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Limitations of Embryo Selection Methods Sjoerd Repping, PhD1
Sebastiaan Mastenbroek, PhD1
1 Center for Reproductive Medicine, Academic Medical Center,
University of Amsterdam, Amsterdam, The Netherlands Semin Reprod Med 2014;32:127–133
Abstract
Keywords
► ► ► ► ►
assisted reproduction embryo transfer embryo selection cryopreservation IVF outcome
Address for correspondence Sebastiaan Mastenbroek, PhD, Center for Reproductive Medicine, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands (e-mail: [email protected]).
In in vitro fertilization (IVF), the selection of embryos for transfer is generally based on the morphology of the available embryos. However, not all embryos with good morphology implant and on average only one in four treatments are successful. This has driven a quest for alternative selection methods. The best known alternative selection method is preimplantation genetic screening (PGS), which has been used for over a decade before it was shown to be inferior to morphological selection. Now, new forms of PGS (performing biopsy at another stage of development and new methods for analysis) are emerging, just like alternative noninvasive embryo selection methods. However, the concept that better selection will lead to improved IVF results is not so certain anymore. Evidence is accumulating that all available embryos in an IVF cycle can be cryopreserved and transferred in subsequent cycles without impairing pregnancy rates or maybe even with an improvement in pregnancy rates. Embryo selection will then no longer be able to improve the live birth rate in IVF; it could even lower the live birth rate. Embryo selection will only be able to improve the time to pregnancy, if embryos with the highest implantation potential are transferred first.
Since the first report of a live born child after an in vitro fertilization (IVF) procedure in 1978, IVF has emerged as a well-established reproductive technique for subfertile couples.1 The International Committee for Monitoring Assisted Reproductive Technology reported a total of 954,743 initiated cycles which resulted in an estimated 237,809 babies born in 2004 worldwide.2 The IVF procedure has changed and improved ever since its introduction. The development of IVF started with the IVF of human oocytes, followed by the culture of human embryos up to morula stage and blastocyst stage.3–7 The retrieval of oocytes was initially performed in a natural cycle, but not much later controlled ovarian hyperstimulation was introduced to mature multiple follicles and to obtain multiple oocytes.4 This allowed transfer of multiple embryos to increase pregnancy rates.8,9 However, as clinical and laboratory protocols became more efficient, this also resulted in an increased number of multiple births and with that adverse perinatal outcomes.10 As a consequence, embryo transfer policies restricting the number of embryos to be transferred to reduce the incidence of multiple pregnancies while main-
Issue Theme Selecting the Best Embryo; Guest Editor, Valerie L. Baker, MD
taining or improving overall pregnancy rates were introduced.11 Methods to select the best embryo(s) with the highest implantation potential for transfer became increasingly important. Embryos have been assessed morphologically ever since the start of IVF to evaluate their development in culture, which resulted in various morphological scoring methods for determining the developmental potential of embryos.12–15 As morphological parameters are correlated with the chance of an embryo leading to a live born child after transfer, morphology became the golden standard for selecting the best embryo for transfer.15 However, not all embryos with good morphology implant and on average only one in four of all IVF treatments are successful.16,17 Alternative methods of embryo selection have, therefore, been introduced over the years in an attempt to improve the success rates of IVF treatments. In this review, we will first briefly evaluate the available evidence on the efficacy of these new methods compared with morphological selection of embryos, and we will then consider the possible changing role and use of embryo selection in the near future of IVF.
Copyright © 2014 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) 584-4662.
DOI http://dx.doi.org/ 10.1055/s-0033-1363554. ISSN 1526-8004.
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Kai Mee Wong, MD1
Limitations of Embryo Selection Methods
Wong et al.
Selection Using Embryo Morphology Morphological evaluation is the most commonly used method for selecting the best embryo for transfer.15,18 There are different embryo scoring variables to predict the potential of an embryo to lead to a live birth after transfer to the uterus. As embryo transfer can be performed at different stages during preimplantation development, that is, at pronuclear (PN) stage, cleavage stage, or blastocyst stage, there are different morphological parameters for each stage. Morphological selection of embryos is largely based on clinical experience and local protocols.15 As a consequence, the European Society of Human Reproduction and Embryology recently provided consensus points to define the minimum criteria for oocyte and embryo morphology assessment.15 Aspects that could be assessed are PN number, PN size, location of nuclear polar bodies, number of blastomeres, blastomere size, degree of fragmentation, multinucleation, compaction, expansion, and trophectoderm (TE) and inner cell mass quality.15 As a large proportion of the embryos do not exactly follow the expected developmental timeline and morphological scoring is often performed on a limited number of fixed time points only, new recording systems that allow 24 hours monitoring have been developed.19 These devices allow 24 hours monitoring by using cameras incorporated in the incubation chamber, without disturbing culture conditions as is the case for assessment of embryo morphology outside the incubator. Observational studies using time lapse monitoring suggest that 24 hours monitoring may introduce new dynamic markers of embryonic competence.20–22 Although recent data from a randomized study showed no differences in clinical pregnancy rate or implantation rate, the efficacy of time-lapse evaluation over more traditional morphological evaluation requires further study.23 In addition, attention has to be paid to the safety of the technique, such as periodic illumination and light exposure of the embryos during development.
