**Introductory Chapter: New Technologies for the Study of Embryo Cleavage**

Bin Wu

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.69382

### **1. Introduction**

After fertilization by sperm into oocyte combination, mammal embryogenesis is the process of cell division and cellular differentiation of the embryo that occurs during the early stages of development. In embryology, cleavage is the division of cells in the early embryo. This divi‐ sion from a one‐celled zygote into 2, 4, 8, and 16 cells; morula stage; and finally into blastocyst stage until implantation in the uterus is called embryo cleavage. The zygotes of many species undergo rapid cell cycles with no significant growth, producing a cluster of cells the same size as the original zygote. The different cells derived from the cleavage are called blastomeres and form a compact mass called the morula. Cleavage ends with the formation of the blastula known as the blastocyst stage embryo that is yet to implant in the uterus and hence is also called preimplantation embryo.

In the last three decades, the development of assisted reproductive technology (ART) has created some new observations and novel discoveries in preimplantation embryos, espe‐ cially during embryo cleavage. Preimplantation embryo development experiences a series of critical events and remarkable epigenetic modifications, and reprogramming of gene expres‐ sion occurs to activate the embryonic genome. The alteration of these events often results in changes of embryo quality and morphology. At the cleavage stage, although morphological scores assigned using traditional criteria have little relationship with chromosome abnormali‐ ties [1], morphological evaluation is a major tool to assess embryo quality. Thus, many new observations and technologies have been developed. For example, in order to observe embryo morphology and to assess embryo quality, time‐lapse imaging, and light‐sheet microscopy have made it possible to visualize early mammalian development in greater detail and over longer time periods than ever before [2–4]. This book collects some new technologies and methods on the study of cleavage embryos to select high‐quality embryos for transfer and to improve embryo implantation and pregnancy.

© 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.

#### **2. Observation of fertilized embryos to cleavage embryos**

Since the first rabbit embryo culture was described in 1912 [5] and mouse zygote could be cul‐ tured in vitro to form blastocyst stage embryos [6, 7], embryo quality has become an impor‐ tant factor for pregnancy after the transfer of in vitro embryo into the uterus because embryo quality has a close correlation with transferred embryo implantation in uterus. Since the birth of the first "test‐tube" baby, Louise Brown in July 1978, for which the 2010 Nobel Prize for Physiology or Medicine was awarded to Robert Edwards for developing in vitro fertiliza‐ tion (IVF) and embryo transfer (ET) to treat infertility in women with non‐patent oviducts, in vitro embryo production (IVP) has been widely used in human infertility treatment and animal population reproduction and expansion. However, the success of assisted reproduc‐ tive technology mainly depends on the production of viable embryos with high implantation potential. More importantly, choosing the best embryo for transfer has become the major challenge in IVF. In the early embryo culture, the embryo quality assessment was mainly based on the morphological criteria of the transferred embryo. Thus, performing a serial observation of embryo morphology is a common technique for embryologists to evaluate embryos and has been considered as a key predictor of implantation and pregnancy [8–10]. For a long term, embryologists performed embryo quality and morphology assessments by taking the embryos out of the incubator and placing under a microscope. Besides morphol‐ ogy observation, the researchers are interested in a series of studies on cell nuclear change, gene activation and expression, cytoplasmic protein expression, blastomere differentiation, and so on. However, these studies often result in the death of embryos. For example, in our early study which observed microspindle change after the sperm entry into the egg or the activation of oocyte, the fertilized zygotes or activated eggs needed to be fixed on the slide and stained with immunocytochemical fluorescein and laser confocal microscopy [11]. Our research clearly showed the alteration of microtubule and chromatin after bovine oocyte activation and introcytoplasmic sperm injection (ICSI; **Figure 1**). The sperm into oocyte or calcium ionophore and ethanol may activate oocyte and cause extrusion of the second polar body. In order to observe the time of the second polar body, we stained various stages of oocytes after activation. The result showed that after 5‐hour postactivation, the second polar body may be completely extruded (**Figure 2**).

The study of gene expression often requires to isolate mRNA or protein from embryos [12–14]; hence, embryos needed to be lysed and no embryo would survive. In order to study the cell dif‐ ferentiation on moral and blastocyst stage embryos, a double staining with fluorescein micros‐ copy method has been used to distinguish inner cell mass (ICM) from trophoectoderm (TE). The numbers of two different cells may be counted based on different colors (ICM as blue and TE as pink, **Figure 3**).

These research methods finally damage all embryos, and it is impossible to apply these meth‐ ods to clinical practice. Thus, current embryo quality assessment is based primarily on the morphological criteria of transferred embryos, which includes three major parameters such as blastomere regularity, fragmentation, and cytoplasmic granularity [15]. Also, embryo cell numbers on different culture day and multinuclearity can be considered to evaluate embryo quality [16, 17]. Several reports have documented the association between the morphological Introductory Chapter: New Technologies for the Study of Embryo Cleavage http://dx.doi.org/10.5772/intechopen.69382 5

**2. Observation of fertilized embryos to cleavage embryos**

body may be completely extruded (**Figure 2**).

TE as pink, **Figure 3**).

4 Embryo Cleavage

Since the first rabbit embryo culture was described in 1912 [5] and mouse zygote could be cul‐ tured in vitro to form blastocyst stage embryos [6, 7], embryo quality has become an impor‐ tant factor for pregnancy after the transfer of in vitro embryo into the uterus because embryo quality has a close correlation with transferred embryo implantation in uterus. Since the birth of the first "test‐tube" baby, Louise Brown in July 1978, for which the 2010 Nobel Prize for Physiology or Medicine was awarded to Robert Edwards for developing in vitro fertiliza‐ tion (IVF) and embryo transfer (ET) to treat infertility in women with non‐patent oviducts, in vitro embryo production (IVP) has been widely used in human infertility treatment and animal population reproduction and expansion. However, the success of assisted reproduc‐ tive technology mainly depends on the production of viable embryos with high implantation potential. More importantly, choosing the best embryo for transfer has become the major challenge in IVF. In the early embryo culture, the embryo quality assessment was mainly based on the morphological criteria of the transferred embryo. Thus, performing a serial observation of embryo morphology is a common technique for embryologists to evaluate embryos and has been considered as a key predictor of implantation and pregnancy [8–10]. For a long term, embryologists performed embryo quality and morphology assessments by taking the embryos out of the incubator and placing under a microscope. Besides morphol‐ ogy observation, the researchers are interested in a series of studies on cell nuclear change, gene activation and expression, cytoplasmic protein expression, blastomere differentiation, and so on. However, these studies often result in the death of embryos. For example, in our early study which observed microspindle change after the sperm entry into the egg or the activation of oocyte, the fertilized zygotes or activated eggs needed to be fixed on the slide and stained with immunocytochemical fluorescein and laser confocal microscopy [11]. Our research clearly showed the alteration of microtubule and chromatin after bovine oocyte activation and introcytoplasmic sperm injection (ICSI; **Figure 1**). The sperm into oocyte or calcium ionophore and ethanol may activate oocyte and cause extrusion of the second polar body. In order to observe the time of the second polar body, we stained various stages of oocytes after activation. The result showed that after 5‐hour postactivation, the second polar

