PGD for all cystic fibrosis carrier couples: novel strategy for preventive medicine and cost analysis.

Over 1000 children affected with cystic fibrosis (CF) are born annually in the USA. Since IVF with preimplantation genetic diagnosis (PGD) is an alternative to raising a sick child or to aborting an affected fetus, a cost-benefit analysis was performed for a national IVF-PGD program for preventing CF. The amount spent to deliver healthy children for all CF carrier-couples by IVF-PGD was compared with the average annual and lifetime direct medical costs per CF patient avoided. Treating annually about 4000 CF carrier-couples with IVF-PGD would result in 3715 deliveries of non-affected children at a cost of $57,467 per baby. Because the average annual direct medical cost per CF patient was $63,127 and life expectancy is 37 years, savings would be $2.3 million per patient and $2.2 billion for all new CF patients annually in lifetime treatment costs. Cumulated net saving of an IVF-PGD program for all carrier-couples for 37 years would be $33.3 billion. A total of 618,714 cumulative years of patients suffering because of CF and thousands of abortions could be prevented. A national IVF-PGD program is a highly cost-effective novel modality of preventive medicine and would avoid most births of individuals affected with debilitating genetic disease.

Human embryonic stem cell models of Huntington disease.

Huntington disease (HD) is an incurable late-onset neurodegenerative disorder caused by a CAG repeat expansion in exon 1 of the HD gene (HTT). The major hallmark of disease pathology is neurodegeneration in the brain. Currently, there are no useful in-vitro human models of HD. Recently, two human embryonic stem cell (hESC) lines carrying partial (CAG(37)) and fully (CAG(51)) penetrant mutant alleles have been derived from affected IVF embryos identified following preimplantation genetic diagnosis (PGD). Fluorescence polymerase chain reaction (F-PCR) and Genescan analysis confirmed the original embryonic HD genotypes. Reverse transcription PCR (RT-PCR) analysis confirmed the expression of mutant transcripts and western blot analysis demonstrated expression of mutant huntingtin protein (HTT). After treatment with noggin, HD hESC formed neurospheres, which could be further differentiated into cells susceptible to neurodegeneration in HD, namely primary neurones and astrocytes. Small pool PCR analysis of neurosphere cells revealed instability of disease-length CAG repeats following differentiation. The presence of active HTT genes, neural differentiation capabilities and evidence of CAG repeat instability indicates these HD hESC lines may serve as valuable in-vitro human models of HD to better understand the mechanisms of neurodegeneration in patients, and for drug screening to identify new therapies for human clinical trials.

Frequency and distribution of chromosome abnormalities in human oocytes.

It was previously shown that more than half of the human oocytes obtained from IVF patients of advanced reproductive age are aneuploid, due to meiosis I and meiosis II errors. The present paper further confirms that 61.8% of the oocytes tested by fluorescent probes specific for chromosomes 13, 16, 18, 21 and 22 are abnormal, representing predominantly chromatid errors, which are the major source of aneuploidy in the resulting embryos. Almost half of the oocytes with meiosis I errors (49.3%) are prone to sequential meiosis II errors, which may lead to aneuploidy rescue in 30.8% of the cases. Half of the detected aneuploidies (49.8%) are of complex nature with involvement of two or more chromosomes, or the same chromosome in both meiotic divisions. The aneuploidy rates for individual chromosomes are different, with a higher prevalence of chromosome 21 and 22 errors. The origin of aneuploidy for the individual chromosomes is also not random, with chromosome 16 and 22 errors originating more frequently in meiosis II, and chromosome 18, 13 and 21 errors in meiosis I. There is an age dependence not only for the overall frequency of aneuploidies, but also for each chromosome error, aneuploidies originating from meiosis I, meiosis II, and both meiosis I and meiosis II errors, as well as for different types of aneuploidies. The data further suggest the practical relevance of oocyte aneuploidy testing for detection and avoidance from transfer of the embryos deriving from aneuploid oocytes, which should contribute significantly to the pregnancy outcomes of IVF patients of advanced reproduction age.

Preembryonic diagnosis for sickle cell disease.

