- Email: [email protected]
S5 What about mitochondrial biopsy?
S32
11th International Conference on Preimplantation Genetic Diagnosis
S3 Polar body biopsy and array CGH for aneuploidy in advanced maternal age M. Montag1 . 1 Dept. Gyn. Endocrinol. & Fertil. Disorders, University Clinics Heidelberg, Heidelberg, Germany Aneuploidy testing has started in the 1990s and still to date it is a topic which is controversial and can provoke vivid discussions especially if it comes to the question if such an approach is beneficial in assisted reproduction. Among the different techniques which are applied in routine since then, biopsy of polar bodies at the oocyte/zygote stage and biopsy of trophectoderm cells at the blastocyst stage are those which are at present in the focus of interest. Both techniques seem to have the highest potential if combined with a diagnostic approach which allows investigating all chromosomes. One such approach is comparative genomic hybridization (CGH). Over the last decade CGH has made substantial progression from the conventional chromosome-based hybridisation template to the first CGH-arrays and to the latest single channel array format of today. If the focus is on maternal aneuploidies which have manifested during the meiotic progression of the developing oocyte, polar body biopsy and array-CGH is an option for investigating chromosomal aberrations without touching the embryo and this strategy clearly avoids the problem of embryo mosaicism. For some patients this approach is ethically better acceptable. Retrieval of polar bodies can be done either sequential or simultaneously, provided that the timing is adapted to the course of meiosis (for biopsy) and to the integrity of the chromosomal content of the polar bodies (for amplification). Several publications have reported on the use of this technology for the investigation of structural as well as numerical chromosome aberrations. In patients with advanced maternal age the results of these studies show that at an average maternal age of 40 the mean rate of aneuploidy is 70%, meaning that out of 10 oocytes 3 are chromosomally intact and suitable for transfer. As there is a decline in ovarian reserve in women with advanced age, the rate of cancelled transfers due to the presence of only aneuploid oocytes is very high among these women. To date there is no systematic study available which clearly states to what extent polar body biopsy and array-CGH benefits assisted reproduction treatment. The main question is if aneuploidy testing in advanced maternal age leads to lower abortion rates and/or if it does support higher take home baby rates. Following a pilot study on polar body biopsy and arrayCGH, a multicentre trial has been initiated by the European Society for Human Reproduction and Embryology (ESHRE) which aims to clarify this point in the near future. S4 The use of polar bodies and cumulus cells to assess oocytes and select embryos E. Fragouli1 . Reprogenetics UK, Oxford, UK Introduction: The oocyte with its stored mRNAs and proteins drives and supports the first few cleavage divisions, until the developing embryo is able to activate its own genome approximately three days after fertilisation. Therefore, the ability to identify and select the most competent oocytes, could be of great benefit to IVF treatments, increasing the likelihood that embryos of high implantation potential, capable of producing a healthy pregnancy, are generated. Currently, the way that most IVF laboratories select competent oocytes is based solely on their morphological assessment. This assessment is of great value, but it is not necessarily capable of accurately predicting either the health of the oocyte or the presence of a chromosome abnormality. Aneuploidy is one of the main factors to negatively influence preimplantation development, leading to spontaneous abortions, embryonic arrest, and implantation failure. Most chromosome abnormalities arise during female
meiosis, and become increasingly common with advancing age. Genetic analysis of oocytes or embryos can identify the presence of potentially lethal chromosome abnormalities, but the invasive nature of the biopsy procedure involved, may affect embryonic viability. Our ongoing research has the following aims: 1. Development of a methodological approach combining comprehensive cytogenetic analysis of the oocyte, and a detailed assessment of the corresponding cumulus cell (CC) transcriptome; 2. Comparison of the gene expression patterns of CCs removed from normal and aneuploid oocytes; 3. Identification of novel non-invasive biomarkers for determining oocyte and embryo quality and chromosome content. Materials and Methods: CCs were removed from human MII oocytes. The participating patients were being treated due to male factor infertility and tubal problems (i.e. no ovarian involvement). The average female age was 38.3 years (age range 32 46 years). The chromosomal status of individual oocytes was determined by analysing the first polar body with array comparative genomic hybridization, a method