The study of gene expression often requires to isolate mRNA or protein from embryos [12–14]; hence, embryos needed to be lysed and no embryo would survive. In order to study the cell dif‐ ferentiation on moral and blastocyst stage embryos, a double staining with fluorescein micros‐ copy method has been used to distinguish inner cell mass (ICM) from trophoectoderm (TE). The numbers of two different cells may be counted based on different colors (ICM as blue and

These research methods finally damage all embryos, and it is impossible to apply these meth‐ ods to clinical practice. Thus, current embryo quality assessment is based primarily on the morphological criteria of transferred embryos, which includes three major parameters such as blastomere regularity, fragmentation, and cytoplasmic granularity [15]. Also, embryo cell numbers on different culture day and multinuclearity can be considered to evaluate embryo quality [16, 17]. Several reports have documented the association between the morphological

**Figure 1.** Laser‐scanning confocal microscopy of spindle and chromatin changes at the various time post‐activation and intracytoplasmic sperm injection (ICSI) in bovine. Capital letters (Left) indicate the change post‐activation and small letters (Right) indicate after ICSI. A/a showed at 0.5 h, B/b is 2 h, C/c is 3 h, and D/d is 7 h post‐activation or ICSI. Prenucleus in activated egg and prenuclei in ICSI egg have appeared with red color.

**Figure 2.** Laser‐scanning confocal microscopy of spindle and chromatin changes at the various times post activation in bovine. At 0.5 h after activation, the chromosomes of spindle start to divide, and the completion of spindle division needs about 3 hours and the second polar body may be extruded at about 5 hours. The red and green together indicate the spindle, and the red point indicates the first polar body.

characteristics of cleavage stage embryos with pregnancy success. Thus, this is currently the basic method for embryo quality assessment in human IVF and animal in vitro embryo pro‐ duction. However, although this is easily practiced, it frequently takes embryos out of the incubator which leads to concerns for the safety and stability of culture conditions [18]. Also, some key points of embryonic development may be missed during observation. Evaluation of cleavage embryos during culture and before embryo transfer is an important clinical practice. Currently, the major assessment of in vitro fertilized embryos is visual observation using microscopy. In recent years, various time‐lapse microscopy incubators are being used in human IVF clinic to monitor all the steps of embryo growth and development. Although preimplantation embryo diagnosis and screen (PGD/PGS) technologies have been applied in human embryo selection practice to improve pregnancy rate, these techniques are invasive for embryos. Finding another noninvasive method to select a good embryo will be very useful in human ART practice. Sallam et al. [19] reviewed noninvasive methods for embryo selec‐ tion and evaluated these methods in the light of the best currently available evidence to find out whether any of them is ripe for replacing or supplementing the time‐honored method of morphological assessment. Thus, we need more powerful tools to estimate the morphokinetic markers of embryos.

#### **2.1. Embryo cleavage morphokinetics based on time‐lapse imaging**

For decades, researchers have attempted to follow the development of multicellular organ‐ isms from fertilized eggs into adults. While scientists had explored individual steps of this process, no method existed to enable them to model the whole process of development live. Currently, advances in light‐sheet microscopy reported in two *Nature Methods* papers have Introductory Chapter: New Technologies for the Study of Embryo Cleavage http://dx.doi.org/10.5772/intechopen.69382 7

characteristics of cleavage stage embryos with pregnancy success. Thus, this is currently the basic method for embryo quality assessment in human IVF and animal in vitro embryo pro‐ duction. However, although this is easily practiced, it frequently takes embryos out of the incubator which leads to concerns for the safety and stability of culture conditions [18]. Also, some key points of embryonic development may be missed during observation. Evaluation of cleavage embryos during culture and before embryo transfer is an important clinical practice. Currently, the major assessment of in vitro fertilized embryos is visual observation using microscopy. In recent years, various time‐lapse microscopy incubators are being used in human IVF clinic to monitor all the steps of embryo growth and development. Although preimplantation embryo diagnosis and screen (PGD/PGS) technologies have been applied in human embryo selection practice to improve pregnancy rate, these techniques are invasive for embryos. Finding another noninvasive method to select a good embryo will be very useful in human ART practice. Sallam et al. [19] reviewed noninvasive methods for embryo selec‐ tion and evaluated these methods in the light of the best currently available evidence to find out whether any of them is ripe for replacing or supplementing the time‐honored method of morphological assessment. Thus, we need more powerful tools to estimate the morphokinetic

**Figure 2.** Laser‐scanning confocal microscopy of spindle and chromatin changes at the various times post activation in bovine. At 0.5 h after activation, the chromosomes of spindle start to divide, and the completion of spindle division needs about 3 hours and the second polar body may be extruded at about 5 hours. The red and green together indicate

For decades, researchers have attempted to follow the development of multicellular organ‐ isms from fertilized eggs into adults. While scientists had explored individual steps of this process, no method existed to enable them to model the whole process of development live. Currently, advances in light‐sheet microscopy reported in two *Nature Methods* papers have

**2.1. Embryo cleavage morphokinetics based on time‐lapse imaging**

the spindle, and the red point indicates the first polar body.

markers of embryos.

6 Embryo Cleavage

**Figure 3.** Distinguishing different cells in bovine blastocyst embryos with double staining. Top figure shows a blastocyst embryo with marked inner cell mass (ICM) and around trophectoderm cells (TE). Bottom figure shows double‐stained bovine blastocyst embryo with blue as ICM and pink as TE cells. The picture on top is from webpage search, and the author greatly appreciates Prof. Fuliang Du's courtesy for the unpublished bottom photo.

enabled researchers to visualize early development in great detail [3, 4]. Recent light‐sheet microscopes use a sheet of laser light to illuminate a thin section of a sample and capture the entire plane in one snapshot. This allows them to use much less light than confocal or two‐ photon microscopes. It is very fast but also very gentle to perform extremely well in multiple critical ways at the same time [20]. For imaging the development of entire embryos like those of *Drosophila*, zebrafish, and mice, this new multiview imaging technique is fantastic.

