Noninvasive polarized light microscopy quantitatively distinguishes the multilaminar structure of the zona pellucida of living human eggs and embryos

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1 FERTILITY AND STERILITY VOL. 81, SUPPL. 1, MARCH 2004 Copyright 2004 American Society for Reproductive Medicine Published by Elsevier Inc. Printed on acid-free paper in U.S.A. Noninvasive polarized light microscopy quantitatively distinguishes the multilaminar structure of the zona pellucida of living human eggs and embryos Cory Pelletier, B.A., David L. Keefe, M.D., and James R. Trimarchi, Ph.D. Division of Reproductive Medicine and Infertility, Department of Obstetrics and Gynecology, Women and Infants Hospital of Rhode Island, Brown University Medical School, Providence, Rhode Island Objective: To characterize the architecture of the zona pellucida in living human eggs and embryos, noninvasively with the PolScope, a digital polarizing light microscope. Design: The PolScope was used to examine zonae pellucida of living human eggs and embryos. Setting: Academic IVF clinic. Patient(s): Patients undergoing fresh, nondonor infertility treatment who underwent egg aspiration, fertilization by intracytoplasmic sperm injection or traditional IVF, and cleavage-stage embryo transfer (day 3). Intervention(s): The PolScope imaged the zona of eggs before intracytoplasmic sperm injection and in cleavage-stage embryos before transfer. Main Outcome Measure(s): Thickness and retardance of three zona layers were measured from eight quadrants. Mean and variance in thickness and retardance were calculated for individual eggs and embryos, between eggs and embryos of a cohort, and across the sample patient population. Result(s): Cleavage-stage (day 3) embryos have thinner zonae ( m) than both immature ( m) and mature ( m) eggs. The zona of embryos is thinner, primarily owing to thinning of the outer layer. The thicker the zona layer, the greater its retardance. Considerable variation exists in the thickness and retardance of zona layers around individual eggs and embryos and between members of a cohort. The zona of eggs and embryos from different patients differ in thickness, retardance, and variation. Conclusion(s): Thickness and organization of zonae pellucida of human eggs and embryos varies considerably and can be quantitatively imaged with the PolScope. (Fertil Steril 2004;81(Suppl 1): by American Society for Reproductive Medicine.) Key Words: Embryo, human, IVF, egg, polarized light microscope, zona pellucida Received May 14, 2003; revised and accepted September 19, Supported by the Women and Infants Hospital Family Research Fund. Reprint requests: James R. Trimarchi, Ph.D., Women and Infants Hospital, 1 Blackstone Place, Providence, Rhode Island (FAX: ); jtrimarc@ wihri.org) /04/$30.00 doi: /j.fertnstert The zona pellucida is an extracellular, multilaminar, glycoprotein coat that envelopes the mammalian egg and preimplantation embryo and serves to bind and activate sperm. The matrix of the zona consists of three glycoproteins (ZP1, ZP2, and ZP3), which are synthesized, secreted, and assembled exclusively by the egg during oogenesis (1, 2). The zona serves as a gatekeeper to sperm entry: sperm must bind and penetrate in to reach and fuse with the egg plasma membrane during fertilization (3). After fertilization, the zona matrix protects the cleaving embryo as it traverses the fallopian tube to the uterus, where the blastocyst-stage embryo hatches from the zona before implantation (4, 5). Previously, we used an orientation-independent polarized light microscope (LC-PolScope; Cambridge Research and Instrumentation, Inc., Woburn, MA) to identify some unique features of the zona s architecture. Because the zona forms during oogenesis, we hypothesized that properties of the human zona could estimate oocyte/embryo quality and therefore sought to characterize zona morphology, particularly its thickness, retardance, and degree of irregularity. First, however, the architectural characteristics of the zona in living human eggs needed 850

