Preimplantation genetic diagnosis: current status and new developments

Preimplantation genetic diagnosis (PGD) is a very early form of prenatal diagnosis aimed at eliminating embryos carrying serious genetic diseases before implantation. To this end, two major technologies are in use: the polymerase chain reaction (PCR) for monogenic diseases and fluorescent in-situ hybridization (FISH) for chromosomal aberrations. In this review, a number of problems arising from the use of these technologies, as well as their possible solutions and new developments, are discussed. Concerning PCR, the phenomenon of allelic drop-out, as well as methods to reduce this problem, such as fluorescent PCR, are described. The advantages and disadvantages of sperm separation by flow cytometry as an adjunct to sex determination for the avoidance of X-linked disease are discussed. The application of FISH for aneuploidy detection is commented upon and the advances in cell recycling, in which PCR and FISH are combined, are analysed. Finally, diseases for which PGD is currently possible are summarized.      

Birth of a healthy boy after a double factor PGD in a couple carrying a genetic disease and at risk for aneuploidy: case report

Preimplantation genetic diagnosis (PGD) for monogenic diseases is widely applied, allowing the transfer to the uterus of healthy embryos. PGD is also employed for the detection of chromosome abnormalities for couples at high risk of producing aneuploid embryos, such as advanced maternal (>35 years). A significant number of patients requesting PGD for monogenic diseases are also indicated for chromosome testing. We optimized and clinically applied a PGD protocol permitting both cytogenetic and molecular genetic analysis. A couple, carriers of two cystic fibrosis (CF) mutations (c.3849 + 10 KbC > T and c.3408C > A) with a maternal age of 38 years and two previously failed IVF-PGD cycles, was enrolled in the study. After ovarian stimulation, six oocytes were obtained. To detect abnormalities for all 23 chromosomes of the oocyte, the first polar body (1PB) was biopsied from five of the oocytes and analyzed using comparative genomic hybridization (CGH). CGH analysis showed that 1PB 1 and 1PB 4 were aneuploid (22X,-9,-13,+19 and 22X,-6, respectively), while 1PB 2, 1PB 3 and 1PB 6 were euploid. Blastomere biopsy was only applicable on embryos formed from Oocyte 3 and Oocyte 6. After whole-genome amplification with multiple displacement amplification, a multiplex PCR, amplifying informative short tandem repeats (D7S1799; D7S1817) and DNA fragments encompassing the mutation sites, was performed. MiniSequencing was applied to directly detect each mutation. Genetic diagnosis showed that Embryo 6 was affected by CF and Embryo 3 carried only the c.3849 + 10 KbC > T mutation. Embryo 3 was transferred achieving pregnancy and a healthy boy was born. This strategy may lead to increased pregnancy rates by allowing preferential transfer of euploid embryos.     

Clinical outcome of preimplantation genetic diagnosis for cystic fibrosis: the Brussels' experience

Preimplantation genetic diagnosis is an alternative for prenatal diagnosis that makes it possible to perform the diagnosis of a chromosomal or monogenic disorder at the preimplantation embryo level. Cystic fibrosis is one of the monogenic diseases for which PGD can be performed. In this study, we looked at the requests and PGD cycles for this particular disorder over an 11-year period. Sixty-eight percent of the requests eventually led to at least one complete PGD cycle. In 80% of the cycles, an embryo transfer was performed and an ongoing pregnancy was obtained in 22.2% of the cycles with oocyte retrieval. After embryo transfer, a couple had 27.8% chance of giving birth to a liveborn child. No misdiagnosis was recorded. The rate of perinatal deaths/stillborn children was relatively high, but no excess of major congenital anomalies was observed in the surviving children.     

Current concepts in preimplantation genetic diagnosis (PGD): a molecular biologist's view

The first clinically applied preimplantation genetic diagnosis (PGD) was reported more than a decade ago and since then PGD has known an exponential growth. This first report described the use of PCR to sex embryos from couples at risk for X-linked diseases. Not surprisingly, in the first years, the development of PCR-based tests led to PGD for well-known monogenic diseases such as cystic fibrosis and thalassaemia. When fluorescent in-situ hybridization (FISH) was introduced it quickly replaced PCR-based methods, which had led to misdiagnoses, for sexing of embryos. FISH was also quickly introduced for aneuploidy screening, which has as its main aim the improvement of IVF results in patients with poor reproductive outcome, and later for PGD in translocation carriers. In this review, PGD for patients with a pre-existing genetic risk will be discussed, i.e. the monogenic diseases and the translocations, as well as different biopsy methods and promising new developments.     

