Introduction

The first clinical application of pre-implantation genetic diagnosis (PGD) was reported in 1990 , and the first PGD cycles for β-thalassaemia were reported in 1998 . PGD characterizes the genetic status of cells (usually single cells) that have been biopsied from oocytes/zygotes or embryos created in vitro during an assisted reproductive treatment (ART). PGD is a multi-step procedure that requires close collaboration between gynaecologists who are experts in assisted reproduction, embryologists who are experts in micromanipulation of germ cells and in embryo biopsy and geneticists who are experts in genetic analysis at the single-cell level . The co-ordination of timing at each stage requires clear communication between all those involved.

PGD can be applied for two different purposes with respect to thalassaemia syndromes and haemoglobinopathies. The first involves the use of PGD as a form of early pre-natal diagnosis that represents an alternative reproductive option to conventional pre-natal diagnosis for couples at risk of transmitting a severe haemoglobinopathy and who wish to have an unaffected child. The principal advantage of PGD over conventional pre-natal diagnosis is that it precludes the dilemma of terminating an ongoing pregnancy when the test reveals an affected genetic status. Therefore, PGD is considered to be a valuable reproductive and treatment option for females who have previously undergone a pregnancy termination following a pathological result after a conventional pre-natal diagnosis. In addition, PGD is an appropriate choice for carrier couples who also have infertility problems and plan to use ART anyway. Finally, PGD is a suitable choice for couples with an ethical or religious objection to pregnancy termination .

PGD is also suitable for couples where one member is affected with thalassaemia or sickle cell syndrome and the other is a carrier. The genetic probability that such couples will have an unaffected conception is reduced to 50% (versus the 75% chance if both members of the couple are simple heterozygotes). It is particularly appropriate if the woman is the member of the couple who has haemoglobinopathy as PGD allows to avoid unnecessary pregnancy terminations, thus reducing additional health risks to the patient. Needless to say, particularly if the thalassaemia patient is the woman, they should undergo full assessment prior to the ART to ensure that the treatment and any resulting pregnancies will not have any detrimental consequences to their already burdened state of health.

The second application is to use pre-implantation genetic testing to determine the human leukocyte antigen (HLA) tissue type of an embryo (PGD-HLA) and identify embryos that are HLA compatible with an existing sibling in need of haematopoietic stem cell transplantation (HSCT). Matched embryos that have been identified to be unaffected for the familial haemoglobinopathy can be selected for transfer. Of course, PGD-HLA is associated with several ethical implications that will be presented below.

Prevention of haemoglobinopathies: PGD or conventional pre-natal diagnosis?

Haemoglobinopathies are the most prevalent group of serious autosomal recessive monogenic disorders worldwide. There are an estimated 300,000–400,000 affected births each year, which include approximately 40,000 patients with transfusion-dependent β-thalassaemia major and approximately 280,000 patients with sickle cell syndromes . Despite tremendous advances in the understanding of the molecular pathophysiology of these disorders, along with substantial improvements in conventional therapies, and the emergence of gene and molecular therapies , the so-called ‘prevention’ remains as the favoured strategy for reducing the burden on families that are at risk of having an offspring with the most clinically severe manifestations of these diseases ( Table 1 ). Prevention includes the timely identification of carrier couples so that they can be informed about their reproductive choices and then proceed to select the reproductive option that is most appropriate for them. The disorders of haemoglobin synthesis are, with very rare exceptions, transmitted through an autosomal recessive mode of inheritance, which means that when both partners of a couple carry either severe HBB gene mutations or severe HBA gene mutations, there is a 1 in 4 chances that each conception (pregnancy) will be affected with a severe haemoglobin disorder. It is strongly recommended that all carrier couples should be counselled by qualified health professionals with expert knowledge regarding the molecular genetics and clinical expression of the haemoglobinopathies . Because most carrier couples do not opt for gamete donation or adoption, the most preferred reproductive options include conventional pre-natal diagnosis or PGD.