Alternative Embryo Selection Methods Preimplantation Genetic Screening One of the most applied and best studied alternatives to morphological selection of embryos in IVF is preimplantation genetic screening (PGS). Numerical chromosome abnormalities appear to be common in embryos that are available after IVF.24 These abnormalities are suggested to hinder implantation or to lead to miscarriage. It seems, therefore, only logical to select only embryos free of such abnormalities for transfer in an attempt to improve the live birth rate after IVF and to discard embryos with an abnormal number of chromosomes. This is the concept of PGS. The first pregnancies after PGS were reported in 1995 and since then, PGS has been increasingly used.25,26 At first, it was mainly offered to women of advanced maternal age as an increased number of aneuploidies was found in both embryos and clinically recognized pregnancies of these women that coincided with a decreased chance of pregnancy.27,28 PGS was then also offered to women with recurrent miscarriage, Seminars in Reproductive Medicine
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women with a history of repeated implantation failure (i.e., several failed IVF cycles), women with a partner with low sperm quality (severe male factor), and even to younger women with a good prognosis since high percentages of aneuploidies were found in the embryos of these women as well.29–39 In the beginning, observational studies demonstrated that PGS was associated with higher implantation rates per transferred embryo, although no increase in ongoing pregnancy rate per initiated cycle or per oocyte retrieval was reported.40–44 After publication of multiple randomized controlled trials, however, it was concluded that PGS did not increase live birth rates after IVF.45–47 In fact, PGS, in the form in which it had been widely practiced for years, added a significant additional cost to the IVF treatment of many women without robust evidence of any benefit, and for some woman, the chance of a live birth has probably even been harmed by adding PGS to the IVF procedure.47 In the majority of PGS cycles, one or two blastomeres were aspirated from a cleavage stage day 3 embryo, followed by chromosomal analysis of the aspirated blastomere(s) with the use of fluorescent in situ hybridization (FISH).26 Reasons why PGS has not lived up to its expectations include both technical aspects, such as harm from the biopsy procedure and limitations and failure rate of the FISH analysis, and the biological feature of embryonic mosaicism. It seems plausible to assume that an embryo would at least not benefit from the invasive biopsy procedure, although the extent to which this could be harmful for an embryo is still under discussion.48–51 FISH is only able to analyze a limited number of chromosomes in each nucleus, and as such aneuploidies in chromosomes that are not tested for will be missed. The estimated accuracy per probe is only 92 to 99%, which leads to misdiagnoses. In addition, FISH misses partial and segmental aneuploidy since the FISH probes hybridize to a specific locus or the centromere and provide information only about that segment of the chromosome.52–54 Finally, cells taken from the embryo, especially at cleavage stage may not be representative for the rest of the embryo. In fact, more than 50% of human preimplantation embryos have been shown to be diploid-aneuploid mosaic at cleavage stage.24,54 This undermines PGS efficacy as it potentially discards and excludes embryos from transfer that could potentially develop into a live born child.
New Forms of Preimplantation Genetic Screening The inefficacy of PGS at the cleavage stage using FISH for the analysis led to the development and clinical introduction of new methods of PGS. This concerns PGS methods where the biopsy is being performed at a different stage of development and/or other, more accurate and comprehensive methods to analyze the biopsied cell(s). Aspiration of polar bodies or TE cells can also be performed to allow the detection of chromosome copy numbers.25,55,56 Analysis of polar bodies seems to be less invasive and avoids the difficulties presented by mosaicism. However, only the genetic status of the oocyte, and not the embryo, can be analyzed from polar body biopsies.25 Analysis of TE cells from the blastocyst, which does include both the maternal and paternal genetic
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component, is suggested to be more accurate since more cells can be analyzed. In addition, the analysis of multiple cells also limits (but does not exclude) the problem of mosaicism.57 To improve the accuracy of the analysis and the ability to analyze all 24 chromosomes, different methods for comprehensive chromosome analysis in PGS, such as comparative genomic hybridization (CGH) arrays and single nucleotide polymorphism arrays, have been introduced.54,58–65 The introduction of these new methods has lead, again, to promising first results regarding accuracy and pregnancy rates.58,66,67 A multicenter randomized controlled trial evaluating the clinical efficacy of PGS using polar body biopsy and array CGH for the analysis is currently underway.68–70 Until such trials are completed, there seems to be insufficient evidence to allow introduction of these new forms of PGS into routine clinical practice.71 Technically improved forms of PGS should be introduced into clinical practice with caution, as the same issues that caused PGS not to live up to its expectations before, could hinder its efficacy again. This should include evaluation of data resulting from well-designed randomized trials that reveal whether these new forms of PGS actually improve the live birth rate per started IVF cycle compared with standard IVF care that involves selection of embryos by morphological evaluation.
Other There are various other selection methods that have been proposed as an alternative to morphological selection or PGS. These include new techniques for evaluating embryo viability by new microscopy systems and embryo assessment based on the analysis of embryo metabolism. These approaches are all based on the hypothesis that an embryo that results in a pregnancy develops differently or alters its environment differently compared with a non- or less-viable embryo. The goal of these new approaches is to accurately predict the developmental potential of an individual embryo. Birefringent imaging uses a polarized microscope to illuminate structures, such as membranes, microtubules, microfilaments, and other cytoskeletal structures, of the meiotic spindle and the zona pellucida for analysis.72 No clinical data are available that properly reports on the efficacy of this technique.73,74 Measurement of oxygen consumption by an ultrasensitive respirometer showed a higher oxygen consumption by those oocytes which generated embryos that implanted compared with those that did not implant.75,76 However, so far, only two cohort studies have compared oxygen consumption rates and reproductive outcome. Changes in pyruvate or glucose concentration in the culture medium can reflect the energy metabolism of the embryo.77–79 The usefulness of measuring these specific metabolites as a tool to predict embryo quality is not clear since contradictive results have been reported.80–82 The turnover of several amino acids seems to be correlated with developmental potential of the embryos.83–85 Consumption and secretion of amino acids can be measured using reversephase high performance liquid chromatography and proton nuclear magnetic resonance, but thus far these methods have not been tested in clinical settings.83–85
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Recently, the analysis of single metabolites has been replaced by a broader approach using metabolomic or protein secrotome profiling.86,87 Several studies have analyzed culture media of embryos that were transferred to the uterus on days 2, 3, and 5 by near-infrared spectroscopy, and showed higher mean viability scores for embryos that resulted in a pregnancy with fetal heart activity, compared with those that failed to achieve pregnancy.85,88,89 However, recent randomized controlled trials found no significant differences between embryo selection based on the metabolome and embryo selection based on morphology.90,91 Of note, in one of these trials for 138 transfers where selection was based on a combination of morphology and metabolomic profile, there was also an independent registration of which embryos would have been selected for transfer based on morphology alone. In 75.4% (104 of 138) of the transfers, the embryo with the best morphology did not have the highest viability score and thus different embryos would have been transferred in the case of selection by morphology alone. Interestingly though, there was no significant difference between the live birth rates of these 104 transferred embryos (30.8%; 32 of 104) and the transferred embryos in the control group (31.9%; 52 of 163); (Relative risk [RR], 0.96, 95% confidence interval [CI] ¼ 0.67– 1.39, p ¼ 0.85).90 This suggests that within a group of morphological good quality embryos, there is more than one embryo able to develop into an ongoing pregnancy, possibly without large differences in chances of ongoing implantation between these embryos. Although studies have reported positive results regarding correlation between metabolic status of the embryo and its viability, available clinical data on successful pregnancy outcomes do not support the use of either proteomic or metabolomics approaches.90–92 In summary, routine use of any of these alternative selection methods seems premature, as only a very limited number of properly designed trials are available and none of these trials showed an increased efficacy for any of the alternative selection methods compared with standard morphological selection.
