**Methods for Spatio-Temporal Analysis of Embryo Cleavage In Vitro**

Anna Leida Mölder, Juan Carlos Fierro-González and Aisha Khan

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.69650

#### **Abstract**

Automated or semiautomated time-lapse analysis of early stage embryo images during the cleavage stage can give insight into the timing of mitosis, regularity of both division timing and pattern, as well as cell lineage. Simultaneous monitoring of molecular processes enables the study of connections between genetic expression and cell physiology and development. The study of live embryos poses not only new requirements on the hardware and embryo-holding equipment but also indirectly on analytical software and data analysis as four-dimensional video sequencing of embryos easily creates high quantities of data. The ability to continuously film and automatically analyze growing embryos gives new insights into temporal embryo development by studying morphokinetics as well as morphology. Until recently, this was not possible unless by a tedious manual process. In recent years, several methods have been developed that enable this dynamic monitoring of live embryos. Here we describe three methods with variations in hardware and software analysis and give examples of the outcomes. Together, these methods open a window to new information in developmental embryology, as embryo division pattern and lineage are studied in vivo.

**Keywords:** embryo cleavage, time-lapse analysis, morphokinetics, embryo profiling, phylogenetics, cell lineage

#### **1. Introduction**

Despite 30 years of practice, the success rate for implantation of embryos into the uterus in in vitro fertilization (IVF) is still only around 30% [1, 2]. Consequently, when transferring embryos from in vitro culture and implanting them, it is critical that only the best embryos are selected. This will not only optimize the chance of live birth but also reduce the need for

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

multiple embryo transfer, with the subsequent risk of twin pregnancy and the neonatal complications and associated maternal pregnancy-related health problems. Though cultivation methods have improved, embryo selection is still largely based on manual evaluation of morphological criteria, and much research has been done in identifying morphological features correlated with embryo health. Other methods such as genetic screening and metabolic profiles of culture media exist, but have not yet proven to increase pregnancy rates [3–9]. There is an ongoing discussion concerning the relevance of embryo morphology in quality assessment [10], but it is likely that it will continue to play a large part in IVF embryo evaluation also in the future. Traditionally, embryo quality assessment has been performed by manual inspection using light microscopy at intermittent time points during embryo development. Novel technical solutions have recently made it possible to monitor embryos continuously using timelapse imaging, opening new possibilities for embryo evaluation based on dynamic properties.

It has been shown that the timing of key occurrences within the embryo can vary greatly between embryos that have similar morphological appearance at the end of the recording period and that embryo morphology can change in a matter of hours [11–14], emphasizing the fact that dynamic monitoring is preferred over intermittent monitoring of embryos. An important endpoint for embryo studies is the timing of embryo cleavage, which has been shown to correlate to embryo viability and implantation potential [15–18]. For research purposes, tracking of cell lineage and cell positioning within the early embryo provides important information to understand pluripotency. Embryos are also a good model for the study of developmental biology and three-dimensional cellular interaction. The ability to continuously film and analyze growing embryos gives new insights into temporal embryo development by studying morphokinetics as well as morphology. Until recently, this was only possible by a tedious manual process. Although currently some human IVF laboratories have started to use time-lapse technology to monitor embryo cleavage and growth, further description of the technology and its potential is needed. The focus of this chapter is on the methods used to study living early embryos over time and the possibilities they render as new tools for embryological research and clinical application.

#### **2. The role of live imaging in embryology**

Conventional microscopy suffers from several drawbacks, such as requiring sample fixing and only providing static information in an intermittent manner. The complete understanding of cell division and development requires a dynamic perspective on an individual cell level as most information on cell response to environment, dynamic gene expression and timing would be missed in a static analysis. In recent years, the imaging technologies have provided new tools in microscopy, sample handling, and hardware and software for live imaging of individual cells. There are several examples of single cell [19–22] and single molecule monitoring in living cells, using both marker-based and marker-free approaches.

Fluorescent tags enable the tracing of specific proteins and measurement of their characteristics to study gene expression, protein localization, and function and protein-protein interaction. By using several markers simultaneously, it is possible to track several proteins or gene expressions at once. With time-lapse microscopy, intracellular events can be linked to external factors such as cell-cell interaction and ultimate cell fate. These methods give us remarkable new insights into the dynamics of gene expression, cellular interactions, and heterogeneous processes. In fluorescence imaging, a laser is used to excite the fluorophores at a particular wavelength. Full field epifluorescence can then be used to measure the light as the fluorescent tags emit light while returning to their unexcited molecular state [23]. In confocal imaging, a pinhole in combination with focused laser light is introduced to effectively reduce background fluorescence and allows optical sectioning of the sample by mechanical scanning. Varying the pinhole will effectively vary the thickness of the sample being imaged, the image resolution, and the acquisition time.

For some applications, the use of fluorescent tags is not feasible. By continuously filming embryo material some important information on cell outline, position, shape, and texture can be extracted from the time-lapse sequences without the use of fluorescent markers. By matching and tracking, this information can be combined to a timing profile of the dividing embryo, detecting temporal location of division and tracking cell lineage over time. Using computer vision in combination with a noninvasive imaging method makes it possible to continuously study embryo growth with minimal sample interference. Fluorescence imaging allows the noninvasive measurement of gene expression and intracellular characteristics, while markerfree light microscopy allows the tracking of cellular size, shape, and behavior over time in response to molecular changes. This combination gives us the possibility to directly monitor cellular responses and changes in gene expression in response to the environment. The result is a cellular model that can bridge the molecular scale to the cellular, mapping the actual connections between the chemical and the biological world.

#### **3. Noninvasive techniques for embryo imaging**

Currently, a set of biotechniques has been successfully applied to mouse and human embryo imaging. This technique includes the addition of a fluorescent marker and marker-free methods. For research purposes, the addition of fluorescent proteins can be considered a noninvasive method, if the protocol used does not significantly disturb embryo growth. For clinical applications in human embryology, no markers of any kind can be used. In this section, we will refer to fluorescent marker methods as noninvasive, and specify the "truly" noninvasive method as "marker free."

#### **3.1. Fluorescence imaging**

multiple embryo transfer, with the subsequent risk of twin pregnancy and the neonatal complications and associated maternal pregnancy-related health problems. Though cultivation methods have improved, embryo selection is still largely based on manual evaluation of morphological criteria, and much research has been done in identifying morphological features correlated with embryo health. Other methods such as genetic screening and metabolic profiles of culture media exist, but have not yet proven to increase pregnancy rates [3–9]. There is an ongoing discussion concerning the relevance of embryo morphology in quality assessment [10], but it is likely that it will continue to play a large part in IVF embryo evaluation also in the future. Traditionally, embryo quality assessment has been performed by manual inspection using light microscopy at intermittent time points during embryo development. Novel technical solutions have recently made it possible to monitor embryos continuously using timelapse imaging, opening new possibilities for embryo evaluation based on dynamic properties. It has been shown that the timing of key occurrences within the embryo can vary greatly between embryos that have similar morphological appearance at the end of the recording period and that embryo morphology can change in a matter of hours [11–14], emphasizing the fact that dynamic monitoring is preferred over intermittent monitoring of embryos. An important endpoint for embryo studies is the timing of embryo cleavage, which has been shown to correlate to embryo viability and implantation potential [15–18]. For research purposes, tracking of cell lineage and cell positioning within the early embryo provides important information to understand pluripotency. Embryos are also a good model for the study of developmental biology and three-dimensional cellular interaction. The ability to continuously film and analyze growing embryos gives new insights into temporal embryo development by studying morphokinetics as well as morphology. Until recently, this was only possible by a tedious manual process. Although currently some human IVF laboratories have started to use time-lapse technology to monitor embryo cleavage and growth, further description of the technology and its potential is needed. The focus of this chapter is on the methods used to study living early embryos over time and the possibilities they render as new tools for

Conventional microscopy suffers from several drawbacks, such as requiring sample fixing and only providing static information in an intermittent manner. The complete understanding of cell division and development requires a dynamic perspective on an individual cell level as most information on cell response to environment, dynamic gene expression and timing would be missed in a static analysis. In recent years, the imaging technologies have provided new tools in microscopy, sample handling, and hardware and software for live imaging of individual cells. There are several examples of single cell [19–22] and single molecule monitoring in living cells, using both marker-based and marker-free approaches.

Fluorescent tags enable the tracing of specific proteins and measurement of their characteristics to study gene expression, protein localization, and function and protein-protein interaction. By using several markers simultaneously, it is possible to track several proteins or gene expressions

embryological research and clinical application.

42 Embryo Cleavage

**2. The role of live imaging in embryology**

Adding fluorescent proteins (FP) is a standard way to selectively study specific intracellular targets [24]. The most common fluorescent tag is the green fluorescent protein (GFP) [25], derived from the jellyfish *Aequorea Victoria* [26]. The FP is introduced by transfection or microinjection of a plasmid DNA expressing vector, carrying the genetic code for the protein. By tagging a biologically functional protein of interest with the FP, a specific pathway can be tracked. The use of FPs enables a straightforward way to locate the protein within the cell, but this can have drawbacks. Phototoxicity may occur at short enough wavelengths and at high laser excitation intensities [27]. Also, a transient expression of FP may result in higher-thannormal levels of the functional protein accompanying it, which may have unforeseen effects on the dynamic behavior of the entire system. Alternatively, the FP can be integrated into the genome using targeted genome editing technologies like CRISPR-Cas9 (M3), in which case the number of plasmid copies per cell will no longer affect the protein concentration. Control experiments are necessary to establish the effect of the FP study method, which may differ for each host system or experimental environment.

FP can also be used to study the dynamics around the FP binding site by fluorescence recovery after photobleaching (FRAP) [28]. In FRAP, a fluorophore is covalently attached to the molecule of interest. The fluorophore is intentionally photobleached using incident laser light. The diffusion of the molecules can now be quantified by studying the gradual brightening of the photobleached spot, as fresh fluorophores migrate into this area. Three closely related techniques are the fluorescence loss in photobleaching (FLIP), fluorescence decay after photoactivation (FDAP), and fluorescence correlation spectroscopy (FCS) [29]. Fluorescence resonance energy transfer (FRET) (sometimes also called Förster resonance energy transfer) can be used to study protein-protein interactions [30]. In this case, a donor fluorophore is placed in an excited state by incident laser light, and the energy held in the excited molecular state is transferred to an acceptor fluorophore which must be in close proximity (typically less than ten nanometers). When two molecules under study are labelled with the donor and acceptor fluorophores, respectively, the detected light from the acceptor fluorophore indicates that the two molecules are in close proximity.

A number of studies have used fluorescent markers using various imaging modalities to study protein movement within the embryo [31–34] and using embryonic stem cells [35, 36].

#### **3.2. Marker-free microscopy**

Currently, IVF centers or clinics are using two main techniques for embryo imaging: Hoffman modulation contrast imaging (HMC) (sometimes referred to as white light) [37, 38] and darkfield imaging (DF) [39]. For research purposes, CARS [40] and light sheet microscopy [41] are also becoming increasingly common. HMC was standard before time-lapse imaging of IVF embryos came in use and is still used in manual microscopy set ups. Consequently, images from time-lapse sequencing resemble the microscopy images to which embryologists are accustomed, an advantage when annotating images and comparing manual and computational approaches. HMC is best suited for imaging internal cell detail. On the other hand, Darkfield gives better detail to edge structures such as cell membranes, and more accurately to detect and track cell outlines.

Darkfield imaging is an imaging method that excludes any unscattered light, causing the samples to appear brighter on a darker background and enhancing the contrast of the imaged and unstained sample [42]. It is a simple yet effective method to noninvasively enhance sample contrast but has the disadvantage of low light levels available for collection. To compensate, the sample must be strongly illuminated and the heavy light exposure can cause sample damage. However, the low light level also means the image is almost entirely free from optical artifacts. Darkfield microscopy is most useful for studying boundary structures with a high difference in refractive index and imaging cell membranes is, for instance, more effective than internal cell structures. It is best suited for thin samples with high differences in refractive index (such as for sharp edges) and for thick samples, artifacts may occur.

HMC Imaging was invented by Hoffman in 1975 [43]. Today, it is a common technique for noninvasive contrast enhancement of biological samples. Its advantages include good contrast, low light exposure, excellent resolution, and a short depth of field, with the opportunity of focal sectioning at a resolution controllable by the numerical aperture of the objective. The ability to section is also influenced by the sample homogeneity. The disadvantages include strong optical artifacts and image appearance unsuitable for computerized image processing. HMC is commonly been used for embryology studies and has been included in a number of commercial products.

#### **4. Challenges in live embryo imaging**

normal levels of the functional protein accompanying it, which may have unforeseen effects on the dynamic behavior of the entire system. Alternatively, the FP can be integrated into the genome using targeted genome editing technologies like CRISPR-Cas9 (M3), in which case the number of plasmid copies per cell will no longer affect the protein concentration. Control experiments are necessary to establish the effect of the FP study method, which may differ for

FP can also be used to study the dynamics around the FP binding site by fluorescence recovery after photobleaching (FRAP) [28]. In FRAP, a fluorophore is covalently attached to the molecule of interest. The fluorophore is intentionally photobleached using incident laser light. The diffusion of the molecules can now be quantified by studying the gradual brightening of the photobleached spot, as fresh fluorophores migrate into this area. Three closely related techniques are the fluorescence loss in photobleaching (FLIP), fluorescence decay after photoactivation (FDAP), and fluorescence correlation spectroscopy (FCS) [29]. Fluorescence resonance energy transfer (FRET) (sometimes also called Förster resonance energy transfer) can be used to study protein-protein interactions [30]. In this case, a donor fluorophore is placed in an excited state by incident laser light, and the energy held in the excited molecular state is transferred to an acceptor fluorophore which must be in close proximity (typically less than ten nanometers). When two molecules under study are labelled with the donor and acceptor fluorophores, respectively, the detected light from the acceptor fluorophore indicates that the

A number of studies have used fluorescent markers using various imaging modalities to study protein movement within the embryo [31–34] and using embryonic stem cells [35, 36].

Currently, IVF centers or clinics are using two main techniques for embryo imaging: Hoffman modulation contrast imaging (HMC) (sometimes referred to as white light) [37, 38] and darkfield imaging (DF) [39]. For research purposes, CARS [40] and light sheet microscopy [41] are also becoming increasingly common. HMC was standard before time-lapse imaging of IVF embryos came in use and is still used in manual microscopy set ups. Consequently, images from time-lapse sequencing resemble the microscopy images to which embryologists are accustomed, an advantage when annotating images and comparing manual and computational approaches. HMC is best suited for imaging internal cell detail. On the other hand, Darkfield gives better detail to edge structures such as cell membranes, and more accurately

Darkfield imaging is an imaging method that excludes any unscattered light, causing the samples to appear brighter on a darker background and enhancing the contrast of the imaged and unstained sample [42]. It is a simple yet effective method to noninvasively enhance sample contrast but has the disadvantage of low light levels available for collection. To compensate, the sample must be strongly illuminated and the heavy light exposure can cause sample damage. However, the low light level also means the image is almost entirely free from optical artifacts. Darkfield microscopy is most useful for studying boundary structures with a high

each host system or experimental environment.

44 Embryo Cleavage

two molecules are in close proximity.

**3.2. Marker-free microscopy**

to detect and track cell outlines.