First successful double‐factor PGD for Lynch syndrome: monogenic analysis and comprehensive aneuploidy screening

Preimplantation genetic diagnosis (PGD) has been applied worldwide for a great variety of single‐gene disorders over the last 20 years. The aim of this work was to perform a double‐factor preimplantation genetic diagnosis (DF‐PGD) protocol in a family at risk for Lynch syndrome. The family underwent a DF‐PGD approach in which two blastomeres from each cleavage‐stage embryo were biopsied and used for monogenic and comprehensive cytogenetic analysis, respectively. Fourteen embryos were biopsied for the monogenic disease and after multiple displacement amplification (MDA), 12 embryos were diagnosed; 5 being non‐affected and 7 affected by the disease. Thirteen were biopsied to perform the aneuploidy screening by short‐comparative genomic hybridization (CGH). The improved DF‐PGD approach permitted the selection of not only healthy but also euploid embryos for transfer. This has been the first time a double analysis of embryos has been performed in a family affected by Lynch syndrome, resulting in the birth of two healthy children. The protocol described in this work offers a reliable alternative for single‐gene disorder assessment together with a comprehensive aneuploidy screening of the embryos that may increase the chances of pregnancy and birth of transferred embryos.     

Preimplantation genetic diagnosis for Huntington's disease with exclusion testing

Huntington's disease is an autosomal dominant, late-onset disorder, for which the gene and the causative mutation have been known since 1993. Some at-risk patients choose for presymptomatic testing and can make reproductive choices accordingly. Others however, prefer not to know their carrier status, but may still wish to prevent the birth of a carrier child. For these patients, exclusion testing after prenatal sampling has been an option for many years. A disadvantage of this test is that unaffected pregnancies may be terminated if the parent at risk (50%) has not inherited the grandparental Huntington gene, leading to serious moral and ethical objections. As an alternative, preimplantation genetic diagnosis (PGD) on embryos obtained in vitro may be proposed, after which only embryos free of risk are replaced. Embryos can then be selected, either by the amplification of the CAG repeat in the embryos without communicating results to the patients (ie non-disclosure testing), which brings its own practical and moral problems, or exclusion testing. We describe here the first PGD cycles for exclusion testing for Huntington's disease in five couples. Three couples have had at least one PGD cycle so far. One pregnancy ensued and a healthy female baby was delivered.     

Real-time PCR for single-cell genotyping in sickle cell and thalassemia syndromes as a rapid, accurate, reliable, and widely applicable protocol for preimplantation genetic diagnosis

Sickle-cell and beta-thalassemia syndromes are priority genetic diseases for prevention programs involving population screening with the option of prenatal diagnosis for carrier couples. Preimplantation genetic diagnosis (PGD) represents a specialized alternative to prenatal diagnosis and is most appropriately used for couples with an unsuccessful reproductive history and/or undergoing assisted reproduction. However, clinical application of PGD has been hindered by difficulties in reliably transferring molecular diagnostic protocols to the single-cell level. We standardized and validated a protocol involving first-round multiplex PCR, amplifying the region of the beta-globin gene containing most of the common disease mutations world-wide and two unlinked microsatellite markers (GABRB3 and D13S314), followed by: 1) analysis of beta-globin genotypes with real-time PCR and 2) microsatellite sizing to exclude chance contamination. The protocol was standardized on 100 single lymphocytes from a beta-thalassemia heterozygote, including 15 artificially contaminated samples, the latter demonstrated through microsatellite analysis. PCR failure and allele drop-out (ADO) were observed in one (uncontaminated) sample each (1.2%). A pilot study in six clinical PGD cycles with five different beta-globin genotype interactions achieved results (in 5-6 hr) in 46 out of 50 single blastomeres (92%), all concordant with results from an established PGD method applied simultaneously; microsatellite analysis detected only parental alleles, excluding contamination. Beta-globin genotypes were also confirmed in two blastomeres through prenatal diagnosis (twin pregnancy), and in 11 out of 12 spare embryos, revealing one incident of ADO. Overall, the protocol proved to be sensitive, accurate, reliable, rapid, and applicable for many genotype interactions, with internal monitoring of contamination, thus fulfilling all requirements for clinical PGD application.     

