High magnitude of light retardation by the zona pellucida is associated with conception cycles

Y. Shen1,2, T. Stalf1, C. Mehnert1, U. Eichenlaub-Ritter3,4 and H.-R. Tinneberg1,2

1 Centre of In-Vitro-Fertilisation (CIF) in the Justus-Liebig-University, 2 Department of Gynaecology and Obstetrics, Women's Hospital, Justus-Liebig-University Giessen, D-35392 Giessen and 3 University of Bielefeld, Faculty of Biology IX, Gene Technology/Microbiology, D-33501 Bielefeld, Germany

4 To whom correspondence should be addressed. Email: eiri{at}uni-bielefeld.de


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Failures in expression of zona proteins correlate to subfertility in animals. Low expression of the zona proteins by the growing human oocyte may indicate reduced developmental potential. Therefore, we non-invasively analysed the thickness and the structure of the zona pellucida (ZP) of human oocytes with respect to embryo fate after ICSI. METHODS: Retardance magnitude and thickness of the inner, middle and outer layers of the ZP were quantitatively analysed by a Polscope in 166 oocytes selected for transfer after ICSI (63 patients; 32.8±4.4 years) on the basis of pronuclear score at day 1. Blastomere number was determined at day 2. Data were compared between conception cycles (CC; 65 oocytes/23 patients) and non-conception cycles (NCC; 101 oocytes/40 patients) and with respect to maternal age. RESULTS: The thickness was slightly elevated (P<0.001), and the mean magnitude of light retardance was nearly 30% higher (P<0.001) in the inner layer of the zona pellucida of oocytes contributing to CC compared to NCC. Embryos in the CC group tended to develop faster. CONCLUSIONS: The magnitude of light retardance by the zona pellucida inner layer appears to present a unique non-invasive marker for oocyte developmental potential.

Key words: conception/human oocyte/ICSI/Polscope/zona pellucida


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
A multi-laminar glycoprotein coat, termed the zona pellucida, surrounds growing and mature oocytes as well as the preimplantation embryo of mammals (Baranska et al., 1975Go; Bercegeay et al., 1995Go; Green, 1997Go; Prasad et al., 2000Go; Herrler and Beier, 2000Go; Carino et al., 2002Go). Three genes encoding three to four zona proteins are highly conserved (Spargo and Hope, 2003Go). The zona glycoproteins appear to be coordinately secreted by the oocyte during folliculogenesis, as shown in the mouse (Epifano et al., 1995Go; Soyal et al., 2000Go), while there is evidence from other species, including the human, that granulosa cells may also contribute to stage-dependent zona protein expression during folliculogenesis (Sinowatz et al., 2001Go; Bogner et al., 2004Go; Gook et al., 2004Go). At the ultrastructural level, the three-dimensional highly ordered filament structure of the zona pellucida has been confirmed by studies in mammalian oocytes, including human oocytes (Familiari et al., 1992Go; Green, 1997Go, Wassarman et al., 1999Go, 2004Go; Oehninger, 2003Go). At the molecular level, the zona pellucida consists of a paracrystalline, three-dimensional network structure composed of heterodimeric filaments of ZP2 and ZP3 proteins, cross-linked by ZP1 proteins (Wassarman, 1988Go; Qi et al., 2002Go; Wassarman et al., 2004Go). The functional significance for a non-random, variable quantity and distribution of differently glycosylated zona proteins within the zona layers is still unknown (for recent discussion, see Bogner et al., 2004Go; Jimenez-Movilla et al., 2004Go). Human oocytes express three highly conserved zona proteins, which change conformation after cortical granule extrusion at fertilization, as in the mouse (e.g. Nikas et al., 1994Go; Moos et al., 1995Go). Another human zona protein (ZPB) may contribute in unknown ways to species-specific sperm–oocyte interactions in human fertilization (Lefievre et al., 2004Go).

The zona proteins form a thick extracellular matrix separating the mammalian oocyte physically from follicle cells. However, it is essential that the zona pellucida is crossed by radially arranged transzonal cytoplasmic projections during oocyte growth and maturation. They provide the conditions for direct junctional coupling, cell–cell signalling, and exchange of molecules between the oocyte and the somatic compartment (Albertini and Rider, 1994Go; Motta et al., 1994Go), which are important for oocyte growth and sustained meiotic arrest (Eppig, 1991Go; Webb et al., 2002Go). Within the zona, the projections may form a hexagonal network together with zona fibrils, and thus may contribute to the three-dimensional organization of the extracellular space (Albertini and Rider, 1994Go; Motta et al., 1994Go; Albertini and Barrett, 2003Go).

ZP1 proteins are required for the structural integrity of the zona pellucida (Greve and Wassarman, 1985Go; Wassarman et al., 2004Go). Although oocytes of mice lacking the ZP1 gene still secrete the ZP2 and ZP3 proteins, they possess a thinner and more loosely organized zona pellucida, relative to the wild type. The diameter of ovulated oocytes of the ZP1 –/– homozygote is on average only half of that of oocytes from the wild type (Rankin et al., 1999Go). This suggests that effective granulosa cell–oocyte signalling may depend on the presence of a functional, highly structured zona, and that disturbances cause subfertility. Apart from this, reduced expression of zona proteins during oocyte growth and folliculogenesis may indicate general problems in the highly orchestrated processes of maturation at the critical phase of oogenesis and folliculogenesis when oocytes acquire full nuclear and developmental competence.

