Human Reproduction Vol.18, No.7 pp. 1494±1503, 2003
DOI: 10.1093/humrep/deg272
Multiparameter analysis of human oocytes at metaphase II
stage after IVF failure in non-male infertility
FaõÈcËal Miyara1, FrancËois-Xavier Aubriot2, AmeÂlie Glissant2, Catherine Nathan2,
SteÂphane Douard2, Alexandre Stanovici2, Florence Herve2, Martine Dumont-Hassan2,3,
Alain LeMeur3, Paul Cohen-Bacrie3 and Pascale Debey1,4
1
UMR1198 Biologie du DeÂveloppement et Reproduction, INRA, Jouy-en-Josas Cedex, 2Centre FIV Pierre Cherest, Neuilly-sur-Seine
Cedex and 3Laboratoire d'Eylau, Paris, France
4
To whom correspondence should be addressed at: UMR1198 Biologie du DeÂveloppement et Reproduction, INRA, 78352,
Jouy-en-Josas Cedex, France. E-mail: [email protected]
BACKGROUND: Using ¯uorescence imaging, an a posteriori multiparametric analysis was performed of human
oocytes which failed to give pronucleated zygotes after IVF in cases of very low rates of fertilization or complete fertilization failure. METHODS: The analysis included: (i) the state of the maternal and paternal chromatin; (ii) quality of the metaphase II oocytes; and (iii) cortical granule (CG) distribution. RESULTS: Most oocytes were arrested
in metaphase II, but they were abnormal in 50% of cases. The incidence of spindle and chromosome aberrations
was strongly in¯uenced by maternal age (69% for 40- to 45-year-old women versus 35% for 26- to 33-year-olds),
and sperm chromatin was always condensed in immature oocytes, and fully decondensed only in normal metaphase
II. The migration of CGs appeared to be associated with achievement of nuclear maturation at the time of puncture.
CONCLUSIONS: These factors, when analysed on a complete set of oocytes from the same patient, provided information about potential causes of IVF failure, and also represented part of an `oocyte quality evaluation' to select the
assisted fertilization technique most suitable for each patient. For example, when the majority of oocytes were
judged non-fertilizable at a ®rst attempt, no pregnancy was registered at any subsequent attempt.
Key words: cortical granules/human metaphase II oocytes/IVF failure/metaphase-promoting factor/nuclear and cytoplasmic
competence
Introduction
Today, IVF is used extensively to bypass certain cases of
infertility such as tubal disease, anovulation, endometriosis and
unexplained infertility, and at present is successful in 50±70%
of cases (Ola et al., 2001). Previously, fertilization failure
following classical IVF was thought to be due to a lack of
sperm penetration through the zona pellucida (ZP) and/or lack
of membrane fusion, but the zona and oolemma barriers can be
bypassed using ICSI (Palermo et al., 1992; 2002). Besides
fertilization failures after conventional IVF (Fishel et al.,
2000), ICSI programmes have been expanded to include
patients with poor quality sperm, azoospermia, immunological
infertility and chemotherapy/radiotherapy treatments (Horne
et al., 2001). Nonetheless, the fertilization rate for ICSI
¯uctuates between 50 and 70%; i.e., it is not higher than the
IVF rate for non-male factor patients (Ola et al., 2001). These
data suggest that factors other than sperm binding and
penetration may limit the fertilization rate. Owing to the
psychological and economical cost of performing repetitive
ICSI, the relevance of carrying out such a technique must be
questioned in each individual case.
1494
The success of clinical IVF is based largely on the ability to
retrieve good quality metaphase II (MII) oocytes at a high
frequency. The quality of oocytes is characterized by interrelated factors, generally classi®ed respectively as nuclear and
cytoplasmic `competence'. Nuclear competence characterizes
the quality of oocyte chromatin and spindle (Mattson and
Albertini, 1990; Albertini, 1992), which is essentially governed
by the level of the metaphase-promoting factor (MPF) activity
(Hashimoto and Kishimoto, 1988; Murray, 1989; Verde et al.,
1990; 1992). MPF governsÐeither directly or indirectlyÐall
processes linked to metaphase entry in eukaryotic cells, such as
nuclear envelope breakdown (Ookata et al., 1992), histone
phosphorylation (Collas, 1999) and microtubule polymerization (Charrasse et al., 2000). Mitogen-activated protein (MAP)
kinase (a serine/threonine kinase) is another principal regulator
of oocyte maturation, and its action in regulating cell cycle
events may be uncoupled from MPF in mammalian oocytes
(Sun et al., 1999). The results of recent studies have shown that
MAP kinase is more important than MPF in controlling
chromatin and microtubule behaviour in both mouse (Verlhac
et al., 1994) and porcine oocytes (Sun et al., 2001; 2002).