Time‐lapse imaging is another noninvasive, emerging technology that allows 24‐hour moni‐ toring of embryo development, offering the possibility of increased quantity and quality of morphological information without disturbing the culture condition [21]. The time‐lapse microscope is very useful for embryo development observation. In the last decade, many human IVF clinics or centers have started to use time‐lapse imaging to monitor embryo growth and division during in vitro culture and finally to select good quality embryo for transfer according to record data and pictures. This technique has been reported to be able to improve transferred embryo implantation and pregnancy [22, 23]. Based on time‐lapse record for embryo cleavage, normal embryo cleavage speed may be determined. Thus, in the second chapter of this book, the timing of embryo cleavage has been outlined based on morphoki‐ netic markers by the time‐lapse monitor. According to this embryo cleavage timing outline, embryologists may clearly know at which stage an embryo should be at various time points. Thus, an optimal quality embryo or a high‐potential implantation embryo may be selected for transfer to obtain a higher pregnancy rate. Using time lapse continuously and frequently recording system, some morphokinetic markers can be revealed in time‐lapse system. For instance, the rapid division of embryo cells at a given time often results in lower implanta‐ tion rate. In the normal situation, the division from zygote into 2–3 cells requires about 10–11 hours of time, but Rubio et al. [21] found that some embryos just spend about 5 hours to com‐ plete this division, and these embryos have much lower implantation rate than normal divi‐ sion embryos (1.2% vs 20%). Also, embryo unequal cleavage which is defined as an abruption of one blastomere into three daughter blastomeres or an interval of cell cycle less than 5 hours often produces significant lower implantation potential [24]. Thus, we may use these more precise morphokinetic markers to distinguish the embryo quality.

The third chapter further examines and verifies whether time‐lapse imaging technology is useful for the selection of "top‐quality" embryos for transfer to improve ART outcome rather than conventional morphological evaluation. Interestingly, the possible correlations between the sex of the embryo, embryo fragmentation, treatment protocols, different culture media, and embryo morphokinetics have been evaluated based on some new researches on time‐ lapse imaging facilities. Furthermore, various algorithms and predictive models designed in ART cycles with time‐lapse imaging are also discussed. For example, a lot of researches on animal and human embryonic development speed by ordinary morphology observation showed that male embryos grow faster than female embryos [25–27]. However, current time‐ lapse imaging observation may provide more detail and exact information on the difference in male and female embryos during early divisions. Although female embryos showed late cleavage (t8), morula (tM), and blastocyst stage morphokinetic parameters, they presented earlier expansion than males. Thus, the key time points of observation is related to embryo gender development. Interestingly, the authors designed a model according to the time of second synchrony and morula formation with four subgroups to predict the probability of an embryo being female.

enabled researchers to visualize early development in great detail [3, 4]. Recent light‐sheet microscopes use a sheet of laser light to illuminate a thin section of a sample and capture the entire plane in one snapshot. This allows them to use much less light than confocal or two‐ photon microscopes. It is very fast but also very gentle to perform extremely well in multiple critical ways at the same time [20]. For imaging the development of entire embryos like those

Time‐lapse imaging is another noninvasive, emerging technology that allows 24‐hour moni‐ toring of embryo development, offering the possibility of increased quantity and quality of morphological information without disturbing the culture condition [21]. The time‐lapse microscope is very useful for embryo development observation. In the last decade, many human IVF clinics or centers have started to use time‐lapse imaging to monitor embryo growth and division during in vitro culture and finally to select good quality embryo for transfer according to record data and pictures. This technique has been reported to be able to improve transferred embryo implantation and pregnancy [22, 23]. Based on time‐lapse record for embryo cleavage, normal embryo cleavage speed may be determined. Thus, in the second chapter of this book, the timing of embryo cleavage has been outlined based on morphoki‐ netic markers by the time‐lapse monitor. According to this embryo cleavage timing outline, embryologists may clearly know at which stage an embryo should be at various time points. Thus, an optimal quality embryo or a high‐potential implantation embryo may be selected for transfer to obtain a higher pregnancy rate. Using time lapse continuously and frequently recording system, some morphokinetic markers can be revealed in time‐lapse system. For instance, the rapid division of embryo cells at a given time often results in lower implanta‐ tion rate. In the normal situation, the division from zygote into 2–3 cells requires about 10–11 hours of time, but Rubio et al. [21] found that some embryos just spend about 5 hours to com‐ plete this division, and these embryos have much lower implantation rate than normal divi‐ sion embryos (1.2% vs 20%). Also, embryo unequal cleavage which is defined as an abruption of one blastomere into three daughter blastomeres or an interval of cell cycle less than 5 hours often produces significant lower implantation potential [24]. Thus, we may use these more

of *Drosophila*, zebrafish, and mice, this new multiview imaging technique is fantastic.

8 Embryo Cleavage

precise morphokinetic markers to distinguish the embryo quality.

The third chapter further examines and verifies whether time‐lapse imaging technology is useful for the selection of "top‐quality" embryos for transfer to improve ART outcome rather than conventional morphological evaluation. Interestingly, the possible correlations between the sex of the embryo, embryo fragmentation, treatment protocols, different culture media, and embryo morphokinetics have been evaluated based on some new researches on time‐ lapse imaging facilities. Furthermore, various algorithms and predictive models designed in ART cycles with time‐lapse imaging are also discussed. For example, a lot of researches on animal and human embryonic development speed by ordinary morphology observation showed that male embryos grow faster than female embryos [25–27]. However, current time‐ lapse imaging observation may provide more detail and exact information on the difference in male and female embryos during early divisions. Although female embryos showed late cleavage (t8), morula (tM), and blastocyst stage morphokinetic parameters, they presented earlier expansion than males. Thus, the key time points of observation is related to embryo gender development. Interestingly, the authors designed a model according to the time of In order to further study and explore morphokinetics of embryo cleavage, the fourth chapter discusses some methods for **spatiotemporal analysis of embryo cleavage in vitro.** 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 pat‐ tern, as well as into cell lineage. Simultaneous monitoring of molecular processes enables the study of connections between genetic expression and cell physiology and development. By time‐lapse imaging data and analytical software, a four‐dimensional video sequencing of embryos can be easily created so that growing embryos display new insights into temporal embryo development. In this chapter, the authors describe three methods with variations in hardware and software analysis by giving some examples of the outcomes to open a window to new information in developmental embryology, as embryo division pattern and lineage are studied in vivo.