Embryos found to be abnormal during preimplantation genetic diagnosis are discarded or analyzed to confirm the diagnosis. The destruction of affected embryos is ethically unacceptable to some couples. We developed a preembryonic genetic diagnosis, that uses sequential first and second polar body removal, followed by oocyte freezing at the pronuclear stage. This was applied in a patient at risk of having a child with sickle cell disease, who suffered hyper-stimulation syndrome. Fourteen oocytes were obtained and tested for the maternal sickle cell allele by PCR analysis of the first and second polar body. Immediately after procedure of polar body removal, the pronuclear-stage oocytes were frozen. Six mutation-free oocytes detected by polar body analysis were then thawed, allowed to cleave, and transferred in the two consecutive clinical cycles, both resulting in clinical pregnancies, one of which resulted in birth of a healthy child. The oocytes predicted to contain abnormal beta-globin gene were not further cultured, to avoid formation and discard of the affected embryos. The results demonstrate feasibility of preembryonic diagnosis for single gene disorders, avoiding the establishment and destruction of mutant embryos.

Reliability of preimplantation diagnosis for single gene disorders.

Reliability of preimplantation genetic diagnosis (PGD) depends on controlling one of the most important limitations of single cell PCR, undetected allele drop out (ADO), which may lead to misdiagnosis. To avoid this we introduced mutation analysis simultaneously with linked polymorphic markers, pre-selecting only those embryos whose unaffected status could be confirmed by at least one linked polymorphic marker. We applied this strategy for testing 1047 oocytes, from which 237 unaffected ones were pre-selected for transfer back to patients, resulting in 34 unaffected pregnancies and birth of 23 healthy children. Embryos originating from mutant oocytes and those with insufficient marker information were followed up by multiplex PCR to confirm single cell PCR diagnosis. Of 75 (8.5%) detected ADO, only seven (under 1%) were missed in the actual PGD, demonstrating high reliability of PGD (98%) based on multiplex single cell PCR.

Chromosomal abnormalities in the first and second polar body.

Aneuploidy free oocytes may be pre-selected by testing the first and second polar bodies removed from oocytes following their maturation and fertilization. We present here our experience on the application of the method in IVF cycles from patients of advanced maternal age. Overall, 5590 oocytes were obtained from 917 cycles and tested by polar body sampling and fluorescent in situ hybridization (FISH) analysis using specific probes for chromosomes 13,16,18,21 and 22. FISH results were available in 4599 (82.2%) of 5590 oocytes studied, from which 2077(45.2%) were with aneuploidies. Thirty six point one percent of aneuploidies were of the first meiotic origin, and 29.3% of the second meiotic origin. Most errors in the first meiotic division were represented by chromatid errors. The transfer of embryos deriving from 2014 of 2520 aneuploidy free oocytes in 821 treatment cycles resulted in 182 (22.2%) clinical pregnancies and 140 healthy children born after confirmation of the polar body diagnosis. Polar body testing of oocytes provides an approach for pre-selection of aneuploidy free embryos, improving pregnancy rate in IVF patents of advanced maternal age.

A simplified and efficient method for obtaining metaphase chromosomes from individual human blastomeres.

OBJECTIVE: To develop a reliable and cost-effective technique for karyotyping single human blastomeres for preimplantation diagnosis of chromosomal translocations. DESIGN: Controlled laboratory study. SETTING: Preimplantation genetic diagnosis and IVF program, Reproductive Genetics Institute/IVF Illinois, Chicago, Illinois. PATIENT(S): Patients undergoing IVF and preimplantation genetic diagnosis. INTERVENTION(S): Individual human blastomeres were fused with enucleated or intact mouse zygotes. After blastomere-cytoplast fusion, heterokaryons were fixed at metaphase of the first cleavage division or treated with okadaic acid to induce premature chromosome condensation. MAIN OUTCOME MEASURE(S): Percentage of analyzable metaphase plates and ease and reliability of the procedure. RESULT(S): The effectiveness of the proposed technique with blastomeres from day 3 diploid embryos was 91%. Sixty-three metaphases were obtained from 69 blastomeres; 3 blastomeres had not fused, 1 heterokaryon had no chromatin (an anucleated cytoplasmic bleb was biopsied and fused), and 2 heterokaryons cleaved before they were fixed. CONCLUSION(S): Human blastomere fusion with an intact mouse zygote is an efficient and technically undemanding method for obtaining metaphase chromosome plates from individual human blastomeres for preimplantation testing for chromosomal translocations and aneuploidy.

