12.2.1 Incubators

Amongst the changes within the confines of the IVF laboratory, one could argue that incubators have perhaps exhibited the greatest change. These laboratory workhorses have reduced in size considerably from large box-type units of ~150 L that were created to hold numerous flasks of adherent somatic cells to much smaller benchtop units optimized for embryo culture of one patient in each individual unit^{3, 4} (Figure 12.1). In addition to the dramatic size reduction, these IVF specific incubators are designed to reduce environmental stressors through:

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  improved temperature recovery/stability

  
  
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  proper gas mixture (low oxygen)

  
  
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  gas/pH stability

  
  
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  better air quality (internal filtration methods)

  
  
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  and other relevant factors, such as cleaning and sterilization, monitoring and data logging capabilities, patient capacity.⁴

The advent of time-lapse imaging in conjunction with IVF-specific benchtop incubators has resulted in even more specialized and complex devices to potentially benefit IVF. These time-lapse incubators permit and promote the increased use of uninterrupted culture, where neither media nor embryo is disturbed via manual removal from the incubator for routine handling or observation. This uninterrupted approach attempts to further improve environmental stability and enhance outcomes, but impeccable quality control is paramount to avoid potential detrimental variables, such as evaporation, VOC accumulation and media degradation.⁵ Validation of a superior culture environment and outcomes for these new incubators has not been verified in all studies,^{4, 6–9} but the increased data available from the imaging offers new opportunities to improve embryo selection using algorithms and artificial intelligence, or research into other novel visual indicators of embryo morphokinetics. It also creates a unique platform on which to build an even more customized system and workflow targeted to the unique needs and demands of the clinical IVF laboratory.

Figure 12.1 IVF culture incubators have evolved over time, reducing in size to units with individualized culture changes. a. Large box incubator, b. Small box incubator, c. various benchtop incubators, some with individualized chambers, d. time lapse incubators with individualized culture chambers. This evolution has results in improved growth conditions due, in part, to improved environmental stability.

As an example of potential advancement, current time lapse imaging systems used in clinical IVF utilize basic brightfield or darkfield imaging to visualize cells and track cell divisions. These are simple imaging technologies with limitations, such as low contrast, low resolution, and limits to magnification. However, more complex imaging approaches exist that could lend additional insight into cell quality, beyond cell division and basic morphology.^{10, 11} Several novel imaging approaches have been used to noninvasively examine gametes and embryos without negatively impacting function or development. Techniques like polarized light microscopy, RAMAN Fourier Transformed Infrared (FTIR), Coherent anti-RAMAN Stokes (CARS),¹² and combinations of these approaches can give information about oocyte spindle location and maturation status, cell lipid content, mitochondrial status, DNA damage, and other attributes.^{10, 11} Features of these approaches are shown in Table 12.1. In research settings, these unique microscopy technologies have revolutionized the study of fine structures (down to the packing arrangement of DNA in sperm) in living cells. Whether these more complex imaging approaches could be miniaturized or made compatible with benchtop IVF incubators is unknown, but they offer an intriguing pathway to expand upon current time-lapse imaging approaches.

12.3.1 Novel Culture Platforms

While advancement in incubators has noticeably progressed, the physical platform on which gametes and embryos are cultured has remained largely unchanged. Since the start of culturing gametes and embryos in vitro, laboratories have utilized glass or plastic petri dishes, typically intended for adherent cell culture, and modified slightly for use with the reproductive cells. Recently, advancements in the physical platform on which embryos are cultured have begun to emerge.^13–17 Novel culture platform prototypes to benefit the unique requirements of oocytes and embryos have been made by 3D printing and other advanced manufacturing technologies. These embryo specific dishes have confined/constrictive designs to help create beneficial microenvironments, taking advantage of the benefits of group embryo culture and embryo spacing, while keeping cells separate for identification or selection purposes. ^{18, 19} These include microfunnels, channels, and other unique depressions to hold the cells (Figure 12.2). Several of these novel culture platforms are the direct result of the new IVF specific incubators and go hand-in-hand with attempting to provide a superior culture environment based on the particular needs of delicate reproductive cells. At least one preliminary study indicates that a microvolume approach using the Well-of-a-Well (WOW) system may be superior for human embryo development compared to larger volume culture.²⁰ Currently, there is no consensus as to a superior culture dish or volume of media used for culture.

Figure 12.2 Various embryo specific culture dishes aimed at creating beneficial microenvironments and/or permitting individual cell separation/identification. Embryos are increasingly being cultured in specific dishes tested for use with embryos for toxicity and customized to create confined microfunnels or wells rather than using generic cell culture/petri dishes and larger volumes of media.