Before the procedures associated with PGD are described in the subsequent sections below, let us discuss about conventional pre-natal diagnosis in a few words. The aim of conventional pre-natal diagnosis is to provide an accurate result as early as possible in pregnancy. Pre-conditions include the prior characterization of the parental disease-causing mutations and the prompt and safe biopsy of foetal material; the latter requires close collaboration with experienced gynaecologists. Presently, there are two procedures for obtaining foetal genetic material: trophoblast sampling (chorionic villi sampling; CVS) from 11 weeks of pregnancy and amniocentesis, usually after 15 weeks . The use of maternal blood as a source of cell-free foetal DNA for determining the foetal genotype is still being validated for clinical application, although it is likely that non-invasive pre-natal diagnosis for monogenic diseases will soon be an additional option for analysing the genetic status of an ongoing pregnancy . The molecular analytical procedures for determining the genotype of foetal DNA are applied in specialist genetic laboratories that use the highest standards of quality, as outlined by recommendations such as those from the European Molecular Quality Network . Numerous DNA analytical methods can support genotype analysis; however, the details of these methods are beyond the scope of this review and are discussed elsewhere . Whatever method(s) are selected for the genotyping of the foetal DNA, they should support the highest level of sensitivity and specificity . In addition to characterizing the globin gene mutations in a pre-natal CVS or amniocentesis sample, it is recommended that the parental DNA samples be analysed alongside the foetal DNA samples and that additional methods be applied to exclude maternal contamination. The final result should be available within a few working days and reported to the couples along with complete genetic counselling . The relative pros and cons of conventional pre-natal diagnosis and PGD are summarized in Table 2 . It is generally agreed that the major disadvantage of conventional pre-natal diagnosis based on the analysis of foetal DNA from any source (CVS, amniocentesis or maternal blood) is the need to terminate an affected ongoing pregnancy if the couple wish to have children unaffected by haemoglobinopathy.

Prevention of haemoglobinopathies: PGD or conventional pre-natal diagnosis?

Haemoglobinopathies are the most prevalent group of serious autosomal recessive monogenic disorders worldwide. There are an estimated 300,000–400,000 affected births each year, which include approximately 40,000 patients with transfusion-dependent β-thalassaemia major and approximately 280,000 patients with sickle cell syndromes . Despite tremendous advances in the understanding of the molecular pathophysiology of these disorders, along with substantial improvements in conventional therapies, and the emergence of gene and molecular therapies , the so-called ‘prevention’ remains as the favoured strategy for reducing the burden on families that are at risk of having an offspring with the most clinically severe manifestations of these diseases ( Table 1 ). Prevention includes the timely identification of carrier couples so that they can be informed about their reproductive choices and then proceed to select the reproductive option that is most appropriate for them. The disorders of haemoglobin synthesis are, with very rare exceptions, transmitted through an autosomal recessive mode of inheritance, which means that when both partners of a couple carry either severe HBB gene mutations or severe HBA gene mutations, there is a 1 in 4 chances that each conception (pregnancy) will be affected with a severe haemoglobin disorder. It is strongly recommended that all carrier couples should be counselled by qualified health professionals with expert knowledge regarding the molecular genetics and clinical expression of the haemoglobinopathies . Because most carrier couples do not opt for gamete donation or adoption, the most preferred reproductive options include conventional pre-natal diagnosis or PGD.

Before the procedures associated with PGD are described in the subsequent sections below, let us discuss about conventional pre-natal diagnosis in a few words. The aim of conventional pre-natal diagnosis is to provide an accurate result as early as possible in pregnancy. Pre-conditions include the prior characterization of the parental disease-causing mutations and the prompt and safe biopsy of foetal material; the latter requires close collaboration with experienced gynaecologists. Presently, there are two procedures for obtaining foetal genetic material: trophoblast sampling (chorionic villi sampling; CVS) from 11 weeks of pregnancy and amniocentesis, usually after 15 weeks . The use of maternal blood as a source of cell-free foetal DNA for determining the foetal genotype is still being validated for clinical application, although it is likely that non-invasive pre-natal diagnosis for monogenic diseases will soon be an additional option for analysing the genetic status of an ongoing pregnancy . The molecular analytical procedures for determining the genotype of foetal DNA are applied in specialist genetic laboratories that use the highest standards of quality, as outlined by recommendations such as those from the European Molecular Quality Network . Numerous DNA analytical methods can support genotype analysis; however, the details of these methods are beyond the scope of this review and are discussed elsewhere . Whatever method(s) are selected for the genotyping of the foetal DNA, they should support the highest level of sensitivity and specificity . In addition to characterizing the globin gene mutations in a pre-natal CVS or amniocentesis sample, it is recommended that the parental DNA samples be analysed alongside the foetal DNA samples and that additional methods be applied to exclude maternal contamination. The final result should be available within a few working days and reported to the couples along with complete genetic counselling . The relative pros and cons of conventional pre-natal diagnosis and PGD are summarized in Table 2 . It is generally agreed that the major disadvantage of conventional pre-natal diagnosis based on the analysis of foetal DNA from any source (CVS, amniocentesis or maternal blood) is the need to terminate an affected ongoing pregnancy if the couple wish to have children unaffected by haemoglobinopathy.

PGD for HLA matching

As already mentioned, an alternative application of PGD is to determine the HLA tissue type of an embryo (PGD-HLA). The ultimate goal is to facilitate a HSCT to cure haemoglobinopathy in an affected child in the family. The first PGD-HLA matching case was reported in 2001 for a case of Fanconi anaemia . Subsequently, the number of reported cases of PGD-HLA steadily increased each year, the most common indication to date being HLA matching combined with PGD to exclude a thalassaemia or sickle cell syndrome .

