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Embryo selection in assisted conception
ARTICLE IN PRESS Current Obstetrics & Gynaecology (2004) 14, 291–293
www.elsevier.com/cuog
Embryo selection in assisted conception Victoria Lamb, Christine Leary, Mary Herbert, Alison Murdoch* Newcastle Fertility Centre at Life, Bioscience Centre, International Centre for Life, Newcastle Upon Tyne NE1 4EP, UK
KEYWORDS Embryo selection; Blastocyst; Aneuploidy screening
Summary One of the most important aspects in the success of an IVF cycle is the selection of those embryos with the highest developmental and implantation potential. A reliable selection procedure will enable fewer embryos to be replaced in each cycle, thereby reducing the incidence of multiple pregnancies whilst maintaining an acceptable pregnancy rate. At present, the most widely used methods of selection are based on morphological criteria. However, these methods may be imprecise. Chromosomal aberrations within embryos are relatively common and may be compatible with development beyond the stage at which embryo transfer normally occurs. More invasive selection methods, such as aneuploidy screening, may improve implantation and pregnancy rates, particularly for older women, but these techniques present their own ethical dilemmas. & 2004 Elsevier Ltd. All rights reserved.
Introduction
Pronuclear scoring
In an IVF programme the ultimate aim is to produce a healthy baby. In order to have a good chance of achieving this goal, superovulation is usually used, with the resulting oocytes being inseminated and generally resulting in numerous embryos. Although practice varies between countries, in the UK two embryos are generally transferred to the patient, to ensure a good chance of pregnancy whilst minimising the risk of a high order multiple pregnancy. However, in order to give the patient the best chance of a pregnancy the embryos with the highest developmental potential must be selected for transfer. Again, practice varies, but different methods of assessing or predicting developmental potential are used.
The first stage at which an embryo may be scored for development is while the pronuclei are still visible in the hours after fertilisation. Embryos are scored according to equality of size and positioning of pronuclei, alignment of nucleoli and appearance of cytoplasm. To date, studies have been unconvincing in demonstrating that pronuclear scoring gives an accurate prediction of development or implantation. However, it has been suggested that combining pronuclear scoring, assessment of early cleavage and grading of cleavage stage embryos can give a good indication of implantation potential. In Germany, where strict regulations allow only those embryos that will be transferred to be cultured beyond the pronuclear stage, developing a reliable scoring system for this stage of development has particular importance.
Early cleavage *Corresponding author. Tel.: þ 44-191-319-4740; fax: þ 44191-219-4747. E-mail address: [email protected] (A. Murdoch).
Numerous studies have reported that embryos which undergo their first cleavage division within
0957-5847/$ - see front matter & 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.curobgyn.2004.04.011
ARTICLE IN PRESS 292
27 h of insemination have a higher blastocyst and implantation potential than those embryos cleaving later. It has been postulated that cells with an aneuploid chromosome status cleave slower than those that are genetically normal. This observation should assist in the selection of genetically normal embryos with high implantation potential for transfer, without necessitating invasive and costly testing. It could also give confidence in a move to single embryo transfers, mirroring practice in Scandinavia, with the aim of further reducing the incidence of multiple pregnancies and their associated complications.
Grading at the cleavage stage This is probably the most widely used method of assessing which embryos to replace and/or freeze. The grading of embryos at the cleavage stage involves measurement of the rate of development, which is judged by the number of cells or number of divisions an embryo has undergone, and an assessment of the morphology, taking into account uneven sized cells, fragmentation and multinucleation. It has been reported that uneven sized blastomeres and fragmentation are associated with multinucleation and chromosomal abnormalities. This means of embryo selection has been shown to give a good indication of the likelihood of an embryo to give rise to a pregnancy. However, there is not always an association between the genetic normality of an embryo and its morphological appearance. Approximately half of human IVF embryos are chromosomally abnormal, and even when strict morphological criteria are used for embryo selection, 25% of the selected embryos may still be abnormal. It is also possible that these genetically abnormal embryos may show normal development to the stage at which embryo transfer commonly occurs (day two or three). Therefore, grading and transfer on day two or three may not detect embryos that are grossly genetically abnormal and therefore have no chance of giving rise to a healthy baby.