Effect of chromosomal translocations on the development of preimplantation human embryos in vitro.

OBJECTIVE: To determine the reliability of a new technique for single human blastomere karyotyping during clinical cases for preimplantation genetic diagnosis of translocations. DESIGN: Controlled clinical study. SETTING: Preimplantation genetic diagnosis and IVF program. PATIENT(S): Nineteen preimplantation genetic diagnosis cases with 11 types of translocations (10 reciprocal and one Robertsonian) involving chromosomes 1, 5, 7, 8, 9, 11, 12, 13, 14, 15, 16, 18, 20, 21, and 22. INTERVENTION(S): Blastomere biopsy followed by blastomere nucleus conversion into metaphase chromosomes. Fluorescent in situ hybridization (whole chromosome painting) was used for the detection of chromosomally unbalanced preimplantation human embryos. MAIN OUTCOME MEASURE(S): Percentage of informative metaphase plates and effect of unbalanced translocations on preimplantation embryo development. RESULT(S): Informative metaphases were obtained for 84% of the blastomeres. Analysis of preimplantation development of the resulting embryos showed that an unbalanced chromosomal complement does not affect embryo ability to reach the blastocyst stage in vitro. CONCLUSION(S): For the translocations tested, there is no evident selection against chromosomally unbalanced embryos at the preimplantation stage of embryo development.

Three-dimensional partial zona dissection for preimplantation genetic diagnosis and assisted hatching.

OBJECTIVE: To develop a new approach to partial zona dissection of oocytes and embryos for facilitating preimplantation genetic sampling and assisted hatching. DESIGN: Controlled clinical study. SETTING: Preimplantation genetic diagnosis and IVF program, Reproductive Genetics Institute/IVF Illinois, Chicago, Illinois. PATIENT(S): Three hundred forty patients undergoing IVF in whom preimplantation genetic diagnosis or assisted hatching was required. INTERVENTION(S): Three-dimensional partial zona dissection and conventional partial zona dissection were performed, with the use of a simple microneedle, on embryos before preimplantation genetic sampling or on day 3 of embryo development before ET. MAIN OUTCOME MEASURE(S): Pregnancy rate, implantation rate, and ease of preimplantation genetic sampling. RESULT(S): A pregnancy rate of 42% and an embryo implantation rate of 17.6% were obtained in the group in which three-dimensional partial zona dissection was performed, compared with a pregnancy rate of 33% and an implantation rate of 14.7% in the control group, which underwent conventional partial zona dissection. Preimplantation genetic sampling can be performed without distortion of the blastomere configuration in the embryo. CONCLUSION(S): Three-dimensional partial zona dissection is a safe and simple mechanical means of creating an opening sufficient for the atraumatic removal of material from oocytes and embryos for preimplantation genetic diagnosis and assisted hatching.

Preimplantation testing for phenylketonuria.

OBJECTIVE: To use preimplantation genetic diagnosis to achieve a phenylketonuria-free pregnancy in a couple at 50% risk for producing an affected child. DESIGN: DNA analysis of the first and second polar bodies (PB1 and PB2) obtained from oocytes of a heterozygous mother in IVF-ET, with the goal of identifying and transferring back to the patient the embryos resulting from mutation-free oocytes. SETTING: IVF program of Reproductive Genetics Institute, Chicago, Illinois. PATIENT(S): A mother carrying the R408W mutation and a father with compound heterozygosity for R408 and Y414C mutations in phenylalanine hydroxylase (PAH) gene. INTERVENTION(S): Removal and testing for maternal mutation in PB1 and PB2 from each oocyte after standard IVF. MAIN OUTCOME MEASURE(S): DNA analysis of PB1 and PB2 indicating whether corresponding oocytes were mutation-free, for the purposes of transferring only unaffected embryos resulting from these oocytes. RESULT(S): Of 11 zygotes with both PB1 and PB2, 6 were predicted to be free of phenylketonuria. Of these, 4 were transferred, resulting in an unaffected twin pregnancy and birth of two healthy children. CONCLUSION(S): Preimplantation genetic diagnosis of phenylketonuria resulted in the birth of phenylketonuria-free children. Preimplantation genetic diagnosis by PB analysis in couples with a compound heterozygous male partner is clinically useful.