ã European Society of Human Reproduction and Embryology
Analysis of human oocytes after IVF failure
Activation of MAP kinases occurs after germinal vesicle
breakdown (GVBD) and keeps microtubules and chromatin
from entering into an interphase con®guration (Sun et al.,
1999). In human oocytes, extrusion of the ®rst polar body and
formation of the MII oocyte occurs at 32 h after ovarian
stimulation, this being 6±8 h before ovulation (BomselHelmreich et al., 1987). The presence of a functional spindle
ensures the ®delity of chromosome segregation, and is thus an
essential feature of healthy MII oocytes (Eichenlaub-Ritter
et al., 1986; Van Blerkom and Henry, 1992; Volarcik et al.,
1998; Van Blerkom and Davis, 2001). Ageing of MII oocytes,
thermal changes and insuf®cient oxygen supply throughout the
entire culture period (Hu et al., 2001; Wang et al., 2001) may
cause concomitantly a deterioration of the spindle and
displacement of chromosomes from the spindle equator
(Eichenlaub-Ritter et al., 1986).
Cytoplasmic maturation can be considered as the sum of the
processes by which the mammalian oocyte changes from a
developmentally incompetent cell to one with the capacity to
support fertilization and early embryonic development. One of
these changes concerns the redistribution of cell organelles
named cortical granules (CGs) (Ducibella et al., 1990; 1993;
Yoshida et al., 1993). CGs are small (0.2±0.6 mm diameter)
secretory granules which were ®rst identi®ed using electron
microscopy and originate from the Golgi body (Gulyas, 1976;
Manna et al., 2001). During oocyte growth, the CGs increase in
number and migrate toward the cortex, assuming a position
0.4±0.6 mm below the plasma membrane (Ducibella et al.,
1988; 1994). In large germinal vesicle (GV) mouse oocytes,
most of the CGs are uniformly localized in the cortex, while a
population of CGs remains in the interior. Meiotic maturation
is accompanied by an additional movement of CGs residing in
the interior to the periphery of the oocyte, as well as the
formation of a CG-free domain (CGFD) overlying the MII
spindle (Ducibella et al., 1988; 1990; Connors et al., 1998).
The cortical accumulationÐbut not formation of the CGFDÐ
is observed during meiotic maturation in others mammals, such
as cat (Byers et al., 1992), pig (Sun et al., 2001), cattle (Izadyar
et al., 1998) and human (Ghetler et al., 1998). CGs contain
mucopolysaccharides and several enzymes such as proteases,
peroxidase and acid phosphatase (Flechon, 1970; Moller and
Wassarman, 1989; Hoodbhoy and Talbot, 1994). Electron
microscopic studies have shown that the CGs positioned
beneath the plasma membrane undergo exocytosis in response
to an inositol triphosphate (IP3)-induced calcium rise generated by fertilization or parthenogenetic activation (Schultz and
Kopf, 1995) and extrude their contents into the perivitellin
space (Chamow and Hedrick, 1986). This release modi®es the
extracellular coat of the oocyte (Connors et al., 1998), thus
inducing an extracellular block to polyspermy.
Another criterion of cytoplasmic maturation is the ability to
decondense the sperm chromatin following sperm penetration
(Navara et al., 1995) and to transform it into the male
pronucleus. Decondensation of the spermatozoon is considered
to occur independently from the resumption of meiosis, under
the in¯uence of a cytoplasmic factor called sperm decondensation factor (SDF), the synthesis of which is induced during
normal oocyte maturation (Moor and Gandol®, 1987). In
contrast, changes in sperm chromatin beyond the initial
decondensation stage depend on cytoplasmic conditions
which also permit female pronucleus formation; that is, the
decrease in MPF activity triggered by sperm entry (Adenot
et al., 1991; Gook et al., 1998; Kovacic and Vlaisavljevic,
2000; Bao et al., 2002). Moreover, in-vitro insemination of
metaphase I oocytes leads to condensation of male chromatin
into premature chromosome condensation (PCC) (Clarke and
Masui, 1986; 1987).
The aim of the present study, which involved the close
collaboration of clinicians and cell biologists, was to evaluate
oocyte and/or sperm defects in cases of very low rates of
fertilization or complete fertilization failure. For that purpose,
an a posteriori multiparametric analysis was undertaken of
human oocytes which failed to produce pronucleated zygotes
after IVF. Patients presented with tubal disease, endometriosis,
polycystic ovary syndrome (PCOS) or unexplained infertility.
The decision was made to analyse both female and male
chromatin, together with the microtubular array, as well as CG
distribution. When used in conjunction with the patient clinical
data, these observationsÐwhich may be gathered using regular
microscopic equipmentÐare aimed towards understanding the
physiological causes of fertilization failure, and represent
valuable tools for the future orientation of therapeutics in the
treatment of infertility.
