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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: [email protected]

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

metaphase II (MII) until ovulation and fertilization.
The success of clinical IVF remains compromised by suboptimal
culture conditions, resulting in impaired embryo development IVM of human oocytes is becoming an emerging application
and subsequent loss of viability. Even though pregnancy rates in IVF laboratories, and has great promise. Methods used
have increased since the first human IVF attempts, this can be in cattle for IVM of primary oocytes have been applied to
primarily attributed to the introduction of ovarian stimulation women suffering from infertility (Cha et al., 1991). To be
and the subsequent replacement of an increasing number of successful, the oocyte should achieve two levels of maturation,
embryos. nuclear maturation through meiosis I and II progression, and
cytoplasmic maturation.
The initiation of oocyte in-vitro maturation (IVM) was first
reported by Pincus and Enzman (1935) and Edwards et al. In the study of Sirard and colleagues (1989) on the timing
(1965, 1969). An interesting feature of the mammalian oocyte of nuclear events during IVM, the germinal vesicle (GV)
is that oocytes undergo spontaneous maturation upon removal was observed from 0 to 6.6 h, GVBD occurred at 6.6–8.0 h,
from the follicle without stimulation factors (Edwards, 1965). chromatin condensation at 8–10.3 h, metaphase I (MI) at 10.3–
Resumption of meiosis leads to germinal vesicle breakdown 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 acquisition of developmental competence is under continuous
relationship between premature chromosome condensation and investigation. The present review describes some of biological
IVM of oocytes was suggested (Santalo et al., 1992). More aspects of oocyte maturation in vitro to date.
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). Oocyte−follicle relationship
Aneuploidy resulting from errors in meiotic chromosome
segregation is the leading cause of pregnancy losses (Li et al., Meiotic and developmental competence
2002). This could explain the reduction found in developmental
competence of IVM human oocytes compared with those In each menstrual cycle, a number of follicles are activated
matured in vivo and supports the need for improving in-vitro to enter a growth phase characterized by granulosa cell
culture conditions. proliferation and an increase in oocyte size. The capacity of
oocyte maturation is closely related to follicular maturation. It
Cytoplasmic maturation in itself describes both the ultrastructural has been found that in the human oocyte there is a decreased
changes that take place in the oocyte from the GV to the MII maturation rate in oocytes from small follicles (3–4 mm) when
stage and the acquisition of developmental competence of the compared with those from larger follicles (9–15 mm) (Tsuji
oocyte (Duranthon and Renard, 2001). Cytoplasmic maturation et al., 1985). The growing follicle must reach a minimum
is indirectly and retroactively assessed as the ability of the mature of 3–4 mm in size to acquire the capacity to respond to a
oocyte to undergo normal fertilization, cleavage and blastocyst developmental signal. This capacity will increase (percentage
development. Other indirect morphological parameters taken of oocytes capable of responding) as the follicle reaches a
into account to evaluate cytoplasmic maturation include plateau or a reduction of its growth rate, either by dominance
cumulus cell expansion, expulsion of the polar body and an or early atresia. Moor and Trounson (1977) observed in sheep
increased perivitelline space (Kruip et al., 1983). Oocyte that as the follicle size increased, the frequency of oocytes
maturation also involves complex interactions of different progressing to the second meiotic metaphase increased as well.
intracellular, paracrine and structural factors, including sterols, Their observation showed that follicle size could be used as an
steroids, growth factors, cyclic adenosine monophosphate and indictor for selecting oocytes with high meiotic competence.