The Changing Role of Embryo Selection The main goal of embryo selection has always been the improvement of pregnancy rates after IVF as cryopreservation of (supernumerary) embryos was considered to lower the implantation potential of these embryos.93,94 In the slow freezing cryopreservation protocols that have been used for many years, lethal ice formation and cell damage was assumed to lower pregnancy rates compared with transfer of fresh embryos.95 Now, two developments challenge this goal of embryo selection. First of all, cryopreservation techniques have improved over the years, and it has been suggested that even better outcomes may be expected with the introduction of vitrification protocols.96 Vitrification prevents ice crystals formation by using high concentrations of cryoprotectants thereby causing less cell damage.97,98 Vitrification indeed showed a higher rate of postthaw embryo survival when compared with slow freezing.99–101 Evaluation of clinical data Seminars in Reproductive Medicine
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in a systematic review and meta-analysis demonstrated that vitrification was superior to slow freezing for clinical and ongoing pregnancy rate, and for implantation rate (odds ratio [OR] ¼ 1.55, 95% CI ¼ 1.03–2.32; OR ¼ 1.82, 95% CI ¼ 1.04– 3.20; and OR ¼ 1.49, 95% CI ¼ 1.03–2.15, respectively).101 However, only four studies were included in these comparisons and data of different embryo stages were taken together in the analysis due to the limitation of studies. Second, there is increasing evidence that the ovarian hyperstimulation used in IVF could have a negative effect on endometrial receptivity and the synchronization between embryo and endometrial development.102–104 High levels of estrogen are suggested to play a role in the impairment of endometrial receptivity.105,106 This advocates for transferring embryos back to the uterus in a nonhyperstimulated cycle. Studies on ovarian hyperstimulation syndrome (OHSS) and oocyte donation cycles, where all embryos were electively cryopreserved and subsequently transferred in nonhyperstimulated cycles, support this hypothesis. A Cochrane review comparing the effectiveness of the elective cryopreservation of all embryos for the prevention of OHSS with fresh embryo transfer showed similar pregnancy rates to the fresh transfer, although this conclusion was based on only two randomized clinical trials.107 Similarly, retrospective studies of shared oocyte cycles, in which the retrieved oocytes were shared between donor and recipient showed significantly higher pregnancy rates in the recipient compared with the donors in the stimulated cycle, whereas no difference in pregnancy rate was observed between the cryocycles of the donor and recipient.103,108 These results were obtained using slow freeze embryo cryopreservation protocols so with the use of vitrification even better results could be expected. It can thus be hypothesized that all available embryos in an IVF cycle can be cryopreserved and transferred in subsequent cycles without impairment of pregnancy rates, or maybe even with improvement of pregnancy rates, as the positive effect of a nonstimulated, more receptive endometrium could outweigh the negative effect of embryo cryopreservation. Two recent randomized controlled trials support this theory.109,110 One well-designed trial showed a higher ongoing pregnancy rate with frozen-thawed embryo transfer after elective vitrification of all embryos when compared with the transfer of fresh embryos (OR ¼ 1.66, 95% CI ¼ 1.07–2.56, p ¼ 0.02), but this trial only included women under the age of 38 years with a high ovarian response to controlled ovarian hyperstimulation.109 Another trial also reported higher ongoing pregnancy rates in cycles with elective frozen-thawed embryo transfers when compared with cycles with fresh embryo transfers, in this case in women under the age of 41 years, but in this study a not so common IVF protocol was used.110 Women in the cryopreservation arm had all their available zygotes (2PN embryos) cryopreserved and the entire cohort of embryos was subsequently thawed in a cycle without ovarian hyperstimulation. After culturing the 2PN embryos to the blastocyst stage, embryos were selected for transfer and supernumerary embryos were again cryopreserved. More well-designed and powered studies are needed to corroborate these findings, also for other groups of women. Seminars in Reproductive Medicine
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Besides efficacy, recent data on the safety of IVF also argues toward implementing a freeze all concept. A meta-analysis of studies on the follow-up of IVF pregnancies showed that women who were pregnant after receiving cryopreserved thawed embryo transfers were less prone to pregnancy complications, such as small for gestational age (RR ¼ 0.45; 95% CI ¼ 0.30–0.66), preterm birth (RR ¼ 0.84; 95% CI ¼ 0.78–0.90), low birth weight (RR ¼ 0.69; 95% CI ¼ 0.62– 0.76), perinatal mortality (RR ¼ 0.68; 95% CI ¼ 0.48–0.96), and antepartum hemorrhage (RR ¼ 0.67; 95% CI ¼ 0.55– 0.81) when compared with women receiving fresh embryo transfers.111 This suggests that in case the efficacy of a freeze all scenario is comparable to fresh transfer, a freeze all scenario is still preferable as it will lead to less comorbidity and more healthy children. The role of embryo selection in IVF needs to be reevaluated in a scenario where all available embryos are cryopreserved and transferred in subsequent cycles. Embryo selection will no longer be able to improve live birth rates in such a situation, as by definition the live birth rate per stimulated IVF cycle simply cannot be improved when all available embryos are serially transferred in optimal conditions. On the contrary, embryo selection could even lower the live birth rate after IVF. If selection is being used to identify and discard nonviable embryos, as for example is being done in PGS, it will lower the live birth rate in case it identifies nonviable embryos with an accuracy that lies below 100 percent. The latter currently holds for all available embryo selection methods. A selection method that identifies nonviable embryos with 100% accuracy is not to be expected in the near future. One role for embryo selection would still remain and that is to select which embryo to transfer first. In case a selection method is not used to discard embryos, but to rank the available embryos according to their viability, to transfer the embryo(s) with highest implantation potential first, it will shorten the time to pregnancy for a woman.