The zona pellucida is also essential for oocyte fertilization and implantation development in vivo (e.g. Rankin et al., 2001bGo). It serves taxon-specific sperm-binding (Tsubamoto et al., 1999Go; Miller et al., 2002Go; Wassarman, 2002Go), prevents polyspermy (Hoodbhoy and Dean, 2004Go; Yanagimachi, 1994Go), and protects embryos from mechanical stress prior to implantation (Herrler and Beier, 2000Go). Accordingly, defects in the zona pellucida are usually associated with fertilization problems or developmental abnormalities. For instance, mice lacking ZP3 or ZP2 genes are unable to form a zona pellucida and are sterile (Liu et al., 1996Go; Rankin et al., 2001aGo).

Up to now, only a few predictive non-invasive markers for oocyte quality have been identified on the basis of morphological criteria, which can be assessed in assisted reproduction by using conventional microscopy prior to insemination (Ebner et al., 2003Go). In this respect, orientation-independent polarizing microscopy (Polscope) was a breakthrough since non-invasive analysis of spindles in human oocytes became possible (Oldenbourg, 1996Go). Polarizing microscopy uses the principle that polarized light passing through crystalline or paracrystalline objects will be altered, for instance, in the plane and phase of vibration. The method can therefore be used to assess crystalline or paracrystalline structures qualitatively and quantitatively. For instance, the relative magnitude of the retardance of the polarized light is an indicator for density, high order alignment, or thickness of an object. Accordingly, the Polscope in combination with the respective software has been used to analyse spindle morphology and texture, as well as the substructure of the zona pellucida of mammalian oocytes qualitatively and quantitatively (e.g. Keefe et al., 1997Go; Wang et al., 2001Go; Eichenlaub-Ritter et al., 2002Go; Pelletier et al., 2004Go). The thickness of the zona and the appearance of the individual layers change according to the stage of maturation of the human oocyte and the post-fertilization development of the embryo (Bertrand et al., 1996Go; Pelletier et al., 2004Go). It has been suggested that the oocyte's zona morphology may be influenced by hormonal homeostasis and reproductive age of the woman (Bertrand et al., 1996Go), and that the thickness of the zona pellucida in individual embryos from a patient might be correlated to developmental capacity and implantation rate (Gabrielsen et al., 2001Go). However, to our knowledge there is no quantitative analysis relating the morphology of the zona pellucida, as seen by the Polscope in unfertilized mature human oocytes, to the fate of the oocyte and embryo after ICSI.

Therefore, we tested the hypothesis that the morphology, thickness and texture of the zona of a human oocyte reflect normal expression and the functional integrity of the respective follicle, for instance, the optimal para- and autocrine signalling between the oocyte and the somatic compartment in the follicle during oocyte growth and maturation, and, thus, may be related to oocyte developmental competence after fertilization. To test our assumption, we analysed the zona pellucida of oocytes from patients undergoing assisted reproduction by quantitative Polscope microscopy. Zona morphology was compared between cohorts of oocytes contributing to conception cycle (CC) or non-conception cycle (NCC) after selection of embryos according to standard morphological criteria (e.g. Stalf et al., 2002Go) and pronuclear score (PN score; Scott and Smith, 1998Go). Furthermore, we examined correlations between maternal age and zona morphology in CC and NCC.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Source of human oocytes
Oocytes from 63 ICSI patients, aged 23–42 years (mean age, 32.8±4.4 years), which had been selected for embryo transfer, were included after informed consent of the patients. The mean age of patients was not significantly different in the groups comprising CC compared to NCC cycles (32.7±3.8 and 32.9±4.7 years respectively; not significant; Table I). Number of attempts was also not significantly different. Male subfertility was the indication in all cases. The study protocol and design were approved by the ethics committee of the University Hospital of Giessen.


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Table I. Summary of data on variables between patients whose oocytes contributed to a conception cycle (CC) or non-conception cycle (NCC)

 
Ovarian stimulation, retrieval and culture of oocytes
Oocytes were obtained from ICSI patients undergoing controlled ovarian stimulation induced by hMG (Menogon®; Ferring, Germany) protocol. Ovulation was trigged by administration of 10 000 IU hCG (Organon, Germany) 36 h prior to puncture, when the dominant follicles were >20 mm in diameter and E2 level in blood was >1000 pg/ml. There was no difference in peak estradiol levels in groups of patients later experiencing CC or NCC (Table I).

Number of follicles was similar in the two groups (Table I). Retrieval of oocytes was performed by ultrasound-guided transvaginal aspiration. Cumulus–oocyte complexes (COC) were collected in HEPES-buffered human tubule fluid medium (HTF medium; Irvine Scientific, USA) supplemented with 10% human serum albumin (HSA, No. 20744411A; Behring, Germany). On average, 9±5.0 oocytes were initially retrieved from patients in CC cycles and 8±3.8 oocytes in NCC cycles respectively (Table I). After brief exposure to 80 IU/ml hyaluronidase (Sigma, Germany), cumulus cells were removed by repeated gentle aspiration. Denuded oocytes were washed twice in fresh HTF+10% HSA. In both groups on average 7.9 mature polar body oocytes were available for ICSI (Table I).