gap junction (Jamnongjit and Hamnes, 2005). Many factors and
regulatory molecules have been shown either to inhibit or to With respect to follicle size, oocytes recovered during
promote nuclear and cytoplasmic oocyte maturation (Chian et the follicular phase show definitive differences in meiotic
al., 2004), either directly or via the cumulus cells. competence. However, oocytes recovered in the luteal phase,
irrespective of follicle size, result in a meiotic competence
Advances made in immature oocyte collection and culture similar to that of oocytes recovered from large follicles during
conditions have increased the clinical feasibility of IVM (Son et the follicular phase (Machatkova et al., 2004). After fertilization,
al., 2005). However, in order to achieve acceptable birth rates, these luteal-phase oocytes also demonstrate a high potential
future studies should focus on characterization and regulation to reach blastocyst stage. Also, in humans, early evidence
of oocyte cytoplasmic maturation, and on how oocyte-derived suggested that immature oocytes collected in the luteal phase
factors influence zygotic genome activation and embryonic have significantly higher maturation rates (Cha et al., 1992). In
developmental competence. addition, aspiration in the mid-follicular phase of antral follicles
(≥8 mm) in women with regular menstrual cycles, prior to the
In clinical application of IVM, nearly 50% of immature emergence of a dominant follicle, offers the best oocytes for
oocytes reach their maturation after 24–28 h (Abdul-Jalil et al., maturation and ultimately pregnancy rates (Mikkelsen et al.,
2001) with nearly 20−25% clinical pregnancy in patients with 1999). The relative influence of follicular and luteal phases on
polycystic ovarian syndrome at risk for ovarian hyperstimulation oocyte meiotic competence was not clearly defined, but may be
syndrome or in normo-ovulatory patients (Le Du et al., 2005; related to follicular atresia.
Mikkelsen, 2005). This result is in contrast to studies in other
species where MII is achieved much more easily. When As expected, the follicle size from which an oocyte is derived
immature oocytes were cultured in suitable conditions in vitro, is also related to its ability to progress from early cleavage
a significant proportion of oocytes reached nuclear maturation to blastocyst stages. Oocyte developmental competence
(bovine, Ali and Sirard, 2002; sheep, Guler et al., 2000; rabbit, characterizes the ability of such oocytes to develop to the
Lorenzo et al., 1996; pig, Ye et al., 2005). However, Barnes et al. blastocyst stage in vitro. This observation is well demonstrated
(1996) reported in humans significantly higher rates of oocyte in mouse, ewe and cow, where oocytes from small growing
maturation and fertilization of immature oocyte from patients follicles lack the ability to progress beyond the 2–8-cell stage.
with regular cycles compared with irregular and anovulatory Conversely, oocytes derived from large follicles (≥8 mm) may
patients. This study also showed that the culture of immature achieve blastulation rates comparable to IVM oocytes (Moor
oocytes for 48 h can increase the maturation rate (57 versus and Trounson 1977; Eppig et al. 1992). It appears that oocytes
82%) but did not affect the fertilization or the cleavage rate. require an additional ‘prematuration’ to express this competence
(Hendriksen et al., 2000). In vivo, this pre-maturation occurs
The optimization of clinical and biological aspects of IVM during pre-ovulatory growth before the LH surge. This suggests
cycles will enhance the take-home baby rate (Papanikolaou, that developmental competence is mainly acquired during
2005). Several factors determine the ultimate competence of oocyte growth within the follicle by an unknown mechanism
the oocyte; these have been investigated and attempts made (Sirard, 2001). Final steps in oocyte maturation are crucial to
to mimic these conditions in vitro. The complexity of the the acquisition of functional properties necessary for further
438 orchestration of the events that control oocyte growth and final 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 1996). Human oocytes in which the outer layers of cumulus
viable embryos, partly due to abnormal oocyte cytoplasmic granulosa are partially expanded are classified as atretic. These
maturation in vitro (Krisher and Bavister, 1998). Embryos oocytes results in high rates of maturation and fertilization
produced after IVM of human oocytes are often arrested at when compared with oocytes with healthy, tightly compacted
pronuclear or 4–8-cell stage when attempting to culture them cumulus granulosa (Blondin and Sirard, 1995; Blondin et al.,
to blastocysts (Barnes et al. 1996). During IVM programmes, 1997). In the final steps of follicular growth, even in atretic
in most cases, embryos obtained after IVM have been replaced follicles, oocytes could undergo a final maturational event at the
at 2–8-cell stage and their developmental potential to the cytoplasmic level that would render them competent to develop.
