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Review
In-vitro maturation of oocytes: biological
aspects
Dr Pierre Miron obtained his medical degree from the University of Sherbrooke (1980),
completed an obstetrics & gynecology specialty at University of Montreal (1985) and
became, the same year, fellow of the Royal College of Physicians and Surgeons of Canada.
He pursued his academic training as a fellow in reproductive endocrinology and infertility at
the Royal Women’s Hospital, University of Melbourne, Australia (1986), under the guidance
and mentorship of Professors Ian Johnston and John McBain. Dr Miron is currently
professor at University of Montreal and founded more recently, FERTILYS Reproductive
Center, Canada.

Dr Pierre Miron
A Ali1, M Benkhalifa1,2, P Miron1,3,4
1
Centre de Fertilité et de Reproduction Fertilys, Laval, Québec, Canada; 2ATL R and D Laboratory, Reproductive
Biology and Genetics, 78320 La Verrière, France; 3Department of Obstetrics and Gynecology, Hôpital Maisonneuve-
Rosemont, Faculté de Médecine, Université de Montréal, Montréal, Canada
4
Correspondence: Department of Obstetrics and Gynecology, Hôpital Maisonneuve-Rosemont, 5415, Boulevard de
l’Assomption, Montréal (QC), Canada H1T 2M4. e-mail: pierre.miron@fertilys.com

Abstract
Natural cycle and in-vitro maturation (IVM) of oocytes are becoming interesting alternatives to classical assisted reproduction
technology approaches for patients, especially in those at high risk for ovarian hyperstimulation syndrome or with poor ovarian
reserve. More than for their clinical and biological indications, natural cycle and IVM of oocytes can also be considered
as good social and economic alternatives to the classical IVF treatment, based on their financial cost–effectiveness with
exclusion of expensive medications. To be successful, IVM must entail both nuclear and cytoplasmic maturation, and its
maturation and success rates are affected by the number of collected cumulus layers, the degree of atresia and the maturation
rate between 24 and 48 h. Endogenous regulation of oocyte maturation is a complex sequence of events regulated by
endocrine parameters, oocyte/follicular cross-talk, and intra-oocyte kinase/phosphatase interactions. This complex process
requires a better definition of each contributing factor affecting oocyte development and the resulting embryo quality. The
clinical aspects of IVM have been documented earlier; the present paper will mainly focus on the biological aspect of oocyte
maturation in vitro and the quality of derived embryos.

Keywords: clinical application, cumulus cells, cytoplasmic maturation, developmental competence, nuclear maturation, oocyte

Introduction (GVBD), the first meiotic reduction division, and arrest at

The success of clinical IVF remains compromised by suboptimal
culture conditions, resulting in impaired embryo development
and subsequent loss of viability. Even though pregnancy rates
have increased since the first human IVF attempts, this can be
primarily attributed to the introduction of ovarian stimulation
and the subsequent replacement of an increasing number of
embryos.
The initiation of oocyte in-vitro maturation (IVM) was first
reported by Pincus and Enzman (1935) and Edwards et al.
(1965, 1969). An interesting feature of the mammalian oocyte
is that oocytes undergo spontaneous maturation upon removal
from the follicle without stimulation factors (Edwards, 1965).
Resumption of meiosis leads to germinal vesicle breakdown
metaphase II (MII) until ovulation and fertilization.
IVM of human oocytes is becoming an emerging application
in IVF laboratories, and has great promise. Methods used
in cattle for IVM of primary oocytes have been applied to
women suffering from infertility (Cha et al., 1991). To be
successful, the oocyte should achieve two levels of maturation,
nuclear maturation through meiosis I and II progression, and
cytoplasmic maturation.
In the study of Sirard and colleagues (1989) on the timing
of nuclear events during IVM, the germinal vesicle (GV)
was observed from 0 to 6.6 h, GVBD occurred at 6.6–8.0 h,
chromatin condensation at 8–10.3 h, metaphase I (MI) at 10.3–
15.4 h, anaphase I (AI) at 15.4–16.6 h, telophase I (TI) at 16.6– 437
 Review - In-vitro maturation of oocytes: biological aspects - A Ali et al.
18.0 h and metaphase II (MII) at 18–24 h. In 1992, a direct
relationship between premature chromosome condensation and
IVM of oocytes was suggested (Santalo et al., 1992). More
recently, Li and collaborators confirmed that IVM could have
deleterious effects on the organization of the meiotic spindle
and chromosome alignment of human oocytes (Li et al., 2006).