Materials and methods
Patients and protocols
A total of 713 oocytes (155 patients) originating from cohorts where
<20% oocytes were fertilized, was included in this study. Indications
for IVF included endometriosis (n = 30), PCOS (n = 6) and idiopathic
infertility (n = 62). The sperm was normal in all cases. Cases of
combined male and female infertility (25 patients) were also included
in the study. Sperm parameters were evaluated according to World
Health Organization (WHO) guidelines (Belsey et al., 1980). For
ovarian stimulation, most patients (n = 137) were down-regulated with
GnRH agonist (3.75 mg Decapeptyl; Ispen-Biotech Laboratories
France, or Enantone; Takeda, France) and subsequently underwent
controlled ovarian hyperstimulation with either FSH (Gonal F, Serono
France; or Puregon; Organon, France) or hMG (Menogon; Ferring,
France). Some patients (n = 8) underwent controlled ovarian
hyperstimulation using gonadotrophins and GnRH antagonist
(Cetrotide; Serono, France). In all cases where at least one follicle
had reached a diameter of 16±18 mm, a single dose of hCG (5000 or
10 000 IU) was administered s.c.
Oocyte collection
At ~36 h after stimulation, oocytes were retrieved using transvaginal
ultrasound-guided aspiration. Cumulus-enclosed oocytes were collected in IVFÔ-20 medium (Vitrolife; MoÈlndalsvaÈgen, Gothenburg,
Sweden) and left intact for conventional IVF. Cumulus±oocyte
complexes were inseminated with ~23105 motile sperm in 1 ml
Scandinavian IVF medium at 37°C in 5% CO2 in an humidi®ed
atmosphere. Fertilization was assessed at 17±20 h after insemination
(day 1) by the presence of two pronuclei and polar bodies. On day 2,
normal embryos were transplanted to the uterine cavity or, if
necessary, frozen for later transplantation. Oocytes remaining at the
MII stage at 48 h after insemination were removed and used for further
multiparametric analysis. Patients from ICSI programmes who had not
1495
F.Miyara et al.
Table I. State of maternal chromatin in non-fertilized oocytes issued from cohorts with very low or zero fertilization rates in IVF procedures
Patient age (years)
Patients (n)
Oocytes (n)
GV/MI
MII
26±33
34±39
40±45
Total
60
54
41
155
281
269
163
713
35
39
11
85
231
192
135
558
(12.5)
(14.5)
(7.0)
(12.0)
Parthenogenetic
(82.0)
(71.5)
(82.5)
(78.0)
15
38
17
70
(5.5)
(14.0)
(10.5)
(10.0)
Values in parentheses are percentages.
GV = germinal vesicle; MI = metaphase I; MII = metaphase II.
been inseminated (e.g. azoospermia due to complete absence of
spermatozoa) donated control oocytes at the MII stage. The MII-stage
controls were analysed on the same day as puncture.
Fluorescence staining of meiotic spindles and chromosomes
Oocytes were ®xed in 2% paraformaldehyde (PFA) in phosphatebuffered saline (PBS) at 37°C for 30 min; this ®xative is known to
preserve cellular structures and antigenic sites (Grootenhuis et al.,
1996). Oocytes were then rinsed in PBS, permeabilized by the
addition of 1% Triton X-100 in PBS for 20 min, and incubated in PBS
+ 2% bovine serum albumin (BSA). Incubation with the primary
antibody, a mouse monoclonal antibody (IgG) raised against a-tubulin
(Clone DM1A; Amersham Pharmacia, France) diluted 1:400 in PBS +
2% BSA, was performed overnight at 4°C. Oocytes were rinsed
several times in PBS, and incubated for 1 h at room temperature with
the secondary antibody, a ¯uoroscein isothiocyanate (FITC)-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch
Laboratories, West Grove, USA) diluted 1:200 in PBS + 2% BSA.
Excess antibody was washed out in PBS. The oocytes were labelled
using 2 mg/ml Hoechst 33342 (Riedel de Haen, Germany) for 20 min,
transferred into PBS, and then mounted under a coverslip in citi¯uor
(Citi¯uor Products, Canterbury, UK).
Stained oocytes were examined using an inverted microscope (Carl
Zeiss, Oberkochen, Germany) that was equipped for epi¯uorescence
and ®tted with appropriate ®lter combinations. Images were recorded
through a charge-coupled device (CCD) camera (Type KAF 1400, 12bit range; Photometrics, Tucson, AZ, USA) cooled to 10°C, and
coupled to the IPLAB spectrum imaging software (Vysis, France).
Acquired images were processed using Adobe Photoshop 6.0 (Adobe
Systems, Mountain View, CA, USA).