which identifies aneuploidy affecting any chromosome. Transcriptomic analysis of CCs was achieved by using RNA amplification (via in vitro transcription) and a microarray which examined over 29,000 genes. This was followed by verification of results using real time polymerase chain reaction (RT-PCR) assays. Results: Data obtained from the initial microarray experiments demonstrated that expression patterns among all CCs were very similar, with 17,388 genes being consistently detected in all samples. There were however, differences in the transcriptome of CCs removed from normal and also chromosomally abnormal oocytes. Both microarray and additional large-scale real-time PCR experiments suggested that CCs associated with aneuploid oocytes were less transcriptionally active, compared to CCs surrounding normal oocytes. Moreover, two genes were identified showing statistically significant (P < 0.05) differences in their expression between CCs of normal and aneuploid oocytes. Of these two genes, one is involved in intracellular signalling and homeostasis, whereas the other regulates carbohydrate metabolism and apoptosis. Additionally, one of these two genes seems to be indicative of the oocyte’s ability to result to a healthy live birth. Conclusions: The comparison of the gene expression patterns seen in CCs surrounding normal and chromosomally abnormal oocytes provides for the first time an insight into the follicular environment of oocytes that become aneuploid. From the obtained results it was evident that CCs of chromosomally abnormal oocytes were less proliferative and transcriptionally quiescent compared to CCs of normal oocytes. This finding suggests that there is a direct link between follicular microenvironment and the genesis of oocvte aneuploidy. We identified 2 genes exhibiting highly significant differences in their expression between CCs of normal and abnormal oocytes, one of which was also capable of possibly predicting the oocyte’s ability to lead to a successful pregnancy. These genes have the potential to serve as non-invasive markers of oocyte aneuploidy, either reducing or even eliminating the necessity for oocyte or embryo biopsy.
Advanced topics in clinical PGD (1) S5 What about mitochondrial biopsy? L. Gianaroli1 , M.C. Magli1 , I. Stanghellini1 , A.M. Crivello1 , A.P. Ferraretti1 . 1 S.I.S.Me.R. Reproductive Medicine Unit, Bologna, Italy Introduction: Processes like oocyte maturation, fertilization, embryo development and implantation require great quantity
SESSIONS, Advanced topics in clinical PGD (1) of energy, which is provided by the thousands of mitochondria accumulated during oogenesis. Failures in any of these processes may depend on mitochondrial defects. More specifically, mutations in mitochondrial DNA (mtDNA) may be associated with the respiratory chain possibly resulting in neuromuscular disorders, which do not have an effective treatment and whose severity is correlated with the amount of mutated molecules inherited. Finding a way to study and evaluate the mutated mtDNA within an oocyte may offer couples at risk for transmitting mitochondrial diseases the possibility to select healthy oocytes. Moreover, the use of a single test able to combine mitochondrial analysis and aneuploidy screening would be of clinical advantage for those couples with advanced age, but could also provide relevant information on the possible correlation between oocyte ageing and loss of mitochondrial metabolic efficiency. The aim of this study was to design a method able to detect both aneuploidy and mtDNA in oocytes and corresponding polar bodies (PBs) with the finality of studying segregation of mutant and wild-type mtDNA molecules during human meiosis. Materials and Methods: The SurePlex™ amplification kit (Rubicon) was used for Whole Genome Amplification (WGA) of oocytes and PBs from 16 patients (mean age 40.1±1.5 years) undergoing ICSI cycles. An aliquot of the amplified product was destined to array-Comparative Genomic Hybridization (CGH). In a second aliquot, the mitochondrial D-loop region was amplified and sequenced. Sequences obtained from oocytes and PBs were compared with those obtained from blood. The DNA_Aligment 1.3.1.1 software was used to align sequences to rCRS (revised Cambridge Reference Sequence) for detecting polymorphic sites. Results: A total of 122 PBs (including first and second PB) and 51 oocytes were amplified using WGA and analyzed by arrayCGH. Aneuploidy was diagnosed in 96 polar bodies (79% vs. 21% euploid) and 35 oocytes (69% vs. 27% euploid and 4% with no result). The amplification of the D-loop region was successful in 46% of PBs, failed in 22% of them, and gave suboptimal results in the remaining 32%. The D-loop region amplified in 80% of oocytes, while in the remaining 20% bands were either weak or aspecific. The comparative analysis of the sequences showed a correspondence between the DNA extracted from blood, oocytes and PBs, with the great majority of polymorphisms detected in oocytes being also present in PBs. There was an exception in 8 cases (6.5%) for