blastocyst stage is relatively unknown. When considering the Moreover, oocytes may retain their developmental competence
low pregnancy rate achieved after IVM of human oocytes, it acquired in the late follicular phase even when atresia is under
appears that human oocytes acquire their ability to mature and way; in that regard, it was shown that the cumulus−oocyte
develop to blastocyst relatively late during folliculogenesis. complex (COC) is the last part of the follicle to be affected by
Allowing such development of IVM of human oocytes to atresia (Kruip and Dieleman, 1982).
blastocysts could be an important selection tool for embryo
transfer (Barnes et al., 1995). Moreover, optimization of IVM It is well known that the follicle can profoundly influence the
protocols is vital not only for generating viable embryos, but quality of the oocyte obtained at ovulation and, as a result, the
also to support the development of subsequent offspring into quality of embryo obtained. Recently, bovine follicular fluid
normal adults (Eppig and O’Brien, 1998). (bFF) of competent follicles (>8 mm) from FSH-stimulated
animals was compared with bFF from small (2–5 mm) follicles
For single embryo selection prior to transfer, more than culture during IVM. The objective was to evaluate if adding bFF to
media development and improvement, there is a need for an the maturation medium level could influence the developmental
indicator of viability. Other visual assessments used to evaluate competency of selected oocytes obtained from 2–5-mm-sized
developmental competence include morphological evaluations follicles (Ali et al., 2004). The conclusion was that follicular
such as blastocoele expansion, number of blastomeres, and fluid originating from competent follicles increased the
trophectoderm to inner-cell mass ratio (Ali et al., 2004). developmental competence of abattoir-derived oocytes and, as
Moreover, functional evaluations such as the ability to resume a result, the quality of embryo obtained.
development after freezing and induce a pregnancy should
also be considered to provide a more complete idea of the In humans, follicular vascularity and the level of intrafollicular
developmental potential of the oocyte (Isachenko et al., 2004; oxygen appear to be important determinants of oocyte
Sirard et al., 2006). competence. Findings from several studies indicate that embryos
with the highest implantation potential originate from follicles
The ovarian follicular population is another parameter used that are well vascularized and oxygenated (Van Blerkom, 1998,
to estimate the developmental competence of the oocyte. The 2000).
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 Metabolic coupling
have at least one follicle larger than 10 mm or with more than
10 follicles of 2−5 mm have a high developmental potential. Within the follicle, there are several different somatic cell
In contrast, oocytes retrieved from ovaries with fewer than phenotypes that surround the oocyte. It is now widely
10 follicles of 2−5 mm or no follicle larger than 10 mm reach recognized that bi-directional communication between the
lower blastocyst rates with lower cell numbers (Gandolfi et al., oocyte and follicular somatic cells is fundamentally important
1997). 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
Follicular quality et al., 1998). The degree of expansion is also considered as a
morphological indicator of oocyte quality and directly related
It was observed that the development to blastocyst of an oocyte to developmental capacity of the oocyte to reach maturation
recovered from an atretic follicle (showing signs of cumulus (Ali and Sirard, 2002a).
expansion in the outer layers of the cumulus granulosa and
slight granulations in the oocyte cytoplasm), irrespective Earlier studies have demonstrated that granulosa cells are
of size, was equally good as that of an oocyte recovered metabolically coupled to oocyte via gap junctions during growth
from large non-atretic follicles. It is believed that some large and initial stages of maturation (Brower and Schultz, 1982; de
follicles contain developmentally incompetent oocytes, while Loos et al., 1991). Cessation of metabolic coupling coincides
some medium-sized follicles contain competent oocytes with the breakdown of the gap junction, which generally
(Blondin and Sirard, 1995). Follicular atresia may promote occurs just prior to the first meiotic metaphase. Nutritional and
the acquisition of developmental competence (Hendriksen regulatory elements responsible for growth, maintenance of
et al., 2000). Blondin et al. (1997) found that bovine oocytes meiotic arrest, and substrates for maturation and development
derived from ovaries maintained at 35°C for 4 h yielded a are able to pass through these gap junctions (Tanghe et al.,
higher frequency of blastocysts. Their data suggested that 2002).
developmental competence was acquired shortly prior to IVM
and depended on the handling conditions of post-mortem In cattle, it is speculated that granulosa cells, in response to LH,
ovaries. Similar conclusions to those of Blondin and Sirard synthesize pyruvate, which is transferred to the maturing oocyte.
