**2. Physiology of the blastocyst**

The transformation of the fertilized oocyte into the blastocyst is not only characterized by major morphological events but also by dramatic changes in its

physiology, reflected in changes in the relative activity of the metabolic pathways which provide not only energy but also the biosynthetic intermediates required to support proliferation [1]. The ability of the cleavage stage embryo to react in the environment during early cleavage is limited because the human genome embryo is still inactive and the system that regulates the balance of the osmotic pressure is not fully functional [1, 2].

The tendency of metabolism to produce energy from pronucleate oocytes until blastocyst stage can be assessed from mitochondrial forms. In the stage of pronucleate oocytes and cleavage stage, their mitochondrial form is still immature, and the production of energy in oocytes is usually low and will be increased tremendously from cleavage stage embryo until blastocyst stage*.* In the stage of pronucleate oocytes, the type of metabolism is oxidative phosphorylation (OXPHOST); then in the cleavage stage embryo, the metabolism uses lactate, pyruvate, specific amino acids, and fatty acids [1–3].

In the blastocyst stage, the metabolism produces energy that mainly depends on the process of glycolysis, with anabolic dominantly seen in the mitochondria [3, 4].

#### **Figure 1.**

*Tricarboxylic acid (TCA) cycle or the Krebs cycle. The citric acid cycle begins with one acetyl-CoA molecule reacting with one molecule of H2O, releasing a coenzyme-A group, and donating the remaining two carbon atoms in the form of an acetyl group to oxaloacetic acid which has molecules with four carbon atoms, to produce citric acid with six carbon atoms. The outcome products of the first turn of the cycle are one GTP (or ATP), three NADH, one QH2, and two CO2. Because two acetyl-CoA molecules are produced from each glucose molecule, two cycles are required per glucose molecule. Therefore, at the end of two cycles, the final products are two GTP, six NADH, two QH2, and four CO2.*

**65**

*of ATP.*

**Figure 2.**

*Glycolysis pathway or Embden-Meyerhof-Parnas (EMP) pathway. (1) In the first stage, glucose will be converted to glucose 6-phosphate by the hexokinase enzyme. This stage requires energy from adenosine triphosphate (ATP). The ATP that has released the stored energy will change to ADP. (2) Glucose 6-phosphate will be converted to fructose 6-phosphate which is catalyzed by the enzyme phosphohexose isomerase. (3) Fructose 6-phosphate will be converted to fructose 1,6-bisphosphate; this reaction is catalyzed by the enzyme phosphofructokinase. In this reaction, energy from ATP is needed. (4) Fructose 1,6-bisphosphate (6 C atoms) will be broken down into glyceraldehyde 3-phosphate (3 atoms C) and dihydroxy acetone phosphate (3 atoms C). The reaction is catalyzed by the enzyme aldolase. (5) One molecule of dihydroxy acetone phosphate that is formed will be converted to glyceraldehyde 3-phosphate by the enzyme triose phosphate isomerase. The enzyme works back and forth, meaning it can also convert glyceraldehyde 3-phosphate to dihydroxy acetone phosphate. (6) Glyceraldehyde 3-phosphate will then be converted to 1,3-bisphosphoglycerate by the enzyme glyceraldehyde 3-phosphate dehydrogenase. In this reaction NADH will be formed. (7) 1,3 bisphosphoglycerate will be converted to 3-phosphoglycerate by the phosphoglycerate kinase enzyme. These reactions will be released as energy in the form of ATP. (8) 3-phosphoglycerate will be converted into 2-phosphoglycerate by the phosphoglycerate mutase enzyme. (9) 2-phosphoglycerate will be converted to phosphoenolpyruvate by the enzyme enolase. (10) Phosphoenolpyruvate will be converted to pyruvate which is catalyzed by the pyruvate kinase enzyme. In this stage also produced energy in the form* 

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

*Embryology - Theory and Practice*

fully functional [1, 2].

acids, and fatty acids [1–3].

physiology, reflected in changes in the relative activity of the metabolic pathways which provide not only energy but also the biosynthetic intermediates required to support proliferation [1]. The ability of the cleavage stage embryo to react in the environment during early cleavage is limited because the human genome embryo is still inactive and the system that regulates the balance of the osmotic pressure is not

