Preimplantation genetic diagnosis of spinocerebellar ataxia 3 by (CAG)(n) repeat detection

Spinocerebellar ataxia 3 (SCA3) is an autosomal dominant neurodegenerative disorder characterized by variable expression and a variable age of onset. SCA3/MJD (Machado-Joseph disease) is caused by an expansion of a (CAG)(n) repeat in the MJD1 gene on chromosome 14q32.1. A single cell PCR protocol has been developed for preimplantation genetic diagnosis (PGD) of SCA3 to select unaffected embryos on the basis of the CAG genotype. Single leukocytes and blastomeres served as a single cell amplification test system to determine the percentage of allelic drop-out (ADO) and PCR efficiency. Out of 105 tested heterozygous single leukocytes, 103 (98.1%) showed a positive amplification signal, while five cells (4.9%) showed ADO. Amplification in single blastomeres was obtained in 13 out of a total of 14, and ADO was observed in two out of the 13 single blastomeres. PGD of SCA3 was performed in a couple with paternal transmission of the SCA3 allele. Seven embryos were available for biopsy, all biopsied blastomeres showed amplification and no ADO occurred. One embryo was diagnosed as affected whereas six embryos were diagnosed as unaffected. Two unaffected embryos were transferred and resulted in a singleton pregnancy and the birth of a healthy girl.     

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。     

Strategies and clinical outcome of 250 cycles of Preimplantation Genetic Diagnosis for single gene disorders

BACKGROUND: We report on our experience with preimplantation genetic diagnosis (PGD) for single gene disorders (SGDs), from 1999 to 2004, describing strategies and overall clinical outcome of 250 cycles in 174 couples for 23 different genetic conditions. METHODS: PGD cycles included 15 for autosomal dominant, 148 for autosomal recessive and 19 for X-linked SGDs. In addition, 68 cycles of PGD for SGDs were performed in combination with HLA matching. The strategy in each case used an initial multiplex PCR, followed by minisequencing to identify the mutation(s) combined with multiplex PCR for closely linked informative markers to increase accuracy. Linkage analysis, using intragenic and/or extragenic polymorphic microsatellite markers, was performed in cases where the disease-causing mutation(s) was unknown or undetectable. RESULTS: In 250 PGD cycles, a total of 1961 cleavage stage embryos were biopsied. PCR was successful in 3409 out of 3149 (92.4%) biopsied blastomeres and a diagnosis was possible in 1849 (94.3%) embryos. Four hundred and twenty-seven embryos were transferred in 211 cycles, resulting in 71 pregnancies (33.6% per embryo transfer), including 15 biochemical pregnancies, six spontaneous miscarriages, two ectopic pregnancies, which were terminated, and nine pregnancies which are still ongoing. The remaining pregnancies were confirmed to be unaffected and went to term without complications, resulting in the birth of 35 healthy babies. CONCLUSIONS: Minisequencing for mutation detection combined with multiplex fluorescence PCR for linkage analysis is an efficient, accurate and widely applicable strategy for PGD of SGDs. Our experience provides a further demonstration that PGD is an effective clinical tool and a useful option for many couples with a high risk of transmitting a genetic disease.     

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.     

Pregnancies following blastocyst stage transfer in PGD cycles at risk for beta-thalassaemic haemoglobinopathies.