Analysis of CG distribution
Cortical granules were labelled with lens culinaris agglutinin (LCA);
this lectin was reported previously to bind speci®cally to the CGs
content and exudate (Ducibella et al., 1994). Oocytes were ®rst ®xed
as above in 2% PFA in PBS for 30 min at 37°C, and washed in PBS
containing 2% BSA. The ZP was then removed mechanically with a
small needle and narrow-mouth glass pipettes. Previous attempts to
use pronase (0.25%) or a-chymotrypsin (0.01%) before ®xation
failed, because: (i) a large number of oocytes, in which the ZP was not
easily detached, were lysed due to over-exposure to enzymes; and (ii)
a-chymotrypsin and pronase caused holes to develop in the plasma
membrane, thereby rendering the results of the CG labelling
uninterpretable. Labelling of CGs was subsequently performed
according to a previously published method (Ghetler et al., 1998).
Brie¯y, oocytes were incubated in 5 mg/ml biotin-conjugated LCA
(Sigma-Aldrich Chimie, France) for 30 min, and stained with 2 mg/ml
rhodamine red±streptavidin (Jackson ImmunoResearch) for 30 min.
Finally, oocytes were labelled with 1 mg/ml Sytox green (Molecular
Probes, Eugene, OR, USA), which stains double-stranded DNA for 15
min, transferred into PBS, mounted under a coverslip in citi¯uor, and
1496
then observed on an inverted microscope (Nikon France, Paris,
France) equipped with Bio-Rad LaserSharp MRC-1024 confocal laser
scanning software (Elexience, Paris, France). The objective lens was a
Nikon Fluor oil-immersion 1003 (NA 1.3), and the pinhole was set at
between 1.5 and 2.8 mm. The 488 nm and 568 nm wavelengths of the
laser were used for excitation of ¯uorescein (or Sytox green) and
rhodamine respectively. Optical sections were imaged at 5 mm
intervals.
Results
Assessment of sperm penetration and female chromatin
status
The maturation status of the oocyte, as well as the presence and
condensation state of the sperm chromatin, can be assessed by
using a-tubulin immunolabelling coupled to DNA staining by
the speci®c ¯uorescent probe, Hoechst 33342. In fact, 85.5% of
the oocytes did not show any presence of sperm. Hence,
oocytes could be classi®ed into three different categories
(Table I):
1. Meiotically immature oocytes (12% of cases), de®ned by
the absence of a polar body (PB0) and the presence of maternal
chromatin at the GV or ®rst meiotic metaphasic plate (MI)
stages. Both of these couldÐor could notÐbe penetrated by
sperm. In the case of MI, the spermatozoon was generally still
condensed in the ooplasm or formed PCC organized in a
spindle characterized by the presence of the sperm tail at one of
the poles (Figure 1a,a¢).
2. Mature oocytes were de®ned by the presence of a polar
body (PB1) and the presence of the second meiotic metaphasic
plate (MII) (78% of cases). If, after 48 h of culture, MII was
localized close to the PB1 (`cortical position'), this suggested
that PB1 extrusion was recent and the oocyte was immature at
the time of retrieval (Van Wissen et al., 1992) (Figure 2A,b and
c). By contrast, `overmature' or aged oocytes were characterized by the migration of MII away from PB1 (`centripetal
position') (Figure 2A,d). In that case, oocytes were probably
mature at retrieval and aged in vitro. In both types of oocytes,
sperm either couldÐor could notÐbe present. Sperm PCC,
which may appear as individual chromosomes or as a dense
chromatin mass, were found in 42 oocytes (61%; Table II;
Figure 1 a,a¢-b,b¢), partly decondensed spermheads in 25
oocytes (36%; Table II; Figure 1 c,c¢), and decondensed
spermheads in only two meiotically mature oocytes (3%;
Table II; Figure 1 d,d¢).
3. This indicates that fertilization does not always constitute
an effective stimulus to restart the cell cycle and complete
meiosis. Oocytes with PB1 and female chromatin as a clumped
Analysis of human oocytes after IVF failure
where 1-cell embryos were characterized by the presence of the
®rst and second polar bodies as well as both male and female
pronuclei, an early blockage of embryonic development could
be considered.
Quality of the metaphasic spindle
The status of the metaphasic plate (assembly of chromosomes
at the spindle equator) represents a more advanced characterization of oocyte quality. Some 53% of all MII oocytes
exhibited a normal spindle (Figure 2A,c,d), though this
proportion was higher in `cortical' (70% on average) than in
`centripetal' MII (46% on average) (Table III).
