Recent Advances in In Vitro Fertilization
Introduction
Chapter 2
In Vitro Fertilization (IVF)
Diego MORENO GARCIA1, Alberto NERA1, Lamia
BRIAND1, Emmanuel TOPIE1, Djemil BENCHARIF1
and Daniel TAINTURIER1
Laboratory of Biotechnology and Pathology of Repro-
1 oocytes [1]. Five years later, the birth of baby rabbits con-
duction, Nantes-Atlantic National College of Veterinary
Medicine, France
*
Corresponding Author: Prof. Daniel TAINTURIER,
Laboratory of Biotechnology and Pathology of Repro-
duction, Nantes-Atlantic National College of Veterinary
Medicine, Food Science and Engineering - Oniris. CS
40706, 44307 NANTES Cedex, France, Email: daniel.
tainturier@oniris-nantes.fr
First Published February 26, 2016
Copyright: © 2016 Daniel TAINTURIER et al.
This article is distributed under the terms of the Creative
Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which
permits unrestricted use, distribution, and reproduction
in any medium, provided you give appropriate credit to
the original author(s) and the source.
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Recent Advances in In Vitro Fertilization
For the past few decades, reproductive biotechnolo-
gies such as artificial insemination (AI), embryo trans-
fer (ET), and embryo production via in vitro fertilisation
(IVF), have undergone incredible advances.
The first IVF of a mammalian oocyte, confirmed cy-
tologically, was performed in a rabbit using spermatozoa
that had capacitated in the uterus and recently ovulated
firmed the biological normality of in vitro fertilisation.
For practical reasons, but also to understand the mecha-
nisms of capacitation, fertilisations were then attempted
using spermatozoa that had capacitated in vitro. The first
IVF under these conditions were achieved in rodents
(hamsters, 1963; mice, 1968), then in man in 1978 [2], and
much later in large domestic mammals: the first IVF calf
was born in 1982 [3], then the first goats in 1985, lambs
and piglets in 1986, and the first foal in 1990 [4]. Some
time later, IVF was achieved using oocytes that had been
matured in vitro in various species, with the first calf born
under these conditions in 1986, the first piglet in 1988,
and the first lamb in 1990 [4].
In reality, IVF is already an integral part of the em-
bryo transfer (ET) process used for the industrial produc-
tion of bovine embryos; this process involves in vitro oo-
cyte maturation, IVF, in vitro embryo development, and
finally the in utero transplantation of the embryo in the
recipient dam. IVF is also a valuable means of learning
more about the mechanisms involved in vivo fertilisation.
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Recent Advances in In Vitro Fertilization
With IVF, one can also study the associated problems: oo-
cyte maturation, spermatozoa capacitation, activation of
the egg, control of the embryo’s genome, mechanisms of
cell division, influence of maternal and paternal genomes,
interaction between the nucleus and cytoplasm, etc. IVF
also offers the possibility of analysing the mechanism of
nuclear differentiation by using nuclear grafts, an essen-
tial part of the process needed to develop embryo cloning
techniques.
In farm animals, IVF presents several important ad-
vantages, such as the production of a large number of em-
bryos in the same time period, the ability to use embryos
from dead cows or those with reproductive problems, the
possibility of producing embryos from a cow during the
first third of gestation as well as the production of em-
bryos in pre-pubertal heifers.
This technique is currently used intensively in cattle,
on pregnant and non-pregnant cows, with or without re-
productive abnormalities. Thus IVF is a low-cost means
of producing embryos on a massive scale from ovaries
obtained at the abattoir. In recent years, this biotechnol-
ogy has been widely used in cattle on a commercial scale;
according to data collected by the IETS [international em-
bryo transfer society), in 2012 a total of 1,143,119 embry-
os produced in vivo and in vitro were transferred in cattle.
Of this total, 699,586 embryos were produced in vivo, and
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Recent Advances in In Vitro Fertilization
443, 533 were fertilised in vitro [5].
In practice, the procedure for producing embryos in
vitro (IVP) involves four distinct phases:
1. Harvesting the oocytes (via OPU, Ovum Pick Up,
ultrasound guided follicular puncture; or via nee-
dle aspiration from ovaries obtained post-mor-
tem).
2. In vitro maturation (IVM)
3. In vitro fertilisation (IVF)
4. In vitro development (IVD)
The last three phases (IVM, IVF, and IVD) are per-
formed in a specific medium in an incubator at 38.5°C
to mimic in utero conditions. Despite numerous studies
performed on the three main phases of in vitro embryo
production (IVM, IVF, IVD), their efficacy is still low,
with only 30 to 40% of oocytes actually developing into
blastocysts [6].
Obtaining Immature Oocytes via
Ovum Pick Up (OPU)
Oocytes can be obtained post-mortem via follicular
aspiration; alternatively, OPU [7], which is directly de-
rived from the method of oocyte collection that is used
routinely in humans, has enabled considerable advances
in the collection of bovine oocytes. This technique enables
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Recent Advances in In Vitro Fertilization
the multiplication of high-performance female descend-
ants using oocytes harvested from animals with a known
genetic potential. The oocytes are harvested via a needle
introduced into the dorsal sac of the vagina under ultra-
sound guidance. The ultrasound probe can be used to
visualise ovarian follicles of 6 to 8 mm in diameter, which
are then punctured. In cows, these probes are usually 5
or 7.5 MHz sectorial probes. The animal is sedated, often
with additional epidural anaesthesia, the probe is inserted
intravaginally and the ovary grasped via the rectum and
held against the head of the probe. The follicles are thus
visible on the screen of the ultrasound allowing a needle
to be guided through the vaginal wall and into the follicle.
An aspiration system into a specific medium (containing
PBS and heparin) allows collection of the follicular flu-
id and the oocyte surrounded by its cumulus oophorus,
composed of follicular cells. This minimally invasive tech-
nique enables a high rate of repeated harvest, up to twice
a week for several weeks, with no negative effects on the
donors [8-9]. As such up to 5 oocytes can be collected at
a time and around 35% of the subsequent blastocysts are
suitable for in vitro transfer [10]. The OPU technique is
also useful for other species such as the bison and horses.
In small ruminants, oocytes are usually recovered via lap-
arotomy or laparoscopy. Currently, the use of high resolu-
tion ultrasound with high frequency transducers (25-70
MHz) enables dynamic examination of the development
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Recent Advances in In Vitro Fertilization
of small antral follicles and to observe the Cumulus Oo-
cyte Complexes (COC) in vivo [11].
In Vitro Oocyte Maturation (IVM)
An oocyte blocked at the germinal vesicle stage re-
quires a maturation phase to enable fertilisation. This in-
volves a certain number of modifications: nuclear matura-
tion culminates in the emission of the first polar globule
and the formation of the second maturation phase (be-
tween 18-21h in cows; 24 in the ewe, and 27 hours in
goats). Membrane maturation, which enables the oocyte
to specifically recognise the spermatozoa of its species,
and cytoplasmic maturation, which prevents polyspermia
via the cortical reaction, and which ensures the synthesis
of the proteins needed for fertilisation [12].
Under natural conditions, the follicle undergoes sig-
nificant modification during maturation. Expansion of
the cumulus is reliant on the presence of FSH in the folli-
cular fluid, whose action is modulated by LH. FSH stimu-
lates the synthesis of hyaluronic acid by the cells of the
cumulus, which results in the formation of an abundant
viscoelastic matrix, which in turn expands the intercellu-
lar spaces and thus the cumulus as a whole [13]. In addi-
tion, once the inhibitory action of OMI (Oocyte Meiotic
Factor) has been lifted by the LH peak, the permeable
junctions that unite the oocyte to the cells of cumulus rup-
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Recent Advances in In Vitro Fertilization
ture. In vitro, this inhibition is not an issue as there are no
OMI-producing cells [14].