(1995) have been observed in human oocytes (Barnes et al., 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), be altered by the in-vitro environment. These alterations can
indicating the interdependence of these cell types (Zuelke and become evident as errors that may be compatible with early
Brackett, 1993). embryonic development but are deleterious for viability.

It is evident that cumulus cells provide many important In most reports, IVM of mammalian oocytes was performed
functions not just for the oocyte but also for zygote, such as in incompletely defined systems containing co-cultured
pronuclei formation, polar body extrusions, and organelle somatic cells, blood serum, bovine serum albumin (BSA) or
redistribution and cytoskeletal rearrangements. The cumulus cell-conditioned medium. Besides being a potential source of
provides not only a microenvironment that would consist in infectious agents, these undefined components make it difficult
low concentrations of glucose and high lactate concentration, to undertake proper quality control and to evaluate the basic
but also provides homeostasis regulation for the oocyte and the requirements for metabolic substrates, nutritional, growth
early embryo (Mori et al., 2000; Tanghe et al., 2002). factors and hormones during IVM.

The beneficial effect of cumulus oophorus during IVM can be Pituitary hormones and oocyte
attributed to the formation of a favourable microenvironment
(biochemical or metabolic) around the oocyte (Tanghe et developmental competence
al., 2002). Cumulus cells participate in oocyte development
during IVM, either by secreting soluble factors, which induce In most mammalian oocytes, supplementation of in-vitro culture
developmental competence or by removing inhibitory or media by gonadotrophins, steroids and growth factors separately
toxic components from the maturation medium (Homa, 1995; or in combination can stimulate or inhibit cumulus expansion
Hashimoto et al., 1998). In addition, cumulus cells might have and/or nuclear and cytoplasm maturation. It is now common
unknown promoting effects on subsequent oocyte development practice in mammalian oocyte media to add these supplements
which might be attributable to intracellular changes such as pH during IVM. Supplementation with recombinant human FSH
or calcium ions (Mori et al., 2000). Another possible influence (rFSH) and 17β-oestradiol during IVM of bovine oocytes results
of cumulus cells during IVM of bovine oocytes might be in a positive effect by increasing the number of embryos after
that cumulus cells decrease oxygen tension in the immediate IVF (Ali and Sirard, 2002b). Media for IVM of human oocytes
vicinity of the oocyte as a result of an active metabolism of contain oestradiol to support cytoplasmic maturation, which is
the cumulus cells (Ali et al., 2003). By removing the cumulus necessary for fertilization and early embryonic development
cells artificially, it has been demonstrated that their presence (Tesarik and Mendoza, 1995). This is in agreement with the finding
during maturation is necessary for most oocytes to express that oocytes matured in the presence of higher concentrations
competency (Ali et al., 2005). In this study, oocyte MII and of oestradiol (1000 ng/ml) improved the developmental rate to
development to the 2–8-cell stage 72 h after insemination blastocyst stage (Ali and Sirard, 2002b).
were not affected by the absence of cumulus cells during
maturation. However, development beyond the blastocyst stage Other observations in cattle suggest the possibility of including
was significantly lower in cumulus-free than in cumulus-intact hyaluronic acid (HA) with oestradiol in culture media to
oocytes, indicating the importance of cumulus cells during increase the efficiency of in-vitro blastocyst production from
IVM of oocytes for cytoplasmic maturation and subsequent IVM oocytes using completely defined conditions (Ali et al.,
early development (Figure 1). 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
Culture conditions presence of cumulus cells during IVM might be necessary to
express the competence of oocytes and that cumulus expansion
It is known that treating the cause instead of the symptoms is not required to improve this competence; at the least, there
produces the most encouraging results. Early embryo is no linear relationship between cumulus expansion and
development appears to be dependant on the maturation cytoplasmic maturation (Ali and Sirard, 2002a,b). Moreover,
microenvironment of the oocyte (Table 1). Significant these results suggest that communication between the oocyte
improvements in the overall rate of oocyte maturation and and surrounding cumulus cells is important in determining
embryo development can be achieved by adding mature which factors are released by the cumulus cells (Ail and Sirard,
mammalian fluid from large follicles (containing oocytes more 2005; Ali et al., 2005).
