Trophectoderm biopsy and WGA
The 36 couples (29 α-thalassemia and 7 β-thalassemia) underwent a total of 41 PGT cycles (31 α-thalassemia and 10 β-thalassemia) (Table 1 and Supplementary Table 1) (Figure 1). Seven hundred and seventy-seven oocytes were retrieved. Of these 631 were fertilized with intracytoplasmic sperm injection (ICSI), and 217 were cultured to blastocysts of good enough quality to perform trophectoderm (TE) biopsy (better than ⅢCC). The average number of blastocysts for each couple was 6.0 (217/36). WGA was successfully performed for all TE cells.
NGS-based SNP haplotyping and mutation detection
Due to these families lacking related relatives and proband, all analyses were based on the blastocysts. Three couples (families 8, 31, and 35) only had two biopsied blastocysts and did not receive conclusive PGT results from NGS. A second PGT cycle produced conclusive results. Therefore, all of the biopsied blastocysts received conclusive PGT results (100%, 217/217) (Table 1).
Using family 13 as an example to analyze α- thalassemia (Figure 2), 138 SNPs within 1 Mb upstream and 132 SNPs within 2 Mb downstream from the HBA1 and HBA2 gene were adopted with sequencing depth >30X. We could deduce whether the mutation allele was present in the embryo by analyzing these SNPs. For instance, we found that embryos 5 and 7 carried the disease allele from the couple according to the sequencing depth of the SEA area (Figure 2a, part of the SNP results). We also found that these two embryos inherited both maternal and paternal disease-associated haplotypes. Next, information SNPs in the SEA area were used to construct the haplotype. In brief, the mother was heterozygous A/C, and the father was A/A on SNP in position 119006. This SNP was considered as the maternal information SNP. While the affected embryos (5 and 7) were homozygous A/A (Figure 2b, part of the SNP results), we could easily deduce that alleles with the base A from the mother were pathogenic, and this was the disease-associated haplotype. At least two upstream and two downstream markers closely linked to the gene underlying the mutation were analyzed, and the disease-associated and non-disease-associated maternal haplotype was successfully distinguished. Hence, we concluded that embryos 1, 5, and 7 carried the disease-associated maternal haplotype. Similarly, in position 207611, the maternal was C/C and the paternal was C/T, this SNPs was considered as the paternal information SNPs (Figure 2b). While the affected embryos (5 and 7) were homozygous C/C. We could easily deduce that alleles with base C from the father were pathogenic, and this was another disease-associated haplotype. Hence, we concluded that embryos 2, 3, 4, 5, and 7 carried the disease-associated paternal haplotype. So, embryos 5 and 7 were homozygous, embryos 1, 2, 3, and 4 were heterozygous, and embryos 6, 8, and 9 were wildtype.
The analysis method for β- thalassemia (using family 30 as an example) is shown in Figure 3. Ninety-five SNPs within 2 Mb upstream and downstream respective from the HBB gene were adopted with sequencing depth >30X. At first, we could deduce embryos 4 and 6 carried the disease allele according to the sequencing depth of the βCD41-42 (Figure 3a). Thus, we deduced that these two embryos inherited both maternal and paternal disease-associated haplotypes (Figure 3b). Next, information SNPs in the HBB were used to construct the haplotype as described above. We could also conclude that embryos 1, 4, 5, and 6 carried the disease-associated maternal haplotype and that embryos 2, 4, and 6 carried the disease-associated paternal haplotype. So, embryos 4 and 6 were homozygous, embryos 1, 2, and 5 were heterozygous, and embryo 3 was wild-type.
PGT results
After NGS-based SNP haplotyping and mutation detection, 160 (73.7%, 160/217) blastocysts were found to be unaffected by either α- thalassemia or β- thalassemia (Table 1). PGT-A results of these blastocysts showed that 112 (70.0%, 112/160) were euploid, which were defined as transferable blastocysts (Table 1). And the average number of transferable blastocysts for each couple was 3.1 (112/36).