Blastocyst scoring Blastocyst formation represents an important milestone in preimplantation development. By the blastocyst stage the embryo has developed two distinct cell lineages: the trophectoderm, which will give rise to the extraembryonic tissue, and the inner cell mass, from which the fetus will develop.
V. Lamb et al.
Human embryos cultured in vitro undergo blastocyst formation between 5 and 7 days after insemination. However, despite improvements to the formulation of culture media, the majority of in vitro cultured human embryos are not capable of developing to the blastocyst stage. Comparisons of the incidence of chromosomal abnormalities in cleavage stage embryos and blastocysts indicate that aneuploidy predisposes to developmental arrest, as the proportion of embryos with chromosomal abnormalities is lower in blastocysts. Therefore, delaying transfer until the blastocyst stage not only selects embryos with the highest developmental competence but should also help select embryos with a normal chromosomal constitution. The method of grading blastocysts is similar to that of grading cleavage stage embryos, in that the blastocysts are scored on how similar they are to the morphological ideal. This will involve assessment of the degree of expansion of the blastocoele, presence of a clear inner cell mass and defined pavement-like trophectoderm. Despite the obvious advantages in helping to select viable embryos for transfer, concerns have been raised about the possible adverse effects of culturing human embryos to the blastocyst stage. For example, there have been numerous case reports of monozygotic twinning following blastocyst transfer, the most recent of which reported a quintuplet pregnancy following transfer of two blastocysts. Recently there have also been concerns that extended culture can have detrimental effects on human embryos due to epigenetic disturbances. This hypothesis remains to be confirmed but clearly raises concerns for patients and health professionals alike.
Aneuploidy screening This is a relatively new area and not an uncontroversial one. It normally involves removal of one or two cells from cleavage stage (six–eight cell) embryos. The cells are analysed to determine whether they contain a normal complement of the chromosomes being tested, these cells being assumed to be representative of the embryo as a whole. However, it is well documented that there may be considerable differences in the chromosomal complement of different cells within an embryo. This mosaicism can tend to lead to incorrect diagnosis of aneuploidy or normality within the remaining embryo. Aneuploidy screening differs from preimplantation genetic diagnosis (PGD) in that PGD was developed to detect specific
ARTICLE IN PRESS Embryo selection in assisted conception
genetic abnormalities, whereas aneuploidy screening tests for a number of the most common constitutive human aneuploidies (e.g. chromosomes 13, 16, 18, 21 and 22). The rationale for aneuploidy screening is that the proportion of embryo transfers resulting in a healthy baby can be increased by avoiding replacement of aneuploid embryos. This method of selecting embryos has been used to help reduce the incidence of trisomic pregnancy in women of advanced reproductive age. Data from a large multi-centre study indicates that aneuploidy screening does not promote increased implantation, however the authors report a significantly higher ongoing pregnancy rate due to reduced incidence of miscarriage. Aneuploidy screening is available in the UK but is not widely used at present. This is partly because of the extra time and hence cost involved in this treatment, the problems associated with mosaicism in embryos, and the availability of suitable genetics laboratories to perform the tests. There is also a lack of convincing evidence that this treatment is genuinely beneficial. Some groups in society would argue that aneuploidy screening is implying that disabled people are undesirable, that it will lead to the production of so-called ‘designer babies’, and that this in turn reduces children to mere commodities.
The future At present there is not widespread adoption of aneuploidy screening for embryo selection, but this situation will probably change with time and may take on a more important role in embryo selection, particularly for older patients or if single embryo transfers become more commonplace. In the future biochemical assessments may be developed that could give a more accurate measurement of an embryo’s developmental potential. It has already been shown that amino acid consumption correlates with embryo quality.
Conclusion Various means of embryo selection at different developmental stages are currently available,
293
with differing opinions on their effectiveness. However, it should not be forgotten that the implantation potential of an embryo, and the assessment of that, is only one of the factors involved in producing a healthy pregnancy. Also, whilst there will always be controversy surrounding such an important and emotive area as this, it should be kept in mind that most couples do not want a ‘designer baby’. What they do want, however, is a healthy baby to start, add to or complete their families. As health professionals we are surely dutybound to provide these couples with the best, both safest and most effective method of achieving this.
