**3.3 TE morphology**

The outer cells of the blastocyst, forming the blastocyst structure itself, are called the trophectoderm (TE) cells. TE cells play a role in the formation of fluid accumulation in the blastocoel which can be a key in ICM determination, but in the early stages of the blastocyst stage, the role of TE cells is unclear. TE cells will develop into extraembryonic tissue such as the placenta. In addition, TE cells are thought to play a role in the implantation process which the TE cells have contributed in the phase of apposition, adhesion, and invasion of the endometrium which can support the implantation of the blastocyst in the uterus. Molecular factors are also produced by TE cells that play a role in the embryo implantation process [6–9]. The TE cells have traditionally been graded in a similar manner to the ICM, i.e., by their number and cohesiveness according to three different grades (A, B, C). According to blastocyst grading by Gardner and Schoolcraft, the best TE cell grade (A) contains many cells that form a cohesive epithelium; the middle TE category (B) is composed of few cells forming a loose epithelium, and the worst category (C) describes a TE that contains very few large cells that struggle to form a cohesive epithelium. However, other publications have found no relationship between TE grade and viability (**Figure 4**) [6, 8, 9].

Gardner and Schoolcraft in 1999 introduced the blastocyst grading system which is adopted by the majority of IVF laboratories in the world. In the blastocyst grading system, they classified the degree of blastocyst into three category,

**69**

(TE) cell quality [6, 10].

**Figure 6.**

**Figure 5.**

must be in the form of blastocyst [9].

Istanbul consensus (**Figures 5**–**9; Table 1**) [6, 9].

*Human Blastocyst Formation and Development DOI: http://dx.doi.org/10.5772/intechopen.82095*

i.e., degree of expansion, inner cell mass (ICM) quality, and the trophectoderm

*Blastocyst (grade 4: C: C) means that blastocyst embryo has been expanded (grade 4), has ICM that is difficult to see and has very few cells (grade C), and has TE which has very little cell (grade C).*

*Blastocyst (grade 4: A: A) means that the blastocyst has been expanded (grade 4), has a large ICM and compact (grade A), and has TE which consists of many cells which are mutually binding (grade A).*

Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology states that a good embryo on day 5 which is examined at 116 ± 2 hours

Embryo, according to the Istanbul consensus, is given a grade based on three things; the first thing is the embryo development stage, the second is ICM, and the third is TE. So that the order of the fifth day embryo assessment is based on the stages of development, then based on ICM morphology, and the last is based on TE. The following is a presentation of the fifth day embryo according to the

**Figure 4.** *Blastocyst morphology. (1) inner cell mass (ICM), (2) blastocoel, and (3) trophectoderm.*

*Human Blastocyst Formation and Development DOI: http://dx.doi.org/10.5772/intechopen.82095*

#### **Figure 5.**

*Embryology - Theory and Practice*

cells that are loosely bound [6, 9].

**3.3 TE morphology**

[6, 8, 9].

packed cells and loosely bound cells that cause the size of the ICM very large and/ or small morphologically [6]. The morphological form of ICM is assessed based on how much the cell compaction is until there is no cell clot at all. A grade of 1 is given to ICM with a very large and dense form of cell clots. A grade of 2 is given to ICM with a slightly diffuse cell form. A grade of 3 is given to ICM with very few cells and which does not even form clots. However, the best ICM grade (A) contains tightly packed and many cells; the middle ICM grade (B) is composed of loosely grouped and several cells, and the worst grade (C) describes an ICM that contains very few

The outer cells of the blastocyst, forming the blastocyst structure itself, are called the trophectoderm (TE) cells. TE cells play a role in the formation of fluid accumulation in the blastocoel which can be a key in ICM determination, but in the early stages of the blastocyst stage, the role of TE cells is unclear. TE cells will develop into extraembryonic tissue such as the placenta. In addition, TE cells are thought to play a role in the implantation process which the TE cells have contributed in the phase of apposition, adhesion, and invasion of the endometrium which can support the implantation of the blastocyst in the uterus. Molecular factors are also produced by TE cells that play a role in the embryo implantation process [6–9]. The TE cells have traditionally been graded in a similar manner to the ICM, i.e., by their number and cohesiveness according to three different grades (A, B, C). According to blastocyst grading by Gardner and Schoolcraft, the best TE cell grade (A) contains many cells that form a cohesive epithelium; the middle TE category (B) is composed of few cells forming a loose epithelium, and the worst category (C) describes a TE that contains very few large cells that struggle to form a cohesive epithelium. However, other publications have found no relationship between TE grade and viability (**Figure 4**)

Gardner and Schoolcraft in 1999 introduced the blastocyst grading system which is adopted by the majority of IVF laboratories in the world. In the blastocyst grading system, they classified the degree of blastocyst into three category,

