Morphological parameters and euploidy rates
Pronuclei evaluation: The embryos were classified into four groups (Z1 to Z4) based on the D1 pronuclei evaluation. A comparison was made between the inter-group euploidy rates to investigate potential differences in the D1 pronuclei evaluation between euploid and aneuploid embryos. To account for the influence of maternal age on embryo euploidy[11, 12], we conducted separate Chi-Square tests for the age groups <35 years and ≥35 years. The results, displayed in Table.3, indicated that all p-values were >0.05, signifying that the variations in euploidy rate across the Z1 to Z4 groups were not statistically significant. Thus, there was no observed distinction in the D1 pronuclei evaluation between euploid and aneuploid embryos.
Table.3 Pronuclei Evaluation and Euploidy Rate
|
Female Age
|
Pronuclei Evaluation
|
Euploidy Rate
|
Chi-Square test
|
|
Z1
|
36.76% (25/68)
|
c2=5.001
P=0.172
|
<35y
|
Z2
|
44.12% (105/238)
|
Z3
|
47.89% (193/403)
|
|
Z4
|
32.00% (8/25)
|
|
Z1
|
30.19% (16/53)
|
c2=0.210
P=0.976
|
≥35y
|
Z2
|
30.93% (60/194)
|
Z3
|
31.09% (74/238)
|
|
Z4
|
25.00% (3/12)
|
Cell Fragment: The embryos were divided into three groups based on the degree of cell fragmentation on D2 and D3 (0% to 5%, 6% to 20%, 21% to 50%), and no embryos with cell fragmentation greater than 50% were collected. As shown in Table.4, all P-values were >0.05, demonstrating that the differences in euploidy rates among the three groups of embryos were not statistically significant. Therefore, the degree of cell fragmentation on D2 and D3 does not reflect the euploidy of embryos.
Table.4 Cell Fragment and Euploidy Rate
Female Age
|
Cell Fragment
|
D2 Euploidy Rate
|
Chi-Square test
|
D3 Euploidy Rate
|
Chi-Square test
|
|
0%-5%
|
44.14% (241/546)
|
c2=0.983
P=0.612
|
43.68% (197/451)
|
c2=1.641
P=0.440
|
<35y
|
6%-20%
|
47.40% (82/172)
|
48.41% (122/252)
|
|
21%-50%
|
53.33% (8/15)
|
41.38% (12/29)
|
|
0%-5%
|
32.19% (122/379)
|
c2=1.212#
P=0.538
|
32.72% (107/327)
|
c2=2.964
P=0.227
|
≥35y
|
6%-20%
|
26.79% (30/112)
|
26.14% (40/153)
|
|
21%-50%
|
28.57% (2/7)
|
41.18% (7/17)
|
#For 33.33% of theoretical frequency <5, Pearson Chi-square test is not applicable, so Monte Carlo approximation is used to calculate Chi-square value and asymptotic significance
D2: The second day after ICSI, D3: The third day after ICSI
Heterogeneity of Blastomere: The embryos were classified into three groups (A, B, C; description shown in Table.1) based on the heterogeneity of blastomere on D2 and D3. The results, as shown in Table.5, indicated that the P-values for the Chi-Square tests for the euploidy rates among the three groups of D2 embryos were all >0.05, demonstrating that the differences were not statistically significant. For the D3 embryos, when the woman's age was ≥35y, the P-values were all >0.05, showing no statistically significant difference. However, for embryos of women aged <35y, the P-value was <0.05, indicating a significant difference in the euploidy rates among the three groups. Comparing the three groups pairwise, the difference in euploidy rate was statistically significant between groups A and B, while the differences between groups A and C, and B and C, were not statistically significant.