The remainder of the MII oocytes (46.5%) presented various
abnormalities in chromosome distribution and spindle organization. Among these were found dispersed chromosomes
(Figure 2A,e,f), clumped female chromatin with interphasic
microtubules (Figure 2A,g) or an abnormal spindle
(Figure 2A,h), spindle with either no (Figure 2A,i) or several
poles (Figure 2A,j±l). Importantly, a spindle which was
disorganized but with well-aligned chromosomes was never
observed, whereas the presence of a well-organized spindle
with dispersed chromosomes was rather frequent. This dispersion, as well as the small cytoplasmic asters often seen in
overmature oocytes (Figure 2) may occur due to oocyte ageing,
as observations take place 48 h after ovulation (EichenlaubRitter et al., 1986; Van Wissen et al., 1991; Battaglia et al.,
1996).
When patients were grouped according to their age, it was
noted that oocytes from older patients (aged >40 years) showed
a higher proportion of aberrant MII, such as chromosome
misalignment or completely disrupted spindles (69% for 40- to
45-year-old women versus 35% for 26- to 33-year-old)
(Table III). Remarkably, this increase was seen to be particularly drastic for oocytes that were mature at the time of retrieval
(centripetal), but was much lower in the case of oocytes that
were not mature at the time of retrieval (cortical) (Table III).
In contrast, control oocytes presented a cortical MII plate
with a symmetric, barrel-shaped meiotic spindle which was
radially oriented, and there were no detectable cytoplasmic
asters (Figure 2A,a). In addition, these control oocytes with a
normal spindle generally did not show any morphological
abnormalities: the ZP was of normal thickness, the perivitellin
space was limited to the site of normal PB1 extrusion, and the
cytoplasm was without vacuoles or dense granules.
Figure 1. Different con®gurations of the sperm chromatin in
various cases of IVF failure. DNA staining using Hoechst 33342
(H) and immuno¯uorescence detection of a-tubulin (a-Tu). (a,a¢)
Dense chromatin mass; (b,b¢) prematurely condensed chromosomes
well organized in the middle of a barrel-shaped spindle; (c,c¢) partly
decondensed chromatin; (d,d¢) completely decondensed chromatin;
(e,e¢) male pronucleus. Labelling of the sperm tail tubulin
(arrowheads) con®rms the presence of sperm within the oocyte.
Scale bar = 5 mm.
mass or an interphasic nucleus, and without evidence of sperm
penetration, were probably parthenogenetically activated (10%
cases; Table I), although ageing and nuclear reconstitution can
also induce such morphology. Lastly, in the rare cases (2%)
Cortical reaction
Migration and redistribution of CGs in the cortex is a common
attribute of cytoplasmic maturation. The CGs may undergo
exocytosis after activation of the oocyte at fertilization, and this
is termed the cortical reaction.
A total of 33 oocytes was analysed for the distribution of
CGs, organization of the meiotic spindle, and alignment of
chromosomes on the metaphasic plate. A good correlation
between nuclear maturation of the oocyte and CG localization
was found, with three cases being identi®ed:
1. CGs distributed as large aggregates in the entire
cytoplasm, with very few at the level of the plasma membrane
(Figure 2B,a). This distribution was similar to that seen in
1497
F.Miyara et al.
Figure 2. (A) Different con®gurations of spindle and chromosomes observed in human metaphase II (MII) oocytes obtained just after
puncture (a) or after IVF failure (b±l). DNA staining using Hoechst 33342 (blue) and immuno¯uorescence detection of a-tubulin (green).
Control oocytes (a) were analysed on the day of puncture, whereas oocytes in (b±l) were analysed after IVF failure (i.e. 48 h after
insemination). (b) In normal oocytes, which seem immature, the ®rst polar body (PB) is extruded after puncture and still linked to the oocyte
by the meiotic midbody (Mb) or is still in proximity to well-aligned chromosomes in the middle of a barrel-shaped spindle (c). (d) In normalaged oocytes, the barrel-shaped spindle has a centripetal position far from the PB. Abnormal MII oocytes exhibit a barrel-shaped spindle and
dispersed chromosomes (arrowheads, e and f), clumped female chromatin with interphasic microtubules (g) or an abnormal spindle (h). (i±l)
Additional examples of MII spindles showing disturbances in the spindle (no or several poles) and in chromosome alignment. Note the
presence of small cytoplasmic aggregates in aged oocytes (arrows), but not in the control. Cc = clumped chromatin. (B) Different distribution
of cortical granules (CGs) in human oocytes obtained after IVF failure. Each oocyte was labelled with an antibody to a-tubulin (green), with
biotin-conjugated LCA (red) or with Sytox green (inserts), and visualized using confocal ¯uorescence microscopy. (a,d) CGs as large
aggregates located over the entire cytoplasm, spindle and chromosomes are completely disorganized. (b,e) CGs localized in the periphery as
individual particles as well as small aggregates. The microtubules (*) and metaphasic plate are well organized in a cortical barrel-shaped
spindle. (c,f) CGs dispersed as individual particles in the cortical cytoplasm aligning the oolemma. The spindle and metaphasic plate are well
organized in a centripetal, barrel-shaped spindle. Scale bars: (A) = 7 mm; (B) = 20 mm.
human GV oocytes (data not shown). In that case, abnormalities were observed in most cases (88%) in the organization of
the meiotic spindle and metaphasic plate (Figure 2B,a).