likely to develop into blastocysts) in the culture media (Blondin
et al., 2002; Ali et al., 2004). In cattle, the concentration of oestradiol in the follicular fluid of
a dominant follicle is higher than in others, indicating a possible
Early embryo development is a complex mechanism, based on role of oestradiol on the cytoplasmic maturation occurring
interactions between intracellular and extracellular cell biology before LH surge. Recently, studies in cattle have postulated
of oocyte and spermatozoa development and maturation. It is that increased developmental competence of bovine oocytes
therefore quite remarkable that oocytes matured and embryos in response to rFSH and oestradiol occurs by the production
produced in vitro still maintain a reasonable degree of functional of factors that reach the oocyte through the cell−cell coupling
and developmental competence. Studies have investigated pathways or that the coupling per se results in physiological
a number of culture modifications, based on physiological changes (Ali and Sirard, 2005; Ali et al., 2005). Moreover,
conditions found in the female reproductive tract, to improve these studies provide novel evidence of a direct role of rFSH in
embryo developmental outcome. In-vitro conditions cannot the regulation of gap junction communication between cumulus
truly replicate in-vivo conditions. Features of oocyte and cells and oocytes and the consequences of such conditions on
440 embryo morphology, metabolic and biochemical properties can 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.

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.

The roles of protein kinase A (PKA) and protein kinase C of LH on oocytes, as shown by an increase in embryo yield
(PKC) (possibly involved in rFSH response), were investigated after IVF and in-vitro culture (Younis et al., 1989; Zuelke and
recently (Ali and Sirard, 2005) using activators (Sp-cAMPS, Brackett, 1992; Gliedt et al. 1996; Choi et al., 2001). However,
PMA) or inhibitors (Rp-cAMPS, sphingosine) of these two recent findings have clearly shown that LH had no effect on
protein kinases respectively. The developmental competence of the developmental potential of bovine oocytes when added
oocytes was measured by the rate of blastocyst formation after to an IVM defined medium (Ali and Sirard, 2002b). These
IVF. This study is one of the first to report such high development results confirmed earlier findings from Izadyar and co-workers
rates of bovine oocytes after IVM using defined conditions, (1996), who reported that when bovine oocytes were cultured
especially after short-term treatment with rFSH. The data show in the presence of FSH and human chorionic gonadotrophin
that the PKC pathway is implicated in r-FSH, which improved (HCG), only FSH, and not LH, influenced IVM of oocytes. The
oocyte competence, especially after short-term treatment. These absence of an in-vitro effect of LH during oocyte maturation
results indicate that the PKA and PKC pathways can modulate can be observed by the fact that the effect of LH on oocyte is
the maturation of oocytes in vitro. A better understanding of not direct. Oocyte donors with low serum LH also have low
the mechanism by which rFSH stimulates the oocytes can have oestradiol concentrations (Tesarik and Mendoza, 2002). In fact,
important implications in the light of clinical interest in the use most COC used for IVM studies originated from small- and
of rFSH in assisted reproduction protocols. medium-sized follicles (2–6 mm), and it has been demonstrated
that receptors for FSH but not LH are transcribed in the cumulus
During the period of folliculogenesis and oocyte maturation in and granulosa cells of these follicles (Van Tol et al., 1996). Such
vivo, growing evidence indicates an important role of both FSH contradictory results could partly be explained, in earlier studies
and LH. In human IVF, Filcori (1999) reported that a minimal demonstrating a beneficial effect of LH supplementation, by the
level of LH activity during exogenous stimulation protocols possible use of LH preparations contaminated by FSH, thyroid
is required to optimize ovulation induction in patients. In stimulating hormone (TSH) or other contaminants. They could
fact, it has been postulated that profound LH suppression also be explained by the fact that LH receptor is not detected in
could affect optimal oocyte maturation and/or endometrial COC from small antral follicles and that in-vitro FSH priming
development (Lévy et al., 2000). Moreover, the final stages of is initially required (Okazaki et al., 2003).