Conclusion New embryo selection methods show promising results, but proper evidence regarding their efficacy, in terms of improved live birth rates per stimulated IVF cycle compared with traditional morphological selection, is lacking. Still many of these techniques already have been implemented in routine clinical practice. When PGS using cleavage stage biopsy and FISH was developed and introduced into clinical practice there was in fact also a lack of properly designed studies. To prevent repetition of harm, it is crucial to provide sufficient evidence on the efficacy of new PGS techniques or other new embryo selection methods in IVF. However, evidence is accumulating that all available embryos in an IVF cycle can be cryopreserved and transferred in subsequent cycles without impairing pregnancy rates or maybe even with an improvement in pregnancy rates. In such a situation embryo selection will no longer be able to improve live birth rates. On the contrary, embryo selection might even lower the live birth rate after IVF, if it is being used to identify and discard nonviable embryos with less than 100% accuracy.
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Comprehensive molecular cytogenetic analysis of the human blastocyst stage. Hum Reprod 2008;23(11):2596–2608 Wells D, Delhanty JD. Comprehensive chromosomal analysis of human preimplantation embryos using whole genome amplification and single cell comparative genomic hybridization. Mol Hum Reprod 2000;6(11):1055–1062 Handyside AH, Robinson MD, Simpson RJ, et al. Isothermal whole genome amplification from single and small numbers of cells: a new era for preimplantation genetic diagnosis of inherited disease. Mol Hum Reprod 2004;10(10):767–772 Le Caignec C, Spits C, Sermon K, et al. Single-cell chromosomal imbalances detection by array CGH. Nucleic Acids Res 2006; 34(9):e68 Spits C, Le Caignec C, De Rycke M, et al. Whole-genome multiple displacement amplification from single cells. Nat Protoc 2006; 1(4):1965–1970 Iwamoto K, Bundo M, Ueda J, et al. Detection of chromosomal structural alterations in single cells by SNP arrays: a systematic survey of amplification bias and optimized workflow. PLoS ONE 2007;2(12):e1306 Forman EJ, Hong KH, Ferry KM, et al. In vitro fertilization with single euploid blastocyst transfer: a randomized controlled trial. Fertil Steril 2013;100(1):100, e1 Capalbo A, Bono S, Spizzichino L, et al. Sequential comprehensive chromosome analysis on polar bodies, blastomeres and trophoblast: insights into female meiotic errors and chromosomal segregation in the preimplantation window of embryo development. Hum Reprod 2013;28(2):509–518 Harper J, Sermon K, Geraedts J, et al. What next for preimplantation genetic screening? Hum Reprod 2008;23(3):478–480 Geraedts J, Collins J, Gianaroli L, et al. What next for preimplantation genetic screening? A polar body approach!. Hum Reprod 2010;25(3):575–577 Geraedts J, Montag M, Magli MC, et al. Polar body array CGH for prediction of the status of the corresponding oocyte. Part I: clinical results. Hum Reprod 2011;26(11):3173–3180 Gleicher N, Barad DH. A review of, and commentary on, the ongoing second clinical introduction of preimplantation genetic screening (PGS) to routine IVF practice. J Assist Reprod Genet 2012;29(11):1159–1166 Montag M, Schimming T, Köster M, et al. Oocyte zona birefringence intensity is associated with embryonic implantation potential in ICSI cycles. Reprod Biomed Online 2008;16(2):239–244 Wang WH, Meng L, Hackett RJ, Odenbourg R, Keefe DL. The spindle observation and its relationship with fertilization after intracytoplasmic sperm injection in living human oocytes. Fertil Steril 2001;75(2):348–353 Molinari E, Evangelista F, Racca C, Cagnazzo C, Revelli A. Polarized light microscopy-detectable structures of human oocytes and embryos are related to the likelihood of conception in IVF. J Assist Reprod Genet 2012;29(10):1117–1122 Tejera A, Herrero J, de Los Santos MJ, Garrido N, Ramsing N, Meseguer M. Oxygen consumption is a quality marker for human oocyte competence conditioned by ovarian stimulation regimens. Fertil Steril 2011;96(3):618–623, e2 Tejera A, Herrero J, Viloria T, Romero JL, Gamiz P, Meseguer M. Time-dependent O2 consumption patterns determined optimal time ranges for selecting viable human embryos. Fertil Steril 2012;98(4):849–857, e1–e3 Hardy K, Hooper MA, Handyside AH, Rutherford AJ, Winston RM, Leese HJ. Non-invasive measurement of glucose and pyruvate uptake by individual human oocytes and preimplantation embryos. Hum Reprod 1989;4(2):188–191 Gott AL, Hardy K, Winston RM, Leese HJ. Non-invasive measurement of pyruvate and glucose uptake and lactate production by single human preimplantation embryos. Hum Reprod 1990;5(1): 104–108
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assessment of human embryo nutrient consumption as a measure of developmental potential. Fertil Steril 2001;76(6): 1175–1180 Conaghan J, Handyside AH, Winston RM, Leese HJ. Effects of pyruvate and glucose on the development of human preimplantation embryos in vitro. J Reprod Fertil 1993;99(1):87–95 Conaghan J, Hardy K, Handyside AH, Winston RM, Leese HJ. Selection criteria for human embryo transfer: a comparison of pyruvate uptake and morphology. J Assist Reprod Genet 1993; 10(1):21–30 Turner K, Martin KL, Woodward BJ, Lenton EA, Leese HJ. Comparison of pyruvate uptake by embryos derived from conception and nonconception natural cycles. Hum Reprod 1994;9(12):2362–2366 Houghton FD, Hawkhead JA, Humpherson PG, et al. Non-invasive amino acid turnover predicts human embryo developmental capacity. Hum Reprod 2002;17(4):999–1005 Brison DR, Houghton FD, Falconer D, et al. Identification of viable embryos in IVF by non-invasive measurement of amino acid turnover. Hum Reprod 2004;19(10):2319–2324 Seli E, Botros L, Sakkas D, Burns DH. Noninvasive metabolomic profiling of embryo culture media using proton nuclear magnetic resonance correlates with reproductive potential of embryos in women