This study included 342 couples who requested PGT-M for α- or β-thalassemia at three reproductive centers between 2019 and 2022. In total, 1778 embryos were tested, with the average maternal age being 31.3 ± 4.3 years. Two hundred twenty-six couples were carriers of α-thalassemia, yielding 1197 blastocysts; 112 couples were carriers of β-thalassemia, contributing 568 blastocysts; and 4 couples were carriers of both α- and β-thalassemia, resulting in 13 blastocysts. The median number of embryos obtained per couple was 5, ranging from a minimum of 1 to a maximum of 21 embryos (Table 1).

Figure 1 details three scenarios of haplotype analysis, from straightforward (Scenario I) to most challenging (Scenario III). Scenario I is the most straightforward when biological samples from probands were available; Scenario II presents moderate difficulty when the couple lacks probands but provides complete samples from both biological parents; Scenario III poses the greatest challenge, marked by the absence of a proband and a lack of parental DNA samples.

Haplotype analysis from straightforward (Scenario I) to most challenging (Scenario III) in 342 couples. Scenario I: proband DNA samples available; Scenario II: parental DNA samples available; Scenario III: no DNA samples from probands or incomplete parental DNA samples

Among the 342 couples included in this study, 59 (17.3%) fit Scenario I as described above. Samples from the probands included peripheral blood, miscarriage tissue, amniotic fluid, and chorionic villi. The SNP haplotypes linked to the defective chromosomes carried by these couples were successfully confirmed and used to guide subsequent embryo selection (Fig. 1).

Two hundred two couples (59.1%) were categorized under Scenario II. Among these 202 couples, 198 couples (98.0%, 198/202) successfully obtained valid SNP loci for linkage analysis by sequencing both parental samples. However, the SNP haplotypes of four couples could not be determined based on the parental SNP loci. In one couple, the female partner had a de novo mutation (–SEA) in the α-thalassemia gene, whose parents had normal α-globin genes. In the other three couples, referred to as heterozygous families, the genotype of one partner was αα/–SEA, and since both parents were αα/–SEA, it was unclear which disease-causing chromosome was inherited. Among these 4 couples, in one couple with a de novo mutation and two couples with αα/–SEA, the haplotypes were successfully determined through reference embryo SNP loci (Fig. 1; Supplementary Fig. 2). In the remaining couple, by providing samples from two unaffected siblings and one carrier sibling, along with valid SNP loci from the parental samples, the haplotype was successfully determined (Supplementary Fig. 3).

The remaining 81 (23.7%) couples were categorized under Scenario III, wherein probands and either one or both parents’ blood samples were unavailable (Fig. 1). Among them, 40 couples provided peripheral blood samples from siblings, as detailed in Supplementary Table 1.

Within this subset of 40 couples (Fig. 2), 31 couples (Supplementary Table 1, ID: A1–A30, B1) were missing a parental sample from one partner. Through the use of sibling samples, successful haplotype analysis was achieved for 25 couples (80.6%, 25/31). However, haplotype analysis failed in 6 couples. For 2 of these couples, the haplotype was successfully constructed through single sperm sequencing, as the male partner’s parental samples were missing (Supplementary Fig. 4A). Additionally, three couples determined their haplotypes through reference embryos (Supplementary Fig. 4B). Unfortunately, haplotype analysis failed in 1 couple due to only one embryo achieved, in which the target gene was tested as normal, rendering it unsuitable as a reference embryo.

Haplotype analysis for 40 couples in Scenario III, classified by the number of missing parental DNA samples. Haplotypes were constructed using SNP sites from siblings’ DNA samples, single sperm, or affected embryos

Two couples lacked parental samples of the male partner (Fig. 2; Supplementary Table 1, ID: A31, B2). In one case, the male partner’s haplotype was successfully determined through SNP linkage analysis using samples from 5 siblings (Fig. 2; Supplementary Table 1, ID: A31). In the other case, paternal sample of the male partner was unavailable, and the female partner belonged to a heterozygous family. The two embryos obtained were found to possess the normal target gene, so it is unsuitable as a reference embryo (Fig. 2; Supplementary Table 1, ID: B2). Seven couples were unable to provide complete family samples from either side (Supplementary Table 1, ID: A32–A38). Ultimately, haplotypes were successfully analyzed in all 7 couples through the sibling samples or reference embryos.

For the 9 couples (ID: A26–A30, A33, A36, and B1–B2) whose haplotype analysis was unsuccessful despite the use of sibling samples, the analysis revealed two primary causes: (1) the siblings’ genotypes were consistent with the carriers, as observed in couples A30 and A33, and (2) the sibling genotypes were normal, and the parental genotypes provided were also normal, making it difficult to rule out the possibility of de novo mutations, as seen in couples A27–A29 (Supplementary Table 1).

For the additional 41 couples in Scenario III, who were unable to provide sibling samples, 40 couples successfully constructed the haplotype through reference embryos (Fig. 1). In one couple, both partners were devoid of paternal samples. The male partner’s haplotype was constructed via single sperm sequencing, while the female partner’s haplotype was accurately constructed through reference embryo analysis (Supplementary Fig. 4C).

In summary, haplotype linkage was successfully established in 99.4% (340/342) of couples. Parental haplotype analysis failed for two couples because the number of embryos obtained was low and the target gene in the embryos was normal, making them unsuitable for use as reference embryos for haplotype construction.

