D., for their support of this work. REFERENCES 1. Burgoyne PS, Holland K, Stephens R. Incidence of numerical chromosome abnormalities in human pregnancy estimated from induced and spontaneous abortion data. 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