Aneuploidy resulting from errors in meiotic chromosome
segregation is the leading cause of pregnancy losses (Li et al.,
2002). This could explain the reduction found in developmental
competence of IVM human oocytes compared with those
matured in vivo and supports the need for improving in-vitro
culture conditions.
Cytoplasmic maturation in itself describes both the ultrastructural
changes that take place in the oocyte from the GV to the MII
stage and the acquisition of developmental competence of the
oocyte (Duranthon and Renard, 2001). Cytoplasmic maturation
is indirectly and retroactively assessed as the ability of the mature
oocyte to undergo normal fertilization, cleavage and blastocyst
development. Other indirect morphological parameters taken
into account to evaluate cytoplasmic maturation include
cumulus cell expansion, expulsion of the polar body and an
increased perivitelline space (Kruip et al., 1983). Oocyte
maturation also involves complex interactions of different
intracellular, paracrine and structural factors, including sterols,
steroids, growth factors, cyclic adenosine monophosphate and
gap junction (Jamnongjit and Hamnes, 2005). Many factors and
regulatory molecules have been shown either to inhibit or to
promote nuclear and cytoplasmic oocyte maturation (Chian et
al., 2004), either directly or via the cumulus cells.
Advances made in immature oocyte collection and culture
conditions have increased the clinical feasibility of IVM (Son et
al., 2005). However, in order to achieve acceptable birth rates,
future studies should focus on characterization and regulation
of oocyte cytoplasmic maturation, and on how oocyte-derived
factors influence zygotic genome activation and embryonic
developmental competence.
In clinical application of IVM, nearly 50% of immature
oocytes reach their maturation after 24–28 h (Abdul-Jalil et al.,
2001) with nearly 20−25% clinical pregnancy in patients with
polycystic ovarian syndrome at risk for ovarian hyperstimulation
syndrome or in normo-ovulatory patients (Le Du et al., 2005;
Mikkelsen, 2005). This result is in contrast to studies in other
species where MII is achieved much more easily. When
immature oocytes were cultured in suitable conditions in vitro,
a significant proportion of oocytes reached nuclear maturation
(bovine, Ali and Sirard, 2002; sheep, Guler et al., 2000; rabbit,
Lorenzo et al., 1996; pig, Ye et al., 2005). However, Barnes et al.
(1996) reported in humans significantly higher rates of oocyte
maturation and fertilization of immature oocyte from patients
with regular cycles compared with irregular and anovulatory
patients. This study also showed that the culture of immature
oocytes for 48 h can increase the maturation rate (57 versus
82%) but did not affect the fertilization or the cleavage rate.
The optimization of clinical and biological aspects of IVM
cycles will enhance the take-home baby rate (Papanikolaou,
2005). Several factors determine the ultimate competence of
the oocyte; these have been investigated and attempts made
to mimic these conditions in vitro. The complexity of the
438 orchestration of the events that control oocyte growth and final
acquisition of developmental competence is under continuous
investigation. The present review describes some of biological
aspects of oocyte maturation in vitro to date.
Oocyte−follicle relationship
Meiotic and developmental competence
In each menstrual cycle, a number of follicles are activated
to enter a growth phase characterized by granulosa cell
proliferation and an increase in oocyte size. The capacity of
oocyte maturation is closely related to follicular maturation. It
has been found that in the human oocyte there is a decreased
maturation rate in oocytes from small follicles (3–4 mm) when
compared with those from larger follicles (9–15 mm) (Tsuji
et al., 1985). The growing follicle must reach a minimum
of 3–4 mm in size to acquire the capacity to respond to a
developmental signal. This capacity will increase (percentage
of oocytes capable of responding) as the follicle reaches a
plateau or a reduction of its growth rate, either by dominance
or early atresia. Moor and Trounson (1977) observed in sheep
that as the follicle size increased, the frequency of oocytes
progressing to the second meiotic metaphase increased as well.
Their observation showed that follicle size could be used as an
indictor for selecting oocytes with high meiotic competence.
With respect to follicle size, oocytes recovered during
the follicular phase show definitive differences in meiotic
competence. However, oocytes recovered in the luteal phase,
irrespective of follicle size, result in a meiotic competence
similar to that of oocytes recovered from large follicles during
the follicular phase (Machatkova et al., 2004). After fertilization,
these luteal-phase oocytes also demonstrate a high potential
to reach blastocyst stage. Also, in humans, early evidence
suggested that immature oocytes collected in the luteal phase
have significantly higher maturation rates (Cha et al., 1992). In
addition, aspiration in the mid-follicular phase of antral follicles
(≥8 mm) in women with regular menstrual cycles, prior to the
emergence of a dominant follicle, offers the best oocytes for
maturation and ultimately pregnancy rates (Mikkelsen et al.,
1999). The relative influence of follicular and luteal phases on
oocyte meiotic competence was not clearly defined, but may be
related to follicular atresia.
