Three births after preimplantation genetic diagnosis for cystic fibrosis with sequential first and second polar body analysis

OBJECTIVE: The purpose of this study was to determine the accuracy and feasibility of sequential polar body removal and analysis for preimplantation genetic diagnosis of mendelian disorders. STUDY DESIGN: Three couples with risk factors for cystic fibrosis had preimplantation genetic diagnosis with the use of sequential polar body analysis. After stimulation, oocytes were harvested and the first polar bodies were removed and analyzed on the day of aspiration. The following day, after fertilization, the second polar bodies were aspirated. Only embryos known to have inherited the normal maternal allele were transferred. RESULTS: All three couples had successful pregnancies resulting in the births of unaffected infants. CONCLUSIONS: Preimplantation diagnosis with the use of sequential polar body removal is feasible and can prevent the establishment of genetically abnormal pregnancies for couples at risk. (Am J Obstet Gynecol 1998;178:1298-306.)     

Birth of Healthy Children After Preimplantation Diagnosis of Thalassemias

Background: Preimplantation genetic diagnosis (PGD) allows couples at risk of having children with thalassemia to ensure the healthy outcome of their pregnancy. Methods: Seventeen PGD clinical cycles were initiated for Cypriot couples at risk of having children with different thalassemia mutations, including IVS1-110, IVSI-6, and IVSII-745. Unaffected embryos for transfer were selected by testing oocytes, using first and second polar body (PB) removal and nested polymerase chain reaction analysis followed by restriction digestion. Results: Unaffected embryos were selected in 16 of 17 PGD cycles. Of 166 oocytes studied from these cycles, 110 were analyzed by sequential analysis of both the first and the second PB, resulting in preselection and transfer of 45 unaffected embryos. This resulted in seven pregnancies and in the birth of five healthy thalassemia-free children. The embryos predicted to have inherited the affected allele were not transferred. Analysis of these embryos confirmed the PB diagnosis. Conclusions: Sequential first and second PB testing of oocytes is reliable for PGD of thalassemia and is a feasible alternative to prenatal diagnosis in high-risk populations.     

Polar body-based preimplantation diagnosis for X-linked disorders

Preimplantation diagnosis for X-linked disorders has been performed predominantly by gender determination, which, however, leads to the discarding of 50% unaffected male embryos. In an attempt to identify X-linked mutation-free embryos for transfer, the present authors introduced preimplantation genetic diagnosis (PGD), using a sequential first and second polar body analysis, as an alternative to gender determination. This method was offered to eight couples at risk for having children with X-linked disorders, including haemophilia B, fragile-X syndrome (FMR1), myotubular myotonic dystrophy (MTMD), ornithine transcarbamylase (OTC) deficiency and X-linked hydrocephalus. The first and second polar bodies were removed following maturation and fertilization of oocytes in a standard IVF protocol and analysed using a multiplex nested polymerase chain reaction (PCR), involving testing for mutations simultaneously with linked markers. Overall, 13 PGD cycles were performed, resulting in the detection of 25 embryos with the predicted mutation-free maternal contribution; these embryos were transferred back to the patients in all cycles, yielding four clinical pregnancies. Four children were born following these pregnancies, including three unaffected and one with misdiagnosis as a result of allele dropout (ADO), which was predictable in the case of FMR1. Presented results demonstrate the clinical usefulness of the specific polar body testing for X-linked disorders as an alternative to PGD by gender determination.     

Pre-embryonic diagnosis for Sandhoff disease

Embryos found to be abnormal during preimplantation genetic diagnosis (PGD) are discarded or analysed to confirm the diagnosis. To overcome this limitation, which is unacceptable in some communities and ethnic groups, pre-embryonic genetic diagnosis has been introduced, involving sequential first and second polar body analysis followed by transfer of embryos deriving from the mutation-free oocytes, while removing from culture and freezing the mutant oocytes at the pronuclear stage. The technique is applied here to PGD of Sandhoff disease caused by 16-kb deletion of the hexosaminidase B gene for a couple with a religious objection to discarding embryos irrespective of embryo genotype. Of 16 oocytes tested in a standard IVF protocol for 16-kb deletion, simultaneously with five linked polymorphic markers, eight were predicted mutant and frozen prior to syngamy, with the remaining eight, found to be free of mutation, further cultured and confirmed unaffected using blastomere biopsy. The transfer of two of these embryos resulted in birth of an unaffected child, demonstrating feasibility of pre-embryonic diagnosis to avoid embryo discard.     

