Multiparameter analysis of human oocytes at metaphase II stage after IVF failure in non-male infertility

Faïçal Miyara1, François-Xavier Aubriot2, Amélie Glissant2, Catherine Nathan2, Stéphane Douard2, Alexandre Stanovici2, Florence Herve2, Martine Dumont-Hassan2,3, Alain Le Meur3, Paul Cohen-Bacrie3 and Pascale Debey1,4

1 UMR1198 Biologie du Développement et Reproduction, INRA, Jouy-en-Josas Cedex, 2 Centre FIV Pierre Cherest, Neuilly-sur-Seine Cedex and 3 Laboratoire d’Eylau, Paris, France

4 To whom correspondence should be addressed at: UMR1198 Biologie du Développement et Reproduction, INRA, 78352, Jouy-en-Josas Cedex, France. e-mail: debey{at}mnhn.fr


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Using fluorescence 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 influenced 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 first 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
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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., 2001Go). 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., 1992Go; 2002). Besides fertilization failures after conventional IVF (Fishel et al., 2000Go), ICSI programmes have been expanded to include patients with poor quality sperm, azoospermia, immunological infertility and chemotherapy/radiotherapy treatments (Horne et al., 2001Go). Nonetheless, the fertilization rate for ICSI fluctuates between 50 and 70%; i.e., it is not higher than the IVF rate for non-male factor patients (Ola et al., 2001Go). 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.

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 inter-related factors, generally classified respectively as nuclear and cytoplasmic ‘competence’. Nuclear competence characterizes the quality of oocyte chromatin and spindle (Mattson and Albertini, 1990Go; Albertini, 1992Go), which is essentially governed by the level of the metaphase-promoting factor (MPF) activity (Hashimoto and Kishimoto, 1988Go; Murray, 1989Go; Verde et al., 1990Go; 1992). MPF governs—either directly or indirectly—all processes linked to metaphase entry in eukaryotic cells, such as nuclear envelope breakdown (Ookata et al., 1992Go), histone phosphorylation (Collas, 1999Go) and microtubule polymerization (Charrasse et al., 2000Go). 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., 1999Go). 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., 1994Go) and porcine oocytes (Sun et al., 2001Go; 2002). Activation of MAP kinases occurs after germinal vesicle breakdown (GVBD) and keeps microtubules and chromatin from entering into an interphase configuration (Sun et al., 1999Go). In human oocytes, extrusion of the first polar body and formation of the MII oocyte occurs at 32 h after ovarian stimulation, this being 6–8 h before ovulation (Bomsel-Helmreich et al., 1987Go). The presence of a functional spindle ensures the fidelity of chromosome segregation, and is thus an essential feature of healthy MII oocytes (Eichenlaub-Ritter et al., 1986Go; Van Blerkom and Henry, 1992Go; Volarcik et al., 1998Go; Van Blerkom and Davis, 2001Go). Ageing of MII oocytes, thermal changes and insufficient oxygen supply throughout the entire culture period (Hu et al., 2001Go; Wang et al., 2001Go) may cause concomitantly a deterioration of the spindle and displacement of chromosomes from the spindle equator (Eichenlaub-Ritter et al., 1986Go).

