Author's personal copy

Provided for non-commercial research and educational use.

Not for reproduction, distribution or commercial use.

This article was originally published in Encyclopedia of Reproduction, Second

Edition, published by Elsevier, and the attached copy is provided by Elsevier for the
author's benefit and for the benefit of the author's institution, for non-commercial
research and educational use including without limitation use in instruction at your
institution, sending it to specific colleagues who you know, and providing a copy to your
institution's administrator.

All other uses, reproduction and distribution, including

without limitation commercial reprints, selling or
licensing copies or access, or posting on open
internet sites, your personal or institution’s website or
repository, are prohibited. For exceptions, permission
may be sought for such use through Elsevier’s
permissions site at:
https://www.elsevier.com/about/our-business/policies/copyright/permissions

From Gardner, D. K. (2018). Human Embryo Development and Assessment of Viability. In M.

K. Skinner (Ed.), Encyclopedia of Reproduction. vol. 5, pp. 176–185. Academic
Press: Elsevier. http://dx.doi.org/10.1016/B978-0-12-801238-3.64857-2
ISBN: 9780128118993
Copyright © 2018 Elsevier Inc. All rights reserved.
Academic Press
 Author's personal copy

Human Embryo Development and Assessment of Viability

David K Gardner, University of Melbourne, Parkville, VIC, Australia; and Melbourne IVF, East Melbourne, VIC, Australia
© 2018 Elsevier Inc. All rights reserved.

Introduction

The preimplantation period of human embryo development is marked by several key morphological, physiological and genetic
changes, all of which occur within a 6-day period, after which the embryo implants into the endometrium of the uterus. From fertil-
ization in the oviduct, to implantation in the uterus, the embryo develops from a totipotent single cell, to a multicellular blastocyst
of over 100 cells, which contains two distinct cell populations. The outer layer of the blastocyst, which subsequently forms the
placenta is referred to as the trophectoderm and represents the first transporting epithelium of the conceptus. Within the fluid cavity,
the blastocoel, resides an eccentrically placed group of cells known as the inner cell mass, and it is from this smaller group of cells
that the fetus develops. During this period of life the embryo is free floating within the female tract and derives all of its nutrients
from the surrounding fluids. This environment is characterized by nutrient and oxygen gradients as the embryo passes through the
oviduct and uterus (Gardner et al., 1996; Ng et al., 2017), and becomes increasingly more complex in composition with the uterine
environment providing an array of cytokines and growth factors (Hannan et al., 2011).
Development of the human embryo in vitro was significantly improved when culture media started to reflect the composition of
the female reproductive tract, rather than being empirically derived (Gardner and Lane, 1997, 2017). Now it is possible to maintain
the human embryo through to the blastocyst stage in culture, resulting in improved outcome for patients undergoing in vitro Fertil-
ization (IVF) procedures. Analysis of embryo morphology, kinetics of development and the analysis of metabolic function have all
been linked, to some degree, to pregnancy outcome. The future of human IVF will see the development and application of further
biomarkers of developmental potential, potentially combined with the introduction of microfluidic devices, which are suited to
single embryo/cell analyses (Thouas et al., 2013).

The Environments Created by the Female Reproductive Tract to Support the First Week of Life

The environments created within the lumen of the female reproductive tract are designed to support the stage-specific needs of the
developing embryo, and are not simple serum-transudates (Leese, 1988). Hence, fluids within the oviduct and uterus are unique,
complex and change with regards to the day of the menstrual cycle. Human oviduct fluid is comprised of high levels of albumin and
mucins, and contains pyruvate (0.32 mM) and lactate (10.5 mM) and low levels of glucose (0.5 mM) (Gardner et al., 1996).
Mammalian oviduct fluid is characterized by very high levels of specific amino acids including alanine, glutamate, glycine and
taurine (Harris et al., 2005; Miller and Schultz, 1987), and has oxygen levels of between 5% and 8% (Gardner and Lane, 2017;
Ng et al., 2017). In contrast, uterine fluid is somewhat more complex, characterized by the presence of glycosaminoglycans, espe-
cially hyaluronan and a rich reservoir of growth factors and cytokines. Uterine fluid has relatively high levels of glucose (3.15 mM),
and low levels of pyruvate (0.1 mM) and lactate (5.87 mM) (Gardner et al., 1996). The level of oxygen in the uterus ranges from

2.5% to 5% and is considered to drop close to 2% around the time of implantation (Gardner and Lane, 2017; Ng et al., 2017). As
well as providing gradients of nutrients to the embryo, which also act as signals to the embryo (Gardner and Harvey, 2015), the
embryo is exposed to numerous cytokines and growth factors, whose levels not only change with the day of the menstrual cycle,
but also differ between fertile and infertile patients (Hannan et al., 2011), thereby indicating they have important roles in regulating
embryonic development, differentiation and implantation. Hence, the environment to which the human preimplantation embryo
is exposed to in vivo is anything but constant. As the biology of the developing embryo is considered in detail, the significance of the
dynamic environment created within the maternal tract becomes evident.

Biology of Human Preimplantation Development

The human oocyte is the largest cell in the female, being
110 um in diameter, and is surrounded by a glycoprotein coat known as
the zona pellucida. The zona serves not only to prevent polyspermy postfertilization, but also prevent premature attachment and
possible invasion of the developing embryo into the wall of the oviduct. The zona is subsequently lost at the blastocyst stage
through the action of both embryo and uterine-derived proteases. After Fertilization, the embryo must undergo the extrusion of
the second polar body, decondensation of the sperm head, pronuclei formation, and finally the breakdown of the pronuclear
membrane. The fertilized oocyte is totipotent, in that it can give rise to every cell type in the body. In the human preimplantation
embryo, the first few divisions are referred to as restrictive mitoses, in which the cells (know as blastomeres) divide but the daughter
cells are smaller. This is required to restore the cytoplasmic: nuclear ratio, which is abnormally high in the fertilized oocyte. The first
of these divisions occurs around 18 h after fertilization, with subsequent divisions occurring approximately every 12 h, although
there is variation between embryos (Meseguer et al., 2011). Up to the 8-cell stage, individual blastomeres are held in proximity
by the zona, but are not joined per se. Indeed, a breach in the zona could lead to a loss of cells. In terms of their physiology, indi-
vidual blastomeres lack sophisticated means of regulating their homeostasis, such as pH control, and are therefore more dependent