Although advances have been achieved in techniques for live single-cell imaging in recent years, several challenges still exist for wider implementation. An experimental design for long-term imaging and analysis must ensure not only high-quality imaging but also longterm support for sample vitality and appropriate computational methods for the analysis. Observing embryo in vitro requires an incubator environment to provide optimal living conditions or the sample during the imaging period. Temperature changes can affect the function of physiological processes as well as reaction kinetics and the challenge will increase with the length of the study sequence. One solution is the installation of an incubation flow chamber on the microscope, reducing the amount of gas and liquid to sustain the sample to a small volume, but suffering from drawbacks such as the risk of introducing condensation on the incubator chamber surfaces. Another approach is to integrate the microscopy optics in an incubator chamber, posing demands on the microscope optics and electronics to function in a humid, temperate atmosphere. A limited number of commercial solutions exist, which combine incubation capabilities with imaging hardware. With any of these solutions, the embryo medium and container must not introduce imaging artifacts such as light reflecting surfaces, auto-fluorescence, or excessive medium volumes in the light path. Another challenge is the loading and retrieval of cells from the mounting chamber, a process that may cause loss of cell identification. For IVF, several combinations of incubators and microscopes exists [44], either as integrated solutions or in the form of a microscope designed for use inside an incubator. So far, no difference has yet been seen in growth and implantation rates of embryos grown in the standard intermittent incubator system and a time-lapse incubator system [45–47]. One study found a higher rate of miscarriage for the time-lapse group, indicating there are reasons for caution. However, the same study noted no effect on pregnancy rates or embryo health prior to implantation [48].

In nonhuman IVF, phase contrast microscopy is commonly utilized instead of HMC. Phase contrast microscopy is similar to HMC in that it gives high level of image detail at the expense of image artifacts in the form of halos around sample objects. The varying appearance of embryos of different species will affect the decision of which optical system to use. Some species have dark, dense-appearing embryos (e.g., pig), while others are more translucent (e.g., mouse). As a consequence the optimal optical system for a given embryo species vary, and any appropriate analytical software must be chosen accordingly. Single-cell studies using darkfield imaging is limited by the hardware to the 4–6 cell stage. Using focal sectioning in HMC, it is possible to image the entire embryo from zygote to blastocyst stage, but any automated analysis becomes increasingly difficult with increasing cell number as the out-offocus image details cannot be removed, despite the sectioning. In humans, the compaction at the 9–16-cell stage involves a reduction in visibility of cell boundaries and may represent a feasible stage for automated detection beyond the 8-cell stage. The cavitation and blastocyst formation stages also offer opportunities for automated analysis of images, covering expansion and collapse events.

In fluorescence time-lapse imaging of nonhuman mammalian embryos, the life time of the fluorophores is limited, an effect referred to as *photobleaching* [27]. Bleaching can be limited by reducing exposure, but ultimately sets a limit to the duration of the imaging sequence. The most severe cause of concern is the toxic effects caused by the exposure to intense laser light for a prolonged period of time. This *phototoxicity* can be limited by minimizing laser exposure using mechanical fast shutters or switching LEDs, but any shutters will quickly reach the end of their life span in a continuous time-lapse imaging set up. Switching at 1 Hz, a shutter will open and close a million times in about 12 days. In all cases, an efficient microscopy control software is necessary.

There is a trade-off between information gathered and potentially harmful sample exposure, and the frequency of image capture must be carefully chosen depending on the study endpoint and the expected frequency of the dynamics under study. In the case of simultaneous monitoring of multiple samples, two solutions exist. In *scanning*, either the imaging hardware or sample is moved and repositioned at each image capture. In this case there is a trade-off (limited by the moving mechanics) between samples imaged and images captured per sample. In *full-field,* the image captured includes all samples simultaneously. In this case, there is instead a trade-off between the number of samples imaged and the image resolution available to each sample.

For two-dimensional imaging, full-field techniques are the most efficient as they capture the entire field of view in one single exposure. However, the stability of the system becomes critical as the time-lapse sequence length increases. *Focal drift* remains a problem and an autofocus mechanism or a method for user input to correct may be needed.

Even with moderate capture frequency, the amount of data from time-lapse studies can quickly build up to terabytes or more, especially if data is recorded simultaneously in multiple dimensions and imaging modalities. Consequently, both *data storage*, efficient *access to data* for analysis and the *post-acquisition analysis* itself must be considered. A small amount of video data may be analyzed manually, but this method quickly becomes cumbersome and time-consuming and automatic or semiautomatic methods are necessary. Manual evaluation of images is also prone to errors and inter-observer variability [49, 50]. It is often beneficial if the intended analysis can be considered already at the image capture stage so that acquisition, image quality, and hardware set up can be optimized upfront. Several open source software applications exist for the analysis of video sequences. Unfortunately, they are generally not suited for more advanced analysis of multidimensional data, which is often the case in embryo studies, where three-dimensional scanning or focal sectioning is used to capture data in multiple dimensions. Specialized solutions tailored to the data are also often both faster and more accurate than a general purpose application. The development of analytical tools hinges on access to *verification data*, for example, in the form of annotated image data for ground truth. With the increasing amount of generated image data, the availability of such training data has become a significant bottleneck. The solution, increased sharing and open access to data and annotations, requires standardized methods for data management, format, and metadata storage. To this end, open-source bioimage database systems such as OMERO [51] are an important step.

embryos of different species will affect the decision of which optical system to use. Some species have dark, dense-appearing embryos (e.g., pig), while others are more translucent (e.g., mouse). As a consequence the optimal optical system for a given embryo species vary, and any appropriate analytical software must be chosen accordingly. Single-cell studies using darkfield imaging is limited by the hardware to the 4–6 cell stage. Using focal sectioning in HMC, it is possible to image the entire embryo from zygote to blastocyst stage, but any automated analysis becomes increasingly difficult with increasing cell number as the out-offocus image details cannot be removed, despite the sectioning. In humans, the compaction at the 9–16-cell stage involves a reduction in visibility of cell boundaries and may represent a feasible stage for automated detection beyond the 8-cell stage. The cavitation and blastocyst formation stages also offer opportunities for automated analysis of images, covering expan-

In fluorescence time-lapse imaging of nonhuman mammalian embryos, the life time of the fluorophores is limited, an effect referred to as *photobleaching* [27]. Bleaching can be limited by reducing exposure, but ultimately sets a limit to the duration of the imaging sequence. The most severe cause of concern is the toxic effects caused by the exposure to intense laser light for a prolonged period of time. This *phototoxicity* can be limited by minimizing laser exposure using mechanical fast shutters or switching LEDs, but any shutters will quickly reach the end of their life span in a continuous time-lapse imaging set up. Switching at 1 Hz, a shutter will open and close a million times in about 12 days. In all cases, an efficient microscopy control

There is a trade-off between information gathered and potentially harmful sample exposure, and the frequency of image capture must be carefully chosen depending on the study endpoint and the expected frequency of the dynamics under study. In the case of simultaneous monitoring of multiple samples, two solutions exist. In *scanning*, either the imaging hardware or sample is moved and repositioned at each image capture. In this case there is a trade-off (limited by the moving mechanics) between samples imaged and images captured per sample. In *full-field,* the image captured includes all samples simultaneously. In this case, there is instead a trade-off between the number of samples imaged and the image resolution available

For two-dimensional imaging, full-field techniques are the most efficient as they capture the entire field of view in one single exposure. However, the stability of the system becomes critical as the time-lapse sequence length increases. *Focal drift* remains a problem and an autofocus

Even with moderate capture frequency, the amount of data from time-lapse studies can quickly build up to terabytes or more, especially if data is recorded simultaneously in multiple dimensions and imaging modalities. Consequently, both *data storage*, efficient *access to data* for analysis and the *post-acquisition analysis* itself must be considered. A small amount of video data may be analyzed manually, but this method quickly becomes cumbersome and time-consuming and automatic or semiautomatic methods are necessary. Manual evaluation of images is also prone to errors and inter-observer variability [49, 50]. It is often beneficial if the intended analysis can be considered already at the image capture stage so that acquisition,

mechanism or a method for user input to correct may be needed.

sion and collapse events.

46 Embryo Cleavage

software is necessary.

to each sample.

The optimal *choice of analysis* differs widely with the experimental set up and the aim of the study. Often, an initial analytical step is the identification of cell outlines in images. There are several ways to detect and track cell outlines in embryo imaging, both segmentation-based (requiring an identification of embryonic cell outlines), segmentation-free [52–55], or a combination of these [56]. Usually, a correctly performed segmentation [54, 57–59] provides the most detailed information on cell position, shape, and outline, but is computationally also the more challenging.

No single set of experimental conditions for long-term imaging can be used universally. Each biological question and model requires its own specific combination of hardware and software tools and must often be customized. Solutions to these challenges will enable important discoveries in embryology in the future. Kang et al. [60] and Turksen [61] provide useful summaries of protocols for fluorescent labelling and the imaging and tracking of stem cell, respectively. The following three sections exemplify successful time-lapse imaging methodologies for both human and nonhuman embryos with solutions to the experimental challenges using three very different approaches.

### **5. Method 1: three-dimensional mouse embryo morphology using fluorescent markers**

To understand compaction, cell lineage, cell rearrangement and dynamic behavior of embryonic cells during the cleavage phase, and dynamic imaging is necessary. This project studied the role of filopodia formation in compaction, apical constriction, pluripotent cell internalization, and cell positioning prior to embryo compaction, which is believed to be important for pluripotent development of embryonic cells. In addition, intracellular processes are monitored using a variety of targeted fluorescently tagged proteins and transcription factors.

With fluorescence microscopy, we can selectively excite and visualize fluorescent proteins as a marker in living tissue. The discovery of genetically encoded fluorescent proteins (FPs) permits the quantitative analysis of most cellular proteins including monitoring of their distribution and dynamics [62]. Fluorescence imaging is a technique that perfectly addresses problems in embryonic development, because of the need to study embryos in vivo. In confocal microscopy, in contrast to widefield fluorescence imaging, the detector pinhole blocks fluorescence from areas that lie out of focus [63]. This allows confocal imaging to reduce some of the scattering effects elicited by widefield fluorescence microscopy. However, scanning a single section implies the excitation and, therefore, damaging off-focus areas above and below the focal plane. In addition, the pinhole will also exclude scattered signal photons emitted from the focal plane as they travel away from the specimen. Therefore, widefield and confocal imaging are methods best suited for thin samples of less than ~40 μm. To study the events occurring deeper in the mouse embryo, which is about 100 μm in diameter (70 m of cellular portion plus the zona pellucida), requires the use of two-photon excitation fluorescence microscopy.

Two-photon excitation (2PE) fluorescence microscopy is a way to limit phototoxicity in the sample and to extend the imaging time and depth at high resolution and contrast [64]. In 2PE, two photons of half the excitation energy are needed to place the FP in the excited state. A focused laser is used in 2PE to generate higher intensity localized in the area of the focal plane, which results in excitation limited to a very small focal volume (typically of ~0.1 μm<sup>3</sup> ). A combination of confocal and two-photon excitation (2PE) fluorescence microscopy can be used to follow and characterize different morphogenetic changes in developing embryos such as cell division, polarity, filopodia formation and dynamics, compaction, and blastocyst cavitation (**Figure 1**). For this aim, specific fluorescently tagged proteins or peptides are used to label nuclear, cytoplasmic, or membrane constituents and optimized confocal and 2PE fluorescence imaging methods [29, 31, 65]. These imaging conditions allow the scan of a single embryo at intervals down to less than 60 s and reconstruction of 3D embryo morphology using Imaris (Bitplane AG) or ZEN (Zeiss) software. For long-term imaging sessions positioning software (Zeiss Zen) is used to image 20–30 embryos cultured next to each other (**Figure 1**). Thanks to the high-sensitive detectors of confocal and 2PE fluorescence microscopes, it is possible to perform long-term imaging sessions lasting more than 24 h, without this affecting the health and integrity of the mouse embryos. Thus it is possible to follow in an overnight imaging session cell dynamics in 20–30 embryos. Images are captured at intervals of 40 min from eight-cell stage to blastocyst (an interval of about 36 h). Capturing fluorescent imaging together with brightfield optics makes it possible to monitor simultaneously cell and molecular dynamics (**Figure 1D**).

For the simultaneous subcellular study of proteins at different stages of development, it is possible to study the dynamics of subcellular markers from zygote to blastocyst stage. For this purpose, DNA constructs in the pCS2+ expression vector [66] and synthesized capped RNA (using the Ambion mMessage mMachine SP6 transcription kit) are used. Capped marker-GFP RNA is injected into one-cell stage embryos. For nulcei, H2B-RFP is commonly used as marker, whereas memb-mCherry, Ecad-RFP, Ecad-GFP, or Ezrin-RFP can be used for membrane monitoring (**Figure 1**) [32, 34, 65]. **Figure 1C** shows an example of using the nuclear marker H2B-GFP and the membrane marker Ecad-GFP. Polarity events can be studied using Ezrin-GFP. Ezrin is expressed homogeneously in all cells before it becomes polarized during embryonic compaction [67] (**Figure 1B**). Hence, colocalization with Ezrin-GFP is an excellent way to study the dynamics and distribution of any protein of interest during compaction and cell polarity.

Methods for Spatio-Temporal Analysis of Embryo Cleavage In Vitro http://dx.doi.org/10.5772/intechopen.69650 49

problems in embryonic development, because of the need to study embryos in vivo. In confocal microscopy, in contrast to widefield fluorescence imaging, the detector pinhole blocks fluorescence from areas that lie out of focus [63]. This allows confocal imaging to reduce some of the scattering effects elicited by widefield fluorescence microscopy. However, scanning a single section implies the excitation and, therefore, damaging off-focus areas above and below the focal plane. In addition, the pinhole will also exclude scattered signal photons emitted from the focal plane as they travel away from the specimen. Therefore, widefield and confocal imaging are methods best suited for thin samples of less than ~40 μm. To study the events occurring deeper in the mouse embryo, which is about 100 μm in diameter (70 m of cellular portion plus the zona pellucida), requires the use of two-photon excitation fluorescence

Two-photon excitation (2PE) fluorescence microscopy is a way to limit phototoxicity in the sample and to extend the imaging time and depth at high resolution and contrast [64]. In 2PE, two photons of half the excitation energy are needed to place the FP in the excited state. A focused laser is used in 2PE to generate higher intensity localized in the area of the focal plane,

bination of confocal and two-photon excitation (2PE) fluorescence microscopy can be used to follow and characterize different morphogenetic changes in developing embryos such as cell division, polarity, filopodia formation and dynamics, compaction, and blastocyst cavitation (**Figure 1**). For this aim, specific fluorescently tagged proteins or peptides are used to label nuclear, cytoplasmic, or membrane constituents and optimized confocal and 2PE fluorescence imaging methods [29, 31, 65]. These imaging conditions allow the scan of a single embryo at intervals down to less than 60 s and reconstruction of 3D embryo morphology using Imaris (Bitplane AG) or ZEN (Zeiss) software. For long-term imaging sessions positioning software (Zeiss Zen) is used to image 20–30 embryos cultured next to each other (**Figure 1**). Thanks to the high-sensitive detectors of confocal and 2PE fluorescence microscopes, it is possible to perform long-term imaging sessions lasting more than 24 h, without this affecting the health and integrity of the mouse embryos. Thus it is possible to follow in an overnight imaging session cell dynamics in 20–30 embryos. Images are captured at intervals of 40 min from eight-cell stage to blastocyst (an interval of about 36 h). Capturing fluorescent imaging together with brightfield optics makes it possible to monitor simultaneously cell and molecular dynamics (**Figure 1D**). For the simultaneous subcellular study of proteins at different stages of development, it is possible to study the dynamics of subcellular markers from zygote to blastocyst stage. For this purpose, DNA constructs in the pCS2+ expression vector [66] and synthesized capped RNA (using the Ambion mMessage mMachine SP6 transcription kit) are used. Capped marker-GFP RNA is injected into one-cell stage embryos. For nulcei, H2B-RFP is commonly used as marker, whereas memb-mCherry, Ecad-RFP, Ecad-GFP, or Ezrin-RFP can be used for membrane monitoring (**Figure 1**) [32, 34, 65]. **Figure 1C** shows an example of using the nuclear marker H2B-GFP and the membrane marker Ecad-GFP. Polarity events can be studied using Ezrin-GFP. Ezrin is expressed homogeneously in all cells before it becomes polarized during embryonic compaction [67] (**Figure 1B**). Hence, colocalization with Ezrin-GFP is an excellent way to study the dynamics and distribution of any protein of interest during compaction and

). A com-

which results in excitation limited to a very small focal volume (typically of ~0.1 μm<sup>3</sup>

microscopy.