1498
2. CGs mainly located at the periphery of the oocyte. Below
this ring of CGs, small CG aggregates still remained in the
cytoplasm (Figure 2B,b). The spindle and metaphasic plate in
Analysis of human oocytes after IVF failure
Table II. Overall percentage of sperm-penetrated oocytes and state of the sperm in metaphase II (MII)-stage oocytes
Patient age (years)
26±33
34±39
40±45
Total
Oocytes without sperm (n)
195/231
153/192
130/135
478/558
(84.5)
(79.5)
(96.0)
(85.5)
Oocytes with sperm (n)
36 (15.5) 8/60 patients
39 (20.5) 12/54 patients
5 (14.0) 3/41 patients
80/558 (14.5)
State of the sperm chromatin in MII-stage oocytes
PCC
Partially decondensed
Decondensed
21/33 (63.5)
18/32 (56.0)
3/4 (75.0)
42/69 (61.0)
11/33 (33.5)
13/32 (40.5)
1/4 (25.0)
25/69 (36.)
1/33 (3.0)
1/32 (3.5)
0 (0)
2/69 (3.0)
Values in parentheses are percentages.
PCC = premature chromosome condensation.
Table III. Analysis of the quality of the metaphases in metaphase II (MII) oocytes obtained after IVF failure
Patient age (years)
26±33
34±39
40±45
Total
MII in cortical position
Total
Normal
64
51
36
151
45
39
22
106
(70)d
(76)d
(61)d
(70)
MII in centripetal position
Total
Abnormal
Total
Normal
Abnormal
Normal
Abnormal
19
12
14
45
167
141
99
407
105 (63)a
66(47)b
20 (20)c
191 (46)
62
75
79
216
150
105
42
297
81
87
93
261
(30)
(24)
(39)
(30)
(37)
(53)
(80)
(54)
(65)
(54.5)
(31)
(53.5)
(35)
(45.5)
(69)
(46.5)
Values in parentheses are percentages.
different values (c2 test, P < 0.05).
dNon-signi®cantly different values (c2 test).
a,b,cSigni®cantly
these oocytes were well organized, but located cortically. Their
position with respect to the PB could not be analysed as it was
detached from the oocyte when the ZP was removed
(Figure 2B,b). In this case, it could be suggested that the
delay of CG translocation and fusion with the plasma
membrane re¯ects an uncompleted nuclear maturation at the
time of puncture. It is believed that fertilization failure and
infertility in those cases might be attributable to uncoupling of
the nuclear and cytoplasmic maturation processes.
3. All CGs fused with the plasma membrane, so that it was
uniformly labelled (Figure 2B). In this case, the spindle and
chromosomes were well organized and in the centripetal
position (Figure 2B,a). Thus, cytoplasmic maturationÐwhen
estimated by the migration of CGsÐseems to be associated
with the achievement of nuclear maturation at the time of
puncture.
Combination of cellular and clinical analyses
From a clinical point of view, the analysis of oocytes not
fertilized after IVF may be of diagnostic value. IVF cycles
without recognized male factor for infertility, but without
cleavage after IVF, may reveal a sperm defectÐespecially
when the majority of oocytes have well-organized chromosomes and spindle and are not penetrated by sperm. On the
other hand, the presence of sperm chromatin in some uncleaved
oocytes excluded the possibility of sperm penetration incapacity; in this case, an oocyte problem could be suspected.
Classi®ed as `fertilizable' were those oocytes which had: (i)
chromosomes that were well aligned on the metaphasic plate
and in the middle of a barrel-shaped spindle; (ii) the
chromosomes and meiotic spindle in a centripetal position;
and (iii) CGs that were exclusively cortical. Classi®ed as
`unfertilizable' were those oocytes which had one of the
following defects: (i) chromosomes totally dispersed on a well-
organized spindle; (ii) chromosomes outside the metaphasic
plate; (iii) aberrations in the spindle; and (iv) still condensed
chromatin with interphasic microtubules. All of these meiotic
defects were generally accompanied by poor migration of the
CGs.
In addition to the quality of the oocytes as assessed above,
data relating to the age of the patients, the pathology as
indicated by clinicians, the number of IVF attempts, and the
protocols of ovarian stimulation were given. The present study
focused especially on endometriosis, PCOS and idiopathic
infertility. When the majority of a patient's oocytes were
fertilizable, but not fertilized at the ®rst attempt, then a new
attempt was recommended. In such cases, a good pregnancy
rate was obtained with this new attempt, irrespective of the
pathology encountered (Table IV), the pregnancy rate being
similar after either IVF or ICSI (38 versus 32%). By contrast,
when the majority of the oocytes was judged to be nonfertilizable at a ®rst attempt, then no pregnancy was registered
at a second attempt (Table IV). These ®ndings clearly
demonstrate the reliability of the a posteriori multiparametric
analysis utilized in the present study.