undergoing in vitro fertilization. Fertil Steril 2008;90(6): 2183–2189 Katz-Jaffe MG, Gardner DK, Schoolcraft WB. Proteomic analysis of individual human embryos to identify novel biomarkers of development and viability. Fertil Steril 2006;85(1):101–107 Nel-Themaat L, Nagy ZP. A review of the promises and pitfalls of oocyte and embryo metabolomics. Placenta 2011;32(Suppl 3): S257–S263 Seli E, Sakkas D, Scott R, Kwok SC, Rosendahl SM, Burns DH. Noninvasive metabolomic profiling of embryo culture media using Raman and near-infrared spectroscopy correlates with reproductive potential of embryos in women undergoing in vitro fertilization. Fertil Steril 2007;88(5):1350–1357 Vergouw CG, Botros LL, Roos P, et al. Metabolomic profiling by near-infrared spectroscopy as a tool to assess embryo viability: a novel, non-invasive method for embryo selection. Hum Reprod 2008;23(7):1499–1504 Vergouw CG, Kieslinger DC, Kostelijk EH, et al. Day 3 embryo selection by metabolomic profiling of culture medium with nearinfrared spectroscopy as an adjunct to morphology: a randomized controlled trial. Hum Reprod 2012;27(8):2304–2311 Hardarson T, Ahlström A, Rogberg L, et al. Non-invasive metabolomic profiling of Day 2 and 5 embryo culture medium: a prospective randomized trial. Hum Reprod 2012;27(1):89–96 Katz-Jaffe MG, Gardner DK. Symposium: innovative techniques in human embryo viability assessment. Can proteomics help to shape the future of human assisted conception? Reprod Biomed Online 2008;17(4):497–501 Edgar DH, Bourne H, Speirs AL, McBain JC. A quantitative analysis of the impact of cryopreservation on the implantation potential of human early cleavage stage embryos. Hum Reprod 2000;15(1): 175–179 Wang JX, Yap YY, Matthews CD. Frozen-thawed embryo transfer: influence of clinical factors on implantation rate and risk of multiple conception. Hum Reprod 2001;16(11):2316–2319 Shaw JM, Jones GM. Terminology associated with vitrification and other cryopreservation procedures for oocytes and embryos. Hum Reprod Update 2003;9(6):583–605
(mini-IVF) for IVF utilizing vitrification and cryopreserved embryo transfer. Reprod Biomed Online 2010;21(4):485–495 Youssry M, Ozmen B, Zohni K, Diedrich K, Al-Hasani S. Current aspects of blastocyst cryopreservation. Reprod Biomed Online 2008;16(2):311–320 Liebermann J, Nawroth F, Isachenko V, Isachenko E, Rahimi G, Tucker MJ. Potential importance of vitrification in reproductive medicine. Biol Reprod 2002;67(6):1671–1680 Kolibianakis EM, Venetis CA, Tarlatzis BC. Cryopreservation of human embryos by vitrification or slow freezing: which one is better? Curr Opin Obstet Gynecol 2009;21(3):270–274 Loutradi KE, Kolibianakis EM, Venetis CA, et al. Cryopreservation of human embryos by vitrification or slow freezing: a systematic review and meta-analysis. Fertil Steril 2008;90(1):186–193 AbdelHafez FF, Desai N, Abou-Setta AM, Falcone T, Goldfarb J. Slow freezing, vitrification and ultra-rapid freezing of human embryos: a systematic review and meta-analysis. Reprod Biomed Online 2010;20(2):209–222 Paulson RJ, Sauer MV, Lobo RA. Embryo implantation after human in vitro fertilization: importance of endometrial receptivity. Fertil Steril 1990;53(5):870–874 Check JH, Choe JK, Katsoff D, Summers-Chase D, Wilson C. Controlled ovarian hyperstimulation adversely affects implantation following in vitro fertilization-embryo transfer. J Assist Reprod Genet 1999;16(8):416–420 Shapiro BS, Daneshmand ST, Garner FC, Aguirre M, Ross R. Contrasting patterns in in vitro fertilization pregnancy rates among fresh autologous, fresh oocyte donor, and cryopreserved cycles with the use of day 5 or day 6 blastocysts may reflect differences in embryo-endometrium synchrony. Fertil Steril 2008;89(1):20–26 Krikun G, Schatz F, Taylor R, et al. Endometrial endothelial cell steroid receptor expression and steroid effects on gene expression. J Clin Endocrinol Metab 2005;90(3):1812–1818 Liu Y, Lee KF, Ng EH, Yeung WS, Ho PC. Gene expression profiling of human peri-implantation endometria between natural and stimulated cycles. Fertil Steril 2008;90(6):2152–2164 D’Angelo A, Amso N. Embryo freezing for preventing ovarian hyperstimulation syndrome. Cochrane Database Syst Rev 2007; (3):CD002806 Check JH, O’Shaughnessy A, Lurie D, Fisher C, Adelson HG. Evaluation of the mechanism for higher pregnancy rates in donor oocyte recipients by comparison of fresh with frozen embryo transfer pregnancy rates in a shared oocyte programme. Hum Reprod 1995;10(11):3022–3027 Aflatoonian A, Oskouian H, Ahmadi S, Oskouian L. Can fresh embryo transfers be replaced by cryopreserved-thawed embryo transfers in assisted reproductive cycles? A randomized controlled trial. J Assist Reprod Genet 2010;27(7):357–363 Shapiro BS, Daneshmand ST, Garner FC, Aguirre M, Hudson C, Thomas S. Evidence of impaired endometrial receptivity after ovarian stimulation for in vitro fertilization: a prospective randomized trial comparing fresh and frozen-thawed embryo transfer in normal responders. Fertil Steril 2011;96(2):344–348 Maheshwari A, Pandey S, Shetty A, Hamilton M, Bhattacharya S. Obstetric and perinatal outcomes in singleton pregnancies resulting from the transfer of frozen thawed versus fresh embryos generated through in vitro fertilization treatment: a systematic review and meta-analysis. Fertil Steril 2012;98(2):368–377, e1–e9
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Limitations of Embryo Selection Methods
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Limitations of Embryo Selection Methods Sjoerd Repping, PhD1
Sebastiaan Mastenbroek, PhD1
1 Center for Reproductive Medicine, Academic Medical Center,
University of Amsterdam, Amsterdam, The Netherlands Semin Reprod Med 2014;32:127–133
Abstract
Keywords
► ► ► ► ►
assisted reproduction embryo transfer embryo selection cryopreservation IVF outcome
Address for correspondence Sebastiaan Mastenbroek, PhD, Center for Reproductive Medicine, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands (e-mail: [email protected]).
