Conversion and non-conversion approach to preimplantation diagnosis for chromosomal rearrangements in 475 cycles.

Due to the limitations of preimplantation genetic diagnosis (PGD) for chromosomal rearrangements by interphase fluorescent in-situ hybridization (FISH) analysis, a method for obtaining chromosomes from single blastomeres was introduced by their fusion with enucleated or intact mouse zygotes, followed by FISH analysis of the resulting heterokaryons. Although this allowed a significant improvement in the accuracy of testing of both maternally and paternally derived translocations, it is still labour intensive and requires the availability of fertilized mouse oocytes, also creating ethical issues related to the formation of interspecies heterokaryons. This method was modified with a chemical conversion procedure that has now been clinically applied for the first time on 877 embryos from PGD cycles for chromosomal rearrangements and has become the method of choice for performing PGD for structural rearrangements. This is presented within the context of overall experience of 475 PGD cycles for translocations with pre-selection and transfer of balanced or normal embryos in 342 (72%) of these cycles, which resulted in 131 clinical pregnancies (38%), with healthy deliveries of 113 unaffected children. The spontaneous abortion rate in these cycles was as low as 17%, which confirms an almost five-fold reduction of spontaneous abortion rate following PGD for chromosomal rearrangements.

Oocyte-like structures arising from cells of follicular fluid are not captured in aspirates.

Recently, cells from ovarian surface epithelium (OSE) of post-menopausal women and women with premature ovarian failure were investigated and oocyte-like cells with diameters up to 95 microm were found to arise after a certain time in culture. In addition, it seems that a mixed population of germ cells and germline stem cells exists in non-follicle ovarian structures. Relating to an earlier publication, where it was shown that pre-antral follicles with immature oocytes could be captured in follicular fluid (FF) aspirates due to the incorporated tissue in the puncture needle, it was reasoned that OSE or otherwise germline stem cells, possibly captured equally through ovarian puncture, might give rise to oocyte-like cells. The aim of this study was therefore to try to derive such oocyte-like cells from FF aspirates of patients undergoing IVF after culture. Additionally, FF-derived cells were aggregated with human embryonic stem cells to see if an embryonic environment had the ability to enable cells from the FF aspirate to acquire an oocyte-like morphology. Investigations could not confirm the development of oocyte-like cells from cells of FF aspirates.

Non-informative results and monosomies in PGD: the importance of a third round of re-hybridization.

The incidence of non-informative results after fluorescence in-situ hybridization (FISH) was analysed in preimplantation genetic diagnosis (PGD). FISH was performed on seven chromosomes (13, 16, 18, 21, 22, X, and Y) in two rounds of hybridization (one biopsied blastomere per day 3 embryo). A third round with telomeric probes was performed in order to analyse the chromosome(s) in question. A total of 702 embryos out of a total of 719 embryos from 95 cycles were analysed. The remaining 17 embryos were anucleated and/or had poor quality and could not be diagnosed. After FISH analysis, 52.7% of blastomeres were found to be abnormal, 27.1% euploid, and 20.2% had non-informative results. Abnormalities considered as non-informative included 'monosomy in question' (46.5%), 'trisomy in question' (40.2%), compound aneuploidy (8.5%), and 'no result' (4.9%) for a tested chromosome. Following re-hybridization with telomeric probes, euploidy was found in 42.4% of 'monosomies in question,' in 82.4% of 'trisomies in question,' in 16.7% of compound aneuploidies, and in 71.4% of 'no results' for a tested chromosome. Only 4.2% of non-informative results could not be rescued. This study clearly demonstrates the importance of re-hybridizing non-informative results and monosomies using a third round of hybridization with telomeric probes for chromosome(s) in question.

Impact of meiotic and mitotic non-disjunction on generation of human embryonic stem cell lines.

At least 50-60% of oocytes derived from IVF procedures are chromosomally abnormal due to meiotic I or II errors. Through the use of polar body and blastomere diagnosis, euploid embryos suitable for transfer can be identified. Those embryos that are aneuploid are usually discarded, or otherwise can be used to generate chromosomally abnormal human embryonic stem cell (hESC) lines. The authors' centre has one of the largest repositories of hESC lines with genetic and chromosomal disorders generated from preimplantation genetic diagnosis (PGD) abnormal embryos. The results, studying hESC lines derived from PGD abnormal zygotes, imply that aneuploidies resulting from meiotic non-disjunction have a greater impact on viability of cells of the human embryos than those originating from post-zygotic mitotic non-disjunction.

Embryonic stem cells from morula.