During in vitro oocyte maturation, the nuclear phase
normally occurs spontaneously, whereas cytoplasmic as-
pects are dependent on the maturation medium [14].
The most widely used medium for maturing oocytes is
TCM-199; this medium is buffered with bicarbonate and
contains mineral salts, sources of carbon and energy (glu-
cose), and amino acids such as cystine and cysteine, im-
portant in the metabolism of glutathione and glutamine,
which with glucose and pyruvate provide the principal
energy sources of the oocyte [15-16]. This medium has
similar characteristics to the follicular fluid; its osmotic
pressure varies between 280 and 310 mOsm/kg and the
pH between 7.0 and 7.6. Normally, the osmotic pressure
of follicular fluid lies between 280 and 320 mOsm/kg and
the pH between 7.3 and 7.4 [17].
High molecular weight molecules are often added
to this medium, such as foetal calf serum [18], or serum
from a cow that is in oestrus [19], whose surfactant effect
prevents the adhesion of the oocyte cumulus complexes.
However, the use of these animal-derived additives pos-
es a sanitary risk and hinders the reproducibility of ex-
periments. Good results have been achieved by replac-
ing these animal proteins with synthetic polymers with
the same surfactant properties, such as polyvinyl alcohol
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(PVA) [13].
In addition, the gonadotrophic hormones, notably
FSH, have been used in oocyte maturation media in cattle
by promoting the expansion of the cells of the cumulus in
vitro [20-21]. FSH potentiates the action of growth fac-
tors such as EGF or PDGF, and stimulates the expression
of the LH receptor by the cells of the cumulus; LH has a
direct action on the metabolism of the oocyte, increasing
glycolyis and oxidative phosphorylation. GH accelerates
nuclear maturation, stimulates the expansion of the cu-
mulus, and improves the rate of development to the blas-
tocyst stage in the absence of serum and gonadotrophic
hormones [22]. In addition to the effect on nuclear matu-
ration, GH also has an effect on cytoplasmic maturation
expressed on the rate of development; This effect results
from an acceleration of the migration of cortical granules
[13].
Other factors can also intervene during IVM. Epider-
mal growth factor (EGF) whose beneficial effect on nu-
clear and cytoplasmic maturation has been demonstrated
in cattle. This effect was reported on denuded oocytes,
suggesting that EGF may act, at least in part, directly on
the oocyte [23]. Factors from the insulin family, largely
involved in the control of folliculogenesis [24]; notably
IGF-1, whose beneficial effect during IVM has been dem-
onstrated in horses [25], cats [26], and cattle, where the
maximal rates of expansion of the cumulus and nuclear
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Recent Advances in In Vitro Fertilization
maturation were obtained in the presence of IGF-1 and
EGF [27]. Another molecule is activin, present in the folli-
cular fluid, and whose receptor is expressed by the cumu-
lus and by the oocyte; their beneficial effect during IVM
has been demonstrated on bovine oocytes [28].
The physical conditions used for in vitro maturation
are generally those of the core temperature of the species
(37°C in primates and in small rodents, 39°C in domes-
tic mammals), in an atmosphere enriched with 5% CO2
saturated in humidity, for a duration of 24 hours (mice,
ruminants) to 44 hours (humans, pigs) [13].
Classification of Oocyte Quality for In
Vitro Maturation (IVM)
Immature bovine oocytes can be classified into 4 cate-
gories depending on the degree of compaction of the cells
of the cumulus and the transparency of the cytoplasm [29-
30].
Category 1
The cumulus oocyte complex (COC) is transparent.
The cumulus (cells of the granulosa) is compact and com-
pletely surrounds the oocyte. The cytoplasm has a homo-
geneous appearance.
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Category 2
The COC has the same appearance as in category 1,
but the cytoplasm is more irregular. It has a darker zone
visible in the periphery.
Category 3
The entire COC is dark, the cumulus is less com-
pact, the cytoplasm is more irregular and presents darker
clumps.
Category 4
The cumulus is completely disorganised or even ab-
sent (naked oocytes). De Loos et al., 1989 [29], studied
the behaviour of 4 categories of immature oocytes during
IVM and observed that only category 4 oocytes showed a
reduced capacity for growth. Oocytes from categories 1 to
3 demonstrated a similar capacity for development in an
IVM system.
In Vitro Fertilisation (IVF)
Several studies have tested in vitro fertilisation me-
dia that mimic the composition of the media in the fal-
lopian tubes, achieving cleavage rates of between 80 and
85% in ruminants. Various factors are important during
IVF: firstly, the elimination of the seminal plasma, which
would normally occur during the passage through the
cervix or by resorption in the uterus; in vitro, it should be
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Recent Advances in In Vitro Fertilization
performed by centrifugation using a density gradient, or
via spontaneous ascending migration in a diluent (swim
up). Next is spermatic capacitation, this is achieved in vit-
ro through the presence of specific substances in the me-
dium in which the spermatozoa are suspended [31].
The most widely used medium for the capacitation
of spermatozoa and IVF is FERT-TALP (Fertilization Ty-
rode’s Albumin Lactate Pyruvate medium) [32]. This me-
dium is often supplemented with heparin in cows or in the
serum of ewes that are in oestrus in small ruminants, to
capacitate the spermatozoa in vitro.
Heparin leads the efflux of cholesterol and phospho-
lipids from the plasma membrane of the spermatozoa,
which is an important step in the capacitation process
[33-35]. In association with heparin, other acceptors of
cholesterol, such as BSA, are often used in the capacita-
tion / fertilisation medium. BSA can act by absorbing the
cholesterol from the membrane, leading to changes in the
plasma membrane [36]. Although capacitation induced
by heparin does not require the presence of BSA, the
capacitated spermatozoa need the latter to undergo the
acrosome reaction, the exact role of BSA during the acro-
some reaction is not fully understood [35].
In order for heparin to capacitate the spermatozoa,
there needs to be a minimum amount of bicarbonate in
the capacitation / fertilisation medium [35]. BSA only in-
tervenes on the sub-population of spermatozoa that have
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undergone modifications of the membrane induced by bi-
carbonate [37]. Bicarbonate alters the plasma membrane
inducing the rapid collapse of the transversal asymmetry
of the phospholipids, facilitating the elimination of cho-
lesterol from the membrane during capacitation [38]. Af-
ter 1 to 10 hours, depending on the species, a significant
percentage of spermatozoa have capacitated. The times
required are similar in vivo and in vitro [31].
Successful in vitro fertilisation is dependent on a high
concentration of spermatozoa in contact with the oocyte,
yet in vivo only a few spermatozoa make it to the oviduct.
Although the spermatozoal density is high, in vitro ca-
pacitation is still less effective than in vivo. If capacitation
is incomplete when the gametes come into contact, ferti-
lisation may not occur until 6 to 8 hours later. This leads
to ageing of the oocytes, which prejudices the success of
fertilisation and subsequent embryo development [31].
The conditions used for IVF are the same as those
used for IVM. Although 37°C is the optimal tempera-
ture to fertilise the oocytes of mice and women, it does
not enable fertilisation in domestic animals with a core
body temperature of 38-39°C. In cattle, IVF is performed
in an atmosphere enriched with 5% CO2 for a duration of
around 20 hours [4].
