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.
The mean ages of the women were identical in the 2 study groups (Table 1). In group A, 189 mature oocytes were fertilized and cultured for biopsy on day 3, and in group B, 330 mature oocytes were fertilized and cultured with the aim of biopsy on day 5.
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.
One of the major advantages of blastocyst biopsy over cleavage biopsy is that a larger quantity of trophectoderm cells with adequate genetic material is obtained for testing, compared with one blastomere on day 3 of development25,32. More DNA available for analysis ensures higher amplification efficiency and assessment as our data shows. PGT may be performed for any genetic disorder provided there is enough sequence information to promote the design of specific primers or probes. Genotyping for monogenic disorders are PCR based. These PCR-based techniques require very sensitive protocols and the application for the diagnosis of monogenic disorders in single cells is susceptible to inherent flaws such as complete amplification failure or allelic drop out where one allele fails to amplify26,33. In this study, PCR product was achieved in only 64.7% of biopsied embryos in group A while 93.4% with successful amplification was recorded in group B. Among the embryos in group A that provided no PCR product but developed into blastocysts, genotyping may have been successful if trophectoerm biopsy took place at the blastocyst stage. For trophectoderm biopsy only 6.6% of cases recorded genotyping failure. Embryo biopsy at the blastocyst stage should have a positive impact on the reduction of PCR failure as well as allelic drop out, in regards to PGD for monogenic disorders, as there is availability of more cells for genetic analysis25.
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.
Extended culture could also play a beneficial role in the preference of trophectoderm biopsy over cleavage biopsy. As highlighted by Adler et al36, increasing the duration of embryo culture with biopsy at the blastocyst stage resulted in lower aneuploidy rates as compared with cleavage stage biopsy. Although aneuploidy screening was not performed in our study, this indicates an added advantage of blastocyst biopsy over cleavage. Worthy of note, is the drop in clinical pregnancy rates (20%) in group B (trophectoderm biopsy) from a 60% positive pregnancy test rate. We speculate this may be a result of embryos not undergoing chromosomal screening in addition to sickle cell diagnosis.
Successful outcomes following trophectoderm biopsy, require reliable and efficient cryopreservation techniques. The application of accurate blastocyst vitrification, coupled with consistent excellent warming results, allows enough time for genetic analysis of biopsied specimen37 as well as reduce the occurrence of ovarian hyper stimulation in patients at risk. Being a specialized technique that requires high efficacy, incorrect application of cryopreservation resulting in the loss of precious embryos may be a limiting factor in the global acceptance of routine blastocyst biopsy technique. However, there is literature documenting high (over 95%) survival rates of embryos following vitrification and subsequent warming19,38. Success with this approach demonstrates that trophectoderm biopsy is an effective method of achieving pregnancy during the application of PGT for sickle cell anemia.
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.
This preliminary study suggests that trophectoderm biopsy provides sufficient genetic material for more efficient diagnosis and does not compromise embryo implantation and pregnancy rates in PGT cycles. We consider it important to publish this because of the high number of enquiries about the process of PGT in this part of the world. Second, the preliminary report will encourage the use of trophectoderm biopsy, which has many advantages. The evolution of this technique as well as proper cryobiology in developing countries such as Nigeria, make it possible for IVF clinics to offer solutions to patients at risk of transmitting hereditary diseases.
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.
Sources of funding
Conflict of interest statement
The authors declare that they have no financial conflict of interest with regard to the content of this report.
1. Harper JC, Sengupta SB. Preimplantation genetic diagnosis: state of the art 2011. Hum Genet 2012;131:175–86.
2. Stern HJ. Preimplantation genesis diagnosis: Prenatal testing for embryos finally achieving its potential. J Clin Med 2014;3:280–309.
3. Handyside AH, Kontogianni EH, Hardy K, et al. Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification. Nature 1990;344:768–770.
4. Xu K, Shi ZM, Veeck LL, et al. First unaffected pregnancy using preimplantation genetic diagnosis for sickle cell anemia
. JAMA 1999;281:1701–6.
5. Kuliev A, Pakhalchuk T, Verlinsky O, et al. Preimplantation genetic diagnosis for hemoglobinopathies. Hemoglobin 2011;34:547–555.
6. Ashiru OA, Fagbohun CF, Abisogun AO, et al. In vitro fertilisation and embryo transfer of human oocytes in Lagos. Medipharm Med J 1986;1:23–6.
7. Okeke C, Ailoje-Ibru K, Olukoya K, et al. Successful pregnancy outcome after in vitro fertilization following Pre-implantation Genetic Diagnosis/Polymerase Chain Reaction screening for single gene disorder (sickle cell anaemia) before embryo transfer: the clinical experience of an in vitro fertilization clinic in Nigeria. Niger Med J 2014;55:87–90.
8. Harper JC, Boelaert K, Geraedts J, et al. ESHRE PGD Consortium data collection V: cycles from January to December 2002 with pregnancy follow-up to October 2003. Hum Reprod 2006;21:3–21.
9. Harper JC, Coonen E, De Rycke M, et al. ESHRE PGD Consortium data collection X: cycles from January to December 2007 with pregnancy follow-up to October 2008. Human Reprod 2010;25:2685–707.
10. Harton G, Magli C, Lundin K, et al. ESHRE PGD Consortium/Embryology Special Interest Group-Best Practice Guidelines for Polar Body and Embryo Biopsy for Preimplantation Genetic Diagnosis/Screening (PGD/PGS). Hum Reprod 2010;26:41–6.
11. Jones GM, Trounson AO, Gardner DK, et al. Evolution of a culture protocol for successful blastocyst development and pregnancy. Hum Reprod 1998;13:169–77.