In addition to the dimensions of the culture device, exploration of novel materials or surface coatings may be useful in improving culture conditions. In vivo, the female reproductive tract provides a moist environment, where the embryo may encounter ciliated epithelium and various crypts and folds within the confines of the oviduct and then the intricate surface of the uterine lining, where various polyhydroxylated compounds, macromolecules, and components of the extracellular matrix are presented to the embryo. This is in stark contrast to the flat and inert surface of a plastic dish in the laboratory, where the embryo is submerged in a relative ocean of culture medium. In a field where the physiological basis has been a driving force in formulation of some culture media as well as the culture atmosphere, both resulting in improved embryo development, perhaps exploration of more physiological culture surfaces may also yield further improvements upon current practices.

Though limited, some research has examined the impact of altering culture surfaces and materials on preimplantation embryo development.¹⁶ For example, surface coatings like Matrigel, hyaluronan, and agarose have been shown to impact embryo development. Mouse embryos cultured on Matrigel coated culture plates increased rates of mouse blastocyst hatching compared to those in media alone, though total rates of blastocyst formation were similar.²¹ A later study using Matrigel yielded higher rates of blastocyst development and increased hatching rates.²² However, subsequent studies examining the ability of Matrigel to support zygote development from random-bred mouse strains, which experience the 2-cell block, demonstrated an inhibitory effect of Matrigel on blastocyst development and hatching.²³ Though conflicting data exists, inclusion of hyaluronic acid in culture media has been shown to be beneficial for mouse and bovine embryo and fetal development,^24–26 and to improve pregnancy and implantation rates when included in human embryo transfer media.^27–31 Thus, the use of hyaluronan coated surfaces may be worthy of exploration.

At least one preliminary abstract has examined the effects of coating the culture surface with a glycosaminoglycan matrix of hyaluronic acid. Though coating of flat polydimethylsiloxane (PDMS) surfaces and microwells with hyaluronic acid hydrogels was able to support mouse blastocyst formation at comparable levels to uncoated flat PDMS surfaces (81.4%,. 72.6%, and 86.3%, respectively), it significantly decreased blastocyst cell numbers compared to no coating.³² Surface coatings using agarose have been used in supporting embryo development in vitro. Microwells made in agarose have been described for use in individual culture zona-free embryos.³³ This method does allow for easy access to and identification of embryos, though no immediate benefit on growth over traditional culture surfaces has been noted. It should be noted that agarose gel tunnels were also used for extended culture of post hatching bovine embryos.³⁴ Interestingly, several types of agarose gel exist and it is unknown whether specific properties of these varying compounds can convey differential effects on embryo development.

Certain limitations exist with 2-dimentional culture, as contact with any specialized surface is minimal. In 3-dimensional culture approaches (Figure 12.3), growing cells are provided structural support and direct physical interaction with their surrounding environment, similar to that experienced in vivo. Additionally, 3-dimensional culture may allow for embedding and orientation of an array of glycoproteins or other macromolecules, compared to culture on a 2-dimensional surface. To date, most of the work with 3-dimensional culture has been performed with follicles or oocytes,^35–37 but similar approaches could be used with embryos, though modifications of the approach may be required to allow embryo visualization and grading, as well as embryo recovery for subsequent embryo transfer. Importantly, any surface coating or 3-dimensional approach must consider the impact of the matrix on altering media composition and the resulting impact on gametes and embryos.

Figure 12.3 Three-dimensional culture approaches may offer a means of providing a more physiological approach to in vitro embryo culture by providing structural support and a more appropriate physical environment via different biomatrices. This method involves encapsulating cells into a bioscaffold or matrix material and has been used successfully for oocytes and follicles. Three-dimensional culture has not received widespread attention for use with embryos, but offers a means to provide structural support, a moist, rather than fluid environment, as well as present potentially beneficial molecules in an oriented fashion. However, 3-dimensional embryo culture does have unique considerations to address to facilitate embryo grading and recovery for subsequent uterine transfer or cryopreservation.

In addition to the surface environment differences experienced in vitro, the physical forces experienced by gametes and embryos in the laboratory also differ from what would be normally encountered in vivo. It is estimated that embryos in the female reproductive tract move and experience a sheer force of 0.1µm/s and 0–3 dyn/mm², respectively.^{38, 39} Thus, subtle or gentle movement could activate beneficial signaling pathways and perhaps benefit embryo development. This “Active Embryo Hypothesis” has some merit when considering the emerging literature.^13–17 On the other hand, embryos can experience excessive forces from procedures such as pipetting, which can impair development.^{40, 41}

Various dynamic culture approaches exist, where cells are actively agitated or moved, rather than sitting in a relatively static state in a drop or well of media (Figure 12.4).

Figure 12.4 Representative images of dynamic embryo culture platforms that have been used clinically for human embryo culture. These platforms have historically required use of small or large box incubator and include a. tilting embryo culture, b. microfluidic culture and c. vibrational culture. Scaling down these approaches for use with smaller, modern benchtop incubators may provide an opportunity to improve upon current culture systems and facilitate more wide-spread implementation of this dynamic approach.