#### **2.2. Gene expression of cleavage embryo and noninvasive assessment of embryo viability via culture media analysis**

Preimplantation embryo development experiences a series of critical events and remarkable epigenetic modifications, and reprogramming of gene expression occurs to activate the embry‐ onic genome. In the early stages of preimplantation embryo development, maternal mRNAs direct embryonic development. Throughout early embryonic development, a differential meth‐ ylation 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 [28]. Also, noncoding RNAs, long noncoding RNAs (lncRNA), and short noncoding RNAs, microRNAs (miRNAs) have been shown to play an important role in the regulation of mRNAs, and there‐ fore their role in preimplantation development has gained significance. Chapter Five reviews the different factors affecting gene expression during preimplantation embryo development, which includes epigenetic factors, focusing on methylation profiles, of gametes and preimplan‐ tation embryos. The effects of noncoding RNAs on gene expression were thoroughly evaluated.

Because gene expression appearance during embryo development in in vitro culture, pre‐ implantation embryos often require rich nutrition culture media. The embryo during its growth and development needs to absorb some important nutritive components from culture medium and metabolically produce some by‐products as gene expression results. From this point of view, in vitro culturing of embryos also provides a very important material for fur‐ ther noninvasive embryo evaluation by means of examining biomarkers in the spent embryo culture medium. Current developed methods concentrate on the measurement of metabolic compounds secreted from developing embryos. These studies mainly utilize the tools of mod‐ ern analytics and proteomics. Some studies suggest that metabolic profiling of embryo cul‐ ture media using optical and nonoptical spectroscopies may provide a useful adjunct to the current embryo assessment strategies and provide insight into the phenotype of embryos with increasing reproductive potential [29].

In the sixth chapter, the authors describe their new discovery, the alpha‐1 chain of the human haptoglobin molecule as a quantitative biomarker of embryo viability. In a series of retro‐ spective, blind experiments achieved more than 50% success rate. This chapter summarizes the currently available metabolic and proteomic approaches as the noninvasive molecular assessment of embryo viability. Recent studies showed that the assessment of the molecular components of nutrient media is a promising area in searching for the markers of successful embryo implantation with the subsequent development of a clinical pregnancy and the birth of a healthy baby to enhance the efficiency of treatment using ART techniques [30]. If the molecular composition of cultivation media can be used as an additional noninvasive proce‐ dure to choose an embryo for selective transfer, it will be very useful to improve human IVF pregnancy outcome.

#### **3. Improving in vitro culture environment for embryo cleavages**

Embryonic quality, cleavage speed, and gene expression have a close relationship with in vitro culture environment, including culture media, incubator type, and gas concentration [31, 32]. Thus, since starting embryo in vitro culture, many studies have concentrated on improving embryo culture condition. For many decades, optimization of culture media for the support of human and animal embryos has been a focus of considerable interest [33]. So far, many commercial embryo culture media are available for human embryo culture, and their effects on embryo culture are varied. The studies comparing these effects of culture media on embryonic development have reported contradictory conclusion. Many studies did not find a significant difference or found just a tiny difference between various culture media [34]. Recently, Mantikou et al. [35] used meta‐analysis to evaluate 31 different comparisons for 20 different culture media and could not find which culture medium leads to the best suc‐ cess rates in IVF/ICSI.

Also, incubators in the IVF laboratory play a pivotal role in providing a stable and appro‐ priate culture environment required for optimizing embryo development and clinical outcomes. With technological advances, several types of incubators have been applied to human IVF laboratory. Recently, Swain [32] did a comparative analysis of embryo cul‐ tural incubators in human IVF laboratories and reviewed some incubator functions and key environmental variables controlled and the technology utilized in various units. This comparison indicates that smaller benchtop/top‐load incubators provide faster recovery of environmental variables, but there is no clear advantage of any particular incubator based on clinical outcomes.

However, based on last decade's IVF practical observation, Dr. Bin Wu's laboratory has found an interesting phenomenon which showed a favorable response of individual patient's embryos to media and incubators. Some patents' embryos grow very well in one kind of medium, but it does not grow well in the other medium. The seventh chapter gives a detailed report on this research result. Thus, in human IVF clinical practice, using two media and two incubators for embryo culture could significantly improve IVF/ICSI embryo quality and increase pregnancy rates.

#### **Author details**

Bin Wu

In the sixth chapter, the authors describe their new discovery, the alpha‐1 chain of the human haptoglobin molecule as a quantitative biomarker of embryo viability. In a series of retro‐ spective, blind experiments achieved more than 50% success rate. This chapter summarizes the currently available metabolic and proteomic approaches as the noninvasive molecular assessment of embryo viability. Recent studies showed that the assessment of the molecular components of nutrient media is a promising area in searching for the markers of successful embryo implantation with the subsequent development of a clinical pregnancy and the birth of a healthy baby to enhance the efficiency of treatment using ART techniques [30]. If the molecular composition of cultivation media can be used as an additional noninvasive proce‐ dure to choose an embryo for selective transfer, it will be very useful to improve human IVF

**3. Improving in vitro culture environment for embryo cleavages**

Embryonic quality, cleavage speed, and gene expression have a close relationship with in vitro culture environment, including culture media, incubator type, and gas concentration [31, 32]. Thus, since starting embryo in vitro culture, many studies have concentrated on improving embryo culture condition. For many decades, optimization of culture media for the support of human and animal embryos has been a focus of considerable interest [33]. So far, many commercial embryo culture media are available for human embryo culture, and their effects on embryo culture are varied. The studies comparing these effects of culture media on embryonic development have reported contradictory conclusion. Many studies did not find a significant difference or found just a tiny difference between various culture media [34]. Recently, Mantikou et al. [35] used meta‐analysis to evaluate 31 different comparisons for 20 different culture media and could not find which culture medium leads to the best suc‐

Also, incubators in the IVF laboratory play a pivotal role in providing a stable and appro‐ priate culture environment required for optimizing embryo development and clinical outcomes. With technological advances, several types of incubators have been applied to human IVF laboratory. Recently, Swain [32] did a comparative analysis of embryo cul‐ tural incubators in human IVF laboratories and reviewed some incubator functions and key environmental variables controlled and the technology utilized in various units. This comparison indicates that smaller benchtop/top‐load incubators provide faster recovery of environmental variables, but there is no clear advantage of any particular incubator based

However, based on last decade's IVF practical observation, Dr. Bin Wu's laboratory has found an interesting phenomenon which showed a favorable response of individual patient's embryos to media and incubators. Some patents' embryos grow very well in one kind of medium, but it does not grow well in the other medium. The seventh chapter gives a detailed report on this research result. Thus, in human IVF clinical practice, using two media and two incubators for embryo culture could significantly improve IVF/ICSI embryo quality and

pregnancy outcome.

10 Embryo Cleavage

cess rates in IVF/ICSI.

on clinical outcomes.

increase pregnancy rates.