In in vitro fertilization (IVF), the selection of embryos for transfer is generally based on the morphology of the available embryos. However, not all embryos with good morphology implant and on average only one in four treatments are successful. This has driven a quest for alternative selection methods. The best known alternative selection method is preimplantation genetic screening (PGS), which has been used for over a decade before it was shown to be inferior to morphological selection. Now, new forms of PGS (performing biopsy at another stage of development and new methods for analysis) are emerging, just like alternative noninvasive embryo selection methods. However, the concept that better selection will lead to improved IVF results is not so certain anymore. Evidence is accumulating that all available embryos in an IVF cycle can be cryopreserved and transferred in subsequent cycles without impairing pregnancy rates or maybe even with an improvement in pregnancy rates. Embryo selection will then no longer be able to improve the live birth rate in IVF; it could even lower the live birth rate. Embryo selection will only be able to improve the time to pregnancy, if embryos with the highest implantation potential are transferred first.
Since the first report of a live born child after an in vitro fertilization (IVF) procedure in 1978, IVF has emerged as a well-established reproductive technique for subfertile couples.1 The International Committee for Monitoring Assisted Reproductive Technology reported a total of 954,743 initiated cycles which resulted in an estimated 237,809 babies born in 2004 worldwide.2 The IVF procedure has changed and improved ever since its introduction. The development of IVF started with the IVF of human oocytes, followed by the culture of human embryos up to morula stage and blastocyst stage.3–7 The retrieval of oocytes was initially performed in a natural cycle, but not much later controlled ovarian hyperstimulation was introduced to mature multiple follicles and to obtain multiple oocytes.4 This allowed transfer of multiple embryos to increase pregnancy rates.8,9 However, as clinical and laboratory protocols became more efficient, this also resulted in an increased number of multiple births and with that adverse perinatal outcomes.10 As a consequence, embryo transfer policies restricting the number of embryos to be transferred to reduce the incidence of multiple pregnancies while main-
Issue Theme Selecting the Best Embryo; Guest Editor, Valerie L. Baker, MD
taining or improving overall pregnancy rates were introduced.11 Methods to select the best embryo(s) with the highest implantation potential for transfer became increasingly important. Embryos have been assessed morphologically ever since the start of IVF to evaluate their development in culture, which resulted in various morphological scoring methods for determining the developmental potential of embryos.12–15 As morphological parameters are correlated with the chance of an embryo leading to a live born child after transfer, morphology became the golden standard for selecting the best embryo for transfer.15 However, not all embryos with good morphology implant and on average only one in four of all IVF treatments are successful.16,17 Alternative methods of embryo selection have, therefore, been introduced over the years in an attempt to improve the success rates of IVF treatments. In this review, we will first briefly evaluate the available evidence on the efficacy of these new methods compared with morphological selection of embryos, and we will then consider the possible changing role and use of embryo selection in the near future of IVF.
Copyright © 2014 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) 584-4662.
DOI http://dx.doi.org/ 10.1055/s-0033-1363554. ISSN 1526-8004.
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Kai Mee Wong, MD1
Limitations of Embryo Selection Methods
Wong et al.
Selection Using Embryo Morphology Morphological evaluation is the most commonly used method for selecting the best embryo for transfer.15,18 There are different embryo scoring variables to predict the potential of an embryo to lead to a live birth after transfer to the uterus. As embryo transfer can be performed at different stages during preimplantation development, that is, at pronuclear (PN) stage, cleavage stage, or blastocyst stage, there are different morphological parameters for each stage. Morphological selection of embryos is largely based on clinical experience and local protocols.15 As a consequence, the European Society of Human Reproduction and Embryology recently provided consensus points to define the minimum criteria for oocyte and embryo morphology assessment.15 Aspects that could be assessed are PN number, PN size, location of nuclear polar bodies, number of blastomeres, blastomere size, degree of fragmentation, multinucleation, compaction, expansion, and trophectoderm (TE) and inner cell mass quality.15 As a large proportion of the embryos do not exactly follow the expected developmental timeline and morphological scoring is often performed on a limited number of fixed time points only, new recording systems that allow 24 hours monitoring have been developed.19 These devices allow 24 hours monitoring by using cameras incorporated in the incubation chamber, without disturbing culture conditions as is the case for assessment of embryo morphology outside the incubator. Observational studies using time lapse monitoring suggest that 24 hours monitoring may introduce new dynamic markers of embryonic competence.20–22 Although recent data from a randomized study showed no differences in clinical pregnancy rate or implantation rate, the efficacy of time-lapse evaluation over more traditional morphological evaluation requires further study.23 In addition, attention has to be paid to the safety of the technique, such as periodic illumination and light exposure of the embryos during development.
Alternative Embryo Selection Methods Preimplantation Genetic Screening One of the most applied and best studied alternatives to morphological selection of embryos in IVF is preimplantation genetic screening (PGS). Numerical chromosome abnormalities appear to be common in embryos that are available after IVF.24 These abnormalities are suggested to hinder implantation or to lead to miscarriage. It seems, therefore, only logical to select only embryos free of such abnormalities for transfer in an attempt to improve the live birth rate after IVF and to discard embryos with an abnormal number of chromosomes. This is the concept of PGS. The first pregnancies after PGS were reported in 1995 and since then, PGS has been increasingly used.25,26 At first, it was mainly offered to women of advanced maternal age as an increased number of aneuploidies was found in both embryos and clinically recognized pregnancies of these women that coincided with a decreased chance of pregnancy.27,28 PGS was then also offered to women with recurrent miscarriage, Seminars in Reproductive Medicine
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women with a history of repeated implantation failure (i.e., several failed IVF cycles), women with a partner with low sperm quality (severe male factor), and even to younger women with a good prognosis since high percentages of aneuploidies were found in the embryos of these women as well.29–39 In the beginning, observational studies demonstrated that PGS was associated with higher implantation rates per transferred embryo, although no increase in ongoing pregnancy rate per initiated cycle or per oocyte retrieval was reported.40–44 After publication of multiple randomized controlled trials, however, it was concluded that PGS did not increase live birth rates after IVF.45–47 In fact, PGS, in the form in which it had been widely practiced for years, added a significant additional cost to the IVF treatment of many women without robust evidence of any benefit, and for some woman, the chance of a live birth has probably even been harmed by adding PGS to the IVF procedure.47 In the majority of PGS cycles, one or two blastomeres were aspirated from a cleavage stage day 3 embryo, followed by chromosomal analysis of the aspirated blastomere(s) with the use of fluorescent in situ hybridization (FISH).26 Reasons why PGS has not lived up to its expectations include both technical aspects, such as harm from the biopsy procedure and limitations and failure rate of the FISH analysis, and the biological feature of embryonic mosaicism. It seems plausible to assume that an embryo would at least not benefit from the invasive biopsy procedure, although the extent to which this could be harmful for an embryo is still under discussion.48–51 FISH is only able to analyze a limited number of chromosomes in each nucleus, and as such aneuploidies in chromosomes that are not tested for will be missed. The estimated accuracy per probe is only 92 to 99%, which leads to misdiagnoses. In addition, FISH misses partial and segmental aneuploidy since the FISH probes hybridize to a specific locus or the centromere and provide information only about that segment of the chromosome.52–54 Finally, cells taken from the embryo, especially at cleavage stage may not be representative for the rest of the embryo. In fact, more than 50% of human preimplantation embryos have been shown to be diploid-aneuploid mosaic at cleavage stage.24,54 This undermines PGS efficacy as it potentially discards and excludes embryos from transfer that could potentially develop into a live born child.
