Aspects of biopsy procedures prior to preimplantation genetic diagnosis

Today, preimplantation genetic diagnosis (PGD) is offered in over 40 centres worldwide for an expanded range of genetic defects causing disease. This very early form of prenatal diagnosis involves the detection of affected embryos by fluorescent in situ hybridization (FISH) (sex determination or chromosomal defects) or by polymerase chain reaction (PCR) (monogenic diseases) prior to implantation. Genetic analysis of the embryos involves the removal of some cellular mass from the embryos (one or two blastomeres at cleavage-stage or some extra-embryonic trophectoderm cells at the blastocyst stage) by means of an embryo biopsy procedure. Genetic analysis can also be performed preconceptionally by removal of the first polar body. However, additional information is then often gained by removal of the second polar body and/or a blastomere from the embryo. Removal of polar bodies or cellular material from embryos requires an opening in the zona pellucida, which can be created in a mechanical way (partial zona dissection) or chemical way (acidic Tyrode's solution). However, the more recent introduction of laser technology has facilitated this step enormously. Different biopsy procedures at different preimplantation stages are reviewed here, including their pros and cons and their clinical applications. The following aspects will also be discussed: safety of zona drilling by laser, use of Ca²⁺/Mg²⁺-free medium for decompaction, and removal of one or two cells from cleavage-stage embryos. Copyright © 2001 John Wiley & Sons, Ltd.     

Preimplantation genetic diagnosis of the fragile X syndrome by use of linked polymorphic markers

Fragile X syndrome is the most common cause of familial mental retardation. The most common mutation is expansion of a triplet (CGG)_n repeat in the 5′ untranslated region of the FMR1 gene on Xq27.3. The expansion is refractory to PCR due to preferential amplification of the smaller allele in heterozygous cells and the high GC content of the repeat and surrounding sequences. Direct detection of the normal parental alleles in preimplantation embryos has been used for preimplantation genetic diagnosis (PGD) of this disorder. However, this approach is only suitable for approximately 63% of couples due to the heterozygosity of the repeat in the normal population. As an alternative we investigated the use of polymorphic markers flanking the mutation to track the normal and premutation carrying maternal chromosomes in preimplantation embryos. Using a panel of 11 polymorphisms, six (CA)_n repeats and five single nucleotide polymorphisms, diagnosis was developed for 90% of referred couples. Multiplex amplification of informative markers was tested in 300 single buccal cells from interested couples with efficiency and allele drop out (ADO) rates ranging from 69% to 96% and 6% to 18%, respectively. Use of this approach is accurate and applicable to a larger number of patients at risk of transmitting fragile X to their offspring. Copyright © 2001 John Wiley & Sons, Ltd.     

Mosaicism of autosomes and sex chromosomes in morphologically normal,monospermic preimplantation human embryos

We have previously detected chromosome abnormalities in human embryos whilst identifying the sex for preimplantation diagnosis of X-linked disease. In this study we assess the incidence of these abnormalities, both for sex chromosomes and autosomes 1 and 17, using dual fluorescent in situ hybridization (FISH). Sixty-nine normally fertilized embryos of good morphology at the 6–10 cell stage (day 3 post-insemination) were examined. The embryos were spread whole using HCl and Tween 20 to dissolve the cytoplasm. Thirty-four embryos were analysed for the sex chromosomes and 35 for autosomes 1 and 17. All probes were directly labelled with fluorochromes allowing analysis in 2 h. Control lymphocytes demonstrated that the probes were of high specificity. For the sex chromosomes, five embryos were mosaic (15 per cent) with the remaining 29 being uniformly XX or XY. In no case was an XX nucleus found in an otherwise XY embryo, indicating that even though mosaicism for the sex chromosomes is present, such abnormalities would not lead to a misdiagnosis of sex. For the autosomes, 16 embryos were abnormal (46 per cent); one embryo was triploid, one was monosomic for chromosome 1, and ten others were diploid mosaics (three diploid/aneuploid, three diploid/polyploid, and four diploid/haploid). A further four embryos had variable chromosome numbers in the majority of nuclei which appeared to be the result of uncontrolled mitotic division. The presence of haploidy or double monosomy, which occurred in 15 per cent of nuclei, has important implications for the diagnosis of trisomies and dominant disorders.     

