Preimplantation genetic testing (PGT) may be offered in combination with assisted reproductive technology to improve reproductive options1. It is most often referred to as preimplantation genetic diagnosis (PGD). This technique uses modern molecular and genetic methods to test for single gene disorders, sex-linked diseases, and chromosomal abnormalities2. The first DNA diagnostic procedure for preimplantation purposes was reported over 20 years ago3 and PGT for sickle cell anemia was first applied successfully nearly a decade after4 as an alternative to prenatal diagnosis to avoid selective termination of pregnancy in a couple at risk of transmitting this genetic defect.
Sickle cell anemia is one of the most common hemoglobinopathies with a frequent indication for PGT5. It is a single gene disorder; and is hereditary as an autosomal recessive trait. This genetic defect is quite common in people of African origin and in Nigeria, affects about 3% of the population. The pioneering work of Ashiru et al6 first introduced in vitro fertilization (IVF) to Nigeria in 1986 and with the evolution of this technology to include PGT; the first live birth of a sickle cell free child after successful PGT in Nigeria was recorded7.
Most couples that frequent fertility clinics for IVF in order to prevent the transmission of hereditary genetic conditions are usually fertile and may already have children naturally. However, excluding the oocyte/zygote or embryo biopsy step, the process of assisted reproductive technology for PGT cases is the same as for infertility treatment, irrespective of whether the couple is fertile or not as IVF is required for the production of embryos to be analyzed. PGT requires the analysis of embryonic genetic material and the 3 possible sources are polar bodies from oocytes, blastomeres from cleavage embryos, and trophectoderm cells from blastocysts. In clinical practice, the most common method for obtaining genetic material for preimplantation genetic analysis is the cleavage stage biopsy where 1, or in some cases, 2 blastomeres are aspirated from 6 to 8 cell embryos on day 3 of development.8–10 There are several ways to breach the zona pellucida but to date embryo biopsy using a laser is the most popular method of choice.
Over the years, significant improvement in laboratory standards, the quality of culture media and the application of extended culture has resulted in an increase in blastocyst quality and quantity11–14 leading to blastocyst culture and transfer performed routinely in many IVF clinics. There are reports showing improved implantation rates after blastocyst transfers compared with earlier stages of transfer15,16. Trophectoderm biopsy has gained increased popularity due to these advances in IVF, enabling many centers to successfully culture embryos to the blastocyst stage of development thus making it a very suitable biopsy stage for PGT1. Clinical successes of PGD following trophectoderm biopsy have previously been reported17–19 following preceding work done by Muggleton-Harris and others in 198820.
In this article, we document our experience using trophectoderm biopsy as well as cleavage biopsy for PGT, to determine the genetic status of embryos produced by assisted reproduction for couples who are heterozygous carriers of the sickle cell mutation. The cases summarized here for the detection of a monogenic disorder date from April 2011 to February 2017
Materials and methods
Before PGT cycles begin, protocol optimization is carried out. Haplotype analysis, which involves the selection of markers by the use of primers for the genetic analysis is performed using information obtained from DNA samples collected from immediate family members (mother, father, and affected child (ren) if available). Karyomapping technology is required to designate a positive or negative reference for each individual with the confirmed mutation.
Patient recruitment and IVF procedures
IVF-PGT cycles were performed for 34 couples; both partners being carriers of the sickle cell anemia trait and requesting for PGT with the goal of preventing the transmission of the genetic defect to their offspring. All cycles took place between April 2011 and February 2017 in collaboration between Medical Art Center, Lagos, Nigeria and Genesis Genetics, Cooper Genomics, USA. Inclusion criteria were all cycles in which one or more embryos underwent day 3 or day5/6 embryo biopsy for sickle cell anemia diagnosis.
All the couples underwent the basic fertility work-up (blood screening, clinical examination, semen analysis, transvaginal ultrasound scan, day 3 female hormonal profile, hysterosalpingography) at Medical Art Center. All couples also received, signed and returned necessary consent forms. IVF cycles commenced following standard IVF protocols in combination with micromanipulation techniques for Intracytoplasmic sperm injection previously described in other studies21. All patients began with controlled ovarian stimulation using gonadotrophin-releasing hormone agonist (short or long) or gonadotrophin-releasing hormone antagonist protocols and follicle-stimulating hormone. Each patient was monitored regularly by ultrasound scans and hormone tests. Transvaginal ultrasound guided oocyte pick up was scheduled for 36 hours after the administration of human chorionic gonadotrophin (HCG) injection as reported previously22. Intracytoplasmic sperm injection and embryo culture takes place thereafter. Subjects were placed in 2 groups (A and B) depending on biopsy type. Group A consisted of patients whose embryos had cleavage biopsy and group B included patients whose embryos had blastocyst biopsy.
All embryos were cultured in vitro in 20 μL micro drops of G1Plus culture medium (Vitrolife Goteborg, Sweden), and on day 3 postfertilization, they were transferred to 30 μL micro drops of G2Plus (Vitrolife Goteborg) for culture to the blastocyst stage. This occurred under standard incubation conditions (37°C and 6% CO2, 5% O2) noted in these journals23,24. All embryo vitrification and transfers took place at the blastocyst stage.
In group A, cleavage stage embryos were evaluated on the morning of day 3 with biopsy of 1–2 blastomeres done on embryos showing 6–8 cells, respectively, as reported in25 (Fig. 1). Blastomeres were rinsed in 0.5 μL of wash buffer, before loading in 0.2 mL RNAse-DNAse free polymerase chain reaction (PCR) tubes containing 2 μL of lysis buffer. Samples were shipped to the genetics laboratory at Genesis Genetics for testing. Following biopsy, embryos were further cultured until day 5 and 6 after fertilization and resulting blastocysts were transferred based on the results obtained. Vitrification of surplus unaffected blastocysts followed thereafter to be warmed for future FET cycles. Unaffected embryos were transferred into patients’ uterus using a Labotect ET catheter (Labotect-Technik-Göttingen GmbH, Germany).
For blastocyst biopsy (group B), assisted hatching took place either on day 3 or on the morning of day 5. Blastocysts after examination were considered ready when 5 or more trophectoderm cells were seen hatching out of the open zona (Fig. 2). Any blastocyst not ready on day 5 was biopsied the next day (day 6). A biopsy pipette with internal diameter of 35 μm (Vitrolife Goteborg) was used to aspirate three to five cells, and the biopsy specimen (Fig. 3) was removed with gentle traction and laser pulsation from a ZILOS-Tk Laser (Hamilton Thorne Biosciences, Beverly, MA) as previously described19. The biopsied specimens, after rinsing in 0.5 μL drops of wash buffer were then loaded into 0.2 mL RNAse-DNAse free PCR tubes with 2 μL of lysis buffer (Fig. 4). Samples were properly sealed, labeled and transported to the genetics laboratory (Genesis Genetics) for analysis. The biopsied blastocysts undergo vitrification. Patients were prepped for FET cycles (endometrial linings of patients were prepared using increasing doses of Estradiolvalerate from 2 mg daily up to 8 mg with continuous monitoring) and unaffected embryos (Fig. 5) transferred using Labotect catheter (Labotect-Technik-Göttingen GmbH). For all patients, serum b-HCG was analyzed 10 days after embryo transfer to determine the onset of pregnancy at 5 weeks of gestation, and viable pregnancy confirmed by ultrasound diagnosis of fetal heartbeat.
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.
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