Birth of healthy children after preimplantation diagnosis of common aneuploidies by polar body fluorescent in situ hybridization analysis. Preimplantation Genetics Group.

OBJECTIVE: To perform preimplantation diagnosis of common aneuploidies by polar body analysis and fluorescent in situ hybridization technique using probes specific for chromosomes X, 18, and 13/21. DESIGN: The first and/or second polar bodies were removed and studied by fluorescent in situ hybridization to detect and avoid fertilization and transfer of oocytes with common aneuploidies. SETTING: The Reproductive Genetics Institute's IVF program at Illinois Masonic Medical Center. PATIENTS: One hundred ninety-three couples of advanced maternal age (34 to 46 years) under-going IVF treatment volunteered to be part of a clinical trial on preimplantation polar body diagnosis of common aneuploidies. INTERVENTIONS: Using micromanipulation procedures, the first and second polar bodies were removed after their extrusion from the oocytes. MAIN OUTCOME MEASURE: Fluorescent in situ hybridization signals specific for chromosomes X, 18, and 13/21. RESULTS: In 235 IVF cycles performed in 193 couples, 1,293 oocytes were biopsied and subjected to fluorescent in situ hybridization analysis, with fluorescent in situ hybridization results available in 993 oocytes (76.8%). Of 993 oocytes with fluorescent in situ hybridization results, 665 (67%) were predicted to be normal based on the chromosomes studied; 460 embryos resulting from these oocytes were transferred in 187 treatment cycles, resulting in 12 births of healthy children and 18 ongoing pregnancies after confirmation of the polar body diagnosis by chorionic villus sampling or amniocentesis. CONCLUSION: Polar body fluorescent in situ hybridization analysis may be used for preimplantation diagnosis of common aneuploidies in IVF patients of advanced maternal age.

Birth of a healthy girl after preimplantation gender determination using a combination of polymerase chain reaction and fluorescent in situ hybridization analysis. Preimplantation Genetics Group.

OBJECTIVE: To perform preimplantation gender determination by a combination of polymerase chain reaction (PCR) sexing and fluorescent in situ hybridization technique using the directly labeled fluorescent alpha-satellite centromeric DNA probes for X and Y chromosomes. SETTING: The IVF program of Illinois Masonic Medical Center. PATIENTS: A couple requested preimplantation diagnosis because the mother is a carrier for hemophilia A. RESULTS: Two blastomeres were aspirated from each of the four- to eight-cell embryos, and only the embryos with both fluorescent in situ hybridization and PCR results indicating female sex chromosomal complement were transferred, resulting in a singleton pregnancy and delivery of a healthy female infant, after prenatal confirmation of the diagnosis as female. The male embryos or embryos diagnosed as females only by PCR were followed up by confirmatory fluorescent in situ hybridization analysis demonstrating a discrepancy of PCR and fluorescent in situ hybridization results in four embryos, presumably because of a possible sperm contamination of the PCR reaction or chromosomal mosaicism. CONCLUSION: The analysis of two blastomeres from the same embryo by a combination of PCR sexing and fluorescent in situ hybridization increases the reliability of preimplantation gender identification at the cleavage stage.

Prepregnancy testing for single-gene disorders by polar body analysis.