It has been shown that it is possible to establish human embryonic stem cell (hESC) lines from morula. Details of the aforementioned injection method of morula under blastocyst are described in this chapter. This chapter also discloses the application of simultaneous staining for two markers, TRA-2-39 and Oct-4, for characteristics of nondifferentiated hESC derived from morula and gives a method. Technical approaches of freezing morula-derived hESC are discussed.

Cytoplasmic cell fusion: Stembrid technology for reprogramming pluripotentiality.

Recent progress in somatic cell nuclear transfer (SCNT) provides the evidence for the presence of reprogramming factors in human embryonic stem cells (hESC). Hybrid hESC with donor human somatic nuclei have been established, but the resulting hybrid hESC contained DNA originating from both hESC and donor somatic cells. There is still no method to completely replace the hESC nuclei by the nuclei of somatic cells and to obtain the pure colonies of hESC with donor genotype. We present here the original technology, which is based on enucleation of h ESC and their fusion with the adult somatic cells, resulting in the establishment of individual-specific hESC with the genotype of the donor somatic cells. The resulting constructs was demonstrated to have the "stemness" of hESC and the genotype of the donor somatic cells. So this "Stembrid technology," may be used for the construction of patient-specific hESC.

Preimplantation genetics: Improving access to stem cell therapy.

There has been progress in the application of stem cell transplantation for treatment of an increasing number of severe congenital and acquired bone marrow disorders, currently restricted by the availability of human leukocyte antigen (HLA)-matched related donors. Preimplantation HLA typing has recently been introduced to improve the access to stem cell therapy for inherited bone marrow failures. Preimplantation genetic diagnosis (PGD) provides an option not only for avoiding an affected pregnancy with thalassemia and other inherited disorders but also for preselection of the HLA-compatible donors for affected siblings. Multiple short tandem repeat markers throughout the HLA region are applied for this purpose, allowing 100% accuracy of HLA typing, through picking up possible recombination in the HLA region, as well as the copy number of chromosome 6, which affect accuracy of preimplantation HLA typing. Present experience of preimplantation HLA typing includes preimplantation HLA typing in 180 cycles, 122 of which were done as part of PGD for Fanconi anemia, thalassemia, Wiscott-Aldrich syndrome, hyper-immunoglobulin M syndrome, hypohidrotic ectodermal dysplasia with immune deficiency, and X-linked adrenoleukodystrophy, and 58 for the sole purpose of HLA typing for leukemias and for aplastic and Diamond-Blackfan anemia. The applied method resulted in the accurate preselection and transfer of 100% HLA-matched embryos, yielding already three dozen clinical pregnancies and the birth of two dozen HLA-matched children to the siblings requiring stem cell transplantation. Successful therapy with HLA-matched stem cells, obtained from these PGD children, has been achieved already for Diamond-Blackfan anemia hypohidrotic ectodermal dysplasia with immune deficiency and thalassemia.

Preimplantation diagnosis for diseases with genetic predisposition and nondisease testing.

Preimplantation genetic diagnosis has become a method of choice in genetic practices. The present experience includes thousands of clinical cases of preimplantation genetic diagnosis, demonstrating the safety, accuracy and reliability of the technique and its clinical relevance for those at-risk couples who cannot accept traditional methods for prenatal diagnosis and termination of pregnancy. Most recently, preimplantation genetic diagnosis has been performed for late-onset disorders, such as genetic predisposition to cancers, Alzhelmer's disease and nondisease testing involving human leukocyte antigen typing, never practiced in prenatal diagnosis. The review of these developments suggests the clinical usefulness of this new approach for avoiding the birth of children with genetic predisposition to cancers and other late-onset disorders, or preimplanation human leukocyte antigen matching to provide the treatment of the affected siblings requiring bone marrow transplantation.

Preimplantation diagnosis for long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency.

Long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency is a fatal autosomal recessive metabolic disorder, presenting during infancy. Preimplantation genetic diagnosis (PGD) provides an option for establishing an unaffected pregnancy, avoiding the risk for termination of pregnancy following prenatal diagnosis. The method for pre-selection of mutation-free oocytes for LCHAD deficiency was developed by testing the first and second polar body removed from oocytes by micromanipulation techniques in the framework of in-vitro fertilization. To avoid misdiagnosis, testing was done using hemi-nested polymerase chain reaction (PCR), with outer primers designed to lie outside the pseudogene, eliminating false priming. Four of 12 tested oocytes were predicted to be unaffected, based on the heterozygous first and mutant second polar body. The embryos resulting from these mutation-free oocytes were replaced, yielding a singleton clinical pregnancy and birth of a healthy child following confirmation by prenatal diagnosis.

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.

Preimplantation genetic diagnosis for the Kell genotype.