Technical and biological constraints limit the overall chance of a successful outcome of PGD-HLA, i.e. the birth of an HLA-matched donor sibling. Technical limitations of PGD-HLA include the genetic analysis of single embryo cells, which are also common to simple PGD and are described below. In addition, there are biological limitations that are related to the probability of finding a genetically suitable embryo and the outcome of fertility treatment. First, with respect to genetic chance, the likelihood that an embryo will be HLA matched to the affected sibling is 25%. If there is a requirement to concurrently exclude a recessive monogenic disease, the chance is diminished to 75% of 25%, i.e. 18.8%. Second, the overall outcome of ART is unpredictable and limited by the reproductive status of the couple, how well the woman responds to hormonal stimulation, the number and quality of embryos produced in the cycle and the implantation receptivity of the woman’s endometrium. A good response to the hormonal stimulation is important to maximize the number of embryos available for diagnosis; this is particularly critical because of the low chance of identifying genetically suitable embryos. Thus, the reproductive status of the couple, particularly the mother, should be carefully evaluated before ART, and all treatment aspects should be optimized to increase the likelihood of producing a large number of oocytes and thus embryos for analysis. In addition, embryo quality is important to ensure the best implantation potential; however, there are currently no methods that efficiently support the selection of the ‘best’ embryo(s) . The overall chance of a pregnancy and birth is near 30% in routine ART for infertility and for PGD in general . It is of note that the rates of pregnancy and live birth tend to be relatively poorer in patients opting for PGD-HLA as women (couples) are often older when they make the decision to take this therapeutic option for their child who is affected with a haemoglobinopathy . 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% for each cycle initiated . Because the clinical utility of HLA-PGD is so limited in practice, couples (families) should be thoroughly counselled before they commence this procedure . It is recommended that alternative treatment options be considered by the family, such as an already existing HLA-compatible sibling or the availability of unrelated matched donor. The physician treating the child for the haemoglobinopathy and the department where the bone marrow transplant will be performed should evaluate the health status of the affected child and confirm that it is satisfactory to proceed with HSCT. With respect to the genetic analysis, the laboratory needs to examine the feasibility of designing an accurate, robust and sensitive genetic test for analysing the embryo biopsy cells (see Section 8 below).

The ethics associated with the practice of PGD-HLA have been extensively debated over the years. Ethical issues include the motives of the parents in creating and selecting a sibling for the sake of saving an affected child, the risks imposed on the mother who will possibly need to undergo several ART cycles to achieve the goal, any potential risks to the child to be born and the psychological impact on all family members . It is currently accepted in most countries that PGD-HLA represents a valuable treatment option for all the family involved, and although it is challenging, it is well worthwhile when it succeeds. However, the need for psychological support to donor and recipients has been emphasized .

Stages in PGD—from patient referral to baby follow-up

As already mentioned above, PGD, whether for disease prevention or HLA matching, involves the close collaboration between experts in assisted reproduction, embryology and genetics, and has many stages ( Figure 1 ). Once a couple decides to investigate the option of undergoing a PGD cycle, they should be provided extensive counselling on all aspects of the procedure by the teams involved (gynaecologists, embryologists and geneticists) before initiating any aspects of the treatment. They should also be evaluated with respect to their overall genetic status, the feasibility of developing a genetic test at the single-cell level for the familial haemoglobinopathy (see Section 6 below) and, of course, their reproductive potential. It is relevant to note that ART as part of PGD is identical to that for infertility treatment, even if the couple is fertile.

Timeline of a typical PGD cycle to exclude a haemoglobinopathy, with or without HLA matching.

Once the couple has been fully evaluated from a genetic and reproductive point of view and the genetics laboratory has confirmed their preparedness with respect to the genotyping protocol, controlled ovarian hyper-stimulation may proceed. This supports the production of multiple mature oocytes with the aim of creating an adequate number of zygotes/embryos to raise the chance of identifying embryos with an acceptable genetic status. The steps of embryo biopsy and genetic analysis of the cells biopsied are exclusive to PGD. These aspects are discussed in more detail below. For PGD applications involving genetic analysis with DNA amplification steps, it is recommended that after oocyte pick-up, all the cumulus cells surrounding each oocyte should be removed before fertilization. This is to preclude the contamination of the embryonic sample by maternally derived DNA . Furthermore, to prevent contamination by the DNA of residual sperm, it is recommended that the fertilization step be performed by intra-cytoplasmic sperm injection (ICSI) rather than simple in vitro fertilization .

It must not be forgotten that the ultimate aim of PGD is the birth of a healthy baby, the decisive testimony of success. Thus, if a pregnancy ensues, it is recommended that there be follow-up of the pregnancy and baby (or babies) by appropriate specialist health practitioners to collect data for the overall evaluation of safety and clinical utility of PGD . A timeline for a typical PGD procedure is presented in Figure 1 .