176 Encyclopedia of Reproduction, 2nd edition, Volume 5 https://doi.org/10.1016/B978-0-12-801238-3.64857-2

Encyclopedia of Reproduction, Second Edition, 2018, 176–185

 Author's personal copy
Embryology j Human Embryo Development and Assessment of Viability 177

upon the maternal tract to provide the appropriate environment (Gardner and Lane, 2005; Lane and Gardner, 2005). Between the 8-
to 16-cell stages, the embryo undergoes a significant change, with the formation of the first transporting epithelium, achieved
through the creation of tight junctions between adjacent cells. This facilitates not only a means to control the internal environment
of the embryo, but also creates an inside and out, with cells developing on the inside of the embryo being destined to become the
fetus, while those cells on the outside become allocated to extra-embryonic membranes such as the placenta (Fleming and Johnson,
1988). Down the microscope, the appearance of individual blastomeres is lost at the 8- to 16-cell stage as the cells flatten or compact
against each other, forming not only tight junctions at the apical surface of the cells, but also forming gap junction between cells.
This process is aptly referred to as compaction, and the embryo becomes known as the morula. At this stage the embryo resembles
a mulberry, and it is from this that the name morula was derived. Rather than continuing to develop as a solid ball of cells, the outer
cells of the morula start to create a cavity through the action of Na/K ATPases on their basolateral membranes, thereby creating an
ionic gradient, inducing the formation of a fluid-filled cavity, known as the blastocoel (Kidder and Watson, 2005). The embryo is
then referred to as the blastocyst. Cells that have been developing on the inside of the embryo develop into a distinct group known
as the inner cell mass (ICM), while the outer cells become the trophectoderm (TE). While the pluripotent ICM cells are destined to
form the fetus, the trophectoderm cells have the incredible task of invading into the maternal endometrium lining the uterus, estab-
lishing a blood supply, and preventing the mother from rejecting the nonself embryo. Fig. 1 shows the spatial positioning of the
human preimplantation embryo as it develops within the lumen of the female reproductive tract, while the detailed characteristics
of human embryo development are illustrated in Fig. 2.
The complex dialogue that exists between the blastocyst and endometrium is far from understood, but what we can deduce is
that cancers employ the same biological strategies as the trophectoderm to invade a host tissue and establish its own vasculature
(Gardner, 1998a,b, 2015). Indeed, analysis of the implantation process has much to inform us about cancers.

Dynamics of Embryo Physiology From the Fertilized Oocyte to the Blastocyst

From Fertilization to the 4 to 8-cell stage the human embryo is dependent upon stable mRNAs and proteins synthesized during
oocyte maturation. Around this time the embryonic genome begins to be transcribed, and between the morula and blastocyst stages

Day 2

Day 1 Day 3 Uterus

2-cell

4-cell Day 4
Early
Syngamy 8-cell Blastocyst
Morula
(Zygote) Day 5
Expanded
Blastocyst

Pronucleate Trophectoderm
Oocyte
Inner Cell Mass Day 6

Ovary Zona Pellucida

Hatched
Fertilization Blastocyst

Day 7–8
MII Oocyte

Implanting
Blastocyst

Epiblast

Fig. 1 Schematic of the human female reproductive tract and the relative position of the preimplantation embryos as it develops and differentiates. From
fertilization in the ampulla of the oviduct, through to implantation in the endometrium 6–7 days later, the human embryo undergoes remarkable
development and differentiation, all the while being exposed to an ever changing environment. The oviduct is characterized by relatively high levels of
pyruvate and lactate and an oxygen concentration of around 8%. In contrast, uterine fluid is characterized by relatively high levels of glucose and
lower concentration of oxygen. Further, the uterine environment is one containing numerous cytokines and growth factors.

Encyclopedia of Reproduction, Second Edition, 2018, 176–185

 Author's personal copy
178 Embryology j Human Embryo Development and Assessment of Viability

Features of the human pronucleate

oocytes:
(i) number of nucleolar precursor bodies
(NPB) in both pronuclei never differed by
more than 3
(ii) NPB are always polarized or not
polarized in both pronuclei but never
polarized in one pronucleus and not in the
other
(iii) angle β from the axis of the pronuclei
and the furthest polar body is less than 50°

Ideal features shared by 2-cell

embryos with high viability:
(i) Mononucleated blastomeres
(ii) Equal cell size
(iii) < 20% fragmentation

Ideal features shared by 4-cell

embryos with high viability:
(i) Mononucleated blastomeres
(ii) Equal cell size
(iii) < 20% fragmentation

Ideal features shared by day 3

embryos with high viability:
(i) Mononucleated blastomeres
(ii) Equal cell size
(iii) < 20% fragmentation
(iv) At least 7 blastomeres

Ideal features shared by morulae with

high viability:
(i) Visibly compacted cells denoted
by the slight reduction in overall
size of the embryo and increase
in space between the embryo
and zona pellucida
(ii) Lack of fragments

Ideal features shared by blastocysts

with high viability:
(i) Expanded blastocoel cavity by
day 5
(ii) Well formed ICM clearly
composed of many cells
(iii) Cohesive epithelium made up
from many cells in the TE
(iv) Signs of the zona pellucida
thinning

Fig. 2 Key morphological features of embryos with high viability. ICM: inner cell mass, TE: trophectoderm. Gardner, D.K., Balaban, B. Assessment of
human embryo development using morphological criteria in an era of time-lapse, algorithms and OMICS: Is looking good still important? Molecular
Human Reproduction 22, 2016, 704–718 with permission.

