Eleven years of preimplantation genetic diagnosis for human leukocyte antigen matching: is there room for improvement?

Keywords::

• assisted reproductive technology
• donor sibling
• embryo biopsy
• hematopoietic stem cell transplantation
• HLA matching
• monogenic disease
• preimplantation genetic diagnosis

Preimplantation testing to determine the HLA tissue type of an embryo (preimplantation genetic diagnosis [PGD]-HLA) aims to identify an embryo that is HLA-compatible with an existing sibling in need of hematopoietic stem cell transplantation (HSCT). Matched embryos can be transferred to the womb, with the hope that a pregnancy is established and a donor sibling born. PGD-HLA requires close collaboration between experts in many fields, including hematology, HSCT, assisted reproductive technology (ART), embryology and genetics. In fact, many aspects of the procedure incorporate the pioneering work of several Nobel prize winners. PGD-HLA was first reported in 2001, 11 years after the first clinical application of PGD to exclude a monogenic disease [1]. Before this, couples seeking a donor sibling to treat an already existing child often risked a natural conception, followed by prenatal testing [2], and PGD-HLA represents a tremendous advancement in this. PGD-HLA is most often performed simultaneously with PGD to diagnose the familial single-gene disease present in the affected sibling, with the dual aim of establishing an unaffected pregnancy which is also HLA compatible with the affected child. HLA typing without mutation analysis is performed in cases where a child requires HSCT to treat an acquired disease, for example certain leukemias [3].

Diagnostic, clinical & ethical parameters

The step of genetic analysis in PGD-HLA diagnosis has evolved through the years as technology has improved and knowledge has broadened. The nature of the HLA region itself presents major challenges for HLA typing, since it is large, highly polymorphic and susceptible to recombination [4]. Various PGD-HLA protocols have been reported. The first of these involved a direct HLA-genotyping approach, using allele-specific PCR or mini sequencing, which required the development of unique protocols for each family. Subsequently, indirect HLA-haplotyping was introduced. On the basis of the linkage analysis, it provides a more generic methodology and has been the preferred approach since 2004 [3,5]. The methodological strategies used vary, some being PCR-based alone and others including an initial step of whole-genome amplification [6]. The largest studies to date report multistep PCR-based protocols, involving several nested PCRs and analytical steps, although lately there is a trend to simplify the diagnostic procedure using single-step highly multiplexed PCR protocols. There are guidelines indicating the minimum number of short tandem repeat markers that should be used in PGD-HLA, and the regions within the HLA locus that should be covered [7]. More recent reports have noted that a higher number of markers may be required for an accurate result, while the necessity of deriving the haplotype over some HLA regions is still not clear [8,9]. Overall, the risk of misdiagnosis reported to date is lower than 0.5% [10].

The chance that the entire PGD-HLA procedure, that is, the birth of a compatible sibling, will be successful is restricted by certain technical but mainly biological limitations. First, take the chance of finding a genetically suitable embryo. For HLA typing alone, the genetic chance embryos will be matched to the affected sibling is 0.25. When concurrently excluding a recessive monogenic disease it falls to 0.188, and when excluding an autosomal dominant or X-linked disease, genetic chance drops to 0.125. Second, the chance of a successful outcome is also influenced by the overall outcome of the ART cycle. The women must respond well to hormonal stimulation to maximize the number of embryos available for diagnosis. Of note, outcomes tend to be poorer in this group of patients due to the often older maternal age, compared with patients undergoing routine fertility treatment [11]. Embryo quality is also important to ensure the best implantation potential, but there are still no certain methods to select the ‘best’ embryo(s). The overall chance of a pregnancy and birth is near 30% in routine ART. Combining all the above, it is no surprise that the reported chance of complete success for a PGD-HLA cycle (the ‘take-home baby rate’) rarely surpasses 10% [12].

Following the birth of a healthy compatible child, there follows the crucial steps of harvesting hematopoietic stem cells and timely HSCT. Finally, the high cost of the procedure may present another limitation, especially when several treatment cycles may be necessary before a matched embryo is identified and a pregnancy achieved.