Address all correspondence to: bwu13@yahoo.com

Arizona Center for Reproductive Endocrinology and Infertility, Tucson, Arizona, USA

#### **References**


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[20] Udan RS, Piazza VG, Hsu CW, Hadjantonakis AK, Dickinson ME. Quantitative imag‐ ing of cell dynamics in mouse embryos using light‐sheet microscopy. Development.

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[22] Meseguer M, Rubio I, Cruz M, Basile N, Marcos J, Requena A. Embryo incubation and selection in a time‐lapse monitoring system improves pregnancy outcome com‐ pared with a standard incubator: A retrospective cohort study. Fertility and Sterility.

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**Embryo Cleavage Morphokinetics**

#### **Chapter 2**

## **Timing of Embryo Cleavage**

#### Meng Ju Lee

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.69437

#### **Abstract**

Time‐lapse system can provide a culture environment to observe the development of embryos continuously. There are many morphokinetic markers to help us to find out the best quality of embryos. We review the studies to clarify the relationship of markers between implantation potential and embryo chromosome status. Surprisingly, most of markers are controversial or no significant effect on implantation potential and preg‐ nancy rate. We suppose that some uncertain factors may influence embryonic implan‐ tation and pregnancy. Here we provide a new method for selecting optimal quality of embryos by many morphokinetic markers in the time‐lapse system. Therefore, we can expect that the time‐lapse system helps us to choose the good quality embryos for sub‐ sequent embryos transfer to improve implantation potential, euploid chromosome and pregnancy rate. Furthermore, studies need to understand the other maternal physical conditions correlation with embryos implantation.

**Keywords:** time‐lapse, cleavage embryo, morphokinetic markers

#### **1. Introduction**

The morphology of embryo is the most widespread method to select the embryo with high implantation potential in assisted reproductive technology (ART). Conventionally, embryo development was daily observed after insemination, which could assist the embryologists to select the optimal embryo to transfer for elevating live birth rate eventually. However, the daily observation is considered as a disadvantage for embryo development because of the frequent transfer between incubator and atmospheric environment. Thus, a new and power‐ ful tool, time‐lapse monitor (TLM), was developed to estimate the morphokinetic markers of embryos. Currently, TLM can be used to evaluate the embryo growing status from the time of insemination to blastocyst formation. The sequential assessment of pronuclear, cleav‐ age stage, and blastocyst morphology can continuously evaluate the morphology of embryos

© 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.

through automatically obtaining images in every 5–20 min. Besides, TLM offers a steady cul‐ ture condition due to bypassing the daily observation. Here, we discuss the timing of embryo cleavage and the following effects of implantation potential in this chapter.

#### **2. Morphokinetic markers**

Generally, there are many milestones (**Figure 1**), including pronucleus appearance, pronu‐ cleus breakdown, first division, second division and blastulation, during the period of fertil‐ ization to blastocyst formation. The TLM fails to obtain the pictures at every minute since the capturing period was limited. Although the limitation of the time lapse is obvious, currently, it is still the most practical manner to evaluate the timing of embryo development rather than daily observation. Here, we listed the morphokinetic markers and discussed the timing of different time point during the development of embryos and the effect of clinical outcomes.

(1) The timing of second polar body extrusion (tPB2): the time of the second polar body extru‐ sion is 2.9 ± 0.1 h after Intra‐cytoplasmic sperm injection (ICSI). The range of extrusion time is around 0.7–10.15 h. If the oocytes from female age >38 years old, the timing of second polar body extrusion was significantly delayed but no other effects were observed in further embryo development [1]. The mean time of tPB2 is 3.9 h in euploid and 4.0 h in aneuploid embryos, respectively. The chromosome integrity of embryos is irrelevant to the timing of second body extrusion [2].

(2) The timing of pronuclear appearance (tPNa): the time of pronuclear appearance is 8.4 ± 2.4 h in the implantation group and 8.2 ± 1.9 h in the non‐implantation group [3]. In euploid embryos, the mean time of tPNa is 10.2 h and 10.1 h in aneuploid embryos [2]. Therefore, the timing of pro‐ nuclear appearance has no significant effect on implantation potential and chromosome status.

(3) The timing of pronuclear fading (tPNf): longer time taken in pronucleus (PN) breakdown might be beneficial for live birth. Azzarello et al. [4] claimed that the timing of tPNf was longer in live birth group (24.9 ± 0.6 vs. 23.3 ± 0.4 h), and there was no live birth if the timing of PN breakdown was less than 20 h. The timing of PN breakdown was equal between implanted and non‐implanted embryos [3, 5]. The mean time of tPNf is 24.4 h in euploid embryos and 24.8 h in aneuploid embryos [2]. The timing of pronuclear fading has no significant difference in embryo implantation and chromosome status but no live birth when tPNf is less than 20 h.

**Figure 1.** The milestones of embryo development. tPB2: the timing of second polar body extrusion, tPNa: the timing of pronuclear appearance, tPNf: the timing of pronuclear fading, t2, 3, 4, 5, 6, 7, 8, 9: time from insemination to the 2, 3, 4, 5, 6, 7, 8, 9 cell stages, tM: time from insemination to morula, tSB: time from insemination to starting blastulation, tEB: time from insemination to expanded blastulation, cc2:t3‐t2, cc3:t5‐t3, s2:t4‐t3, s3:t8‐t5.

(4) Time from insemination to the 2‐cell stage (t2): it is still controversial in the period. Meseguer et al. [6] presented that the t2 of implanted embryos group was shorter than non‐ implanted embryos (25.6 ± 2.2 vs. 26.7 ± 3.8 h). Chamayou et al. [3] showed no significant difference in implanted and non‐implanted embryos (26.9 ± 3.2 vs. 27.0 ± 4.0 h). Kirkegaard et al. [5] claimed that t2 was similar in the pregnancy and non‐pregnant groups. Curiously, t2 is shorter when embryo was incubated in single culture medium than sequential culture medium (27.36 ± 4.12 vs. 29.09 ± 4.86 h) [7]. The mean time of t2 is no significant between euploid (28 h) and aneuploid embryos (28.4 h) [2]. The development of the 2‐cell stage may be faster in implanted embryos but no significant in chromosome status.

through automatically obtaining images in every 5–20 min. Besides, TLM offers a steady cul‐ ture condition due to bypassing the daily observation. Here, we discuss the timing of embryo