Encyclopedia of Reproduction, Second Edition, 2018, 176–185

 Author's personal copy
Embryology j Human Embryo Development and Assessment of Viability 179

there is a dramatic increase in embryonic gene expression, so that by the blastocyst stage the embryonic genome is active (Hamatani
et al., 2006; Jukam et al., 2017). As the human embryo progresses through the preimplantation period, it undergoes changes in
morphology, exhibits an exponential increase in cell number, and forms its blastocoel. Underlying all of this are remarkable changes
in metabolism; the embryo changes the relative activity of specific pathways as a means of fulfilling the changing requirements as
development proceeds. The metabolic transformation has typically been perceived as a means to an end with regards to develop-
mental processes. However, it is now perceived that in some cases it is the metabolism of the embryo that regulates specific processes
(Gardner, 2015; Gardner and Harvey, 2015), as will be elucidated.
The human oocyte is a truly remarkable cell given that it can reside in the ovary for up to 40 years and still give rise to a viable
pregnancy. In order to achieve this, the oocyte exhibits a unique metabolism, one based primarily on low levels of oxidation of
pyruvate, with little to no ability to utilize glucose. Oxygen consumption by the embryo remains low until after compaction (Kur-
osawa et al., 2016). The quiescent nature of the oocyte’s existence means that its energy demands are extremely low. When a cell
does not utilize energy, any ATP synthesized is not immediately used, and hence the cell is characterize by a high ATP:ADP ratio,
and this is true of the oocyte (Leese et al., 1984). A direct consequence of this high ratio is the allosteric inhibition of a rate-limiting
enzyme in glycolysis, phosphofructokinase (PFK), hence explaining why the oocyte cannot metabolize glucose. The fertilized oocyte
shares the same metabolism as the unfertilized egg. Indeed, it is not until the 4- to 8-cell stage that the embryo can utilize glucose as
an energy source. As the embryo commences its divisions, and cell number increases, there is a concomitant increase in the demand
for energy, which is required not solely for cell proliferation, but also for the formation and maintenance of the blastocoel (the latter
representing a constant energetic demand). Consequently, the absolute levels of ATP decline as it is rapidly utilized, resulting in
a reduction in the ATP:ADP ratio, and a concomitant increase in AMP, both of which have a positive allosteric effect on PFK,
and thereby facilitating a high flux of glucose through glycolysis. Oxygen consumption subsequently rises significantly upon forma-
tion of the blastocyst. However, rather than oxidizing all of the glucose consumed, the blastocyst exhibits an idiosyncratic trait
referred to as aerobic glycolysis, where lactate is produced from glucose even when there is sufficient oxygen for its complete oxida-
tion. This type of glucose metabolism was first described in cancers by Otto Warburg in 1923, and is hence referred to as the War-
burg Effect. Warburg proposed that the high levels of lactate produced by tumors were a result of impaired mitochondrial function,
however this does not appear to be the case. Certainly the mitochondria of the blastocyst have the capacity for oxidative metabo-
lism. So what is the significance of this rather intriguing situation?
Although energetically unfavorable, aerobic glycolysis ensures that the biosynthetic arm of the pentose phosphate pathway
(PPP) has maximum substrate availability at all times. As with all proliferating cells, there is a demand for biosynthetic precursor
provision for membrane and nucleic acid synthesis. Glucose has a significant role in cellular biosynthesis, its carbon being required
for the synthesis of triacylglycerols, phospholipids and as a precursor for complex sugars of mucopolysaccharides and glycoproteins.
Hence, glucose is required by the morula and blastocysts stages not only for energy but to provide the intermediates for biosynthesis
and the ribose moieties for RNA and DNA synthesis (Gardner, 1998a,b; Gardner and Wale, 2013). The complexities and dynamics
of human embryo metabolism are highlighted in Fig. 3. Recently, the significance of aerobic glycolysis for the blastocyst implan-
tation has been re-visited (Gardner, 2015). It is proposed that the human blastocyst has the capacity to create a microenvironment,
characterized by high lactate and reduced pH, both of which facilitate the breakdown of the endometrial extracellular matrix, are
involved in angiogenesis and actively help to modulate the local immune response to the blastocyst. It seems that embryonic
metabolism is far more than energy production.

Human Embryo Culture

Given the dramatic differences in the physiology and metabolism of the embryo before and after compaction, optimizing the
culture of the human preimplantation embryo has been challenging. However, through analysis of human female oviduct and
uterine fluid, sequential media were developed, capable of supporting the human embryo throughout the preimplantation period
(Gardner and Lane, 1997), leading to increased implantation rates (Gardner et al., 1998). Today, blastocyst transfer is considered
the standard of care in human IVF. Of significance, oxygen is a powerful regulator of the preimplantation embryo, and hence when-
ever possible, human embryos should not be exposed to atmospheric oxygen (
20%), but rather cultured in the presence of more
physiological levels, i.e.,
5%.

Analysis of Development and Viability: Morphology

What are the key morphological parameters during the cleavage stages that are associated with subsequent embryo viability and
implantation potential post transfer? The percentage and localization of fragmentation, evenness of the blastomeres, multinuclea-
tion and rate of cleavage are features typically assessed to evaluate the quality of a cleavage stage embryo on day 2 and day 3 (Gard-
ner and Balaban, 2016). The common characteristics of embryos with high viability include 4 or 5 blastomeres on day 2, and at least
7 blastomeres on day 3 after fertilization, with < 20% fragmentation and no signs of multinucleation. Rienzi et al. (2005) suggested
a cumulative classification scheme based on the most essential morphological features that should be examined to evaluate
a cleavage stage embryo. Morphological parameters selected were cleavage rate, blastomere symmetry, cytoplasmic appearance,
extent of fragmentation and blastomere nucleus status. Such a grading scheme effectively predicted blastocyst formation and

Encyclopedia of Reproduction, Second Edition, 2018, 176–185

 Author's personal copy
180 Embryology j Human Embryo Development and Assessment of Viability

(A)
Glucose Cleavage stages

GLUTs Glycolysis

Glucose NADPH Biosynthesis

ATP NADP
ADP
Glucose 6-phosphate PPP Glycine
GSH

Fructose 6-phosphate pHi

ATP
PFK
-ve
ADP ATP: ADP
Fructose 1, 6-bisphosphate Aspartate
NAD
Aspartate
2 ATP NADH
PEP
Pyruvate PK
NADH Oxaloacetate
Pyruvate NAD
NADH
NAD

