Paving the way for a gold standard of care for infertility treatment: improving outcomes through standardization of laboratory procedures

Commercialized culture media has improved IVF success and is a vital factor influencing IVF outcome, affecting pre- and post-implantation stages (). Extended embryo culture prolongs growth, enables more advanced embryos to be selected, and has been linked to improved IVF success in young patients with a low body mass index (). As with any embryo selection technique, extended culture to the blastocyst stage may shorten time to pregnancy (with higher pregnancy rates per transfer and implantation rate; , ), but does not improve cumulative pregnancy rate (, ). Further research is required to establish optimum culture conditions for embryo development and the optimal time for transfer ().

The proliferation of commercially available culture media has seen significant improvements and optimizations in recent years and now consists of two different approaches to embryo culture: sequential, where embryos are moved part way through the culture period to a medium with a different composition; and single-step, in which embryos are held in the same dish throughout and culture medium is not replenished at any point.

In sequential culture, the different requirements of an early versus late-stage embryo are considered. For example, the energy source provided in an early-stage medium (for embryo culture from the two pronuclear [2PN] stage until approximately day 3) is almost exclusively focused towards pyruvate and lactate, as opposed to glucose for embryo culture from day 3 onwards until embryo transfer. In addition, essential amino acids are almost completely omitted from media used for early embryo culture but are included in media used for embryos that have undergone compaction. Finally, the chelating agent ethylenediaminetetraacetic acid (EDTA) is included in media for early embryo culture but omitted from media used for extended culture, in line with the hypothesis that this compound is toxic to blastocyst stage embryos ().

In single-step culture, the needs of the early (2PN stage) and late (blastocyst stage) embryos are balanced with the use of a single medium. This method is particularly advantageous when used in conjunction with continuous embryo monitoring systems, as it enables uninterrupted embryo culture and assessment from the 2PN stage until embryo transfer. This minimizes stress to embryos as a result of changes in pH, light and temperature outside of the incubator when performing static morphology grading or exposure to a medium of different composition.

Despite these different culture methods, several authors (most recently ) do not consider that either is ‘superior’ to the other. Rather, the approach selected depends on individual laboratory preferences and needs to dovetail with existing local practices. Ideally a single-step culture should be selected when using a continuous embryo monitoring system, in order to entirely benefit from uninterrupted culture and fully access the functionality of the device. With the growth in popularity of continuous embryo monitoring through time-lapse technology, one might expect to see an increase in the use of single-step versus sequential culture. Either way, the benefits of modern culture media, which are the result of multi-faceted quality control testing alongside accredited and audited manufacturing facilities, should ensure that clinics continue to purchase media from commercial sources.

Cryopreservation, which is mostly used to preserve surplus embryos and cryopreserve donated gametes or embryos for future cycles, is an integral part of today's IVF process (). Although useful, particularly in countries with directives to reduce multiple pregnancy rates by means of single embryo transfer, cryopreservation comes at a cost. Even the best cryopreservation protocol can generate cellular injury in embryos, which may reduce their potential to survive when compared with fresh embryos (). Over the last 6–7 years, it has been demonstrated that vitrification, when performed correctly, achieves higher survival rates than slow cooling, simply by eliminating the process of crystal ice formation that can damage cellular structures and cellular membranes (, ). While vitrification has been shown to result in significantly higher clinical pregnancy and live birth rates when compared with slow freezing of embryos (), there are limited direct and randomized comparisons between vitrification and slow freezing in a clinical setting. However, the general trend observed in clinical practice alongside several meta-analyses (, ) suggests that, in competent hands, vitrification has a significantly lower damage rate to eggs and embryos than slow freezing, with clinical outcomes comparable to fresh embryo transfer. The growing trend towards freeze-all cycles, separating the oocyte collection process from the embryo transfer procedure, has made the safety and efficacy of cryopreservation of paramount importance. There is accumulating evidence that this strategy provides a safer (eliminating risks of hyperstimulation) approach, with at least equivalent results () and better obstetric and perinatal outcomes versus fresh embryo transfer ().