Preventive measures for single-gene disorders are currently based on carrier screening in pregnancy and prenatal diagnosis. Although this has been extremely effective for preventing new cases of common inherited conditions, the major limitation is still termination of 25% of wanted pregnancies following detection of affected fetuses. To overcome this important problem, we developed a method for prepregnancy genetic testing that involves DNA analysis of the first and second polar bodies, which are extruded during maturation and fertilization of oocytes. We offered this option to 28 couples at risk for having children with single-gene disorders. Fifty clinical cycles were performed from these patients for the following conditions: 20 for cystic fibrosis, 18 for thalassemia, 6 for sickle cell disease, 2 each for Gaucher disease and LCHAD (long-chain 3-hydroxyacyl-COA dehydrogenase deficiency), and 1 each for hemophilia B and phenylketonuria. Oocytes obtained from these patients using in vitro fertilization procedures (IVF) were tested by a sequential multiplex nested PCR analysis of the first and second polar body to detect the gene involved simultaneously with linked polymorphic markers. A total of 191 of 399 oocytes with predicted genotype were mutation free and preselected for fertilization and transfer. In all but three cycles, one to three unaffected embryos with predicted unaffected genotypes were transferred, resulting in 20 pregnancies, from which 19 healthy children have been born. The follow-up analysis of embryos resulting from oocytes with predicted affected genotype, confirmed the diagnosis in 97% of cases, demonstrating the reliability of prepregnancy diagnosis of single-gene defects by polar body analysis.

Prepregnancy genetic testing for age-related aneuploidies by polar body analysis.

Current practice for prevention of chromosomal aneuploidies involves prenatal screening and termination of pregnancy, a procedure that is not universally acceptable. We introduced prepregnancy genetic testing by sampling and fluorescence in situ hybridization (FISH) analysis of the first and second polar body (PB), to avoid fertilization and transfer of embryos resulting from aneuploid oocytes. In 395 in vitro fertilization (IVF) patients of advanced maternal age, the first and second PBs were removed following their extrusion from oocytes and studied by FISH, using probes specific for chromosomes 13, 18, and 21, to detect and avoid the transfer of oocytes with common aneuploidies. Overall, 3,651 oocytes obtained from 598 IVF cycles were available for FISH analysis, with 2,952 showing interpretable FISH results (80.9%). The analysis revealed 1,271 (43.1%) oocytes with aneuploidy, which were excluded from transfer and subjected to follow-up FISH analysis to confirm PB diagnosis in the cleavage or blastocyst stage embryos. Only embryos originating from 1,681 aneuploidy-free oocytes were transferred back to patients, resulting in 119 pregnancies overall, from which 78 healthy children have already been born, 35 were spontaneously aborted, and 16 are ongoing, after confirming PB diagnosis by prenatal diagnosis. The results demonstrate that PB-based preimplantation diagnosis may be used for prepregnancy screening in women with age-related risk for common aneuploidies.

Decreasing risk of pregnancy loss following chorionic villus sampling. Elimination of transabdominal chorionic villus sampling during the ninth week of pregnancy.

Chorionic villus sampling (CVS) is a method of obtaining fetal cells in the first trimester of pregnancy for genetic analysis. The transcervical (TC) approach was the first technique to be widely used. In the National Institute of Child Health and Human Development collaborative study the absolute loss rate following CVS (the total number of spontaneous abortions and neonatal deaths following CVS) was 4%. More recently the transabdominal (TA) approach has been introduced. This study compares the loss rates for the two approaches at various gestational ages for three 6-month periods following the addition of the TA approach with each other and with the loss rates prior to the introduction of TA CVS. We found that the percentage of pregnancy losses following TA CVS during the ninth week of gestation (63-69 days) was consistently higher than for TC CVS performed at the same gestational age. The loss rate for TC CVS has steadily decreased since the introduction of TA CVS after remaining the same for the two years prior to the introduction of the TA approach. After minimizing the number of TA CVS performed during the ninth week of gestation, the overall loss rate during the most recent 6-month period has been reduced to 0.94%. We conclude that the lowest loss rate following CVS can be obtained if both the TA and TC methods are available, and that the number of TA procedures performed during the ninth week of gestation is minimized.

Preimplantation diagnosis for aneuploidies in assisted reproduction.

