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.

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.

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.

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.

Preimplantation testing for chromosomal disorders improves reproductive outcome of poor-prognosis patients.

The clinical impact of PGD was evaluated through the analysis of the reproductive outcome before and after PGD in the same group of poor prognosis IVF patients, undergoing PGD for chromosomal abnormalities. Based on a series of 2359 PGD cycles, resulting in the establishment of 498 chromosomal abnormality-free clinical pregnancies, the reproductive history prior to PGD was analysed. Of 483 previous pregnancies analysed in patients with 432 pregnancies generated after PGD for aneuploidies, 328 (68%) ended in spontaneous abortions, in contrast to 28.4% after PGD, with only 155 (32%) resulting in deliveries, compared with 71.9% take-home baby rates after PGD. The patients experienced 315 previous IVF attempts, resulting in the transfer of 706 embryos in 308 cycles, of which only 49 (6.9%) implanted, compared with a 34.9% implantation rate observed in the same patients after PGD. Similar analysis of the previous reproductive outcomes of 45 carriers of balanced translocations achieving pregnancies following PGD, showed even stronger clinical impact, with a reduction of spontaneous abortions from 87.8% to 17.8%, and improvement of take-home baby rate from 11.5% to 81.4% after PGD. The results demonstrate a strong clinical impact of PGD, resulting in improvement of implantation rate, reduction of spontaneous abortions and increase in the take-home baby rate.

Preimplantation diagnosis and HLA typing for haemoglobin disorders.

Haemoglobin disorders are among the most frequent indications for preimplantation genetic diagnosis (PGD), introduced as an important option to couples at risk for producing offspring with thalassaemia and sickle cell disease. Previous experience mainly included PGD for beta-thalassaemia, while PGD for alpha-thalassaemia resulting in an unaffected pregnancy has not been reported. This study presents the results of the world's largest experience of 197 PGD cycles for haemoglobin disorders, which includes PGD for alpha-thalassaemia, resulting in 53 clinical pregnancies and birth of 45 healthy children, with five still ongoing. Fifty-four of these cycles were performed in combination with HLA typing, allowing the birth of thalassaemia-free children who were also HLA identical to the affected sibling, with successful stem cell transplantation in one case. As an increasing proportion of patients requesting PGD with HLA typing are of advanced reproductive age, aneuploidy testing was performed simultaneously with PGD. The results show that PGD has now become a practical approach for prevention of haemoglobin disorders, and is gradually being used also for improving access to HLA compatible stem cell transplantation for this group of diseases.

Cytogenetic analysis of human somatic cell haploidization.

Despite recent interest in the derivation of female and male gametes through somatic cell nuclear transfer, there is still insufficient data on chromosomal analysis of these gametes resulting from haploidization, especially involving a human nuclear donor and recipient oocytes. The objective of this study was to investigate the fidelity of chromosomal separation during haploidization of human cumulus cells by in-vitro matured human enucleated MII oocytes. A total of 129 oocytes were tested 4-7, 8-14, or 15-21 h after nuclear transfer (NT) followed by electro-stimulation, resulting in 71.3% activation efficiency on average. Haploidization was documented by the formation of two separate groups of chromosomes, originating from either polar body/pronucleus (PB/PN), or only 2PN, which were tested by 5-colour FISH, or DNA analysis for copy number of chromosomes 13, 16, 18, 21, 22 and X. Two PN were formed more frequently than PB/PN, irrespective of incubation time. In agreement with recent reports on mouse oocytes, as many as 90.2% of the resulting haploid sets tested showed abnormal chromosome segregation, suggesting unsuitability of the resulting artificial gametes for practical application at the present time.

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.

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 genetic diagnosis for early-onset torsion dystonia.

Early-onset primary torsion dystonia (DYT1) is the most severe and common form of hereditary movement disorders, characterized by sustained twisting contractures that begin in childhood, which is caused in majority of cases by a 3-bp deletion of the DYT1 gene on chromosome 9q34 at the heterozygote state. As there is no effective treatment of this disease, preimplantation genetic diagnosis (PGD) may be a useful option for at-risk couples to establish an DYT1 mutation-free pregnancy. PGD was performed for two obligate carriers of the DYT1 3-bp deletion, using blastomere testing to preselect the mutation-free embryos, based on mutation analysis with simultaneous testing of the three closely linked markers, D9S62, D9S63 and ASS. Of 19 tested blastomeres in three cycles, 17 had conclusive information about the mutation and linked markers, of which eight were predicted to be free of 3-bp deletion. Six of these embryos were transferred back to patients, two in each cycle, yielding singleton DYT1 3-bp deletion-free clinical pregnancies in two. One of these pregnancies was terminated due to severe anencephaly and the other resulted in birth of a mutation-free child. This is the first PGD for primary torsion dystonia, providing an alternative for those at-risk couples who cannot accept prenatal diagnosis and termination of pregnancy as an option for avoiding early onset torsion dystonia.