Further reading De Rycke M, Liebaers I, Van Steirteghem A. Epigenetic risks related to assisted reproductive technologies: risk analysis and epigenetic inheritance. Hum Reprod Update 2002; 17:2487–94. Ebner T, Moser M, Sommergruber M, Tews G. Selection based on morphological assessment of oocytes and embryos at different stages of preimplantation deveopment: a review. Hum Reprod Online 2003;9:251–62. Findikli N, Kahraman S, Kumtepe Y, et al. Assessment of DNA fragmentation and aneuploidy on poor quality human embryos. Reprod Biomed Online 2004;8:196–206. Hassold T, Hunt P. To err (meiotically) is human: the genesis of human aneuploidy. Nat Rev Genet 2001;2:280–91. Munne S, Magli C, Cohen J, et al. Positive outcome after preimplantation diagnosis of aneuploidy in human embryos. Hum Reprod 1999;14:2191–9. Nagy ZP, Dozortsev D, Diamond M, et al. Pronuclear morphology evaluation with subsequent evaluation of embryo morphology significantly increases implantation rates. Fertil Steril 2003;80:67–74. Van Royen E, Mangelschots K, Vercruyssen M, et al. Multinucleation in cleavage stage embryos. Hum Reprod 2003; 18:1062–9. Wharf E, Dimitrakopoulos A, Khalaf Y, Pickering S. Early embryo development is an indicator of implantation potential. Reprod Biomed Online 2004;8:212–8. Ziebe S, Lundin K, Loft A, et al. FISH analysis for chromosomes 13, 16, 18, 21, 22, X and Y in all blastomeres of IVF preembryos from 144 randomly selected donated human oocytes and impact on pre-embryo morphology. Hum Reprod 2003; 18:2575–81. Zikopoulos K, Platteau P, Kolibianakis E, Albano C, Van Steirteghem A, Devroey P. Quintuplet pregnancy following transfer of two blastocysts: case report. Hum Reprod 2004; 19:325–7.
www.elsevier.com/cuog
Embryo selection in assisted conception Victoria Lamb, Christine Leary, Mary Herbert, Alison Murdoch* Newcastle Fertility Centre at Life, Bioscience Centre, International Centre for Life, Newcastle Upon Tyne NE1 4EP, UK
KEYWORDS Embryo selection; Blastocyst; Aneuploidy screening
Summary One of the most important aspects in the success of an IVF cycle is the selection of those embryos with the highest developmental and implantation potential. A reliable selection procedure will enable fewer embryos to be replaced in each cycle, thereby reducing the incidence of multiple pregnancies whilst maintaining an acceptable pregnancy rate. At present, the most widely used methods of selection are based on morphological criteria. However, these methods may be imprecise. Chromosomal aberrations within embryos are relatively common and may be compatible with development beyond the stage at which embryo transfer normally occurs. More invasive selection methods, such as aneuploidy screening, may improve implantation and pregnancy rates, particularly for older women, but these techniques present their own ethical dilemmas. & 2004 Elsevier Ltd. All rights reserved.
Introduction
Pronuclear scoring
In an IVF programme the ultimate aim is to produce a healthy baby. In order to have a good chance of achieving this goal, superovulation is usually used, with the resulting oocytes being inseminated and generally resulting in numerous embryos. Although practice varies between countries, in the UK two embryos are generally transferred to the patient, to ensure a good chance of pregnancy whilst minimising the risk of a high order multiple pregnancy. However, in order to give the patient the best chance of a pregnancy the embryos with the highest developmental potential must be selected for transfer. Again, practice varies, but different methods of assessing or predicting developmental potential are used.
The first stage at which an embryo may be scored for development is while the pronuclei are still visible in the hours after fertilisation. Embryos are scored according to equality of size and positioning of pronuclei, alignment of nucleoli and appearance of cytoplasm. To date, studies have been unconvincing in demonstrating that pronuclear scoring gives an accurate prediction of development or implantation. However, it has been suggested that combining pronuclear scoring, assessment of early cleavage and grading of cleavage stage embryos can give a good indication of implantation potential. In Germany, where strict regulations allow only those embryos that will be transferred to be cultured beyond the pronuclear stage, developing a reliable scoring system for this stage of development has particular importance.