OBJECTIVE: To use preimplantation genetic diagnosis (PGD) to achieve a Kell 1 (K1) allele-free pregnancy in couples at risk for producing a child with hemolytic disease of the newborn (HDN) caused by maternofetal incompatibility in sensitized mothers. DESIGN: DNA analysis of biopsied blastomeres from cleavage-stage embryos in IVF-ET with the goal of identifying and transferring back to patients the K1 allele-free embryos. SETTING: IVF program at the Reproductive Genetics Institute, Chicago, Illinois, and IVF Michigan, Rochester Hills, Michigan. PATIENT(S): Two at-risk couples with a history of neonatal death caused by HDN due to K1/K2 genotype in a male partner. INTERVENTION(S): Biopsy of single blastomeres and testing for paternal K1 allele in each embryo after standard IVF. MAIN OUTCOME MEASURE(S): DNA analysis of blastomeres indicating whether corresponding embryos were K1 allele-free for the purpose of transferring only embryos without the K1 allele. RESULT(S): Of 36 embryos tested in five cycles from two couples, 18 were predicted to be K1 allele-free. Of these, 9 were transferred, resulting in a K1 allele-free twin pregnancy and the birth of two healthy children. CONCLUSION(S): PGD of the K1 genotype resulted in the birth of healthy twins confirmed to be free of the K1 allele. PGD in couples with a heterozygous K1/K2 male partner provides an option for avoiding HDN in sensitized mothers.

Over a decade of experience with preimplantation genetic diagnosis: a multicenter report.

OBJECTIVE: To review a 12-year experience of the world's three largest preimplantation genetic diagnosis (PGD) centers. DESIGN: Multicenter analysis of the clinical outcome of PGD. SETTING: In vitro fertilization programs at the Reproductive Genetics Institute, Chicago, Illinois; Saint Barnabas Medical Center, West Orange, New Jersey; and SISMER, Bologna, Italy. PATIENT(S): Poor-prognosis IVF patients, patients carrying balanced chromosomal translocations, and couples at risk for producing children with Mendelian disorders. INTERVENTION(S): In vitro fertilization, intracytoplasmic sperm injection, polar body removal, blastomere biopsy, and ET. MAIN OUTCOME MEASURE(S): DNA or chromosomal analysis of biopsied polar bodies or blastomeres, implantation and clinical pregnancy rates, and live-born pregnancy outcome. RESULT(S): A total of 754 babies have been born as a result of 4,748 PGD attempts, which shows the expanded application and the practical relevance of PGD for single-gene disorders, chromosomal aneuploidies and translocations, late-onset diseases with genetic predisposition, and nondisease testing in couples at need for human leukocyte antigens-matched offspring for treatment of affected siblings. CONCLUSION(S): Preimplantation genetic diagnosis is evolving to become a clinical option for couples at risk for producing offspring with Mendelian diseases, has a positive numerical impact in standard assisted reproduction practices through aneuploidy testing, and reduces by at least fourfold the spontaneous abortion rate in couples carrying translocations.

Over a decade of experience with preimplantation genetic diagnosis.

The three respondents provide additional support for preimplantation genetic diagnosis (PGD) having the pivotal place it now has in prenatal genetic diagnosis: chromosomal abnormalities (e.g., unbalanced translocations), Mendelian disorders, and HLA typing for transfer of compatible, genetically normal, embryos. Transferring euploid embryos has decreased the clinical abortion rate and increased the implantation rate in assisted reproductive technologies (ART), but it has not necessarily improved the live-birth rate. Safer embryo biopsy, more extensive diagnostic efforts (i.e., microarray analysis), and more refined patient selection may be required before shifting from preselection of embryos based solely on morphological parameters to transfer of only aneuploidy-free embryos.

Preimplantation genetic diagnosis for polycystic kidney disease.

OBJECTIVE: To use preimplantation genetic diagnosis for achieving a polycystic kidney disease (PKD)-free pregnancy for a couple in which the female partner was affected by PKD but whose PKD1 or PKD2 carrier status was not established. DESIGN: Case report. SETTING: The IVF program of Reproductive Genetics Institute, Chicago, Illinois. PATIENT(S): An at-risk couple with the female partner affected by PKD, whose PKD1 or PKD2 carrier status was not established. INTERVENTION(S): Removal of PB1 and PB2 and testing for three closely linked markers to PKD1 (Kg8, D16S664, and SM7) and four closely linked markers to PKD2 (D4S2922, D4S2458, D4S423, and D4S1557) after standard IVF. MAIN OUTCOME MEASURE(S): Deoxyribonucleic acid analysis of PB1 and PB2 indicating whether corresponding oocytes were PKD1 or PKD2 allele free, for the purpose of transferring only embryos resulting from mutation-free oocytes. RESULT(S): Of 11 oocytes tested by PB1 and PB2 DNA analysis, 7 were predicted to contain PKD1 or PKD2, with the remaining 4 free of both mutations. Three embryos resulting from these oocytes were transferred, yielding a twin pregnancy and the birth of two unaffected children. CONCLUSION(S): This is the first preimplantation genetic diagnosis for PKD, which resulted in the birth of healthy twins confirmed to be free of PKD1 and PKD2. Preimplantation genetic diagnosis based on linked marker analysis provides an alternative for avoiding the pregnancy and birth of children with PKD, even in at-risk couples without exact PKD1 or PKD2 carrier information.

