Advances in embryo selection methods.

Despite many recent advances in the field of reproductive biology and medicine, the efficiency of in vitro fertilization procedures remains relatively low. There is a need for a reliable and non-invasive method of embryo selection to ensure that only embryos with the highest developmental potential are chosen for transfer to mothers-to-be. Here, we compare various methods currently used for assessing embryonic viability, such as examination of embryonic morphology, quality of the genetic material, or metabolism. Additionally, we discuss novel procedures for embryonic assessment based on advanced time-lapse imaging techniques, which show great promise and may lead to increased in vitro fertilization efficiencies.

Introduction

Since 1978, when the first baby conceived in vitro was born, in vitro fertilization (IVF) has become a major method for treating infertility. Although IVF procedures have been greatly improved over the years, their efficiency, as defined by live birth rate, is still only about 30-40%. Consequently, many couples have to undergo treatment several times before they succeed and this increases emotional and financial costs, as well as additional health risks for women. The efficiency of IVF can be increased by transferring multiple embryos in a single cycle, but this may lead to multiple-pregnancies, and consequently serious health complications for mothers and babies. There is, therefore, a strong need for single embryo transfers, and so the development of reliable methods of selecting embryos is crucial [1,2]. Here, we provide a short review of current techniques for embryo assessment, with a focus on new ways of distinguishing high-quality embryos based on recent advances in time-lapse imaging technology.

Static assessment of embryo morphology by light microscopy

Visual assessment of embryo morphology is the most traditional and popular method of embryo selection. Several parameters can be assessed at different developmental stages, providing valuable information about the quality of embryos [3]. Embryos can be graded according to the morphology of their pronuclei on day 1 after fertilization [4-6], the number and shape of blastomeres and degree of fragmentation on day 2 or 3 [7-9], or by the morphology of the blastocyst at day 5 or 6 [10-12]. Depending on the procedure, embryos are evaluated at one or several developmental stages.

Although morphological assessment is inexpensive and easy to implement in a clinical environment, it has its drawbacks. Firstly, the visual grading of embryos is subjective and requires significant expertise. Moreover, even when such expertise is available, the technique is not always accurate: low-graded embryos often prove to have high developmental potential and can develop to term. Therefore, there has been a drive to develop an alternative method of embryo evaluation that provides more detailed information about the developmental status of the embryo and, most importantly, is quantitative and objective.

Preimplantation genetic diagnosis and screening

Preimplantation genetic diagnosis has been developed to detect inherited genetic disorders. It uses polymerase chain reaction (PCR)-based techniques to diagnose specific genetic mutations [13] or fluorescence in situ hybridization (FISH) to diagnose chromosomal abnormalities or to sex the embryos for patients carrying X-chromosome-linked diseases [14-16]. Preimplantation genetic diagnosis can be applied to embryos at different stages: zygotes (polar body biopsy), cleavage stage embryos (blastomere biopsy) or blastocysts (trophectoderm biopsy).

An alternative approach is preimplantation genetic screening, which was developed to improve IVF outcomes in mothers of advanced age, those presenting with repeated miscarriages or implantation failure, or in cases of severe male factor infertility. The classic form of preimplantation genetic screening, involving FISH assessment of a limited number of chromosomes in one blastomere of a cleavage stage embryo, has been shown to be ineffective and has been gradually replaced by technologies based on comparative genomic hybridization (CGH) or single nucleotide polymorphism (SNP) arrays [17,18]. Both of these approaches allow the whole embryonic genome to be analysed and, as for preimplantation genetic diagnosis, used at different embryonic stages.

Metabolomics and proteomics in screening

Another method of embryo selection is based on the analysis of embryo metabolism. Changes in pyruvate or glucose concentration in the culture medium can reflect the energy metabolism of the embryo, although their usefulness as a tool to predict the embryo's quality is not clear [19-23]. On the other hand, oxygen consumption [24-27] and amino-acid turnover [28-30] have proved to be more reliable indicators of an embryo's viability. Recently, the analysis of single metabolites has been gradually replaced by a broader approach: metabolomic or protein secrotome profiling [31], which can provide a complete picture of an embryo's metabolism and gene expression patterns. Although many reports have shown a correlation between metabolic status of the embryo and its viability, establishing its potential value for the IVF clinics, for the moment these techniques remain difficult to implement in a clinical environment. This is because metabolomic or secreted protein analyses involve spectroscopic/spectrometric and chromatographic techniques, which currently require expensive equipment and highly skilled staff [31].