oocyte maturation in vivo are induced by a rise in serum LH
concentrations. Schoolcraft and co-workers (1999) reported Another pituitary hormone that has been proposed in assisted
that including LH in ovarian stimulation protocols using highly reproduction is growth hormone (GH). The role of GH in
purified FSH preparations would improve blastocyst quality ovarian function, follicular growth, and steroidogenesis is
as reflected by increased embryo implantation and pregnancy well known and evidence shows a positive effect of GH on
rate. However, LH supplementation, particularly in ovarian oocyte maturation. GH concentration in FF has been shown
stimulation protocols using GnRH agonists, and the concept to be positively related to both normal fertilization and pre-
of ‘window’ for LH requirement, still remain today a matter implantation embryo morphology and cleavage speed (Mendoza
of debate (Tesarik and Mendoza, 2002; Humaidan, 2006; et al., 1999). Moreover, GH is known to enhance intrafollicular
Kolibianakis et al., 2006). metabolic events required for oocyte maturation. A study
by Mendoza and co-authors (2002) also reported that GH
The effect of LH during IVM on oocyte maturation and concentration in FF is significantly higher in conception IVF
subsequent development has also been questioned. Initially, as compared with non-conception cycles. Other observations in
442 there have been many studies reporting the beneficial effects 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 induced cumulus expansion in hamsters and cattle (Leibfried-
delivery and live birth rates in women of ≥40 years (Tesarik Rutledge et al. 1986). Additionally, bovine oocytes matured in
et al., 2005). The addition of GH during IVM has been shown the presence of FCS had higher levels of sperm penetration.
to accelerate nuclear maturation and promote subsequent FCS contains a protein called fetuin that inhibits zona pellucida
cleavage and embryonic development (Iga et al., 1998). As for hardening initiated by cortical granule release (Schroeder et
FSH, GH exerts its effects during oocyte maturation through a al., 1990). Low rates of fertilization have been documented
cyclic adenosine monophosphate (cAMP) signal transduction following IVM of human oocytes (Barnes et al., 1995, 1996).
pathway. The promotory effect of GH on the developmental While zona hardening has yet to be defined for IVM oocytes,
competence of the oocyte is due to a higher fertilization rate as there is no doubt that the zona pellucida of day 3−5 human
a consequence of an improved cytoplasmic maturation (Izadyar embryos following IVM is extremely hard (Trounson et al.,
et al., 1998). These results were confirmed later in humans by 1994)
Menezo and co-workers (2003), who reported that GH receptor
is present in oocytes and early preimplantation embryos.
Serum and oocyte developmental
competence
Protein sources
Protein supplementation during IVM can affect the efficiency of
Follicular fluid the procedure. In-vitro-matured human and bovine oocytes have
been shown to reduce protein content compared with in-vivo-
Follicular fluid composition changes during follicle growth. A matured oocytes (Trounson et al., 2001), suggesting that proteins
study by Sirard and co-authors demonstrated that addition of play a critical role in acquisition of developmental competence.
selected follicular fluid influences the developmental potential FCS and maternal serum have been widely used and accepted
of oocytes obtained from unselected ovaries (Sirard et al., as protein supplements in IVF. Although oocytes can be cultured
1995). In fact, the beneficial effect on development is present without protein or hormone supplementation (Ali and Sirard,
in dominant follicles but not in all growing or regressing 2002a,b), embryo production seems to be improved when a
dominant follicles, suggesting a fine tuning between growth and protein supplementation is included in the culture medium. The
differentiation signals. Therefore, follicular supplementation, role of FCS in oocyte developmental competence is still unclear.