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Recent Advances in In Vitro Fertilization
In Vitro Embryo Development (IVD)
The secretions of the fallopian tubes and uterus play
an essential role during in vivo embryo development. The
liquid in the fallopian tubes has an osmotic pressure of
around 290 mOsm/kg and its pH varies between 7.2 and
7.6. It is rich in potassium and bicarbonate ions and has
lower concentrations of Na+, Ca2+, Mg2+, and Cl-; it is
also rich in energy sources such as glucose, lactate, and
pyruvate, amino acids, vitamins A and E, which protect
against free radicals, lipids, proteins such as albumin,
transferrin, and immunoglobulins G, and growth factors
[17]. Under these conditions, the embryo is therefore ca-
pable of balancing its endogenous metabolism pools by
incorporating certain external components and via the
metabolic “turn over” from its own reserves.
In vitro, the pre-implantation development of the em-
bryo occurs in close relation with the culture medium.
This demands specific balanced conditions in the medium
to allow embryonic metabolism. With the exception of
man and rabbits, there is an in vitro blockade of the devel-
opment of the segmented egg in numerous mammals: at
the 2-cell stage in mice, 4-cell stage in pigs, 8-16-cell stage
in cows. These blockades correspond to poor synchroni-
sation between the fall of maternal mRNA and the start
of mRNA transcription from the embryonic genome. This
stoppage is linked to inadequate culture conditions for the
development of the embryo [39].
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Numerous studies have been conducted into culture
systems to improve the conditions of in vitro embryo de-
velopment. Tervit el al., 1972 [40] proposed SOF (Syn-
thetic Oviductal Fluid), a culture system that enables the
production of viable blastocysts from oocytes fertilised in
ruminants. However, this system does not make it pos-
sible to remove the blockade of development observed in
ruminants at the 8-16 cell stage. This lead to the creation
of co-culture systems. The first experiments with embryo
co-culture with tubular or uterine cells were conducted in
ewes [41]. This system made it possible to eliminate the
blockade in ruminant eggs. The tubular cells act on em-
bryo development by reducing the oxygen concentration,
by eliminating certain toxic components present in the
culture medium such as metal ions and bivalent cations
[41]. They also supply growth factors that are required for
the in vitro development of the embryo [2].
Subsequently, co-culture was performed in the pres-
ence of epithelial cell lines of non-genital origin such as
BRL (Buffalo Rat Liver) or Vero cells (renal cells of green
monkeys) in an atmosphere enriched with 5% CO2. These
cell lines were used successfully for the co-culture of mu-
rine [43], bovine [44-45], and human embryos [46].
Later on, these positive effects obtained in co-culture,
notably the low oxygen concentration, were conceptual-
ised using systems defined under low-oxygen culture con-
ditions to limit oxidation reactions [47]. Defined media
were created from SOF, such as SOFaa supplemented with
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Recent Advances in In Vitro Fertilization
amino acids [48], KSOM [49], or CR1aa [50]. Currently,
the most widely used culture medium is SOF as modified
by Holm et al., 1997 [51]. It is supplemented with sodium
citrate, myo-inositol, and amino acids. In this system the
eggs are cultured at 38.5°C in a humid atmosphere in the
presence of serum or bovine serum albumin (BSA) in mi-
crodrops covered with mineral oil under 5% CO2, 5% O2,
and 90% N2 [12].
Synthetic Media for Embryo Culture
On a practical level, one problems remains, which
has yet to be fully resolved, concerning the use of animal-
based products as a source of protein for in vitro culture
media (foetal calf serum (FCS), serum from cows in oes-
trus, bovine serum albumin, etc.). Despite the beneficial
effect of FCS and BSA on embryo development [52], these
biological products are associated with certain sanitary
disadvantages [53]; they can be a source of viral or other
pathogenic agents and can provoke alterations during in
vitro embryo development.
Despite the rich composition of FCS, with growth fac-
tors, proteins, vitamins, trace elements, hormones, etc. all
of which are essential for cell growth, their use is contro-
versial for several reasons. Various studies have demon-
strated that the presence of FCS in the culture medium
has a noxious effect on the embryo during development.
These embryos have high concentrations of lipids in the
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cells of the embryonic bud and the cells of the trophecto-
derm [54, 55]. This accumulation of lipids can affect the
tolerance of the embryo to cryopreservation [56-58]. In
certain cases, a phenomenon characterised by high birth-
weight calves, prolonged gestation, frequent dystocias,
high rates of abortion, and perinatal mortality were ob-
served [59,60]. From a sanitary point of view, FCS can be
contaminated by viruses such as bovine viral diarrhoea
[61]. This contamination can be limited by the irradiation
of commercial serums, but this procedure is not always
applied. Seasonal and continental variation in the compo-
sition of FCS results in variations from one batch to an-
other provoking differences in the cultures, which leads to
variable results [62].
The beneficial effect of BSA on embryo development
has been proven [63], which is not surprising given that
albumin is an extracellular protein, the most widespread
in the mammalian reproductive system. BSA may have a
nutritional role by supplying amino acids after hydroly-
sis. It can also bind to several low molecular weight com-
pounds such as heavy metal ions, free radicals, citrate,
steroids, etc. It can protect cell components against the ef-
fects of toxins, and regulate the oxydoreductive potential,
pH, and osmolality [57, 64]. Yet, despite these advantages,
there are variations between batches of BSA, and it also
presents a health risk.
Furthermore, it is difficult to elucidate the specific
functions of each growth factor or other stimulants, given
its undefined composition [52].
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Recent Advances in In Vitro Fertilization
For in vitro embryo production, it has been clearly
established that these embryos are morphologically and
physiologically different from those fertilised in vivo. For
example, there may be differences in the structure of the
zona pellucida [65]. The zona pellucida of embryos pro-
duced by IVF can be considered as immature and certain
pathogenic microbes seem to adhere more easily to the
zona pellucida of embryos that are produced in vitro [66-
68]. Using electron microscopy, Vanroose et al., 2000 [65],
concluded that the intact zona pellucida of an embryo
produced in vitro is made in such a way that the bovine vi-
ral diarrhoea virus (BVDV) or bovine herpes virus (BHV-
1) should not be able to cross it and infect the embryonic
cells. However, small viruses such as BVDV can bind to
the outer layers of the zona pellucida and contaminate the
embryo as it hatches.
For all of these reasons, in addition to the hygiene
conditions associated with handling embryos during in
vitro production, it is essential to have an appropriate cul-
ture medium for the development of the embryo after in
vitro fertilisation. Several recent studies have therefore at-
tempted to establish defined culture media to replace all
molecules of animal origin (FCS, BSA) using synthetic
molecules to standardise the embryo production chain
and reduce health risks. Several different molecules have
been tested for use in culture media.
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Molecules Used to Replace Molecules
of Animal Origin in In Vitro Embryo
Culture Media
Polyvinyl alcohol (PVA) has been proposed as a pre-
ferred additive, especially given that this synthetic poly-
mer has a similar surfactant activity to albumin. Despite
this similarity, the majority of studies have reported gen-
erally lower development rates with PVA in comparison
with BSA and with BSA and FCS [52].
Polyvinylpyrrolidone (PVP) is another synthetic
polymer that has been used as a substitute for BSA in em-
bryo culture media. In cattle, blastocyst rates are compa-
rable to those obtained with BSA [70, 71]. This molecule
has been used more recently as a replacement for FCS in
bovine oocyte maturation media, indicating that PVP has
a similar effect to FCS as a function of the maturation of
oocytes and the development of the embryo [72, 73].