Allele Dropout in Polar Bodies and Blastomeres

Purpose: Because allele dropout (ADO) is frequently observed in single-cell polymerase chain reaction analysis, it is important to develop a method for efficient detection of ADO, in order to avoid possible misdiagnosis in preimplantation diagnosis. Methods: We introduced a simultaneous amplification of mutant genes and linked polymorphic markers, such as a 4-bp repeat (GATT) at the 3 end of intron 6 in the cystic fibrosis (CF) gene and a short tandem repeat at the 5 end of the -globin gene. Three types of single heterozygous cells were studied for the amplification of both alleles, including 150 blastomeres, 1615 fibroblasts, and 170 first polar bodies, obtained from patients at risk for having children with cystic fibrosis (delta F-508 mutation) or sickle cell disease. Results: ADO rates of as high as 33.3% for delta F-508 mutation and 22.8% for -globin gene were observed in single blastomeres, compared to 7.1 and 7.7% in single fibroblasts and 5.9 and 9.6% in first polar bodies, respectively. The application of simultaneous amplification of the above linked polymorphic markers allowed detection of more than half of the cases of ADO in blastomeres (19.4% for cystic fibrosis and 12.3% for -globin gene) and almost all ADOs in polar bodies, particularly when the two-step sequential analysis of the first and second polar body was applied in preimplantation diagnosis of single gene disorders. Conclusions: Simultaneous amplification of linked polymorphic markers in single-cell DNA analysis of single-gene defects is an efficient method for avoiding the risk of misdiagnosis in preimplantation diagnosis.     

First clinical application of comparative genomic hybridization and polar body testing for preimplantation genetic diagnosis of aneuploidy

OBJECTIVE: To develop a preimplantation genetic diagnosis (PGD) protocol that allows any form of chromosome imbalance to be detected.DESIGN: Case report employing a method based on whole-genome amplification and comparative genomic hybridization (CGH).SETTING: Clinical IVF laboratory.PATIENT(S): A 40-year-old IVF patient.INTERVENTION(S): Polar body and blastomere biopsy.MAIN OUTCOME MEASURE(S): Detection of aneuploidy.RESULT(S): Chromosome imbalance was detected in 9 of 10 polar bodies. A variety of chromosomes were aneuploid, but chromosomal size was found to be an important predisposing factor. In three cases, the resulting embryos could be tested using fluorescence in situ hybridization, and in each case the CGH diagnosis was confirmed. A single embryo could be recommended for transfer on the basis of the CGH data, but no pregnancy ensued.CONCLUSION(S): Evidence suggests that preferential transfer of chromosomally normal embryos can improve IVF outcomes. However, current PGD protocols do not allow analysis of every chromosome, and therefore a proportion of abnormal embryos remains undetected. We describe a method that allows every chromosome to be assessed in polar bodies and oocytes. The technique was accurate and allowed identification of aneuploid embryos that would have been diagnosed as normal by standard PGD techniques. As well as comprehensive cytogenetic analysis, this protocol permits simultaneous testing for multiple single-gene disorders.     

Preimplantation Diagnosis of Thalassemias

Purpose: Preimplantation genetic diagnosis (PGD) is an important option for couples at risk of having children with -globin mutations to avoid selective abortions of affected fetuses following prenatal diagnosis. Methods: We performed PGD for thalassemia in 12 clinical cycles (IVS1-110, and IVS-745 mutations) using biopsy of the first and second polar bodies (PBs) extruded from oocytes during maturation and fertilization, coupled with nested polymerase chain reaction analysis and restriction digestion. Results: A total of 118 oocytes was obtained, of which 78 had results for both the first and the second PBs. This resulted in the selection and transfer of 30 unaffected embryos (2.5 embryos per cycle). To avoid a possible misdiagnosis due to allele dropout (ADO), we have also introduced simultaneous detection of two highly polymorphic linked markers, a short tandem repeat immediately at the 5 end of the globin gene and HUMTH01 which is a syntenic short tandem repeat. The application of multiplex polymerase chain reaction of the -globin gene and linked polymorphic markers enabled detection of ADO in five first PBs, thus avoiding the transfer of potentially affected embryos resulting from their corresponding oocytes. Conclusions: Confirmation studies of the embryos resulting from the oocytes predicted to contain an affected gene confirmed the diagnosis in 98% of the cases, thus demonstrating the accuracy and reliability of PB PGD of thalassemia mutations. The application of PB analysis in six patients resulted in two ongoing pregnancies with a thalassemia-free fetus already confirmed in both of them by prenatal diagnosis.     