As expected, the follicle size from which an oocyte is derived
is also related to its ability to progress from early cleavage
to blastocyst stages. Oocyte developmental competence
characterizes the ability of such oocytes to develop to the
blastocyst stage in vitro. This observation is well demonstrated
in mouse, ewe and cow, where oocytes from small growing
follicles lack the ability to progress beyond the 2–8-cell stage.
Conversely, oocytes derived from large follicles (≥8 mm) may
achieve blastulation rates comparable to IVM oocytes (Moor
and Trounson 1977; Eppig et al. 1992). It appears that oocytes
require an additional ‘prematuration’ to express this competence
(Hendriksen et al., 2000). In vivo, this pre-maturation occurs
during pre-ovulatory growth before the LH surge. This suggests
that developmental competence is mainly acquired during
oocyte growth within the follicle by an unknown mechanism
(Sirard, 2001). Final steps in oocyte maturation are crucial to
the acquisition of functional properties necessary for further
development (Hyttel et al., 1997).
 Review - In-vitro maturation of oocytes: biological aspects - A Ali et al.
Current IVM protocols are generally inefficient at producing
viable embryos, partly due to abnormal oocyte cytoplasmic
maturation in vitro (Krisher and Bavister, 1998). Embryos
produced after IVM of human oocytes are often arrested at
pronuclear or 4–8-cell stage when attempting to culture them
to blastocysts (Barnes et al. 1996). During IVM programmes,
in most cases, embryos obtained after IVM have been replaced
at 2–8-cell stage and their developmental potential to the
blastocyst stage is relatively unknown. When considering the
low pregnancy rate achieved after IVM of human oocytes, it
appears that human oocytes acquire their ability to mature and
develop to blastocyst relatively late during folliculogenesis.
Allowing such development of IVM of human oocytes to
blastocysts could be an important selection tool for embryo
transfer (Barnes et al., 1995). Moreover, optimization of IVM
protocols is vital not only for generating viable embryos, but
also to support the development of subsequent offspring into
normal adults (Eppig and O’Brien, 1998).
For single embryo selection prior to transfer, more than culture
media development and improvement, there is a need for an
indicator of viability. Other visual assessments used to evaluate
developmental competence include morphological evaluations
such as blastocoele expansion, number of blastomeres, and
trophectoderm to inner-cell mass ratio (Ali et al., 2004).
Moreover, functional evaluations such as the ability to resume
development after freezing and induce a pregnancy should
also be considered to provide a more complete idea of the
developmental potential of the oocyte (Isachenko et al., 2004;
Sirard et al., 2006).
The ovarian follicular population is another parameter used
to estimate the developmental competence of the oocyte. The
number and size of the follicles present in the ovary at the
time of aspiration may be used to select oocytes with higher
developmental competence. Oocytes retrieved from ovaries that
have at least one follicle larger than 10 mm or with more than
10 follicles of 2−5 mm have a high developmental potential.
In contrast, oocytes retrieved from ovaries with fewer than
10 follicles of 2−5 mm or no follicle larger than 10 mm reach
lower blastocyst rates with lower cell numbers (Gandolfi et al.,
1997).
Follicular quality
It was observed that the development to blastocyst of an oocyte
recovered from an atretic follicle (showing signs of cumulus
expansion in the outer layers of the cumulus granulosa and
slight granulations in the oocyte cytoplasm), irrespective
of size, was equally good as that of an oocyte recovered
from large non-atretic follicles. It is believed that some large
follicles contain developmentally incompetent oocytes, while
some medium-sized follicles contain competent oocytes
(Blondin and Sirard, 1995). Follicular atresia may promote
the acquisition of developmental competence (Hendriksen
et al., 2000). Blondin et al. (1997) found that bovine oocytes
derived from ovaries maintained at 35°C for 4 h yielded a
higher frequency of blastocysts. Their data suggested that
developmental competence was acquired shortly prior to IVM
and depended on the handling conditions of post-mortem
ovaries. Similar conclusions to those of Blondin and Sirard
(1995) have been observed in human oocytes (Barnes et al.,
1996). Human oocytes in which the outer layers of cumulus
granulosa are partially expanded are classified as atretic. These
oocytes results in high rates of maturation and fertilization
when compared with oocytes with healthy, tightly compacted
cumulus granulosa (Blondin and Sirard, 1995; Blondin et al.,
1997). In the final steps of follicular growth, even in atretic
follicles, oocytes could undergo a final maturational event at the
cytoplasmic level that would render them competent to develop.