Vitrification is much more hands-on than slow freezing, and requires a higher skill base from the technician in order to ensure the best possible outcome. Variation in outcomes between users and clinics has been reported. In order to combat this, concerted efforts need to be put into training and it has been suggested that limits should be imposed on the number of embryologists that complete the vitrification process to maintain quality (). However, this may not be necessary with additional training, benchmarking, accepted key performance indicators (), published standards and automation.

A number of variables have an impact on the outcome of vitrification. These include the type and concentration of cryoprotectants, temperature and timing of embryo exposure, the rate of cooling and subsequent warming, and whether or not the embryo comes into direct contact with liquid nitrogen (). As in slow freezing, where the use of a controlled-rate freezer enables the repeatable and standardized slow freezing rate that is required for successful cryopreservation, control through automation of some of these vitrification variables may further enhance repeatability and reduce the variation in outcomes.

Automation potentially provides a series of advantages over manual cryopreservation: (i) improved workflows by allowing users to process multiple embryos simultaneously, (ii) reduced training requirements and (iii) reduced intra- and inter-operator variability. Currently, there is one automated vitrification instrument, the Gavi (Genea Biomedx, Australia), commercially available in some markets, with others in development. The Gavi uses a closed system to vitrify up to four embryos simultaneously. Vitrification of mouse and human research blastocysts using automated vitrification has been shown to be equivalent in recovery, survival and development potential to that of the popular Cryotop manual vitrification method (, ).

Currently, most embryos are selected based upon stringent morphological factors. However, despite recent attempts to provide expert consensus regarding standardized timing and nomenclature for embryo morphological classification (), single-point morphology remains a subjective assessment (, ). Morphological embryo selection is imprecise, subjective, has low predictive value for embryo implantation potential and displays relatively high inter-observer variability (even within the same centre) (, ). Together, these findings indicate that current selection methods, employed in the vast majority of IVF cycles globally, are inadequate.

There is a strong need to develop objective biomarkers of gamete viability and embryo implantation potential that are independent of embryo appearance. Combining selection based on morphological factors with metabolic, protein and genetic markers in culture media may optimize pregnancy and live birth rates and become the standard of care by consistently selecting embryos with the highest implantation potential (). When any new technology is being applied for embryo assessment, we must evaluate its efficacy by taking into consideration the implantation potential of individual embryos by calculating the implantation rate, clinical and ongoing pregnancy rate when performing single embryo transfers, or known implantation data on patients with 0 or 100% implantation when transferring two or more embryos. In the end, the final measure of success in assisted reproductive technologies is a successful, healthy, live birth following the transfer of a single embryo (, ).

The most common genetic abnormality in humans is the presence of numerical chromosome abnormalities (aneuploidy) in gametes and embryos. Aneuploidy rates increase with maternal age and can be as high as 85% in women over 42 years of age, but are also present (20–25%) in young, good-prognosis patients and oocyte donors (). Currently, no method exists to reduce the occurrence of aneuploidy in women undergoing IVF. However, the hypothesis that eliminating or deprioritizing the transfer of aneuploid embryos in favour of euploid embryos will improve implantation rates, reduce miscarriage rates, and reduce the time to pregnancy is sound, and has a growing evidence base to support it (, , , ). Indeed, in the absence of aneuploidy screening, embryo selection is predominantly based on morphological appearance with the risk of transferring aneuploid embryos and concomitant failed implantation, miscarriage or birth of a child with a viable trisomy (, ).