Hyaluronic Acid is the most abundant glycosami-
noglycan in the follicular fluid, oviduct, and uterus of
the cow [74], mouse [75], and woman [76]. Its receptor
(CD44) is expressed on the surface of oocytes, cells of the
cumulus, and of the bovine [77] and human [78] embryo,
at different stages of development. It is involved in
several processes, the proliferation and differentiation of
various cell types, dynamic processes carried by the in-
teraction with the extracellular matrix, the regulation of
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Recent Advances in In Vitro Fertilization
protein secretion, gene expression [19], and the fixation
of several molecules such as lipases or growth factors that
affect cell function and morphology [79]. There appears
to be a close relationship between hyaluronic acid and GF.
Klominek et al., 1989 [80], demonstrated that the pres-
ence of PDGF-BB, EGF, bFGF, and TGF-f31, during the
culture of human fibroblasts, produce a marked stimula-
tion of hyaluronic acid synthesis, an effect that is not ob-
served when using these GF and CYK separately. The ef-
fect of hyaluronic acid in culture media was first studied
several years ago; Hamashima 1982 [81], demonstrated
that this molecule promotes the differentiation of the ex-
tra-embryonic tissues of mice embryos. More recently, its
beneficial effect on embryo development has been proven
in pigs where it produced improved blastocyst rates [82].
In cattle, through higher blastocyst rates [83], hyaluronic
acid improves the survival rate of embryos after vitrifica-
tion, resulting in increased gestation rates after embryo
transfer [84]. A reduction in the expression of the genes
coding for apoptosis (Bax - SOX) has also been observed
in embryos frozen with hyaluronic acid [85].
Preparations of recombinant albumin have recently
become available. They are free from all potential biologi-
cal contamination such as viruses and prions. In addition,
their composition is purer than that of traditional albu-
mins, which are purified from blood, and which often con-
tain unknown molecules. This is the case of citrate, which
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was isolated in batches of BSA that showed embryotrophic
activity in the rabbit [86]. In cattle, Lane et al., 2003 [87],
used recombinant albumin in association with hyaluronic
acid in the culture medium, obtaining blastocyst rates that
were comparable to those obtained with BSA.
Tween-80 is a synthetic surfactant with the ability to
reduce the surface tension of the culture medium. This
polymer can replace the surfactant properties of FCS and
BSA in culture media, but does not have their embryo-
trophic properties. Palasz et al., 2000 [88], improved the
rate of bovine blastocysts produced in vitro thanks to the
addition of tween-80.
The effect of ITS was studied by George et al., 2008
[57]. This association of molecules contains an anti-apop-
totic agent (insulin), a protein that detoxifies the medium
by binding metals (transferrin), and an antioxidant (sele-
nium). In addition to SOF, ITS gives a blastocyst rate at 8
days after fertilisation that is comparable to that obtained
with SOF supplemented with FCS. The addition of BSA to
this SOF-ITS medium gives similar hatching rates to SOF-
FCS. This SOF-BSA-ITS combination therefore increases
the cryotolerance of embryos comprised between 160 and
180 µm and reduces the quantity of lipids in the embryo in
comparison with the medium containing FCS.
Plant peptones have been used as a substitute for pro-
teins of animal origin. These peptones have anti-oxidant
properties. George et al., 2009 [89], obtained post-thaw
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Recent Advances in In Vitro Fertilization
development, hatching, and survival rates similar to those
obtained with BSA. However, they reported a significant
reduction in the concentration of glutathione when the
embryos were produced with plant peptones. After sup-
plementation of the medium with glutathione precursors
(cystine and P-mercaptoethanol), the glutathione concen-
tration increased significantly.
Growth factors and cytokines used in
in vitro embryo culture media
The beneficial effect of growth factors and cytokines
for the culture of embryos produced in vitro is linked to
the natural production of these molecules in the repro-
ductive tract during the peri-implantation period of the
embryo.
EGF (Epidermal Growth factor) has been used very
little in culture media. Their efficacy during in vitro oo-
cyte maturation has been widely demonstrated in the rat
[90], man [91], pigs [92], and in cattle [23, 93].
The IGF family (Insulin-like growth factors) has been
widely used in culture media with satisfactory results.
Several studies have demonstrated a positive effect of IGF-
1 in embryo development in the cow [94-97] and in man
[98]. An anti-apoptotic effect of IGF-1 in the culture me-
dium has been observed in murine [99], porcine [100],
bovine [101], and human [98] embryos. An increase in
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gestation rates and parturitions following transfers of bo-
vine embryos produced in vitro has been demonstrated
[102-104]. A reduction in the apoptotic index was also
observed in murine embryos cultured in the presence of
IGF-11 [105].
FGF (Fibroblast growth Factor) promotes in vitro
embryo development in cows [106, 107], the migratory
activity of the cells of the trophoblast in sheep [108], and
increases the production of interferon-tau by the tropho-
blast in cows [109-111].
PDGF-BB (Platelet Derived Growth Factor-SB), stim-
ulates in vitro embryo development in cows [112-113].
TG-F131 (Transforming Growth Factor-131), improves
the embryo development rate and the rates of embryo im-
plantation in mice produced in vitro [114] and promotes
in vitro embryo development in cows [107].
Several studies have also demonstrated the beneficial
effect of certain cytokines during embryo development.
This is the case of GM-CSF (Granulocyte Macrophage
Colony Stimulating Factor), which improves the in vitro
development potential of bovine [115] and porcine em-
bryos [116], increases the number of cells in the inner cell
mass and gestation rates in cows [117], and stimulates the
production of interferon-tau by the trophectoderm in cat-
tle [110] and sheep [118].
LIF (Leukocyte Inhibitory Factor) is another cy-
www.avidscience.com 23
Recent Advances in In Vitro Fertilization
tokine. it does not improve the blastocyst rate, but increas-
es the number of cells in the inner cell mass and the total
number of cells of bovine [119] and ovine [120] embryos
produced in vitro.
Larson et al., 1992 [107] and Neira et al., 2010 [60],
demonstrated a beneficial effect by combining certain
growth factors and cytokines with a synergistic action
during early in vitro embryo development.
More recently, Moreno et al., 2015 [121], demonstrat-
ed the beneficial effect of an association of growth factors
and cytokines (IGF-1, IGF-11, bFGF, GM-CSF, PDGF-BB,
LIF, and TGF-1), supplemented with hyaluronic acid and
recombinant albumin, on in vitro embryo development
in cattle. This combination has a synergistic action, with
higher blastocyst rates 7 days post-fertilisation in compar-
ison with the commonly used culture medium containing
FCS, BSA, and ITS. The number of cells in the embryonic
bud and the post-thaw survival obtained with this syn-
thetic medium made with growth factors and cytokines
were compared to those of the medium containing BSA.
Finally, the viability of embryos produced in this synthet-
ic medium has been demonstrated by the production of
pregnancies and neonates born after the transfer of fresh
and frozen embryos into recipient heifers.
24 www.avidscience.com
Recent Advances in In Vitro Fertilization
References
1. Dauzier L, Thibault C, Wintenberger S. La fecun-
dation in vitro de l’oeuf de lapine. C.R. Acad. Sci.
1954; 38:1063-1064.
2. Martal J. Les biotechnologies de la reproduction
animale. In: Scriban R, editor. Dans Biotechnolo-
gie, 5th edn. Paris: Tec. & Doc. 1999; 627-759.
3. Brackett BG, Bousquet D, Boice MI, Donawick
WG, Evans JF, et al. Normal development follow-
ing in vitro fertilization in the cow. Biol Reprod.
1982; 27:147-158.