Table 1
The preimplantation genetic testing outcomes of the 36 families.
|
Cycles
|
Embryo state
|
PGT results
|
Pregnancy results
|
Genetic testing
|
PGT
|
FET
|
Oocyte
|
MⅡ
|
Biopsy blastocyst
|
Unaffected1
|
Transferrable2
|
CP
|
LB
|
PGT-M
|
Amniocentesis
|
After born
|
Family 1
|
1
|
1
|
26
|
25
|
9
|
6
|
3
|
1
|
1
|
Wild type
|
Wild type
|
Wild type
|
Family 2
|
1
|
1
|
22
|
17
|
8
|
6
|
4
|
1
|
1
|
Heterozygote
|
Heterozygote
|
Heterozygote
|
Family 3
|
1
|
1
|
13
|
12
|
3
|
2
|
1
|
1
|
1
|
Wild type
|
NA
|
Wild type
|
Family 4
|
1
|
1
|
11
|
10
|
5
|
2
|
2
|
1
|
1
|
Wild type
|
NA
|
Wild type
|
Family 5
|
1
|
1
|
12
|
12
|
9
|
6
|
3
|
1
|
1
|
Heterozygote
|
Heterozygote
|
Heterozygote
|
Family 6
|
1
|
2
|
15
|
14
|
7
|
6
|
2
|
1
|
1
|
Heterozygote
|
Heterozygote
|
Heterozygote
|
Family 7
|
1
|
1
|
24
|
20
|
4
|
4
|
1
|
1
|
1
|
Heterozygote
|
Heterozygote
|
Heterozygote
|
Family 83
|
2
|
2
|
44
|
31
|
8
|
6
|
4
|
1
|
1
|
Heterozygote
|
NA
|
Heterozygote
|
Family 9
|
1
|
2
|
35
|
27
|
6
|
6
|
6
|
1
|
0
|
Wild type
|
Miscarriage
|
NA
|
Family 10
|
1
|
1
|
28
|
16
|
4
|
1
|
1
|
1
|
0
|
Wild type
|
Miscarriage
|
NA
|
Family 11
|
1
|
1
|
15
|
11
|
5
|
3
|
2
|
1
|
1
|
Wild type
|
Wild type
|
NA
|
Family 12
|
1
|
3
|
21
|
16
|
4
|
4
|
3
|
1
|
1
|
Heterozygote
|
NA
|
Heterozygote
|
Family 13
|
1
|
2
|
26
|
20
|
9
|
7
|
5
|
1
|
1
|
Wild type
|
Wild type
|
NA
|
Family 14
|
1
|
2
|
7
|
5
|
3
|
1
|
1
|
0
|
0
|
Heterozygote
|
NA
|
NA
|
Family 154
|
2
|
1
|
48
|
36
|
11
|
7
|
2
|
1
|
2
|
Wild type
|
NA
|
Wild type
|
Family 16
|
1
|
1
|
19
|
12
|
4
|
3
|
2
|
1
|
1
|
Heterozygote
|
Heterozygote
|
NA
|
Family 17
|
1
|
0
|
6
|
4
|
2
|
0
|
0
|
0
|
0
|
NA
|
NA
|
NA
|
Family 18
|
1
|
1
|
19
|
10
|
3
|
2
|
2
|
1
|
1
|
Heterozygote
|
Heterozygote
|
NA
|
Family 19
|
1
|
3
|
40
|
33
|
15
|
11
|
11
|
1
|
1
|
Heterozygote
|
NA
|
Heterozygote
|
Family 20
|
1
|
2
|
25
|
21
|
4
|
4
|
3
|
1
|
1
|
Heterozygote
|
NA
|
Heterozygote
|
Family 21
|
1
|
1
|
19
|
14
|
8
|
8
|
4
|
1
|
1
|
Wild type
|
Wild type
|
NA
|
Family 22
|
1
|
1
|
19
|
13
|
5
|
5
|
3
|
1
|
1
|
Heterozygote
|
Heterozygote
|
NA
|
Family 23
|
1
|
1
|
29
|
27
|
6
|
4
|
4
|
1
|
0
|
Heterozygote
|
NA
|
NA
|
Family 24
|
1
|
1
|
16
|
15
|
7
|
4
|
4
|
1
|
1
|
Heterozygote
|
Heterozygote
|
NA
|
Family 25
|
1
|
1
|
19
|
19
|
5
|
3
|
2
|
1
|
0
|
Wild type
|
Miscarriage
|
NA
|
Family 26
|
1
|