48 Embryo Cleavage

cell polarity.

**Figure 1.** (A) Injection of nuclear (H2B-RFP) and cytoplasmic or membrane markers (marker-GFP) RNA at one cell stage, showing morphogenetic changes during mouse embryo development. (B) Cell polarity events (arrowheads) are observed at eight-cell stage visualized with the protein Ezrin-GFP. (C) Monitoring cytokinesis and timing of cell division using the membrane marker Ecad-GFP and nuclear marker H2B-RFP; Chromatin condensation is highlighted by dotted arrowheads, and cell division by plain arrowheads. (D) Cavitation (arrowhead) during blastocyst formation is observed with bright field optics combined with fluorescence imaging of membrane and nuclear markers (Ecad-GFP and H2B-RFP). Scale bar, 10 μm.

### **6. Method 2: cell lineage studies of human embryos using machine learning**

This method focuses on automated monitoring of human embryonic cells in dark field timelapse microscopy images of embryos with the goal to develop methods to segment, detect, localize the embryonic cells at each time step, and perform cell lineage analysis on a complete sequence. The result is a helpful tool for embryologists and IVF clinicians to understand the development of human embryo and more accurately select viable embryos.

In contrast to other cells (e.g., stem cells and embryonic cells of other species), automated analysis of nonstained human embryonic cells is challenged by complex development patterns such as compact growth and overlapping cells. These challenges are further complicated by the limitations of the single plane imaging limitations imposed by the dark field imaging mode, causing intensity variance and loss of depth information.

An important and first step in automated analysis is being able to efficiently and reliably segment the embryo from background noise. To this end, a framework to segment the developing embryo by estimating the contour around the embryo was developed by defining segmentation as an energy minimization problem and solving it via graph cuts [68]. Second, cells are spatially localized and divisions subsequently detected. For localization purposes, cells are modeled as ellipses fitted to the segmented outlines for each time step (**Figure 2**).

Predicting the number of cells is a fundamental task in cell biology analysis, and an indirect way to temporally locate embryo cleavage events. In the context of human embryonic cells, cell number is of prime importance as current embryo viability biomarkers require accurate cells counts. The prediction of cell numbers can either be performed directly from the microscopy images [69] or by detecting (localizing) cells [70, 71]. Both approaches can also be used in combination. In this method, a framework that combines both approaches in a conditional random field (CRF) [72] is used. The result is a model of the cell division ancestry by recording cell associations between adjacent frames, resulting in a complete lineage tree for the time-lapse sequence. Cell lineage analysis is vital in understanding dynamics of developing embryos and is a fundamental step in cell biology analysis. The cell lineage tree and segmented shapes can now be studied for various attributes of the growing embryo such as timing of cell cleavage, abnormal division patterns, and cell symmetry (**Figure 3**).

**Figure 2.** Example of (a) dark field microscopy image of a two-cell stage human embryo; (b) cell localization with fitted ellipses; (c) three to four cell division association for lineage tree construction.

**Figure 3.** Proposed system for automated monitoring of early stage human embryo development.

#### **7. Method 3: human embryo profiling using video image processing**

embryo by estimating the contour around the embryo was developed by defining segmentation as an energy minimization problem and solving it via graph cuts [68]. Second, cells are spatially localized and divisions subsequently detected. For localization purposes, cells are

Predicting the number of cells is a fundamental task in cell biology analysis, and an indirect way to temporally locate embryo cleavage events. In the context of human embryonic cells, cell number is of prime importance as current embryo viability biomarkers require accurate cells counts. The prediction of cell numbers can either be performed directly from the microscopy images [69] or by detecting (localizing) cells [70, 71]. Both approaches can also be used in combination. In this method, a framework that combines both approaches in a conditional random field (CRF) [72] is used. The result is a model of the cell division ancestry by recording cell associations between adjacent frames, resulting in a complete lineage tree for the time-lapse sequence. Cell lineage analysis is vital in understanding dynamics of developing embryos and is a fundamental step in cell biology analysis. The cell lineage tree and segmented shapes can now be studied for various attributes of the growing embryo such as tim-

**Figure 2.** Example of (a) dark field microscopy image of a two-cell stage human embryo; (b) cell localization with fitted

modeled as ellipses fitted to the segmented outlines for each time step (**Figure 2**).

50 Embryo Cleavage

ing of cell cleavage, abnormal division patterns, and cell symmetry (**Figure 3**).

ellipses; (c) three to four cell division association for lineage tree construction.

**Figure 3.** Proposed system for automated monitoring of early stage human embryo development.

HMC imaging is superior when it comes to image detail of human embryos. However, optical artifacts introduced by the optical modulation causes edge structures to appear with multiple gradients. Objects in focus commonly appear clearly, but at the same time, superimposed light from out-of-focus objects will often introduce "shadows" in the image. The result

**Figure 4.** (a) Illustration of computational pipeline of the captured image series of an embryo. The optimal focal plane from the image stack was selected. A region of interest (ROI) was selected within each individual image, and one value of the variance in image intensity was computed for each ROI. This process was repeated for each capture in the image series, resulting in a function v(t) describing the variance as a function of time. v(t) was then further analyzed for the occurrence of detectable key events, profiling the embryo development. Finally, the profiles for embryos forming blastocysts and for those not forming blastocysts were compared. (b) Image intensity variance of an embryo during the course of 280 frame captures, normalized to the first image in the series. Divisions during the cleavage stage are detectable as sudden increases in image variance, due to the number of increased edges in the image, as blastomeres undergo mitosis. At the onset of compaction, individual blastomere membranes are no longer distinguishable, and the variance drops and remains at a low level during the morula stage. The variance increases once more as blastocoel expansion sets may fluctuate strongly during the blastocyst stage, if the embryo displays several cycles of collapse and re-expansion. The growth of the embryo has been considered in five stages. (A) Initial divisions from fertilization to onset of compaction, (B) onset to completion of compaction, (C) morula, (D) cavitation, (E) blastocyst. The mean and change in variance has been calculated for each section. Dashed trend lines have been added for illustrative purpose [75].

**Figure 5.** Profile of three representative embryos showing decreasing quality (a–c). Variance was calculated from the image intensity at a circular region encompassing the center of the embryo. A few example images are shown at points where characteristic changes are visible in the variance profile. For a good quality embryo (a) mitotic divisions are visible as successive increases in image variance, and the morula stage as a period of lowered variance; (b) illustrates a clearly expressed pronuclear breakdown, but experiences fragmentation during the cleavage stage, even though a blastocyst iseventually formed. In (c), the pronuclear breakdown is also apparent, but the embryo develops early fragments, never reaching a blastocyst stage [75].

is an image where it is inherently difficult to segment cell outlines, but with a high degree of detail in internal cell structures, despite the fact that the technique is completely marker free. Attempting to segment such an image is possible, but since subsequent analysis is often Methods for Spatio-Temporal Analysis of Embryo Cleavage In Vitro http://dx.doi.org/10.5772/intechopen.69650 53

**Figure 6.** Detection of zygote and pronucleus in human embryo. (a) Original image. (b) Edge detection. (c) Five most significant circular structures selected. (d) 10 most significant circular structures selected. (e) Overlap of circular structures selected from the same image rotated 6 60°. (f) Outline of pronucleus indicated, overlap of three calculations at separate angles. (g) Outline of pronucleus selected. (h) Outline of zygote selected [74].

**Figure 5.** Profile of three representative embryos showing decreasing quality (a–c). Variance was calculated from the image intensity at a circular region encompassing the center of the embryo. A few example images are shown at points where characteristic changes are visible in the variance profile. For a good quality embryo (a) mitotic divisions are visible as successive increases in image variance, and the morula stage as a period of lowered variance; (b) illustrates a clearly expressed pronuclear breakdown, but experiences fragmentation during the cleavage stage, even though a blastocyst iseventually formed. In (c), the pronuclear breakdown is also apparent, but the embryo develops early fragments, never

is an image where it is inherently difficult to segment cell outlines, but with a high degree of detail in internal cell structures, despite the fact that the technique is completely marker free. Attempting to segment such an image is possible, but since subsequent analysis is often

reaching a blastocyst stage [75].

52 Embryo Cleavage

dependent on the resulting segmented outline, it is easy to introduce cumulative errors. This method focuses on the detection of developmentally relevant events in the embryo such as compaction, blastocoel formation, nuclei localization, cell cleavage, and embryo fragmentation without the need for complete segmentation.

Raw images are spatially filtered to embryo location and a set of image features are extracted from the embryo interior [73, 74]. As the embryo grows, characteristics of the image will change also the image features, making it possible to profile embryo development without the complete image data [75]. One example is shown in **Figure 4**, where the gray-level variance of the image of the embryo interior is used to plot a development sequence of the embryo in two dimensions. The gray-level variance is a measure of the contrast in the image and will increase for each cell division, as each division introduces a new cell, and thus a new set of darker cell membrane into the image, contributing to a rise in image variance. As a consequence, each cell division can be detected as a sudden steep gradient in the variance profile. The compaction is detected as a massive loss of variance as the cell membranes becomes less apparent, followed by a new increase in variance as the embryo forms a blastocoel. The ideal development of an embryo follows a predictable pattern over time, where events such as cleavage can be more easily and automatically detected than using images directly (**Figure 4**) and abnormal development will differ clearly (**Figure 5**).

Simultaneously to feature detection, segmentation of intracellular structures such as nuclei and pronuclei is possible due to the high level of image detail (**Figure 6**). The segmentation is constricted in shape and size, ensuring the located structures are of the predefined biological shape. A slight disturbance is introduced in the form of a rotation and serves to effectively average out the located structures and preventing the detection of false positives [74]. The result is a framework where the entire development from zygote to blastocyst can be profiled and combined with the visibility of relevant intracellular compartments such as nuclei, without the need for any fluorescent markers.

#### **8. Conclusion**

It has been shown that embryos can grow outside the womb for longer than 14 days, a limitation set by legal requirements [76]. This period of early embryo development has yet been little studied, due to technical constraints. New combinations of software analysis, imaging, and incubator technologies will soon make it possible to study embryo development from a whole new set of perspectives.

Using specific FP-tagged protein markers for the nucleus and plasma membrane it is possible to follow the dynamics of important morphogenetic changes during mammalian embryo development, including cell division, cell polarity, and cavitation during blastocyst formation. The quantitative analysis of these developmental hallmarks pave the way for the design of functional and phenotypical studies such as silencing (knocking down), overexpressing, or blocking using inhibitors of selected genes of interest. These method combinations can lead to the crucial understanding of developmental function and disease.

Methods for automated or semiautomated label-free analysis of embryos in vivo make it possible to study embryo development over longer times than previously possible—opening up a new set of insights into especially early human development, where ethical considerations are important for the choice of study method. By time-lapse sequence studies of routinely growing embryos in IVF, the research data can be gathered in a clinical context, and methods can simultaneously contribute to better IVF embryo monitoring.

In conclusion, these noninvasive methods open a window to increase the understanding of general developmental embryology as well as specific medical questions such as embryo division patterns, lineage, and the reasons behind the low human fertility rates.

#### **Acknowledgements**

dependent on the resulting segmented outline, it is easy to introduce cumulative errors. This method focuses on the detection of developmentally relevant events in the embryo such as compaction, blastocoel formation, nuclei localization, cell cleavage, and embryo fragmenta-

Raw images are spatially filtered to embryo location and a set of image features are extracted from the embryo interior [73, 74]. As the embryo grows, characteristics of the image will change also the image features, making it possible to profile embryo development without the complete image data [75]. One example is shown in **Figure 4**, where the gray-level variance of the image of the embryo interior is used to plot a development sequence of the embryo in two dimensions. The gray-level variance is a measure of the contrast in the image and will increase for each cell division, as each division introduces a new cell, and thus a new set of darker cell membrane into the image, contributing to a rise in image variance. As a consequence, each cell division can be detected as a sudden steep gradient in the variance profile. The compaction is detected as a massive loss of variance as the cell membranes becomes less apparent, followed by a new increase in variance as the embryo forms a blastocoel. The ideal development of an embryo follows a predictable pattern over time, where events such as cleavage can be more easily and automatically detected than using images directly (**Figure 4**) and abnormal devel-

Simultaneously to feature detection, segmentation of intracellular structures such as nuclei and pronuclei is possible due to the high level of image detail (**Figure 6**). The segmentation is constricted in shape and size, ensuring the located structures are of the predefined biological shape. A slight disturbance is introduced in the form of a rotation and serves to effectively average out the located structures and preventing the detection of false positives [74]. The result is a framework where the entire development from zygote to blastocyst can be profiled and combined with the visibility of relevant intracellular compartments such as nuclei, with-

It has been shown that embryos can grow outside the womb for longer than 14 days, a limitation set by legal requirements [76]. This period of early embryo development has yet been little studied, due to technical constraints. New combinations of software analysis, imaging, and incubator technologies will soon make it possible to study embryo development from a

Using specific FP-tagged protein markers for the nucleus and plasma membrane it is possible to follow the dynamics of important morphogenetic changes during mammalian embryo development, including cell division, cell polarity, and cavitation during blastocyst formation. The quantitative analysis of these developmental hallmarks pave the way for the design of functional and phenotypical studies such as silencing (knocking down), overexpressing, or blocking using inhibitors of selected genes of interest. These method combinations can lead to

the crucial understanding of developmental function and disease.

tion without the need for complete segmentation.

54 Embryo Cleavage

opment will differ clearly (**Figure 5**).

out the need for any fluorescent markers.

**8. Conclusion**

whole new set of perspectives.

Anna Leida Mölder is grateful for the help and support of Manchester Metropolitan University. Juan Carlos Fierro-González is grateful for the support received from the Swedish Society for Medical Research.

#### **Author details**

Anna Leida Mölder<sup>1</sup> \*, Juan Carlos Fierro-González<sup>2</sup> and Aisha Khan<sup>3</sup>

\*Address all correspondence to: mail@annaleida.com

1 Manchester Metropolitan University, Manchester, UK

2 Chalmers University of Technology, Department of Biology and Biological Engineering, Gothenburg, Sweden

3 Research & Engineering, Progyny Inc, New York, USA

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**Gene Expression of Cleavage Embryo and Noninvasive Assessment**

## **Control of Embryonic Gene Expression and Epigenetics**

Pinar Tulay

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67851

#### **Abstract**

Preimplantation embryo development follows a series of critical events. Remarkable epigenetic modifications and reprogramming of gene expression occur to activate the embryonic genome. In the early stages of preimplantation embryo development, maternal mRNAs direct embryonic development. Throughout early embryonic development, a differential methylation pattern is maintained although some show stage‐specific changes. Recent studies have shown that differential demethylation process results in differential parental gene expression in the early developing embryos that may have an impact on the correct development. In the recent years, noncoding RNAs, long noncoding RNAs (lncRNA) and short of mRNAs and therefore their role in preimplantation devel‐ opment has gained significance.