Prevention of Age-Related Aneuploidies by Polar Body Testing of Oocytes

Purpose: We previously demonstrated that aneuploidy-free oocytes may be preselected by testing the first and second polar bodies removed from oocytes following their maturation and fertilization. The present paper describes the results of the application of the method in 659 in vitro fertilization cycles from patients of advanced maternal age. Methods: Using micromanipulation techniques, 3943 oocytes were tested by polar body sampling and fluorescent in situ hybridization analysis using specific probes for chromosomes 13, 18, and 21. Results: Fluorescent in situ hybridization results were available for 3217 (81.6%) of 3943 oocytes studied, of which 1388 (43.1%) had aneuploidies; 35.7% of the aneuploidies were of first meiotic division origin, and 26.1% of second meiotic division origin. Most errors in the first meiotic division were represented by chromatid malsegregation. The transfer of embryos deriving from 1558 of 1829 aneuploidy-free oocytes in 614 treatment cycles resulted in 131 clinical pregnancies and 88 healthy children born after confirmation of the polar body diagnosis. Conclusions: Polar body testing of oocytes provides an accurate and reliable approach for prevention of age-related aneuploidies in in vitro fertilization patients of advanced maternal age.     

Preimplantation Diagnosis of Common Aneuploidies by the First- and Second-Polar Body FISH Analysis

Purpose: A low pregnancy rate in in vitro fertilization (IVF) patients of advanced maternal age may be caused by aneuploidies originating from non disjunction in the first or second meiotic divisions. We introduced genetic testing of oocytes by sampling and fluorescent in situ hybridization (FISH) analysis of the first and second polar bodies, to avoid fertilization and transfer of aneuploid oocytes in IVF patients of advanced maternal age. Methods: Three hundred and sixty-three IVF patients 34 years and older participated in the study. Using micromanipulation procedures, the first and second polar bodies were removed following their extrusion from the oocytes and studied by FISH, using probes specific for chromosomes 13, 18, and 21 to detect oocytes with common aneuploidies. Results: Of a total of 538 IVF cycles, 3250 oocytes were available for FISH analysis, with conclusive FISH results in 2742 oocytes (84.3%). As many as 1102 (40%) of oocytes were predicted to be aneuploid and not transferred. Of 1640 embryos predicted to be normal, 1145 were transferred in 467 treatment cycles, resulting in 107 pregnancies (23%), from which 67 healthy children have been born, 32 pregnancies spontaneously aborted, and 15pregnancies are ongoing after being confirmed normal by prenatal diagnosis. Conclusions: Preimplantation diagnosis by first- and second-polar body FISH analysis allows us to avoid the age-related risk of common aneuploidies in IVF patients of advanced maternal age.     

Viability of embryos following second polar body removal in a mouse model

Purpose Our purpose was to evaluate the effect of the second polar body removal on the viability and developmental potential of the resulting embryos.Method The second polar body was removed in 343 mouse oocytes and the developmental potential of the resulting embryos was compared in vitro to that in 223 intact mouse embryos and 222 controls which were placed under the same experimental conditions as those micromanipulated.Results The proportion of morphologically normal blastocysts formed by polar body biopsied oocytes (70.8%) was not significantly different from that obtained from the control (81.1%) and intact (85%) oocytes. Cell counts of blastocysts obtained from the manipulated and nonmanipulated oocytes were also not statistically different.Conclusion The second polar body biopsy does not have a significant adverse effect on preimplantation development and may be applied for genetic evaluation of oocytes.     