Ethical parameters of the PGD-HLA practice have been extensively debated during the past 12 years. Main issues have included the parental motives in creating and selecting a child to be ‘used’ in order to save a sibling, the risks imposed to the mother who will possibly need to undergo several ART cycles, any potential risks to the child to be born and the psychological impact on all family members [2,13]. Today, it is acknowledged that PGD-HLA represents a valuable treatment option for all involved (the parents, the healthy baby born and sick child cured), and although difficult, it is well worth the effort when it succeeds. However, the need for psychological support of donor and recipients has been underlined [14,15].

Worldwide experience

Probably due to the complexity of the whole procedure, which requires a highly dedicated multidisciplinary team of clinicians and health scientists, worldwide, there are relatively few PGD centers offering HLA typing on human preimplantation embryos (15% of centers report data to the European Society of Human Reproduction and Embryology), and published data on PGD-HLA are limited. On the basis of these data from the European Society of Human Reproduction and Embryology, more than 500 PGD-HLA cycles have been performed overall to date. The most common indication is PGD combined with exclusion of β-thalassemia and/or sickle-cell syndromes (~70% of cycles performed) [12]. The largest published series of PGD-HLA cycles reports 262 cycles combining HLA typing and disease exclusion and 62 cycles for HLA typing alone, reporting a 30–35% clinical pregnancy rate per embryo transfer, with 61 babies born and 21 children cured [11]. However, the outcomes of PGD-HLA reported by other groups vary, illustrating the unpredictability of overall success with the technique. Reports of successful births of healthy HLA donors are limited, and the exact number of children cured globally is not known, but estimated to be a few hundred [16,17].

Expected developments & future aims

In the past few years, continued advancements in HSCT, including the introduction of novel transplantation protocols, the possibility of using haploidentical family donors and the increasing number of donor registries and cord blood banks, have led to an increase in the number of transplant procedures performed worldwide with an increasing number of patients receiving this effective therapy [18,19]. Transplantation success continues to be higher with matched sibling transplants versus unrelated transplants and thus PGD-HLA remains an advantageous approach. However, one possibility is that future improvements in HSCT, which ensure safer transplant procedures overall, may prevail, making PGD-HLA redundant.

So, is there room for improving PGD-HLA? The major limitations to success include the limited genetic chance and the low rates of live births after ART. The genetic chance cannot be changed per se. However, there are approaches on the horizon that may maximize the quality of genetic diagnosis in each single embryo cell that is sent for analysis. The optimization of newer molecular approaches at the single-cell level, such as single nucleotide polymorphism arrays and next-generation sequencing technologies, will offer more generic approaches for genetic diagnosis, simplifying protocol work-up and allowing wider application. They will additionally provide considerably more genetic information, which may facilitate a better understanding of some pertinent issues, such as recombination events in the HLA region. This, together with improved knowledge about the HLA regions most important for successful HSCT, may allow for the identification of more embryos that are appropriate for embryo transfer.

Future developments in ART are also expected to improve live birth rates. At present, a main research focus is the search for biomarkers to enable noninvasive embryo assessment for selection of oocytes and embryos with the highest implantation potential [20]. In the PGD-HLA procedure, due to the low proportion of embryos that are genetically suitable for transfer, a better evaluation of embryo potential could improve outcomes. Other aspects to improve ART results include the personalization of hormonal stimulation protocols, to obtain better-quality oocytes, and consequently a higher number and improved quality of embryos available for biopsy and diagnosis. This will, therefore, increase the chance of identifying good-quality embryos for transfer, and selecting those embryos will be critical for improving cycle outcome.

Thus, there is some room for improvement for PGD-HLA. However, we still do not know the overall clinical utility of the procedure, and there is a need to initiate a more holistic follow-up of cycles performed, preferably worldwide. The most notable gap, currently, is the lack of reports referring to HLA-matched siblings born and children successfully cured. To assist in this, experts involved in PGD and HSCT should support the development of a PGD-HLA cycle database together to include complete details for each cycle performed (ART, diagnosis, pregnancy and transplantation outcome and complications arising at any stage). This will enable a better assessment of the procedure and a better understanding of the laboratory and clinical issues involved, as well as revealing the impact on families, supporting a patient-centered view and an overall assessment of the true benefits of the PGD-HLA procedure.

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.