Zona pellucida imaging in living human oocytes
Prior to oocyte fertilization by ICSI (2 h post retrieval), oocytes were individually analysed by Polscope, and data were stored without using them for selection of embryos. To avoid any prolonged handling and viewing of the oocytes before ICSI, the images of oocytes obtained by standard illumination and microscope setting (see below) were saved for quantitative analysis before or after transfer of embryos.

Oocytes were imaged non-invasively by enhanced polarizing microscopy (Polscope) using circularly polarized light and electronically controlled liquid crystal analyser optics (Oldenbourg, 1996Go). Polscope filters and a COHU CCD camera were installed on a Nikon Eclipse TE-2000 inverted microscope equipped with Hoffman interference optics, x10, x20 and x40 strain-free objective lenses, and the SpindleViewTM imaging system with the software for quantitative assessment of birefringence and retardance of light (CRI Inc., USA). The object stage of the microscope was heated by a temperature control system at 37 °C (Minitub HT300; Tiefenbach, Germany).

To image the zona, oocytes were transferred individually to a pre-warmed 5 ml droplet of injection medium (HEPES-buffered human tubule fluid medium) overlaid with equilibrated mineral oil (Sigma, Germany) in a WillCo Wells BV dish (Ref. no.: GWSt-5040; The Netherlands). Alignment of the microscope and calibration of the software was performed before oocyte imaging. Using the automatic setting for contrast enhancement, images through the oocyte equator were saved and later quantitatively analysed with the SpindleView image processing system. Quantitative analysis of the thickness and retardance magnitude of the zona layers of oocytes used for transfer was performed along a line scan across the entire zona pellucida, which was perpendicular to the cell membrane on a cord from the oocyte centre towards the oolemma and zona (Figure 1D). All the measurements were performed by the same person and blindly with respect to the age of the patient, numbers of additional oocytes or contribution to a CC or a NCC. The average retardance rate was calculated for each oocyte from three cross-section scans of each individual oocyte (see below).



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Figure 1. Images of human oocytes selected for embryo transfer. (A) The zona pellucida of oocytes observed by light microscopy appears transparent. (B) The three-laminar architecture of zona pellucida with a highly birefringent inner layer (IL), followed by a dark-appearing middle layer (ML, arrow) and a greyish-appearing outer layer (OL) can be detected by Polscope microscopy. (C) Most oocytes of the conception cycle (CC) group contained a bright and thick zona pellucida. (D) Measurements of the thickness and retardance magnitude of the zona pellucida in each individual oocyte were done in three areas. The first and second measurements were done on a cross-sectional region of the zona on a line scan through the centre of the oocyte (dotted line). The third line scan was perpendicular to the two first measurements at an angle of ~90°. (D–F) Oocytes from patients in the non-pregnant group with aberrant zona pellucida, which was either relatively thin (D), irregular (E) or sub-split into two layers (F, arrows). (G) Typical retardance curve of a line scan through the zona pellucida of an oocyte from the CC group (solid line) and the non-conception cycle (NCC) group (dotted line) respectively. Vertical dotted lines depict outer boundaries of layers measured on the line scan of the oocyte contributing to a CC or NCC. Open arrows show the outer measurement points included in the calculation of the magnitude of retardance of the outer and inner layers. For the middle layer, calculation was from the boundaries defined by the dotted vertical lines. (H) Scheme of the thickness, and retardance magnitude of the three zona layers in CC versus NCC. RT = retardance. Bar = 25 µm.

 
After imaging by Polscope, oocytes were transferred to another coded dish for microinjection by ICSI, so that the fate of each individual oocyte could be followed. Microinjection of sperm in ICSI was blind with respect to data obtained by Polscope (position of spindle, expression of spindle, zona morphology etc.), and vice versa; evaluation by Polscope was initially done prospectively without the aim of selecting cohorts of oocytes for ICSI or transfer. The selection of embryos for transfer was entirely based on standard scoring criteria evaluating embryo morphology at the pronuclear stage, 18 h after fertilization (see below).

Since thickness and texture of the human zona pellucida may be heterogeneous for individual oocytes and within each zona layer, one line scan was usually performed in an area away from the first polar body providing a profile of thickness and magnitude of retardance of the tri-layered structure at 0.5 µm steps (Figure 1D, G). A second line scan was located ~180° away from the first one, extending the line from the first measurement through the oocyte centre outwards (Figure 1D). Initially, only these two line scans served to obtain information on the medium zona thickness or the medium magnitude of retardance within each layer. To obtain more information, later a third area on a line scan of a line perpendicular to the first two scans was performed (at an angle of ~90°, Figure 1D). Thus, three measurements were performed in different areas of the zona of each oocyte. Maximal differences (in percentage of the highest value) in zona retardance of the inner layer between the three measurements in each oocyte were compared statistically.