Moreover, oocytes may retain their developmental competence
acquired in the late follicular phase even when atresia is under
way; in that regard, it was shown that the cumulus−oocyte
complex (COC) is the last part of the follicle to be affected by
atresia (Kruip and Dieleman, 1982).
It is well known that the follicle can profoundly influence the
quality of the oocyte obtained at ovulation and, as a result, the
quality of embryo obtained. Recently, bovine follicular fluid
(bFF) of competent follicles (>8 mm) from FSH-stimulated
animals was compared with bFF from small (2–5 mm) follicles
during IVM. The objective was to evaluate if adding bFF to
the maturation medium level could influence the developmental
competency of selected oocytes obtained from 2–5-mm-sized
follicles (Ali et al., 2004). The conclusion was that follicular
fluid originating from competent follicles increased the
developmental competence of abattoir-derived oocytes and, as
a result, the quality of embryo obtained.
In humans, follicular vascularity and the level of intrafollicular
oxygen appear to be important determinants of oocyte
competence. Findings from several studies indicate that embryos
with the highest implantation potential originate from follicles
that are well vascularized and oxygenated (Van Blerkom, 1998,
2000).
Metabolic coupling
Within the follicle, there are several different somatic cell
phenotypes that surround the oocyte. It is now widely
recognized that bi-directional communication between the
oocyte and follicular somatic cells is fundamentally important
for folliculogenesis and oocyte growth and maturation (Eppig,
2001). The morphology of the cumulus surrounding an oocyte
is commonly used as a selection criterion prior to IVM (Goud
et al., 1998). The degree of expansion is also considered as a
morphological indicator of oocyte quality and directly related
to developmental capacity of the oocyte to reach maturation
(Ali and Sirard, 2002a).
Earlier studies have demonstrated that granulosa cells are
metabolically coupled to oocyte via gap junctions during growth
and initial stages of maturation (Brower and Schultz, 1982; de
Loos et al., 1991). Cessation of metabolic coupling coincides
with the breakdown of the gap junction, which generally
occurs just prior to the first meiotic metaphase. Nutritional and
regulatory elements responsible for growth, maintenance of
meiotic arrest, and substrates for maturation and development
are able to pass through these gap junctions (Tanghe et al.,
2002).
In cattle, it is speculated that granulosa cells, in response to LH,
synthesize pyruvate, which is transferred to the maturing oocyte.
These events do not occur in the absence of an LH surge or in 439
 Review - In-vitro maturation of oocytes: biological aspects - A Ali et al.
COC in which the oocyte has been removed (oocytectomized),
indicating the interdependence of these cell types (Zuelke and
Brackett, 1993).
It is evident that cumulus cells provide many important
functions not just for the oocyte but also for zygote, such as
pronuclei formation, polar body extrusions, and organelle
redistribution and cytoskeletal rearrangements. The cumulus
provides not only a microenvironment that would consist in
low concentrations of glucose and high lactate concentration,
but also provides homeostasis regulation for the oocyte and the
early embryo (Mori et al., 2000; Tanghe et al., 2002).
The beneficial effect of cumulus oophorus during IVM can be
attributed to the formation of a favourable microenvironment
(biochemical or metabolic) around the oocyte (Tanghe et
al., 2002). Cumulus cells participate in oocyte development
during IVM, either by secreting soluble factors, which induce
developmental competence or by removing inhibitory or
toxic components from the maturation medium (Homa, 1995;
Hashimoto et al., 1998). In addition, cumulus cells might have
unknown promoting effects on subsequent oocyte development
which might be attributable to intracellular changes such as pH
or calcium ions (Mori et al., 2000). Another possible influence
of cumulus cells during IVM of bovine oocytes might be
that cumulus cells decrease oxygen tension in the immediate
vicinity of the oocyte as a result of an active metabolism of
the cumulus cells (Ali et al., 2003). By removing the cumulus
cells artificially, it has been demonstrated that their presence
during maturation is necessary for most oocytes to express
competency (Ali et al., 2005). In this study, oocyte MII and
development to the 2–8-cell stage 72 h after insemination
were not affected by the absence of cumulus cells during
maturation. However, development beyond the blastocyst stage
was significantly lower in cumulus-free than in cumulus-intact
oocytes, indicating the importance of cumulus cells during
IVM of oocytes for cytoplasmic maturation and subsequent
early development (Figure 1).