**Keywords:** gene expression, methylation, miRNA

#### **1. Introduction**

Preimplantation embryo development follows a series of critical events. These events start at gametogenesis, formation of mature gametes, and lasts until parturition. Male and female gametes are derived from primordial germ cells (PGCs) by the processes of spermatogenesis and oogenesis, respectively. PGCs have unique properties of gene expression, epigenetics, morphology and behaviour. Once the PGCs undergo mitosis, spermatogenesis and oogenesis progress differently. In spermatogenesis, spermatogonia undergo mitosis starting at puberty until death and each primary spermatocyte produces four spermatids at the end of meiosis. In oogenesis, PGCs differentiate into oogonia, they enter meiosis and arrest until puberty. Unlike meiosis II in spermatogenesis, secondary oocyte does not complete meiosis II until fertilisation. With completion of meiosis II, each oogonia produce a single viable oocyte [1].

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

At fertilisation, the oocyte completes meiosis and the fertilised oocyte is called the zygote. Oocyte and sperm nuclei fuse resulting in syngamy (**Figure 1**). The zygote undergoes a series of cleavage divisions, forming two‐cell, four‐cell, eight‐cell morula and blastocyst stages [2] (**Figure 1**). During cleavage stage divisions, programming of maternal and paternal chromosomes takes place to create the embryonic genome (embryonic genome activation, EGA) and to start the preimplantation embryo development. If the EGA fails, the development does not continue because of the inability of the embryo to have cellular functions [3]. This activation is initiated by the degradation of maternal nucleic acids, specific RNAs stored in oocytes, proteins and other macromolecules [4]. Upon EGA, which starts at the two‐cell stage in mouse and four‐ to eight‐cell stage in human [5], remarkable reprogramming of expres‐ sion occurs in the preimplantation embryo. These reprogramming events are controlled by DNA methylation, histone acetylation, transcription, translation and miRNA regulation [6]. Therefore, the development of preimplantation embryos includes continuous molecular, cel‐ lular and morphological events. These events would eventually form a multilineage embryo that has a capability to implant and continue the foetal development.

In this chapter, different factors affecting gene expression during preimplantation embryo development will be discussed. Epigenetic factors, focusing on methylation profiles, of gam‐ etes and preimplantation embryos will be reviewed. The effects of noncoding RNAs on gene expression will be thoroughly evaluated.

**Figure 1.** Schematic diagram outlining the main stages of preimplantation embryo development. Fertilisation followed by syngamy, cleavage divisions results in two, three, four, and so on cell embryos which eventually form the morula and the blastocyst.

#### **2. Gene expression and epigenetics**

For a normal developing embryo, the expression of both maternal and paternal genes is required. An intense epigenetic change occurs upon fertilisation to establish pluripotency [7]. Although there are a number of post‐translational modifications within chromatin including acetylation, ubiquitination, SUMOylation and phosphorylation; methylation of histone lysine and arginine residues is the main focus in preimplantation embryos.

Methylation and chromatin modification not only play crucial roles in determining the transcriptional state but also are capable of determining the transcriptional repression [8–10]. The mechanism leading to the changes in methylation is not well established, but it has been suggested that the reprogramming takes place by either passive or active demeth‐ ylation. Indirect pathways of demethylation are associated with DNA repair [11–14]. Two main stages, PGCs and preimplantation embryos, are important in the regulation by methylation.

#### **2.1. Epigenetic modification of the zygote and the preimplantation embryos**

At fertilisation, the oocyte completes meiosis and the fertilised oocyte is called the zygote. Oocyte and sperm nuclei fuse resulting in syngamy (**Figure 1**). The zygote undergoes a series of cleavage divisions, forming two‐cell, four‐cell, eight‐cell morula and blastocyst stages [2] (**Figure 1**). During cleavage stage divisions, programming of maternal and paternal chromosomes takes place to create the embryonic genome (embryonic genome activation, EGA) and to start the preimplantation embryo development. If the EGA fails, the development does not continue because of the inability of the embryo to have cellular functions [3]. This activation is initiated by the degradation of maternal nucleic acids, specific RNAs stored in oocytes, proteins and other macromolecules [4]. Upon EGA, which starts at the two‐cell stage in mouse and four‐ to eight‐cell stage in human [5], remarkable reprogramming of expres‐ sion occurs in the preimplantation embryo. These reprogramming events are controlled by DNA methylation, histone acetylation, transcription, translation and miRNA regulation [6]. Therefore, the development of preimplantation embryos includes continuous molecular, cel‐ lular and morphological events. These events would eventually form a multilineage embryo

In this chapter, different factors affecting gene expression during preimplantation embryo development will be discussed. Epigenetic factors, focusing on methylation profiles, of gam‐ etes and preimplantation embryos will be reviewed. The effects of noncoding RNAs on gene

For a normal developing embryo, the expression of both maternal and paternal genes is required. An intense epigenetic change occurs upon fertilisation to establish pluripotency [7]. Although there are a number of post‐translational modifications within chromatin including acetylation, ubiquitination, SUMOylation and phosphorylation; methylation of histone lysine

**Figure 1.** Schematic diagram outlining the main stages of preimplantation embryo development. Fertilisation followed by syngamy, cleavage divisions results in two, three, four, and so on cell embryos which eventually form the morula

Methylation and chromatin modification not only play crucial roles in determining the transcriptional state but also are capable of determining the transcriptional repression

and arginine residues is the main focus in preimplantation embryos.

that has a capability to implant and continue the foetal development.

expression will be thoroughly evaluated.

**2. Gene expression and epigenetics**

and the blastocyst.

66 Embryo Cleavage

In mammals (human, bovine, rat, pig and mouse), the zygote undergoes genome‐wide demethylation [15–17] with the exception of imprinted genes [18]. The male pronucleus of the zygote undergoes selective demethylation due to the loss of DNA replication leading to asymmetric methylated sister chromatids [15, 16, 19, 20]. These events start following the sperm decondensation in humans and in mouse with some variations [17, 21, 22]. The female pronucleus of the zygote remains highly methylated at this stage [17, 21, 22]. Demethylation of the maternal genome starts with the first cleavage divisions [19, 23, 24]. By the morula stage, the mouse preimplantation embryos become undermethylated. Polarisation and com‐ paction of individual blastomeres start at around eight‐cell stage of the developing embryo. Many factors are involved in these processes including E‐cadherin (CDH1), partitioning defective homologue 3 (PARD3), PARD6B and protein kinase C zeta [25–27].

The blastocyst stage embryo has a fluid‐filled cavity and two cell populations consisting of inner cell mass (ICM) and trophectoderm (TE). All the blastomeres are believed to be totipotent in cleavage embryos until four‐ to eight‐cell stage since these cells form both the ICM and TE lineage [28]. ICM develops into epiblast, whereas TE forms the extraembry‐ onic tissues such as placenta. ICM is composed of pluripotent cells that have the capacity to develop into any cell type of the foetus. Transcriptional and epigenetic events strictly regulate these differentiation events. A number of transcriptional factors play a crucial role in blastocyst formation. These include caudal type homeobox 2 (CDX2) for TE specification, octamer 3/4 (OCT4) and NANOG for the establishment of ICM pluripotency [29–31]. CDX2 is extensively expressed in eight‐ and 16‐cell stage and it is expressed only in TE cells of the blastocyst [32]. Although OCT4 and NANOG are also expressed broadly at eight‐ and 16‐cell stage embryos, they are only expressed in ICM in blastocysts [32]. A number of transcription factors are required for blastocyst formation. Embryos lacking CDX2 expression cannot form blastocoel cavity but they have the ability to implant [30]. Lack of OCT4 or NANOG expres‐ sion causes failure of ICM and the development of these embryos is arrested at the blastocyst stage [31, 32]. TEAD4 is another transcription factor that has a role in blastocyst transition in which the lack of TEAD4 nuclear localisation impairs TE‐specific transcriptional programme in inner blastomeres [33]. Furthermore, the aberrant expression of TCFAP2C transcription factor also leads to embryonic arrest during morula to blastocyst transition [34] and Klf5 mouse‐mutant embryos arrest at the blastocyst stage [35].

The remethylation process starts shortly after implantation [16, 22, 23, 36]. This *de novo* methylation occurs asymmetrically, such that ICM is hypermethylated possibly due to the Dnmt3b methylase [37], whereas TE remains hypomethylated due to the active demethyl‐ ation by enzyme catalysis and passive demethylation [11, 14, 22]. Alteration of the methyla‐ tion profiles in embryos has been shown to cause alterations of ICM and TE differentiation. Variations of the H3 arginine 26 residue (H3R26me) were shown to lead to changes of TE and ICM differentiation of a blastomere [38].

X‐chromosome inactivation is an epigenetic phenomenon in which the activity of X chromo‐ somes is strictly regulated to equalise X‐chromosome expression and gene dosage between males and females and relative to autosome chromosomes [39]. For correct development, X‐chromosome dosage compensation is crucial. The inactivation of X chromosome occurs in at least two phases: initiation and maintenance. X‐inactivation mouse model systems have shown that the inactivation of X chromosome takes place during early embryogenesis of the female embryo by undergoing transcriptional silencing of genes along the X chromosome [40]. In human preimplantation embryos, it has been shown that the reduced expression of X chromosomes in females ensures the dosage compensation [41]. LncRNA *XIST* expres‐ sion activates the X‐chromosome inactivation by engaging proteins functioning in chromatin remodelling [3, 42]. With the advanced technologies, including single‐cell RNA sequencing, it has emerged that lncRNAs *XACT* and *XIST* are expressed on the active X chromosome in the early human preimplantation embryos [43]. Furthermore, the expression of these two RNAs has never been shown to overlap. Introducing *XACT* into heterologous systems caused the accumulation of *Xist* RNA in *cis* and therefore it may be involved in the control of *XIST* association to chromosome in *cis* and may temper its ability of silencing. It is also possible that *XACT* functions in balancing the X‐chromosome inactivation at the early stages of preimplantation embryo development [43, 44]. Recently, the dosage compensation was shown to be driven by a CAG promoter of a new Xist allele (Xist(CAG)) [45]. Furthermore, Xist(CAG) upregulation in preimplantation embryos showed variation depending on the parental origin and the paternal expression was suggested to be preferentially inactivated with the paternal Xist(CAG) transmission [45].

#### **2.2. Epigenetic modification of the gametes**

In germ cells, methylation is maintained in a sex‐specific manner. Methylation in PGCs dimin‐ ishes as they migrate to the gonads. Studies suggest that in females, remethylation occurs after birth when the oocytes are in the process of development. When demethylation is com‐ pleted, the PGCs either enter mitosis in males or arrest at meiosis in females [46].

Reprogramming of the methylation in the embryo is necessary for parent‐specific expression of genes [14]. Gene expression varies during preimplantation embryo development due to these reprogramming events and appropriate gene expression determines the survival of the embryo [6]. Recently, short noncoding RNAs, microRNAs (miRNAs) and long noncoding RNAs (lncRNA) have gained importance in their potential function to affect numerous path‐ ways by targeting multiple genes [47, 48].

#### **3. Gene expression and small noncoding RNAs: microRNAs**

MiRNAs are a large family of short noncoding RNAs between 17 and 25 nucleotides (nt) in length [49]. MiRNAs were first identified in *Caenorhabditis elegans* over two decades ago [50] and since then many have been identified in multiple organisms, such as worms, flies, fish, frogs, mammals and plants, by molecular cloning and bioinformatics [51]. Most miRNA sequences are conserved among a wide range of mammalians [52], though there are some that differ from each other only by a single nucleotide [53]. The conserved miRNA sequences among different species can be distinguished by the nomenclature such that when only the first three letters differ this indicates the same sequence in different species, that is, hsa‐ miR‐145 in *Homo sapiens* and mmu‐miR‐145 in *Mus musculus* [54].

MiRNAs have been shown to be of great importance in a wide variety of biological processes involving cell cycle regulation, apoptosis, cell differentiation, imprinting, homeostasis and development, including limb development [55], morphogenesis of lung epithelial [56], embryonic angiogenesis [57], formation of hair follicle and proliferation of T‐cell [58, 59]. They play key roles in regulating transcriptional and post‐transcriptional gene silencing in many organisms by targeting mRNAs for translational inhibition, cleavage, degradation or destabilisation [53, 60–64]. Each miRNA has multiple mRNA targets that may regulate up to 30% protein‐coding genes and shape protein production from hundreds to thousands of genes [65–67]. MiRNAs recognise their targets through base pairing of the complementary sequence of their seed sequence (2–8 nt of miRNAs) within the open reading frame (ORF) and 3′untranslated region (UTR) of target mRNA [68]. Although the targets of miRNAs are not fully known, bioinformatics studies show a range of possible target genes [69]. The functional activities and the predicted/observed targets of miRNAs can be identified using miRNA databases. These databases can be accessed using the following URL: (http://www.targetscan. org/, http://www.microrna.org/microrna/home.do and http://mirdb.org/miRDB/).

#### **3.1. MiRNA biogenesis**

Variations of the H3 arginine 26 residue (H3R26me) were shown to lead to changes of TE and

X‐chromosome inactivation is an epigenetic phenomenon in which the activity of X chromo‐ somes is strictly regulated to equalise X‐chromosome expression and gene dosage between males and females and relative to autosome chromosomes [39]. For correct development, X‐chromosome dosage compensation is crucial. The inactivation of X chromosome occurs in at least two phases: initiation and maintenance. X‐inactivation mouse model systems have shown that the inactivation of X chromosome takes place during early embryogenesis of the female embryo by undergoing transcriptional silencing of genes along the X chromosome [40]. In human preimplantation embryos, it has been shown that the reduced expression of X chromosomes in females ensures the dosage compensation [41]. LncRNA *XIST* expres‐ sion activates the X‐chromosome inactivation by engaging proteins functioning in chromatin remodelling [3, 42]. With the advanced technologies, including single‐cell RNA sequencing, it has emerged that lncRNAs *XACT* and *XIST* are expressed on the active X chromosome in the early human preimplantation embryos [43]. Furthermore, the expression of these two RNAs has never been shown to overlap. Introducing *XACT* into heterologous systems caused the accumulation of *Xist* RNA in *cis* and therefore it may be involved in the control of *XIST* association to chromosome in *cis* and may temper its ability of silencing. It is also possible that *XACT* functions in balancing the X‐chromosome inactivation at the early stages of preimplantation embryo development [43, 44]. Recently, the dosage compensation was shown to be driven by a CAG promoter of a new Xist allele (Xist(CAG)) [45]. Furthermore, Xist(CAG) upregulation in preimplantation embryos showed variation depending on the parental origin and the paternal expression was suggested to be preferentially inactivated

In germ cells, methylation is maintained in a sex‐specific manner. Methylation in PGCs dimin‐ ishes as they migrate to the gonads. Studies suggest that in females, remethylation occurs after birth when the oocytes are in the process of development. When demethylation is com‐

Reprogramming of the methylation in the embryo is necessary for parent‐specific expression of genes [14]. Gene expression varies during preimplantation embryo development due to these reprogramming events and appropriate gene expression determines the survival of the embryo [6]. Recently, short noncoding RNAs, microRNAs (miRNAs) and long noncoding RNAs (lncRNA) have gained importance in their potential function to affect numerous path‐

MiRNAs are a large family of short noncoding RNAs between 17 and 25 nucleotides (nt) in length [49]. MiRNAs were first identified in *Caenorhabditis elegans* over two decades ago

pleted, the PGCs either enter mitosis in males or arrest at meiosis in females [46].