Table.5 Heterogeneity of Blastomere and Euploidy Rate
|
Female Age
|
Heterogeneity of Blastomere
|
D2 Euploidy Rate
|
Chi-Square test
|
D3 Euploidy Rate
|
Chi-Square test
|
|
A
|
50.00% (40/80)
|
c2=2.679
P=0.250
|
62.71% (37/59) a
|
c2=8.202
P=0.017*
|
<35y
|
B
|
43.55% (253/581)
|
43.24%(243/562)b
|
|
C
|
52.05% (38/73)
|
45.95%(51/111)a,b
|
|
A
|
34.29% (24/70)
|
c2=0.886
P=0.642
|
40.48% (17/42)
|
c2=4.349
P=0.114
|
≥35y
|
B
|
29.87% (115/385)
|
31.28% (127/406)
|
|
C
|
34.88% (15/43)
|
20.41% (10/49)
|
*Significant differences by two-tailed Pearson chi-square test
a,b Different superscript letters indicate significant differences among groups (P < 0.05)
D2: The second day after ICSI, D3: The third day after ICSI
Gardner Criteria: Blastocysts were categorized into four groups based on Gardner Criteria of inner cell mass (ICM) and trophectoderm (TE) for comparison of their euploidy rates. The groups included Top group = AA (n=595), Good group = AB, BA, BB (n=823), Fair group = AC, BC, CA, CB (n=465), and Poor group = CC (n=5). For the small number of embryos in the poor group (n=5), they were excluded from the analysis. As shown in Table.6, regardless of maternal age, the euploidy rate of embryos decreased with a decrease in Gardner grading levels, with all P-values < 0.001. There was a significant difference in euploidy rates among the three groups. Further pairwise comparisons revealed that when the woman was <35y, the differences between the Top and Good groups, as well as between the Good and Fair groups, were statistically significant, while the difference between the Top and Good groups was not statistically significant. When the woman was ≥35y, the differences between the Top and Fair groups, as well as between the Top and Good groups, were both statistically significant, while the difference between the Good and Fair groups was not statistically significant. In conclusion, the Gardner Criteria of blastocysts is associated with the euploidy of embryos, especially for older women. The euploidy rate for the Fair group (AC, BC, CA, CB) was 15.75%. When either the ICM or TE is graded as C, the euploidy rate significantly decreases.
Table.6 Gardner Criteria and Euploidy Rate
|
Female Age
|
Gardner Criteria
|
Euploidy Rate
|
Chi-Square test
|
|
Top
|
53.25% (131/246)a
|
c2=17.410
P<0.001*
|
<35y
|
Good
|
45.10% (152/337)a
|
|
Fair
|
31.79% (48/151)b
|
|
Top
|
55.78% (82/147)a
|
c2=62.451
P<0.001*
|
≥35y
|
Good
|
23.21% (52/224)b
|
|
Fair
|
15.75% (20/127)b
|
*Significant differences by two-tailed Pearson chi-square test
a,b Different superscript letters indicate significant differences among groups (P < 0.05)
Morphokinetic parameters and euploidy rates
Female Age: The female age was grouped by whether it was advanced maternal age or not. The differences in 18 morphokinetic parameters and 2 AI scores between groups were compared. As shown in Table.7, the differences in parameters such as tPB2, tPNa, t3, t4, t5, t6, t7, t8, tSB, tB, tB-tSB, KIDScore, iDAScore, were statistically significant with P < 0.05. Combined with the median, it can be seen that embryos from females aged <35y develop faster at time points such as tPB2, tPNa, t3, t4, t5, t6, t7, t8, tSB, tB, with the time interval from blastocyst formation to blastocyst formation being shorter compared to embryos from females aged ≥35y, and both KIDScore and iDAScore scores are higher.