Preimplantation HLA testing.

CONTEXT: Preimplantation genetic diagnosis (PGD) has become an option for couples for whom termination of an affected pregnancy identified by traditional prenatal diagnosis is unacceptable and is applicable to indications beyond those of prenatal diagnosis, such as HLA matching to affected siblings to provide stem cell transplantation. OBJECTIVE: To describe preimplantation HLA typing, not involving identification of a causative gene, for couples who had children with bone marrow disorders at need for HLA-matched stem cell transplantation. DESIGN, SETTING, AND PARTICIPANTS: HLA matching procedures conducted at a single site during 2002-2003 in an in vitro fertilization program for 9 couples with children affected by acute lymphoid leukemia, acute myeloid leukemia, or Diamond-Blackfan anemia requiring HLA-matched stem cell transplantation. In 13 clinical cycles, DNA in single blastomeres removed from 8-cell embryos following in vitro fertilization was analyzed for HLA genes simultaneously with analysis for short tandem repeats in the HLA region to select and transfer only those embryos that were HLA matched to affected siblings. MAIN OUTCOME MEASURES: Results of HLA matching and pregnancy outcome. RESULTS: As a result of testing a total of 199 embryos, 45 (23%) HLA-matched embryos were selected, of which 28 were transferred in 12 clinical cycles, resulting in 5 singleton pregnancies and birth of 5 HLA-matched healthy children. CONCLUSION: This is the first known experience of preimplantation HLA typing performed without PGD for a causative gene, providing couples with a realistic option of having HLA-matched offspring to serve as potential donors of stem cells for their affected siblings.

Correlation between preimplantation genetic diagnosis for chromosomal aneuploidies and the efficiency of establishing human ES cell lines.

There are several sources from which human embryonic stem cell (hESC) lines can be generated: surplus embryos after in vitro fertilization procedures, one- and three-pronuclear zygotes, early arrested or highly fragmented embryos that have reached the blastocyst stage, or otherwise chromosomally or genetically abnormal embryos after preimplantation genetic diagnosis (PGD). We report on the efficiency of establishing hESC lines from blastocysts with proven meiotic or mitotic errors after sequential testing of both polar bodies and blastomere analysis on day 3. The success rate of establishing hESC lines originating from blastocysts carrying a meiotic error was as low as 2.4% and differed significantly from the success rate of establishing hESC lines originating from blastocysts with balanced meiotic errors (21.6%) or mitotic errors (after sequential testing (9.1%) and after blastomere testing alone (12.2%)). This suggests that it may be reasonable to apply sequential PGD prior to the initiation of hESC culture. Information about the karyotype may in the future help refine the methods and possibly improve the efficiency by which hESC lines are derived from embryos with prezygotic abnormalities. Additionally, it may in general prove very difficult to obtain abnormal hESC lines for scientific study from aneuploid PGD embryos, which will limit our ability to study the biological consequences of chromosomal abnormalities. Furthermore, the success rates for generating aneuploid cell lines originating from fertilized oocytes carrying a prezygotic nondisjunction error seem to mirror the miscarriage rates during pregnancy of embryos carrying such errors.

Impact of preimplantation genetic diagnosis for chromosomal disorders on reproductive outcome.

Despite recent controversy, existing experience suggests that aneuploidy testing has had a significant impact on the reproductive outcome of poor prognosis IVF patients, which is based on the experience of over 20,000 cases of PGD for chromosomal disorders. The clinical impact was demonstrated in the improved implantation and pregnancy rates, reduction of spontaneous abortions and improved take-home baby rate in patients of advanced reproductive age, those with repeated IVF failures and recurrent spontaneous abortions. The lack of positive effect of aneuploidy testing in a few smaller series may be due to potential detrimental effect of two blastomere removal, reducing the implantation potential of the biopsied embryos, or exclusion from testing a few key chromosomes and poor outcome of aneuploidy testing, affecting the appropriate pre-selection of embryos for transfer. Despite the need for randomized controlled studies to quantify in more detail the clinical impact of the pre-selection of aneuploidy-free zygotes, the positive impact of PGD is particularly obvious from the comparison of reproductive outcome in the same patients with and without PGD, revealing the actual benefits of PGD.