For quantitative comparisons between retardance of zona layers in germinal vesicle oocytes, metaphase II oocytes and preimplantation embryos, Pelletier et al. (2004)Go defined the retardance magnitude of individual layers of the zona by taking one value at the midpoint of eight cords through the layer and then calculating the average of the eight points. However, the zona pellucida is an extremely heterogeneous, net-like structure surrounding oocytes (Magerkurth et al., 1999Go). We therefore calculated the mean values of the retardance magnitude using all measurement points spaced 0.5 µm apart along the whole cross-section curve for each layer (Figure 1G), except for the two outermost points (indicated by the area between the arrows in Figure 1G). Since the retardance may steeply increase or rise more gradually going from the inside towards the outside of the zona, and retardance in the perivitelline space may not be close to zero, the outer measurement points of the layers defining boundaries for determination of thickness of each zona layer were usually designated at the site of an increase over two measurement points (indicated by vertical stippled lines in Figure 1G). The middle layer was defined by the dark area between the boundaries of the outer and inner layer (between the dotted vertical lines in the middle of Figure 1G). The average retardance was calculated initially from the three line scans for each individual oocyte. For comparison between the CC and NCC groups, average retardance and thickness was normalized from two to three oocytes used in transfer for each patient to avoid bias between two and three embryo transfers. We avoided measuring an area with an abnormal zona phenotype, e.g. in cases where a zona layer was split into two sublayers (Figure 1F, arrows). In these cases, the three measurements were performed in areas close to the normal cross-section regions but adjacent to the split zona area, such that the zona was still in close proximity to the oolemma. Only three such oocytes were examined (Table I). In each case only one single oocyte with a split zona was used in transfers including one to two additional embryos (Table I).

A few oocytes used for transfer could not be evaluated for zona morphology because granulosa cells were still attached after isolation, and the boundaries of the zona layers could not be unambiguously identified. However, all of the 65 oocytes comprising embryos transferred after ICSI in the CC group and 101 oocytes of the 104 embryos later used for transfer in the NCC group were included in the calculations. Of the 65 oocytes used later in embryo transfer in the CC group, 93.2% possessed a birefringent spindle, and similarly, 95.0% of the 101 oocytes in the NCC possessed a birefringent spindle (Figure 1C, D), not significantly different from each other (Table I). A similarly high percentage of oocytes with birefringent spindles was reported previously by Rienzi et al. (2004)Go. However, the overall numbers of oocytes exhibiting a spindle in all oocytes from our patients, including those not used in transfer, was <90% (data not shown), as in most other published reports (e.g. Wang et al., 2001Go). Statistical analysis of zona and spindle data from all oocytes of the CC and NCC patients, including those that were not used for transfer, is currently in progress, but has not been the subject of the present study.

Assessment of PN score for embryo selection, development of embryos at day 2 and pregnancy
After Polscope analysis, oocytes were transferred to another dish and subjected to standard ICSI by independent persons, who were not involved in Polscope imaging. The selection of oocytes used for embryo transfer was performed at the pronuclear stage, 18 h post-insemination. The criteria of PN score (Figure 2) were according to standard criteria used in the IVF centre at Giessen University. Selection was based on the position of both pronuclei and the alignment of nucleoli in the pronuclei (modified scoring criteria from Scott and Smith, 1998Go) (see Figure 2) as well as the overall morphology of the pro-embryo.



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Figure 2. Pronuclear scoring system (modified from Scott and Smith, 1998Go). (A–D) Score AD: pronuclei closely apposed, further designation according to the position of nucleoli as follows: (A) Score A: three to 10 nucleoli aligned at the pronuclear junction. (B) Score B: nucleoli polarized but not highly apposed at pronuclear junction. (C) Score C: More than seven nucleoli scattered in nucleoplasm. (D) Score D: asymmetric distribution of nucleoli and/or numbers of nucleoli >10 or <5. (E) Score E: pronuclei spatially separated, unequal in size and/or not clearly distinguishable.

 
In embryo selection for transfer, the following factors were especially considered: (i) PN score of the embryo should be as high as possible, usually A–D according to our criteria, but in a few cases, where no other embryos were available, embryos with PN score E, including an embryo with one more distinct and one diffuse-appearing PN, were also used for transfer. (ii) The cytoplasm should be homogeneous and the oocyte without deformations. (iii) The localization of the nucleoli should be similar in both pronuclei (Nagy et al., 2003Go). A peripheral accumulation of nucleoli in only one PN would be considered as an indicator of poor quality of the embryo (see Figure 2). (iv) The presence of a halo was considered as an indicator of high quality of the embryo (Stalf et al., 2002Go), and was therefore used as additional indicator in embryo selection, especially when selection occurred between embryos of otherwise similar morphology.