The tendency of metabolism to produce energy from pronucleate oocytes until blastocyst stage can be assessed from mitochondrial forms. In the stage of pronucleate oocytes and cleavage stage, their mitochondrial form is still immature, and the production of energy in oocytes is usually low and will be increased tremendously from cleavage stage embryo until blastocyst stage*.* In the stage of pronucleate oocytes, the type of metabolism is oxidative phosphorylation (OXPHOST); then in the cleavage stage embryo, the metabolism uses lactate, pyruvate, specific amino

In the blastocyst stage, the metabolism produces energy that mainly depends on the process of glycolysis, with anabolic dominantly seen in the mitochondria [3, 4].

*Tricarboxylic acid (TCA) cycle or the Krebs cycle. The citric acid cycle begins with one acetyl-CoA molecule reacting with one molecule of H2O, releasing a coenzyme-A group, and donating the remaining two carbon atoms in the form of an acetyl group to oxaloacetic acid which has molecules with four carbon atoms, to produce citric acid with six carbon atoms. The outcome products of the first turn of the cycle are one GTP (or ATP), three NADH, one QH2, and two CO2. Because two acetyl-CoA molecules are produced from each glucose molecule, two cycles are required per glucose molecule. Therefore, at the end of two cycles, the final products are* 

**64**

**Figure 1.**

*two GTP, six NADH, two QH2, and four CO2.*

#### **Figure 2.**

*Glycolysis pathway or Embden-Meyerhof-Parnas (EMP) pathway. (1) In the first stage, glucose will be converted to glucose 6-phosphate by the hexokinase enzyme. This stage requires energy from adenosine triphosphate (ATP). The ATP that has released the stored energy will change to ADP. (2) Glucose 6-phosphate will be converted to fructose 6-phosphate which is catalyzed by the enzyme phosphohexose isomerase. (3) Fructose 6-phosphate will be converted to fructose 1,6-bisphosphate; this reaction is catalyzed by the enzyme phosphofructokinase. In this reaction, energy from ATP is needed. (4) Fructose 1,6-bisphosphate (6 C atoms) will be broken down into glyceraldehyde 3-phosphate (3 atoms C) and dihydroxy acetone phosphate (3 atoms C). The reaction is catalyzed by the enzyme aldolase. (5) One molecule of dihydroxy acetone phosphate that is formed will be converted to glyceraldehyde 3-phosphate by the enzyme triose phosphate isomerase. The enzyme works back and forth, meaning it can also convert glyceraldehyde 3-phosphate to dihydroxy acetone phosphate. (6) Glyceraldehyde 3-phosphate will then be converted to 1,3-bisphosphoglycerate by the enzyme glyceraldehyde 3-phosphate dehydrogenase. In this reaction NADH will be formed. (7) 1,3 bisphosphoglycerate will be converted to 3-phosphoglycerate by the phosphoglycerate kinase enzyme. These reactions will be released as energy in the form of ATP. (8) 3-phosphoglycerate will be converted into 2-phosphoglycerate by the phosphoglycerate mutase enzyme. (9) 2-phosphoglycerate will be converted to phosphoenolpyruvate by the enzyme enolase. (10) Phosphoenolpyruvate will be converted to pyruvate which is catalyzed by the pyruvate kinase enzyme. In this stage also produced energy in the form of ATP.*

In the cleavage stage embryo, pyruvate uptake increases continuously until the blastocyst stage. In the blastocyst stage, glucose uptake is higher than pyruvate uptake, and O2 consumption will increase in the initial development stage before compaction (pre-compaction). In the pre-compaction stage, we can observe low biosynthetic activity, low O2 consumption, and ovoid form of mitochondria; the main nutrition is pyruvate, with dominant maternal genome where cells divide in the similar shape [1–3].

In the post-compaction stage, we can observe high biosynthetic activity, higher oxygen consumption, and elongated form of mitochondria; the main nutrition is glucose, and with dominant human embryo genome. In this stage, cells will be differentiated into trophectoderm (TE) and inner cell mass (ICM) [1–3, 5].