which the possibility of preferential segregation of mutated mitochondrial sequences in PBs is currently under study. Conclusion: The present results demonstrate the applicability of WGA for the analysis of mitochondrial sequences on DNA extracted from different tissues, more specifically oocytes and corresponding PBs. From a clinical perspective, this makes it possible to approach the study of mtDNA mutations and segregation in a preimplantation genetic diagnosis perspective. From the scientific side, the concomitant analysis of PBs, corresponding oocyte and blood, will contribute additional knowledge to the understanding of mitochondrial segregation during oocyte maturation. S6 Polar body analysis by array CGH and follow up at cleavage stages D. Christopikou1 . 1 Embryogenesis, Athens, Greece Chromosome aneuploidy is a major cause of IVF failure and pregnancy loss. Until recently, preimplantation genetic screening for aneuploidy by cleavage stage biopsy and interphase fluorescence in situ hybridisation (FISH) with chromosome specific probes in one or more hybridisations was in widespread clinical use. However, a number of randomised clinical trials
S33 demonstrated that there is no increase, and in one case a decrease, in live birth rates in women of advanced maternal age. There are several potential explanations for these results. Theoretically, testing for aneuploidy can only improve the chance of a healthy livebirth, where there are some euploid embryos to select, in a fresh transfer. Also, embryo biopsy at cleavage stages is invasive, chromosomal mosaicism can result in false positive results, preventing transfer of an otherwise euploid embryo, and analysis of limited numbers of chromosomes may limit the effectiveness of the testing. Microarray based comparative genomic hybridisation (array CGH) is now possible at the single cell level following whole genome amplification. Furthermore, polar body analysis is less invasive and allows errors in female meiosis to be identified without the complication of possible chromosomal mosaicism at later stages. Use of this approach has revealed that human oocytes collected from women of advanced maternal age have a high incidence of aneuploidy, involving all chromosomes, and shown that many oocytes have multiple aneuploidies. Analysis of both the first and second polar bodies (PB1 and PB2) and the corresponding oocyte also demonstrated that almost all errors are caused by premature predivision of sister chromatids in the first meiotic division and that there is a high incidence of errors in the second division, some of which balance errors in the first division. Using this data as a basis for interpretation, we have used polar body biopsy and array CGH analysis in a series of couples requesting aneuploidy testing, mainly for advanced maternal age, but also in one case for risk of maternal translocation chromosome imbalance. With patients’ informed consent, we have then followed up zygotes diagnosed as aneuploid, at cleavage stages, by lysing the whole embryo, amplifying the whole genome and analysing by array CGH. This has demonstrated that almost all the aneuploidies, predicted on the basis of the interpretation of copy number changes in the two polar bodies, are present in a majority of cells in the corresponding cleavage stage embryos and there are only a small number of additional aneuploidies, possibly of paternal or postzygotic origin. This data, therefore, does not support the possibility of frequent or extensive ‘self correction’ of maternal meiotic errors, although it does not eliminate the possibility of changes in a minority of cells and validates polar body analysis as a strategy for identifying the most frequent aneuploidies affecting the human preimplantation embryo. S7 Blastocyst biopsy for aneuploidy screening S. Munn´ e1 , P. Colls1 , G. Harton1 , E. Fragouli2 , D. Wells2 . 1 Reprogenetics, Livingston, NJ, USA, 2 Reprogenetics UK, Oxford, UK Preimplantation Genetic Diagnosis at least in the US has transitioned from FISH technology to microarrays and from day three biopsy to blastocyst biopsy. Only small centers with no laser are still using day 3 biopsy. Mounting evidence indicates that trophectoderm (TE) biopsy is less detrimental to implantation than day three biopsy when replacing on day 5 or 6 of a fresh cycle. However, most data coupling arrays and blastocyst biopsy has used vitrification and replacement in a warmed cycle, which introduces the confounding factor of altered uterine receptivity in stimulated cycles. It is well known that freeze-all cycle embryos implant better than those replaced on a fresh cycle. Evidence from Japan indicates that day 3 biopsy coupled with vitrification and replacement on a warmed cycle yields similar implantation rates as TE biopsy and better than day 3 biopsy and day 4 5 biopsy. Transport PGS with array CGH (aCGH) permits TE biopsy and day-6 morning transfer. We have preliminary data showing equal to higher implantation rates using this approach than TE biopsy and vitrification or day 3 biopsy and fresh transfer. Thus it seem