**3. Gene expression and small noncoding RNAs: microRNAs**

ICM differentiation of a blastomere [38].

68 Embryo Cleavage

with the paternal Xist(CAG) transmission [45].

**2.2. Epigenetic modification of the gametes**

ways by targeting multiple genes [47, 48].

MiRNA biogenesis involves multiple important steps. MiRNAs are first transcribed from genomic DNA into primary miRNA (pri‐miRNA), which contains a stem‐loop structure, by RNA polymerase II. These pri‐miRNAs are then processed by Drosha, which is a 30–160 kDa protein with one dsRNA‐binding and two catalytic domains [70]. In the presence of DGCR8, both strands of the hairpin are cut generating a pre‐miRNA product of approximately 70 nt in size [71]. These pre‐miRNAs are carried from the nucleus into the cytoplasm by Exportin‐5 (Exp5), which is a nucleocytoplasmic transporter in karyopherin family that has binding sites for pre‐miRNAs in the presence of RAs‐related nuclear protein (Ran) and guanosine triphosphate (GTP) [72, 73]. These miRNAs are further cleaved by cytoplasmic RNase endonuclease, Dicer, making 21–22 nt double‐stranded structure. Although one of the strands is usually degraded, both strands of the pre‐miRNA may be associated with Argonaute (Ago)‐protein‐containing complex and they are mediated by RISC/miRNP (RNA‐induced silencing complex/mi‐ribonucleoprotein) to form single‐stranded mature miRNAs. MiRNAs associated with RISC mainly target mRNAs and they either inhibit their translation or cause degradation of mRNA that results in reduced protein synthesis [70, 74].

Studies showed that processing of miRNAs by Dicer was vital and any defects, such as deletion of Dicer in the developing animals, caused aberrations [75, 76]. Lack of Dicer in Drosophila germ line stem cells postponed the G1/S phase transition [77], suggesting that miRNAs may be vital for stem cells to bypass this checkpoint. Reduced and disorganised spindles, incor‐ rect chromosome alignment and defects in gastrulation were observed with the Dicer‐mutant oocytes in mouse and in *C. elegans*, respectively [50, 78]. Injection of miR‐430 in zebrafish and *C. elegans* partially repaired the gastrulation, retinal development and somatogenesis [78]. Dicer deletion in zebrafish, mouse and hippocampal initiated problems in the nervous sys‐ tem and led to the inability of forming mature miRNAs that resulted in variations of brain morphogenesis and differentiation of neurons [79, 80]. Although the axis formation and early differentiation of maternal‐zygotic Dicer‐mutant zebrafish and mouse embryos were normal, they still triggered defects in somitogenesis, morphogenesis that affected the brain formation, gastrulation, heart development and apoptosis in limb mesoderm, respectively [78, 81–83]. Apoptosis was enhanced in the developing limb mesoderm of Dicer null mouse [84]. Dicer deficiency mainly led to embryo death in mouse around embryonic day 7.5 [50, 78, 85] and in zebrafish [86] that may indicate the importance of miRNA‐mediated gene silencing at mater‐ nal to zygotic transition.

Complete loss of Dicer1 in somatic cells of mouse reproductive tract not only showed reduced expression of miRNAs but also caused the female mice to become infertile with compromised oocyte and embryo integrity [50, 87]. Dicer‐deficient male mice were shown to have poor prolif‐ eration of spermatogonia. Loss of Dicer1 in the germ line of male mice (homozygote Dicer1) led to decreased fertility due to abnormal spermatogenesis. The number of germ cells was reduced with abnormal spermatids, abnormal phenotype of spermatocytes with condensed nucleus, abnormal sperm motility and mutant testes with Sertoli tubules [88]. Studies suggest that the transfer of maternal cytoplasmic Dicer disguised the early abnormal phenotypes [78, 89].

Knock‐out of Ago2 in mouse embryonic fibroblasts and haematopoietic cells caused decreased levels of mature miRNAs [61, 90, 91]. Ago2‐deficient oocytes were observed to develop the mature oocytes with abnormal spindles and chromosomes were not able to unite properly with reduced expression levels of miRNAs (more than 80%). Loss of Ago2 function leads to embryo death around embryonic day 9.5 in mouse [92].

#### **3.2. Expression of miRNAs in preimplantation embryos**

The expression of miRNAs in preimplantation embryos has been mainly studied by knock‐ out experiments, by cloning experiments and by identifying individual miRNAs by microar‐ ray analysis and real‐time polymerase chain reaction [93]. The expression studies have been carried out using animal models and tissues, cultured cells; that is, cancer cells and human embryonic stem cells; and mouse/bovine/human gametes and embryos. Human embryonic stem cells, which are derived from the inner cell mass of an embryo at the blastocyst stage and are characterised by their ability of self‐renewal and multipotency, are the key in gene expression research since the access of human embryos is difficult and these cells are one of the closest representations of human embryos. Studying miRNA expression in stem cells not only gives insight into potential miRNAs expressed in human embryos but also may show the important role of miRNAs in the stem cell functioning [94].

MiRNA expression has been observed as early as oogenesis and spermatogenesis in mouse, bovine and human [95, 96]. Differences in the miRNA expression have been observed between immature and mature oocytes that may represent the natural turnover and indicate that each embryonic stage is defined by a specific miRNA. Similar miRNA expression profiles in mature mouse oocytes and early developing embryos indicate that at these stages the zygote has maternally inherited miRNAs [50]. Similar to oocyte, sperm carries a range of miRNAs. Approximately 20% of these miRNAs are located in the nuclear or perinuclear part of the sperm indicating that these miRNAs are transferred to the zygote at the time of fertilisation [97]. It was suggested that the sperm‐borne miRNAs may down‐regulate the maternal tran‐ scripts in mammals. However, when this hypothesis was tested using microarray analysis, it was shown that none of these miRNAs in the sperm have significant importance since all of them were already present in the oocytes (meiosis II) [98].

be vital for stem cells to bypass this checkpoint. Reduced and disorganised spindles, incor‐ rect chromosome alignment and defects in gastrulation were observed with the Dicer‐mutant oocytes in mouse and in *C. elegans*, respectively [50, 78]. Injection of miR‐430 in zebrafish and *C. elegans* partially repaired the gastrulation, retinal development and somatogenesis [78]. Dicer deletion in zebrafish, mouse and hippocampal initiated problems in the nervous sys‐ tem and led to the inability of forming mature miRNAs that resulted in variations of brain morphogenesis and differentiation of neurons [79, 80]. Although the axis formation and early differentiation of maternal‐zygotic Dicer‐mutant zebrafish and mouse embryos were normal, they still triggered defects in somitogenesis, morphogenesis that affected the brain formation, gastrulation, heart development and apoptosis in limb mesoderm, respectively [78, 81–83]. Apoptosis was enhanced in the developing limb mesoderm of Dicer null mouse [84]. Dicer deficiency mainly led to embryo death in mouse around embryonic day 7.5 [50, 78, 85] and in zebrafish [86] that may indicate the importance of miRNA‐mediated gene silencing at mater‐

Complete loss of Dicer1 in somatic cells of mouse reproductive tract not only showed reduced expression of miRNAs but also caused the female mice to become infertile with compromised oocyte and embryo integrity [50, 87]. Dicer‐deficient male mice were shown to have poor prolif‐ eration of spermatogonia. Loss of Dicer1 in the germ line of male mice (homozygote Dicer1) led to decreased fertility due to abnormal spermatogenesis. The number of germ cells was reduced with abnormal spermatids, abnormal phenotype of spermatocytes with condensed nucleus, abnormal sperm motility and mutant testes with Sertoli tubules [88]. Studies suggest that the transfer of maternal cytoplasmic Dicer disguised the early abnormal phenotypes [78, 89].

Knock‐out of Ago2 in mouse embryonic fibroblasts and haematopoietic cells caused decreased levels of mature miRNAs [61, 90, 91]. Ago2‐deficient oocytes were observed to develop the mature oocytes with abnormal spindles and chromosomes were not able to unite properly with reduced expression levels of miRNAs (more than 80%). Loss of Ago2 function leads to

The expression of miRNAs in preimplantation embryos has been mainly studied by knock‐ out experiments, by cloning experiments and by identifying individual miRNAs by microar‐ ray analysis and real‐time polymerase chain reaction [93]. The expression studies have been carried out using animal models and tissues, cultured cells; that is, cancer cells and human embryonic stem cells; and mouse/bovine/human gametes and embryos. Human embryonic stem cells, which are derived from the inner cell mass of an embryo at the blastocyst stage and are characterised by their ability of self‐renewal and multipotency, are the key in gene expression research since the access of human embryos is difficult and these cells are one of the closest representations of human embryos. Studying miRNA expression in stem cells not only gives insight into potential miRNAs expressed in human embryos but also may show the

MiRNA expression has been observed as early as oogenesis and spermatogenesis in mouse, bovine and human [95, 96]. Differences in the miRNA expression have been observed between

embryo death around embryonic day 9.5 in mouse [92].

**3.2. Expression of miRNAs in preimplantation embryos**

important role of miRNAs in the stem cell functioning [94].

nal to zygotic transition.

70 Embryo Cleavage

Multiple miRNAs were involved in the formation of germ cell layers. MiR‐290, which was expressed at different levels during preimplantation embryo development of mouse embryos, had a negative effect on the germ cell and mesoderm differentiation in the mouse ES cells *via* targeting Nodal inhibitors [99]. In zebrafish, however, miR‐290 cluster played an important role in regulating the mesoderm induction [100]. Therefore, it is not clear if miR‐290 has an inhibitory effect on the mesoderm differentiation. Other miRNAs have been shown to have an effect in mesoderm differentiation in zebrafish, such as miR‐15 and miR‐16 [100], which were also expressed in mouse preimplantation embryos [50].

Mainly, the same miRNAs are expressed during the cleavage divisions of the embryo in mouse and bovine. However, their expression levels often vary during these stages. In murine embryos, the level of miRNA expression is reduced by as much as 60% between one‐ and two‐ cell stages. At the end of four‐cell stage, mouse embryos have approximately twice as much miRNA compared to the two‐cell stage embryo. This implies that the maternally inherited miRNAs degrade at this stage and the EGA starts between the one‐cell and four‐cell stages [50]. Even though the synthesis and degradation of miRNAs coexists during the preimplan‐ tation embryo development in mice, the overall miRNA expression increased towards the blastocyst stage [101].

More than 700 miRNAs have been identified in humans [87, 95, 96, 102]. The level of expres‐ sion for the majority of these miRNAs stayed the same between the oocyte and the blastocyst stage [87]. More than 50% of the miRNAs expressed in human oocytes and blastocysts were shown to be involved in tumourigenesis, that is, let‐7 family, miR‐19a, miR‐21 and miR‐34 [103–109].

#### **4. Gene expression and long noncoding RNAs**

In the last few years, in addition to short noncoding RNAs, the lncRNA have gained impor‐ tance in their roles to affect gene expression. The mammalian genomes consist of long inter‐ genic noncoding RNAs (lincRNAs) that have been suggested to take a role in the regulation of pluripotency during preimplantation embryo development [110]. Human pluripotency transcripts 2, 3 and 5 (HPAT2, HPAT3 and HPAT5) were reported to adjust the pluripotency and ICM formation in preimplantation embryos. Furthermore, HPAT5 was shown to interact with let‐7 family of miRNAs [110].

Implantation of embryos involves complex mechanisms and many different genetic and physiological factors are involved during the process. Developing preimplantation embryo must have a good coordinated interaction with the maternal uterine endometrium. LncRNAs were shown to be differentially expressed in endometrial tissues obtained from pigs with pregnancy and non‐pregnancy with two lncRNAs, TCONS\_01729386 and TCONS\_01325501, with potential roles in implantation [111].

#### **5. Gene expression and assisted reproductive technologies**

In Western world, approximately 1% of children are born with assisted reproductive technol‐ ogy (ART) treatments. The infertile couples have the best possibility to conceive a child with these treatments. Although these techniques have been considered to be safe in terms of foetal and post‐natal development [112, 113], there is an increased risk for morbidities, especially imprinting disorders [114]. Furthermore, the global gene expression profiles vary due to *in vitro* culture of zygotes [115, 116] and *in vitro* fertilisation processes [117]. Following *in vitro* culture, apoptotic and morphogenetic pathways have shown to be altered [118].

Intra‐cytoplasmic sperm injection (ICSI), one of the widely used ART techniques, provides infertile couples with sperm motility problems a great chance to have a baby. ICSI is a unique process in which the sperm is injected into the ooplasm [119]. However, ICSI bypasses a number of physiological processes that would normally take place. These embryos derived from ICSI were shown to be cleaved at a slower rate. Furthermore, a reduced number of embryos become hatched with a fewer number of cells and the calcium oscillations are shorter with different patterns [120]. Mice embryos generated by ICSI were shown to be obese and have anomalies of the organs [121].

#### **6. Conclusion**

Normal development of preimplantation embryos involves complex mechanisms. For a normal developing embryo, the expression of both maternal and paternal genes is required. Several factors are involved in the regulation of parental genes in preimplantation embryos. Epigenetic modifications are one of the most important factors that are involved in the regulation of gene expression during preimplantation embryos. Extensive research studies have been performed throughout the years to establish the methylation profiles of the mammalian gametes and embryos. In the more recent years, the importance of noncoding RNAs in the regulation of genes has become clear. A handful of studies have been performed to analyse the expression of microRNAs, which have been shown to regulate mRNAs that encode up to 30% human pro‐ tein‐coding genes. The expression of miRNAs has been observed in mouse, bovine and human gametes and embryos. Furthermore, in the last couple of years, expression of long noncoding RNAs and their roles in embryonic development and implantation have been investigated. The extensive research studies have provided crucial understanding of the development of preimplantation embryos and the regulation of gene expression, and with the advancing tech‐ nologies more molecular studies will help to comprehend the mechanisms better.

### **Author details**

#### Pinar Tulay

Implantation of embryos involves complex mechanisms and many different genetic and physiological factors are involved during the process. Developing preimplantation embryo must have a good coordinated interaction with the maternal uterine endometrium. LncRNAs were shown to be differentially expressed in endometrial tissues obtained from pigs with pregnancy and non‐pregnancy with two lncRNAs, TCONS\_01729386 and TCONS\_01325501,

In Western world, approximately 1% of children are born with assisted reproductive technol‐ ogy (ART) treatments. The infertile couples have the best possibility to conceive a child with these treatments. Although these techniques have been considered to be safe in terms of foetal and post‐natal development [112, 113], there is an increased risk for morbidities, especially imprinting disorders [114]. Furthermore, the global gene expression profiles vary due to *in vitro* culture of zygotes [115, 116] and *in vitro* fertilisation processes [117]. Following *in vitro*

Intra‐cytoplasmic sperm injection (ICSI), one of the widely used ART techniques, provides infertile couples with sperm motility problems a great chance to have a baby. ICSI is a unique process in which the sperm is injected into the ooplasm [119]. However, ICSI bypasses a number of physiological processes that would normally take place. These embryos derived from ICSI were shown to be cleaved at a slower rate. Furthermore, a reduced number of embryos become hatched with a fewer number of cells and the calcium oscillations are shorter with different patterns [120]. Mice embryos generated by ICSI were shown to be obese and

Normal development of preimplantation embryos involves complex mechanisms. For a normal developing embryo, the expression of both maternal and paternal genes is required. Several factors are involved in the regulation of parental genes in preimplantation embryos. Epigenetic modifications are one of the most important factors that are involved in the regulation of gene expression during preimplantation embryos. Extensive research studies have been performed throughout the years to establish the methylation profiles of the mammalian gametes and embryos. In the more recent years, the importance of noncoding RNAs in the regulation of genes has become clear. A handful of studies have been performed to analyse the expression of microRNAs, which have been shown to regulate mRNAs that encode up to 30% human pro‐ tein‐coding genes. The expression of miRNAs has been observed in mouse, bovine and human gametes and embryos. Furthermore, in the last couple of years, expression of long noncoding RNAs and their roles in embryonic development and implantation have been investigated. The extensive research studies have provided crucial understanding of the development of

**5. Gene expression and assisted reproductive technologies**

culture, apoptotic and morphogenetic pathways have shown to be altered [118].

with potential roles in implantation [111].