4. Crozet N. La fécondation in vivo et in vitro. Dans
La reproduction chez les mammifères et l’homme,
Thibault C, Levasseur MC, INRA. Edit. Ellipses,
Paris:315-338.
5. Perry G. 22nd Annual report of the International
Embryo Transfer Society (IETS). 2013.
6. Camargo LSA, Viana JHM, Sá WF, Ferreira AM,
Ramos AA, et al. Factors influencing in vitro em-
bryo production. Anim. Reprod. 2006; 3: 19-28.
7. Pieterse MC, Kappen KA, Kruip Th. AM, Taverne
MAM. Aspiration of bovine oocytes during trans-
vaginal ultrasound scanning of the ovaries. The-
riogenology. 1988; 30: 751-762.
8. Galli C, Crotti G, Notari C, Turini P, Duchi R, et
www.avidscience.com 25
Recent Advances in In Vitro Fertilization
al. Embryo production by ovum pick up from live
donors. Theriogenology. 2001; 55: 1341-1357.
9. Gibbons JR, Beal WE, Krisher RL, Faber EG, Pear-
son RE, et al. Effects of one versus twice weekly
transvaginal aspiration on bovine oocyte recovery
and embryo development. Theriogenology. 1994;
42: 405-419.
10. De Roover R, Genicot G, Leonard S, Bols P, Dessy
F. Ovum pick up and in vitro embryo produc-
tion in cows superstimulated with an individually
adapted superstimulation protocol. Anim Reprod
Sci. 2005; 86: 13-25.
11. Pfeifer LFM, Adams GP, Pierson RA, Singh J. Ul-
trasound biomicroscopy: a non-invasive approach
for in vivo evaluation of oocytes and small antral
follicles in mammals. Reprod Fert Dev. 2014; 26:
48-54.
12. Chemineau P, Cognié Y, Thimonier J. La maîtrise
de la reproduction des mammifères domestiques.
Dans La reproduction chez les mammifères et
l’homme, Thibault C, Levasseur MC, INRA. Edit.
Ellipses, Paris. 2001; 792-817.
13. Mermillod P. Croissance et maturation de l’ovocyte
in vivo et in vitro. Dans La reproduction chez les
mammifères et l’homme, Thibault C, Levasseur
26 www.avidscience.com
Recent Advances in In Vitro Fertilization
MC, INRA. Edit. Ellipses, Paris. 2001; 348-366.
14. Mermillod P, Marchal R. La maturation de
l’ovocyte de mammifères. médecine/sciences.
1999; 15 : 148-156.
15. Mermillod P, Wils C, Massip A, Dessy F. Collec-
tion of oocytes and production of blastocysts in
vitro from individual slaughtered cows. J Reprod
Fertil. 1992; 96: 717-723.
16. Steeves TE, Gardner DK. Metabolism of glucose,
pyruvate, and glutamine during the maturation
of oocytes derived from pre-pubertal and adult
cows. Mol Reprod Dev. 1999; 54: 92-101.
17. Menezo Y, Guérin P. Biochimie de l’environnement
in vivo et in vitro de l’ovocyte et du jeune embry-
on. Dans La reproduction chez les mammifères et
l’homme, Thibault C, Levasseur MC, INRA. Edit.
Ellipses, Paris. 2001; 410-424.
18. Nedambale TL, Dinnyés A, Groen W, Dobrinsky
JR, Tian XC, et al. Comparison on in vitro ferti-
lized bovine embryos cultured in KSOM or SOF
and cryopreserved by slow freezing or vitrifica-
tion. Theriogenology. 2004; 62: 437-449.
19. Stojkovic M, Kölle S, Peinl S, Stojkovic P,
Zakhartchenko V, et al. Effects of high concen-
trations of hyaluronan in culture medium on
development and survival rates of fresh and fro-
www.avidscience.com 27
Recent Advances in In Vitro Fertilization
zen–thawed bovine embryos produced in vitro.
Reproduction. 2002; 124: 141–153.
20. Calder MD, Caveney AN, Smith LC, Watson AJ.
Responsiveness of bovine cumulus-oocyte-com-
plexes (COC) to porcine and recombinant human
FSH, and the effect of COC quality on gonadotro-
pin receptor and Cx43 marker gene mRNAs dur-
ing maturation in vitro. Reprod Biol Endocrinol.
2003; 1: 14.
21. Tol HM, Eijk MG, Mummery CL, van den Hurk
R, Bevers MM. Influence of FSH and hCG on
the resumption of meiosis of bovine oocytes sur-
rounded by cumulus cells connected to membra-
na granulose. Mol Reprod Dev. 1996; 45: 218–224.
22. Izadyar F, Hage WJ, Colenbrander B, Bevers MM.
The promotory effect of growth hormone on the
developmental competence of in vitro matured
bovine oocytes is due to improved cytoplasmic
maturation. Mol Reprod Dev. 1998; 49: 444-453.
23. Lonergan P, Carolan C, Van Langendonckt A,
Donnay I, Khatir H, et al. Role of epidermal
growth factor in bovine oocyte maturation and
preimplantation embryo development. Biol Re-
prod. 1996; 54: 1412-1421.
24. Monget P, Monniaux D. Growth factors and the
28 www.avidscience.com
Recent Advances in In Vitro Fertilization
control of folliculogenesis. J Reprod Fertil. 1995;
49: 321-333.
25. Carneiro G, Lorenzo PL, Pimentel C, Pegoraro L,
Bertolini M, et al. Influence of insulin-like growth
factor-I and its interaction with gonadotropins,
estradiol, and fetal calf serum on in vitro matura-
tion and parthenogenetic development in equine
oocytes. Biol Reprod. 2001; 65: 899-905.
26. Kitiyanant Y, Saikhun J, Pavasuthipaisit K. So-
matic cell nuclear transfer in domestic cat oocytes
treated with IGF-I for in vitro maturation. Theri-
ogenology. 2003; 59: 1775-1786.
27. Lorenzo PL, Illera MJ, Illera JC, Illera M. En-
hancement of cumulus expansion and nuclear
maturation during bovine oocyte IVM with the
addition of epidermal growth factor and insulin-
like growth factor-I. J Reprod Fertil. 1994; 101:
697-701.
28. Silva CC, Knight PG. Modulatory actions of ac-
tivin-A and follistatin on the developmental com-
petence of in vitro-matured bovine oocytes. Biol
Reprod. 1998; 58: 558-565.
29. De Loos F, Van Vliet C, Maurik P, Kruip AM.
Morphology of immature bovine oocytes. Gamete
Research. 1989; 24: 197-204.
30. Hanzen Ch. Biotechnologie : La production
www.avidscience.com 29
Recent Advances in In Vitro Fertilization
d’embryons in vitro. Université de Liège. Méde-
cine Vétérinaire. 2013.
31. Thibault C. La fécondation. Dans La reproduc-
tion chez les mammifères et l’homme, Thibault C,
Levasseur MC, INRA. Edit. Ellipses, Paris. 2001;
366-389.
32. Parrish JJ, Suski-Parrish JL, Leibried-Rutledge
ML, Critser ES, Eyestone WH, et al. Bovine in vit-
ro fertilization with frozen-thawed semen. Theri-
ogenology. 1986; 35: 547-555.
33. Manjunath P, Therien I. Role of seminal plasma
phospholipid binding proteins in sperm mem-
brane lipid modification that occurs during ca-
pacitation. J Reprod Immunol. 2002; 53: 109–119.