*Blastocyst morphology. (1) inner cell mass (ICM), (2) blastocoel, and (3) trophectoderm.*

**68**

**Figure 4.**

*Blastocyst (grade 4: A: A) means that the blastocyst has been expanded (grade 4), has a large ICM and compact (grade A), and has TE which consists of many cells which are mutually binding (grade A).*

#### **Figure 6.**

*Blastocyst (grade 4: C: C) means that blastocyst embryo has been expanded (grade 4), has ICM that is difficult to see and has very few cells (grade C), and has TE which has very little cell (grade C).*

i.e., degree of expansion, inner cell mass (ICM) quality, and the trophectoderm (TE) cell quality [6, 10].

Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology states that a good embryo on day 5 which is examined at 116 ± 2 hours must be in the form of blastocyst [9].

Embryo, according to the Istanbul consensus, is given a grade based on three things; the first thing is the embryo development stage, the second is ICM, and the third is TE. So that the order of the fifth day embryo assessment is based on the stages of development, then based on ICM morphology, and the last is based on TE. The following is a presentation of the fifth day embryo according to the Istanbul consensus (**Figures 5**–**9; Table 1**) [6, 9].

#### **Figure 7.**

*Early blastocyst is the fifth day embryo development stage which has a blastocoel less than 50% of the embryo volume, and the formation of ICM and TE is not clear.*

#### **Figure 8.**

*Blastocyst (grade 2: A: A) embryos that have embryo development stage with a grade of 2 are blastocyst embryos which still have thick ZP.*

#### **Figure 9.**

*Blastocyst (grade 5: A: A) means that blastocyst embryo which has come out of its shell (grade 5) has ICM whose cells are large and easy to observe (grade A) and has TE consisting of many cells (grade A).*

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plantation embryos [11–13].

chromatin remodeling [12–16].

*Human Blastocyst Formation and Development DOI: http://dx.doi.org/10.5772/intechopen.82095*

is thinner 5 Hatching out of the shell 6 Hatched out of the shell **ICM grade Inner cell mass quality** A Tightly packed and many cells B Loosely grouped and several cells

C Very few cells

**TE grade Trophectoderm quality**

C Very few large cells

**Table 1.**

A Many cells forming a cohesive epithelium B Few cells forming a loose epithelium

*The blastocyst grading system. Modified from Gardner and Schoolcraft.*

**Blastocyst development and stage status**

3 Full blastocyst—Blastocoel completely filling the embryo

1 Early blastocyst—Blastocoel cavity less than half the volume of the embryo 2 Blastocyst—Blastocoel cavity more than half the volume of the embryo

4 Expanded blastocyst—Blastocoel volume is now larger than that of early embryo and zona

**Expansion grade**

**4. Gene expression of human preimplantation embryo**

The human embryo preimplantation development is characterized by reprogramming and programming that encompasses fusion of the egg and sperm pronuclei, epigenetic reprogramming, an extensive wave of degradation of maternal transcripts, and activation of the nascent human embryonic genome [11, 12]. There are 1909 genes expressed in only oocytes (maternal genome) and 3122 genes expressed in only blastocyst (embryonic genome). The main difference in observed profile expression between oocytes and embryos is reflected in blastocyst and oocyte specific gene expression analysis. Specific oocyte and blastocyst genes are separated in groups by expression levels. There are 270 specific oocyte genes and 308 blastocyst-specific genes that show high levels of expression. In the panther pathway analysis of high expression genes, the MII oocyte pathway is Wnt signaling pathway, where blastocyst-specific genes are expressed according to integrin signaling pathway, cytoskeletal regulation. The scarcity of the materials, however, both in size (10.1 mm diameter) and in quantity (only a few to tens of oocytes from each ovulation), have been almost the molecular analysis of preim-

Some interesting patterns in embryo preimplantation based on transcriptional genome-wide analysis include: (1) several genes that experience up- or downregulation during human oocyte maturation from immature germinal vesicle oocyte to the oocyte metaphase II stage; (2) partially expressed transcripts of the MII oocytes will be partially downregulated or degraded during the development to the 4-cell stage; (3) many genes are upregulated after the 4-cell stage, which reflects the main wave Embryonic Genome Activation (EGA); (4) genes involved in lineage commitment are regulated in the development of preimplantation; and (5) many expressed genes dynamically encode transcription factors, epigenetic modifying factors, and

*Human Blastocyst Formation and Development DOI: http://dx.doi.org/10.5772/intechopen.82095*


**Table 1.**

*Embryology - Theory and Practice*

*volume, and the formation of ICM and TE is not clear.*

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**Figure 9.**

**Figure 8.**

**Figure 7.**

*embryos which still have thick ZP.*

*Blastocyst (grade 2: A: A) embryos that have embryo development stage with a grade of 2 are blastocyst* 