Amino acids in the metabolism of blastocyst can be used as a source of energy, and some amino acids such as aspartate through malate aspartate shuttle enter in the tricarboxylic acid (TCA) cycle to produce energy. However, glutamine can also

#### **Figure 3.**

*Metabolism of the blastocyst. After compaction, there is an increase in oxygen consumption and utilization of glucose as a source of energy (glycolysis). The increase in oxygen consumption reflects the considerable energy required for the formation and maintenance of the blastocoel, but the increase in glucose utilization reflects an increased demand for biosynthetic process. In TCA cycles, it produces NADH, GTP, QH, and CO2 and 34 ATP. PEP, phosphoenolpyruvate.*

**67**

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

through the activity of Na<sup>+</sup>

**3. Morphology of the blastocyst**

ity of the blastocyst [6, 7].

**3.1 Degree of expansion**

**3.2 ICM morphology**

dant [1–3, 5].

enter as glutamate in the TCA cycle to produce energy. Amino acid in the blastocyst stage also plays a role in the regulation of intracellular pH buffer, as a material

needed for the degradation of the zona pellucida with protease enzyme. Pyruvate as a source of energy reserves other than carbohydrates also functions as an antioxi-

The human blastocyst uses amino acids as a source of energy in the catabolism process and produces ammonium 30 pmol/hour. The most used amino acid is aspartate, besides consuming arginine, serine, methionine, valine, and leucine [1–3, 5]. Metabolism of the blastocyst occurs in two different places: in trophectoderm (TE) cells where glucose consumption occurs and half is converted to lactate, whereas glycolysis process occurs in inner cell mass (ICM) (**Figures 1**–**3**) [1–3, 5].

In in vitro fertilization (IVF), the blastocyst culture was important to increase the success rate of IVF because of better embryo selection after better genomic activation and endometrial receptivity [6]. The blastocyst comprises two cell types: the inner cell mass (ICM, from which the fetal tissues develop) and the trophectoderm (which will form mostly extraembryonic tissues such as the placenta [1]). This morphological differentiation was thought to represent the developmental capabil-

The fluid accumulation presence between cells at the morulae stage is the phase that determines embryonic development. The accumulated fluid will form blastocoel gradually which usually occurs on day 4 and/or on the beginning of day 5 human embryo stage which marks the development of new embryos known as the blastocyst stage. An increase in fluid volume and the number of cells in the blastocyst causes an enhancement in the size cavity of the blastocyst and its cavity with depletion of the zona pellucida (ZP) [6]. The number of cells that comprise a blastocyst can vary considerably as shown in one study to range between 24 and 322

The stages of blastocyst embryo development are divided into four grades. A grade of 1 is given to the embryo with blastocoel cavity less than 50% of the embryo volume. A grade of 2 is given to the embryo with blastocoel cavity as much as 50% of the embryo volume or even more than that. A grade of 3 was given to the embryo that had a blastocoel cavity which had fulfilled that all embryos and zona pellucida (ZP) appeared to be thinner than embryo on day 3. A grade of 4 is given to the

A collection of cells located within the blastocoel in one pole of the blastocyst cavity is called ICM. ICM will develop into fetal tissue. ICM consists of tightly

cells, which is often reflected in the blastocyst's morphology [8].

embryo that has successfully hatched from ZP [6, 9].

After compaction, the embryo exhibits increased of O2 consumption and glucose usage capacity as an energy source. This oxygen consumption increase shows that the energy is needed for the formation and maintenance of blastocoel [1–3, 5]. Increased metabolism of the blastocyst occurs due to the increased release of blastomere to 150–200 cells with the formation and maintenance of blastocoel

ATPase pump which produces energy. Energy is

development process and as antioxidants and chelators [1–3, 5].

/K+

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

*Embryology - Theory and Practice*

the similar shape [1–3].