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., 1990Go; 1993; Yoshida et al., 1993Go). CGs are small (0.2–0.6 µm diameter) secretory granules which were first identified using electron microscopy and originate from the Golgi body (Gulyas, 1976Go; Manna et al., 2001Go). During oocyte growth, the CGs increase in number and migrate toward the cortex, assuming a position 0.4–0.6 µm below the plasma membrane (Ducibella et al., 1988Go; 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., 1988Go; 1990; Connors et al., 1998Go). The cortical accumulation—but not formation of the CGFD—is observed during meiotic maturation in others mammals, such as cat (Byers et al., 1992Go), pig (Sun et al., 2001Go), cattle (Izadyar et al., 1998Go) and human (Ghetler et al., 1998Go). CGs contain mucopolysaccharides and several enzymes such as proteases, peroxidase and acid phosphatase (Flechon, 1970Go; Moller and Wassarman, 1989Go; Hoodbhoy and Talbot, 1994Go). 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, 1995Go) and extrude their contents into the perivitellin space (Chamow and Hedrick, 1986Go). This release modifies the extracellular coat of the oocyte (Connors et al., 1998Go), 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., 1995Go) and to transform it into the male pronucleus. Decondensation of the spermatozoon is considered to occur independently from the resumption of meiosis, under the influence of a cytoplasmic factor called sperm decondensation factor (SDF), the synthesis of which is induced during normal oocyte maturation (Moor and Gandolfi, 1987Go). 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., 1991Go; Gook et al., 1998Go; Kovacic and Vlaisavljevic, 2000Go; Bao et al., 2002Go). Moreover, in-vitro insemination of metaphase I oocytes leads to condensation of male chromatin into premature chromosome condensation (PCC) (Clarke and Masui, 1986Go; 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 observations—which may be gathered using regular microscopic equipment—are 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
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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., 1980Go). 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 IVFTM-20 medium (Vitrolife; Mölndalsvägen, Gothenburg, Sweden) and left intact for conventional IVF. Cumulus–oocyte complexes were inseminated with ~2x105 motile sperm in 1 ml Scandinavian IVF medium at 37°C in 5% CO2 in an humidified 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 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 fixed in 2% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) at 37°C for 30 min; this fixative is known to preserve cellular structures and antigenic sites (Grootenhuis et al., 1996Go). 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 {alpha}-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 fluoroscein 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 µg/ml Hoechst 33342 (Riedel de Haen, Germany) for 20 min, transferred into PBS, and then mounted under a coverslip in citifluor (Citifluor Products, Canterbury, UK).

Stained oocytes were examined using an inverted microscope (Carl Zeiss, Oberkochen, Germany) that was equipped for epifluorescence and fitted with appropriate filter combinations. Images were recorded through a charge-coupled device (CCD) camera (Type KAF 1400, 12-bit 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 specifically to the CGs content and exudate (Ducibella et al., 1994Go). Oocytes were first fixed 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 {alpha}-chymotrypsin (0.01%) before fixation 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) {alpha}-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., 1998Go). Briefly, oocytes were incubated in 5 µg/ml biotin-conjugated LCA (Sigma-Aldrich Chimie, France) for 30 min, and stained with 2 µg/ml rhodamine red–streptavidin (Jackson ImmunoResearch) for 30 min. Finally, oocytes were labelled with 1 µg/ml Sytox green (Molecular Probes, Eugene, OR, USA), which stains double-stranded DNA for 15 min, transferred into PBS, mounted under a coverslip in citifluor, and 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 100x (NA 1.3), and the pinhole was set at between 1.5 and 2.8 µm. The 488 nm and 568 nm wavelengths of the laser were used for excitation of fluorescein (or Sytox green) and rhodamine respectively. Optical sections were imaged at 5 µm intervals.


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 Materials and methods
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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 {alpha}-tubulin immunolabelling coupled to DNA staining by the specific fluorescent probe, Hoechst 33342. In fact, 85.5% of the oocytes did not show any presence of sperm. Hence, oocytes could be classified into three different categories (Table I):


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Table I. State of maternal chromatin in non-fertilized oocytes issued from cohorts with very low or zero fertilization rates in IVF procedures
 
1. Meiotically immature oocytes (12% of cases), defined by the absence of a polar body (PB0) and the presence of maternal chromatin at the GV or first meiotic metaphasic plate (MI) stages. Both of these could—or could not—be 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').



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Figure 1. Different configurations of the sperm chromatin in various cases of IVF failure. DNA staining using Hoechst 33342 (H) and immunofluorescence detection of {alpha}-tubulin ({alpha}-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) confirms the presence of sperm within the oocyte. Scale bar = 5 µm.

 
2. Mature oocytes were defined 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., 1992Go) (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 could—or could not—be 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').



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Figure 2. (A) Different configurations 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 immunofluorescence detection of {alpha}-tubulin (green). Control oocytes (a) were analysed on the day of puncture, whereas oocytes in (bl) were analysed after IVF failure (i.e. 48 h after insemination). (b) In normal oocytes, which seem immature, the first 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 normal-aged 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). (il) 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 {alpha}-tubulin (green), with biotin-conjugated LCA (red) or with Sytox green (inserts), and visualized using confocal fluorescence 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 µm; (B) = 20 µm.

 

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Table II. Overall percentage of sperm-penetrated oocytes and state of the sperm in metaphase II (MII)-stage oocytes
 
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 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%) where 1-cell embryos were characterized by the presence of the first 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).