The results of aneuploidy screening in 276 couples undergoing assisted reproductive techniques

Preimplantation genetic diagnosis for aneuploidy screening (PGD-AS) using sequential in situ hybridization was applied for aneuploidy testing in 276 couples with 282 ART cycles. Patients with advanced maternal age (AMA, n = 147), recurrent implantation failure (RIF, n = 48), repeated early spontaneous abortion (RSA, n = 32) and abnormal gamete cell morphology (AGCM, n = 55) including macrocephal sperm forms or cytoplasmic granular oocytes were included. Embryo biopsy was performed on day 3 in a calcium–magnesium–free medium by using a noncontact diode laser system. After fixation and enzymatic treatment, fluorescent in situ hybridization (FISH) was carried out on 1147 blastomeres with specific probes for chromosomes 13, 16, 18, 21 and 22 for AMA group, 13, 18, 21, X and Y for AGCM group and 13, 16, 18, 21, 22, X and Y for RIF and RSA groups respectively. The overall chromosomal abnormality rate in analyzed embryos was 40.9%, with no significant difference between AMA, RIF and RSA groups (p > 0.05). However, AGCM group presented a higher rate of chromosomal aneuploidies (57.4%) than the other three groups (p < 0.01). A total of 84% biopsied embryos presented cleavage in 24 h and embryo transfer was realized in 278 cycles. In four cycles, no chromosomally normal embryo was found for embryo transfer. A total of 88 pregnancies (31.6%) were achieved, 19.3% resulted in abortion and 63 healthy births were obtained, with a total of 93 babies born. Aneuploidy testing in couples with poor prognosis undergoing ART cycles is a useful tool to increase the chance of ART success. Furthermore, abnormal gamete cell morphology should be considered one of the major indications for PGD in ART programs as high aneuploidy rates were observed in this group. Copyright © 2004 John Wiley & Sons, Ltd.     

Normal pregnancy after preimplantation DNA diagnosis of a dystrophin gene deletion

To perform preimplantation DNA diagnosis for Duchenne muscular dystrophy (DMD) in a female carrier of a dystrophin gene deletion of exons 3–18, we developed a polymerase chain reaction (PCR)-based assay of exon 17 sequences. Exon 17 was efficiently amplified in all 50 single blastomeres of normal control embryos and in five blastomeres of one male embryo of the DMD carrier obtained after a first preimplantation diagnosis (PID) for gender determination. In ten blastomeres of another two male embryos of the DMD carrier, no PCR signals were observed, probably as a result of the deletion. After intracytoplasmic sperm injection, embryos were analysed for exon 17 and three of the four embryos showing normal PCR signals were replaced, resulting in a singleton pregnancy. Prenatal diagnosis showed a female karyotype and DNA analysis indicated that the fetus was not a DMD carrier.     

First application of preimplantation genetic diagnosis to neurofibromatosis type 2 (NF2)

Neurofibromatosis type 2 (NF2) is a dominantly inherited cancer predisposition syndrome that is caused bymutations in the NF2 gene. We report here the first clinical preimplantation genetic diagnosis (PGD) forNF2. A protocol was developed to simultaneously amplify the mutation and a single nucleotide polymorphism (SNP) located within the gene. The mutation and polymorphism were analysed by simultaneous fluorescent single-strand conformation polymorphism (SSCP) on an automated DNA sequencer. The mutation, carried by the male partner, was a single base pair substitution affecting a splice site in intron 4 of the gene. The female partner was infertile due to polycystic ovary syndrome and would require IVF to conceive. The couple was found to be informative at a linked intragenic SNP situated in the 5′ untranslated region of the gene. The SNP was included in the assay to reduce the risk of misdiagnosis due to allele dropout (ADO). The couple underwent three cycles of treatment during which a total of 43 blastomeres were biopsied from 31 embryos. Amplification at both loci was obtained in 35 cells (81%). A total of five embryos were transferred, two in the first cycle, two in the second and one in the third. No pregnancy ensued. The results of the diagnoses indicated that, in this couple, the inheritance of the mutation may be non-Mendelian. Out of a total of 32 embryos tested only four were found not to carry the mutation. The reasons for this apparent skew remain unknown. Copyright © 2002 John Wiley & Sons, Ltd.     