Lactate LDH Pyruvate Malate

OAA Glutamine Glutamine
NAD
PDC NADH Aspartate
Acetyl CoA
Lactate
TCA
cycle

NADH +H

34ATP
CO2

Fig. 3 (A) Metabolism of the pronucleate oocyte and cleavage stage embryo. Prior to compaction the embryo has a metabolism based around low
levels of oxidation of pyruvate, lactate and specific amino acids. The ovulated oocyte is surrounded by, and is directly connected to, cumulus cells
which actively produce pyruvate and lactate from glucose. This creates a high concentration of pyruvate and lactate and a low concentration of
glucose around the fertilized oocyte. Upon dispersal of the cumulus cells, the human zygote and cleavage stage embryo still find themselves in a rela-
tively high concentration of pyruvate (0.32 mM) and lactate (10.5 mM), and low levels of glucose (0.5 mM) within the ampulla. The early embryo is
characterized by a high ATP:ADP level, which in turn allosterically inhibits PFK, thereby limiting the flux of glucose through the glycolytic pathway
prior to compaction. Significantly, the relative abundance of nutrients affects the metabolism of the embryo. For example, the ratio of pyruvate:
lactate in the surrounding environment directly affects the ratio of NADH:NADþ in the embryo, which in turn controls the redox state of the cells and
hence the flux of nutrients through specific energy generating pathway. Amino acids fill several niches in embryo physiology, such as the use of
glycine as buffer of intracellular pH (pHi). Several amino acids are also utilized as energy sources by the early embryo such as glutamine and aspar-
tate. Aspartate can be utilized through the malate-aspartate shuttle, the significance of which during embryo development we are only beginning to
understand. The thickness of the lines represents the relative flux of metabolites through that pathway. GLUTs, glucose transporters; GSH, reduced
glutathione; LDH, lactate dehydrogenase; OAA, oxaloacetate; PDC, pyruvate dehydrogenase complex; pHi, intracellular pH; PFK, phosphofructokinase;
PK, pyruvate kinase; PPP, pentose phosphate pathway. (B) Metabolism of the blastocyst. After compaction the embryo exhibits greatly increased
oxygen consumption and an increased capacity to use glucose as an energy source. The increase in oxygen consumption plausibly reflects the
considerable energy required for the formation and maintenance of the blastocoel, while the increase in glucose utilization reflects an increased
demand for biosynthetic precursors. Consequently, there is a reduction in the ATP:ADP ratio, and a concomitant increase in AMP, which will have
a positive allosteric effect on PFK, thereby facilitating a higher flux of glucose through glycolysis. Rather than oxidize the glucose consumed, the
blastocyst exhibits high levels of aerobic glycolysis. Although this may appear energetically unfavorable, it does ensure that the biosynthetic arm of
the pentose phosphate pathway (PPP) has maximum substrate availability at all times. Activity of the PPP will ensure reducing equivalents are avail-
able for biosynthesis and ensure production of glutathione (reduced), a key intracellular antioxidant. In order for high levels of glycolysis to proceed
the blastomeres need to regenerate cytosolic NADþ. This can be achieved through the generation of lactate from pyruvate. A second means of gener-
ating cytosolic NADþ is through the activity of the malate-aspartate shuttle. Although it is evident that blastocysts do use aerobic glycolysis, it is
proposed here that the significant increase in oxygen utilization at this stage of development could be largely attributed to the activity of the malate-
aspartate shuttle and the resulting demand for oxygen to convert intramitochondiral NADH to ATP. Indeed many tumors that exhibit aerobic glycol-
ysis also have high levels of malate-aspartate shuttle activity. Furthermore, inhibition of this shuttle has dire consequences for subsequent fetal
development. The thickness of the lines represents the relative flux of metabolites through that pathway. ACL, acetyl-citrate lyase; GLUTs, glucose
transporters; GSH, reduced glutathione; LDH, lactate dehydrogenase; OAA, oxaloacetate; PDC, pyruvate dehydrogenase complex; pHi, intracellular pH;
PFK, phosphofructokinase; PK, pyruvate kinase; PPP, pentose phosphate pathway. Adapted from Gardner, D.K. and Wale P.L. Analysis of metabolism
to select viable human embryos for transfer. Fertility and Sterility 99, 2013, 1062–1072 with permission.

Encyclopedia of Reproduction, Second Edition, 2018, 176–185

 Author's personal copy
Embryology j Human Embryo Development and Assessment of Viability 181

(B)
Glucose Blastocyst

GLUTs Glycolysis
Glucose NADPH Biosynthesis
ATP NADP +
ADP
Glucose 6-phosphate PPP
GSH
Fructose 6-phosphate
ATP +ve
ADP
PFK
ATP: ADP
Fructose 1, 6-bisphosphate
NAD
Aspartate
Aspartate
2 ATP NADH

PEP
PKM2
NADH Oxaloacetate
Pyruvate
NADH NAD
NAD
Glutamine
Lactate LDH
Glutamine
Lactate Pyruvate
Malate
NAD OAA
PDC NADH Aspartate
Acetyl CoA
TCA Glutamate
cycle

NADH +H
CO2
34ATP
Citrate Citrate
ACL
ATP
ADP
Acetyl CoA

Lipid synthesis

Fig. 3 (continued).