Novel universal approach for preimplantation genetic diagnosis of beta-thalassaemia in combination with HLA matching of embryos

BACKGROUND: Beta-Thalassaemia results from co-inheritance of two mutant beta-globin alleles. Allogeneic cord blood cell transplantation (CBT) from an HLA-identical sibling donor is an excellent treatment option for beta-thalassaemia. In families with an affected child and willing to have another child, IVF followed by preimplantation genetic diagnosis (PGD) can be applied to exclude affected embryos. Furthermore, healthy embryos could be HLA matched with the affected child so that cord blood from the future newborn can be used to transplant the affected sibling. METHODS: We developed an indirect single-cell HLA typing technique based on the use of a bank of seven microsatellite markers within the HLA locus from which four informative and evenly distributed markers were selected. RESULTS: The methodology was validated in three beta-thalassaemia families having six ovarian stimulation cycles in view of IVF and PGD. Six PGD cycles were performed in two families. On 58 embryos tested, the combined PCR was successful in 54 (93%). Two transfers were done and one clinical pregnancy was obtained. Using confirmatory analysis on 50 embryos, the accuracy for HLA typing was 100%. CONCLUSION: This strategy offers a new therapeutic option for patients with beta-thalassaemia and other monogenic diseases that can be cured with CBT.     

Pregnancy after preimplantation genetic diagnosis for Ataxia Telangiectasia

Ataxia Telangiectasia (AT) is an autosomal recessive disorder with an incidence estimated at 1 in 40 000 to 1 in 100 000 live births. More than 100 different somatic and germ-line mutations have been identified in the AT gene, the majority of which cause premature protein truncation. The immense size of the AT gene (66 exons) complicates the detection of mutations. A Saudi family with three affected children suffering from AT consulted our IVF centre for preimplantation genetic diagnosis (PGD). Despite advanced maternal age and unknown mutation, the family was screened for AT mutations. A large deletion in the gene was found to be responsible for the phenotype of AT. The mutation detection permitted us to perform PGD on AT for the first time. Single cell PCR consisted of amplifying one of the deleted exons, exon 19. Homozygous affected embryos show an absence of the exon, while in heterozygous or normal embryos the exon is amplified successfully. After ICSI, three embryos were suitable for embryo biopsy. After biopsy only one embryo showed exon amplification and was transferred. A singleton pregnancy ensued and prenatal diagnosis confirmed the presence of exon 19. This report demonstrates that PGD is feasible despite advanced maternal age and poor response to follicle stimulation.     

Preimplantation genetic diagnosis: patients' experiences and attitudes

BACKGROUND: This study aims to report the experiences and attitudes of patients who have undergone preimplantation genetic diagnosis (PGD). The extent to which this technique is acceptable to the individuals for whom it is intended is relatively unexplored, and remains a crucial issue that may ultimately determine the value of PGD as an alternative to prenatal diagnosis in high-risk couples. METHODS: An information sheet and questionnaire was distributed to 67 couples who had been treated at the Hammersmith Hospital, London and the Dexeus Institute, Barcelona. RESULTS: One-third of patients had an affected child, over half had previous experience of conventional prenatal diagnosis and over one-third had had terminations of pregnancy because of a genetic risk. Patients perceive the main advantage of PGD to be that only unaffected embryos are transferred to the uterus and thus therapeutic termination of pregnancy can be avoided; the main disadvantage is the low success rate. A total of 41% of patients found the treatment cycle extremely stressful, and, of the 20 patients who had experienced both prenatal diagnosis and PGD, 40% of patients found PGD less stressful, although 35% experienced more stress. Of those couples who contemplated a further pregnancy 76% would choose PGD, 16% would opt for prenatal diagnosis, and 8% no tests at all. CONCLUSIONS: The experience of prenatal diagnosis and termination of pregnancy can be an unwelcome memory and this leads to a demand for an alternative approach. Our data suggest that PGD is acceptable to patients and is a valuable alternative to prenatal diagnosis.     

