Of the 272 biopsied embryos analysed, 58 blastocysts were subjected to aCGH analysis in 18 cycles and 214 blastocysts were subjected to NGS in 69 cycles. There were no significant differences in the mean maternal age, body mass index, basal follicle stimulating hormone, luteinizing hormone, prolactin, and oestradiol levels between the groups. The median number of retrieved, fertilized, and cleaved oocytes between groups did not differ significantly. The mean number of biopsied blastocysts and ratio of no embryo-transfer per cycle were also similar. The detailed data are summarized in Table 1.
[Table 1 near here]
The molecular karyotype was analysed in the cycles to identify reciprocal and Robertsonian translocations. Of the 58 blastular molecular karyotypes obtained from aCGH, 67.24% abnormal embryos were identified. They include 36.21% with a translocation-related abnormality, 17.24% with a non-translocation-related abnormality, and 15.52% with a combination of abnormalities. Among the 214 blastocysts analysed by NGS, 59.81% of the embryos showed a chromosomal abnormality, including 28.50% due to parental-derived translocation, 13.08% with other abnormalities, and 18.22% with combined abnormalities. The ratios of euploidy and aneuploidy, including translocation-related, non-translocation-related, and combined abnormalities were not significantly different between the aCGH and NGS groups (Table 2). In the NGS group, chromosomal abnormalities not related to translocations were reanalysed. Among the 39 chromosomes that were abnormal, 10.26% of the abnormalities occurred in chromosomes X, 16, and 7; followed by 7.69% abnormalities in chromosomes 10, 6, 2, and 1; 5.13% abnormalities in chromosomes 21, 13, 11, 4, and 3; and 2.56% abnormalities in chromosomes Y, 17, 12, 9, and 8 (Fig. 1). Among the 10 chromosomal abnormalities not related to translocations found in the aCGH group, 20% occurred in chromosome 4, followed by 10% for chromosomes X, 22, 19, 18, 11, 8, and 3 (Fig. 2). The frequency of abnormalities in chromosome 4 was higher overall for both aCGH and NGS.
Table 1
Basic parameters of samples used for aCGH and NGS testing.
Cycle parameters
|
aCGH
|
NGS
|
P-value
|
Biopsied cycles
|
18
|
69
|
|
Maternal age (years)
|
30.3 ± 3.96
|
29.04 ± 4.10
|
0.23
|
BMI (kg/m2)
|
20.10 ± 2.50
|
21.50 ± 2.91
|
0.07
|
bFSH (IU/L)
|
5.0 ± 1.32
|
5.82 ± 1.38
|
0.81
|
bLH (IU/L)
|
4.28 ± 2.47
|
4.12 ± 2.31
|
0.80
|
PRL (IU/L)
|
15.61 ± 7.16
|
17.54 ± 7.83
|
0.36
|
E2 (pg/mL)
|
41.78 ± 17.95
|
41.28 ± 16.89
|
0.91
|
Duration of stimulation (days)
|
11.06 ± 1.59
|
11.42 ± 1.79
|
0.43
|
Total gonadotropin doses (IU)
|
2425.00 ± 644.15
|
2474.82 ± 791.23
|
0.81
|
Number of oocytes
|
15.67 ± 7.05
|
15.09 ± 6.61
|
0.74
|
Number of metaphases II
|
13.06 ± 7.44
|
12.75 ± 5.78
|
0.85
|
Number of fertilizations
|
11.33 ± 5.66
|
11.54 ± 5.38
|
0.89
|
Number of cleavages
|
10.17 ± 5.70
|
10.78 ± 5.06
|
0.66
|
Number of biopsied blastocysts
|
58
|
214
|
|
Mean of biopsied blastocysts per cycle
|
3.22 ± 2.56
|
3.10 ± 1.96
|
0.83
|
Number of biopsied blastocysts on day 5
|
1.94 ± 2.1
|
1.70 ± 1.88
|
0.63
|
Number of biopsied blastocysts on day 6
|
1.23 ± 1.13
|
1.41 ± 1.19
|
0.68
|
Proportion of balanced blastocysts (%)
|
32.76% (19/58)
|
40.19% (86/214)
|
0.30
|
Proportion of no embryo-transfer (%)
|
44.44% (8/18)
|
31.88% (22/69)
|
0.32
|
† Data are mean ± standard deviation unless otherwise indicated.