In the cleavage stage embryo, pyruvate uptake increases continuously until the blastocyst stage. In the blastocyst stage, glucose uptake is higher than pyruvate uptake, and O2 consumption will increase in the initial development stage before compaction (pre-compaction). In the pre-compaction stage, we can observe low biosynthetic activity, low O2 consumption, and ovoid form of mitochondria; the main nutrition is pyruvate, with dominant maternal genome where cells divide in

In the post-compaction stage, we can observe high biosynthetic activity, higher oxygen consumption, and elongated form of mitochondria; the main nutrition is glucose, and with dominant human embryo genome. In this stage, cells will be dif-

Amino acids in the metabolism of blastocyst can be used as a source of energy, and some amino acids such as aspartate through malate aspartate shuttle enter in the tricarboxylic acid (TCA) cycle to produce energy. However, glutamine can also

*Metabolism of the blastocyst. After compaction, there is an increase in oxygen consumption and utilization of glucose as a source of energy (glycolysis). The increase in oxygen consumption reflects the considerable energy required for the formation and maintenance of the blastocoel, but the increase in glucose utilization reflects an increased demand for biosynthetic process. In TCA cycles, it produces NADH, GTP, QH, and CO2 and 34* 

ferentiated into trophectoderm (TE) and inner cell mass (ICM) [1–3, 5].

**66**

**Figure 3.**

*ATP. PEP, phosphoenolpyruvate.*

enter as glutamate in the TCA cycle to produce energy. Amino acid in the blastocyst stage also plays a role in the regulation of intracellular pH buffer, as a material development process and as antioxidants and chelators [1–3, 5].

After compaction, the embryo exhibits increased of O2 consumption and glucose usage capacity as an energy source. This oxygen consumption increase shows that the energy is needed for the formation and maintenance of blastocoel [1–3, 5].

Increased metabolism of the blastocyst occurs due to the increased release of blastomere to 150–200 cells with the formation and maintenance of blastocoel through the activity of Na<sup>+</sup> /K+ ATPase pump which produces energy. Energy is needed for the degradation of the zona pellucida with protease enzyme. Pyruvate as a source of energy reserves other than carbohydrates also functions as an antioxidant [1–3, 5].

The human blastocyst uses amino acids as a source of energy in the catabolism process and produces ammonium 30 pmol/hour. The most used amino acid is aspartate, besides consuming arginine, serine, methionine, valine, and leucine [1–3, 5].

Metabolism of the blastocyst occurs in two different places: in trophectoderm (TE) cells where glucose consumption occurs and half is converted to lactate, whereas glycolysis process occurs in inner cell mass (ICM) (**Figures 1**–**3**) [1–3, 5].

#### **3. Morphology of the blastocyst**

In in vitro fertilization (IVF), the blastocyst culture was important to increase the success rate of IVF because of better embryo selection after better genomic activation and endometrial receptivity [6]. The blastocyst comprises two cell types: the inner cell mass (ICM, from which the fetal tissues develop) and the trophectoderm (which will form mostly extraembryonic tissues such as the placenta [1]). This morphological differentiation was thought to represent the developmental capability of the blastocyst [6, 7].

#### **3.1 Degree of expansion**

The fluid accumulation presence between cells at the morulae stage is the phase that determines embryonic development. The accumulated fluid will form blastocoel gradually which usually occurs on day 4 and/or on the beginning of day 5 human embryo stage which marks the development of new embryos known as the blastocyst stage. An increase in fluid volume and the number of cells in the blastocyst causes an enhancement in the size cavity of the blastocyst and its cavity with depletion of the zona pellucida (ZP) [6]. The number of cells that comprise a blastocyst can vary considerably as shown in one study to range between 24 and 322 cells, which is often reflected in the blastocyst's morphology [8].

The stages of blastocyst embryo development are divided into four grades. A grade of 1 is given to the embryo with blastocoel cavity less than 50% of the embryo volume. A grade of 2 is given to the embryo with blastocoel cavity as much as 50% of the embryo volume or even more than that. A grade of 3 was given to the embryo that had a blastocoel cavity which had fulfilled that all embryos and zona pellucida (ZP) appeared to be thinner than embryo on day 3. A grade of 4 is given to the embryo that has successfully hatched from ZP [6, 9].

#### **3.2 ICM morphology**

A collection of cells located within the blastocoel in one pole of the blastocyst cavity is called ICM. ICM will develop into fetal tissue. ICM consists of tightly

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 cells that are loosely bound [6, 9].