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Table III. Analysis of the quality of the metaphases in metaphase II (MII) oocytes obtained after IVF failure
 
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 (Eichenlaub-Ritter et al., 1986Go; Van Wissen et al., 1991Go; Battaglia et al., 1996Go).

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.

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 identified:

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 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).

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 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 reflects 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 maturation—when estimated by the migration of CGs—seems 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 defect—especially 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.

Classified 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. Classified 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 first 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 non-fertilizable at a first attempt, then no pregnancy was registered at a second attempt (Table IV). These findings clearly demonstrate the reliability of the a posteriori multiparametric analysis utilized in the present study.


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Table IV. Pregnancy rate according to oocyte quality after analysis of the IVF failure
 

    Discussion
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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 fails—either completely or partially—then 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 significant increase in the frequency of meiotic spindle abnormalities as the number of stimulations has been increased (Van Blerkom and Davis, 2001Go). Disorganization of the spindle in these oocytes may cause dispersion of the chromosomes, thereby preventing extrusion of the first 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, 1992Go). 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., 2001Go). 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., 1996Go), 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 age—a 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., 1996Go; Battaglia and Miller, 1997Go; Volarcik et al., 1998Go), together with a higher incidence of errors in chromosome segregation in the first meiotic division (Volarcik et al., 1998Go). 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., 1996Go). This led to the consideration that more oocytes from 40- to 45-year-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. chromosome misalignment and spindle disruption) do not prevent meiotic progression from MI to MII, but rather meiosis seems to proceed by default (Liu and Keefe, 2002Go). Although ageing-associated 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., 1988Go; Van Blerkom and Davis, 2001Go). In addition, the aneuploidy rate does not increase significantly with mouse age (Zuccotti et al., 1998Go). Much higher rates of meiotic aberration were found in old, senescence-accelerated mouse (SAM) oocytes (Liu and Keefe, 2002Go). SAM mice exhibit mitochondrial dysfunction and oxidative damage early during ageing (Mori et al., 1998Go). Oxidative stress has been shown to induce disturbances in chromosomal distribution in the MII spindle of mouse oocytes (Tarin et al., 1998Go), whereas mitochondrial dysfunction might compromise the activity of microtubule motor proteins as a consequence of a reduced energy supply (Liu and Keefe, 2002Go). 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., 1996Go; Abbott et al., 2001Go), and a role for microfilaments has also been proposed in mouse (Tahara et al., 1996Go; Connors et al., 1998Go), hamster (DiMaggio et al., 1997Go) and porcine (Kim et al., 1996Go) oocytes. An alternate hypothesis is that unfertilized MII-stage oocytes are deficient in a Ca2+-dependent signalling component necessary for CG translocation and subsequent extrusion (Abbott et al., 2001Go). In mouse oocytes, artificial CG extrusion was induced by a Ca2+ ionophore (Tatone et al., 1999Go), while inhibition of calcium-dependent protein kinase II (CAMK II) negatively affected CG exocytosis and MPF inactivation (Tatone et al., 2002Go), and blocked transit from MII to anaphase II (Johnson et al., 1998Go). 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 confirmation 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., 1985Go; Bedford and Kim, 1993Go). 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 pre-ovulatory oocytes (Rousseau et al., 1977Go), fertilized oocytes (Lopata et al., 1980Go) and IVF fertilization-failed oocytes (Ducibella et al., 1995Go). 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, 1982Go; Dolci et al., 1991Go). For example, one group (Talevi et al., 1997Go) 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 modifications of the zona components which might block sperm penetration.

Although the zona and oolemma barriers may be bypassed with ICSI (Palermo et al., 1992Go), 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 significant (38 versus 32%). In turn, ICSI is often associated with reduced blastocyst formation (Dumoulin et al., 2000Go; Griffiths et al., 2000Go), sperm DNA fragmentation (Sakkas et al., 1999Go) and an increase in the rate of aneuploidy (Bernardini et al., 1997Go). The technique itself may have negative effects on embryonic development (Griffiths et al., 2000Go; Dumoulin et al., 2001Go). 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 components—DNA, spindle and CGs—requires only the use of a fluorescence microscope, yet permits rapid assessment of oocyte quality. Further simplification of these protocols would render the analyses both rapid and amenable to the clinical situation. Moreover, all three factors—when analysed on a complete set of oocytes from the same patient—provide 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 financed 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.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on January 8, 2003; accepted on March 13, 2003.