Dynamic embryo culture approaches used with human embryos include ^14–16:

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  motorized tilting devices

  
  
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  vibrating platforms

  
  
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  methods to move media and cells though microfluidic devices using syringes or piezo-actuated pin systems.

A promising and widely trialed dynamic embryo culture approach appears to be subtle and periodic vibration. Initial studies in mouse, bovine, and human indicate that short bursts of vibration of around 5 seconds every ~60 min around 44Hz, improve embryo development and outcomes.^42–47 However, success rates of control samples were poor and a randomized clinical trial results demonstrated no improvement in usable blastocyst rate or sustained implantation rate between embryos cultured with a microvibration platform versus static culture.⁴⁸

Notably, all of these dynamic culture systems require standard box-type (large or small) incubators for placement. With improved scaling and customization, these innovations may be applied in similar fashion to embryo specific benchtop incubators.

Microfluidic approaches lend themselves to combining several steps onto a single, novel platform to reduce handling and associated cell stress^{15, 16} (Figure 12.5). Various procedures have been performed on microfluidic devices such as:

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  oocyte maturation

  
  
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  oocyte denuding

  
  
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  cell orientation for ICSI or biopsy

  
  
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  embryo culture

  
  
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  vitrification.

Figure 12.5 Various microfluidic platforms aimed at improving procedural steps involved in IVF have been examined, though most have not been widely implemented in clinical settings. a. Simple devices used for sperm sorting have received the most clinical use: semen is loaded into an inlet port and motile sperm collected from out an outlet port after traversing some arrangement of channels/obstacles to aid in sperm selection. While many experimental designs exist, at least two microfluidic systems have been tested on culturing human embryos: b. a simple static system and c. using actuated pins to drive fluid flow though microfluidic channels in a pulsatile fashion across embryos held in microfunnels.

12.5.1 Electronic Witnessing

Double witnessing of key procedural steps in the IVF process is a requirement for an adequate quality control system. This is meant to avoid sample mix-ups and errors. However, manual witnessing is still subject to error. Electronic witnessing systems now exist for use in the IVF laboratory. Using RFID tags or printed barcodes or other image capture approaches, this necessary procedural step can be automated to create a more efficient and effective workflow. This technology is discussed in more detail in Chapter 7.

12.5.2 Digital Quality Control

It is widely accepted that laboratory quality control and assurance must be performed routinely, but appropriate levels of monitoring, what to monitor, and the best ways to monitor it are not clear. This has led to increasing demands on laboratory staff to monitor (sometimes multiple times a day), record, and ensure that the laboratory equipment and environment stays within preset operational parameters. Until recently, the dynamics/timing of the response of a liquid nitrogen dewar to physical tank failure was largely unknown (i.e., how a storage vessel behaves when the vacuum is breached) was unknown.⁵⁸ To address issues like this and others, digital and cloud-based solutions to discover malfunctioning instruments or environments are being adapted to ART from other industries where they are already best practice tools. In the prior example of dewar failure, new monitoring approaches not only include internal temperature, but external temperature measurement via probes or thermosensitive cameras and weight monitoring approaches that can all be monitored remotely. New cloud-based applications to collect, store, retrieve, and analyze instrument quality control data are available and lend support to standardization of quality control parameters and the opportunity to integrate with electronic health records to relate measured parameters to clinical outcomes.⁵⁹ Staff-based competency assessment is also in development to further digitalize staff-related competency assessments, training documentation, annual procedure evaluations, and real-time “in-cycle” embryologist statistics.

12.5.3 Artificial Intelligence

Artificial intelligence (AI), machine learning, and deep learning use big data to look for subtle outcomes or patterns that humans cannot detect. The applications for IVF range from automation of follicle counts, to gamete and embryo grading and selection, to determination of genetic status, quality control and embryologist KPIs, prediction of live birth, and even to donor: recipient gamete phenotype matching. With the increased adoption of imaging systems in IVF, application of AI to help analyze these images or perhaps more useful, videos, for key selection criteria may help improve quality of prediction and decrease time to clinical pregnancy. The achievement of a successful pregnancy is highly dependent on embryo quality. Numerous preliminary studies have emerged using AI for embryo image/video analysis as a possible selection tool and show great promise to predict which embryo can maximize the likelihood of a singleton pregnancy, for example, by age group. These studies have further demonstrated that AI systems do not require experts to annotate images (raw video can be used without human blastocyst assessment), that AI systems can reveal hidden significant details that human embryologists cannot evaluate, and that ploidy of an embryo can be predicted by AI.^60–67

In addition to image analysis for embryo selection, AI can be used to look for other patterns in images and data to further improve laboratory operations.