BACKGROUND: Preimplantation genetic diagnosis (PGD) usually involves blastomere biopsy 3 days post-insemination (p.i.), followed by genetic analysis and transfer of unaffected embryos later on day 3 or 4. We evaluate a strategy involving embryo biopsy on day 3 p.i., genetic analysis on day 4 and, following culture in blastocyst sequential media, transfer of unaffected embryos on day 5 p.i. METHODS: PGD cycles were initiated in 15 couples at risk of transmitting beta-thalassaemia major. Oocyte retrieval and ICSI were performed according to standard protocols. Embryo culture used blastocyst sequential media. Embryos were biopsied on day 3 p.i. using acid Tyrode's for zona drilling, and the single blastomeres were genotyped by a protocol involving nested polymerase chain reaction and denaturing gradient gel electrophoresis analysis. RESULTS: Forty of 109 (37%) embryos biopsied on day 3 p.i. developed to blastocysts by day 5 p.i., with at least one blastocyst available for transfer in 12 cycles (80%). Genotype analysis characterized 51/109 (47%) embryos unaffected for beta-thalassaemia major, of which 28 were blastocysts. Transfer of 37 day 5 p.i. embryos (blastocysts and non blastocysts) initiated eight clinical pregnancies. Implantation rate per embryo transferred was 12/37 (32%). CONCLUSIONS: Embryo biopsy on day 3, followed by delayed transfer until day 5 p.i. offers a novel and effective strategy to overcome the time limit encountered when performing PGD, without compromising embryo implantation.     

The role of preimplantation genetic diagnosis in the management of severe rhesus alloimmunization: first unaffected pregnancy: case report

Rhesus (Rh) D alloimmunization may cause haemolytic disease of the fetus and newborn if the fetal Rh blood type is positive. Although the incidence of severe RhD alloimmunization has decreased with prophylactic anti-D immunoglobulin administration during and after pregnancy, sensitization still occurs in a small group of women. In such women, Rh disease will continue to be significant problem and for their babies who may be affected. Preimplantation genetic diagnosis (PGD) may be utilized to avoid materno-fetal blood group incompatibility in an RhD-sensitized woman. Biopsy of a single cell from early cleavage-stage embryos screening for RhD-negative embryos allows the transfer of only RhD-negative embryo(s) into the uterus. This avoids any complications related to haemolytic disease of the fetus and newborn. This article describes the first reported case of an unaffected pregnancy using PGD for Rh disease. IVF and embryo transfer resulted in a clinical pregnancy and the birth of a healthy girl confirmed to be blood type RhD negative. PGD in couples with a heterozygous RhD-positive male partner provides an option for avoiding haemolytic disease of the newborn in RhD alloimmunized mothers.     

Single intragenic microsatellite preimplantation genetic diagnosis for cystic fibrosis provides positive allele identification of all CFTR genotypes for informative couples

This study is part of a strategy aimed at using fluorescent polymerase chain reaction (PCR) on informative genetic microsatellite markers as a diagnostic tool in preimplantation genetic diagnosis (PGD) of severe monogenic disease. Two couples, both of whom had previously had children who were compound heterozygote for severe cystic fibrosis mutations, were offered PGD using fluorescent PCR of the highly polymorphic cystic fibrosis transmembrane conductance regulator (CFTR) intragenic microsatellite marker IVS17bTA. Cleavage-stage embryo biopsy followed by PCR resulted in transfer of one unaffected carrier embryo for each couple. This approach eliminates the need for single cell multiplex PCR strategies to detect CF compound heterozygotes. It also provides a control of chromosome 7 ploidy in the blastomeres and a selection against allele dropout by positive detection of each CFTR copy of all genotypes in preimplantation embryos from genetically informative families.     

A modified cryopreservation method increases the survival of human biopsied cleavage stage embryos

BACKGROUND: The relatively poor survival rate of human biopsied cleavage stage embryos following cryopreservation is a significant obstacle in the application of preimplantation genetic diagnosis (PGD). We have attempted to improve cryosurvival of biopsied embryos by modifying the standard embryo cryopreservation technique. METHODS: Biopsied embryos were cryopreserved in 1.5 mol/l 1,2-propanediol in the presence of an elevated concentration of sucrose (0.2 mol/l) and human serum albumin was replaced by maternal serum (20% vol:vol). An additional initial thawing step in the presence of 0.3 mol/l sucrose was also included. RESULTS: The proportion of biopsied embryos which survived cryopreservation with > or =50% of their blastomeres intact was significantly higher using the modified method (138/185; 75%) than that observed using the standard propanediol method (20/46; 43%; P = 0.022). Total blastomere survival was also significantly increased as a result of the modifications (1010/1513; 67% versus 177/385; 46%; P < 0.001). Six fetal hearts have been detected to date following replacement of biopsied embryos cryopreserved with the modified method. CONCLUSIONS: Survival of human biopsied cleavage stage embryos can be restored to a level similar to that of non-biopsied controls by modification of the cryopreservation procedure. Embryos which have been cryopreserved using the modified method can implant following replacement in utero.     