2
|
8
|
7
|
4
|
4
|
4
|
1
|
0
|
Wild type
|
Miscarriage
|
NA
|
Family 27
|
1
|
2
|
11
|
9
|
6
|
5
|
3
|
1
|
0
|
Wild type
|
Miscarriage
|
NA
|
Family 28
|
1
|
1
|
11
|
11
|
3
|
2
|
1
|
0
|
0
|
Wild type
|
NA
|
NA
|
Family 29
|
1
|
2
|
17
|
13
|
5
|
3
|
3
|
0
|
0
|
Wild type
|
NA
|
NA
|
Family 30
|
2
|
4
|
47
|
40
|
10
|
7
|
6
|
2
|
0
|
Wild type
|
Miscarriage
|
NA
|
Family 313
|
2
|
3
|
27
|
26
|
7
|
6
|
5
|
1
|
1
|
Heterozygote
|
Heterozygote
|
Heterozygote
|
Family 32
|
1
|
2
|
21
|
15
|
6
|
5
|
5
|
1
|
1
|
Heterozygote
|
Heterozygote
|
Heterozygote
|
Family 33
|
1
|
0
|
15
|
14
|
3
|
1
|
0
|
0
|
0
|
NA
|
NA
|
NA
|
Family 34
|
1
|
1
|
12
|
11
|
9
|
7
|
7
|
1
|
1
|
Wild type
|
NA
|
Wild type
|
Family 353
|
2
|
1
|
28
|
28
|
4
|
4
|
1
|
1
|
0
|
Heterozygote
|
Heterozygote
|
NA
|
Family 36
|
1
|
1
|
22
|
17
|
6
|
5
|
2
|
1
|
0
|
Heterozygote
|
Heterozygote
|
NA
|
PGT: preimplantation genetic testing, FET: Frozen embryo transfer, CP: clinical pregnancy, LB: live birth, NA: not applicable
1 Unaffected embryos, including non-carrier and carrier embryos
2 Transferable embryos diagnosed as unaffected and euploid
3 Not enough biopsied blastocysts for analysis and a second oocyte pick-up was needed.
4 Single blastocyst transfer which developed to monochorionic diamniotic.
|
Clinical outcomes
Two couples had no transferable blastocysts after the first PGT cycle, and did not perform another PGT cycle. The other 34 couples were transferred with a single blastocyst (53 FET cycles). Thirty-two cycles resulted in clinical pregnancy and the clinical pregnancy rate was 60.1% (32/53) per FET cycle. Family 15 developed a monochorionic diamniotic twin pregnancy after a single blastocyst transfer and resulted in the birth of two healthy babies.
Twenty-two cycles (22 couples) resulted in 23 live births and the live birth rate was 43.4% (23/53, 3 cycles were ongoing pregnancy). The prenatal diagnosis results and/or thalassemia gene analysis after the delivery were concordant with the NGS-PGT results for all 25 cycles. Seven cycles resulted in miscarriage before 12 weeks’ gestation (7/32, 21.9%), and the abortion villus from 4 of the cycles showed normal karyotype and thalassemia results consistent with the NGS-PGT results. However, samples from the aborted fetuses’ in 3 cycles were not available because the pregnancy lasted less than 5 weeks.