New tools for preimplantation genetic diagnosis of Huntington's disease and their clinical applications

Huntington's disease (HD) is a late-onset neurodegenerative disorder transmitted as an autosomal dominant trait. The causative mutation was characterised in 1993. For HD carriers willing to create a family, prenatal diagnosis (PND) or preimplantation genetic diagnosis (PGD) based on the mutation identification can be offered. For at-risk persons who do not want to undergo presymptomatic testing (PT), an exclusion test can be proposed. With such a test, only foetuses or embryos that inherit an allele from the unaffected grandparent are considered as unaffected. In cases of PND, if the foetus has one allele of the affected grandparent, termination of pregnancy is proposed. In cases of PGD, only not at-risk embryos are transferred. Since the beginning of our PGD activity, we have had 43 PGD referrals for HD, of which 24 were from patients who know their genetic status and 19 from patients who do not wish to perform PT. We have developed 12 multiplex fluorescent PCR protocols applied at the single-cell level for PGD, some of which target the CAG repeat while others use two different polymorphic microsatellites. We present here these different protocols and their clinical applications, as well as the characterisation and use of a new highly polymorphic intragenic marker. Between May 2001 and December 2003, 39 PGD cycles have been performed for 17 couples, 11 of whom had a known genetic status and six who did not wish to perform PT, resulting in four pregnancies.     

紧密连锁的多态性位点在β地中海贫血植入前遗传学诊断中的应用

目的 探讨与β珠蛋白基因紧密连锁的多态性位点HumTH01在β地中海贫血（β地贫）植入前遗传学诊断（preimplantation genetic diagnosis，PGD）中的作用。方法 对4例已出生重型β地贫患儿的、双方均为β地贫基因携带者的夫妇进行了6个周期的PGD治疗，应用多重巢式PCR同时检测β珠蛋白基因及HumTH01基因，选择健康的胚胎移植入子宫。结果 6个周期共活检44个胚胎，获得44个卵裂球，其中41个卵裂球扩增成功，35个胚胎经PCR分析后获得明确诊断，移植了14个胚胎，获得1例临床妊娠。孕17周时经脐带血穿刺，证实为完全正常胚胎，现已出生一正常女婴。单个卵裂球平均扩增效率为89．7％，等位基因脱扣（allele drop-out，ADO）率为14．4％。HumTH01基因可以帮助检测出ADO及污染的发生。结论 本研究为国内首次报道应用多重巢式PCR同时检测β珠蛋白基因及HumTH01基因对β地贫进行植入前遗传学诊断并成功获得临床妊娠。在PGD中同时检测与β珠蛋白基因紧密连锁的多态性位点可以降低PGD中由于ADO及污染造成的误诊的风险。     

The Brussels' experience of more than 5 years of clinical preimplantation genetic diagnosis

This paper describes the 5 years' experience of preimplantation genetic diagnosis (PGD) at the Brussels Free University. Our first PGD was carried out in February 1993. Up to October 1998, we carried out 183 PGD cycles on fresh cleavage embryos of 92 couples for 25 different conditions. Patients were treated for autosomal recessive (n = 39), autosomal dominant (n = 65) and X-linked recessive (n = 47) monogenic disorders as well as for autosomal structural aberrations (n = 10), sex chromosome numerical and structural aberrations (n = 21) and a combination of the two latter (n = 1). Specific diagnosis was carried out by polymerase chain reaction (n = 108). Fluorescence in-situ hybridization was used for sexing (n = 64) and structural aberrations (n = 11). We transferred 1.6 +/- 1.1 embryos per cycle, resulting in an implantation rate of 12.0% per replaced embryo. Ongoing pregnancies were achieved in 29 cycles, i.e. 23 singletons, five twins and one dichorionic triplet with an acardius acranius. The ongoing pregnancy rates per cycle, per transfer and per couple were 16.4, 19.9 and 31.5% respectively. While 28 ongoing pregnancies resulted in the births of 34 infants, one pregnancy was terminated after misdiagnosis. The results of 24 PGD were confirmed by prenatal diagnosis or after birth while no information was available in four pregnancies. Our series demonstrates that PGD is a feasible technique by which to avoid the birth of genetically affected children to couples at risk.     