PGD for autosomal dominant polycystic kidney disease type 1

Autosomal dominant polycystic kidney disease (ADPKD) is primarily characterized by renal cysts and progression to renal failure. It is a genetically heterogeneous disease, with mutations in the PKD1 gene accounting for the majority of cases. Direct mutation detection for PKD1-linked ADPKD or type 1 is complicated by the large size and complex genomic structure of PKD1. This paper describes a microsatellite marker-based assay for PGD in couples at risk of transmitting ADPKD type 1. During PGD, genetic analysis is carried out on single blastomeres biopsied from preimplantation embryos obtained after IVF, and only embryos unaffected by the disease under investigation are selected for transfer. Single-cell genetic analysis relied on a fluorescent duplex-PCR of linked polymorphic markers followed by fragment length determination on an automated sequencer. The co-amplification of the intragenic KG8 and the extragenic D16S291 marker at the single-cell level was evaluated in pre-clinical tests on lymphoblasts and research blastomeres. The developed assay proved to be efficient (96.1% amplification) and accurate (1.4% allele drop-out and 4.3% contamination), and can be applied in all informative ADPKD type 1 couples. From five clinical cycles carried out for three couples, two pregnancies ensued, resulting in the birth of two healthy children.     

An evaluation of PGD in clinical genetic services through 3 years application for prevention of beta-thalassaemia major and sickle cell thalassaemia

PGD represents an alternative within prenatal diagnosis services, which avoids terminating affected on-going pregnancies. In Greece, prevention programmes for haemoglobinopathies, including the option of prenatal diagnosis, are well established. Following optimization of a single-cell genotyping strategy (designed to be applicable for the majority of beta-thalassaemia major or sickle thalassaemia genotype interactions) along with close collaboration with an IVF unit, we integrated the option of PGD for at-risk couples with a problematic reproductive history. A total of 59 couples requesting PGD were counselled, of whom 41 initiated 63 PGD cycles. Following standard assisted reproduction treatment for oocyte retrieval, 20 cycles were cancelled (too few oocytes and/or poor quality embryos), but in 43 cycles single blastomeres were biopsied from 3 day embryos and genotyped (total 302). Diagnosis was achieved for 236 embryos, and 100 of 125 unaffected embryos were transferred. Sixteen pregnancies were established, although six were lost within the first trimester. Ten pregnancies underwent second trimester prenatal diagnosis, with nine pregnancies (13 babies: six singletons, two twins and one triplet) confirmed unaffected, although one singleton was a PGD misdiagnosis and terminated. The triplet pregnancy was selectively reduced to twins, and nine pregnancies went to term, with 12 healthy babies born. This report highlights advantages, limitations and approaches towards improvement when incorporating PGD within genetic services for a common recessive disease.     

The development of cytogenetically normal, abnormal and mosaic embryos: a theoretical model