New Forms of Preimplantation Genetic Screening The inefficacy of PGS at the cleavage stage using FISH for the analysis led to the development and clinical introduction of new methods of PGS. This concerns PGS methods where the biopsy is being performed at a different stage of development and/or other, more accurate and comprehensive methods to analyze the biopsied cell(s). Aspiration of polar bodies or TE cells can also be performed to allow the detection of chromosome copy numbers.25,55,56 Analysis of polar bodies seems to be less invasive and avoids the difficulties presented by mosaicism. However, only the genetic status of the oocyte, and not the embryo, can be analyzed from polar body biopsies.25 Analysis of TE cells from the blastocyst, which does include both the maternal and paternal genetic
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component, is suggested to be more accurate since more cells can be analyzed. In addition, the analysis of multiple cells also limits (but does not exclude) the problem of mosaicism.57 To improve the accuracy of the analysis and the ability to analyze all 24 chromosomes, different methods for comprehensive chromosome analysis in PGS, such as comparative genomic hybridization (CGH) arrays and single nucleotide polymorphism arrays, have been introduced.54,58–65 The introduction of these new methods has lead, again, to promising first results regarding accuracy and pregnancy rates.58,66,67 A multicenter randomized controlled trial evaluating the clinical efficacy of PGS using polar body biopsy and array CGH for the analysis is currently underway.68–70 Until such trials are completed, there seems to be insufficient evidence to allow introduction of these new forms of PGS into routine clinical practice.71 Technically improved forms of PGS should be introduced into clinical practice with caution, as the same issues that caused PGS not to live up to its expectations before, could hinder its efficacy again. This should include evaluation of data resulting from well-designed randomized trials that reveal whether these new forms of PGS actually improve the live birth rate per started IVF cycle compared with standard IVF care that involves selection of embryos by morphological evaluation.
Other There are various other selection methods that have been proposed as an alternative to morphological selection or PGS. These include new techniques for evaluating embryo viability by new microscopy systems and embryo assessment based on the analysis of embryo metabolism. These approaches are all based on the hypothesis that an embryo that results in a pregnancy develops differently or alters its environment differently compared with a non- or less-viable embryo. The goal of these new approaches is to accurately predict the developmental potential of an individual embryo. Birefringent imaging uses a polarized microscope to illuminate structures, such as membranes, microtubules, microfilaments, and other cytoskeletal structures, of the meiotic spindle and the zona pellucida for analysis.72 No clinical data are available that properly reports on the efficacy of this technique.73,74 Measurement of oxygen consumption by an ultrasensitive respirometer showed a higher oxygen consumption by those oocytes which generated embryos that implanted compared with those that did not implant.75,76 However, so far, only two cohort studies have compared oxygen consumption rates and reproductive outcome. Changes in pyruvate or glucose concentration in the culture medium can reflect the energy metabolism of the embryo.77–79 The usefulness of measuring these specific metabolites as a tool to predict embryo quality is not clear since contradictive results have been reported.80–82 The turnover of several amino acids seems to be correlated with developmental potential of the embryos.83–85 Consumption and secretion of amino acids can be measured using reversephase high performance liquid chromatography and proton nuclear magnetic resonance, but thus far these methods have not been tested in clinical settings.83–85
Wong et al.
Recently, the analysis of single metabolites has been replaced by a broader approach using metabolomic or protein secrotome profiling.86,87 Several studies have analyzed culture media of embryos that were transferred to the uterus on days 2, 3, and 5 by near-infrared spectroscopy, and showed higher mean viability scores for embryos that resulted in a pregnancy with fetal heart activity, compared with those that failed to achieve pregnancy.85,88,89 However, recent randomized controlled trials found no significant differences between embryo selection based on the metabolome and embryo selection based on morphology.90,91 Of note, in one of these trials for 138 transfers where selection was based on a combination of morphology and metabolomic profile, there was also an independent registration of which embryos would have been selected for transfer based on morphology alone. In 75.4% (104 of 138) of the transfers, the embryo with the best morphology did not have the highest viability score and thus different embryos would have been transferred in the case of selection by morphology alone. Interestingly though, there was no significant difference between the live birth rates of these 104 transferred embryos (30.8%; 32 of 104) and the transferred embryos in the control group (31.9%; 52 of 163); (Relative risk [RR], 0.96, 95% confidence interval [CI] ¼ 0.67– 1.39, p ¼ 0.85).90 This suggests that within a group of morphological good quality embryos, there is more than one embryo able to develop into an ongoing pregnancy, possibly without large differences in chances of ongoing implantation between these embryos. Although studies have reported positive results regarding correlation between metabolic status of the embryo and its viability, available clinical data on successful pregnancy outcomes do not support the use of either proteomic or metabolomics approaches.90–92 In summary, routine use of any of these alternative selection methods seems premature, as only a very limited number of properly designed trials are available and none of these trials showed an increased efficacy for any of the alternative selection methods compared with standard morphological selection.