Preimplantation diagnosis of genetic diseases

One of the landmarks in clinical genetics is prenatal diagnosis of genetic disorders. The recent advances in the field have made it possible to diagnose the genetic conditions in the embryos before implantation in a setting of in vitro fertilization. Polymerase chain reaction and fluorescence in situ hybridization are the two common techniques employed on a single or two cells obtained via embryo biopsy. The couple who seek in vitro fertilization may screen their embryos for aneuploidy and the couple at risk for a monogenic disorder but averse to abortion of the affected fetuses after prenatal diagnosis, are likely to be the best candidates to undergo this procedure. This article reviews the technique, indications, benefits, and limitations of pre-implantation genetic testing in clinical practice.     

Place of preimplantation diagnosis in genetic practice

Preimplantation genetic diagnosis (PGD) is currently one of the practical options available for couples at-risk to avoid the birth of children with genetic and chromosomal disorders. Despite its novelty, PGD has already become an alternative to traditional prenatal diagnosis, allowing establishing only unaffected pregnancies avoiding the risk for pregnancy termination. Indications for PGD have currently expanded beyond those practices in prenatal diagnosis, such as late-onset diseases with genetic predisposition, and preimplantation HLA typing with the purpose of establishing potential donor progeny for stem cell treatment of siblings, which makes PGD also an important compliment to prenatal diagnosis. The fact that more than 1,000 apparently healthy unaffected children have been born after PGD suggests its accuracy, reliability, and safety. PGD is presently an excellent option for carriers of balanced translocations, and appears to be of special value for avoiding age-related aneuploidies in patients of advanced reproductive age. The accumulated experience of thousands of PGD cycles for poor prognosis in vitro fertilization (IVF) patients provides strong evidence of the improvement of clinical outcome, particularly obvious from the reproductive history of patients. This makes of practical relevance to inform couples at-risk about availability of PGD option, so they make their own choice in avoiding the birth of affected offspring and having healthy children of their own.     

Single embryo transfer in preimplantation genetic diagnosis cycles for women <36 years does not reduce delivery rate

BACKGROUND: The Belgian legislation imposes single embryo transfer (SET) on women of <36 years in their first treatment cycle to avoid multiple pregnancies. The aim of this study is to assess the impact of this legislation on the outcome of preimplantation genetic diagnosis (PGD) for inherited diseases in young women undergoing SET. METHODS: A retrospective analysis of PGD cycles for monogenic disorders and translocations in women <36 years on their first treatment cycle. Two groups of patients were defined according to the implementation of the Belgian legislation: (i) double embryo transfer (DET), January 2001-June 2003 (ii) SET, July 2003-June 2005. The primary and secondary outcome measures were delivery per embryo transfer and multiple pregnancy rates, respectively. A subgroup analysis for monogenic disorders and translocations was performed. RESULTS: 62 cycles were included in the DET group and 73 cycles in the SET group. The mean age, number of cumulus-oocyte complexes, number of fertilized oocytes, number of biopsied and cryopreserved embryos were comparable between both groups. There was no significant difference in the delivery rates between the DET and the SET groups (33.9% versus 27.4%, respectively). Multiple pregnancies were avoided when SET was performed. When monogenic disorders and chromosomal translocations were separately evaluated, no significant difference in the delivery rate after SET was observed. CONCLUSIONS: The implementation of a SET policy in young women undergoing PGD for monogenic disorders and translocations enables a significant reduction of multiple pregnancies without significantly affecting the delivery rate.     