Assisted reproduction and preimplantation genetic diagnosis (PGD) involve various complicated techniques, each of them with its own problems. However, the greatest problem with PGD for chromosome abnormalities is not of a technical nature but is a biological phenomenon: chromosomal mosaicism in the cleavage stage embryo. Here, we present a hypothetical, quantitative model for the development of chromosomally normal, abnormal and mosaic embryos. The arising of mosaicism in 2-8-cell embryos was described by a binomial probability model on the occurrence of mitotic events inducing chromosomal changes in the blastomeres. This model converted the 'mean' rate of mosaicism found in cross-sectional studies (60%) into an equal rate of mosaic embryos at arrival at the 8-cell stage (59.8%). The disappearance of > 90% of the mosaic embryos or the mosaicism itself from surviving embryos during the morula stage was explained by mitotic arrest of most of the mitotically changed cells under increasing cell cycle control. In our model, 25.9 and 14.3% of the embryos at the 8-cell stage are normal and abnormal respectively. The remaining 59.8% of the embryo shows mosaicism: 34.6% of abnormal/normal cells and 25.2% of abnormal/abnormal cells. The high proportion of abnormal and mosaic embryos together explains the high rate of abnormal laboratory findings in PGD for chromosomal abnormalities and aneuploidy screening. The poor representation of a 1- or 2-cell biopsy for the 7- or 6-cell post-biopsy embryo in the case of mosaicism explains the high rate of false-negative and false-positive results.     

Reduced survival after human embryo biopsy and subsequent cryopreservation.

Preimplantation genetic diagnosis (PGD) is performed in couples at risk of genetic disease, so as to avoid transfer of embryos which are affected by a monogenic disease or which carry chromosomal aberrations. As in all in-vitro fertilization (IVF) cycles, supernumerary non-affected good-quality embryos may be available after PGD. These embryos can be cryopreserved. So far, limited data on survival after cryopreservation of biopsied human embryos are available. In this study, human embryos of good morphological quality derived from abnormal fertilization were used to evaluate the influence of the embryo biopsy procedure on survival after cryopreservation. Embryos were allocated to three different groups: control (n = 20), drilling-only (n = 16), and biopsy (n = 29). After freezing and thawing, a significantly lower number of blastomeres was intact in the drilling-only group (46/118, i.e. 39.0%, P < 0.01) and in the embryo biopsy group (46/156, i.e. 29.5%, P < 0.0001) than in the control group (85/151, i.e. 56.3%). This difference was reflected in survival rates of embryos. Fifty-five per cent of the control embryos, 37.5% of the drilling-only group, and 33.3% of the biopsy group had at least 50% of their blastomeres intact. After further in-vitro culture, four blastocysts, three from the drilling-only group and one from the biopsy group, developed from the surviving embryos. From this study it can be concluded that current cryopreservation procedures are less successful when biopsied human embryos are cryopreserved, but that surviving embryos can develop to the blastocyst stage and thus may have the potential to develop to term.     

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。     

Preimplantation genetic diagnosis for neurofibromatosis type 1

PGD is an alternative to prenatal diagnosis that circumvents therapeutic abortion. Diagnosis is carried out on single cells obtained from three-day-old embryos, and only those that are free of the disease under consideration are transferred to the mother. Neurofibromatosis type 1 (NF1) is a common neurocutaneous disorder, inherited as an autosomal dominant trait and caused by mutations in the NF1 gene. For some patients, PGD may be the only acceptable manner to ensure the birth of unaffected children. Because of the large number of known NF1 mutations, the development of mutation-specific single-cell protocols is impractical, labour-intensive and expensive. This paper discusses the development of five PGD protocols, three of which are based on multiplex PCR for microsatellite-markers linked to the NF1 gene. After a linkage study, the diagnosis can be established through the markers, thereby obviating the need to detect the mutation itself. This not only ensures the accurate diagnosis of the embryos, but also a prompt acceptance of PGD referrals since one protocol can be useful for several couples. In addition, two mutation-specific PCRs were developed for two couples where a marker-based protocol was not applicable. In total, 16 PGD cycles were carried out for six couples, which resulted in one ongoing pregnancy and the delivery of a healthy unaffected boy.     