72 Embryo Cleavage

have anomalies of the organs [121].

**6. Conclusion**

Address all correspondence to: pinar.tulay@neu.edu.tr

Department of Medical Genetics, Faculty of Medicine, Near East University, Nicosia, North Cyprus

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78 Embryo Cleavage


**Provisional chapter**

#### **Non‐Invasive Assessment of the Embryo Viability via the Analysis of the Culture Media the Analysis of the Culture Media**

**Non**‐**Invasive Assessment of the Embryo Viability via** 

DOI: 10.5772/intechopen.69436

Gergely Montskó, Zita Zrínyi, Ákos Várnagy, József Bódis and Gábor L. Kovács József Bódis and Gábor L. Kovács Additional information is available at the end of the chapter

Gergely Montskó, Zita Zrínyi, Ákos Várnagy,

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.69436

#### **Abstract**

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[121] Fernandez‐Gonzalez, R., et al., Long‐term effects of mouse intracytoplasmic sperm injection with DNA‐fragmented sperm on health and behavior of adult offspring. Biol

of mouse preimplantation embryos. Reproduction 2007. **134**: pp. 63‐72.

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mouse. Mol Hum Reprod, 2007. **13**: pp. 265‐72.

opment. Mol Hum Reprod, 2003. **9**: pp. 523‐33.

Reprod, 2008. **78**: pp. 761‐72.

80 Embryo Cleavage

Infertility in recent years is a growing public health issue throughout the developed world. Assisted reproductive techniques, especially *in vitro* fertilization, have the poten‐ tial to partially overcome the low natural reproductive ratio. Nowadays, single embryo transfer gains grounds in clinical practice, urging the development of more reliable methods for selecting the best embryo. In the traditional clinical practice, embryos are selected for transfer based on morphological evaluation. *In vitro* culturing of embryos also provides a very important material for further non‐invasive evaluation by means of examining a biomarker in the spent culture medium (SEC). Current measure methods concentrate on the metabolomic activity of the developing embryos none compounds. These studies are mainly utilizing the tools of modern analytics and proteomics. In a paper published by Montskó et al. in 2015, the alpha‐1 chain of the human haptoglobin molecule was described as a quantitative biomarker of embryo viability. In a series of retrospective, blind experiments achieved more than 50% success rate. This chapter sum‐ marizes the currently available metabolomic and proteomic approaches as the non‐inva‐ sive molecular assessment of embryo viability.

**Keywords:** *in vitro* fertilization, embryo viability, non‐invasive analysis, proteomics, mass spectrometry, haptoglobin alpha‐1 chain

#### **1. Introduction**

Nowadays, infertility is a major public health issue affecting couples in the developed world. With the widespread use of assisted reproductive techniques (ARTs), especially *in vitro* fertiliza‐ tion (IVF), there are more and more pregnancies conceived. Currently, approximately 3–4% of

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

all deliveries are IVF pregnancy and this number continues increasing. Availability of ART is a very relevant topic. The cultural and legal conditions, insurance/public funding systems and structure of data collection can influence not only the amount of treatment cycles per inhabitant but also success rates. The Assisted Reproductive Technology National Summary Report of the USA showed a total of 142.000 IVF cycles in 2007 [1], while the most current results in 2014 was 208.604 cycles [2]. The type of ART cycle applied (non‐donor or donor egg cycle) is highly var‐ ied based on the woman's age. The women younger than 35 years of age often used their own eggs (non‐donor) in the majority of cases and just about 4% used donor eggs. However, 38% of women aged 43–44 and 73% of women older than 44 needed to use donor eggs [2]. Similar to the USA, the numbers of ART cycles in Europe show a growing tendency. In 2007, the reported number [3] of treatment cycles was 493.134, while the latest available report describes 640.144 cycles in 2012 [4]. Among the 452.578 fresh cycles reported in 2012, the fraction of IVF and intra‐ cytoplasmic sperm injection (ICSI) was 139.978 (31%) and 312.600 (69%), respectively [4]. Despite evolving microsurgical technologies—such as ICSI and some new embryo culturing materials the rate of successful delivery is far below expectations. In the European IVF monitoring report published in 2016 [4], this rate was 27.8‐33.8% depending on the technique of the cycles.

Successful implantation is a complex and bilateral process that requires the selection of a viable embryo and the effective interaction with a receptive endometrium. It is highly unlikely, how‐ ever, that for the low delivery rate following IVF only maternal reasons would be responsible. In Europe, the total proportion of single embryo transfers (SETs) was 30%. Double embryo transfers occurred in 55% of the cycles, triple embryo transfers were reported in 13% and four or more embryos were transferred in 1% of the cycles. The highest proportions of SETs in 2012 were found in Sweden (76.3%), Finland (75.0%), Norway (60.8%), Belgium (51.1%), Iceland (49.4%), the Czech Republic (47.4%), Austria (46.5%) and Denmark (46.4%) [4]. Nowadays, SET gains grounds in clinical practice. The adoption of an elective SET policy is spreading, urging the development of a reliable method for selecting the most viable embryo, that is, the embryo with the best implanta‐ tion potential. In the traditional clinical practice of ART, embryos are selected for transfer based on non‐invasive morphological evaluation. Several new morphological parameters such as the cleavage rate, blastomere shape and symmetry, and the presence of an adequate trophectoderm layer (TL) or an inner cell mass (ICM) are considered as indicators of implantation potency.

#### **2. Embryo morphology**

The most obvious approach for the viability assessment of *in vitro* fertilized embryos is the visual inspection using microscopy. The main reason is the use of any invasive technique such as genetic screening following on‐cell embryo biopsy may raise a series of ethical questions. One must not forget that any impact, which affects the embryo during the first days of devel‐ opment, might have undesired late consequences. The choice of morphological parameter depends partly on the time spent after fertilization.

On the first day of development, the morphology of the two pronuclei (the interphase zygotic nuclei) can be graded at 1‐cell stage zygotes. Zygote has two pronuclei as the female from the oocyte and the male one from the spermium. Until the end of the interphase of the first embryonic cell cycle, the two pronuclei remain separated. Though on the first day of *in vitro* embryonic development, nucleoli screening is reported to be predictive of pregnancy rate, there are still some disagreements about the usefulness of this morphological marker [5].

all deliveries are IVF pregnancy and this number continues increasing. Availability of ART is a very relevant topic. The cultural and legal conditions, insurance/public funding systems and structure of data collection can influence not only the amount of treatment cycles per inhabitant but also success rates. The Assisted Reproductive Technology National Summary Report of the USA showed a total of 142.000 IVF cycles in 2007 [1], while the most current results in 2014 was 208.604 cycles [2]. The type of ART cycle applied (non‐donor or donor egg cycle) is highly var‐ ied based on the woman's age. The women younger than 35 years of age often used their own eggs (non‐donor) in the majority of cases and just about 4% used donor eggs. However, 38% of women aged 43–44 and 73% of women older than 44 needed to use donor eggs [2]. Similar to the USA, the numbers of ART cycles in Europe show a growing tendency. In 2007, the reported number [3] of treatment cycles was 493.134, while the latest available report describes 640.144 cycles in 2012 [4]. Among the 452.578 fresh cycles reported in 2012, the fraction of IVF and intra‐ cytoplasmic sperm injection (ICSI) was 139.978 (31%) and 312.600 (69%), respectively [4]. Despite evolving microsurgical technologies—such as ICSI and some new embryo culturing materials the rate of successful delivery is far below expectations. In the European IVF monitoring report

published in 2016 [4], this rate was 27.8‐33.8% depending on the technique of the cycles.

**2. Embryo morphology**

82 Embryo Cleavage

depends partly on the time spent after fertilization.

Successful implantation is a complex and bilateral process that requires the selection of a viable embryo and the effective interaction with a receptive endometrium. It is highly unlikely, how‐ ever, that for the low delivery rate following IVF only maternal reasons would be responsible. In Europe, the total proportion of single embryo transfers (SETs) was 30%. Double embryo transfers occurred in 55% of the cycles, triple embryo transfers were reported in 13% and four or more embryos were transferred in 1% of the cycles. The highest proportions of SETs in 2012 were found in Sweden (76.3%), Finland (75.0%), Norway (60.8%), Belgium (51.1%), Iceland (49.4%), the Czech Republic (47.4%), Austria (46.5%) and Denmark (46.4%) [4]. Nowadays, SET gains grounds in clinical practice. The adoption of an elective SET policy is spreading, urging the development of a reliable method for selecting the most viable embryo, that is, the embryo with the best implanta‐ tion potential. In the traditional clinical practice of ART, embryos are selected for transfer based on non‐invasive morphological evaluation. Several new morphological parameters such as the cleavage rate, blastomere shape and symmetry, and the presence of an adequate trophectoderm layer (TL) or an inner cell mass (ICM) are considered as indicators of implantation potency.

The most obvious approach for the viability assessment of *in vitro* fertilized embryos is the visual inspection using microscopy. The main reason is the use of any invasive technique such as genetic screening following on‐cell embryo biopsy may raise a series of ethical questions. One must not forget that any impact, which affects the embryo during the first days of devel‐ opment, might have undesired late consequences. The choice of morphological parameter

On the first day of development, the morphology of the two pronuclei (the interphase zygotic nuclei) can be graded at 1‐cell stage zygotes. Zygote has two pronuclei as the female from The time point of the breakdown of the pronuclear membranes or the time of the first cleav‐ age following fertilization is considered as an indicator of reproductive potential of embryos. Fancsovits et al. reported the relationship of the time point of the pronuclear breakdown with clinical pregnancy and implantation rates. The earliest pronuclear breakdown was at 18 hours after fertilization and the latest time was 31 hours post‐insemination. Transferring embryos with the early pronuclear breakdown resulted in a significantly higher clinical pregnancy rate (48.3 vs. 27.3%) and the implantation rates (26.5 vs. 15.1%) [6].

On the second and other later days, the blastomere size, cleavage rate, and pattern of the developing embryo may be evaluated. The best quality embryos supposed to have developed to the four to five blastomere stage on day 2 and have seven or more blastomeres on the third day [5]. Along the number of blastomers, the symmetry of the cleavage is also considered as an indicator of embryo quality. The embryos with symmetric cleavage patterns have a tendency for significantly higher implantation than asymmetric blastomeric shape. Thus, the acceptable cleavage pattern can also be a predictor of implantation outcome [7].

Another important morphological parameter is the grade of fragmentation at the early embry‐ onic development. Cytoplasmic fragments can be found in any human embryo irrespective whether they were fertilized *in vitro* or *in vivo*. The amounts of fragments vary highly, ranging from a few small fragments to a notably high extent of fragmentation involving even blasto‐ mere number loss in early cleavage stage embryos. The degree of fragmentation is widely used as an indicator of embryo quality and a predictor of implantation potential. Extensive fragmentation is commonly associated with reduced blastocyst formation and implantation potential. If the degree of fragmentation is below 15%, it seems no effect on blastocyst forma‐ tion, but more that 15% fragmentation will quickly declines blastocyst formation [8].

The morphological scoring of embryos on 5 and 6 days is also possible by the populations of inner cells (inner cell mass precursors) and outer cells (trophoblast precursors) segregating at about 16‐cell stage [9]. An appropriate quality blastocyst has a blastocoel, a trophectoderm layer (TE) and an inner cell mass (ICM). Therefore, the examination of the cell number or the area covered by these cells might be an important factor correlating with embryo viability [5].

It can be seen even on these highlighted examples that there are several options to study the morphology of *in vitro* fertilized embryos and to use these observations to predict implantation potential. It is advised not to select a single parameter, the combination of more than one serves as a better option. The full history of embryo development combining grading of zygotes, cleav‐ age stages and if possible, blastocysts is required to maximize the reliability [5]. Morphological evaluation is an inexpensive method which can be easily implemented in the clinical environ‐ ment. The biggest drawback of morphological evaluation is that it is a highly subjective method. Therefore, there was a need to form a consensus on these parameters, namely which morpho‐ logical markers need to be used, what is the weighing of these parameters in the final score, and a scale on which all individual parameters are graded. An international consensus was created in 2011 by the Alpha Scientists in Reproductive Medicine and the European Society of Human Reproduction and Embryology (ESHRE) Special Interest Group of Embryology, based on sev‐ eral morphology markers in different stages of development. The result of this agreement is known as the Istanbul Consensus scoring system. It was expected that standardization of labo‐ ratory practice related to embryo morphology assessment will result in more effective com‐ parisons of treatment outcomes worldwide. The document set by the Alpha Scientists group intended to refer as a global standardized consensus for the accurate description of embryo development [10]. The scoring system is composed of several morphological aspects and also considers time spent after fertilization. Nowadays, the guideline sets here serve as the accepted methodology of viability assessment of *in vitro* fertilized embryos.

### **3. Analysis of the embryo culture medium**

Because of ethical reasons, a huge effort is made to find ways of non‐invasive viability assess‐ ment. The most obvious approach is to study the metabolomic activity of the embryo through the analysis of secreted compounds or by studying the alterations made by the embryo within the culture medium. Due to the importance of the surrounding environment of the embryo and the goal of single embryo transfer concept, and the maintenance of acceptable pregnancy rates, selecting the most optimal culture medium is a crucial point.

First human embryos were cultured in simple salt solutions or in more complex media originally designed for tissue culturing. These early media consisted of physiological salt solutions with added glucose, pyruvate and lactate, and was also supplemented with the patient's serum. Later, it was also revealed that the addition of amino acids to the culture medium increases reproduc‐ tive potential. Research papers described in both animal and human models that the introduc‐ tion of amino acids has a positive effect on embryo development and increases viability [11].

Using the experiences published in the literature, several clinics started to develop 'in‐house' embryo culturing media, but this way the standardization of culturing circumstances is not an easy task [11]. Therefore, shortly, commercially produced media specifically designed for use in clinical IVF applications was developed satisfying the growing needs. These media are aseptically produced in a specialized factory under standardized conditions, regulations and quality control, and therefore an attractive alternative of 'in‐house' embryo culturing media. Nowadays, two types of media exist: sequential culture systems and monoculture systems. Monoculture systems use a single medium composition to support zygote development to the blastocyst stage. The limitation of monoculture systems is that they do not adapt to the alter‐ ing biochemical needs of the embryo during its development. A medium composition suit‐ able for early cleavage state embryos might not be optimal for the blastocyst stage embryos. Therefore, the majority of IVF clinics use sequential culture systems. It has been determined that conditions that support blastocyst development might inhibit the development of early cleavage stage embryos. If the practice of the clinic covers blastocyst transfer, the sequential medium is the best choice [11].