Culture conditions
It is known that treating the cause instead of the symptoms
produces the most encouraging results. Early embryo
development appears to be dependant on the maturation
microenvironment of the oocyte (Table 1). Significant
improvements in the overall rate of oocyte maturation and
embryo development can be achieved by adding mature
mammalian fluid from large follicles (containing oocytes more
likely to develop into blastocysts) in the culture media (Blondin
et al., 2002; Ali et al., 2004).
Early embryo development is a complex mechanism, based on
interactions between intracellular and extracellular cell biology
of oocyte and spermatozoa development and maturation. It is
therefore quite remarkable that oocytes matured and embryos
produced in vitro still maintain a reasonable degree of functional
and developmental competence. Studies have investigated
a number of culture modifications, based on physiological
conditions found in the female reproductive tract, to improve
embryo developmental outcome. In-vitro conditions cannot
truly replicate in-vivo conditions. Features of oocyte and
440 embryo morphology, metabolic and biochemical properties can
be altered by the in-vitro environment. These alterations can
become evident as errors that may be compatible with early
embryonic development but are deleterious for viability.
In most reports, IVM of mammalian oocytes was performed
in incompletely defined systems containing co-cultured
somatic cells, blood serum, bovine serum albumin (BSA) or
cell-conditioned medium. Besides being a potential source of
infectious agents, these undefined components make it difficult
to undertake proper quality control and to evaluate the basic
requirements for metabolic substrates, nutritional, growth
factors and hormones during IVM.
Pituitary hormones and oocyte
developmental competence
In most mammalian oocytes, supplementation of in-vitro culture
media by gonadotrophins, steroids and growth factors separately
or in combination can stimulate or inhibit cumulus expansion
and/or nuclear and cytoplasm maturation. It is now common
practice in mammalian oocyte media to add these supplements
during IVM. Supplementation with recombinant human FSH
(rFSH) and 17β-oestradiol during IVM of bovine oocytes results
in a positive effect by increasing the number of embryos after
IVF (Ali and Sirard, 2002b). Media for IVM of human oocytes
contain oestradiol to support cytoplasmic maturation, which is
necessary for fertilization and early embryonic development
(Tesarik and Mendoza, 1995). This is in agreement with the finding
that oocytes matured in the presence of higher concentrations
of oestradiol (1000 ng/ml) improved the developmental rate to
blastocyst stage (Ali and Sirard, 2002b).
Other observations in cattle suggest the possibility of including
hyaluronic acid (HA) with oestradiol in culture media to
increase the efficiency of in-vitro blastocyst production from
IVM oocytes using completely defined conditions (Ali et al.,
2002; Figure 2). Interestingly, when oestradiol and HA were
added to the maturation medium, cumulus expansion was never
observed. These results confirmed recent findings that the
presence of cumulus cells during IVM might be necessary to
express the competence of oocytes and that cumulus expansion
is not required to improve this competence; at the least, there
is no linear relationship between cumulus expansion and
cytoplasmic maturation (Ali and Sirard, 2002a,b). Moreover,
these results suggest that communication between the oocyte
and surrounding cumulus cells is important in determining
which factors are released by the cumulus cells (Ail and Sirard,
2005; Ali et al., 2005).
In cattle, the concentration of oestradiol in the follicular fluid of
a dominant follicle is higher than in others, indicating a possible
role of oestradiol on the cytoplasmic maturation occurring
before LH surge. Recently, studies in cattle have postulated
that increased developmental competence of bovine oocytes
in response to rFSH and oestradiol occurs by the production
of factors that reach the oocyte through the cell−cell coupling
pathways or that the coupling per se results in physiological
changes (Ali and Sirard, 2005; Ali et al., 2005). Moreover,
these studies provide novel evidence of a direct role of rFSH in
the regulation of gap junction communication between cumulus
cells and oocytes and the consequences of such conditions on
further competence.
 Review - In-vitro maturation of oocytes: biological aspects - A Ali et al.

Figure 1. Effect of the presence or absence of cumulus cells during different periods of in-vitro maturation on further development
of bovine oocytes in vitro. Pooled data from three replicates (mean ± SEM). Values with different superscripts (a−c) are significantly
different within a column (P < 0.05). All treatments were replicated at the same time. Recombinant human FSH (rFSH) = synthetic
oviductal fluid/bovine serum albumin (BSA + rFSH) (Ali et al., 2005). COC = cumulus−oocyte complexes; DO = denuded oocyte;
MII = metaphase II.

Table 1. Some factors added to in-vitro maturation media to enhance oocyte maturation
and subsequent embryo development.