*Early blastocyst is the fifth day embryo development stage which has a blastocoel less than 50% of the embryo* 

*Blastocyst (grade 5: A: A) means that blastocyst embryo which has come out of its shell (grade 5) has ICM whose cells are large and easy to observe (grade A) and has TE consisting of many cells (grade A).*

*The blastocyst grading system. Modified from Gardner and Schoolcraft.*

## **4. Gene expression of human preimplantation embryo**

The human embryo preimplantation development is characterized by reprogramming and programming that encompasses fusion of the egg and sperm pronuclei, epigenetic reprogramming, an extensive wave of degradation of maternal transcripts, and activation of the nascent human embryonic genome [11, 12].

There are 1909 genes expressed in only oocytes (maternal genome) and 3122 genes expressed in only blastocyst (embryonic genome). The main difference in observed profile expression between oocytes and embryos is reflected in blastocyst and oocyte specific gene expression analysis. Specific oocyte and blastocyst genes are separated in groups by expression levels. There are 270 specific oocyte genes and 308 blastocyst-specific genes that show high levels of expression. In the panther pathway analysis of high expression genes, the MII oocyte pathway is Wnt signaling pathway, where blastocyst-specific genes are expressed according to integrin signaling pathway, cytoskeletal regulation. The scarcity of the materials, however, both in size (10.1 mm diameter) and in quantity (only a few to tens of oocytes from each ovulation), have been almost the molecular analysis of preimplantation embryos [11–13].

Some interesting patterns in embryo preimplantation based on transcriptional genome-wide analysis include: (1) several genes that experience up- or downregulation during human oocyte maturation from immature germinal vesicle oocyte to the oocyte metaphase II stage; (2) partially expressed transcripts of the MII oocytes will be partially downregulated or degraded during the development to the 4-cell stage; (3) many genes are upregulated after the 4-cell stage, which reflects the main wave Embryonic Genome Activation (EGA); (4) genes involved in lineage commitment are regulated in the development of preimplantation; and (5) many expressed genes dynamically encode transcription factors, epigenetic modifying factors, and chromatin remodeling [12–16].

Human genome activation begins in the 4- and 8-cell stages or even in the early 2-cell stage. Data suggest that TE or ICM cell lineage-associated genes are expressed in human embryos later than in mice at around early blastocyst stage, but it is still unclear. Human embryos can be cultured in vitro for 7–8 days post-fertilization. Genes unique in the blastocysts included annexins A2 and A3 (ANXA2, ANXA3), gap junction protein alpha 1 (GJPA1), guanosine triphosphate-binding protein 4 (GTPBP4), and adenosine triphosphatase H.–transporting, lysosomal accessory protein 1 (ATP6AP1). The blastocyst-specific genes were associated with oxidative phosphorylation, glycolysis, and sterol metabolism and were rich in RNA-binding proteins, methyltransferases, gap junction proteins, and intermediate filaments. Oxidative phosphorylation and glycolysis processes control ATP generation during pre-compaction and cavitation stages, respectively. Highly expressed blastocyst genes were involved in the Rho GTPase, control pathway that regulates cytoskeletal changes occurring during cell growth and development; the PDGF pathway, which plays a critical role in cellular proliferation and metabolism, and the integrin signaling pathway are also important in actin reorganization [12–19].

Studies on gene expression during the preimplantation period have identified transcription factors from housekeeping genes, transcription and growth factor genes, sex-determining genes, tissues specifics, novel genes, and genes of unknown functions [12, 13].

#### **4.1 Housekeeping gene**

Housekeeping gene is a gene that plays a role in regulating basal cell function which is important in cell maintenance and also has a specific role in tissues or organisms. Therefore, housekeeping gene is estimated to be expressed in all cells of organism normally, including tissue, external signal, cell cycle, or cell development stage [12, 13, 20].

Beta-actin, keratin-18, ubiquitous cytoskeletal elements, cell adhesion molecules, and alpha tubulin have been detected, as well as such housekeeping genes as hypoxanthine phosphoribosyl transferase (HPRT), adenosine phosphoribosyl transferase (APRT), glucose-6-phosphate dehydrogenase, hexokinase I, and adenosine deaminase. Overall, out of the 536 housekeeping genes investigated, 427 were detected in the oocyte and 452 in the blastocyst [12, 13, 20].