A very important additive of any type of embryo culturing medium is human serum albumin, which is the most abundant soluble protein constituent of blood described with several physi‐ ological roles. In culture medium, albumin serves as pH buffer, an osmotic regulator, mem‐ brane stabilizator, a surfactant and a scavenger of metals or toxic substances. Earlier, albumin supplementation was done using human or maternal serum but it has now shifted towards the use of purified albumin products, mainly because of the risk of transferring infectious diseases. With the use of purified albumin products this risk can be eliminated. However, the batch‐to‐batch stability of different lots of albumin products is sometimes questionable. The use of recombinant albumin might solve all the issues discussed above, but their use is not as widespread as the use of purified albumin products [12].

When dealing with purified albumin products, one must consider that these products are not a 100% pure. In Dyrlund et al.'s recent study [13], 110 proteins other than albumin were identified in commercially available unconditioned culture media supplemented with puri‐ fied human serum albumin products. Probably it is not an issue in clinical practice since these products have been proven themselves for decades. However, if we use the culture medium as a material for research purpose, it is a very important question.

The measurement of the spent culture medium (SEC) may be served as an exceptional non‐ invasive alternative in the search of markers of embryo viability. In SEC, the interesting com‐ pounds can be divided into two major groups. One consists of compounds present in the unconditioned medium and these compounds may be quantitatively altered by the devel‐ oping embryo (e.g. nutrients or peptide/protein compounds) [14]. The other group contains embryo‐related molecules (e.g. proteins and metabolic end products) secreted by the embryo into the surrounding medium. In order to analyse the secretome of the developing embryo, especially the proteome, it has to be cleared which identified protein originates from the embryo and which was present (or altered in concentration) in the unconditioned medium.

#### **4. Metabolomic studies**

a scale on which all individual parameters are graded. An international consensus was created in 2011 by the Alpha Scientists in Reproductive Medicine and the European Society of Human Reproduction and Embryology (ESHRE) Special Interest Group of Embryology, based on sev‐ eral morphology markers in different stages of development. The result of this agreement is known as the Istanbul Consensus scoring system. It was expected that standardization of labo‐ ratory practice related to embryo morphology assessment will result in more effective com‐ parisons of treatment outcomes worldwide. The document set by the Alpha Scientists group intended to refer as a global standardized consensus for the accurate description of embryo development [10]. The scoring system is composed of several morphological aspects and also considers time spent after fertilization. Nowadays, the guideline sets here serve as the accepted

Because of ethical reasons, a huge effort is made to find ways of non‐invasive viability assess‐ ment. The most obvious approach is to study the metabolomic activity of the embryo through the analysis of secreted compounds or by studying the alterations made by the embryo within the culture medium. Due to the importance of the surrounding environment of the embryo and the goal of single embryo transfer concept, and the maintenance of acceptable pregnancy

First human embryos were cultured in simple salt solutions or in more complex media originally designed for tissue culturing. These early media consisted of physiological salt solutions with added glucose, pyruvate and lactate, and was also supplemented with the patient's serum. Later, it was also revealed that the addition of amino acids to the culture medium increases reproduc‐ tive potential. Research papers described in both animal and human models that the introduc‐ tion of amino acids has a positive effect on embryo development and increases viability [11].

Using the experiences published in the literature, several clinics started to develop 'in‐house' embryo culturing media, but this way the standardization of culturing circumstances is not an easy task [11]. Therefore, shortly, commercially produced media specifically designed for use in clinical IVF applications was developed satisfying the growing needs. These media are aseptically produced in a specialized factory under standardized conditions, regulations and quality control, and therefore an attractive alternative of 'in‐house' embryo culturing media. Nowadays, two types of media exist: sequential culture systems and monoculture systems. Monoculture systems use a single medium composition to support zygote development to the blastocyst stage. The limitation of monoculture systems is that they do not adapt to the alter‐ ing biochemical needs of the embryo during its development. A medium composition suit‐ able for early cleavage state embryos might not be optimal for the blastocyst stage embryos. Therefore, the majority of IVF clinics use sequential culture systems. It has been determined that conditions that support blastocyst development might inhibit the development of early cleavage stage embryos. If the practice of the clinic covers blastocyst transfer, the sequential

methodology of viability assessment of *in vitro* fertilized embryos.

rates, selecting the most optimal culture medium is a crucial point.

**3. Analysis of the embryo culture medium**

84 Embryo Cleavage

medium is the best choice [11].

The current goal of IVF is to reduce the number of transferred embryos in a single cycle, prefer‐ ably to only one. Therefore, there is an increasing need for new markers of viability. Numerous factors have been identified as suitable markers of implantation potential, started by the mea‐ surement of glucose uptake rate or the determination of pyruvate concentration in the culture medium. Papers reporting such applications in mouse and human models [15, 16] described that blastocysts implanted and developed properly after transferring to the uterus had a sig‐ nificantly higher rate of glucose consumption *in vitro* than those that failed to implant. During the *in vitro* development of human embryos, pyruvate and glucose uptakes were found to be significantly higher by embryos forming normal blastocysts than embryos failing to develop properly. In the first group, an average 22.1 pmol per embryo per hour glucose uptake was recorded, while in the latter group this was only 10.2 pmol per embryo per hour. Comparison of glucose uptakes with morphological embryo grading revealed that the highest glucose uptake was seen in blastocysts of highest grade. Among blastocysts of the same grade from the same patient, there was a notable spread of glucose uptake, indicating that glucose consump‐ tion during *in vitro* development may report additional information on embryo viability. It is also described that the measurement of glucose in the medium is more important than that of pyruvate since pyruvate uptakes were similar irrespective of blastocyst grade.

Another option is the examination of amino acid turnover during the early embryonic devel‐ opment by analysing quantitative changes in the amino acid profile of the medium. Amino acids have numerous biological functions during the early period of embryo development. Houghton et al. [17] quantitatively analysed amino acid turnover using high‐performance liquid chromatography of individual human embryos. Quantitatively different patterns of amino acid utilization were found between embryos that went on to form a blastocyst and those that failed to develop to blastocyst stage. In the group of normally developing embryos, an increased consumption of leucine from the culture medium was determined. It was also found that the profiles of alanine, arginine, glutamine, methionine, and asparagine predicted developmental potential significantly. Brison et al. [18] revealed alterations in the amino acid concentration of the medium of human zygotes cultured to the 2‐cell stage. The turnover of three amino acids, that is, asparagine, glycine, and leucine, was found to be significantly asso‐ ciated with clinical pregnancy and live birth.

Not only selected metabolomic compounds can be examined, but also the analysis of the total metabolome is possible. This area of metabolomic experiments examines the overall metabolic content of the surrounding medium, rather than measuring known nutrients or metabolites. Using analytical techniques such as Raman or near‐infrared (NIR) spectroscopy, it is possible to obtain the whole spectral profile of the culture medium surrounding the embryo. It has to be highlighted that it is not possible to identify specific components, it is only possible to detect specific changes to the obtained spectrum. The potential advantage of this approach is an overall analysis for the culture environment [19]. The concept is that after performing spectroscopy at multiple wavelengths in the medium samples of embryos with different implantation outcome, spectral alterations are searched for. These differences are calculated into viability scores or indexes using mathematical algorithms. The observed alterations in the spectra are due to differences in the amount of chemical groups which is a consequence of the metabolic activity of the embryo. The methodology cannot identify the compounds responsible for the spectral differences but indirectly reports information on the metabolomic activity of the developing embryo. For example, if spectral signatures from the near‐infrared show differences through the 750–950‐nm spectral region, it reports a change in the relative amounts of –OH, –CH and –NH groups [20]. Both Raman and NIR spectroscopic analyses of spent culture media of embryos with known implantation potential demonstrated signifi‐ cantly higher viability indices for embryos representing transfers resulting in clinical preg‐ nancy. When embryos with similar morphology were examined using infrared spectroscopy, viability scores varied remarkably indicating that the analysis of the total metabolome also reports additional information on embryo viability [19]. When calculated viability scores were compared with live birth rates, it was found that embryos having viability scores <0.45 resulted in 19.4% live birth rate, while embryos having viability scores >0.578 resulted in 46.9% live birth rate. This is a very important observation because it clearly indicates that non‐inva‐ sive metabolomic analysis of the medium of *in vitro* fertilized embryos has its place in the process of viability assessment. Probably a new and additional method cannot replace the existing methodology. However, it can add some new information by identifying markers of low implantation potential unnoticed by the morphological evaluation.

#### **5. Proteomic studies**

same patient, there was a notable spread of glucose uptake, indicating that glucose consump‐ tion during *in vitro* development may report additional information on embryo viability. It is also described that the measurement of glucose in the medium is more important than that of

Another option is the examination of amino acid turnover during the early embryonic devel‐ opment by analysing quantitative changes in the amino acid profile of the medium. Amino acids have numerous biological functions during the early period of embryo development. Houghton et al. [17] quantitatively analysed amino acid turnover using high‐performance liquid chromatography of individual human embryos. Quantitatively different patterns of amino acid utilization were found between embryos that went on to form a blastocyst and those that failed to develop to blastocyst stage. In the group of normally developing embryos, an increased consumption of leucine from the culture medium was determined. It was also found that the profiles of alanine, arginine, glutamine, methionine, and asparagine predicted developmental potential significantly. Brison et al. [18] revealed alterations in the amino acid concentration of the medium of human zygotes cultured to the 2‐cell stage. The turnover of three amino acids, that is, asparagine, glycine, and leucine, was found to be significantly asso‐

Not only selected metabolomic compounds can be examined, but also the analysis of the total metabolome is possible. This area of metabolomic experiments examines the overall metabolic content of the surrounding medium, rather than measuring known nutrients or metabolites. Using analytical techniques such as Raman or near‐infrared (NIR) spectroscopy, it is possible to obtain the whole spectral profile of the culture medium surrounding the embryo. It has to be highlighted that it is not possible to identify specific components, it is only possible to detect specific changes to the obtained spectrum. The potential advantage of this approach is an overall analysis for the culture environment [19]. The concept is that after performing spectroscopy at multiple wavelengths in the medium samples of embryos with different implantation outcome, spectral alterations are searched for. These differences are calculated into viability scores or indexes using mathematical algorithms. The observed alterations in the spectra are due to differences in the amount of chemical groups which is a consequence of the metabolic activity of the embryo. The methodology cannot identify the compounds responsible for the spectral differences but indirectly reports information on the metabolomic activity of the developing embryo. For example, if spectral signatures from the near‐infrared show differences through the 750–950‐nm spectral region, it reports a change in the relative amounts of –OH, –CH and –NH groups [20]. Both Raman and NIR spectroscopic analyses of spent culture media of embryos with known implantation potential demonstrated signifi‐ cantly higher viability indices for embryos representing transfers resulting in clinical preg‐ nancy. When embryos with similar morphology were examined using infrared spectroscopy, viability scores varied remarkably indicating that the analysis of the total metabolome also reports additional information on embryo viability [19]. When calculated viability scores were compared with live birth rates, it was found that embryos having viability scores <0.45 resulted in 19.4% live birth rate, while embryos having viability scores >0.578 resulted in 46.9% live birth rate. This is a very important observation because it clearly indicates that non‐inva‐ sive metabolomic analysis of the medium of *in vitro* fertilized embryos has its place in the

pyruvate since pyruvate uptakes were similar irrespective of blastocyst grade.

ciated with clinical pregnancy and live birth.

86 Embryo Cleavage

It is hypothesized that secretory compounds found in the culture medium might provide a characteristic molecular fingerprint. This pattern informs us about embryo growth, devel‐ opmental competences, and implantation potential. With the emerging of sensitive and spe‐ cific new analytical techniques, it is possible to carry out a comprehensive analyses of the surrounding environment of pre‐implantation embryos [21]. These molecular profiles are supposed to utilize with high accuracy the differentiation of viable and non‐viable embryos [22]. The identification of new biomarkers of the embryonic secretome can result in signifi‐ cant improvements in the efficiency of IVF cycles, increasing pregnancy rate per transfer and decreasing the costs of the procedure. There is also a more subjective aspect of more reliable viability assessment: the reduction of the patient's emotional stress [23]. Biological functions are often regulated or carried out by proteins, therefore to understand how a cell or in this case a small population of cells function can be crucial. The analysis of the proteome reports us how the embryo responds to external and also internal conditions. The analysis of the embryonic protein production into the surrounding medium provides a new, molecular per‐ spective of the biochemical pathways activated during the early embryonic development [21].

The proteomic analysis of the embryonic secretome covers the use of the latest analytical tools, very often mass spectrometry (MS) or liquid chromatography‐coupled mass spectrome‐ try (LC‐MS). MS is probably the most promising technique to study the embryonic secretome. The standard proteomic approach involves separation of intact proteins using 2D gel electro‐ phoresis followed by immediate MS analysis or more likely by digestion and the analysis of the resulting peptide profile. The LC‐MS analysis of tryptic digests of control and conditioned embryo culture media, characterization of embryo‐related peptides and proteins is now also possible. More recent advances like involving nano‐ultra‐high pressure chromatography (nano‐UPLC) and label‐free quantification with mass spectrometry allows the use of mini‐ mal amounts of sample and the efficient identification of numerous peptides and proteins in a single analytical run [24]. Matrix‐assisted laser‐desorption ionization‐time‐of‐flight mass spectrometry (MALDI‐TOF) and surface‐enhanced laser‐desorption ionization‐time‐of‐flight mass spectrometry (SELDI‐TOF) are also used to detect different proteins in embryo culture media. SELDI‐TOF is a highly sensitive and more importantly a high‐throughput method for proteomic analysis, especially for proteins having low molecular weight [21].

Candidates of markers of viability secreted by the embryo cover a broad range of molecules. Sher et al. [25] used the soluble human leukocyte antigen G (sHLA‐G) as a predictor of implanta‐ tion and pregnancy rate. sHLA‐G was quantified using an immunoassay and two groups were made according to the quantitative results. Embryos producing sHLA‐G above the geometric mean were considered as sHLA‐G+ while the ones producing the antigen below the geometric mean were considered as sHLA‐G−. In the previous group, significantly higher pregnancy and implantation rates were observed. In the sHLA‐G+ group, the pregnancy and implantation rates were 75 and 44%, compared to 23 and 14% of the sHLA‐G− group, respectively.

The role of apolipoprotein A1 was also described in Ref. [26] after identification by gel elec‐ trophoresis followed by MALDI‐TOF MS. Quantification was also performed by ELISA and by quantitative reverse transcriptase polymerase chain reaction of mRNA of apolipoprotein A1. It was found that the level of apolipoprotein A1 correlates with blastocyst grade, but it does not correlate with implantation and pregnancy rates. Contradictory to those findings, Nyalwidhe et al. [22] used MS, Western‐blot, and ELISA to identify 14 differentially regulated peptides that were then used to generate genetic algorithms being able to identify embryo transfer cycles resulting in pregnancy and cycles with failed implantation. These genetic algo‐ rithms were able to recognize with 71–84% accuracy embryo transfer cycles, which resulted in pregnancy. Several of the 14 peptides were identified as fragments of apolipoprotein A‐1, showing reduced expression in media samples representing transfer cycles resulting in viable pregnancies. McReynolds et al. reported an interesting approach based on proteomic analysis [27]. Potential biomarker candidates were selected using an Linear Trap Quadropole‐Fourier Transform (LTQ‐FT) ultra hybrid mass spectrometer operated in tandem mass spectromet‐ ric (MS/MS) mode. Using this proteomic platform, we identified lipocalin‐1 to be associated with chromosome aneuploidy. The concentration of lipocalin‐1 was determined using a com‐ mercially available lipocalin‐1 ELISA kit. A clear discrimination of euploid and aneuploid embryos may be determined based on change of lipocalin‐1 concentration in micro‐drops of culture media. The lipocalin‐1 concentration from aneuploid blastocysts showed more signifi‐ cant increase than euploid blastocysts. Pooled micro‐drops of euploid embryos contained 3–4 ng/ml of lipocalin‐1, while aneuploid embryos contained this compound in a concentration of 6–7 ng/ml. When analysing individual micro‐drops of euploid and aneuploid embryos in the spent culture media samples, the results were 4–5 vs. 5–6 ng/ml of lipocalin‐1, respectively.