Discussion
Within the present study, an analysis was made of those
oocytes which failed to fertilize after IVF and which belonged
to cohorts where most (or all) oocytes failed, the object being to
identify the possible causes of fertilization failure. When IVF
failsÐeither completely or partiallyÐthen several factors can
be suspected which involve the ovarian stimulation used, the
age of the patients, and the meiotic maturation stage at the
moment of sperm penetration. With regard to the effect of
ovarian stimulation, several studies using mouse oocytes have
shown a progressive and signi®cant increase in the frequency
1499
F.Miyara et al.
Table IV. Pregnancy rate according to oocyte quality after analysis of the
IVF failure
No. of patients*
75
23
Oocyte quality
Fertilizable
Unfertilizable
New attempt
Pregnancy
IVF
ICSI
IVF
ICSI
21
8
41
9
8 (38)a
0
13 (32)b
0
Values in parentheses are percentages.
*Idiopathic, endometriosis and PCOS. Cases of combined male and female
fertility problems were excluded from this survey.
aFertilization rates were between 45 and 70% in all cases, except for one
case of 20%.
bFertilization rates were between 50 and 75% all cases.
PCOS = polycystic ovary syndrome.
of meiotic spindle abnormalities as the number of stimulations
has been increased (Van Blerkom and Davis, 2001).
Disorganization of the spindle in these oocytes may cause
dispersion of the chromosomes, thereby preventing extrusion
of the ®rst or second polar body, possibly leading to aneuploidy
of the zygote. Indeed, a cytogenetic analysis of mouse 1-cell
embryos after several ovarian stimulations showed chromosomal aneuploidy mainly at the level of the female pronucleus
(Vogel and Spielmann, 1992). These are not natural cycles,
however, and from human oocyte donation programmes it does
not appear that repetitive stimulations are accompanied by a
reduction in oocyte quality (Caligara et al., 2001). In the
present study, in all cases (n = 38) where oocytes from the same
patient after three different stimulation cycles could be
analysed, no reduction was observed in the percentage of
oocytes considered as normal. The present data are in
agreement with those reported by others (Dor et al., 1996), in
which no crucial changes were indicated in the rates of
pregnancy after IVF, even after eight cycles of ovarian
stimulation.
In contrast, in the present study an incidence of 35±45.5%
abnormalities was found in organization of the spindle and
metaphasic plate in patients aged between 26 and 33 years,
while an incidence of 69% was found in those women aged
40±45 years. This difference suggests a strong dependence on
patient ageÐa result which is in line with earlier reports which
showed more frequent defects in chromosome alignment and
spindle formation in MII oocytes from aged, non-stimulated
women (Battaglia et al., 1996; Battaglia and Miller, 1997;
Volarcik et al., 1998), together with a higher incidence of
errors in chromosome segregation in the ®rst meiotic division
(Volarcik et al., 1998). The 69% incidence of abnormalities
found in the present study among non-fertilized MII oocytes
from 40- to 45-year old women was slightly less than the value
of 79% observed among MII oocytes from women of the same
age but without ovarian stimulation (Battaglia et al., 1996).
This led to the consideration that more oocytes from 40- to 45year-old women, after being fertilized, present with defects in
their meiotic spindle and chromosome organization. Indeed,
91% of these oocytes (n = 22) did not achieve pregnancy after
implantation, most likely as a consequence of the bad
organization of the spindle and chromosomes before fertilization. In fact, it appears that meiotic aberrations (i.e. chromo1500
some misalignment and spindle disruption) do not prevent
meiotic progression from MI to MII, but rather meiosis seems
to proceed by default (Liu and Keefe, 2002). Although ageingassociated infertility has also been found in mice, age-related
abnormalities in meiotic spindle organization and chromosome
alignment have not been recognized (Eichenlaub-Ritter et al.,
1988; Van Blerkom and Davis, 2001). In addition, the
aneuploidy rate does not increase signi®cantly with mouse
age (Zuccotti et al., 1998). Much higher rates of meiotic
aberration were found in old, senescence-accelerated mouse
(SAM) oocytes (Liu and Keefe, 2002). SAM mice exhibit
mitochondrial dysfunction and oxidative damage early during
ageing (Mori et al., 1998). Oxidative stress has been shown to
induce disturbances in chromosomal distribution in the MII
spindle of mouse oocytes (Tarin et al., 1998), whereas
mitochondrial dysfunction might compromise the activity of
microtubule motor proteins as a consequence of a reduced
energy supply (Liu and Keefe, 2002). In this respect, the severe
disturbances seen in old SAM oocytes may mimic better the
human situation.