Generally, there are many milestones (**Figure 1**), including pronucleus appearance, pronu‐ cleus breakdown, first division, second division and blastulation, during the period of fertil‐ ization to blastocyst formation. The TLM fails to obtain the pictures at every minute since the capturing period was limited. Although the limitation of the time lapse is obvious, currently, it is still the most practical manner to evaluate the timing of embryo development rather than daily observation. Here, we listed the morphokinetic markers and discussed the timing of different time point during the development of embryos and the effect of clinical outcomes. (1) The timing of second polar body extrusion (tPB2): the time of the second polar body extru‐ sion is 2.9 ± 0.1 h after Intra‐cytoplasmic sperm injection (ICSI). The range of extrusion time is around 0.7–10.15 h. If the oocytes from female age >38 years old, the timing of second polar body extrusion was significantly delayed but no other effects were observed in further embryo development [1]. The mean time of tPB2 is 3.9 h in euploid and 4.0 h in aneuploid embryos, respectively. The chromosome integrity of embryos is irrelevant to the timing of

(2) The timing of pronuclear appearance (tPNa): the time of pronuclear appearance is 8.4 ± 2.4 h in the implantation group and 8.2 ± 1.9 h in the non‐implantation group [3]. In euploid embryos, the mean time of tPNa is 10.2 h and 10.1 h in aneuploid embryos [2]. Therefore, the timing of pro‐ nuclear appearance has no significant effect on implantation potential and chromosome status. (3) The timing of pronuclear fading (tPNf): longer time taken in pronucleus (PN) breakdown might be beneficial for live birth. Azzarello et al. [4] claimed that the timing of tPNf was longer in live birth group (24.9 ± 0.6 vs. 23.3 ± 0.4 h), and there was no live birth if the timing of PN breakdown was less than 20 h. The timing of PN breakdown was equal between implanted and non‐implanted embryos [3, 5]. The mean time of tPNf is 24.4 h in euploid embryos and 24.8 h in aneuploid embryos [2]. The timing of pronuclear fading has no significant difference in embryo implantation and chromosome status but no live birth when tPNf is less than 20 h.

**Figure 1.** The milestones of embryo development. tPB2: the timing of second polar body extrusion, tPNa: the timing of pronuclear appearance, tPNf: the timing of pronuclear fading, t2, 3, 4, 5, 6, 7, 8, 9: time from insemination to the 2, 3, 4, 5, 6, 7, 8, 9 cell stages, tM: time from insemination to morula, tSB: time from insemination to starting blastulation, tEB: time

from insemination to expanded blastulation, cc2:t3‐t2, cc3:t5‐t3, s2:t4‐t3, s3:t8‐t5.

cleavage and the following effects of implantation potential in this chapter.

**2. Morphokinetic markers**

18 Embryo Cleavage

second body extrusion [2].

(5) Time from insemination to the 3, 4, 5 cells (t3, t4, t5): some studies have shown that the enhanced implantation potential has been observed in shorter t3, t4 and t5. The time periods of t3, t4 and t5 were significantly shorter in implanted embryos than non‐implanted embryos. The times of t3 (37.4 ± 2.8 h), t4 (38.2 ± 3.0 h) and t5 (52.3 ± 4.2 h) are significant difference in implanted embryos compared with the times of t3 (38.4 ± 5.2 h), t4 (40.0 ± 5.4 h) and t5 (52.6 ± 6.8 h) in non‐implanted embryos [6]. However, Chamayou et al. [3] and Kirkegaard et al. [5] demonstrated that there was no difference in embryo implantation and pregnancy rate. The embryo development is faster in single culture medium than in sequential culture medium (t3, 37.75 ± 6.64 vs. 39.53 ± 6.15 h; t4, 40.07 ± 5.98 vs. 41.45 ± 6.07 h; t5, 48.77 ± 9.49 vs. 52.22 ± 9.34 h) [7]. The mean time of t3 (37.4 vs. 37.2 h) and t5 (50.4 vs. 50.6 h) is no significant difference between euploid and aneuploid embryos, but the mean time of t4 (40 h) is significant difference between the euploid (40 h) and aneuploidy (41.1 h) blastocysts [2]. Consequently, faster embryo development of t3, 4, 5 is beneficial for implantation, but only t4 might influence the euploid rate of blastocysts.

(6) Time from insemination to the 6, 7, 8, 9 cells (t6, t7, t8, t9): according to the previous report, although the time from insemination to the 8 cells exhibited faster in implanted embryos (54.9 ± 5.2 vs. 58.0 ± 7.2 h) [8], the other report showed that there are no statistical difference between the implanted and nonimplanted embryos at t6 (54.3 ± 5.8 vs. 54.5 ± 8.2 h), t7 (57.4 ± 8.6 vs. 57.6 ± 9.8 h), t8 (61.0 ± 10.8 vs. 60.8 ± 11.5 h) and t9 (77 ± 8.5 vs. 76 ± 11.3 h) [3]. In addi‐ tion, Kirkegaard et al. [5] also proved that the pregnant rate was irrelevant to the period. In euploid embryos, the t6 (53.9 h), t7 (57.8 h), t8 (61.9 h) and t9 (76.1 h) are similar to the time in aneuploid embryos [2]. Statistically, the t6, t7, t8 and t9 have no significant difference between the implanted and non‐implanted embryos and between the euploid and aneuploid embryos.

(7) Time from insemination to morula (tM): morula is defined as all cells fused together. There is no difference that the tM is 86 ± 9.1 and 84.4 ± 11.4 h in implanted and non‐implanted embryos, respectively [3]. The tM of euploid (94.4 h) and aneuploid (95.3 h) are insignificant [2]. Therefore, statistically, the tM does not involve in the implantation potential and chromo‐ some status.

(8) Time from insemination to starting blastulation (tSB): the initiation of blastulation means the time point of the blastocoel cavity observation. There is no significant difference in the mean time of tSB in implantation and pregnancy [3, 5]. Therefore, the time from insemina‐ tion to starting blastulation does not affect embryo implantation potential and pregnancy rate. However, the mean time of tSB (103.4 h) in euploid embryos is significant shorter than aneuploid embryos (103.4 h, *p* = 0.007) [2]. Furthermore, the shorter tSB refers to more chance of euploid embryos for embryo transfer.

(9) Time period from insemination to expanded blastulation (tEB): expanded blastulation means the diameter of blastocyst had increased by more than 30%, the expanding results in a thin zona pellucida [9]. There is no *statistical significance* between implanted embryos and non‐ implanted embryos (111.7 vs. 110.5 h) [3]. Kirkegaard et al. [5] also indicated that there is no sig‐ nificant difference in pregnancy and non‐pregnancy groups (104 h). However, the mean time of tEB is significant shorter in euploid embryos than that in aneuploid embryos (118.7 vs. 122.1 h) [2]. In addition, the shorter time of embryos achieved expanded blastulation is more likely to be euploid embryo. The faster embryos of expanded blastulation have more euploid embryos but the meant time of tEB has no difference between implantation potential and pregnancy.