These examples clearly indicate that the non‐invasive proteomic analysis of spent culture medium samples has a great potential to determine embryo developmental potency. Thus, this method can be integrated to the existing viability assessing concepts.

### **6. Viability assessment using quantitative determination of the haptoglobin alpha‐1 chain**

By LC‐MS analysis of spent culture medium samples incubated for 3 days, four different polypeptides were detected and the mass spectra revealed that the monoisotopic masses of the four molecules were 4787.4, 4464.6, 4622.4, and 9186.5 Da, respectively. These numbers showed quantitative difference between the viable (successful pregnancy) and the non‐viable (no pregnancy) embryo groups [28]. As the result of various proteomic and statistical consid‐ erations, the number of biomarker candidates was reduced to a 9186.5 Da polypeptide. The respective mass spectrum is depicted in **Figure 1**.

Only this compound differed significantly in quantity between the viable and non‐viable embryo groups (*p* = 0.005). Proteomic identification was carried out after digestion of the respec‐ tive chromatographic fraction. By database search using MS data and manual investigation of

Non‐Invasive Assessment of the Embryo Viability via the Analysis of the Culture Media http://dx.doi.org/10.5772/intechopen.69436 89

implantation rates were observed. In the sHLA‐G+ group, the pregnancy and implantation

The role of apolipoprotein A1 was also described in Ref. [26] after identification by gel elec‐ trophoresis followed by MALDI‐TOF MS. Quantification was also performed by ELISA and by quantitative reverse transcriptase polymerase chain reaction of mRNA of apolipoprotein A1. It was found that the level of apolipoprotein A1 correlates with blastocyst grade, but it does not correlate with implantation and pregnancy rates. Contradictory to those findings, Nyalwidhe et al. [22] used MS, Western‐blot, and ELISA to identify 14 differentially regulated peptides that were then used to generate genetic algorithms being able to identify embryo transfer cycles resulting in pregnancy and cycles with failed implantation. These genetic algo‐ rithms were able to recognize with 71–84% accuracy embryo transfer cycles, which resulted in pregnancy. Several of the 14 peptides were identified as fragments of apolipoprotein A‐1, showing reduced expression in media samples representing transfer cycles resulting in viable pregnancies. McReynolds et al. reported an interesting approach based on proteomic analysis [27]. Potential biomarker candidates were selected using an Linear Trap Quadropole‐Fourier Transform (LTQ‐FT) ultra hybrid mass spectrometer operated in tandem mass spectromet‐ ric (MS/MS) mode. Using this proteomic platform, we identified lipocalin‐1 to be associated with chromosome aneuploidy. The concentration of lipocalin‐1 was determined using a com‐ mercially available lipocalin‐1 ELISA kit. A clear discrimination of euploid and aneuploid embryos may be determined based on change of lipocalin‐1 concentration in micro‐drops of culture media. The lipocalin‐1 concentration from aneuploid blastocysts showed more signifi‐ cant increase than euploid blastocysts. Pooled micro‐drops of euploid embryos contained 3–4 ng/ml of lipocalin‐1, while aneuploid embryos contained this compound in a concentration of 6–7 ng/ml. When analysing individual micro‐drops of euploid and aneuploid embryos in the spent culture media samples, the results were 4–5 vs. 5–6 ng/ml of lipocalin‐1, respectively.

These examples clearly indicate that the non‐invasive proteomic analysis of spent culture medium samples has a great potential to determine embryo developmental potency. Thus,

By LC‐MS analysis of spent culture medium samples incubated for 3 days, four different polypeptides were detected and the mass spectra revealed that the monoisotopic masses of the four molecules were 4787.4, 4464.6, 4622.4, and 9186.5 Da, respectively. These numbers showed quantitative difference between the viable (successful pregnancy) and the non‐viable (no pregnancy) embryo groups [28]. As the result of various proteomic and statistical consid‐ erations, the number of biomarker candidates was reduced to a 9186.5 Da polypeptide. The

Only this compound differed significantly in quantity between the viable and non‐viable embryo groups (*p* = 0.005). Proteomic identification was carried out after digestion of the respec‐ tive chromatographic fraction. By database search using MS data and manual investigation of

this method can be integrated to the existing viability assessing concepts.

**haptoglobin alpha‐1 chain**

88 Embryo Cleavage

respective mass spectrum is depicted in **Figure 1**.

**6. Viability assessment using quantitative determination of the** 

rates were 75 and 44%, compared to 23 and 14% of the sHLA‐G− group, respectively.

**Figure 1.** Mass spectrum of the haptoglobin alpha‐1 fragment. The horizontal axis represents the measured mass to charge ratio values, displayed as m/z. Absolute peak intensity is shown on the vertical axis. The most intensive peak at m/z 1149.6 corresponds to the [M+8H]8+ ion of the molecule. The peaks at m/z 1021.9 and m/z 1313.7 represent the [M + 9H]9+ and [M + 7H]7+ molecular ions, respectively.

sequence annotations of entries, the protein was identified as the alpha‐1 chain of human hap‐ toglobin. The alpha‐1 form of this subunit has a monoisotopic mass of 9186.4 Da. All enzymatic fragments identified by tandem mass spectrometry correspond to this region of the haptoglobin precursor protein.

In a set of blind and retrospective experiments including 161 haptoglobin alpha‐1 chain measurements, 62 samples were found to be biochemically non‐viable and 99 samples were biochemically viable. The biochemically non‐viable 62 embryos did not result in any suc‐ cessful baby delivery, while in the biochemically viable group showed 55% pregnancy rate (**Figure 2**). This result revealed a significant difference between viable and non‐viable embryo groups (*p* < 0.001) on the basis of the amount of the alpha‐1 chain. Moreover, we have found a significant correlation (*p* < 0.001) between the amount of the peptide fragment and the preg‐ nancy outcome.

The probable source of human haptoglobin in the unconditioned medium is the protein con‐ tamination of various purified albumin products. The sources of the haptoglobin alpha‐1 chain in the culture medium are due to the reduction of the disulphide bonds connecting the chains of the matured haptoglobin molecule. The explanation for the increased amount of alpha‐1 chain in the samples of non‐viable embryos might be the fact that abnormally devel‐ oping or damaged embryos often show the characteristics of apoptosis in a larger extent than normal embryos. Apoptosis later might be followed by secondary necrosis accompanied by increased membrane permeability. We hypothesize that these processes might result in the release of enzymes or other chemical factors from the cells of abnormally developing embryos altering the chemical environment in the medium.

**Figure 2.** Results of the blinded analysis of embryo culture medium after 3 days of incubation (*n* = 161). In the group assessed as biochemically non‐viable, no pregnancy was found. Embryos assessed as biochemically viable, showed an 55% pregnancy rate.

### **7. Apoptosis during early embryonic development**

Programmed cell death (PCD)—also called apoptosis—is a well‐known biological phenom‐ enon. It is characterized by cell membrane blebbing, chromatin condensation and DNA frag‐ mentation, involving several membrane receptors and the activation of signal transduction pathways. Classic signs of apoptosis are cell shrinkage, nuclear condensation, and the forma‐ tion of vesicles called 'apoptotic bodies'. The most significant biochemical event associated with apoptosis is DNA fragmentation producing a specific gel electrophoresis picture called the DNA ladder. Apoptosis *in vivo* occurs in every multicellular organism and is an essential biological process [29].

Normal apoptosis in early embryos is crucial for proper development. In blastocysts, for exam‐ ple, both the inner cell mass and the trophectoderm layer undergo apoptosis [29]. Apoptosis during the normal development of the pre‐implantation embryos has several functions. It is hypothesized that the cell number in the inner mass of the blastocyst follows an equilibrium and apoptosis helps to maintain cellular homeostasis. The other possible reason for PCD dur‐ ing early development is the elimination of cells with an abnormally altered genetic constitu‐ tion or cells having other abnormalities or inadequate developmental potential. For example, within the inner cell mass, the appearance of aneuploid cells is well known. The markers of apoptosis are also considered as additional features for oocyte and embryo quality assess‐ ment. Arrested embryos tend to have a high grade of apoptosis [30].

Apoptotic cells should be normally phagocytosed, however, if it is not possible they may undergo secondary necrosis, which differs from apoptosis by an increase in membrane permeability and excretion of cytosolic structures. These events are observed in a variety of different cell types [31]. The apoptotic program provides two alternative ways of cell elimination. Early surface signals can allow scavenger phagocytes to recognize apoptotic cells and remove them with a 'silent' elimination process. Secondary necrosis occurs in the absence of scavenger cells leading to a final autolytic disintegration. These cells exhibit spe‐ cific apoptotic signs and also necrotic features, for example, the degradation of the cyto‐ plasmic membrane. Secondary necrosis might also occur *in vivo* accompanying several pathological cases when functioning scavenger cells are not available [32]. *In vitro* apoptosis tends to proceed in a similar way involving the activation of hydrolytic enzymes and a damage of the cytoplasmic membrane, resulting in cell disruption. This process occurs if the removal of the apoptotic cells or apoptotic bodies fails. The events described in the process of primary necrosis are operating during secondary necrosis, too. The mechanism of cell death involves proteolysis due to the activity of proteinases causing an additional release of cytosolic compounds [33]. Studies on animal models indicate that *in vitro* cultur‐ ing increases PCD and that the composition of the medium can affect the incidence of the process. The reason is that the culture medium lacks some crucial maternal 'survival' factors [31]. We hypothesize that during the *in vitro* culturing an increased PCD is observed, result‐ ing in secondary necrosis because of the absence of scavenging cells in the artificial *in vitro* environment. The described phenomenon of haptoglobin cleavage might be a result of fac‐ tors released from the embryonic cells due to secondary necrosis and increased membrane permeability.

#### **8. Concluding remarks**

**7. Apoptosis during early embryonic development**

ment. Arrested embryos tend to have a high grade of apoptosis [30].

biological process [29].

55% pregnancy rate.

90 Embryo Cleavage

Programmed cell death (PCD)—also called apoptosis—is a well‐known biological phenom‐ enon. It is characterized by cell membrane blebbing, chromatin condensation and DNA frag‐ mentation, involving several membrane receptors and the activation of signal transduction pathways. Classic signs of apoptosis are cell shrinkage, nuclear condensation, and the forma‐ tion of vesicles called 'apoptotic bodies'. The most significant biochemical event associated with apoptosis is DNA fragmentation producing a specific gel electrophoresis picture called the DNA ladder. Apoptosis *in vivo* occurs in every multicellular organism and is an essential

**Figure 2.** Results of the blinded analysis of embryo culture medium after 3 days of incubation (*n* = 161). In the group assessed as biochemically non‐viable, no pregnancy was found. Embryos assessed as biochemically viable, showed an

Normal apoptosis in early embryos is crucial for proper development. In blastocysts, for exam‐ ple, both the inner cell mass and the trophectoderm layer undergo apoptosis [29]. Apoptosis during the normal development of the pre‐implantation embryos has several functions. It is hypothesized that the cell number in the inner mass of the blastocyst follows an equilibrium and apoptosis helps to maintain cellular homeostasis. The other possible reason for PCD dur‐ ing early development is the elimination of cells with an abnormally altered genetic constitu‐ tion or cells having other abnormalities or inadequate developmental potential. For example, within the inner cell mass, the appearance of aneuploid cells is well known. The markers of apoptosis are also considered as additional features for oocyte and embryo quality assess‐

Our detailed study showed that the alpha‐1 chain of the human haptoglobin molecule may be used as a biomarker to distinguish the *in vitro* culture embryo implantation ability, which yet has not been proven earlier by others to be an indicator of embryo viability. The embryos diagnosed as biochemically non‐viable did not lead to pregnancy at all. However, the embryos that were classified as biochemically viable showed a 55% pregnancy rate, while the control only showed the 30% pregnancy rate without the measurement of the haptoglobin alpha‐1 fragment. The authors think that non‐invasive metabolomic and proteomic approaches might have a place in the process of routine IVF but cannot substitute the process of morphological assessment. An ideal practice of IVF might contain a step ruling out the morphologically worst embryos followed by a laboratory measurement of the haptoglobin alpha‐1 chain of media of the remaining ones. The main disadvantage of this technique is the application of mass spec‐ trometry in the routine process of IVF, which requires an expensive laboratory background and is usually not available in the reproductive units. The developing field of lab‐on‐a‐chip concept in combination with already existing point‐of‐care medical instruments can be a possible end point [34].

### **Acknowledgements**

The presented work was supported by Hungarian Scientific Research Fund – OTKA/115394/2015/ HU "Early biochemical indicators of embryo viability", EDIOP‐2.3.2‐15‐2016‐00021/HU "The use of chip‐technology in increasing the effectiveness of human *in vitro* fertilization", EFOP‐ 3.6.1.‐16‐2016‐00004 Comprehensive Development for Implementing Smart Specialization Strategies at the University of Pécs and by the ÚNKP‐17‐4‐III "New National Excellence Program of the Ministry of Human Capacities" grants.

#### **Author details**

Gergely Montskó1,2, Zita Zrínyi1 , Ákos Várnagy2,3, József Bódis2,3 and Gábor L. Kovács1,2,4\*

\*Address all correspondence to: kovacs.l.gabor@pte.hu

1 Szentágothai Research Centre, University of Pécs, Pécs, Hungary

2 MTA‐PTE Human Reproduction Scientific Research Group, University of Pécs, Pécs, Hungary

3 Department of Obstetrics and Gynecology, University of Pécs, Pécs, Hungary

4 Department of Laboratory Medicine, University of Pécs, Pécs, Hungary

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**Acknowledgements**

92 Embryo Cleavage

**Author details**

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Gergely Montskó1,2, Zita Zrínyi1

Program of the Ministry of Human Capacities" grants.

\*Address all correspondence to: kovacs.l.gabor@pte.hu

1 Szentágothai Research Centre, University of Pécs, Pécs, Hungary

The presented work was supported by Hungarian Scientific Research Fund – OTKA/115394/2015/ HU "Early biochemical indicators of embryo viability", EDIOP‐2.3.2‐15‐2016‐00021/HU "The use of chip‐technology in increasing the effectiveness of human *in vitro* fertilization", EFOP‐ 3.6.1.‐16‐2016‐00004 Comprehensive Development for Implementing Smart Specialization Strategies at the University of Pécs and by the ÚNKP‐17‐4‐III "New National Excellence

2 MTA‐PTE Human Reproduction Scientific Research Group, University of Pécs, Pécs, Hungary

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, Ákos Várnagy2,3, József Bódis2,3 and Gábor L. Kovács1,2,4\*


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94 Embryo Cleavage


**Improving Embryo Cleavage Technology**