Duplex PCR for preimplantation genetic diagnosis (PGD) of spinal muscular atrophy

The main difficulty in developing a molecular diagnosis of spinal muscular atrophy (SMA) resides in the specific genomic structure of the locus. Indeed, two highly homologous survival motor neurone genes, SMN1 and SMN2, are present at the locus. The detection of the homozygous deletion of exons 7 and 8 of the SMN1 gene, which is present in 90 to 98% of the patients, is based on methods highlighting 1 of the 8 nucleotidic mismatches existing between these 2 genes. In order to offer preimplantation genetic diagnosis (PGD) for SMA, we developed a new allele-specific amplification method. The main disadvantage of our previously described strategy resided in the possibility of diagnosing, in case of amplification failure, an unaffected embryo as affected. We present here a new PGD-SMA method. We established the conditions for three different duplex PCRs, allowing the specific detection of the SMN1 gene and one polymorphic marker, either D5S629, D5S1977, or D5S641. Of the 60 to 90 single cells tested, the PCR efficiency varied from 98 to 100% with a complete genotype obtained in a range between 81 and 87% with a global allele drop-out rate of 9%. Such a test was used to perform 1 PGD cycle for which 7 embryos could be analysed. All the embryos were fully diagnosed, six as unaffected and one as affected. Four embryos were transferred, but no pregnancy ensued. Copyright © 2003 John Wiley & Sons, Ltd.     

β-Glucuronidase activity in mouse oocytes,mouse preimplantation embryos,and human gametes

A microfluorimetric assay was designed to monitor β-glucuronidase (β-gluc) activity during the early development of mouse embryos and was also applied to measure β-gluc activity in human sperm cells and single human oocytes. Mouse oocytes and preimplantation embryos at different stages of development, failed fertilized human oocytes, and human sperm cells were collected, and β-gluc activity was analysed. In the mouse, β-gluc activity could be measured starting from the morula stage on in a pooled sample of 15 embryos and at blasto-cyst formation in one single embryo. β-Gluc activity was low in human sperm cells. The enzyme could be readily demonstrated in 87 per cent of non-fertilized human oocytes. Considerable variation in β-gluc activity was noticed between individual oocytes.     

Rapid sexing of human embryos by non-radioactive in situ hybridization: Potential for preimplantation diagnosis of X-linked disorders

Sixty spare human embryos at various stages of preimplantation development were prepared for cytogenetic analysis. Fluorescent staining of those with metaphases allowed scoring for the presence of a Y chromosome. In situ hybridization was then performed using a biotinylated Y-specific sequence, and the probe was detected by a standard streptavidinlinked alkaline phosphatase system. This enabled comparison of the chromosomal sex with that obtained after in situ hybridization in 28 embryos, and the sexing result obtained by the two methods was concordant in all cases. A further 21 embryos in which no metaphase chromosomes were obtained were sexed by biotinylated in situ hybridization only. Overall, 66 per cent of male interphase nuclei demonstrated a Y-specific hybridization signal. Results were obtained in under 24 h, which may permit the sexing of an embryo biopsied during cleavage and the transfer of sexed embryos at the blastocyst stage to the mother's uterus in the same cycle as oocytes are collected for in vitro fertilization.     

Strategies for prenatal and preimplantation genetic diagnosis in Marfan syndrome (MFS)

Marfan syndrome (MFS) is an autosomal dominant disorder with a prevalence of 2–3 per 10 000 individuals. Symptoms range from skeletal overgrowth, cutaneous striae to ectopia lentis and aortic dilatation leading to dissection. Prenatal diagnosis was until recently mainly performed in familial cases by linkage analysis. However, mutation detection has become available with thorough screening methods. The phenotypic variability observed in MFS makes reproductive options difficult, as molecular diagnosis cannot predict clinical severity of the disease. Data are presented on 15 prenatal and/or preimplantation genetic diagnoses (PGD) in nine families, originating from Belgium, the Netherlands, Spain and France. In four families data from linkage analysis were used, whereas in five other families the causative FBN1 mutation was characterised. Four PGD cycles in two couples led to one ongoing pregnancy. In addition, two amniocenteses and nine chorionic villus (CV) samplings were performed. In five pregnancies an affected fetus was diagnosed. In one of them, the couple chose to continue the pregnancy and an affected child was born, whereas the other four couples decided to terminate the pregnancy. It is expected that the greater availability of mutation testing of the FBN1 gene will increase requests for prenatal diagnosis. PGD appears to be an acceptable alternative for couples facing ethical reproductive dilemmas. Copyright © 2002 John Wiley & Sons, Ltd.     