34. Medeiros CMO, Parrish JJ. Changes in lectin
binding to bovine sperm during heparin-induced
capacitation. Molec Reprod Develop. 1996; 44:
525–532.0
35. Parrish JJ. Bovine in vitro fertilization: In vitro
oocyte maturation and sperm capacitation with
heparin. Theriogenology. 2014; 81: 67-73.
36. Gadella BM, Luna C. Cell biology and functional
dynamics of the mammalian sperm surface. The-
riogenology. 2014; 81: 74-84.
37. Flesch FM, Brouwers JF, Nievelstein PF, Verkleij
30 www.avidscience.com
Recent Advances in In Vitro Fertilization
AJ, van Golde LM, et al. Bicarbonate stimulated
phospholipid scrambling induces cholesterol re-
distribution and enables cholesterol depletion in
the sperm plasma membrane. J Cell Sci. 2001; 114:
3543–3555.
38. Harrison RA, Gadella BM. Bicarbonate-induced
membrane processing in sperm capacitation. The-
riogenology. 2005; 63: 342-351.
39. Martal J, Chene N, Huynh L, Zourbas S, Rogez
C, et al. Les interactions immuno-endocrines en-
tre la mère et l’embryon. Dans L’embryon Chez
L’homme et l’animal. INSERM – INRA, Paris.
2002; 159-201.
40. Tervit HR, Whittingham DG, Rowson LEA. Suc-
cessful culture in-vitro of sheep and cattle ova. J
Reprod Fertil. 1972; 30: 493–497.
41. Gandolfi F, Moor R. Stimulation of early embry-
onic development in sheep by co-culture with
oviduct epthelial cells. J Reprod Fertil. 1987; 81:
23–28.
42. Mclaughlin KJ, Mclean DM, Stevens G, Ashman
RA, Lewis PA, et al. Viability of one-cell bovine
embryos cultured in synthetic ovidut fluid medi-
um. Theriogenology. 1990; 33: 1191-1199.
43. Ouhibi N, Hamadi J, Guillaud J, Ménézo Y. Co-
culture of 1-cell mouse embryos on different cell
www.avidscience.com 31
Recent Advances in In Vitro Fertilization
supports. Human Reprod. 1990; 5: 737-743.
44. Myers MW, Broussard JR, Ménézo Y, Prough SG,
Blackwell J, et al. Established cell lines and their
conditioned media support bovine embryo devel-
opment during in vitro culture. Human Reprod.
1994; 9: 1927-1931.
45. Reed WA, Suh TK, Bunch TD, White KL. Culture
of in vitro fertilized bovine embryos with bovine
oviductal epithelial cells, buffalo rat liver (BRL)
cells, or BRL-cell-conditionned medium. Theri-
ogenology. 1996; 45: 439-449.
46. Ménézo Y, Hazout A, Herbaut N, Nicollet B. Co-
culture of embryos on Vero cells and transient
transfer of blastocysts in humans. Human Reprod.
1992; 7: 101-106.
47. Watson AJ, Watson PH, Warnes D, Walker SK,
Armstrong DT, et al. Preimplantation develop-
ment of in vitro-matured and in vitro-fertilized
ovine zygotes: comparison between coculture on
oviduct epithelial cell monolayers and culture un-
der low oxygen atmospheres. Biol Reprod. 1994;
50: 715–724.
48. Gardner DK, Lane M, Spitzer A. Enhanced rates
of cleavage and development for sheep zygotes
cultured to the blastocyst stage in vitro in the ab-
32 www.avidscience.com
Recent Advances in In Vitro Fertilization
sence of serum and somatic cells: amino acids, vi-
tamins and culturing embryos in groups stimulate
development. Biol Reprod. 1994; 50: 390-400.
49. Lawitts JA, Biggers JD. Culture of preimplantation
embryos. Methods Enzymol. 1993; 225: 153-164.
50. Rosenkrans CF, Zeng GQ, McNamara GT, Schoff
P, First NL. Development of embryos in vitro as
affected by energy substrates. Biol Reprod. 1993;
49: 459.462.
51. Holm P, Booth P, Vajta G, Callesen H. A protein-
free SOF system supplemented with amino ac-
ids, sodium citrate and myo-inosito for bovine
culture. Report of the 13th scientific Meeting of
the European Embro Transfer Association, Lyon,
France (abstract 158). 1997.
52. Lim K, Jang G, Ko K, Lee W, Park H, et al. Im-
proved in vitro bovine embryo development and
increased efficiency in producing viable calves us-
ing defined media. Theriogenology. 2007; 67: 293-
302.
53. Krisher RL, Lane M, Bavister B. Developmental
competence and metabolism of bovine embryos
cultured in semi-defined and defined culture me-
dia. Biol Reprod. 1999; 60: 1345–1352.
54. Abe H, Yamashita S, Itoh T, Satoh T, Hoshi H. Ul-
trastructure of bovine embryos developed from in
www.avidscience.com 33
Recent Advances in In Vitro Fertilization
vitro–matured and–fertilized oocytes: Compara-
tive morphological evaluation of embryos cul-
tured either in serum-free medium or in serum-
supplemented medium. Mol Reprod Dev. 1999;
53: 325-335.
55. Abe H, Hoshi H. Evaluation of bovine embryos
produced in high performance serum-free media.
J Reprod Dev. 2003; 49: 193-202.
56. Abe H, Yamashita S, Satoh T, Hoshi H. Accumula-
tion of cytoplasmic lipid droplets in bovine em-
bryos and cryotolerance of embryos developed
in different culture systems using serum-free or
serum-containing media. Mol Rep Dev. 2002; 61:
57-66.
57. George F, Daniaux C, Genicot G, Verhaeghe B,
Lambert P, Donnay I. Set up of a serum-free cul-
ture system for bovine embryos: Embryo develop-
ment and quality before and after transient trans-
fer. Theriogenology. 2008; 69: 612-623.
58. Rizos D, Gutiérrez-Adán A, Pérez-Garnelo S, De
la Fuente J, Boland MP, et al. Bovine embryo cul-
ture in the presence or absence of serum: Implica-
tions for blastocyst development, cryotolerance,
and messenger RNA expression. Biol Reprod.
2003; 68: 236-243.
34 www.avidscience.com
Recent Advances in In Vitro Fertilization
59. Van Wagtendonk-de Leeuw AM, Aerts BJG, Den
Daas JHG. Abnormal offspring following in vitro
production of bovine preimplantation embryos: a
field study. Theriogenology. 1998; 49: 883-894.
60. Neira JA, Tainturier D, Peña MA, Martal J. Effect
of the association of IGF-I, IGF-II, bFGF, TGF-β1,
GM-CSF, and LIF on the development of bovine
embryos produced in vitro. Theriogenology. 2010;
73: 595-604.
61. Guérin B, Nibart M, Marquant-Leguienne B,
Humblot P. Sanitary risks related to embryo trans-
fert in domestic species. Theriogenology. 1997; 47:
33-42.
62. Van der Valk J, Brunner D, De Smet K, Fex Sven-
ningsen A, Honegger P, et al. Optimization of
chemically defined cell culture media – Replacing
fetal bovine serum in mammalian in vitro meth-
ods. Toxicology in Vitro. 2010; 24: 1053-1063.
63. Thompson JG. In vitro culture and embryo me-
tabolism of cattle and sheep embryos—a decade
of achievement. Anim Reprod Sci. 2000; 60: 263–
275.
64. Bavister BD. Culture of preimplantation embryos:
facts and artifacts Hum Reprod Update. 1995; 1:
91–148.