Early work using fluorescence in situ hybridization for PGS on cleavage-stage embryos at day 3 of development was shown to be ineffective and potentially harmful to a patient's chance of success (). This was due to the reliance on optimal preparation of interphase nuclei, analysis of a limited number of chromosomes, and evaluation of cleavage-stage embryos that had a higher rate of mosaicism (potentially leading to increased risk of false-positives) and greater susceptibility to damage during biopsy (, ). However, this landmark study clearly demonstrated that for PGS to be successful the diagnostic test would need to be highly accurate, sensitive, specific, reliable and reproducible, while the embryo biopsy would need to be as safe and effective as possible. Next-generation sequencing (NGS) solutions are predicted to overtake array comparative genomic hybridization (, ) as the gold standard for PGS as they appear to demonstrate improved accuracy, reproducibility and scalability, thereby reducing the cost per sample and making PGS accessible to more patients (, ). All of these techniques use DNA extracted from embryonic cells and examine all 24 chromosomes simultaneously, which is now considered essential for optimal selection, as changes in copy numbers have been seen for all chromosomes in human IVF embryos (, , ).

PGS can be performed at different stages of embryo development by extracting DNA from the oocyte polar bodies, blastomeres at the cleavage stage, and trophectoderm from the blastocyst stage for evaluation (, ). Mosaicism is the presence of two or more chromosomally different cell lineages in an embryo and occurs as a result of errors in cell division. With the trend towards more multicellular biopsy at the blastocyst stage and the emergence of sensitive NGS tools, more embryos are now being identified as exhibiting mosaicism. While this is nothing new, there is uncertainty and inconsistency as to whether embryos identified as having a single mosaic chromosome should be transferred and whether some aneuploidy and mosaicism can be attributed to the clinic and/or laboratory practice (). While some embryos identified as mosaic (and thus classified as ‘abnormal’ by some clinicians and laboratories) can result in live births (), others can result in higher rates of failed implantation or miscarriage (), leading to the dilemma of whether to discard a mosaic embryo or not. Clearly the specific chromosome(s) and aneuploidy involved, combined with the degree to which mosaicism is present, are relevant factors. Recent clinical recommendations () and guidelines published by the PGD International Society provide some reassurance in this regard and recommend that a suitably accurate and sensitive test, such as NGS, should ideally be used to assess mosaicism ().

While all of these methods are invasive, in experienced hands there is little or no detrimental effect on the developing embryo (). Novel non-invasive techniques for aneuploidy evaluation are being investigated. For example, differentially secreted proteins in the culture medium can be detected via proteomic technologies and mass spectrometry (, ). In addition, blastocoel fluid containing either cells, cell-free DNA, or both has been collected in a minimally invasive procedure to evaluate aneuploidy, with promising results in terms of concordance with the developing embryo (, ). Non-invasive techniques would be ideally positioned to facilitate the rapid adoption of PGS, as a major barrier in the IVF laboratory is the need for skilled biopsy practitioners. Further, the need for a non-contact laser could be reduced or completely eliminated.

Despite the failure of FISH-based PGS, recent evidence supports the use of 24-chromosome PGS to improve outcomes by deselecting aneuploid embryos (, , ) and other larger studies are ongoing (see In summary, it is fair to say that PGS continues to court controversy and there is real concern over whether embryos with an essentially ‘normal’ chromosomal complement are being unnecessarily discarded as a result of technical false-positives or biological mosaicism. Furthermore, successful PGS can only be achieved on a solid foundation of optimized IVF (including culture, biopsy and vitrification) using validated laboratory screening tests. As with any embryo selection, PGS cannot increase cumulative pregnancy rates but instead may reduce time to pregnancy, financial and emotional costs of failed procedures and miscarriage. If PGS is to be routinely adopted, all component processes must be optimized to ensure that any beneficial effects seen as part of clinical trials can be transferred to all clinics worldwide.

While genetic analysis is being proposed as a technology that can improve IVF outcomes by determining genetic status, there are other factors that need to be considered outside of the number of chromosomes present in an embryo. For an embryo to implant, it also needs to have cells with healthy cytoplasm. In some circumstances, cytoplasmic quality may be sufficient to support normal chromosome segregation but may not result in a fully competent embryo. As the embryo divides, each blastomere receives a smaller and smaller share of everything the oocyte has managed to accumulate. The fetal genome does not become capable of adding cytoplasmic factors until the cleavage stage, and the oocyte's remarkable cache of mitochondria do not start to replicate again until blastulation (, ).