12. Jones GM, Trounson AO. Blastocyst stage transfer: pitfalls and benefits. The benefits of extended culture. Hum Reprod 1999;14:1405–8.
13. Gardner DK, Lane M, Schoolcraft WB. Physiology and culture of the human blastocyst. J Reprod Immun 2002;55:85–100.
14. Gardner DK, Lane M, Stevens J, et al. Ongoing development of a human blastocyst culture system. Fertil Steril 2002;78:S8.
15. Karaki RZ, Samarraie SS, Younis NA, et al. Blastocyst culture and transfer: a step toward improved in vitro fertilization outcome. Fertil Steril 2002;77:114–8.
16. Abdelmassih V, Balmaceda JP, Nagy ZP, et al. ICSI and day 5 embryo transfers: higher implantation rates and lower rate of multiple pregnancy with prolonged culture. Reprod Biomed Online 2001;3:216–220.
17. de Boer KA, Catt JW, Jansen RPS, et al. Moving to blastocyst biopsy for PGD and single embryo transfer at Sydney IVF. Fertil Steril 2004;82:295–8.
18. Kokkali G, Vrettou C, Traeger-Synodinos J, et al. Birth of a healthy infant following trophectoderm biopsy
from blastocysts for preimplantation diagnosis of b-thalassaemia major. Hum Reprod 2005;20:1855–9.
19. McArthur SJ, Leigh D, Marshall JT, et al. Pregnancies and live births after trophectoderm biopsy
and preimplantation genetic testing
of human blastocysts. Fertil Steril 2005;84:1628–36.
20. Monk M, Muggleton-Harris A, Rawlings E, et al. Preimplantation diagnosis of HPRT-deficient male, female, and carrier female mouse embryos by trophectoderm biopsy
. Hum Reprod 1988;3:377–81.
21. Yanagimachi R. Intracytoplasmic injection of spermatozoa and spermatogenic cells: its biology and applications in humans and animals. Reprod Biomed Online 2005;10:247–88.
22. Jansen RPS. The effect of female age on the likelihood of a live birth from one in-vitro fertilisation treatment. Med J Aust 2003;178:258–61.
23. Boone WR, Lee Higdon H III, Johson JE. Quality management issues in the assisted reproduction laboratory. J Reprod Stem Cell Biotechnol 2010;1:30–107.
24. Swain JE. Decisions for the IVF laboratory: comparative analysis of embryo culture incubators. Reprod Biomed Online 2014;28:535–47.
25. Kokkali G, Traeger-Synodinos J, Vrettou C, et al. Blastocyst biopsy versus cleavage stage biopsy and blastocyst transfer for preimplantation genetic diagnosis of b-thalassaemia: a pilot study. Hum Reprod 2007;22:1443–9.
26. Thornhill AR, Snow K. Molecular diagnostics in preimplantation genetic diagnosis. J Mol Diagn 2002;4:11–29.
27. Malcov M, Naiman T, Yosef DB, et al. Preimplantation genetic diagnosis for fragile X syndrome using multiplex nested PCR. Reprod Biomed Online 2007;14:515–21.
28. Kanavakis E, Traeger-Synodinos J. Preimplantation genetic diagnosis in clinical practice. J Med Genet 2002;39:6–11.
29. Sandalinas M, Sadowy S, Alikani M, et al. Developmental ability of chromosomally abnormal human embryos to develop to the blastocyst stage. Hum Reprod 2001;16:1954–8.
30. Clouston HJ, Herbert M, Fenwick J, et al. Cytogenetic analysis of human blastocysts. Prenat Diagn 2002;22:1143–52.
31. Grifo JA, Flisser E, Adler A, et al. Programmatic implementation of blastocyst transfer in a university-based in vitro fertilization clinic: maximizing pregnancy rates and minimizing triplet rates. Fertil Steril 2007;88:294–300.
32. Schoolcraft WB, Fragouli E, Stevens J, et al. Clinical application of comprehensive chromosomal screening at the blastocyst stage. Fertil Steril 2010;94:1700–6.
33. Sermon K, Van Steirteghem A, Liebaers I. Preimplantation genetic diagnosis. Lancet 2004;363:1633–9.
34. Cohen J, Wells D, Munné S. Removal of 2 cells from cleavage stage embryos is likely to reduce the efficacy of chromosomal tests that are used to enhance implantation rates. Fertil Steril 2007;87:496–503.
35. Cimadomo D, Capalbo A, Ubaldi FM, et al. The impact of biopsy on human embryo developmental potential during preimplantation genetic diagnosis. Bio Med Res Int 2016;2016:7193075. Available at:http://dx.doi.org/10.1155/2016/7193075
36. Adler A, Lee HL, McCulloh DH, et al. Blastocyst culture selects for euploid embryos: comparison of blastomere and trophectoderm biopsies. Reprod Bio Med Online 2014;28:485–91.
37. Kuwayama M. Highly efficient vitrification
for cryopreservation of human oocytes and embryos: The Cryotop method. Theriogenology 2007;67:73–80.
38. Capalbo A, Treff NR, Cimadomo D, et al. Comparison of array comparative genomic hybridization and quantitative real-time PCR-based aneuploidy screening of blastocyst biopsies. Eur J Hum Genet 2015;23:901–6.
Keywords:Copyright © 2018 The Authors. Published by Wolters Kluwer on behalf of the International Federation of Fertility Societies. All rights reserved.
Sickle cell anemia; Preimplantation genetic testing; Cleavage biopsy; Trophectoderm biopsy; Vitrification; Frozen embryo transfer