Preimplantation Genetic Diagnosis of Inherited Cancer: Familial Adenomatous Polyposis Coli

Purpose: Our purpose was to achieve preimplantation genetic diagnosis (PGD) of the dominant cancer predisposition syndrome, familial adenomatous polyposis coli (FAPC), as an alternative to prenatal diagnosis. Methods: The affected patient was superovulated and oocytes were retrieved and fertilized by intracytoplasmic sperm injection (ICSI). Two cells were biopsiedfrom each embryo and the whole genome was amplified by primer extension preamplification (PEP). Nested PCR was then used to amplify two APC fragments: one including the APC mutation site and the other an informative intragenic polymorphism. Both were detected by simultaneous singlestrand conformation polymorphism and heteroduplex analysis. Results: Four normally fertilized embryos were biopsied on day 3 post ICSI, and two cells were successfully removed from each embryo. Following PEP the APC mutation was successfully amplified in 7 of 8 cells, and the polymorphism in 6 of 8 cells. The APC mutation was detected in three embryos. This result was confirmed by identification of the mutation associated polymorphism in two cases. A single embryo was diagnosed as homozygous normal for the mutation and the polymorphism in both cells sampled. This unaffected embryo was transferred to the mother, but no pregnancy resulted. Conclusions: We report here the first diagnosis of a cancer predisposition syndrome in human preimplantation embryos. Our results indicate that difficulties associated with single-cell PCR, allele-specific amplification failure in particular, need not prevent preimplantation diagnosis of diseases with a dominant mode of inheritance, provided appropriate strategies are applied.     

Preimplantation Genetic Diagnosis of Human Embryos for Marfan's Syndrome

Purpose: Single-cell nested polymerase chain reaction (PCR) and Dde1 endonuclease digestion were used to detect the presence of a Marfan's syndrome mutation in human preimplantation embryos derived from in vitro fertilization (IVF). These procedures were conducted to eliminate the possibility of transmission of the affected allele from the father to his offspring. The mutation on chromosome 15 is transmitted as an autosomal dominant trait, and the chance of having a child affected with the disease is 50%. Methods: A couple presented to the Program for In Vitro Fertilization, Reproductive Surgery and Infertility for preimplantation genetic diagnosis. IVF was performed and embryo biopsy was done on day 3 embryos, Single blastomeres were removed from embryos and subjected to nested PCR analysis and endonuclease digestion to detect a Marfan's syndrome mutation located on chromosome 15 inherited from the father. Results: Thirteen oocytes were injected with spermatozoa using intracytoplasmic sperm injection, and nine fertilized normally. Following embryo biopsy and polymerase chain reaction amplification-Dde1 endonuclease digestion, five embryos were detected that were positive for the mutation. The four non-affected embryos were transferred to the uterus, resulting in a healthy and normal ongoing pregnancy.     

Preimplantation diagnosis for p53 tumour suppressor gene mutations

Preimplantation genetic diagnosis (PGD) was introduced for high-risk couples to avoid establishing affected pregnancies potentially requiring termination following prenatal diagnosis. This opens the possibility for PGD for late onset disorders with genetic predisposition, including inherited cancer predisposition, because only embryos free from the predisposing gene may be transferred back to the patient, with no potential risk for pregnancy termination. PGD was performed for two couples, one with maternally and one with paternally derived p53 tumour-suppressor mutations, 902insC in exon 8 and G524A in exon 5, respectively. This involved a standard IVF protocol, allowing oocytes or embryos to be tested prior to their transfer back to uterus. Maternal mutation was tested by sequential PCR analysis of the first and second polar bodies, removed following maturation and fertilization of oocytes, while paternal mutation analysis required embryo biopsy at the cleavage stage. To avoid misdiagnosis due to allele drop out, multiplex nested PCR was applied, involving p53 mutation analysis simultaneously with the linked short tandem repeats in intron 1. Of 10 oocytes tested in two PGD cycles for 902insC mutation, four unaffected oocytes were pre-selected for transfer yielding no clinical pregnancy. Of 18 embryos analysed in two cycles for G524A mutation, seven mutation-free embryos were detected, two of which were transferred in each cycle, resulting in a singleton pregnancy and birth of a mutation-free child. This is the first PGD for inherited cancer predisposition determined by p53 tumour suppressor mutations, resulting in a clinical pregnancy and birth of a child free from inherited cancer predisposition.     