Besides defects in the extent of nuclear maturation and/or
organization of the meiotic chromosomes, defects in the oocyte
cytoplasm might also account for fertilization failures. One
outstanding feature of correct cytoplasmic maturation is
migration of the CGs. In fact, a strong correlation between
CG translocation and quality of the metaphasic plate was
observed, as no CG translocation was seen in oocytes with
abnormalities in the meiotic spindle and metaphasic plate
organization, whereas CGs were translocated in all normal
centripetal MII-stage oocytes. Microtubules have been suggested to be involved in correct CG translocation (Kim et al.,
1996; Abbott et al., 2001), and a role for micro®laments has
also been proposed in mouse (Tahara et al., 1996; Connors
et al., 1998), hamster (DiMaggio et al., 1997) and porcine (Kim
et al., 1996) oocytes. An alternate hypothesis is that
unfertilized MII-stage oocytes are de®cient in a Ca2+-dependent signalling component necessary for CG translocation and
subsequent extrusion (Abbott et al., 2001). In mouse oocytes,
arti®cial CG extrusion was induced by a Ca2+ ionophore
(Tatone et al., 1999), while inhibition of calcium-dependent
protein kinase II (CAMK II) negatively affected CG exocytosis
and MPF inactivation (Tatone et al., 2002), and blocked transit
from MII to anaphase II (Johnson et al., 1998). The simultaneous effect of the CAMK II on CG migration and MPF activity
suggests that disorganization of the spindle, blocking of the
cortical reaction and premature condensation of the sperm
chromatin that was seen in many of the oocytes analysed might
all be related to anomalies in CAMK II activity. The
con®rmation of a physiological role for CAMK II during
human fertilization requires further investigation, however.
Finally, the absence of sperm in 85.5% of analysed oocytes
after IVF failure is very surprising, and hardening of the ZP
during insemination is the most suspected cause (De Felici
et al., 1985; Bedford and Kim, 1993). The released CG content
is thought to be responsible for ZP hardening. In humans, a
reduction in the number of CGs has been reported in preovulatory oocytes (Rousseau et al., 1977), fertilized oocytes
(Lopata et al., 1980) and IVF fertilization-failed oocytes
Analysis of human oocytes after IVF failure
(Ducibella et al., 1995). On the other hand, the possibility that
the ZP may undergo changes that are not dependent on CG
exocytosis has been previously reported (De Felici and
Siracusa, 1982; Dolci et al., 1991). For example, one group
(Talevi et al., 1997) reported that fertilization-failed human
oocytes showed alterations in carbohydrate distribution of the
ZP that were not related to cortical reaction. In this case,
hardening is probably a consequence of intrinsic modi®cations
of the zona components which might block sperm penetration.
Although the zona and oolemma barriers may be bypassed
with ICSI (Palermo et al., 1992), the results of the present study
show that the difference between pregnancy rates obtained
with a new attempt of IVF or ICSI in the case of non-male
sterility and good quality oocytes, is not signi®cant (38 versus
32%). In turn, ICSI is often associated with reduced blastocyst
formation (Dumoulin et al., 2000; Grif®ths et al., 2000), sperm
DNA fragmentation (Sakkas et al., 1999) and an increase in the
rate of aneuploidy (Bernardini et al., 1997). The technique
itself may have negative effects on embryonic development
(Grif®ths et al., 2000; Dumoulin et al., 2001). These arguments
do not support the use of the ICSI in all treatments of assisted
reproductive technologies.
Based on the results of the present study, it may be
concluded that an analysis of three complementary
componentsÐDNA, spindle and CGsÐrequires only the use
of a ¯uorescence microscope, yet permits rapid assessment of
oocyte quality. Further simpli®cation of these protocols would
render the analyses both rapid and amenable to the clinical
situation. Moreover, all three factorsÐwhen analysed on a
complete set of oocytes from the same patientÐprovide
information relating to potential causes of IVF failure. They
should therefore be considered as part of an `oocyte quality
evaluation' needed to select the clinically assisted fertilization
method best suited to an individual patient.
Acknowledgements
The authors are grateful to the anonymous patients who donated their
oocytes after IVF failures, in order that this research might be
conducted. They also acknowledge the support of the Gynaecology
attending, surgical and technical staff at clinic Pierre Cherest for their
participation in the ovarian aspirations and for human oocyte
treatments. These studies were ®nanced by funds from the French
Institut National de Recherche Agronomique (INRA), from the
Museum National d'Histoire Naturelle (MNHN). F.Miyara was a
fellow from RECREPRO, a doctors association.
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Submitted on January 8, 2003; accepted on March 13, 2003
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