应用全基因组扩增技术对β-地中海贫血进行胚胎植入前遗传学诊断

目的 探讨对β-地中海贫血进行胚胎植入前遗传学诊断的方法。方法 夫妇双方分别为β41-42(-TCTT)及IVS-I 654(C→T)突变杂合子，在本中心进行体外受精-胚胎移植和胚胎植入前遗传学诊断。结果 13个胚胎中共有11个胚胎经PCR分析后获得明确诊断，正常胚胎2个(18．1％)；杂合子胚胎6个(54．5％)；双重杂合子胚胎3个(27．3％)。共移植3个胚胎，其中2个正常胚胎、1个杂合子胚胎。在胚胎移植后5周B超示三胎妊娠，孕8周自然减一胎，并于孕20周时经产前诊断，证实均为健康胎儿。现已分娩双胎分别为正常和杂合子。结论 成功应用全基因组扩增技术对β-地中海贫血进行胚胎植入前遗传学诊断，并分娩健康双胎。     

Successful preimplantation genetic diagnosis is related to the number of available cumulus-oocyte complexes

The inheritance pattern of monogenic inheritable disorders influences the proportion of unaffected embryos after preimplantation genetic diagnosis (PGD). We aimed to investigate the influence of the number of cumulus-oocyte complexes (COC) on the outcome after PGD. Eighty-four cycles of 47 couples were included in our analysis. All couples were at risk of transmitting autosomal recessive, autosomal dominant, X-linked single gene disorders or sexaneuploidies to their offspring. One PGD cycle was carried out for a Yq-deletion of the man. The correlation between the numbers of COC and biopsied embryos and between the numbers of COC and unaffected embryos was highly significant (P <0.05). A pregnancy occurred in 15 cycles and a minimum of six COC were needed to achieve a pregnancy. Thirteen pregnancies were observed in cycles with at least 9 COC. The transfer rate and number of transferred embryos per cycle in the subgroups with <9 COC and > or =9 COC were significantly higher in the latter. Although pregnancy rates did not differ significantly between the two subgroups (probably due to the low number of pregnancies), our data indicate that it is justifiable to cancel PGD cycles in which it is expected that <6 COC will be retrieved and that the couple should be informed about the poor prognosis if <9 COC are retrieved.      

Multiple displacement amplification improves PGD for fragile X syndrome

We report an improvement in the PGD test for fragile X syndrome (FXS). Recently, multiple displacement amplification (MDA) has been reported to yield large amounts of DNA from single cells. Taking into account this technique, we developed a new PGD test for FXS, enabling combined analysis of linked polymorphic markers with the study of the non-expanded CGG repeat. Single cell amplification efficiency was first assessed on single lymphocytes. Amplification rate of the different markers ranged from 85 to 95% with an allele drop-out (ADO) rate comprised between 7 and 34%. Using this test, eight PGD cycles were carried out for six couples, and 37 embryos were analysed after preliminary MDA. Amplification rate was increased by this technique from 41 to 66% so that embryos with no results were rarer (14 versus 45% without MDA). Reliability of the test was considerably improved by combining direct with indirect genetic analysis. Furthermore, in cases of fully expanded alleles too large to be amplified by PCR, this test gives an internal amplification control. Embryonic transfers were carried out in all but one PGD cycles. One biochemical and one clinical pregnancy resulted, and a healthy child was born. This single diagnosis procedure could be suitable to most patients carrying FXS.     

The minisequencing method: an alternative strategy for preimplantation genetic diagnosis of single gene disorders

We have applied a new method of genetic analysis, called 'minisequencing', to preimplantation genetic diagnosis (PGD) of monogenic disorders from single cells. This method involves computer-assisted mutation analysis, which allows exact base identity determination and computer-assisted visualization of the specific mutation(s), and thus facilitates data interpretation and management. Sequencing of the entire PCR product is unnecessary, yet the same qualitative characteristics of sequence analysis are maintained. The main benefit of the minisequencing strategy is the use of a mutation analysis protocol based on a common procedure, irrespective of the mutations involved. To evaluate the reliability of this method for subsequent application to PGD, we analysed PCR products from 887 blastomeres including 55 PGD cases of different genetic diseases, such as cystic fibrosis, beta-thalassaemia, sickle cell anaemia, haemophilia A, retinoblastoma, and spinal muscular atrophy. Minisequencing was found to be a useful technique in PGD analysis, due to its elevated sensitivity, automation, and easy data interpretation. The method was also efficient, providing interpretable results in 96.5% (856/887) of the blastomeres tested. Fifteen clinical pregnancies resulted from these PGD cases; conventional prenatal diagnosis confirmed all the PGD results, and 10 healthy babies have already been born. Its applicability to PGD could be helpful, particularly in cases in which the mutation(s) involved are difficult to assess by restriction analysis or other commonly used methods.