Factors Authors Year

FSH Izadyar et al. 1998

Ali and Sirard 2002b, 2005
Sp-cAMPS (PKA activator) Ali and Sirard 2005
PMA (PKC activator) Ali and Sirard 2005
Oestradiol Tesarik and
Mendoza 1995
Ali and Sirard 2002b
Growth hormone (GH) Iga et al. 1998
Izadyar et al. 1998
Hyaluronic acid (HA) Ali et al. 2002
Follicular fluid (FF) Sirard et al. 1995
Blondin et al. 2002
Ali et al. 2004
Serum Trounson et al. 2001
Intracellular antioxidants (cysteine, cysteamine, Jeong and Yang 2001
glutamine, β-mercaptoethanol) Ali et al. 2003

441
 Review - In-vitro maturation of oocytes: biological aspects - A Ali et al.
The roles of protein kinase A (PKA) and protein kinase C
(PKC) (possibly involved in rFSH response), were investigated
recently (Ali and Sirard, 2005) using activators (Sp-cAMPS,
PMA) or inhibitors (Rp-cAMPS, sphingosine) of these two
protein kinases respectively. The developmental competence of
oocytes was measured by the rate of blastocyst formation after
IVF. This study is one of the first to report such high development
rates of bovine oocytes after IVM using defined conditions,
especially after short-term treatment with rFSH. The data show
that the PKC pathway is implicated in r-FSH, which improved
oocyte competence, especially after short-term treatment. These
results indicate that the PKA and PKC pathways can modulate
the maturation of oocytes in vitro. A better understanding of
the mechanism by which rFSH stimulates the oocytes can have
important implications in the light of clinical interest in the use
of rFSH in assisted reproduction protocols.
During the period of folliculogenesis and oocyte maturation in
vivo, growing evidence indicates an important role of both FSH
and LH. In human IVF, Filcori (1999) reported that a minimal
level of LH activity during exogenous stimulation protocols
is required to optimize ovulation induction in patients. In
fact, it has been postulated that profound LH suppression
could affect optimal oocyte maturation and/or endometrial
development (Lévy et al., 2000). Moreover, the final stages of
oocyte maturation in vivo are induced by a rise in serum LH
concentrations. Schoolcraft and co-workers (1999) reported
that including LH in ovarian stimulation protocols using highly
purified FSH preparations would improve blastocyst quality
as reflected by increased embryo implantation and pregnancy
rate. However, LH supplementation, particularly in ovarian
stimulation protocols using GnRH agonists, and the concept
of ‘window’ for LH requirement, still remain today a matter
of debate (Tesarik and Mendoza, 2002; Humaidan, 2006;
Kolibianakis et al., 2006).
The effect of LH during IVM on oocyte maturation and
subsequent development has also been questioned. Initially,
442 there have been many studies reporting the beneficial effects
Figure 2. Effect of hyaluronic acid (HA) added to the in-vitro
maturation medium on the response to oestradiol on subsequent
bovine embryo development. Pooled data from three replicates
(mean ± SEM) (Ali et al., 2002). SOF = synthetic oviductal
fluid.
of LH on oocytes, as shown by an increase in embryo yield
after IVF and in-vitro culture (Younis et al., 1989; Zuelke and
Brackett, 1992; Gliedt et al. 1996; Choi et al., 2001). However,
recent findings have clearly shown that LH had no effect on
the developmental potential of bovine oocytes when added
to an IVM defined medium (Ali and Sirard, 2002b). These
results confirmed earlier findings from Izadyar and co-workers
(1996), who reported that when bovine oocytes were cultured
in the presence of FSH and human chorionic gonadotrophin
(HCG), only FSH, and not LH, influenced IVM of oocytes. The
absence of an in-vitro effect of LH during oocyte maturation
can be observed by the fact that the effect of LH on oocyte is
not direct. Oocyte donors with low serum LH also have low
oestradiol concentrations (Tesarik and Mendoza, 2002). In fact,
most COC used for IVM studies originated from small- and
medium-sized follicles (2–6 mm), and it has been demonstrated
that receptors for FSH but not LH are transcribed in the cumulus
and granulosa cells of these follicles (Van Tol et al., 1996). Such
contradictory results could partly be explained, in earlier studies
demonstrating a beneficial effect of LH supplementation, by the
possible use of LH preparations contaminated by FSH, thyroid
stimulating hormone (TSH) or other contaminants. They could
also be explained by the fact that LH receptor is not detected in
COC from small antral follicles and that in-vitro FSH priming
is initially required (Okazaki et al., 2003).