Early cleavage *Corresponding author. Tel.: þ 44-191-319-4740; fax: þ 44191-219-4747. E-mail address: [email protected] (A. Murdoch).
Numerous studies have reported that embryos which undergo their first cleavage division within
0957-5847/$ - see front matter & 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.curobgyn.2004.04.011
ARTICLE IN PRESS 292
27 h of insemination have a higher blastocyst and implantation potential than those embryos cleaving later. It has been postulated that cells with an aneuploid chromosome status cleave slower than those that are genetically normal. This observation should assist in the selection of genetically normal embryos with high implantation potential for transfer, without necessitating invasive and costly testing. It could also give confidence in a move to single embryo transfers, mirroring practice in Scandinavia, with the aim of further reducing the incidence of multiple pregnancies and their associated complications.
Grading at the cleavage stage This is probably the most widely used method of assessing which embryos to replace and/or freeze. The grading of embryos at the cleavage stage involves measurement of the rate of development, which is judged by the number of cells or number of divisions an embryo has undergone, and an assessment of the morphology, taking into account uneven sized cells, fragmentation and multinucleation. It has been reported that uneven sized blastomeres and fragmentation are associated with multinucleation and chromosomal abnormalities. This means of embryo selection has been shown to give a good indication of the likelihood of an embryo to give rise to a pregnancy. However, there is not always an association between the genetic normality of an embryo and its morphological appearance. Approximately half of human IVF embryos are chromosomally abnormal, and even when strict morphological criteria are used for embryo selection, 25% of the selected embryos may still be abnormal. It is also possible that these genetically abnormal embryos may show normal development to the stage at which embryo transfer commonly occurs (day two or three). Therefore, grading and transfer on day two or three may not detect embryos that are grossly genetically abnormal and therefore have no chance of giving rise to a healthy baby.
Blastocyst scoring Blastocyst formation represents an important milestone in preimplantation development. By the blastocyst stage the embryo has developed two distinct cell lineages: the trophectoderm, which will give rise to the extraembryonic tissue, and the inner cell mass, from which the fetus will develop.
V. Lamb et al.
Human embryos cultured in vitro undergo blastocyst formation between 5 and 7 days after insemination. However, despite improvements to the formulation of culture media, the majority of in vitro cultured human embryos are not capable of developing to the blastocyst stage. Comparisons of the incidence of chromosomal abnormalities in cleavage stage embryos and blastocysts indicate that aneuploidy predisposes to developmental arrest, as the proportion of embryos with chromosomal abnormalities is lower in blastocysts. Therefore, delaying transfer until the blastocyst stage not only selects embryos with the highest developmental competence but should also help select embryos with a normal chromosomal constitution. The method of grading blastocysts is similar to that of grading cleavage stage embryos, in that the blastocysts are scored on how similar they are to the morphological ideal. This will involve assessment of the degree of expansion of the blastocoele, presence of a clear inner cell mass and defined pavement-like trophectoderm. Despite the obvious advantages in helping to select viable embryos for transfer, concerns have been raised about the possible adverse effects of culturing human embryos to the blastocyst stage. For example, there have been numerous case reports of monozygotic twinning following blastocyst transfer, the most recent of which reported a quintuplet pregnancy following transfer of two blastocysts. Recently there have also been concerns that extended culture can have detrimental effects on human embryos due to epigenetic disturbances. This hypothesis remains to be confirmed but clearly raises concerns for patients and health professionals alike.