Two to three embryos with the highest scores from the cohort obtained from each patient were selected for transfer (2.8±0.4 and 2.6±0.6 embryos per transfer in the CC and NCC groups respectively) (Table I). Of the 169 oocytes from 63 patients, the 166 selected for embryo transfers after ICSI were analysed for development to a 1-, 2–3- or 4–5-cell embryo at 42 h post insemination. The majority of the embryos (100% in the CC and 95% in the NCC group; Table I) were then transferred to the uterus on day 2 after oocyte retrieval. Only five of the 101 embryos in the NCC group (in two treatment cycles) were transferred on day 3, entirely for practical reasons (Table I). Two weeks after transfer, 23 patients had positive results in the pregnancy test three times, while the other 40 patients failed to become pregnant after ICSI treatment. The thickness and the retardance of the ZP and the individual zona layers as assessed prior to insemination by Polscope were compared between the oocytes transferred after ICSI in the pregnant and non-pregnant patients (65 in CC and 101 in NCC).

Statistical analysis
Two-tailed Student's t-test was used for the quantitative analysis of the retardance and thickness of zona layers. Linear regression test was used to calculate the relationship between retardance and thickness of the inner zona layer. Two-way analysis of variance was used for assessing the correlation between zona thickness and retardance and reproductive age within the CC and NCC groups. {chi}2-Test was performed to compare the number of oocytes with a high magnitude of retardance of light (>3 nm), intermediate magnitude of retardance (3–2 nm), and low magnitude of retardance (>2 nm) of the inner layer of the zona between the CC and NCC groups. U-Test was used for the analysis of the heterogeneous properties of the zona pellucida. Logistic regression analysis was performed to assess the sensitivity of the retardance data in comparison to PN score and embryo development at day 2. Significance was considered as P<0.05 for each statistical analysis. All the analyses were performed with SPSS 12.0 and SAS software.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Characteristic texture of the human zona pellucida in oocytes of the CC and NCC group
The human zona pellucida appears comparatively transparent when viewed with a conventional light microscope equipped with Hoffmann interference optics (Figure 1A). Enhanced polarized light microscopy reveals three distinctly different layers of the extracellular coat surrounding the human oocyte (Figure 1B). An inner layer appears as the most birefringent, bright and thickest layer of the human zona pellucida in all oocytes observed by Polscope imaging in this study. The inner (IL) and the outermost layer facing the medium or cumulus (OL) are separated by a thin middle layer (ML), which appears dark and without much birefringence (Figure 1B, G). As noticed already by Pelletier et al. (2004)Go, the morphology of the zona pellucida may be rather heterogeneous around individual oocytes (Figure 1DF), and within cohorts of oocytes from individual patients.

The variability in zona morphology within cohorts of oocytes from individual patients and between oocytes from different patients appeared mainly related to differences in the relative homogeneity and birefringence and brightness of the inner layer of the zona pellucida (Figure 1CF and H). In a few oocytes the zona pellucida appeared especially irregular or thin (Figure 1D and E), and in three cases the inner layer of the zona was subdivided into two layers with a hollow-appearing space in between (Figure 1F, arrows; Table I). There was a maximum difference in zona retardance of the IL between the three line scans in an individual oocyte of 17.2% in the CC and of 44.5% in the NCC group. On average, the maximal difference between the three measurements in all the oocytes of the CC group was 8.5±8.5%, significantly lower than in the NCC group (16.5±14.6%; U-test: P<0.05).

Figure 1G shows two characteristic examples of cross-sections along a line scan at 0.5 µm-spaced measurement points to determine light retardance across the entire zona pellucida. One characteristic scan is from the cohort of oocytes in the CC group (upper solid line), the other is characteristic for an oocyte of the NCC group (lower dotted line). The inner layer (IL) exhibits the highest retardance magnitude and cross-section thickness for both examples. The retardance magnitude of the outer layer (OL) tends to be overall lower than the inner layer as demonstrated in both line scans, while the middle layer (ML) consistently appears dark. As can be seen in all images of oocytes in Figure 1BF, which were later used in transfers after ICSI, the majority of oocytes used for transfer in both groups contained a bi-polar, barrel-shaped meiotic spindle, which was localized close to the first polar body in most cases (double arrows; Figure 1).

The development of embryos used for transfer in CC and NCC cycles
Number of mature oocytes and rate of fertilization was similar in CC and NCC (Table I). After ICSI of all oocytes, selection of embryos was performed according to PN score and the general morphological criteria described above (Figure 2). From the 166 oocytes included in the study and transferred to patients after ICSI, >60% in both groups comprised embryos with PN scores A–C, while around one-third of the oocytes used for transfer in both groups had developed to a pro-embryo with a PN score of D (Figure 3A). Only two oocytes with score E, and one embryo with PN score E in which 1PN had a diffuse nuclear membrane were selected for transfers; these were later shown to belong to the non-pregnant group. Therefore, the average PN score of pro-embryos used for transfer in CC and NCC groups did not differ much. For instance, from the average 2.8 and 2.6 embryos transferred (Table I), the number of embryos selected with PN score A–C was 1.8±0.75 and 1.6±0.85 in CC and NCC group respectively (Figure 3A). Retrospective comparison of embryo development on day 2 after fertilization between those embryos comprising the CC and NCC groups revealed striking differences in mitotic cycles (Figure 3B). In total, 53.8% of the embryos in the CC group had developed to the 4–5-cell stage at 42 h post insemination, whereas only 34.7% of embryos giving rise to the NCC group had reached this advanced stage ({chi}2-test, P<0.05). In contrast, ~60% of the embryos in the NCC group had only ≤3 cells on day 2 of development after oocyte retrieval (Figure 3B).