perhaps in the form of follicular fluid, influences oocyte In the authors’ experience, bovine oocytes are often cultured
competence and subsequent developmental capacity as well as in groups of 10 and, under these conditions, the effect of FCS
embryonic quality does not appear to be beneficial (Ali and Sirard, 2002b). In this
study, the authors suggested that the effect of FCS as a protein
In follicular fluid, oestradiol concentrations are higher in large supplement during IVM of bovine oocytes depends on the kind of
than in small follicles (Gastal et al., 1999; Belin et al., 2000). culture medium used. The same authors demonstrated that when
In cattle, before LH surge, the oestradiol concentration in the FCS is replaced by BSA as the only protein source during IVM of
follicular fluid is high (about 1 μg/ml) but sharply decreases oocytes, BSA not only delays nuclear maturation but also decreases
subsequently (Fortune and Hansel, 1985). Although there the developmental capacity of oocytes. These oocytes yielded the
is no evidence that oestradiol is involved in the resumption lowest frequency of blastocyst development when compared with
of meiosis, it might be that oestradiol is involved in the maturation medium without BSA supplementation.
cytoplasmic changes that occur before LH surge. Follicular
fluid supplementation of IVM medium has been proven to Influence of antioxidants on oocyte
provide a beneficial microenvironment for further development
of the immature oocyte. High developmental rates after IVM, developmental competence
especially after supplementation of bFF were obtained (Ali et
al., 2004). In this study, modified synthetic oviductal fluid (m- Antioxidants may be beneficial additives to synthetic culture
SOF) was used successfully for bovine oocyte maturation and media. In well-defined media, there is a possible lack of serum
gave good embryo development with respect to rate and quality, factors or of other macromolecules that serve as reactive
closer to that observed in vivo. 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
Serum and IVM and fertilization conditions (Mastrioanni, 1965). Culture of embryos with a high
oxygen tension in vitro (20%) can produce more free radicals
Historically, to support sperm capacitation and/or fertilization (Fowler and Callingham, 1995) than embryo culture under
ability, it was necessary to support basic media with proteins. 5% O2 or 7% O2 (Liu and Foote, 1995). Detrimental effects of
Initially, follicular fluid was used because it contains factors oxygen-derived free radicals during in-vitro culture have been
that maintain sperm motility in vitro as well as components that demonstrated in several species. ROS can induce mitochondrial
support capacitation and stimulate acrosome reaction, which dysfunction, DNA, RNA and protein damage (Comporti, 1989)
are prerequisites for sperm penetration of the zona pellucida as well as inhibiting sperm–oocyte fusion (Aitken, 1993). To
(Yanagimachi, 1969). Later, it was discovered that follicular protect oocytes and embryos from oxidative stress during in-vitro
fluid could be replaced by serum albumin (Bavister, 1969). culture, various antioxidants can be added to culture media (Ali
This protein source has been universally used ever since for et al. 2003).
sperm capacitation in IVF. Fetal calf serum (FCS) has long
been recognized as an important constituent for in-vitro oocyte Correct conditions for oocyte and embryo culture are not well
maturation. It is well known that FCS is necessary for FSH 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. follicle stimulating hormone. Reproduction 130, 303–310.
Cytoplasmic maturation of oocytes can be improved by Ali A, Sirard MA 2002a Effect of the absence or presence of various
reducing oxidative stress caused by COC production of reactive protein supplements on further development of bovine oocytes
during in vitro maturation. Biology of Reproduction 66, 901–905.
oxygen species due to the in-vitro culture environment (Ali et
Ali A, Sirard MA 2002b The effects of 17beta-estradiol and protein
al., 2003). Metabolic pathways, mediated by enzymes, such supplement on the response to purified and recombinant follicle
as glutathione, can control ROS cellular concentrations and stimulating hormone in bovine oocytes. Zygote 10, 65–71.
protect the oocyte against damaging effects of oxidative stress. Ali A, Paradis F, Vigneault C et al. 2005 The potential role of gap
Glutathione content of the oocyte can be increased by adding junction communication between cumulus cells and bovine
thiol compounds such as cysteine, cysteamine, glutamine, oocytes during in vitro maturation. Molecular Reproduction and
β-mercaptoethanol and/or follicular fluid to the maturation Development 71, 358–367.
medium (Jeong and Yang, 2001; Ali et al., 2003). Apart from Ali A, Coenen K, Bousquet D, Sirard MA 2004 Origin of bovine
follicular fluid and its effect during in vitro maturation on the
its protective action, glutathione also increases amino acid
developmental competence of bovine oocytes. Theriogenology 62,
transport and stimulates DNA and protein synthesis (Lafleur et 1596–1606.
al., 1994). Ali A, Bilodeau J-F, Sirard M-A 2003 Antioxidants requirement for
bovine oocytes varies during in vitro maturation, fertilization and
development. Theriogenology 59, 939–949.