Preimplantation genetic diagnosis for familial dysautonomia.

Familial dysautonomia (FD) is the most common congenital sensory neuropathy in Ashkenazi Jews, caused by a single major mutation in the IKBKAP gene. Effective management for this severe debilitating disease is still not available, making preimplantation genetic diagnosis (PGD) a useful option for at-risk couples to establish an FD free pregnancy from the outset. PGD was performed for a couple with a previous affected child with FD, using first and second polar body testing to preselect mutation-free oocytes, based on mutation analysis with simultaneous testing of two closely linked markers, D9S58 and D9S1677. Of 15 tested oocytes, 11 carried information about both polar bodies' genotype, of which seven were predicted to be free of the FD gene. Three embryos resulting from these oocytes were transferred back to the patient, resulting in a triplet pregnancy and the birth of three unaffected children confirmed to be free of FD. This is the first PGD for FD, providing an alternative for those at-risk couples who cannot accept prenatal diagnosis and termination of pregnancy as an option for avoiding FD.

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.

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.

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.

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.

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.

Preimplantation diagnosis for Fanconi anemia combined with HLA matching.

CONTEXT: The advent of single-cell polymerase chain reaction (PCR) has presented the opportunity for combined preimplantation genetic diagnosis (PGD) and HLA antigen testing. This is a novel and useful way to preselect a potential donor for an affected sibling requiring stem cell transplantation. OBJECTIVE: To perform in vitro fertilization (IVF) and preimplantation HLA matching combined with PGD for Fanconi anemia (FA). DESIGN: DNA analysis for the IVS 4 + 4 A-->T (adenine to thymine) mutation in the FA complement C (FANCC) gene in single blastomeres, obtained by biopsy of embryos, to identify genetic status and HLA markers of each embryo before intrauterine transfer. SETTING: In vitro fertilization programs at large medical centers in Chicago, Ill, and Denver, Colo. PARTICIPANTS: A couple, both carriers of the IVS 4 + 4 A-->T mutation in the FANCC gene with an affected child requiring an HLA-compatible donor for cord blood transplantation. MAIN OUTCOME MEASURES: DNA analysis of single blastomeres to preselect unaffected embryos representing an HLA match for the affected sibling. RESULTS: Of 30 embryos tested in 4 IVF attempts, 6 were homozygous affected and 24 were unaffected. Five of these embryos were also found to be HLA-compatible, of which 2 were transferred in the first and 1 in each of the other 3 cycles, resulting in a pregnancy and birth of an unaffected child in the last cycle. CONCLUSION: To our knowledge, this is the first PGD with HLA matching, demonstrating feasibility of preselecting unaffected embryos that can also be an HLA-compatible source for stem cell transplantation for a sibling.

Crown-rump lengths in missed miscarriages and trisomy 21.

OBJECTIVES: To compare crown-rump lengths with karyotypes of missed miscarried fetuses and to determine a relationship between crown-rump length and trisomy 21. STUDY DESIGN: Chorionic villus sampling was performed on 129 consecutive missed miscarriages between 10 and 12 weeks by last menstrual period in patients >or= 35 years of age. Crown-rump length was correlated with the karyotype. Statistical analysis was performed using Student's t-test. RESULTS: Twenty-one of 129 missed miscarriages involved fetuses affected by trisomy 21. The crown-rump length was < 22 mm in 77% of missed miscarriages. Using a crown-rump length of >or= 22 mm for the prediction of trisomy 21 had a sensitivity of 86%, specificity of 89%, positive predictive value of 60% and negative predictive value of 97%. At 10-12 weeks, the crown-rump lengths of missed miscarried fetuses with trisomy 21 was significantly larger (P or= 35 years of age, with a missed miscarriage, in whom pregnancies reached >or= 10 weeks from the last menstrual period, a fetal crown-rump length of >or= 22 mm has a high probability that the etiology of the loss will be secondary to trisomy 21.

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.