Dynamic assessment of embryonic development by time-lapse imaging

Due to recent advances in non-invasive time-lapse imaging established in mouse embryos [32], we can now follow the dynamics of embryo divisions and other fertilization-triggered events and correlate them with the developmental potential of the embryos. The last two years have seen two studies employing this technique in a very different way. A team at Stanford University has shown that the timing and synchrony of the first two embryonic divisions are predictive of developmental potential of human embryos (Figure 1) [33]. The authors reported that embryos with a very long first cytokinesis, with a prolonged or an atypically short interval between first and second division or with highly asynchronous divisions of two-cell blastomeres, fail to reach the blastocyst stage. This accords with previous observations, in which timely pronuclear formation and subsequent first cleavage correlated with higher quality of human embryos [34-36]. A completely different approach has been developed by our team at University of Cambridge working collaboratively with teams at Oxford and Cardiff Universities (Figure 2) [37]. In this work, we showed that fertilization of mouse eggs triggers abrupt, repetitive cytoplasmic movements that correlated with Ca2+ oscillations (also triggered by sperm) and depended on the functionality of the cytoskeleton. Moreover, the cytoplasmic flows were predictive of the developmental potential of the embryos. Embryos that showed very frequent increases in cytoplasmic movements (indicating very frequent Ca2+ oscillations) and low cytoplasmic speeds in the intervening periods (reflecting low quality of the actin cytoskeleton) were three-fold less successful in developing to pups than embryos displaying average values of these parameters. Although both methods still have to be tested in a clinical environment and subjected to randomised controlled trials to demonstrate improved live birth outcomes, they offer great hope for more reliable assessment of embryonic quality.

Figure 1.

Novel methods of predicting mammalian embryo viability involving an advanced time-lapse imaging

The method described by Wong et al. involves time-lapse imaging of embryos every 5 min for several hours (from 1- to 4-cell stage) [33]. In comparison, the method established by Ajduk et al. requires a much shorter period of recording (2.5 hours for mouse embryos) but with images taken every 10 seconds [37].

Figure 2.

Analysis of fertilization-triggered cytoplasmic movements in mouse zygotes

Mouse eggs are subjected to time-lapse imaging (1 frame every 10 seconds for 2.5 hours) immediately after fertilization [37]. Acquired images are analysed by the particle image velocimetry method that follows patterns of contrasts between subsequent images and calculates how they move. The sum of all displacement vectors calculated for the zygote in a given time-point (i.e. mean cytoplasmic speed) is plotted over time. The graph shows when fast cytoplasmic movements occurred in the embryo. Mean interval between the fast movements (in red) and mean speed in periods inbetween the fast movements (mean basal speed, in blue) are indicative of developmental potential of the embryo.

Conclusion

Research carried out over the past three decades has provided a broad repertoire of possible improved embryo selection methods. Some of them, such as evaluation of pronuclear morphology, preimplantation genetic diagnosis and preimplantation genetic screening, focus on the nuclear component of the embryo, looking for potential chromosomal abnormalities. Others (e.g. analysis of metabolites, secreted proteins or cytoplasmic flows) concentrate on the cytoplasmic component, analysing the quality of embryo metabolism, the cytoskeleton or Ca2+ homeostasis. We believe that the ultimate selection method should combine these approaches. This can be achieved for instance by following the timing of embryonic cell divisions, as their duration and synchrony can be affected by improper chromosome segregation, cytoskeletal properties and energy levels. An alternative approach could combine preimplantation genetic screening with the analysis of embryo metabolism or cytoplasmic movements. Combination of genetic testing of the polar body and examination of the cytoplasmic flows is especially promising, as it would provide information about embryo quality within several hours after fertilization, and, therefore, significantly quicken the selection process (Figure 1). Indeed, recent publications suggest that a shorter period between in vitro fertilization and embryo transfer may be beneficial, as prolonged in vitro culture of embryos alters their epigenetic modifications and gene expression [38,39]. Through a combination of these new methods, it is reasonable to expect that embryo selection for IVF will become much more reliable in the coming years.