At least one half of oocytes and preimplantation embryos are aneuploid and have to be avoided from transfer in in vitro fertilization (IVF) patients of advanced reproductive age. This can now be done by preimplantation genetic diagnosis, which has recently become an integral part of assisted reproduction technologies and was shown to improve implantation rate and reduce spontaneous abortions after implantation. The experience of approximately 5000 preimplantation genetic diagnosis (PGD) cycles performed for poor prognosis IVF patients have already resulted in birth of approximately 1000 apparently healthy children, suggesting that this novel technique is safe and reliable, and may in future replace the current IVF practice of preselection of embryos for transfer based on morphological parameters.

Preimplantation diagnosis of single disorders.

Preimplantation diagnosis of inherited and chromosomal diseases provides an option for couples at risk for conceiving genetically abnormal fetus to avoid a birth of an affected child without the need for a prenatal diagnosis and selective abortion of affected fetus (1-3). In some countries this might be the only way to the prevention of genetic disease, as abortion is not acceptable procedure. Even in those countries where prenatal diagnosis is practiced for many years, there is also concern that existing genetic programs based on prenatal screening will lead to increasing number of abortions. On the other hand, the possibilities for genotyping oocytes and cleaving embryos open a new prospect for genetic diagnosis before pregnancy, making genetic programs more ethically acceptable in any social setting. This will make preimplantation diagnosis the method of choice in the community based programs for prevention of genetic disease in the future, as well as a useful addition to assisted reproduction technologies, at least for IVF patients of advanced maternal age.

Preimplantation genetic diagnosis for Pelizaeus-Merzbacher disease with testing for age-related aneuploidies.

Pelizaeus-Merzbacher disease (PMD) is an X-linked recessive demyelinating disorder of the central nervous system, caused by mutations of the proteolipid protein 1 gene (PLP1 gene). As no specific therapy is available for PMD, preimplantation genetic diagnosis (PGD) may be a useful option for couples carrying this mutation. PGD was performed for a couple who had had one child with the L86P mutation in exon 3 of the PLP1 gene. Because of advanced maternal age, PGD for this single-gene disorder was performed together with testing for chromosomal abnormalities. Polar bodies and blastomeres were tested for the presence of maternal mutation and closely linked markers DXS8020 and PLP5' (CA)n. The same blastomeres were also tested for the copy number of chromosomes 13, 16, 18, 21, 22, X and Y, and five chromosomally abnormal embryos were identified. A total of three embryos predicted to be unaffected and free of chromosomal disorder were transferred back to the patient, resulting in a twin pregnancy and the birth of two healthy female infants confirmed to be free of PMD, representing the first PGD for PMD combined with aneuploidy testing.

Repository of human embryonic stem cell lines and development of individual specific lines using stembrid technology.

A human embryonic stem cell (HESC) line repository has been established, containing HESC lines with normal and abnormal genotypes, providing the source for studying the primary mechanisms of genetic disorders at the cellular level. Because the outcome of HESC transplantation treatment depends on access to human leukocyte antigen identical stem cells, the development of individual specific HESC was initiated, using the original stembrid technology, which is based on the hybridization of adult somatic cells with cytoplast of HESC lines. The data presented here demonstrate feasibility of this approach in the future development of HESC transplantation treatment of genetic and acquired disorders. The established HESC repository presently contains 166 HESC lines, including 127 with normal genotype and 39 with genetic and chromosomal disorders.

Human embryonic stem cell lines with genetic disorders.

A previous study described the establishment of human embryonic stem cell (ESC) lines from different sources of embryonic material, including morula, whole blastocyst and isolated inner cell mass. Using these methods, a repository of ESC lines has been established with different genetic abnormalities, which provides an unlimited source of disease cells in culture for undertaking research on the primary disturbances of the cellular processes in the genetically abnormal cells. ESC lines with genetic disorders were derived from the mutant embryos detected and avoided from transfer in the ongoing practice of preimplantation genetic diagnosis (PGD). The current repository contains 18 ESC lines with genetic disorders, including adrenoleukodystrophy, Duchenne and Becker muscular dystrophy, Fanconi anaemia, complementation group A, fragile-X syndrome, Huntington disease (three lines), Marfan syndrome, myotonic dystrophy (two lines), neurofibromatosis type I (five lines) and thalassaemia (two lines). These ESC lines are presently used for research purposes and may be available on request.