Aneuploidy screening This is a relatively new area and not an uncontroversial one. It normally involves removal of one or two cells from cleavage stage (six–eight cell) embryos. The cells are analysed to determine whether they contain a normal complement of the chromosomes being tested, these cells being assumed to be representative of the embryo as a whole. However, it is well documented that there may be considerable differences in the chromosomal complement of different cells within an embryo. This mosaicism can tend to lead to incorrect diagnosis of aneuploidy or normality within the remaining embryo. Aneuploidy screening differs from preimplantation genetic diagnosis (PGD) in that PGD was developed to detect specific
ARTICLE IN PRESS Embryo selection in assisted conception
genetic abnormalities, whereas aneuploidy screening tests for a number of the most common constitutive human aneuploidies (e.g. chromosomes 13, 16, 18, 21 and 22). The rationale for aneuploidy screening is that the proportion of embryo transfers resulting in a healthy baby can be increased by avoiding replacement of aneuploid embryos. This method of selecting embryos has been used to help reduce the incidence of trisomic pregnancy in women of advanced reproductive age. Data from a large multi-centre study indicates that aneuploidy screening does not promote increased implantation, however the authors report a significantly higher ongoing pregnancy rate due to reduced incidence of miscarriage. Aneuploidy screening is available in the UK but is not widely used at present. This is partly because of the extra time and hence cost involved in this treatment, the problems associated with mosaicism in embryos, and the availability of suitable genetics laboratories to perform the tests. There is also a lack of convincing evidence that this treatment is genuinely beneficial. Some groups in society would argue that aneuploidy screening is implying that disabled people are undesirable, that it will lead to the production of so-called ‘designer babies’, and that this in turn reduces children to mere commodities.
The future At present there is not widespread adoption of aneuploidy screening for embryo selection, but this situation will probably change with time and may take on a more important role in embryo selection, particularly for older patients or if single embryo transfers become more commonplace. In the future biochemical assessments may be developed that could give a more accurate measurement of an embryo’s developmental potential. It has already been shown that amino acid consumption correlates with embryo quality.
Conclusion Various means of embryo selection at different developmental stages are currently available,
293
with differing opinions on their effectiveness. However, it should not be forgotten that the implantation potential of an embryo, and the assessment of that, is only one of the factors involved in producing a healthy pregnancy. Also, whilst there will always be controversy surrounding such an important and emotive area as this, it should be kept in mind that most couples do not want a ‘designer baby’. What they do want, however, is a healthy baby to start, add to or complete their families. As health professionals we are surely dutybound to provide these couples with the best, both safest and most effective method of achieving this.
Further reading De Rycke M, Liebaers I, Van Steirteghem A. Epigenetic risks related to assisted reproductive technologies: risk analysis and epigenetic inheritance. Hum Reprod Update 2002; 17:2487–94. Ebner T, Moser M, Sommergruber M, Tews G. Selection based on morphological assessment of oocytes and embryos at different stages of preimplantation deveopment: a review. Hum Reprod Online 2003;9:251–62. Findikli N, Kahraman S, Kumtepe Y, et al. Assessment of DNA fragmentation and aneuploidy on poor quality human embryos. Reprod Biomed Online 2004;8:196–206. Hassold T, Hunt P. To err (meiotically) is human: the genesis of human aneuploidy. Nat Rev Genet 2001;2:280–91. Munne S, Magli C, Cohen J, et al. Positive outcome after preimplantation diagnosis of aneuploidy in human embryos. Hum Reprod 1999;14:2191–9. Nagy ZP, Dozortsev D, Diamond M, et al. Pronuclear morphology evaluation with subsequent evaluation of embryo morphology significantly increases implantation rates. Fertil Steril 2003;80:67–74. Van Royen E, Mangelschots K, Vercruyssen M, et al. Multinucleation in cleavage stage embryos. Hum Reprod 2003; 18:1062–9. Wharf E, Dimitrakopoulos A, Khalaf Y, Pickering S. Early embryo development is an indicator of implantation potential. Reprod Biomed Online 2004;8:212–8. Ziebe S, Lundin K, Loft A, et al. FISH analysis for chromosomes 13, 16, 18, 21, 22, X and Y in all blastomeres of IVF preembryos from 144 randomly selected donated human oocytes and impact on pre-embryo morphology. Hum Reprod 2003; 18:2575–81. Zikopoulos K, Platteau P, Kolibianakis E, Albano C, Van Steirteghem A, Devroey P. Quintuplet pregnancy following transfer of two blastocysts: case report. Hum Reprod 2004; 19:325–7.