Preimplantation diagnosis

Research towards preimplantation diagnosis of genetic disease was initiated in the UK. in the mid 1980s with the aim of helping those couples who would prefer selection to occur at this stage rather than during pregnancy. Following in vitro fertilisation, (IVF), biopsy and removal of 1 or 2 of the totipotent cells from the cleavage stage 3 day old embryo provides the material for molecular genetic diagnosis without interfering with development. Earliest applications were in the avoidance of X-linked disease by sexing embryos and selecting females for transfer to the mother. Initially, polymerase chain reaction (PCR) amplification of DNA from the biopsied blastomeres was performed using primers specific for sequences derived from the Y chromosome and this led to the birth of several normal girls. To reduce the risk of misdiagnosis due to amplification failure, PCR based methods for sexing the embryo now employ both X and Y specific sequences, but the preferred method is currently considered to be fluorescent in situ hybridisation (FISH) with fluorochrome labelled DNA probes to the embryonic nuclei that have been fixed and spread on slides. Dual FISH with probes from X and Y chromosomes allows unequivocal diagnosis of sex and determination of chromosome copy number, avoiding transfer of embryos with abnormal numbers of sex chromosomes, including those with only the maternal X that would be at 50% risk for the X-linked disease. The application of FISH for preimplantation diagnosis has also led to the realisation that chromosomal mosaicism is common at the cleavage stage of development, a finding that has important implications for diagnosis of both dominant single gene disorders and trisomies, as well as for our understanding of early human development. Cloning and sequencing of the relevant genes has enabled the development of methods for the diagnosis of certain recessive single gene disorders in cleavage stage embryos. PCR based methods have to be developed for each condition, sometimes for each family if there is heterogeneity. Preimplantation diagnosis has been successful so far for cystic fibrosis, Tay Sachs disease, and Lesch-Nyhan syndrome. Worldwide, 32 pregnancies have been established following all types of preimplantation diagnosis and with 29 babies born, there is no evidence for any adverse effect on development.     

Double locus analysis of chromosome 21 for preimplantation genetic diagnosis of aneuploidy

Preimplantation genetic diagnosis (PGD) of numerical chromosome abnormalities significantly reduces spontaneous abortions and may increase pregnancy rates in women of advanced maternal age undergoing in vitro fertilization. However, the technique has an error rate of around 10% and trisomy 21 conceptions have occurred after PGD. To further reduce the risk of transferring trisomy 21 embryos to the patient, we designed a protocol that analyzes chromosome 21 twice by targeting two different loci. This protocol was applied to 388 embryos from 60 cycles of PGD of aneuploidy. The scoring criterion used was based on giving equal importance to both probe results. Of the 242 embryos diagnosed as abnormal, 125 were re-biopsied to assess the rate of false positives and false negatives of the protocol and their clinical relevance. The results of the present study showed no reduction in the overall fluorescent in situ hybridization (FISH) error rate for single cells. However, by using a different scoring criterion, the incidence of false negative can be reduced to 1.6% without missing any trisomy 21. In addition, the present study suggests that if two or more loci from the same chromosome could be simultaneously analyzed in single cells, errors caused by false monosomies could be reduced. Copyright © 2001 John Wiley & Sons, Ltd.     