Preimplantation genetic diagnosis for ornithine transcarbamylase deficiency by simultaneous analysis of duplex-nested PCR and fluorescence in situ hybridization: a case report

Ornithine transcarbamylase (OTC) deficiency is an X-linked co-dominant disorder. A couple, with a previous history of a neonatal death and a therapeutical termination due to OTC deficiency, was referred to our center for preimplantation genetic diagnosis (PGD). The female partner has a nonsense mutation in the exon 9 of the OTC gene (R320X). We carried out nested polymerase chain reaction (PCR) for R320X mutation and fluorescence in situ hybridization (FISH) for aneuploidy screening. Among a total of 11 embryos, two blastomeres per embryo from 9 embryos were biopsied and analyzed by duplex-nested PCR and FISH, and one blastomere per embryo from 2 embryos by only duplex-nested PCR. As a result of PCR and restriction fragment length polymorphism analysis, four embryos were diagnosed as unaffected embryos having the normal OTC gene. Among these embryos, only one embryo was confirmed as euploidy for chromosome X, Y and 18 by FISH analysis. A single normal embryo was transferred to the mother, yielding an unaffected pregnancy and birth of a healthy boy. Based on our results, PCR for mutation loci and FISH for aneuploidy screening with two blastomeres from an embryo could provide higher accuracy for the selection of genetically and chromosomally normal embryos in the PGD for single gene defects.     

Cryopreservation of biopsied embryos at the blastocyst stage

BACKGROUND: The availability of an efficient cryopreservation program is especially important in the case of embryos that have undergone blastomere biopsy for PGD. Unfortunately, the freezing/thawing of biopsied embryos has given disappointing results when performed at the cleavage stage. In this study, embryos diagnosed as normal after PGD were grown to the blastocyst stage, frozen and thawed for successive frozen embryo transfer. METHODS: A total of 34 patients performed a thawing cycle in which 47 blastocysts were thawed. The cryopreservation solutions were based on HEPES-buffered medium supplemented with human serum albumin (HSA), sucrose and 1,2-propanediol. The same protocol was applied to embryos from 88 IVF/ICSI patients, which underwent 92 thawing cycles with 150 thawed blastocysts. RESULTS: The survival rate was similar in the two groups (53% after PGD and 58% in IVF/ICSI cycles), as well as the cumulative pregnancy rate per patient (59% after PGD versus 47% in IVF/ICSI cycles), despite a higher maternal age and a lower proportion of embryos available for transfer or cryopreservation in the PGD group. CONCLUSIONS: Neither the survival rate nor the subsequent development and chances of implantation, differed between embryos frozen at the blastocyst stage following biopsy and those frozen intact.     

Successful application of preimplantation genetic diagnosis for beta-thalassaemia and sickle cell anaemia in Italy

BACKGROUND: In Italy, the autosomal recessive diseases beta-thalassaemia and sickle cell anaemia are so widespread that in some regions they can be defined as 'social diseases'. In this study, nine clinical applications of preimplantation genetic diagnosis (PGD) were performed for beta-thalassaemia and sickle cell anaemia on seven Sicilian couples and carriers of beta-globin gene mutations. METHODS AND RESULTS: The studied mutations were: Cd39, HbS, IVS1 nt1, IVS1 nt6 and IVS1 nt110. ICSI was performed with partner's sperm on 131 out of 147 retrieved oocytes, and this resulted in 72 zygotes; 32 embryos were successfully biopsied on day 3. The biopsied blastomeres were lysed and the beta-globin alleles amplified by nested PCR. The mutation diagnosis was performed by restriction enzyme digestion and reverse dot-blot. The amplification efficacy was 97.2%. The genotype study of non-transferred and surplus embryos showed that the allele drop-out rate was 8.6%. Seventeen embryos were transferred in utero on day 4. All couples received an embryo transfer; of the four pregnancies obtained, three resulted in live births and one miscarried at 11 weeks. Prenatal diagnosis at the 11th week and miscarriage material analysis confirmed the PGD results. CONCLUSIONS: These studies represent the first successful application of PGD for beta-thalassaemia and sickle cell anaemia in Italy.     