#### **4.2 Transcription and growth factor genes**

Transcription regulators, growth factors, proto-oncogenes cycle gene cells, and receptors are several genes that expressed during the preimplantation period, including CD44, a cell surface glycoprotein that can play a role in implantation; OCT 4 and OCT 6, transcriptional regulators; cyclin B1, a cell cycle gene; colony stimulating factor 1 receptor, c-fms; tumor necrosis factor and its receptors; interleukin-1 type I receptor (IL-1R tI); growth factors such as insulin and their receptors; epidermal growth factor receptor (EGF-R), epidermal growth factor (EGF), and transformation of growth factor-alpha (TGF-α) [12, 13, 17–19].

#### **4.3 Tissue-specific genes, novel genes, and genes of unknown function**

A major histocompatibility complex molecule was detected in preimplantation human embryos and implicated tissue-specific genes as globin and interleukin-10 and also human transposable element, LINE-1, and known expressed sequence tags

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metabolism.

this book.

**Acknowledgements**

**Conflict of interest**

*Human Blastocyst Formation and Development DOI: http://dx.doi.org/10.5772/intechopen.82095*

showed high expression levels [12, 13, 15–17, 21].

occurred in more than 80% of embryos [11, 12, 17, 22].

**5. Conclusions**

(ESTs) in the GenBank and dbEST databases. Of the 46 peptide hormone genes investigated, 13 were detected in the human blastocysts (SPAG9, TMSB10, OXT, POMC, PPY, SCT, LHB, TRH, PRLH, GNRH2, GIP, CCK, and GPHB5) of which four, namely, SPAG9, TMSB10, OXT, and POMC, showed high expression levels. Thirty-two out of the 162 investigated genes coding for human HRs were detected in the human blastocysts, of which NPM1, ATP6AP2, LEPROTL1, and HTR1D

Aneuploidy is common in humans and is the leading cause of all human birth defects as well as miscarriage; errors can arise in meiosis during generation of the oocyte and sperm and in the mitotic divisions of the nascent embryo. A recent study used array-based technology to examine the genome-wide copy number of distinct loci in the cleavage stage human embryos. This study identified several types of chromosomal abnormalities that occurred in human embryos and observed that mosaicism for whole chromosomes (aneuploidies) in one or more blastomeres

Human preimplantation embryos will become an integral and essential part of such endeavors by setting the genetic foundation that determines the course of human development. It is characterized by reprogramming and programming that encompasses fusion of the egg and sperm pronuclei, epigenetic reprogramming, an extensive wave of degradation of maternal transcripts, and activation of the nascent human embryonic genome. The main objective of blastocyst culture was to increase the success rate of in vitro fertilization (IVF) because of better embryo selection after endometrial synchronicity and/or better genomic activation. Metabolism of the blastocyst occurs in two different places: in trophectoderm cells where glucose consumption occurs and half is converted to lactate, whereas glycolysis occurs in ICM. Morphology of the blastocyst depends on the degree of expansion and ICM and TE morphology. This morphological differentiation was thought to represent the developmental capability of the blastocyst. Gene expression during the preimplantation period has identified transcription factors from housekeeping genes, transcription and growth factor genes, sex-determining genes, tissues specifics, novel genes, and genes of unknown functions. Genes unique in human blastocysts included annexins A2 and A3 (ANXA2, ANXA3), gap junction protein alpha 1 (GJPA1), guanosine triphosphate-binding protein 4 (GTPBP4), and adenosine triphosphatase H.– transporting, lysosomal accessory protein 1 (ATP6AP1). The blastocyst-specific genes were associated with oxidative phosphorylation, glycolysis, and sterol

The authors thank all of team Halim fertility center for their help and support to

There is no conflict of interests in this manuscript.

*Human Blastocyst Formation and Development DOI: http://dx.doi.org/10.5772/intechopen.82095*

(ESTs) in the GenBank and dbEST databases. Of the 46 peptide hormone genes investigated, 13 were detected in the human blastocysts (SPAG9, TMSB10, OXT, POMC, PPY, SCT, LHB, TRH, PRLH, GNRH2, GIP, CCK, and GPHB5) of which four, namely, SPAG9, TMSB10, OXT, and POMC, showed high expression levels. Thirty-two out of the 162 investigated genes coding for human HRs were detected in the human blastocysts, of which NPM1, ATP6AP2, LEPROTL1, and HTR1D showed high expression levels [12, 13, 15–17, 21].

Aneuploidy is common in humans and is the leading cause of all human birth defects as well as miscarriage; errors can arise in meiosis during generation of the oocyte and sperm and in the mitotic divisions of the nascent embryo. A recent study used array-based technology to examine the genome-wide copy number of distinct loci in the cleavage stage human embryos. This study identified several types of chromosomal abnormalities that occurred in human embryos and observed that mosaicism for whole chromosomes (aneuploidies) in one or more blastomeres occurred in more than 80% of embryos [11, 12, 17, 22].