Chromosome 18 analysis by fluorescence in situ hybridization (fish) in human blastomeres of abnormal embryos after in vitro fertilization (ivf) attempt

We performed fluorescence in situ hybridization (FISH) with a chromosome 18-specific probe on human abnormal cleaved embryos, fertilized either by two spermatozoa and exhibiting three pronuclei (3 PN) or normally fertilized and exhibiting two pronuclei (2 PN) with subsequent severe fragmentation and/or blocking. The aim of the study was to evaluate the incidence of chromosome 18 anomalies among these embryos, in order to evaluate the FISH efficiency on such material and to obtain more precise and complete data than those obtained with classical cytogenetic analysis. For the 3 PN cleaved embryos, FISH confirmed the frequent regulation towards diploidy (25 per cent) and the high frequency of mosaics (53 per cent). For the 2 PN blocked or damaged embryos, FISH permitted chromosome evaluation, which was otherwise impossible with classical cytogenetic techniques: we also found a high mosaic frequency (45 per cent) with these embryos. If this frequency were the same for normally developing embryos, it would be a major obstacle to the reliability of either chromosomal or genetic preimplantation diagnosis.     

Preimplantation diagnosis: A patient perspective

A new dimension in the prevention of birth defects will be achieved when genetic diseases can be routinely diagnosed in embryos prior to implantation. The impressions and attitudes towards preimplantation diagnosis were studied in prospective patients, women at high reproductive risk for a genetic disease. Their perspective highlighted not only the advantages and disadvantages of this new approach, but also those changes necessary in order for preimplantation diagnosis to become a useful and practical technique. The data presented are based on information obtained by a mailed questionnaire answered by 58 women. The main benefit of preimplantation diagnosis for these high-risk women would be the ability to undertake a pregnancy without having to be subjected to the physical and/or emotional trauma of elective termination. Their major concerns related to possible damage to the embryo following biopsy, the cost of the procedure, and the low success rate of completed pregnancies. Other issues to be addressed before preimplantation diagnosis could begin to compare favourably with existing forms of prenatal testing were that the methods of obtaining oocytes or embryos should be simple, well tolerated, highly efficient, and low in maternal risk, and that the genetic analysis of embryonic or extraembryonic cells should be unequivocally accurate.     

A comparison of different lysis buffers to assess allele dropout from single cells for preimplantation genetic diagnosis

Single cell polymerase chain reaction (PCR) for preimplantation genetic diagnosis (PGD) requires high efficiency and accuracy. Allele dropout (ADO), the random amplification failure of one of the two parental alleles, remains the most significant problem in PCR-based PGD testing since it can result in serious misdiagnosis for compound heterozygous or autosomal dominant conditions. A number of different strategies (including the use of lysis buffers to break down the cell and make the DNA accessible) have been employed to combat ADO with varying degrees of success, yet there is still no consensus among PGD centres over which lysis buffer should be used (ESHRE PGD Consortium, 1999 ). To address this issue, PCR amplification of three genes (CFTR, LAMA3 and PKP1) at different chromosomal loci was investigated. Single lymphocytes from individuals heterozygous for mutations within each of the three genes were collected and lysed in either alkaline lysis buffer (ALB) or proteinase K/SDS lysis buffer (PK). PCR amplification efficiencies were comparable between alkaline lysis and proteinase K lysis for PCR products spanning each of the three mutated loci (ΔF508 in CFTR 90% vs 88%; R650X in LAMA3 82% vs 78%; and Y71X in PKP1 91% vs 87%). While there was no appreciable difference between ADO rates between the two lysis buffers for the LAMA3 PCR product (25% vs 26%), there were significant differences in ADO rates between ALB and PK for the CFTR PCR product (0% vs 23%) and the PKP1 PCR product (8% vs 56%). Based on these results, we are currently using ALB in preference to PK/SDS buffer for the lysis of cells in clinical PGD. Copyright © 2001 John Wiley & Sons, Ltd.     

Personal desires of patients and social obligations of geneticists: applying preimplantation genetic diagnosis for non-medical sex selection

The arguments against the use of preimplantation genetic diagnosis (PGD) for non-medical sex selection are analysed. It is concluded that the distinction between medical and non-medical reasons is difficult to maintain, that the disproportionality of means and end is not a decisive counterargument and that the fear of damage to the reputation of PGD does not justify the refusal of controversial applications. Moreover, since non-medical sex selection does not belong to basic health care, it should not be equally accessible to all. The position defended in this article is founded on two basic principles: (1) medical reasons have priority on non-medical reasons, and (2) personal reasons do not qualify for public funding. In order to respect both principles, it is proposed that restrictions should be installed to control the number of requests for social sexing and that a tax should be imposed on these elective services. The tax should compensate the society for the investment it made in the training and education of the physician. Copyright © 2002 John Wiley & Sons, Ltd.     