Human preimplantation development in vitro is not adversely affected by biopsy at the 8-cell stage

Normally fertilized human embryos biopsied 3 days after in-vitro fertilization (IVF) have been examined for effects on viability and development in vitro after removal of one or two cells at the 8-cell stage (1/8 and 2/8) from each embryo. A high proportion of 7/8 and 6/8 biopsied and unmanipulated embryos developed to the blastocyst stage between days 5 and 6 (79, 71 and 59%, respectively), and many biopsied embryos (56%) hatched from the zona pellucida in vitro. The viability of biopsied embryos which developed to the blastocyst stage was assessed by daily non-invasive measurement of the uptake of two energy substrates, glucose and pyruvate. Uptake of both substrates was generally lower in 7/8 and 6/8 biopsied embryos but only in proportion to the reduced cellular mass. The total cell number and the numbers of both trophectoderm (TE) and inner cell mass (ICM) cells in biopsied embryos at the blastocyst stage, counted by differential labelling of their nuclei, were also reduced in proportion but the ratio of ICM to TE cells was maintained in both 7/8 and 6/8 biopsied embryos. We conclude that removal of one or two cells at the 8-cell stage, while reducing the cellular mass, does not adversely affect the preimplantation/development of biopsied embryos in vitro and suggest that this approach could be used for preimplantation diagnosis of genetic defects.     

Preimplantation genetic diagnosis for achondroplasia: genetics and gynaecological limits and difficulties

BACKGROUND: We report the first attempts at preimplantation genetic diagnosis (PGD) and IVF and their accompanying difficulties for achondroplasia (ACH) patients. METHODS: A PGD test was developed using fluorescent single cell PCR on lymphoblasts from patients and controls and from blastomeres from surplus IVF embryos. A specific digestion control based on the use of two fluorochromes was elaborated. Ovarian stimulation and oocyte retrieval were carried out using conventional protocols. RESULTS: We performed 88 single cell tests for which amplification was obtained in 86 (97.7%) single lymphoblasts. Allele drop out (ADO) was observed in two out of 53 (3.7%) heterozygous lymphoblasts. If we combine the results from the blastomere testing from surplus embryos with those from PGD cycles and re-analysis after PGD, we obtained a PCR signal in 84% of cases of which 91% were correctly diagnosed at the G380 locus. A total of six cycles were performed resulting in three embryo transfers. We observed difficulties in ovarian stimulation and oocyte retrieval with affected female patients. No pregnancy was obtained. CONCLUSION: A PGD test for ACH is now available at our centre but our initial practice raises questions on the feasibility of such a test, specially with affected female patients.     

Prospect of preimplantation genetic diagnosis for heritable mitochondrial DNA diseases

To perform preimplantation genetic diagnosis for women carrying heteroplasmic mitochondrial DNA (mtDNA) mutations, it is necessary to ensure that the proportion of mutant mtDNA diagnosed in the biopsied cell gives an accurate indication of the mutant load in the remaining embryo. A heteroplasmic mouse model, carrying NZB and BALB mtDNA genotypes, was used to study the relative proportions of each mtDNA genotype in the ooplasm and first polar body of mature oocytes, and between blastomeres of early cleavage stage embryos. The levels of heteroplasmy varied widely in the gametes compared with the maternal genotype. However, the distribution of the two mtDNA genotypes was virtually identical between the ooplasm and polar body of a mature oocyte, and also between the blastomeres of each 2-, 4- and 6-8-cell embryo. Therefore, the level of heteroplasmy diagnosed from the polar body of an unfertilized oocyte or from a single blastomere of an embryo is representative of the level in the embryo as a whole. Reliable results were obtained from both polar bodies and blastomeres, but the efficiency of diagnosis was greater with blastomeres. We conclude that preimplantation genetic diagnosis is feasible for mtDNA diseases, although it should be approached with caution, as it is possible that transmission of some pathogenic mutations could behave in a different manner.