DNA samples obtained from either cleavage stage or blastocyst stage biopsy went through the same analytic process. Analysis of the biopsied samples involved PCR protocols. A 3-step nested PCR with or without amplified DNA for both STR and mutation analysis was used. This PCR reaction was followed up with Sangar Sequencing to analyze mutations. These processes have been previously described elsewhere26,27.
In group A, 131 (84%) embryos reached the 6–8 cell stage and were biopsied. Of the 131 blastomeres removed, 86 (65.7%) gave a PCR product representing both alleles allowing an accurate diagnosis. In the remaining 45 (34.3%) blastomeres, “No Result” was indicated which could mean either of the following: no intact DNA for analysis, PCR failure, or PCR product, which did not meet assay quality control criteria (the laboratory does not report separately on these conditions). The total number of embryos diagnosed as unaffected for sickle cell anemia was 53/86 (40.4%). Of the 53 unaffected embryos, 36 developed to the blastocyst stage and were available for transfer.
A total of 27 blastocysts were transferred (average of 2.1 embryos) in group A. Of 18 patients, 15 had embryos that reached the blastocyst stage of which, 13 had blastocysts that were unaffected. One patient had a positive serum b-HCG pregnancy test (7.7% pregnancy rate). Pregnancy was confirmed at 5 weeks by ultrasound diagnosis with a single fetal heartbeat representing a 3.7% implantation and 7.7% clinical pregnancy rate. The single pregnancy was delivered at 37 weeks of gestation and 1 live birth was recorded (3.7%).
In group A, a single patient had a prior pregnancy resulting in the birth of an affected child. The other 17 had no previous affected pregnancy and only went through PGT as a screening test to achieve an unaffected pregnancy.
Group B, 106 (47.3%) embryos made it to the blastocyst stage for biopsy. Of the 106 blastocysts that were biopsied, 99 (93.4%) gave complete genotype analysis with 72 blastocysts diagnosed as unaffected for sickle cell anemia (68%). Only 7 (6.6%) biopsies indicated “No Result.”
Twenty-eight blastocysts were transferred (average of 1.9 embryos) in frozen embryo cycles. Only one patient of 16 had no embryos transferred as all available blastocysts were diagnosed with sickle cell anemia. Surplus unaffected embryos remained vitrified, including blastocycts with no result, as they will be rebiopsied and retested in a future cycle. Embryos diagnosed as affected were discarded. Nine patients had a positive serum b-HCG pregnancy 10 days after embryo transfer (60% pregnancy rate). Pregnancies were confirmed at 5 weeks of gestation by ultrasound diagnosis with 4 fetal heartbeats (2 singletons and 1 set of twins), representing a 32.1% implantation and 20% clinical pregnancy rate. One singleton pregnancy spontaneously aborted and 5 pregnancies were biochemical. Three pregnancies were delivered at 37.3+1.97 weeks, with 4 babies born (20%).
In group B, only 4 of the 16 patients had a previous pregnancy which resulted in the birth of an affected child. The other 12 had no previous affected pregnancy and only went through PGT as a screening test to achieve an unaffected pregnancy.
The live birth of a healthy unaffected child following accurate genetic diagnosis and positive outcomes of all aspects of assisted reproduction depicts the overall success of PGT25,28. This study is the first account of the routine use of blastocysts for PGT and diagnosis of sickle cell anemia in Nigeria. Although both procedures were carried out at different times, the genetic analysis performed for both techniques was the same. Thus we demonstrate here that blastocyst biopsy may produce better outcomes with respect to pregnancy and implantation rates if compared with cleavage biopsy. This may be because of little to no damage to the embryos during trophectoderm biopsy as the blastocysts survived better and continued the developmental process. Our data may also support the evidence that blastocysts have a lower degree of chromosomal abnormalities29,30, thus making embryo biopsy and diagnosis very viable at this stage19. Our observations may also coincide with proof of reduction in the level of mosaicism found compared with that of cleavage stage embryos previously reported here31.
Embryo survival with respect to the implantation potential after biopsy must be considered. To counter the problem of mosaicism in cleavage embryos, 2 blastomere biopsy may be implemented although this can deplete embryonic mass resulting in less favorable clinical outcomes19,34. In blastocyst biopsy, the inner cell mass that eventually become the fetus will unlikely be damaged, as there has been preferential removal of the more accessible trophectoderm cells, which contribute only to placental tissues, thereby reducing trauma to the embryo. This, in turn, reduces probable ethical issues associated with trophectoderm biopsy2,35. However small this study is, the implantation rates (group A: 3.7%; group B: 32.1%), suggest that trophectoderm biopsy favors a higher implantation rate compared to cleavage biopsy.
In summary we have successfully demonstrated the use of both techniques of embryo biopsy. Our study indicates that trophectoderm biopsy incorporated in PGT protocols is a feasible method of improving outcomes in the prevention of pregnancies affected by this hereditary disorder. Although therapeutic trials are being performed for the “correction” of the hemoglobin S abnormality, PGT offers a prophylactic therapy to sickle cell anemia.
Further studies with a greater patient pool and more published data on the process of trophectoderm biopsy should be encouraged as this may even demonstrate an overall advantage of such an approach.
An abstract on this study was presented at the 2017 ASRM Scientific Congress & Expo.
The authors declare that they have no financial conflict of interest with regard to the content of this report.
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