Another pituitary hormone that has been proposed in assisted
reproduction is growth hormone (GH). The role of GH in
ovarian function, follicular growth, and steroidogenesis is
well known and evidence shows a positive effect of GH on
oocyte maturation. GH concentration in FF has been shown
to be positively related to both normal fertilization and pre-
implantation embryo morphology and cleavage speed (Mendoza
et al., 1999). Moreover, GH is known to enhance intrafollicular
metabolic events required for oocyte maturation. A study
by Mendoza and co-authors (2002) also reported that GH
concentration in FF is significantly higher in conception IVF
as compared with non-conception cycles. Other observations in
women suggest the possibility of including GH during ovarian
 Review - In-vitro maturation of oocytes: biological aspects - A Ali et al.
stimulation in an ICSI programme to increase the efficiency of
delivery and live birth rates in women of ≥40 years (Tesarik
et al., 2005). The addition of GH during IVM has been shown
to accelerate nuclear maturation and promote subsequent
cleavage and embryonic development (Iga et al., 1998). As for
FSH, GH exerts its effects during oocyte maturation through a
cyclic adenosine monophosphate (cAMP) signal transduction
pathway. The promotory effect of GH on the developmental
competence of the oocyte is due to a higher fertilization rate as
a consequence of an improved cytoplasmic maturation (Izadyar
et al., 1998). These results were confirmed later in humans by
Menezo and co-workers (2003), who reported that GH receptor
is present in oocytes and early preimplantation embryos.
Protein sources
Follicular fluid
Follicular fluid composition changes during follicle growth. A
study by Sirard and co-authors demonstrated that addition of
selected follicular fluid influences the developmental potential
of oocytes obtained from unselected ovaries (Sirard et al.,
1995). In fact, the beneficial effect on development is present
in dominant follicles but not in all growing or regressing
dominant follicles, suggesting a fine tuning between growth and
differentiation signals. Therefore, follicular supplementation,
perhaps in the form of follicular fluid, influences oocyte
competence and subsequent developmental capacity as well as
embryonic quality
In follicular fluid, oestradiol concentrations are higher in large
than in small follicles (Gastal et al., 1999; Belin et al., 2000).
In cattle, before LH surge, the oestradiol concentration in the
follicular fluid is high (about 1 μg/ml) but sharply decreases
subsequently (Fortune and Hansel, 1985). Although there
is no evidence that oestradiol is involved in the resumption
of meiosis, it might be that oestradiol is involved in the
cytoplasmic changes that occur before LH surge. Follicular
fluid supplementation of IVM medium has been proven to
provide a beneficial microenvironment for further development
of the immature oocyte. High developmental rates after IVM,
especially after supplementation of bFF were obtained (Ali et
al., 2004). In this study, modified synthetic oviductal fluid (m-
SOF) was used successfully for bovine oocyte maturation and
gave good embryo development with respect to rate and quality,
closer to that observed in vivo.
Serum and IVM and fertilization
Historically, to support sperm capacitation and/or fertilization
ability, it was necessary to support basic media with proteins.
Initially, follicular fluid was used because it contains factors
that maintain sperm motility in vitro as well as components that
support capacitation and stimulate acrosome reaction, which
are prerequisites for sperm penetration of the zona pellucida
(Yanagimachi, 1969). Later, it was discovered that follicular
fluid could be replaced by serum albumin (Bavister, 1969).
This protein source has been universally used ever since for
sperm capacitation in IVF. Fetal calf serum (FCS) has long
been recognized as an important constituent for in-vitro oocyte
maturation. It is well known that FCS is necessary for FSH
induced cumulus expansion in hamsters and cattle (Leibfried-
Rutledge et al. 1986). Additionally, bovine oocytes matured in
the presence of FCS had higher levels of sperm penetration.
FCS contains a protein called fetuin that inhibits zona pellucida
hardening initiated by cortical granule release (Schroeder et
al., 1990). Low rates of fertilization have been documented
following IVM of human oocytes (Barnes et al., 1995, 1996).
While zona hardening has yet to be defined for IVM oocytes,
there is no doubt that the zona pellucida of day 3−5 human
embryos following IVM is extremely hard (Trounson et al.,
1994)
Serum and oocyte developmental
competence
Protein supplementation during IVM can affect the efficiency of
the procedure. In-vitro-matured human and bovine oocytes have
been shown to reduce protein content compared with in-vivo-
matured oocytes (Trounson et al., 2001), suggesting that proteins
play a critical role in acquisition of developmental competence.