‡ Abbreviations: aCGH, array comparative genomic hybridization; NGS, next-generation sequencing; BMI, body mass index; bFSH, basal follicle stimulating hormone; PRL, prolactin; E2, oestradiol
|
Table 2
Abnormal embryonic chromosomes determined by aCGH and NGS.
|
aCGH
|
NGS
|
χ2 value
|
P-value
|
Total blastocysts
|
58
|
214
|
|
|
Blastocysts of normal chromosomes
|
32.76% (19/58)
|
40.19% (86/214)
|
1.06
|
0.30
|
Blastocysts of abnormal chromosomes
|
67.24% (39/58)
|
59.81% (128/214)
|
Blastocysts with translocation-related abnormality
|
36.21% (21/58)
|
28.50% (61/214)
|
1.29
|
0.26
|
Blastocysts without translocation-related
|
17.24% (10/58)
|
13.08% (28/214)
|
0.66
|
0.42
|
Blastocysts with combined abnormality
|
15.52% (9/58)
|
18.22% (39/214)
|
0.23
|
0.63
|
† Data are mean ± standard deviation unless otherwise indicated |
‡ Abbreviations: aCGH, array comparative genomic hybridization; NGS, next-generation sequencing |
[Table 2 near here]
[Figures 1 and 2 near here]
Maternal age was available for 272 blastocysts and permitted allocation to the ≥ 35-years group (n = 31) and < 35-years group (n = 241). The rate of chromosome abnormalities was higher in the ≥ 35-years group than in the < 35-years group for both methods, with no statistically significant difference (P > 0.05, Table 3). Comparison of the rate of the normal blastocysts in samples from women < 35-years old using aCGH and NGS revealed more normal blastocysts in the NGS group than in the aCGH group, but without statistical significance (P = 0.53).
Table 3
Chromosome abnormalities in both maternal age groups.
|
aCGH
|
NGS
|
Total
|
Maternal age (years)
|
≥ 35
|
< 35
|
≥ 35
|
< 35
|
≥ 35
|
< 35
|
Number of blastocysts
|
10
|
48
|
21
|
193
|
31
|
241
|
Normal blastocysts
|
20% (2/10)
|
35.41% (17/48)
|
38.10% (8/21)
|
40.41%
(78/193)
|
32.26% (10/31)
|
39.41% (95/241)
|
Abnormal blastocysts
|
80% (8/10)
|
64.58% (31/48)
|
61.90% (13/21)
|
59.59% (115/193)
|
67.74% (21/31)
|
60.58% (146/241)
|
χ2
|
0.33
|
|
0.04
|
|
0.59
|
|
P-value
|
0.47
|
0.83
|
0.44
|
† Data are mean ± standard deviation unless otherwise indicated |
‡ Abbreviations: aCGH, array comparative genomic hybridization; NGS, next-generation sequencing |
[Table 3 near here]
High-quality blastocysts were defined according to the combination of internal cell mass (ICM) or trophectoderm (TE) scores of A or B. Low-quality blastocysts had an ICM or TE score of C. A higher proportion of high-quality blastocysts had normal chromosomal patterns than aneuploidy, without statistical significance (P = 0.12, Table 4). In the NGS group, significantly more normal than abnormal embryos were observed among high-quality blastocysts (P = 0.03). There were more abnormal than normal chromosomes among high-quality embryos in the aCGH group (P = 0.22). There was a significant difference in the overall proportions of normal blastocysts among high-quality embryos detected using NGS and aCGH (P = 0.045).