implantation. Day 1 pronuclear (PN) morphology and nucleolar precursor body (NPB) ratio, day 2 cell number, blastomere
symmetry and nucleation and the ability to cleave from day 2 to day 3 appear to be the six most significant factors associated
with subsequent fetal development.
Several studies have considered the relative weight of such cleavage stage morphological parameters and revealed that cell
number, equal blastomere size and the number of mono-nucleated blastomeres on day 2 were the most predictive markers of blas-
tocyst development and implantation potential. Rienzi et al., reported that cell number is the most critical factor determining the
viability fate of the embryo, not only on day 2 but also on day 3, inferring that the optimal development can be defined as 4 or 5 cell
on day 2 at 44–46 h post insemination, and more than 6 cells (7–9 cell) on day 3 at 66–68 h postinsemination (Rienzi et al., 2005).
In agreement with such reports, the latest Cochrane review on cleavage stage versus blastocyst stage embryo transfer suggested that
the most favorable group for blastocyst transfers were the good prognosis patient group that had higher number of 8 cell embryos
on day 3 (Glujovsky et al., 2012). Evidence based data clearly demonstrates that several morphological features of the cleavage
stages are associated with implantation potential and/or successful blastocyst formation.
Analysis of blastocyst morphology initially focused on the rate of expansion, and the determination of cell allocation to the two
lineages of the inner cell mass (ICM) and trophectoderm (TE). An advantage of assessing the blastocyst is that one is assessing the
embryo proper (cleavage stage morphology reflects primarily oocyte competence). Determining the rate of expansion alone has
physiological value, as the formation of the blastocoel is energetically demanding, and hence the embryo must possess a degree
of metabolic competency to create the ionic gradient required to allow fluid to accumulate within the morula and coalesce to
form a blastocoel. Analysis of the degree of expansion can be difficult after a blastocyst collapses. Interestingly, the thickness of
the zona reflects each blastocyst’s “expansion history,” as an expanding blastocyst will induce changes to the shape of the
surrounding zona, which does not revert back to its original form when the TE collapses.

Encyclopedia of Reproduction, Second Edition, 2018, 176–185

 Author's personal copy
182 Embryology j Human Embryo Development and Assessment of Viability

Gardner and Schoolcraft (1999) therefore developed a comprehensive alphanumeric system, designed to incorporate the degree
of blastocoel expansion, as well as the quality and cell number of the two cell lineages. Herein, blastocyst expansion rate is char-
acterized by numbers from the early blastocyst to the hatched stage. Analysis of both the ICM and TE quality can then be performed
for expanded blastocysts in which both cell types are clearly evident. ICM and TE quality were characterized by three grades, A to C.
ICM grades: A, a tightly packed ICM with many cells; B, loosely grouped ICM with several cells, or C, very few cells and disorganized.
TE grades: A, denoting TE with many cells forming a cohesive epithelium; B, few cells forming a loose epithelium; and C, very few
large cells. (see Fig. 2 for the morphological characteristics of a 4AA blastocyst). Using this scoring system a strong correlation
between the clinical outcome (implantation rates, pregnancy and twinning rates) and the morphological grade of blastocysts
was established (Gardner et al., 2000). Subsequently, Ahlstrom and colleagues determined that although blastocyst expansion
and ICM grade were significant predictors in both univariate and multivariate analyses, they were not predictors of live birth
when analyzed by stepwise logistic regression, rather the TE was the most important determinant of successful transfer outcome
(Ahlstrom et al., 2011). Such observations have been further supported by Ebner et al. (2016).
The physiology underlying the significance of the TE grade may reside in the significance of the TE during the initiation and
progression of implantation, and that a healthy TE ensures acceptable implantation. A larger number of TE cells will be able to
ensure greater signaling and interaction with the endometrium. Interestingly, although the TE produces human chorionic gonad-
otropin (hCG) as one of the earliest signals to the mother, it has recently been proposed that lactate production by the blastocyst,
which will be derived primarily from the TE, has several key functions in regulating the implantation process (Gardner, 2015). These
physiological roles endorse importance of a good TE grade in predicting implantation potential.
In spite of the importance of the TE grade, it remains important to include the ICM grade in order to determine true blastocyst
quality, as it transpires that all three parameters of the blastocyst (degree of expansion, ICM and TE quality) are significantly asso-
ciated with pregnancy and live birth rates (Van den Abbeel et al., 2013). Transfer of blastocysts with an “A” (top quality) grade ICM
further reduces the incidence of pregnancy loss. Further, studies have also determined that ICM grade is positively associated with
birth weight (Licciardi et al., 2015). Considering the quality of both the ICM and TE makes physiological sense as they do not exist
in isolation, but rather coexist as a functional unit, whereby the TE which creates a unique environment for the ICM by the synthesis
of blastocoel fluid, and the ICM itself that regulates the proliferation and activity of the TE (Ansell and Snow, 1975).

Analysis of Development and Viability: Screening for Aneuploidy

Through the development of blastocyst culture, it is now feasible to analyze biopsies taken from the TE of human embryos in order
to determine the genetic constitution of the embryo (Schoolcraft et al., 2010). Technologies for the analysis of chromosome
complement from a single or small group of cells has advanced significantly over recent years, and are now used worldwide to iden-
tify aneuploid embryos, thereby decreasing the time to pregnancy for patients of ages 38 and older (Gardner et al., 2015). However,
even though this approach is able to identify aneuploid embryos to a high level of accuracy, not all euploid embryos are viable, and
hence there remains considerable research into identifying biomarkers of viability (Sanchez et al., 2017).

Analysis of Development and Viability: Metabolism

Given the dynamics and complexity of embryonic metabolism, it is not surprising that should an embryo at any stage of develop-
ment lose control of its energy production, i.e., if the flux of a specific nutrient through a metabolic pathway is altered to a significant
degree, even for a brief period, then this is associated with significantly impaired development in culture and reduction of viability
post transfer (Lane and Gardner, 2005). Proof of concept data was first reported in 1980 by Renard et al., who observed a positive
relationship between glucose uptake by day 10 cow blastocysts and subsequent pregnancy after transfer (Renard et al., 1980). This
relationship between blastocyst glucose uptake and the establishment of viable pregnancies was later confirmed in the mouse
(Gardner and Leese, 1987). Early attempts to quantitate human embryo metabolism focused predominantly on the cleavage stages,
given that it was not feasible to culture the human embryo to the blastocyst stage until the late 1990s. Hardy et al. (1989) reported
that pyruvate uptake of embryos which arrested was lower than those which developed normally. With the development of more
physiological media in the 1990s (Barnes et al., 1995; Gardner, 1994), it became possible to analyze the metabolism of the later
stage embryo prior to transfer. Early studies revealed that blastocysts with the same alpha-numeric score, e.g., 3AA, from the same
patient exhibited a wide range of glucose uptakes, indicating that morphological grade was not directly related to physiological
status of the embryo (Gardner et al., 2001). Given the significance of glucose uptake by blastocysts in animal models, the uptake
of glucose by individual human embryos on day 4 and 5, followed by single embryo transfer, allowed for the relationship between
nutrient uptake and subsequent pregnancy to be established. Consistent with the animal data, glucose uptake by those embryos
which were considered viable by the delivery of a healthy baby, exhibited a significantly higher glucose uptake on both day 4
and day 5 of development, thereby establishing in the human that embryo viability post compaction is positively associated
with a high glucose uptake (Gardner et al., 2011) (Fig. 4). Furthermore, it was observed that female embryos consume significantly
more glucose than males on day 4 of development. This phenomenon was previously reported in the mouse and attributed to the
window in development in which both X-chromosomes are active in female embryos (Gardner et al., 2010; Gutierrez-Adan et al.,
2006).