Several technologies are being proposed to define the quality of the oocyte but none is yet clinically applicable. One must consider that the early embryonic divisions are a reflection of the quality of the cytoplasmic material of the oocytes, and this information can be objectively collected through CEM systems. Non-invasive embryo evaluation using CEM systems to improve pregnancy outcomes remains controversial. There are some robust data indicating an improvement over standard single-point morphological assessment (). However, there remains a lack of further prospective studies on the use of CEM (, , ). The standard method of CEM captures images of the developing embryos for up to 7 days at 5–20 minute intervals, obtaining an unprecedented level of morphological and morphokinetic information with no interruption in culture for the embryo, and thereby maintaining the physiological environment throughout the culture period (, ). Such systems provide a method of embryo assessment that diminishes observer subjectivity, reveals hitherto unseen processes and anomalies, and limits exposure to non-physiological conditions. Standard static morphological assessment requires embryologists to remove embryos from the incubator to visually assess cleavage and morphology at single points in time over the course of several days, which disturbs the culture conditions and can impact embryo development (). These snapshots may well represent false readings, as embryonic development is a dynamic process and key events may be missed if they occur between observations (). Together these factors limit the value of current static morphological assessment. CEM may provide the solution to this significant problem as 24-hour monitoring can be achieved without removing embryos from the culture medium. Several types of equipment exist, some including high-definition cameras and the incubation system (Geri, EmbryoScope, Miri TL), others consisting only of cameras that can be included inside box incubators (Embryo Evaluation Viability Assessment [Eeva], Primo-Vision). The Eeva system differs from other available systems as it uses a proprietary validated algorithm that automatically predicts blastocyst development based on timings of specific events during the first few cleavage divisions, and has been shown to be more effective in this regard than experienced embryology observers alone (). Conflicting results on the impact of undisturbed embryo culture have been reported, but a 1-year retrospective study indicated that undisturbed embryo culture in the EmbryoScope incubator may improve live birth rates, and that conventional culture methods may negatively impact embryo growth and implantation potential (). Several randomized controlled trials are under way to evaluate the clinical value of having an objective CEM system and uninterrupted culture conditions for early embryonic stages. Nevertheless, current retrospective and prospective studies underline the utility of this technology in a clinical setting with promising results (, , ). CEM has been adopted by many clinics and several studies have been completed demonstrating the incremental utility of combining innovative technologies such as PGS with CEM, to objectively identify genetically and cytoplasmically healthy day 3 and day 5 embryos (, , , ). As with so many technological advances, CEM may not yield immediate benefits to all laboratories and successful implementation probably requires some standardization (e.g. with respect to embryo stage annotation). Indeed, CEM has not universally shown embryo selection benefits in clinical trials () and it can be difficult to tease apart potential clinical benefits from an uninterrupted culture system from those obtained via improved embryo selection. However, its additional value in embryo deselection (, ), facilitating uninterrupted culture, as a sensitive quality control tool () and an aid to appropriate timing of procedures in the embryology laboratory (e.g. embryo biopsy) are not without incremental value. Additional large, robust clinical trials will help to guide the future utility of this exciting technology ().

Cross-talk between the transferred embryo and the endometrium is critical for implantation success (). Endometrial receptivity is limited to 4 days during the mid-secretory phase of the menstrual cycle (). As the window of implantation is small, assessing endometrial receptivity is vital in order to establish the optimum time for embryo transfer (). The current method is to evaluate receptivity through assessment of morphology-based histological features. However, this technique is questionable as the endometrial tissue is highly dynamic and changes according to menstrual phase, and with repeated implantation and stimulation (, ). Initiatives to develop reproducible methods for determining endometrial receptivity are ongoing. These include the investigation into the use of lipids, mRNA, genes, peptides and cytokines (, , , , , ).