FCS and maternal serum have been widely used and accepted
as protein supplements in IVF. Although oocytes can be cultured
without protein or hormone supplementation (Ali and Sirard,
2002a,b), embryo production seems to be improved when a
protein supplementation is included in the culture medium. The
role of FCS in oocyte developmental competence is still unclear.
In the authors’ experience, bovine oocytes are often cultured
in groups of 10 and, under these conditions, the effect of FCS
does not appear to be beneficial (Ali and Sirard, 2002b). In this
study, the authors suggested that the effect of FCS as a protein
supplement during IVM of bovine oocytes depends on the kind of
culture medium used. The same authors demonstrated that when
FCS is replaced by BSA as the only protein source during IVM of
oocytes, BSA not only delays nuclear maturation but also decreases
the developmental capacity of oocytes. These oocytes yielded the
lowest frequency of blastocyst development when compared with
maturation medium without BSA supplementation.
Influence of antioxidants on oocyte
developmental competence
Antioxidants may be beneficial additives to synthetic culture
media. In well-defined media, there is a possible lack of serum
factors or of other macromolecules that serve as reactive
oxygen species scavengers (ROS). It is well known that oxygen
concentration within the lumen of the female reproductive tract
is about one-third (3–9%) that found under standard in-vitro
conditions (Mastrioanni, 1965). Culture of embryos with a high
oxygen tension in vitro (20%) can produce more free radicals
(Fowler and Callingham, 1995) than embryo culture under
5% O2 or 7% O2 (Liu and Foote, 1995). Detrimental effects of
oxygen-derived free radicals during in-vitro culture have been
demonstrated in several species. ROS can induce mitochondrial
dysfunction, DNA, RNA and protein damage (Comporti, 1989)
as well as inhibiting sperm–oocyte fusion (Aitken, 1993). To
protect oocytes and embryos from oxidative stress during in-vitro
culture, various antioxidants can be added to culture media (Ali
et al. 2003).
Correct conditions for oocyte and embryo culture are not well
defined and any potential effect of a controlled O2 environment 443
 Review - In-vitro maturation of oocytes: biological aspects - A Ali et al.
can be observed when optimum conditions are maintained.
Cytoplasmic maturation of oocytes can be improved by
reducing oxidative stress caused by COC production of reactive
oxygen species due to the in-vitro culture environment (Ali et
al., 2003). Metabolic pathways, mediated by enzymes, such
as glutathione, can control ROS cellular concentrations and
protect the oocyte against damaging effects of oxidative stress.
Glutathione content of the oocyte can be increased by adding
thiol compounds such as cysteine, cysteamine, glutamine,
β-mercaptoethanol and/or follicular fluid to the maturation
medium (Jeong and Yang, 2001; Ali et al., 2003). Apart from
its protective action, glutathione also increases amino acid
transport and stimulates DNA and protein synthesis (Lafleur et
al., 1994).
Conclusion
Human oocyte maturation in vitro is becoming an effective
alternative to classical IVF treatment. The procedure avoids
ovarian stimulation, side effects of medical treatment and
other risks such as ovarian hyperstimulation syndrome (Chian
et al., 2004; Rao and Tan, 2005). Moreover, natural cycle and
IVM of oocytes can be considered as an economic alternative
to classical assisted reproduction based solely on its financial
cost–effectiveness in excluding the need to pharmacologically
stimulate the ovaries.
Following experience with the IVM procedure, the efficiency
of producing homogene embryo cohort with good quality and
viability is less than in classical IVF and intracytoplasmic sperm
injection. When comparing oocytes matured in vivo versus in
vitro, no apparent differences are seen at the level of nuclear
maturation, in the rates of fertilization or cleavage, but rather
in the developmental competence of the oocytes as exemplified
by poor embryonic developmental competence and pregnancy
rate. The clinical data have shown a biochemical pregnancy
rate of approximately 18%, with a 40% early miscarriage rate
(personal communication, Dr Benkhalifa). These observations
indicate that the cytoplasmic competence must be different
between in-vitro- and in-vivo-maturated oocytes. Advances
made in immature oocyte isolation and maturation, zygote and
embryo culture conditions have increased the clinical feasibility
of IVM. However, in order to achieve acceptable birth rates,
future studies should focus on characterization and regulation
of oocyte cytoplasmic maturation, and on how oocyte-derived
factors influence zygotic genome activation and embryonic
developmental competence. Simple maturation systems now
exist, using a fully defined medium in which the search for
factors improving acquisition of both nuclear and cytoplasmic
competences will be possible. Using these systems, IVM should
be performed in the light of clinical interest and at the laboratory
level to achieve the best possible chance of success.
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