Encyclopedia of Reproduction, Second Edition, 2018, 176–185

 Author's personal copy
Embryology j Human Embryo Development and Assessment of Viability 183

(A) 160 (B) 160

140 140 ∗

Glucose uptake (pmol/embryo/h)

Glucose uptake (pmol/embryo/h)

120 120

100 100

80 ∗∗ 80

60 60

40 40

20 20

0 0
Pregnant Non-pregnant Male Female
Fig. 4 Glucose uptake on day 4 of embryonic development and pregnancy outcome (positive fetal heart beat). Notches represent the interquartile range
(50% of the data), whiskers represent the 5% and 95% quartiles. The line across the box is the median glucose consumption. Significantly different
from pregnant; ** P < 0.01. (B) Glucose uptake by male and female embryos on day 4 of development. Significantly different from male embryos; *
P < 0.05. From Gardner, D.K., Wale, P.L., Collins, R. and Lane M. (2011). Glucose consumption of single post-compaction human embryos is
predictive of embryo sex and live birth outcome. Human Reproduction 26, 1981–1986 with permission.

As well as analysis of carbohydrates, Houghton et al. (2002) measured amino acid turnover by individual human embryos and
observed different patterns of amino acid utilization between embryos that went on to form a blastocyst and those embryos that
failed to develop. Those embryos that did develop, consumed more leucine from the culture medium, and the profiles of alanine,
arginine, glutamine, methionine and asparagine flux were related to blastocyst formation (but not necessarily blastocyst quality). In
a follow up study, Brison et al. observed changes in the levels of amino acids in the spent medium of human zygotes cultured for
24 h to the 2-cell stage. Asparagine, glycine and leucine were all significantly associated with clinical pregnancy and live birth (Bri-
son et al., 2004). Subsequently it was determined that there also appear to be gender differences with regards to amino acid utili-
zation (Picton et al., 2010; Sturmey et al., 2010). Similar to the data on carbohydrate use, these data await validation in a larger
patient population. Further, it is possible to use both data from time-lapse incubation, together with metabolic analysis of the
embryo, to gain further insight into development and to facilitate selection models based on multiple parameters (Lee et al., 2015).

Conclusions

The preimplantation embryo resides within a changing environment in vivo, and undergoes dramatic morphological and physio-
logical changes within the first 5 days of life, transforming from a single totipotent cell, to a blastocyst comprising of two distinct cell
lineages. Underlying these changes are the transition from maternal to embryonic genome control, together with a shift from
a metabolism based on low levels of oxidation of pyruvate, to one based on high levels of glucose uptake, which is subsequently
metabolized by aerobic glycolysis (the Warburg effect). The commonality of glucose metabolism shared between blastocysts and
cancers reflects their proliferation, and their ability to invade the endometrial tissue and establish a blood supply, while avoiding
the maternal immune system (Gardner, 2015). Using physiological data, it has been possible to construct better culture media to
support the development and viability of human embryos conceived through IVF, thereby increasing the overall success of this
procedure. The ability to culture the human embryo to the blastocyst stage has not only led to higher success rates but facilitated
the introduction of trophectoderm biopsy for preimplantation genetic screening. Analysis of embryonic kinetics and metabolism
holds promise for identification of a viability marker to select embryos for transfer, and together with genetic screening, can
help to reduce time to pregnancy for infertile couples.

References

Ahlstrom, A., Westin, C., Reismer, E., Wikland, M., & Hardarson, T. (2011). Trophectoderm morphology: An important parameter for predicting live birth after single blastocyst
transfer. Human Reproduction, 26, 3289–3296.
Ansell, J. D., & Snow, M. H. (1975). The development of trophoblast in vitro from blastocysts containing varying amounts of inner cell mass. Journal of Embryology and Experimental
Morphology, 33, 177–185.

Encyclopedia of Reproduction, Second Edition, 2018, 176–185

 Author's personal copy
184 Embryology j Human Embryo Development and Assessment of Viability