Among the 342 couples included in the study, haplotype analysis ultimately failed for two couples with α-thalassemia, leading to the exclusion of three embryos from these two couples in the CNV analysis of embryos. Therefore, the study analyzed 1775 embryos from the remaining 340 couples.

(1) CNV results:

CNV detection is used to identify fragments with deletion/duplication ≥ 4 Mb, and mosaicism ≥ 30% of chromosome. The overall embryo amplification success rate was 97.7% (1734/1775). Of the amplified samples, 55.0% (953/1734) were euploid embryos, 32.9% (570/1734) were aneuploid embryos, and 12.2% (211/1734) were mosaic embryos (Table 2). Comparing the two WGA methods, the results showed that the MALBAC method outperformed the MDA method in terms of amplification success rate (98.8% vs. 96.2%), with a higher incidence of euploid embryos (57.9% vs. 50.6%, p = 0.003), and a reduced incidence of mosaicism (9.4% vs. 16.2%, p < 0.001) (Table 2).

(2) SNP linkage analysis and direct detection of target pathogenic mutation sites:

Due to the unavoidable occurrence of ADO during the WGA process, there is a certain risk of error in the direct detection of pathogenic loci in embryos. Therefore, the SNP linkage analysis method has been used simultaneously for more reliable results. It was worthwhile to investigate whether it was necessary to detect the target pathogenic mutation sites directly in WGA products derived from embryo biopsies.

Among the 340 couples, there were a total of 224 couples with α-thalassemia, accounting for 1194 embryos. Amplification failed in 32 (2.7%, 32/1194) embryos, and while amplification was successful, SNP linkage analysis failed in 42 (3.6%, 42/1162) embryos. Common factors for unsuccessful SNP linkage analysis included insufficient DNA samples or the number of available SNP sites. Thus, a total of 1120 embryos were included for analyzing the consistency between SNP linkage results and locus detection outcomes (Table 3). The success rate of detecting α-thalassemia loci was 96.3% (1078/1120), and the concordance between SNP linkage analysis results and locus detection results was 93.8% (1011/1078).

The study included 112 couples with beta-thalassemia, encompassing a total of 568 embryos. Nine (1.6%, 9/568) embryos failed amplification, and eleven (2.0%, 11/559) embryos exhibited successful amplification but had failed SNP linkage analysis. Consequently, 548 embryos were included for analyzing the consistency between SNP linkage results and sites detection results (Table 3). The success rate of detecting β-thalassemia loci was 100.0% (548/548), and the concordance between SNP linkage analysis results and locus detection results was 98.2% (538/548).

Additionally, among the four couples with α-/β-thalassemia, there were a total of 13 embryos. WGA amplification was successful for all embryos. The detection of the -α4.2 locus failed in two embryos. The locus detection results were also consistent with the analysis of the remaining 11 embryos for SNP linkage results, which was completed successfully.

In this study, the PGT-M results were reported based on SNP linkage analysis results. A total of 256 couples, with an average maternal age of 31.4 ± 4.1 years, underwent embryo transfer guided by PGT-M combined with PGT-A. Three hundred two single blastocyst transfer cycles were tracked to clinical pregnancy follow-up. Among these cycles, one resulted in an ectopic pregnancy, with the remaining 301 cycles achieving a clinical pregnancy rate of 69.4% (209/301), which was close to the rate of 68.8% (11/16) reported by Chen et al. in their small-scale thalassemia PGT-M study [4]. Currently, 24 cycles have culminated in miscarriage, while 100 cycles have resulted in live births (Table 4). Notably, in 4 cycles, patients received the transfer of low-level mosaic embryos (excluding high-risk chromosomes) due to a lack of euploid embryos, with these transfers leading to 3 clinical pregnancies.

Of the remaining 86 couples, 51 did not have any viable embryos for transfer, accounting for 14.9% (51/342) of the total couples. The 35 couples have not undergone a transfer yet.

Amniocentesis results for 99 transfer cycles were collected, demonstrating a 100% concordance with the PGT-M results for the HBA and HBB genes.

To further validate the accuracy of the SNP linkage analysis, embryos with failed locus detection or inconsistent results with SNP linkage analysis were analyzed in relation to clinical outcomes. According to the results of CNV and SNP linkage analysis, 19 of the 42 embryos with successful SNP linkage analysis but unsuccessful locus detection in α-thalassemia (Table 3, Fig. 3) were eligible for transfer. Five embryos were transferred, resulting in four live births. Amniocentesis results were available for three patients, all of which were consistent with the SNP linkage analysis results (highlighted in orange in Supplementary Table 2).

Clinical outcomes for patients with successful embryo SNP analysis but discrepancies in mutation site detection versus SNP linkage analysis

There were 67 α-thalassemia embryos in which SNP linkage analysis was successful, but the mutation site testing results are inconsistent with the SNP linkage analysis (Table 3, Fig. 3). Among these, 34 embryos were identified as transferrable, and ultimately 10 embryos were transferred. Clinical pregnancies were established in six (60.0%, 6/10) of these cases. Amniocentesis was performed for three patients, with results corroborating the SNP linkage analysis (highlighted in dark green in Supplementary Table 2).