Table.7 Female Age and Morphokinetic Parameters
|
Morphokinetic Parameters
|
<35y(n=1044)
|
≥35y(n=844)
|
Mann-Whitney U test
|
M(P25,P75)
|
M(P25,P75)
|
Z-value
|
P-value
|
tPB2(h)
|
3.54(2.97,4.40)
|
3.71(3.06,4.52)
|
-2.685
|
0.007*
|
tPNa(h)
|
18.85(17.82,19.93)
|
19.33(18.20,20.44)
|
-5.972
|
<0.001*
|
tPNf(h)
|
23.22(21.44,25.12)
|
23.44(21.79,25.11)
|
-1.832
|
0.067
|
t2(h)
|
25.73(23.84,27.62)
|
25.83(24.11,27.59)
|
-1.452
|
0.147
|
t3(h)
|
35.91(33.00,38.48)
|
36.41(33.99,38.69)
|
-2.678
|
0.007*
|
t4(h)
|
37.25(33.00,38.48)
|
37.62(35.32,40.03)
|
-2.074
|
0.038*
|
CC2(h)
|
11.57(10.69,12.49)
|
11.69(10.86,12.64)
|
-1.774
|
0.076
|
S2(h)
|
0.42(0.21,1.26)
|
0.42(0.21,1.12)
|
-0.444
|
0.657
|
t5(h)
|
48.72(43.80,53.00)
|
49.45(45.17,53.49)
|
-2.785
|
0.005*
|
t6(h)
|
55.30(50.96,61.68)
|
55.96(51.62,63.05)
|
-2.153
|
0.031*
|
t7(h)
|
55.57(51.20,61.91)
|
56.23(51.91,63.32)
|
-2.148
|
0.032*
|
t8(h)
|
55.63(51.22,61.95)
|
56.28(51.94,63.32)
|
-2.132
|
0.033*
|
CC3(h)
|
17.58(14.82,23.09)
|
17.77(15.36,23.87)
|
-1.891
|
0.059
|
S3(h)
|
5.47(2.50,15.47)
|
5.18(2.52,14.79)
|
-0.545
|
0.586
|
tSB(h)
|
97.20(91.80,103.45)
|
98.31(92.69,104.09)
|
-2.212
|
0.027*
|
tB(h)
|
105.80(100.13,113.54)
|
107.74(102.09,114.63)
|
-3.290
|
0.001*
|
tB-t8(h)
|
49.36(43.67.55.78)
|
50.27(444.36,57.03)
|
-1.952
|
0.051
|
tB-tSB(h)
|
8.85(6.42,11.13)
|
9.40(6.50,11.95)
|
-2.812
|
0.005*
|
KIDScore
|
6.20(4.60,8.00)
|
5.80(4.40,7.60)
|
-2.741
|
0.006*
|
iDAScore
|
8.50(7.30,9.10)
|
8.30(7.00,9.10)
|
-2.118
|
0.034*
|
*Significant differences by two-tailed Mann-Whitney U test
M= Median, P25= Lower Quartile, P75= Upper Quartile
Embryonic Euploidy:As shown in Table.8, comparing the 18 parameters and 2 AI scores between euploidy embryos and aneuploidy embryos, the results indicate that the euploid develops faster at time points such as t5, t6, t7, t8, tSB, and tB compared to aneuploid, with a shorter time interval from the 8-cell stage to blastocyst formation, and from the initiation of blastocyst formation to formation. The third cleavage cycle (CC3) also takes less time, and the differences are statistically significant. The KIDScore and iDAScore scores are also significantly higher.
Table.8 Embryonic Euploidy and Morphokinetic Parameters
|
Morphokinetic Parameters
|
Euploid(n=724)
|
Aneuploid(n=1164)
|
Mann-Whitney U test
|
M(P25,P75)
|
M(P25,P75)
|
Z-value
|
P-value
|
tPB2(h)
|
3.63(3.07,4.45)
|
3.59(3.00,4.42)
|
-0.934
|
0.35
|
tPNa(h)
|
19.03(17.99,20.02)
|
19.05(18.00,20.12)
|
-0.927
|
0.354
|
tPNf(h)
|
23.30(21.58,25.05)
|
23.35(21.63,25.19)
|
-1.074
|
0.283
|
t2(h)
|
25.77(23.94,27.50)
|
25.82(23.90,27.66)
|
-0.643
|
0.52
|
t3(h)
|
35.93(33.18,38.44)
|
36.35(33.70,38.67)
|
-1.790
|
0.073
|
t4(h)
|
37.19(35.06,39.77)
|
37.51(35.15,40.08)
|
-1.519
|
0.129
|
CC2(h)
|
11.56(10.71,12.51)
|
11.66(10.82,12.60)
|
-1.584
|
0.113
|
S2(h)
|
0.42(0.21,1.23)
|
0.42(0.21,1.18)
|
-0.485
|
0.627
|
t5(h)
|
48.53(43.82,52.79)
|
49.42(44.85,53.44)
|
-2.476
|
0.013*
|
t6(h)
|
54.94(50.96,60.49)
|