Barnes, F. L., Crombie, A., Gardner, D. K., Kausche, A., Lacham-Kaplan, O., Suikkari, A. M., Tiglias, J., Wood, C., & Trounson, A. O. (1995). Blastocyst development and birth after
in-vitro maturation of human primary oocytes, intracytoplasmic sperm injection and assisted hatching. Human Reproduction, 10, 3243–3247.
Brison, D. R., Houghton, F. D., Falconer, D., Roberts, S. A., Hawkhead, J., Humpherson, P. G., Lieberman, B. A., & Leese, H. J. (2004). Identification of viable embryos in IVF by
non-invasive measurement of amino acid turnover. Human Reproduction, 19, 2319–2324.
Ebner, T., Tritscher, K., Mayer, R. B., Oppelt, P., Duba, H. C., Maurer, M., Schappacher-Tilp, G., Petek, E., & Shebl, O. (2016). Quantitative and qualitative trophectoderm grading
allows for prediction of live birth and gender. Journal of Assisted Reproduction and Genetics, 33, 49–57.
Fleming, T. P., & Johnson, M. H. (1988). From egg to epithelium. Annual Review of Cell Biology, 4, 459–485.
Gardner, D. K. (1994). Mammalian embryo culture in the absence of serum or somatic cell support. Cell Biology International, 18, 1163–1179.
Gardner, D. K. (1998a). Changes in requirements and utilization of nutrients during mammalian preimplantation embryo development and their significance in embryo culture.
Theriogenology, 49, 83–102.
Gardner, D. K. (1998b). Embryo development and culture techniques. In J. Clark (Ed.), Animal breeding: Technology for the 21st century (pp. 13–46). London: Harwood Academic.
Gardner, D. K. (2015). Lactate production by the mammalian blastocyst: Manipulating the microenvironment for uterine implantation and invasion? BioEssays, 37, 364–371.
Gardner, D. K., & Balaban, B. (2016). Assessment of human embryo development using morphological criteria in an era of time-lapse, algorithms and OMICS: Is looking good still
important? Molecular Human Reproduction, 22, 704–718.
Gardner, D. K., & Harvey, A. J. (2015). Blastocyst metabolism. Reproduction, Fertility, and Development.
Gardner, D. K., & Lane, M. (1997). Culture and selection of viable blastocysts: A feasible proposition for human IVF? Human Reproduction Update, 3, 367–382.
Gardner, D. K., & Lane, M. (2005). Ex vivo early embryo development and effects on gene expression and imprinting. Reproduction, Fertility, and Development, 17, 361.
Gardner, D. K., & Lane, M. (2017). Embryo culture systems. In D. K. Gardner, & C. Simon (Eds.), Handbook of In Vitro Fertilization (4th edn., pp. 205–244). Boca Raton: CRC Press.
Gardner, D. K., & Leese, H. J. (1987). Assessment of embryo viability prior to transfer by the noninvasive measurement of glucose uptake. The Journal of Experimental Zoology,
242, 103–105.
Gardner, D. K., & Schoolcraft, W. B. (1999). In vitro culture of human blastocyst. In R. Jansen, & D. Mortimer (Eds.), Towards reproductive certainty: fertility and genetics beyond
1999 (pp. 378–388). Carnforth, UK: Parthenon Publishing.
Gardner, D. K., & Wale, P. L. (2013). Analysis of metabolism to select viable human embryos for transfer. Fertility and Sterility, 99, 1062–1072.
Gardner, D. K., Lane, M., Calderon, I., & Leeton, J. (1996). Environment of the preimplantation human embryo in vivo: Metabolite analysis of oviduct and uterine fluids and
metabolism of cumulus cells. Fertility and Sterility, 65, 349–353.
Gardner, D. K., Schoolcraft, W. B., Wagley, L., Schlenker, T., Stevens, J., & Hesla, J. A. (1998). Prospective randomized trial of blastocyst culture and transfer in in-vitro fertilization.
Human Reproduction, 13, 3434–3440.
Gardner, D. K., Lane, M., Stevens, J., Schlenker, T., & Schoolcraft, W. B. (2000). Blastocyst score affects implantation and pregnancy outcome: Towards a single blastocyst
transfer. Fertility and Sterility, 73, 1155–1158.
Gardner, D. K., Lane, M., Stevens, J., & Schoolcraft, W. B. (2001). Noninvasive assessment of human embryo nutrient consumption as a measure of developmental potential.
Fertility and Sterility, 76, 1175–1180.
Gardner, D. K., Larman, M. G., & Thouas, G. A. (2010). Sex-related physiology of the preimplantation embryo. Molecular Human Reproduction, 16, 539–547.
Gardner, D. K., Wale, P. L., Collins, R., & Lane, M. (2011). Glucose consumption of single post-compaction human embryos is predictive of embryo sex and live birth outcome.
Human Reproduction, 26, 1981–1986.
Gardner, D. K., Meseguer, M., Rubio, C., & Treff, N. R. (2015). Diagnosis of human preimplantation embryo viability. Human Reproduction Update, 21, 727–747.
Glujovsky, D., Blake, D., Farquhar, C., & Bardach, A. (2012). Cleavage stage versus blastocyst stage embryo transfer in assisted reproductive technology. Cochrane Database of
Systematic Reviews, 7, CD002118.
Gutierrez-Adan, A., Perez-Crespo, M., Fernandez-Gonzalez, R., Ramirez, M., Moreira, P., Pintado, B., Lonergan, P., & Rizos, D. (2006). Developmental consequences of sexual
dimorphism during pre-implantation embryonic development. Reproduction in Domestic Animals, 41(Suppl 2), 54–62.
Hamatani, T., Ko, M., Yamada, M., Kuji, N., Mizusawa, Y., Shoji, M., Hada, T., Asada, H., Maruyama, T., & Yoshimura, Y. (2006). Global gene expression profiling of preimplantation
embryos. Human Cell, 19, 98–117.
Hannan, N. J., Paiva, P., Meehan, K. L., Rombauts, L. J., Gardner, D. K., & Salamonsen, L. A. (2011). Analysis of fertility-related soluble mediators in human uterine fluid identifies
VEGF as a key regulator of embryo implantation. Endocrinology, 152, 4948–4956.
Hardy, K., Hooper, M. A., Handyside, A. H., Rutherford, A. J., Winston, R. M., & Leese, H. J. (1989). Non-invasive measurement of glucose and pyruvate uptake by individual human
oocytes and preimplantation embryos. Human Reproduction, 4, 188–191.
Harris, S. E., Gopichandran, N., Picton, H. M., Leese, H. J., & Orsi, N. M. (2005). Nutrient concentrations in murine follicular fluid and the female reproductive tract. Theriogenology,
64, 992–1006.
Houghton, F. D., Hawkhead, J. A., Humpherson, P. G., Hogg, J. E., Balen, A. H., Rutherford, A. J., & Leese, H. J. (2002). Non-invasive amino acid turnover predicts human embryo
developmental capacity. Human Reproduction, 17, 999–1005.
Jukam, D., Shariati, S. A. M., & Skotheim, J. M. (2017). Zygotic genome activation in vertebrates. Developmental Cell, 42, 316–332.
Kidder, G. M., & Watson, A. J. (2005). Roles of Na,K-ATPase in early development and trophectoderm differentiation. Seminars in Nephrology, 25, 352–355.
Kurosawa, H., Utsunomiya, H., Shiga, N., Takahashi, A., Ihara, M., Ishibashi, M., Nishimoto, M., Watanabe, Z., Abe, H., Kumagai, J., et al. (2016). Development of a new clinically
applicable device for embryo evaluation which measures embryo oxygen consumption. Human Reproduction, 31, 2321–2330.
Lane, M., & Gardner, D. K. (2005). Understanding cellular disruptions during early embryo development that perturb viability and fetal development. Reproduction, Fertility, and
Development, 17, 371–378.
Lee, Y. S., Thouas, G. A., & Gardner, D. K. (2015). Developmental kinetics of cleavage stage mouse embryos are related to their subsequent carbohydrate and amino acid utilization
at the blastocyst stage. Human Reproduction, 30, 543–552.
Leese, H. J. (1988). The formation and function of oviduct fluid. Journal of Reproduction and Fertility, 82, 843–856.
Leese, H. J., Biggers, J. D., Mroz, E. A., & Lechene, C. (1984). Nucleotides in a single mammalian ovum or preimplantation embryo. Analytical Biochemistry, 140, 443–448.
Licciardi, F., McCaffrey, C., Oh, C., Schmidt-Sarosi, C., & McCulloh, D. H. (2015). Birth weight is associated with inner cell mass grade of blastocysts. Fertility and Sterility, 103,
382–387. e382.
Meseguer, M., Herrero, J., Tejera, A., Hilligsoe, K. M., Ramsing, N. B., & Remohi, J. (2011). The use of morphokinetics as a predictor of embryo implantation. Human Reproduction,
26, 2658–2671.
Miller, J. G., & Schultz, G. A. (1987). Amino acid content of preimplantation rabbit embryos and fluids of the reproductive tract. Biology of Reproduction, 36, 125–129.
Ng, K. Y. B., Mingels, R., Morgan, H., Macklon, N., & Cheong, Y. (2017). In vivo oxygen, temperature and pH dynamics in the female reproductive tract and their importance in
human conception: A systematic review. Human Reproduction Update, 1–20.
Picton, H. M., Elder, K., Houghton, F. D., Hawkhead, J. A., Rutherford, A. J., Hogg, J. E., Leese, H. J., & Harris, S. E. (2010). Association between amino acid turnover and
chromosome aneuploidy during human preimplantation embryo development in vitro. Molecular Human Reproduction, 16, 557–569.
Renard, J. P., Philippon, A., & Menezo, Y. (1980). In-vitro uptake of glucose by bovine blastocysts. Journal of Reproduction and Fertility, 58, 161–164.
Rienzi, L., Ubaldi, F., Iacobelli, M., Romano, S., Minasi, M. G., Ferrero, S., Sapienza, F., Baroni, E., & Greco, E. (2005). Significance of morphological attributes of the early embryo.
Reproductive Biomedicine Online, 10, 669–681.
Sanchez, T., Seidler, E. A., Gardner, D. K., Needleman, D., & Sakkas, D. (2017). Will noninvasive methods surpass invasive for assessing gametes and embryos? Fertility and
Sterility, 108, 730–737.