11th International Conference on Preimplantation Genetic Diagnosis
S3 Polar body biopsy and array CGH for aneuploidy in advanced maternal age M. Montag1 . 1 Dept. Gyn. Endocrinol. & Fertil. Disorders, University Clinics Heidelberg, Heidelberg, Germany Aneuploidy testing has started in the 1990s and still to date it is a topic which is controversial and can provoke vivid discussions especially if it comes to the question if such an approach is beneficial in assisted reproduction. Among the different techniques which are applied in routine since then, biopsy of polar bodies at the oocyte/zygote stage and biopsy of trophectoderm cells at the blastocyst stage are those which are at present in the focus of interest. Both techniques seem to have the highest potential if combined with a diagnostic approach which allows investigating all chromosomes. One such approach is comparative genomic hybridization (CGH). Over the last decade CGH has made substantial progression from the conventional chromosome-based hybridisation template to the first CGH-arrays and to the latest single channel array format of today. If the focus is on maternal aneuploidies which have manifested during the meiotic progression of the developing oocyte, polar body biopsy and array-CGH is an option for investigating chromosomal aberrations without touching the embryo and this strategy clearly avoids the problem of embryo mosaicism. For some patients this approach is ethically better acceptable. Retrieval of polar bodies can be done either sequential or simultaneously, provided that the timing is adapted to the course of meiosis (for biopsy) and to the integrity of the chromosomal content of the polar bodies (for amplification). Several publications have reported on the use of this technology for the investigation of structural as well as numerical chromosome aberrations. In patients with advanced maternal age the results of these studies show that at an average maternal age of 40 the mean rate of aneuploidy is 70%, meaning that out of 10 oocytes 3 are chromosomally intact and suitable for transfer. As there is a decline in ovarian reserve in women with advanced age, the rate of cancelled transfers due to the presence of only aneuploid oocytes is very high among these women. To date there is no systematic study available which clearly states to what extent polar body biopsy and array-CGH benefits assisted reproduction treatment. The main question is if aneuploidy testing in advanced maternal age leads to lower abortion rates and/or if it does support higher take home baby rates. Following a pilot study on polar body biopsy and arrayCGH, a multicentre trial has been initiated by the European Society for Human Reproduction and Embryology (ESHRE) which aims to clarify this point in the near future. S4 The use of polar bodies and cumulus cells to assess oocytes and select embryos E. Fragouli1 . Reprogenetics UK, Oxford, UK Introduction: The oocyte with its stored mRNAs and proteins drives and supports the first few cleavage divisions, until the developing embryo is able to activate its own genome approximately three days after fertilisation. Therefore, the ability to identify and select the most competent oocytes, could be of great benefit to IVF treatments, increasing the likelihood that embryos of high implantation potential, capable of producing a healthy pregnancy, are generated. Currently, the way that most IVF laboratories select competent oocytes is based solely on their morphological assessment. This assessment is of great value, but it is not necessarily capable of accurately predicting either the health of the oocyte or the presence of a chromosome abnormality. Aneuploidy is one of the main factors to negatively influence preimplantation development, leading to spontaneous abortions, embryonic arrest, and implantation failure. Most chromosome abnormalities arise during female
meiosis, and become increasingly common with advancing age. Genetic analysis of oocytes or embryos can identify the presence of potentially lethal chromosome abnormalities, but the invasive nature of the biopsy procedure involved, may affect embryonic viability. Our ongoing research has the following aims: 1. Development of a methodological approach combining comprehensive cytogenetic analysis of the oocyte, and a detailed assessment of the corresponding cumulus cell (CC) transcriptome; 2. Comparison of the gene expression patterns of CCs removed from normal and aneuploid oocytes; 3. Identification of novel non-invasive biomarkers for determining oocyte and embryo quality and chromosome content. Materials and Methods: CCs were removed from human MII oocytes. The participating patients were being treated due to male factor infertility and tubal problems (i.e. no ovarian involvement). The average female age was 38.3 years (age range 32 46 years). The chromosomal status of individual oocytes was determined by analysing the first polar body with array comparative genomic hybridization, a method which identifies aneuploidy