Preimplantation genetic diagnosis using FISH for carriers of Robertsonian translocations: the Portuguese experience

Preimplantation genetic diagnosis (PGD) is an alternative to prenatal diagnosis for couples at risk of transmitting genetic disorders to their offspring. We present a fluorescence in situ hybridization (FISH) analysis of embryos obtained after seven PGD cycles in six couples with Robertsonian translocations and male factor infertility: 4 der(13;14), 1 der(14;21) and 1 der(15;21). Of 74 metaphase II (MII) injected oocytes, 61 (82.4%) fertilized normally and cleaved. Of these, 37/61 (60.7%) embryos were of high morphological quality with ≥6 blastomeres. After biopsy of 44 embryos at day 3 of development, seven degenerated, seven arrested in development and 30/44 (68.2%) evolved, of which 25/30 (83.3%) reached the morula/blastocyst stage. Analysis of biopsied blastomeres showed 23/44 (52.3%) of normal/balanced embryos, of which 15 (11 at the morula/blastocyst stage) were transferred in six cycles. One term pregnancy was achieved, which ended by cesarean section at 37 weeks of gestation, giving birth to two healthy newborn. Analysis of 49 embryos (excluding 12 inconclusive cases) showed a predominance of alternate segregation (38/49, 77.6%) over adjacent segregation (7/49, 14.3%), with one (2%) being a polyploid mosaic and three (6.1%) chaotic. Copyright © 2002 John Wiley & Sons, Ltd.     

Preimplantation genetic diagnosis at 20 years

First reported in 1990, PGD has evolved into a complementary form of prenatal diagnosis offering novel indications. DNA for PGD can be recovered with equal safety and facility from polar bodies I and II, blastomere (8 cell embryo) and trophectoderm (5–6 day blastocyst). Diagnostic accuracy is very high (>99%) for both chromosomal abnormalities and single gene disorders. Traditional application of FISH with chromosome specific probes for detecting aneuploidy and translocations may be replaced or complemented by array comparative genome hybridization (array CGH); biopsied embryos can now be cryopreserved (vitrification) while analysis proceeds in orderly fashion. PGD has been accomplished for over 200 different single gene disorders. Novel indications for PGD not readily applicable by traditional prenatal genetic diagnosis include avoiding clinical pregnancy termination, performing preconceptional diagnosis (polar body I), obtaining prenatal diagnosis without disclosure of prenatal genotype (nondisclosure), diagnosing adult-onset disorders particularly cancer, and identifying HLA compatible embryos suitable for recovering umbilical cord blood stem cells. Copyright © 2010 John Wiley & Sons, Ltd.     

Copy number analysis of meiotic and postzygotic mitotic aneuploidies in trophectoderm cells biopsied at the blastocyst stage and arrested embryos

Preimplantation genetic testing for aneuploidy (PGT-A) by copy number analysis is now widely used to select euploid embryos for transfer. Whole or partial chromosome aneuploidy can arise in meiosis, predominantly female meiosis, or in the postzygotic, mitotic divisions during cleavage and blastocyst formation, resulting in chromosome mosaicism. Meiotic aneuploidies are almost always lethal, however, the clinical significance of mitotic aneuploidies detected by PGT-A is not fully understood and healthy live births have been reported following transfer of mosaic embryos. Here, we used single nucleotide polymorphism genotyping of both polar bodies and embryo samples to identify meiotic aneuploidies and compared copy number changes for meiotic and presumed mitotic aneuploidies in trophectoderm cells biopsied at the blastocyst stage and arrested embryos. PGT-A detected corresponding full copy number changes (≥70%) for 36/37 (97%) maternal meiotic aneuploidies. The number of presumed mitotic copy number changes detected exceeded those of meiotic origin. Although mainly in the mosaic range, some of these mitotic aneuploidies had copy number changes ≥70% and would have been identified as full aneuploidies. Interestingly, many arrested embryos had multiple mitotic aneuploidies across a broad range of copy number changes, which may have arisen through tripolar spindle and other mitotic abnormalities.