Encyclopedia of Reproduction, Second Edition, 2018, 176–185

 Author's personal copy
Embryology j Human Embryo Development and Assessment of Viability 185

Schoolcraft, W. B., Fragouli, E., Stevens, J., Munne, S., Katz-Jaffe, M. G., & Wells, D. (2010). Clinical application of comprehensive chromosomal screening at the blastocyst stage.
Fertility and Sterility, 94, 1700–1706.
Sturmey, R. G., Bermejo-Alvarez, P., Gutierrez-Adan, A., Rizos, D., Leese, H. J., & Lonergan, P. (2010). Amino acid metabolism of bovine blastocysts: A biomarker of sex and
viability. Molecular Reproduction and Development, 77, 285–296.
Thouas, G. A., Potter, D. L., & Gardner, D. K. (2013). Microfluidic devices for the analysis of gamete and embryo physiology. In D. K. Gardner, D. Sakkas, E. Seli, & D. Wells (Eds.),
Human gametes and Preimplantation embryos: Assessment and diagnosis (pp. 281–299). New York: Springer.
Van den Abbeel, E., Balaban, B., Ziebe, S., Lundin, K., Cuesta, M. J., Klein, B. M., Helmgaard, L., & Arce, J. C. (2013). Association between blastocyst morphology and outcome
of single-blastocyst transfer. Reproductive Biomedicine Online, 27, 353–361.

Further Reading

Edwards, R. G. (2000). The role of embryonic polarities in preimplantation growth and implantation of mammalian embryos. Human Reproduction, 15(Suppl 6), 1–8.
Gardner, D. K., & Kelley, R. L. (2017). Impact of the IVF laboratory environment on human preimplantation embryo phenotype. Journal of Developmental Origins of Health and
Disease, 1–18.
Gardner, D. K., & Lane, M. (2014). Culture of viable mammalian embryos. In J. Cibelli, R. Lanza, K. Campbell, & M. D. West (Eds.), Principles of cloning (pp. 63–84). San Diego:
Academic Press.
Gardner, D. K., & Simon, C. (Eds.). (2017). Handbook of in vitro Fertilization (4th edn.). New York: CRC Press. ISBN-13: 978-1498729390.
Gardner, D. K., Weissman, A., Howles, C., & Shoham, Z. (2017). Textbook of assisted reproductive technologies: Laboratory and clinical perspectives (5th edn.). London: Informa.
Harvey, A. J., Rathjen, J., & Gardner, D. K. (2016). Metaboloepigenetic regulation of pluripotent stem cells. Stem Cells International, 2016, 1816525.
Leese, H. J. (2012). Metabolism of the preimplantation embryo: 40 years on. Reproduction, 143, 417–427.
Swain, J. E., Lai, D., Takayama, S., & Smith, G. D. (2013). Thinking big by thinking small: Application of microfluidic technology to improve ART. Lab on a Chip, 13, 1213–1224.
Wale, P. L., & Gardner, D. K. (2016). The effects of chemical and physical factors on mammalian embryo culture and their importance for the practice of assisted human
reproduction. Human Reproduction Update, 22, 2–22.

Encyclopedia of Reproduction, Second Edition, 2018, 176–185