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Figure 3. Pronuclear (PN) scores at 18 h post-insemination (A) and embryo development at day 2 (B). (A) Two to three 1-cell embryos with the best PN scores were selected for embryo transfer from the cohort of each patient. Scores of A–E are shown. Embryos contributing to the conception cycle (CC) or non-conception cycle (NCC) group had similar PN scores on day 1. (B) Development at day 2 was significantly different (a{chi}2-test: significant differences between CC and NCC groups; P<0.05) between the groups. Embryos of the CC group tended to possess more cells and more rapidly divided compared to embryos of the NCC group. Numbers on top of bars are percentages.

 
Quantitative analysis of retardance magnitude of zona layers in CC and NCC
The quantitative analysis of the zona pellucida by Polscope revealed that the average retardance magnitude of the zona pellucida differed considerably between cohorts of oocytes in CC and NCC cycles (Table II). While differences in mean retardance were insignificant in the middle and outer layer, the mean magnitude of retardance of the inner layer of the zona pellucida was significantly higher in the oocytes of the CC compared to the NCC group (2.81±0.60 versus 2.15±0.41 nm, P<0.001; Table II; Figure 1H).


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Table II. Mean retardance magnitude and thickness of the individual zona layers as assessed by Polscope microscopy in oocytes contributing to conception cycle (CC) and non-conception cycle (NCC) groups

 
Moreover, we classified the 166 oocytes into three groups based on their mean retardance magnitude of the first layer of the zona pellucida. Group 1 had a retardance of >3.0 nm; group 2 between 2.0 and 3.0 nm, and group 3 <2.0 nm (Figure 4). Nearly 90% of the oocytes in the group 1 (25 oocytes) comprising oocytes with a retardance >3.0 in the zona inner layer from a total of 28 oocytes were associated with a clinical pregnancy (Figure 4). In contrast, a retardance magnitude of >3 nm was observed in only three oocytes of the NCC group ({chi}2-test: P<0.001). With the decrease of the magnitude of retardance in the zona inner layer, the percentage of oocytes resulting in a pregnancy cycle was considerably reduced. Only 11.4% of the oocytes in the lowest retardance group belonged to a CC after ICSI and embryo transfer while the majority did not contribute to a conception (Figure 4; {chi}2-test: P<0.001).



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Figure 4. Quantitative assessment of average light retardance of the inner layer of the zona pellucida in oocytes contributing to the conception cycle (CC) or non-conception cycle (NCC) group (asignificant difference between NCC and CC groups; P<0.001). Retardance magnitude >3 nm is predictive of CC, while retardance <2 nm of the inner layer of the zona pellucida appears to be associated with reduced developmental potential and pregnancy rate.

 
Quantitative analysis of thickness of the zona layers in CC and NCC
Similar to the magnitude of retardance, thickness of the middle and outer layers did not differ significantly between groups, but the thickness of the inner layer was significantly increased in the pregnant group (11.25±1.44 µm) compared to the non-pregnant group (9.36±1.74 µm, P<0.001, Table II). It appears that it is the relative thickness of layer 1 that leads to an overall increase in thickness of the entire zona pellucida in the CC group. The thickness of the zona pellucida was significantly different between the two groups although the magnitude of the absolute difference in thickness was low, on average only ~1.3 µm (Table II).

Relationship between thickness and retardance
The relationship between retardance and thickness of the inner layer of the zona pellucida in human oocytes, which is not a deterministic (or causal) one, is represented in Figure 5. Obviously, data for individual oocytes do not distribute randomly, supporting the suggestion of Pelletier et al. (2004)Go that the retardance of the zona pellucida, especially the inner layer of the zona pellucida, is positively correlated with zona thickness. The correlation coefficients of the retardance–thickness curves are slightly different with 0.43 and 0.56 for the CC and NCC groups respectively.



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Figure 5. Relationship between retardance and thickness of the inner layer of the zona pellucida in human oocytes contributing to the conception cycle (CC) or non-conception cycle (NCC) group.

 
Influence of maternal age, and differences in zona thickness and retardance between transfer and non-transfer oocytes
The mean retardance and thickness of the zona inner layer and total zona were also compared between oocytes in CC and NCC from younger and more reproductively aged women (Table III). In total, the number of patients in the young, middle and aged groups was 28, 21 and 14 and that of oocytes 77, 54 and 35 respectively (Table III). Among the total of 23 CC patients and 40 NCC patients, no significance was found in the mean retardance and thickness between the zona of oocytes from women of different age belonging to the groups <32, 32–37 and >37 years, while values for mean retardance and thickness were lower in all oocytes from the NCC compared to the CC group at each age (Table III).