affecting any chromosome. Transcriptomic analysis of CCs was achieved by using RNA amplification (via in vitro transcription) and a microarray which examined over 29,000 genes. This was followed by verification of results using real time polymerase chain reaction (RT-PCR) assays. Results: Data obtained from the initial microarray experiments demonstrated that expression patterns among all CCs were very similar, with 17,388 genes being consistently detected in all samples. There were however, differences in the transcriptome of CCs removed from normal and also chromosomally abnormal oocytes. Both microarray and additional large-scale real-time PCR experiments suggested that CCs associated with aneuploid oocytes were less transcriptionally active, compared to CCs surrounding normal oocytes. Moreover, two genes were identified showing statistically significant (P < 0.05) differences in their expression between CCs of normal and aneuploid oocytes. Of these two genes, one is involved in intracellular signalling and homeostasis, whereas the other regulates carbohydrate metabolism and apoptosis. Additionally, one of these two genes seems to be indicative of the oocyte’s ability to result to a healthy live birth. Conclusions: The comparison of the gene expression patterns seen in CCs surrounding normal and chromosomally abnormal oocytes provides for the first time an insight into the follicular environment of oocytes that become aneuploid. From the obtained results it was evident that CCs of chromosomally abnormal oocytes were less proliferative and transcriptionally quiescent compared to CCs of normal oocytes. This finding suggests that there is a direct link between follicular microenvironment and the genesis of oocvte aneuploidy. We identified 2 genes exhibiting highly significant differences in their expression between CCs of normal and abnormal oocytes, one of which was also capable of possibly predicting the oocyte’s ability to lead to a successful pregnancy. These genes have the potential to serve as non-invasive markers of oocyte aneuploidy, either reducing or even eliminating the necessity for oocyte or embryo biopsy.
Advanced topics in clinical PGD (1) S5 What about mitochondrial biopsy? L. Gianaroli1 , M.C. Magli1 , I. Stanghellini1 , A.M. Crivello1 , A.P. Ferraretti1 . 1 S.I.S.Me.R. Reproductive Medicine Unit, Bologna, Italy Introduction: Processes like oocyte maturation, fertilization, embryo development and implantation require great quantity
SESSIONS, Advanced topics in clinical PGD (1) of energy, which is provided by the thousands of mitochondria accumulated during oogenesis. Failures in any of these processes may depend on mitochondrial defects. More specifically, mutations in mitochondrial DNA (mtDNA) may be associated with the respiratory chain possibly resulting in neuromuscular disorders, which do not have an effective treatment and whose severity is correlated with the amount of mutated molecules inherited. Finding a way to study and evaluate the mutated mtDNA within an oocyte may offer couples at risk for transmitting mitochondrial diseases the possibility to select healthy oocytes. Moreover, the use of a single test able to combine mitochondrial analysis and aneuploidy screening would be of clinical advantage for those couples with advanced age, but could also provide relevant information on the possible correlation between oocyte ageing and loss of mitochondrial metabolic efficiency. The aim of this study was to design a method able to detect both aneuploidy and mtDNA in oocytes and corresponding polar bodies (PBs) with the finality of studying segregation of mutant and wild-type mtDNA molecules during human meiosis. Materials and Methods: The SurePlex™ amplification kit (Rubicon) was used for Whole Genome Amplification (WGA) of oocytes and PBs from 16 patients (mean age 40.1±1.5 years) undergoing ICSI cycles. An aliquot of the amplified product was destined to array-Comparative Genomic Hybridization (CGH). In a second aliquot, the mitochondrial D-loop region was amplified and sequenced. Sequences obtained from oocytes and PBs were compared with those obtained from blood. The DNA_Aligment 1.3.1.1 software was used to align sequences to rCRS (revised Cambridge Reference Sequence) for detecting polymorphic sites. Results: A total of 122 PBs (including first and second PB) and 51 oocytes were amplified using WGA and analyzed by arrayCGH. Aneuploidy was diagnosed in 96 polar bodies (79% vs. 21% euploid) and 35 oocytes (69% vs. 27% euploid and 4% with no result). The amplification of the D-loop region was successful in 46% of PBs, failed in 22% of them, and gave suboptimal results in the remaining 32%. The D-loop region amplified in 80% of oocytes, while in the remaining 20% bands were either weak or aspecific. The comparative analysis of the sequences showed a correspondence between the DNA extracted from blood, oocytes and PBs, with the great majority of polymorphisms detected in oocytes being also present in PBs. There was an exception in 8 cases (6.5%) for which the possibility of preferential segregation of mutated mitochondrial sequences in PBs is currently under study. Conclusion: The present results demonstrate the applicability of WGA for the analysis of mitochondrial sequences on DNA extracted from different tissues, more specifically oocytes and corresponding PBs. From a clinical perspective, this makes it possible to approach the study of mtDNA mutations and segregation in a preimplantation genetic diagnosis perspective. From the scientific side, the concomitant analysis of PBs, corresponding oocyte and blood, will contribute additional knowledge to the understanding of mitochondrial segregation during oocyte maturation. S6 Polar body analysis by array CGH and follow up at cleavage stages D. Christopikou1 . 