65. Vanroose G, Nauwynck H, Soom AV, Ysebaert
www.avidscience.com 35
Recent Advances in In Vitro Fertilization
MT, Charlier G, et al. Structural aspects of the
zona pellucid of in vitro-produced bovine embry-
os: a scanning electron and confocal laser scan-
ning microscopic study. Biol Reprod. 2000; 62:
463–469.
66. Le Tallec B, Ponsart C, Marquant-Le Guienne B,
Guérin B. Risks of transmissible diseases in rela-
tion to embryo transfer. Reprod Nutr Dev. 2001;
41: 439-450.
67. Stringfellow DA, Givens MD, Waldrop JG. Bios-
ecurity issues associated with current and emerg-
ing technologies. Reprod Fert Dev. 2004; 16: 1-10.
68. Van Soom A, Nauwynck H. Risk associated with
bovine embryo transfer and their containment.
CAB reviews: Perspectives in Agriculture and
Veterinary Science. 2008.
69. Eckert J, Pugh PA, Thompson JG, Niemann H,
Tervit HR. Exogenous protein affects develop-
mental competence and metabolic activity of bo-
vine pre-implantation embryos in vitro. Reprod
Fertil Dev. 1998; 10: 327-332.
70. Bredbacka K, Bredbacka P. Effect of polyvinylpyr-
rolidone, serum albumin and fetal calf serum on
early cleavage of bovine embryos. Theriogenol-
ogy. 1995; 43: 174-174.
36 www.avidscience.com
Recent Advances in In Vitro Fertilization
71. Iwasaki T, Kimura E, Totsukawa K. Studies on a
chemically defined medium for in vitro culture of
in vitro matured and fertilized porcine oocytes.
Theriogenology. 1999; 51: 709-720.
72. Chung JT, Tosca L, Huang TH, Xu L, Niwa K, et al.
Effect of polyvinylpyrrolidone on bovine oocyte
maturation in vitro and subsequent fertilization
and embryonic development. Reprod Biomed
Online. 2007; 15: 198-207.
73. Vireque AA, Camargo LS, Serepiao RV, Rosa E
Silva AA, Watanabe YF, et al. Preimplantation
development and expression of Hsp-70 and Bax
genes in bovine blastocyst derived from oocytes
matured in alpha-MEM supplemented with
growth factors and synthetic macromolecules.
Theriogenology. 2009; 71: 620-627.
74. Lee CN, Ax RL. Concentration and composition
of glycoaminoglycans in the female bovine repro-
ductive tract. J Dairy Sci. 1984; 67: 2006-2009.
75. Sato E, Ishibashi T, Koide SS. Prevention of spon-
taneous degeneration of mouse oocytes in cul-
ture by ovarian glycosaminoglycans. Biol Reprod.
1987; 37: 371-376.
76. Suchanek E, Simunic V, Juretic D, Grizelj V. Fol-
licular fluid contents of hyaluronic acid, follicle-
stimulating hormone and steroids relative to the
www.avidscience.com 37
Recent Advances in In Vitro Fertilization
success of in vitro fertilization of human oocytes.
Fertil Steril. 1994; 62: 347-352.
77. Furnus C, Valcarcel A, Dulout F, Errecalde A. The
hyaluronic acid receptor (CD44) is expressed in
bovine oocytes and early stage embryos. Theriog-
enology. 2003; 60: 1633-1644.
78. Campbell S, Swann HR, Aplin JD, Seif MW, Kim-
ber SJ, et al. CD44 is expressed throughout preim-
plantation human embryo development Human
Reproduction. 1995; 10: 425-430.
79. Tzanakakis GN, Karamanos NK, Hjerpe A. Ef-
fects on glycosaminoglycan synthesis in cultured
human mesothelioma cells of transforming, epi-
dermal, and fibroblast growth factors and their
combinations with platelet-derived growth factor.
Exp Cell Res. 1995; 220: 130-137.
80. Klominek J, Robert KH, Hjerpe A, Wikstrom B,
Gahrton G. Serum-dependent Growth Patterns
of Two, Newly Established Human Mesothelioma
Cell Lines. Cancer Res. 1989; 49: 6118-6122.
81. Hamashima N. Effect of hyaluronic acid on the
preimplantational development of mouse embryo
in vitro. Dev Growth Differ. 1982; 24: 353-357.
82. Miyoshi K, Umezu M, Sato E. Effect of hyaluronic
acid on the development of porcine 1-cell em-
38 www.avidscience.com
Recent Advances in In Vitro Fertilization
bryos produced by a conventional or new in vitro
maturation/fertilization system. Theriogenology.
1999; 51: 777–784.
83. Furnus CC, de Matos DG, Martinez AG. Effect of
hyaluronic acid on development of in vitro pro-
duced bovine embryos. Theriogenology. 1998; 49:
1489-1499.
84. Block J, Bonilla L, Hansen PJ. Effect of addition
of hyaluronan to embryo culture medium on sur-
vival of bovine embryos in vitro following vit-
rification and establishment of pregnancy after
transfer to recipients. Theriogenology. 2009; 71:
1063-1071.
85. Palasz AT, Breña PB, Martinez MF, Perez-Garnelo
SS, Ramirez MA, et al. Development, molecular
composition and freeze tolerance of bovine em-
bryos cultured in TCM-199 supplemented with
hyaluronan. Zygote. 2008; 16: 39-47.
86. Gray CW, Morgan P, Kane MT. Purification of an
embryotrophic factor from commercial bovine
serum albumin and its identification as citrate. J
Reprod Fert. 1992; 94: 471-480.
87. Lane M, Maybach JM, Hooper K, Hasler JF, Gard-
ner DK. Cryo-survival and development of bovine
blastocysts are enhanced by culture with recombi-
nant albumin and hyaluronan. Mol Reprod Dev.
www.avidscience.com 39
Recent Advances in In Vitro Fertilization
2003; 64: 70-78.
88. Palasz AT, Thundathil J, Verrall RE, Mapletoft RJ.
The effect of macromolecular supplementation
on the surface tension of TCM-199 and the uti-
lization of growth factors by bovine oocytes and
embryos in culture. Anim Reprod Sci. 2000; 58:
229–240.
89. George F, Kerschen D, Van Nuffel A, Rees JF, Don-
nay I. Plant protein hydrolysates (plant peptones)
as substitutes for animal proteins in embryo cul-
ture medium. Reprod Fert Dev. 2009; 21: 587–598.
90. Ben-Yosef D, Galiani D, Dekel N, Shalgi R. Rat
oocytes induced to mature by epidermal growth
factor are successfully fertilized. Mol Cell Endo-
crinol. 1992; 88: 135-141.
91. Das K, Stout LE, Hensleigh HC, Tagatz GE, Phi-
pps WR, et al. Direct positive effect of epidermal
growth factor on the cytoplasmic maturation of
mouse and human oocytes. Fertil Steril. 1991; 55:
1000-1004.
92. Reed ML, Estrada JL, Illera MJ, Petters RM. Ef-
fects of epidermal growth factor, insulin-like
growth factor-I, and dialyzed porcine follicular
fluid on porcine oocyte maturation in vitro. J Exp
Zool. 1993; 266: 74-78.
40 www.avidscience.com
Recent Advances in In Vitro Fertilization
93. Mermillod P, Lonergan P, Carolan C, Khatir H,
Poulin N, et al. Maturation ovocytaire in vitro
chez les ruminants domestiques. Contraception
Fert Sexualité. 1996; 24: 552-558.
94. Bonilla AQ, Ozawa M, Hansen PJ. Timing and de-
pendence upon mitogen-activated protein kinase
signaling for pro-developmental actions of insu-
lin-like growth factor 1 on the preimplantation
bovine embryo. Growth Horm IGF Res. 2011; 21:
107-111.