The Changing Role of Embryo Selection The main goal of embryo selection has always been the improvement of pregnancy rates after IVF as cryopreservation of (supernumerary) embryos was considered to lower the implantation potential of these embryos.93,94 In the slow freezing cryopreservation protocols that have been used for many years, lethal ice formation and cell damage was assumed to lower pregnancy rates compared with transfer of fresh embryos.95 Now, two developments challenge this goal of embryo selection. First of all, cryopreservation techniques have improved over the years, and it has been suggested that even better outcomes may be expected with the introduction of vitrification protocols.96 Vitrification prevents ice crystals formation by using high concentrations of cryoprotectants thereby causing less cell damage.97,98 Vitrification indeed showed a higher rate of postthaw embryo survival when compared with slow freezing.99–101 Evaluation of clinical data Seminars in Reproductive Medicine
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Limitations of Embryo Selection Methods
Limitations of Embryo Selection Methods
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in a systematic review and meta-analysis demonstrated that vitrification was superior to slow freezing for clinical and ongoing pregnancy rate, and for implantation rate (odds ratio [OR] ¼ 1.55, 95% CI ¼ 1.03–2.32; OR ¼ 1.82, 95% CI ¼ 1.04– 3.20; and OR ¼ 1.49, 95% CI ¼ 1.03–2.15, respectively).101 However, only four studies were included in these comparisons and data of different embryo stages were taken together in the analysis due to the limitation of studies. Second, there is increasing evidence that the ovarian hyperstimulation used in IVF could have a negative effect on endometrial receptivity and the synchronization between embryo and endometrial development.102–104 High levels of estrogen are suggested to play a role in the impairment of endometrial receptivity.105,106 This advocates for transferring embryos back to the uterus in a nonhyperstimulated cycle. Studies on ovarian hyperstimulation syndrome (OHSS) and oocyte donation cycles, where all embryos were electively cryopreserved and subsequently transferred in nonhyperstimulated cycles, support this hypothesis. A Cochrane review comparing the effectiveness of the elective cryopreservation of all embryos for the prevention of OHSS with fresh embryo transfer showed similar pregnancy rates to the fresh transfer, although this conclusion was based on only two randomized clinical trials.107 Similarly, retrospective studies of shared oocyte cycles, in which the retrieved oocytes were shared between donor and recipient showed significantly higher pregnancy rates in the recipient compared with the donors in the stimulated cycle, whereas no difference in pregnancy rate was observed between the cryocycles of the donor and recipient.103,108 These results were obtained using slow freeze embryo cryopreservation protocols so with the use of vitrification even better results could be expected. It can thus be hypothesized that all available embryos in an IVF cycle can be cryopreserved and transferred in subsequent cycles without impairment of pregnancy rates, or maybe even with improvement of pregnancy rates, as the positive effect of a nonstimulated, more receptive endometrium could outweigh the negative effect of embryo cryopreservation. Two recent randomized controlled trials support this theory.109,110 One well-designed trial showed a higher ongoing pregnancy rate with frozen-thawed embryo transfer after elective vitrification of all embryos when compared with the transfer of fresh embryos (OR ¼ 1.66, 95% CI ¼ 1.07–2.56, p ¼ 0.02), but this trial only included women under the age of 38 years with a high ovarian response to controlled ovarian hyperstimulation.109 Another trial also reported higher ongoing pregnancy rates in cycles with elective frozen-thawed embryo transfers when compared with cycles with fresh embryo transfers, in this case in women under the age of 41 years, but in this study a not so common IVF protocol was used.110 Women in the cryopreservation arm had all their available zygotes (2PN embryos) cryopreserved and the entire cohort of embryos was subsequently thawed in a cycle without ovarian hyperstimulation. After culturing the 2PN embryos to the blastocyst stage, embryos were selected for transfer and supernumerary embryos were again cryopreserved. More well-designed and powered studies are needed to corroborate these findings, also for other groups of women. Seminars in Reproductive Medicine
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Besides efficacy, recent data on the safety of IVF also argues toward implementing a freeze all concept. A meta-analysis of studies on the follow-up of IVF pregnancies showed that women who were pregnant after receiving cryopreserved thawed embryo transfers were less prone to pregnancy complications, such as small for gestational age (RR ¼ 0.45; 95% CI ¼ 0.30–0.66), preterm birth (RR ¼ 0.84; 95% CI ¼ 0.78–0.90), low birth weight (RR ¼ 0.69; 95% CI ¼ 0.62– 0.76), perinatal mortality (RR ¼ 0.68; 95% CI ¼ 0.48–0.96), and antepartum hemorrhage (RR ¼ 0.67; 95% CI ¼ 0.55– 0.81) when compared with women receiving fresh embryo transfers.111 This suggests that in case the efficacy of a freeze all scenario is comparable to fresh transfer, a freeze all scenario is still preferable as it will lead to less comorbidity and more healthy children. The role of embryo selection in IVF needs to be reevaluated in a scenario where all available embryos are cryopreserved and transferred in subsequent cycles. Embryo selection will no longer be able to improve live birth rates in such a situation, as by definition the live birth rate per stimulated IVF cycle simply cannot be improved when all available embryos are serially transferred in optimal conditions. On the contrary, embryo selection could even lower the live birth rate after IVF. If selection is being used to identify and discard nonviable embryos, as for example is being done in PGS, it will lower the live birth rate in case it identifies nonviable embryos with an accuracy that lies below 100 percent. The latter currently holds for all available embryo selection methods. A selection method that identifies nonviable embryos with 100% accuracy is not to be expected in the near future. One role for embryo selection would still remain and that is to select which embryo to transfer first. In case a selection method is not used to discard embryos, but to rank the available embryos according to their viability, to transfer the embryo(s) with highest implantation potential first, it will shorten the time to pregnancy for a woman.
Conclusion New embryo selection methods show promising results, but proper evidence regarding their efficacy, in terms of improved live birth rates per stimulated IVF cycle compared with traditional morphological selection, is lacking. Still many of these techniques already have been implemented in routine clinical practice. When PGS using cleavage stage biopsy and FISH was developed and introduced into clinical practice there was in fact also a lack of properly designed studies. To prevent repetition of harm, it is crucial to provide sufficient evidence on the efficacy of new PGS techniques or other new embryo selection methods in IVF. However, evidence is accumulating that all available embryos in an IVF cycle can be cryopreserved and transferred in subsequent cycles without impairing pregnancy rates or maybe even with an improvement in pregnancy rates. In such a situation embryo selection will no longer be able to improve live birth rates. On the contrary, embryo selection might even lower the live birth rate after IVF, if it is being used to identify and discard nonviable embryos with less than 100% accuracy.
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Limitations of Embryo Selection Methods
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