Polar body diagnosis of common aneuploidies by FISH

Purpose: The purpose of this work was to investigate the reliability and accuracy of polar body analysis for preimplantation diagnosis of common aneuploidies in IVF patients of advanced maternal age. Design: We have previously introduced polar body analysis as an approach for nondestractive evaluation of the genotype of human oocytes. The method has recently been applied in a clinical trial involving 45 infertile patients, demonstrating the feasibility of preconception diagnosis of common aneuploidies by fluorescent in situ hybridization (FISH). The present paper describes the experience of polar body diagnosis in 135 IVF patients (161 cycles) of advanced maternal age. Results: FISH results of the first and/or second polar bodies were available in 648 (72.4%) of 895 biopsied oocytes subjected to FISH analysis. Of 648 oocytes with FISH results, 208 demonstrated chromosomal abnormalities. Of 440 oocytes predicted to be free from monosomy or trisomy of chromosomes X, 18, and/or 13/21, 314 were normally fertilized, cleaved, and transferred in 122 treatment cycles, resulting in 6 healthy deliveries and 12 ongoing pregnancies following confirmation of the polar body diagnosis by CVS or amniocentesis. Conclusion: The method may be useful for detection of oocytes with common chromosomal trisomies in IVF patients of advanced maternal age.Presented at the 5th Annual Meeting of the International Working Group on Preimplantation Genetics, Hamburg, Germany, June 28, 1995.     

Relationship of the Human Cumulus-Free Oocyte Maturational Profile with In Vitro Outcome Parameters After Intracytoplasmic Sperm Injection

Purpose: We investigated whether the human oocyte maturational profile at the removal of cumulus/corona cells affects the fertilization rate and subsequent embryo quality after intracytoplasmic sperm injection. Methods: A total of 1011 oocytes from 150 cycles was included in this retrospective analysis. Cumulus-free oocytes that were in prophase or metaphase I of meiosis at the removal of cumulus/corona cells were incubated in vitro until they reached metaphase II (in vitro-matured oocytes) and were then immediately injected with a single spermatozoa. Oocytes that were in metaphase II at the removal of cumulus/corona cells (MII oocytes) received sperm injection after 3–4 hr of preinjection incubation. Results: The fertilization rate of the MII oocytes was significantly higher than that of in vitro-matured oocytes (81 vs 62%; P < 0.001). The cleavage rates were similar in the two groups (MII oocytes, 94%; in vitro-matured oocytes, 91%). However, MII oocytes had significantly higher percentages of good-quality embryos (grade 1–3 embryos, 87 vs 58%, P < 0.001) and embryos with high cumulative embryo scores (score 10–32 embryos, 62 vs 33%, P < 0.001). The mean cumulative embryo score of MII oocytes after fertilization was also higher than that of in vitro-matured oocytes (12.1 ± 3.8 vs 8.8 ± 3.4; P = 0.014). Conclusions: MII oocytes that extruded the first polar body at the removal of cumulus/corona cells had better fertilization rates and embryo morphology than in vitro-matured oocytes that extruded the first polar body following the removal of cumulus/corona cells and in vitro culture.     

Reduced allele dropout in single-cell analysis for preimplantation genetic diagnosis of cystic fibrosis

Background: For couples at risk of transmitting a known single-gene defect, preimplantation genetic diagnosis (PGD) allows the identification and transfer of only unaffected embryos followingin vitro fertilisation (IVF), single-cell biopsy at about the eight-cell stage, and genetic analysis by PCR. This technique therefore avoids the risk of terminating an affected pregnancy diagnosed later in gestation. Methods and Results: Using nested PCR, the F508 mutation causing cystic fibrosis can be detected in single cells and we previously reported successful PGD in a couple in whom both partners carry the F508 mutation. To date we have treated 12 couples in a total of 18 cycles. This resulted in five singleton births confirmed to be homozygous normal. Single blastomeres from disaggregated embryos which had not been transferred were analysed to confirm the original diagnosis and assess reliability in clinical practice. Amplification efficiency and accuracy were high, with blastomeres from embryos diagnosed as homozygous normal or affected. In a proportion of blastomeres from presumed carrier embryos, one of the parental alleles failed to amplify, apparently at random (allele dropout, ADO). A possible explanation is the relative inaccessibility of one of the target allele early in the PCR. To test this we have used single lymphocytes from F508 carriers and investigated the effects of various denaturation temperatures in the early cycles of amplification. Conclusion: Increasing the denaturation temperature reduced the rate of ADO without affecting amplification efficiency.Presented at the 5th Annual Meeting of the International Working Group on Preimplantation Genetics, Hamburg, Germany, June 28, 1995.     