95. Moreira F, Paula-Lopes FF, Hansen PJ, Badinga
L, Thatcher WW. Effects of growth hormone and
insulin-like growth factor-I on development of in
vitro derived bovine embryos. Theriogenology.
2002; 57: 895–907.
96. Palma GA, Muller M, Brem G. Effect of insulin-
like growth factor I (IGF-I) at high concentrations
on blastocyst development of bovine embryos
produced in vitro. J Reprod Fertil. 1997; 110: 347–
353.
97. Prelle K, Stojkovic M, Boxhammer K, Motlik J,
Ewald D, et al. Insulin-like growth factor I (IGF-
I) and long R(3) IGF-I differently affect develop-
ment and messenger ribonucleic acid abundance
for IGF-binding proteins and type I IGF receptors
in in vitro produced bovine embryos. Endocrinol-
ogy. 2001; 142: 1309-1316.
www.avidscience.com 41
Recent Advances in In Vitro Fertilization
98. Spanos S, Becker DL, Winston RM, Hardy K. An-
ti-apoptotic action of insulin-like growth factor-I
during human preimplantation embryo develop-
ment. Biol. Reprod. 2000; 63: 1413-1420.
99. Fabian D, Il’kova G, Rehak P, Czikkova S, Baran V,
et al. Inhibitory effect of IGF-I on induced apop-
tosis in mouse preimplantation embryos cultured
in vitro. Theriogenology. 2004; 61: 745-755.
100. Cui XS, Jeong YJ, Jun JH, Kim NH. Insu-
lin-like growth factor-I alters apoptosis related
genes and reduces apoptosis in porcine parthe-
notes developing in vitro. Theriogenology. 2005;
63: 1070-1080.
101. Byrne AT, Southgate J, Brison DR, Leese
HJ. Analysis of apoptosis in the preimplantation
bovine embryo using TUNEL. J Reprod Fertil.
1999; 117: 97-105.
102. Block J, Drost M, Monson RL, Rutledge JJ,
Rivera RM, et al. Use of insulin-like growth fac-
tor-I during embryo culture and treatment of re-
cipients with gonadotropin-releasing hormone to
increase pregnancy rates following the transfer of
in vitro-produced embryos to heat-stressed, lac-
tating cows. J Anim Sci. 2003; 81: 1590-1602.
103. Block J, Hansen PJ. Interaction between
42 www.avidscience.com
Recent Advances in In Vitro Fertilization
season and culture with insulin-like growth fac-
tor-1 on survival of in vitro produced embryos
following transfer to lactating dairy cows. Theri-
ogenology. 2007; 67: 1518-1529.
104. Block J, Hansen P, Loureiro B, Bonilla L.
Improving post-transfer survival of bovine em-
bryos produced in vitro: Actions of insulin-like
growth factor-1, colony stimulating factor-2 and
hyaluronan. Theriogenology. 2011; 76: 1602-1609.
105. Desai N, Kattal N, AbdelHafez F, Szep-
tycki-Lawson J, Goldfarb J. Granulocyte-mac-
rophage colony stimulating factor (GM-CSF) and
co-culture can affect post-thaw development and
apoptosis in cryopreserved embryos. J Assist Re-
prod Genet. 2007; 24: 215-222.
106. Fields SD, Hansen PJ, Ealy AD. Fibroblast
growth factor requirements for in vitro develop-
ment of bovine embryos. Theriogenology. 2011;
75: 1466-1475.
107. Larson RC, Ignotz GG, Currie WB. Trans-
forming growth factor beta and basic fibroblast
growth factor synergistically promote early bo-
vine embryo development during the fourth cell
cycle. Mol Reprod Dev. 1992; 33: 432-435.
108. Yang QE, Giassetti MI, Ealy AD. Fibroblast
growth factors activate mitogen activated protein
www.avidscience.com 43
Recent Advances in In Vitro Fertilization
kinase pathways to promote migration in ovine
trophoblast cells. Reproduction. 2011; 141: 707-
714.
109. Cooke FN, Pennington KA, Yang Q, Ealy
A. Several fibroblast growth factors are expressed
during pre-attachment bovine conceptus develop-
ment and regulate interferon-tau expression from
trophectoderm. Reproduction. 2009; 137: 259-
269.
110. Michael D, Alvarez I, Ocón O, Powell A,
Talbot N, et al. Fibroblast Growth Factor-2 is ex-
pressed by the bovine uterus and stimulates inter-
feron-production in bovine trophectoderm. En-
docrinology. 2006; 147: 3571-3579.
111. Yang QE, Johnson SE, Ealy AD. Protein
kinase C delta mediates fibroblast growth factor-
2-induced interferon-tau expression in bovine
trophoblast. Biol Reprod. 2011; 84: 933-943.
112. Eckert J, Niemann H. Effects of Plateled
Derived Growth Factor (PDGF) on the in vitro
production of bovine embryos in protein-free me-
dia. Theriogenology. 1996; 46: 307-320.
113. Larson RC, Ignotz GG, Currie WB. Platelet
derived growth factor (PDGF) stimulates devel-
opment of bovine embryos during the fourth cell
44 www.avidscience.com
Recent Advances in In Vitro Fertilization
cycle. Development. 1992; 115: 821-826.
114. Luo S, Yin HN, Li SW. Effect of TGF-be-
ta1 on embryo implantation and development in
mice in vitro. J Sichuan Univ. 2010; 41: 265-268.
115. Loureiro B, Bonilla L, Block J, Fear J, Bonil-
la A, et al. Colony-Stimulating Factor 2 (CSF-2)
improves development and post-transfer survival
of bovine embryos produced in vitro. Endocrinol-
ogy. 2009; 150: 5046-5054.
116. Kwak SS, Jeung SH, Biswas D, Jeon YB,
Hyun S. Effects of porcine granulocyte-mac-
rophage colony-stimulating factor on porcine in
vitro-fertilized embryos. Theriogenology. 2012;
77: 1186-1197.
117. Loureiro B, Block J, Favoreto M, Caram-
bula S, Pennington KA, et al. Consequences of
conceptus exposure to colony-stimulating factor
2 on survival, elongation, interferon-τ secretion,
and gene expression. Reproduction. 2011; 141:
617-624.
118. Rooke J, Ewen M, McEvoy T, Entrican G,
Ashworth C. Effect of inclusion of serum and
granulocyte-macrophage colony stimulating fac-
tor on secretion of interferon-tau during the in
vitro culture of ovine embryos. Reprod Fert Dev.
2005; 17: 513-521.
www.avidscience.com 45
Recent Advances in In Vitro Fertilization

119. Sirisathien S, Hernandez-Fonseca HJ,

Bosch P, Hollet BR, Lott JD, et al. Effect of leu-
kemia inhibitory factor on bovine embryos pro-
duced in vitro under chemically defined condi-
tions. Theriogenology. 2003; 59: 1751-1763.
120. Ptak G, Lopes F, Matsukawa K, Tischner
M, Loi P. Leukaemia inhibitory factor enhances
sheep fertilization in vitro via an influence on the
oocyte. Theriogenology. 2006; 65: 1891-1899.
121. Moreno D, Neira A, Dubreil L, Liegeois L,
Destrumelle S, et al. In vitro bovine embryo pro-
duction in a synthetic medium: Embryo develop-
ment, cryosurvival, and establishment of preg-
nancy. Theriogenology. 2015; 84: 1053-1060.

46 www.avidscience.com