(10) Time period between 2‐cell and 3‐cell stage (t3‐t2, cc2): cleavage cycle 2, time of the sec‐ ond cycle is also known as the time between 2‐cell and 3‐cell stage. The mean of cc2 is 11.4 h in implanted embryos and 11.8 h in non‐implanted embryos [3]. Meseguer et al. [6] found the same cc2 (11.8 h) in implanted and non‐implanted embryos. The mean of cc2 (11 h) is also no *statistical significance* between pregnancy and non‐pregnancy group [5]. There is no difference in the mean of cc2 in euploid and aneuploid embryos (10.5 vs. 10.4 h) [2]. Therefore, cc2 can‐ not predict the implantation potential, pregnancy rate and chromosome status.

(11) Time period between 5‐cell and 3‐cell stages (t5‐t3, cc3): it is also defined as cleavage cycle 3 by Chamayou et al. [3]. They presented that the median of cc3 was significant longer in implanted embryos than nonimplanted embryos (14.4 and 13.0 h, respectively). As a result, longer cc3 may be beneficial for embryo development.

(12) Time of synchrony of the second cell cycle (s2, t4‐t3): time between 4‐cell and 3‐cell stages or 3‐cell stage also means s2. The mean of s2 is 2 h in implanted embryos and 1.7 h in non‐ implanted embryos [3]. It also has no significant difference between pregnancy and non‐ pregnancy groups [5]. However, the mean of s2 is significant smaller in euploid embryos than aneuploid embryos (2.6 vs. 4.2 h) [2]. Therefore, the mean of s2 might be used for predicting the chromosome status of embryos.

(13) Time of synchrony of the third cell cycle (s3, t8‐t5): S3 also signifies the time between 8‐cell and 5‐cell stages. It includes the sum of 5‐cell, 6‐cell and 7‐cell stages. There is no difference in the mean of s3 between implanted embryos and non‐implanted embryos (8.0 vs. 8.1 h) [3]. Kirkegaard et al. [5] also found no difference between pregnancy and non‐pregnancy groups. There are no data compared with the mean of s3 in aneuploid and non‐aneuploid embryos. Hence, the effect of s3 on implanted potential and pregnancy rate remains no significantly.

#### **3. Special markers in time‐lapse system**

Some morphokinetic markers are only revealed in the time‐lapse system because the continu‐ ously and frequently recording system. Traditional observation has difficulty in observing these transitory phenomena. Following this, we listed these morphokinetic markers and conclude the effect of embryos.

**Direct cleavage** (≦5 h from 2 to 3 cells): generally, the time from 2 to 3 cells is around 10–11 h [2, 3, 5, 6]. Rubio et al. [10] found that embryos with direct cleavage (≦5 h) have lower implan‐ tation rate than embryos with normal cleavage pattern (1.2 vs. 20%). The incidence rate of direct cleavage is 14%. What is the reason causing direct cleavage is still obscure. Based on the announcement of Rubio et al. [10], the centrioles introduced by the sperm control the first mitotic divisions of the oocytes. Therefore, the impairment of sperm neck, the location of centrioles, during ICSI procedure may alter the timing of first embryos cleavage. Rejection of direct cleavage embryos for transfer could enhance the implantation rate.

**Direct unequal cleavage (DUC)**: actually, direct cleavage could occur at any cleavage cycle. Zhan et al. [11] defined as the abrupt cleavage of one blastomere into three daughter blas‐ tomeres or an interval of cell cycles less than 5 h. Therefore, they describe direct unequal cleavage at first cleavage as DUC‐1, at second cleavage as DUC‐2, at third cleavage as DUC‐3 and embryos exhibiting multiple DUCs as DUC‐Plus. They found that the embryos fertilized with the sperm from epididymis, and testicles have significant higher DUP‐1 percentage (13.6 vs. 11.4%). However, the incidence of DUS‐1 is 9.1% in embryos fertilized with sperm from ejaculation. Besides, the embryos with multinucleation blastomere (MNB) have 2–3 times of incidence compared to non‐MNB embryos. They conclude that blastocyst rate, implanta‐ tion potential and euploid rate are significantly lower in DUC embryos. Non‐DUC embryos should be the first choice for embryos transfer.

**Reverse cleavage**: reverse cleavage can be divided into two types. Reverse cleavage type 1 (complete): blastomeres rejoin after completely separating. Reverse cleavage type 2 (incomplete): zygote or blastomere fails to separate (type I, Supplemental Video 1; type 2, Supplemental Videos 2 are available online at www.fertstert.org). It could occur up to three times in 27.4% of embryos during the first three cleavage cycles [12]. They found GnRH antagonist protocol and ICSI procedure had higher incidence of reverse cleavage compared with GnRH agonist protocol and IVF procedure. Embryos fertilizing with poor sperm motil‐ ity (<21%) also have higher rate of reverse cleavage. Besides, embryos with reverse cleavage are associated with poor grade embryos and lower implantation potential. Therefore, reverse cleavage is a negative factor for embryos selection.

#### **4. Conclusion**

aneuploid embryos (103.4 h, *p* = 0.007) [2]. Furthermore, the shorter tSB refers to more chance

(9) Time period from insemination to expanded blastulation (tEB): expanded blastulation means the diameter of blastocyst had increased by more than 30%, the expanding results in a thin zona pellucida [9]. There is no *statistical significance* between implanted embryos and non‐ implanted embryos (111.7 vs. 110.5 h) [3]. Kirkegaard et al. [5] also indicated that there is no sig‐ nificant difference in pregnancy and non‐pregnancy groups (104 h). However, the mean time of tEB is significant shorter in euploid embryos than that in aneuploid embryos (118.7 vs. 122.1 h) [2]. In addition, the shorter time of embryos achieved expanded blastulation is more likely to be euploid embryo. The faster embryos of expanded blastulation have more euploid embryos but the meant time of tEB has no difference between implantation potential and pregnancy.