Conclusion Ali A, Massicotte L, Sirard MA 2002 Effects of hyaluronic acid during
in vitro maturation on further development of bovine oocytes.
Human oocyte maturation in vitro is becoming an effective Biology of Reproduction 66, 207–212.
alternative to classical IVF treatment. The procedure avoids Barnes FL, Kausche A, Tiglias J et al. 1996 Production of embryos
ovarian stimulation, side effects of medical treatment and from in vitro-matured primary human oocytes. Fertility and
other risks such as ovarian hyperstimulation syndrome (Chian Sterility 65, 1151–1156.
Barnes FL, Crombie A, Gardner DK et al. 1995 Blastocyst
et al., 2004; Rao and Tan, 2005). Moreover, natural cycle and
development and birth after in-vitro maturation of human primary
IVM of oocytes can be considered as an economic alternative oocytes, intracytoplasmic sperm injection and assisted hatching.
to classical assisted reproduction based solely on its financial Human Reproduction 10, 3243–3247.
cost–effectiveness in excluding the need to pharmacologically Bavister BD 1969 Environmental factors important for in vitro
stimulate the ovaries. fertilisation in the hamster. Journal of Reproduction and Fertility
18, 544–545.
Following experience with the IVM procedure, the efficiency Belin G, Goudet G, Duchamp N 2000 Gerard, Intrafollicular
of producing homogene embryo cohort with good quality and concentrations of steroids and steroidogenic enzymes in relation
to follicular development in the mare. Biology of Reproduction 62,
viability is less than in classical IVF and intracytoplasmic sperm
1335–1343.
injection. When comparing oocytes matured in vivo versus in Blondin P, Sirard MA 1995 Oocyte and follicular morphology as
vitro, no apparent differences are seen at the level of nuclear determining characteristics for developmental competence in
maturation, in the rates of fertilization or cleavage, but rather bovine oocytes. Molecular Reproduction and Development 41,
in the developmental competence of the oocytes as exemplified 54–62.
by poor embryonic developmental competence and pregnancy Blondin P, Bousquet D, Twagiramungu H et al. 2002 Manipulation
rate. The clinical data have shown a biochemical pregnancy of follicular development to produce developmentally competent
rate of approximately 18%, with a 40% early miscarriage rate bovine oocytes. Biology of Reproduction 66, 38–43.
Blondin P, Coenen K, Sirard MA 1997 The impact of reactive oxygen
(personal communication, Dr Benkhalifa). These observations
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between in-vitro- and in-vivo-maturated oocytes. Advances Brower PT, Schultz RM 1982 Intercellular communication between
made in immature oocyte isolation and maturation, zygote and granulosa cells and mouse oocytes: existence and possible
embryo culture conditions have increased the clinical feasibility nutritional role during oocyte growth. Developmental Biology 9,
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Cha KY, Koo JJ, Ko JJ et al. 1991 Pregnancy after in vitro fertilization
developmental competence. Simple maturation systems now of human follicular oocytes collected from nonstimulated cycles,
exist, using a fully defined medium in which the search for their culture in vitro and their transfer in a donor oocyte program.
factors improving acquisition of both nuclear and cytoplasmic Fertility and Sterility 55, 109–113.
competences will be possible. Using these systems, IVM should Chian RC, Lim J, Tan SL 2004a State of the art in in vitro oocyte
be performed in the light of clinical interest and at the laboratory maturation. Current Opinion in Obstetrics and Gynecology 16,
level to achieve the best possible chance of success. 211–219.
Chian RC, Buckett WM, Tan SL 2004b In-vitro maturation of human
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