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Table III. Comparison of the mean retardance and the thickness of the zona inner layer and the total zona thickness in oocytes from young and aged women contributing to conception cycle (CC) and non-conception cycle (NCC) groups

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Although more than one embryo is usually transferred, rates of implantation and pregnancies in assisted reproduction are still fairly low (Fauser et al., 2002Go). When ethical and legal considerations constrain selection after fertilization, it appears especially important to identify non-invasive markers of oocyte quality to obtain reasonable implantation and pregnancy rates (e.g. Zollner et al., 2002Go; Ebner et al., 2003Go). Invasive methods such as biopsy and chromosomal analysis of the first and second polar body (Gitlin et al., 2003Go; Kuliev et al., 2003Go) are costly, time-consuming and also not acceptable by some patients. Therefore, the search for cheap and fast, non-invasive methods to identify factors associated with structural and functional integrity of the oocyte has been intensified (Wang et al., 2001Go; Eichenlaub-Ritter et al., 2002Go; Keefe et al., 2003Go; Rienzi et al., 2003Go; Shen et al., 2005Go). Although invasive morphological analysis, mainly on oocytes that failed to be fertilized or those with fertilization abnormalities from IVF programmes, provided a large source for information on the ultrastructure of the zona pellucida (e.g. Phillips and Shalgi, 1980Go; Takagi et al., 1989Go; Magerkurth et al., 1999Go; Vanroose et al., 2000Go; Familiari et al., 2001Go; Bogner et al., 2004Go), it has not been possible to relate this to oocyte developmental potential. Due to the paracrystalline network structure of the zona (Wassarman et al., 2004Go), we could use orientation-independent polarization microscopy in the present study to assess zona morphology quantitatively.

This and other Polscope studies confirm the bilaminar, tripartite nature of the zona (Keefe et al., 1997Go; Pelletieret al., 2004)Go as identified in several species (Baranska et al., 1975Go; Andrews et al., 1992Go; Gilchrist et al., 1997Go; Green, 1997Go; Keefe et al., 1997Go; Dunbar et al., 2001Go; Eberspaecher et al., 2001Go; Sinowatz et al., 2001Go; El-Mestrah et al., 2002Go; Jimenez-Movilla et al., 2004Go). In cases with zona splitting, the patterning or the secretion of protein may have been temporarily interrupted during the formation of this part of the extracellular coat during oocyte growth, or the zona was ruptured by mechanical stress at retrieval or separation from cumulus. This was apparently a rare event in our sample and associated with an NCC. The mean±SD thicknesses of the zona are similar to those found by Pelletier et al. (2004)Go using the same methodology measuring zona thickness and retardance in unselected oocytes (19.5±2.2 µm) but are different from those determined by Shiloh et al. (2004) in human oocytes of non-smoking women using photos of human oocytes imaged by Nomarski contrast optics (15.32±0.10 µm). In the latter study, increased zona thickness was examined mainly in oocytes from smokers (20.15±0.12 µm) but the fate of these oocytes has not been followed after fertilization. In this study, the number of smokers did not differ significantly between CC and NCC (Table I).

The thickness of the zona increases progressively during maturation but becomes thinner after fertilization prior to hatching (Dirnfeld et al., 2003Go; Pelletier et al., 2004Go). Accordingly, the retardance magnitude of the zona pellucida also changes stage-specifically at the immature and mature stages of oogenesis and the preimplantation period (Pelletier et al., 2004Go). It has been suggested that the overall variability in zona thickness at day 2 of human embryogenesis is an indicator for implantation potential (Gabrielsen et al., 2001Go). To our knowledge this is the first report demonstrating that there is a positive correlation between the texture of the zona, as quantitatively assessed by the magnitude of retardance of light in Polscope microscopy in unfertilized, freshly retrieved human oocytes, and pregnancy rate after ICSI. Although significant in the comparison, the differences in thickness between CC and NCC groups were not extensive (on average 1.3 µm). Accordingly, it does not appear to be useful to measure zona thickness for oocyte selection. In contrast, we show here that the magnitude of retardance in the inner layer of the zona is >30% higher in oocytes of the CC compared to NCC group before actual IVF by ICSI, and before zona hardening and thinning had been initiated. Furthermore, oocytes from the CC group appear to have a more homogeneous zona morphology compared to oocytes in the NCC group. Although the embryos in the CC and NCC groups had a quite different developmental rate and fate, the PN-stage oocytes selected by Scott's score criteria at day 1 after ICSI were similar between the two groups at the 1-cell stage. In fact, embryos with a Scott's score of A–C had the same chance (~50%) of contributing to a conception after implantation. Therefore, the currently used PN score criteria appear not to be highly predictive for embryo quality, in particular, to assess embryo capacity for implantation. Our observations imply that high quality embryos, which can facilitate implantation and induce an increase in hCG level in blood, had a significantly higher retardance in the zona IL and developed faster than embryos, which failed to induce a pregnancy, in agreement with other reports in the literature (e.g. Lundin et al., 2001Go; Gianaroli et al., 2003Go; Nagy et al., 2003;Go Neuber et al., 2003Go; Ziebe et al., 2003Go). The retardance magnitude appears therefore to be a new predictive indicator of the high quality and developmental potential of an oocyte to contribute to a conception after ICSI. Since there was also no striking difference in the expression of a spindle, the magnitude of zona retardance appears to present a novel, unique marker for oocytes and embryos with high developmental potential, which possess an otherwise similar morphology.