(10) Time period between 2‐cell and 3‐cell stage (t3‐t2, cc2): cleavage cycle 2, time of the sec‐ ond cycle is also known as the time between 2‐cell and 3‐cell stage. The mean of cc2 is 11.4 h in implanted embryos and 11.8 h in non‐implanted embryos [3]. Meseguer et al. [6] found the same cc2 (11.8 h) in implanted and non‐implanted embryos. The mean of cc2 (11 h) is also no *statistical significance* between pregnancy and non‐pregnancy group [5]. There is no difference in the mean of cc2 in euploid and aneuploid embryos (10.5 vs. 10.4 h) [2]. Therefore, cc2 can‐

(11) Time period between 5‐cell and 3‐cell stages (t5‐t3, cc3): it is also defined as cleavage cycle 3 by Chamayou et al. [3]. They presented that the median of cc3 was significant longer in implanted embryos than nonimplanted embryos (14.4 and 13.0 h, respectively). As a result,

(12) Time of synchrony of the second cell cycle (s2, t4‐t3): time between 4‐cell and 3‐cell stages or 3‐cell stage also means s2. The mean of s2 is 2 h in implanted embryos and 1.7 h in non‐ implanted embryos [3]. It also has no significant difference between pregnancy and non‐ pregnancy groups [5]. However, the mean of s2 is significant smaller in euploid embryos than aneuploid embryos (2.6 vs. 4.2 h) [2]. Therefore, the mean of s2 might be used for predicting

(13) Time of synchrony of the third cell cycle (s3, t8‐t5): S3 also signifies the time between 8‐cell and 5‐cell stages. It includes the sum of 5‐cell, 6‐cell and 7‐cell stages. There is no difference in the mean of s3 between implanted embryos and non‐implanted embryos (8.0 vs. 8.1 h) [3]. Kirkegaard et al. [5] also found no difference between pregnancy and non‐pregnancy groups. There are no data compared with the mean of s3 in aneuploid and non‐aneuploid embryos. Hence, the effect

Some morphokinetic markers are only revealed in the time‐lapse system because the continu‐ ously and frequently recording system. Traditional observation has difficulty in observing these transitory phenomena. Following this, we listed these morphokinetic markers and conclude the

of s3 on implanted potential and pregnancy rate remains no significantly.

not predict the implantation potential, pregnancy rate and chromosome status.

longer cc3 may be beneficial for embryo development.

**3. Special markers in time‐lapse system**

the chromosome status of embryos.

effect of embryos.

of euploid embryos for embryo transfer.

20 Embryo Cleavage

The continuously morphokinetic change of embryo development is the main characteristic of time‐lapse system. We can observe many milestones of embryos development and calcu‐ late the time intervals to understand the relationship of implantation potential, chromosome status and pregnancy rate. Unfortunately, all the morphokinetic markers could not predict implantation potential, chromosome status and pregnancy rate exactly. Most of markers are controversial or no significant effect. Conventionally, embryos with quicker development would be recommended for transfer to raise the pregnancy rate. However, after reviewing all the data, not all markers can support this principle.

The reason of controversial descriptions of the markers is very incomprehensive. We suppose that some factors might influence embryos implantation and pregnancy. Obviously, maternal and physical conditions, such as endometrial receptivity, endometrial polyps, endometrial or endocervical infection, hydrosalpinx, immune disorder, subclinical hypothyroidism etc., can also impede the embryos implantation and the following pregnancy. We also know that aneuploid embryos show poor implantation rate or result in spontaneous abortion. Although some markers correlate with higher rate of euploid embryos, it still cannot be used for predict‐ ing euploid embryos precisely. If people want to know the chromosome status of embryos, pre‐implantation genetic screening (PGS) is still the first choice.

Therefore, the time‐lapse system can help us to evaluate the quality of embryos. We can use more precise morphokinetic markers to distinguish the embryos quality. The embryos with good quality have higher rate of implantation potential and normal chromosome. Currently, PGS is the optimal manner to find out the euploid embryos. However, the good quality of euploid embryo is not a guarantee of embryo implantation and pregnancy. It is the basic condi‐ tion for better embryo implantation. We have to consider many other maternal and physical sit‐ uations which greatly affect embryo implantation to promote the implantation and pregnancy rate. It also needs further studies to clarify the mystery of implantation process.

#### **Author details**

Meng Ju Lee

Address all correspondence to: swrh1214@gmail.com

Stork Fertility Center, Stork Ladies Clinic, Hsinchu, Taiwan

#### **References**


[6] Meseguer M, Herrero J, Tejera A, Hilligsoe KM, Ramsing NB, Remohi J. The use of morphokinetic as a predictor of embryo implantation. Human Reproduction. 2011;**26**:2658‐2671

and physical conditions, such as endometrial receptivity, endometrial polyps, endometrial or endocervical infection, hydrosalpinx, immune disorder, subclinical hypothyroidism etc., can also impede the embryos implantation and the following pregnancy. We also know that aneuploid embryos show poor implantation rate or result in spontaneous abortion. Although some markers correlate with higher rate of euploid embryos, it still cannot be used for predict‐ ing euploid embryos precisely. If people want to know the chromosome status of embryos,

Therefore, the time‐lapse system can help us to evaluate the quality of embryos. We can use more precise morphokinetic markers to distinguish the embryos quality. The embryos with good quality have higher rate of implantation potential and normal chromosome. Currently, PGS is the optimal manner to find out the euploid embryos. However, the good quality of euploid embryo is not a guarantee of embryo implantation and pregnancy. It is the basic condi‐ tion for better embryo implantation. We have to consider many other maternal and physical sit‐ uations which greatly affect embryo implantation to promote the implantation and pregnancy

[1] Liu Y, Chapple V, Roberts P, Ali J, Matson P. Time‐lapse videography of human oocytes following intracytoplasmic sperm injection: Events up to the first cleavage division.

[2] Minasi MG, Colasante A, Riccio T, Ruberti A, Casciani V, Scarselli F, Spinella F, Fiorentino F, Varricchio MT, Greco E. Correlation between aneuploidy, standard morphology eval‐ uation and morphokinetic development in 1730 biopsied blastocysts: A consecutive case

[3] Chamayou S, Patrizio P, Storaci G, Tomaselli V, Alecci C, Ragolia C, Crescenzo C, Guglielmino A. The use of morphokinetic parameters to select all embryos with full capacity to implant. Journal of Assisted Reproduction and Genetics. 2013;30:703‐710 [4] Azzarello A, Hoest T, Mikkelsen AL. The impact of pronuclei morphology and dynamicity on live birth outcome after time‐lapse culture. Human Reproduction. 2012;27:2649‐2657

[5] Kirkegaard K, Kesmodel US, Hindkjaer JJ, Ingerslev HJ. Time‐lapse parameters as pre‐ dictors of blastocyst development and pregnancy outcome in embryos from good prog‐ nosis patients: A prospective cohort study. Human Reproduction. 2013;**28**(10)**:**2643‐2651

rate. It also needs further studies to clarify the mystery of implantation process.

pre‐implantation genetic screening (PGS) is still the first choice.

Address all correspondence to: swrh1214@gmail.com

Reproductive Biology. 2014;**14**(4):249‐256

series study. Human Reproduction. 2016;31(10):2245‐2254

Stork Fertility Center, Stork Ladies Clinic, Hsinchu, Taiwan

**Author details**

Meng Ju Lee

22 Embryo Cleavage

**References**