Table 4
Chromosome abnormalities with different qualities in aCGH and NGS.
Chromosome constitution
|
High-quality embryo
|
Low-quality embryo
|
χ2
|
P-value
|
aCGH
|
normal
|
25% (7/28)
|
40% (12/30)
|
1.48
|
0.22
|
abnormal
|
75% (21/28)
|
60% (18/30)
|
NGS
|
normal
|
45.45% (65/143)
|
29.58% (21/71)
|
4.98
|
0.03
|
abnormal
|
54.55% (78/143)
|
70.42% (50/71)
|
Total
|
normal
|
42.11% (72/171)
|
32.67% (33/101)
|
2.38
|
0.12
|
abnormal
|
57.89% (99/171)
|
67.33% (68/101)
|
† Data are mean ± standard deviation unless otherwise indicated |
‡ Abbreviations: aCGH, array comparative genomic hybridization; NGS, next-generation sequencing |
[Table 4 near here]
The TE scores of blastocysts reflects the formation of a cohesive epithelium. Thus, a cell with a higher TE grade has a greater chance of becoming a normal blastocyst. In the NGS group, decreased TE quality score was associated with an increase in the proportion of abnormal blastocysts, although the difference was not statistically significant (Table 5). No such tendency was observed in the aCGH group, in which there was a higher proportion of abnormal blastocysts associated with a higher TE grade, without a significant difference. Nevertheless, there were more blastocysts of higher TE grades (A and B) with normal chromosomes in the NGS group than in the aCGH group (P = 0.13) (Table 5).
Table 5
Chromosome abnormalities with different trophectoderm score grades in aCGH and NGS.
Chromosome constitution
|
Grade A or B
|
Grade C
|
χ2
|
P-value
|
aCGH
|
normal
|
29.41% (10/34)
|
37.5% (9/24)
|
0.42
|
0.52
|
abnormal
|
70.58% (24/34)
|
62.5% (15/24)
|
NGS
|
normal
|
43.51% (67/154)
|
31.67% (19/60)
|
2.52
|
0.11
|
abnormal
|
56.49% (87/154)
|
68.33% (41/60)
|
Total
|
normal
|
73.33% (77/188)
|
26.67% (28/84)
|
1.42
|
0.23
|
abnormal
|
66.47% (111/188)
|
33.53% (56/84)
|
† Data are mean ± standard deviation unless otherwise indicated |
‡ Abbreviations: aCGH, array comparative genomic hybridization; NGS, next-generation sequencing |
[Table 5 near here]
Next, we analysed the relationship between ICM scores and chromosome abnormalities. A large majority of blastocysts were ICM grade A or B for both normal and abnormal embryos. In the NGS group, there was no difference in the rates of ICM grade A or B normal and abnormal blastocytes of (P = 0.53), whereas there were significantly more abnormal than normal embryos with grade A or B in the aCGH group (P = 0.06). In addition, there were significantly more normal embryos with ICM grade A or B in the NGS group than in the aCGH group (P = 0.05).
[Table 6 near here]
Table 6
Chromosome abnormalities with different inner cell mass scores in aCGH and NGS.
Chromosome constitution
|
Grade A or B
|
Grade C
|
χ2
|
P-value
|
aCGH
|
normal
|
25% (11/44)
|
57.14% (8/14)
|
3.63
|
0.06
|
abnormal
|
75% (33/44)
|
42.86% (6/14)
|
NGS
|
normal
|
40.89% (83/203)
|
27.27% (3/11)
|
0.34
|
0.53
|
abnormal
|
59.11% (120/203)
|
72.73% (8/11)
|
Total
|
normal
|
38.06% (94/247)
|
44% (11/25)
|
0.34
|
0.56
|
abnormal
|
61.94% (153/247)
|
56% (14/25)
|
† Data are mean ± standard deviation unless otherwise indicated |
‡ Abbreviations: aCGH, array comparative genomic hybridization; NGS, next-generation sequencing |