What is the reason for the striking correlation between zona texture and the potential of the embryo to support pregnancy? A robust zona with high-order structured fibres probably reflects the healthiness of an oocyte and its full maturation to metaphase II (Pelletier et al., 2004Go). It tentatively implies that the oocyte, and possibly also the granulosa cells within the follicle (Sinowatz et al., 2001Go; Gook et al., 2004Go), were able to secrete coordinately large amounts of glycosylated zona proteins, which were assembled into a high-order network during folliculogenesis and oocyte growth (Nikas et al., 1994Go). Expression of zona proteins may contribute in unknown ways to the establishment of polarity gradients, and to improving oocyte/somatic cell signalling, e.g. by transzonal projections during growth and maturation (Albertini and Barrett, 2003Go).

A thick and solid zona could also be of advantage in ICSI, because it protects the oocyte particularly well from mechanical stress during the microinjection procedure, and stress-related changes during preimplantation development (e.g. Leese, 2004Go). However, ICSI has been successful in cases of zona-free human oocytes (Ding et al., 1999Go; Takahashi et al., 1999Go; Hsieh et al., 2001Go; Stanger et al., 2001Go), while implantation appeared more jeopardized by absence of a zona in some patients (Hsieh et al., 2001Go). Zona pellucida thickness also appears positively correlated to embryo quality after IVF (Dirnfeld et al., 2003Go).

Furthermore, the accumulation of autocrine and paracrine factors at the zona could enhance oocyte or embryo development (Seshagiri et al., 1994Go; Celik-Ozenci et al., 2003Go; for discussion see Herrler and Beier, 2000Go; Herrler et al., 2003Go; Liu and Armant, 2004Go). For instance, growth hormone can alter the structure and pore size of the zona pellucida of blastocysts in bovine (Kolle et al., 2004Go), and may act in concert with insulin-like growth factor I to optimize blastocyst development (Markham and Kaye, 2003Go). Importantly, our study suggests that a robust zona with a highly structured inner layer per se does not compromise implantation after ICSI.

We did not find a clear correlation between zona thickness and retardance and reproductive age within the CC and NCC groups in our limited sample. Analysis of the zona pellucida may not greatly improve pregnancy rate in aged patients, in which aneuploidy of the embryo may be the primary source of implantation failure (Pellestor et al., 2003Go; Munné et al., 2004Go). Nearly 50% of the embryos transferred in the youngest age group involved conception cycles in contrast to ~30% of the embryos transferred in the age groups >32 years involved in this study. Since nearly all oocytes used for transfer in this study contained birefringent spindles as assessed by Polscope, they probably did not suffer from major spindle damage and aneuploidy, and may therefore have developed into an embryo with a good PN score (Magli et al., 2001Go; Gamiz et al., 2003Go; Gianaroli et al., 2003Go; Balaban et al., 2004Go). The study supports the concept that selection for morphological criteria may reduce the risk of transferring a developmentally compromised embryo (Ziebe et al., 2003Go). We observed that the magnitude of zona retardance in the inner layer of the zona of the unfertilized oocyte correlated predictively with a CC, and also with a comparatively rapid first cleavage. We calculated that 60% of all oocytes in the CC comprised oocytes with a retardance of ≥2.5 nm of the zona IL, and only 25.7% of the NCC oocytes; thus such a value may be rather predictive ({chi}2-test, P<0.001). Results of a logistic regression analysis using SAS system showed that retardance described the quality of oocytes and their fate after insemination more clearly than the PN score and embryo score system. Nevertheless, the numbers of oocytes analysed in this study are low, and further analysis is required. Currently, studies are underway to analyse also the magnitude of retardance of light in the zona of the non-transferred oocytes in our cohorts. This should provide information on inter-individual and inter-oocyte specific differences in zona morphology and texture, which may be used to study age effects, identify adverse lifestyle factors (e.g. smoking, Shiloh et al., 2004Go), or to optimize treatment and to counsel patients.

Clearly, controlled prospective studies are now required to validate whether quantitative zona imaging can improve pregnancy rate or help to identify embryos with high developmental capacity in order to reduce numbers of transferred embryos. In view of the dynamic changes in zona morphology at maturation, fertilization, and, possibly, post-ovulatory oocyte ageing (Dirnfeld et al., 2003Go; Pelletier et al., 2004Go), it is essential that strict timing of imaging is ensured. Especially in cases where legal and ethical considerations prohibit selecting at a more advanced stage of embryogenesis, including screening at the pronuclear stage, zona imaging may offer new options in oocyte selection since the procedure is fast, provides immediate information and has no adverse effects on the oocytes.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors are most grateful to the staff in the Giessen IVF centre for their cooperation throughout the study. We also thank Wolfgang Pabst, Institute for Medical Informatics of the University Hospital of the Justus-Liebig-University, for his contributions on the statistical analysis of the data. We thank Dr R.Eichenlaub for critical reading of the manuscript. The study was supported by the EU (contract number QCRT-2000–00058).


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 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on September 9, 2004; resubmitted on January 14, 2005; accepted on January 21, 2005.





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Articles by Shen, Y.
Articles by Tinneberg, H.-R.