Is there any predictive value of first polar body morphology for embryo genotype or developmental potential?

The first polar body (PB1) was previously used for genotyping oocytes in preimplantation genetic diagnosis (PGD) for Mendelian and chromosomal disorders. A possible use of PB1 morphology for predicting developmental potential was also explored in a few retrospective studies, which showed controversial results. PB1 morphology was evaluated in a prospective study of 831 oocytes, which were then followed up through preimplantation development, with analysis of fertilization rate, embryo quality, survival to blastocyst, and outcome of embryo transfer, demonstrating no correlation with PB1 morphology. There was also no evidence for a PB1 predictive value for these parameters either in good or poor responder patients. An additional 358 oocytes and embryos from patients requesting PGD were tested for aneuploidies, revealing no relationship with PB1 morphology either. This suggests that PB1 morphology may not be a reliable predictor of genotype or the developmental potential of the corresponding oocytes or embryos.     

Cytogenetic analysis of spontaneously activated noninseminated oocytes and parthenogenetically activated failed fertilized human oocytes--implications for the use of primate parthenotes for stem cell production

Purpose: Spontaneous parthenogenetically activated noninseminated oocytes and failed fertilized oocytes after ART activated by puromycin were studied to assess cleavage ability and the cytogenetic constitution of the resulting embryos. Methods: Failed fertilized oocytes were exposed to puromycin, and whenever activation occurred, they were further cultured until arrest of development. FISH was used to assess the ploidy of spontaneous (group A) and induced parthenotes (group B). Results: The mean number of oocytes exposed to puromycin and the percentage and type of activation were identical in IVF and ICSI patients. The more frequent types of activation were one or two pronuclei and one polar body suggesting that retention of the second polar body is a common event after parthenogenetic activation. Conclusions: Retention of the second polar body and chromosome malsegregation were observed after parthenogenetic activation, either spontaneous or induced by puromycin. This means that using parthenogenetic embryos for stem cell research will require great care and attention.     

Advantages of multiple markers and polar body analysis in preimplantation genetic diagnosis for Alagille disease

OBJECTIVE: The development of a preimplantation genetic diagnosis (PGD) protocol for Alagille syndrome (AGS), a rare autosomal dominant disorder with hepatic, cardiac and ophthalmologic involvement. METHODS: We developed a polar body (PB)-based multiplex fluorescent PCR reaction for a female affected with AGS. The protocol included analysis of the Jagged 1 (JAG1) familial mutation and five closely linked highly polymorphic markers (D20S162, D20S901, D20S894, and D20S186). RESULTS: In two cycles of PGD 9 of ten embryos were accurately diagnosed by assessment of first and second PBs, one embryo required additional blastomere biopsy. CONCLUSIONS: This protocol takes advantage of the larger window of opportunity for transfer and the increased accuracy of diagnosis afforded by the combination of PB biopsy and multiple marker analysis. Two cycles resulted in the transfer of two and three mutation-free embryos and a subsequent pregnancy as measured by the rising hCG levels.     

Thirteen years' experience of preimplantation diagnosis: report of the Fifth International Symposium on Preimplantation Genetics

Preimplantation genetic diagnosis (PGD) has been further developed into a practical option for avoiding the birth of affected children, representing an important complement to traditional prenatal diagnosis. More than 1000 unaffected children have been born after PGD, suggesting the accuracy and safety of the procedure, which is currently also used with the establishment of potential donor progeny for stem cell treatment of siblings. Together with progress in the establishment of embryonic stem (ES) cells, this may contribute to the development and application of stem cell therapy. The accumulated experience of thousands of PGD cycles for poor prognosis IVF patients provides further evidence of the improvement of clinical outcome, particularly obvious from the reproductive history of PGD patients. A high prevalence of aneuploidies in oocytes and embryos may affect the accuracy of PGD for single gene disorders, making aneuploidy testing an important part of PGD for causative genes and preimplantation human leukocyte antigen (HLA) typing. A sequential sampling of both oocytes and the resulting embryos may improve accuracy of aneuploidy testing and may also allow the detection and avoidance of transfer of embryos with uniparental disomies. Current developments and application of nuclear transfer and sperm duplication techniques, and microarray technology, may also contribute to the improvement of PGD and help in the development of PGD for genetic expression disorders.