56.06(51.29,63.47)
|
-3.107
|
0.002*
|
t7(h)
|
55.16(51.10,60.76)
|
56.37(51.62,63.80)
|
-3.491
|
<0.001*
|
t8(h)
|
55.20(51.14,60.81)
|
56.40(51.66,63.85)
|
-3.531
|
<0.001*
|
CC3(h)
|
17.15(14.84,21.63)
|
18.22(15.17,25.54)
|
-3.568
|
<0.001*
|
S3(h)
|
5.04(2.46,14.75)
|
5.71(2.56,15.30)
|
-1.479
|
0.139
|
tSB(h)
|
96.56(91.18,102.53)
|
98.36(92.70,104.62)
|
-4.421
|
<0.001*
|
tB(h)
|
105.44(99.49,111.63)
|
107.86(101.72,115.42)
|
-5.782
|
<0.001*
|
tB-t8(h)
|
48.65(43.75,54.86)
|
50.48(44.14,57.31)
|
-3.131
|
0.002*
|
tB-tSB(h)
|
8.71(6.45,10.62)
|
9.41(6.45,12.10)
|
-4.127
|
<0.001*
|
KIDScore
|
6.7(4.9,8.3)
|
5.6(4.3,7.3)
|
-7.804
|
<0.001*
|
iDAScore
|
8.8(7.8,9.2)
|
8.2(6.7,9.)
|
-7.887
|
<0.001*
|
*Significant differences by two-tailed Mann-Whitney U test
M= Median, P25= Lower Quartile, P75= Upper Quartile
Single Chromosome Abnormal: The development process of the embryo is controlled by the genome. As indicated in Table.8, it is known that the non-ploidy of embryonic chromosomes has an impact on the early development of the embryo. However, not all genes affect the early development of the embryo. We want to further explore the effects of specific chromosome aneuploidy on pre-implantation embryos. Therefore, the morphokinetic parameters of embryos with aneuploidy in single chromosomes were analyzed and compared with euploidy embryos.
The morphokinetic parameters of embryos with aneuploidy in single chromosomes were separately compared with euploid using the Mann-Whitney U test. Due to the large number of original data tables, it is not possible to present them one by one, so the statistically significant differences in morphokinetic parameters are summarized in Table. 9. As shown in Table. 9, based on the data included in this study, there is no evidence to suggest that single chromosome aneuploidies, such as chr-3, chr-7, chr-8, chr-10, chr-11, chr-12, chr-13, chr-15, chr-18, chr-20, and chr-21, have any influence on the morphokinetic of embryos from fertilization to the preimplantation stage.
Table.9 Morphokinetic Parameters between Euploid and Single Chromosome Abnormal Embryos
|
Abnormal Chromosome
|
Morphokinetic Parameters that Have Significant Differences by Mann-Whitney U test Between Groups
|
Chromosome 1(n=18)
|
t7、t8、CC3、S3、tSB、tB、
|
Chromosome 2(n=21)
|
tPNf、t2、t4、tSB、tB
|
Chromosome3(n=17)
|
None
|
Chromosome 4(n=22)
|
t6、t7、t8、CC3、tB
|
Chromosome 5(n=14)
|
t5、t7、t8、CC3、
|
Chromosome 6(n=6)
|
t6、tB-t8、KIDScore
|
Chromosome 7(n=20)
|
None
|
Chromosome 8(n=12)
|
None
|
Chromosome 9(n=19)
|
t6、t7、t8、CC3
|
Chromosome10(n=10)
|
None
|
Chromosome 11(n=21)
|
None
|
Chromosome 12(n=8)
|
None
|
Chromosome 13(n=25)
|
None
|
Chromosome 14(n=27)
|
tSB、tB、tB-t8、tB-tSB
|
Chromosome15(n=27)
|
None
|
Chromosome16(n=45)
|
tB-tSB
|
Chromosome17(n=9)
|
tPNa、tB、tB-t8、tB-tSB
|
Chromosome18(n=7)
|
None
|
Chromosome19(n=24)
|
tB-tSB
|
Chromosome20(n=7)
|
None
|
Chromosome21(n=27)
|
None
|
Chromosome22(n=49)
|
tB-tSB
|
Chromosome X(n=9)
|
tPB2、t8、CC3
|
Chromosome Y(n=3)
|
S3
|