1 Embryogenesis, Athens, Greece Chromosome aneuploidy is a major cause of IVF failure and pregnancy loss. Until recently, preimplantation genetic screening for aneuploidy by cleavage stage biopsy and interphase fluorescence in situ hybridisation (FISH) with chromosome specific probes in one or more hybridisations was in widespread clinical use. However, a number of randomised clinical trials
S33 demonstrated that there is no increase, and in one case a decrease, in live birth rates in women of advanced maternal age. There are several potential explanations for these results. Theoretically, testing for aneuploidy can only improve the chance of a healthy livebirth, where there are some euploid embryos to select, in a fresh transfer. Also, embryo biopsy at cleavage stages is invasive, chromosomal mosaicism can result in false positive results, preventing transfer of an otherwise euploid embryo, and analysis of limited numbers of chromosomes may limit the effectiveness of the testing. Microarray based comparative genomic hybridisation (array CGH) is now possible at the single cell level following whole genome amplification. Furthermore, polar body analysis is less invasive and allows errors in female meiosis to be identified without the complication of possible chromosomal mosaicism at later stages. Use of this approach has revealed that human oocytes collected from women of advanced maternal age have a high incidence of aneuploidy, involving all chromosomes, and shown that many oocytes have multiple aneuploidies. Analysis of both the first and second polar bodies (PB1 and PB2) and the corresponding oocyte also demonstrated that almost all errors are caused by premature predivision of sister chromatids in the first meiotic division and that there is a high incidence of errors in the second division, some of which balance errors in the first division. Using this data as a basis for interpretation, we have used polar body biopsy and array CGH analysis in a series of couples requesting aneuploidy testing, mainly for advanced maternal age, but also in one case for risk of maternal translocation chromosome imbalance. With patients’ informed consent, we have then followed up zygotes diagnosed as aneuploid, at cleavage stages, by lysing the whole embryo, amplifying the whole genome and analysing by array CGH. This has demonstrated that almost all the aneuploidies, predicted on the basis of the interpretation of copy number changes in the two polar bodies, are present in a majority of cells in the corresponding cleavage stage embryos and there are only a small number of additional aneuploidies, possibly of paternal or postzygotic origin. This data, therefore, does not support the possibility of frequent or extensive ‘self correction’ of maternal meiotic errors, although it does not eliminate the possibility of changes in a minority of cells and validates polar body analysis as a strategy for identifying the most frequent aneuploidies affecting the human preimplantation embryo. S7 Blastocyst biopsy for aneuploidy screening S. Munn´ e1 , P. Colls1 , G. Harton1 , E. Fragouli2 , D. Wells2 . 1 Reprogenetics, Livingston, NJ, USA, 2 Reprogenetics UK, Oxford, UK Preimplantation Genetic Diagnosis at least in the US has transitioned from FISH technology to microarrays and from day three biopsy to blastocyst biopsy. Only small centers with no laser are still using day 3 biopsy. Mounting evidence indicates that trophectoderm (TE) biopsy is less detrimental to implantation than day three biopsy when replacing on day 5 or 6 of a fresh cycle. However, most data coupling arrays and blastocyst biopsy has used vitrification and replacement in a warmed cycle, which introduces the confounding factor of altered uterine receptivity in stimulated cycles. It is well known that freeze-all cycle embryos implant better than those replaced on a fresh cycle. Evidence from Japan indicates that day 3 biopsy coupled with vitrification and replacement on a warmed cycle yields similar implantation rates as TE biopsy and better than day 3 biopsy and day 4 5 biopsy. Transport PGS with array CGH (aCGH) permits TE biopsy and day-6 morning transfer. We have preliminary data showing equal to higher implantation rates using this approach than TE biopsy and vitrification or day 3 biopsy and fresh transfer. Thus it seem