2 FIGURE 1 The multilaminar structure of the zona pellucida can be quantified with PolScope imaging. Human egg, imaged with differential interference contrast optics (A) and with the PolScope (B). (C) Cleavage-stage (day 3) embryo imaged with the PolScope. Distinct zona layers can be imaged with the PolScope. (D) Thickness and retardance of the zona and each of its layers were measured with spindle view in eight octants around the egg and embryo. (E) Contributions of individual zona layers to total zona thickness of eggs and cleavage stage embryos. Scale bars 20 m. to be carefully described; this was the purpose of this study. The multilaminar structure of the zona is only marginally perceptible with conventional Hoffman (Nikon, Melville, NY) or differential interference contrast optics; with the PolScope, electro-optical hardware and digital processing are used to image macromolecular structures on the basis of their birefringence (6), a unique optical property of highly ordered molecules, such as zona pellucida fibers. Birefringent objects retard one of the two refractive indices of polarized light, the magnitude of which can be quantified by the PolScope and displayed on a grey scale in computerized images (6). Two structures in the mammalian egg exhibit molecular order when imaged with polarized optics: the meiotic spindle and the zona pellucida (2, 7 17). The meiotic spindle is crucial for normal chromosome alignment and separation of maternal chromosomes during meiosis. The PolScope has also been used to examine spindle dynamics, detect spindle morphology, predict chromosome misalignment, monitor thermal control, and perform spindle transfer (8 12, 14 16), but zona architecture has been less well characterized. The PolScope reveals three layers that differ (6). Relative to the oocyte, filaments of the inner zona (layer 1) are oriented radially, whereas filaments of the outer zona (layer 3) are oriented tangentially (3, 6). Owing to this organization, these layers appear birefringent with the PolScope (Fig. 1). The inner and outer layers are separated by a middle layer (layer 2) exhibiting minimal birefringence, which suggests a random orientation of filaments (7). Both the spindle and zona pellucida of living mammalian eggs can be observed noninvasively with the PolScope without the aid of dyes or harmful light wavelengths. Recently, epitope tagging of the zona proteins demonstrated that the various layers of the zona are laid down in temporal sequence, with the inner layer laid down last. Therefore, properties of the zona layers might reflect the history of follicular development and/or oocyte cytoplasmic maturation (1). Zona morphology, particularly its thickness and degree of irregularity, has been proposed to predict subsequent implantation (18), and puncturing the zona (assisted hatching), either mechanically with acid or with a laser, has been proposed to improve implantation rates. However, the value of assisted hatching and the relationship between zona properties and outcomes remains controversial (19 24). It is helpful that the value of assisted hatching remains debatable, because indications remain poorly defined. The purpose of the experiments reported here was to characterize the architecture of the human zona, with use of the PolScope, to provide normative standards for future measures of the human zona. Better characterization of the FERTILITY & STERILITY 851

3 zona layers with the PolScope might clarify which patients would benefit from assisted hatching and whether zona imaging could serve to diagnose oocyte and embryo quality. MATERIALS AND METHODS Sources of Oocytes and Embryos Approval was obtained from the Women and Infants Hospital Institutional Review Committee to collect and analyze images of human eggs and embryos collected routinely during IVF/intracytoplasmic sperm injection (ICSI). Eggs were aspirated from stimulated ovaries of 29 consenting patients, aged years (mean SD, years), who were undergoing egg retrieval for IVF. After retrieval, eggs were cultured in P1 medium (Irvine Scientific, Santa Ana, CA) containing 6% synthetic serum substitute (SSS; Irvine Scientific) for 5 to 6 hours. Before examination with the PolScope, cumulus cells were removed by pipetting cumulus egg complexes in modified human tubule fluid (Irvine Scientific) containing 80 IU/mL hyaluronidase (Sigma Chemical Co., St. Louis, MO). Eggs that had released the first polar body were considered for ICSI, and eggs without a first polar body were further cultured in vitro. In vitro maturation was conducted in P1 medium supplemented with 6% SSS at 37 C, 5% CO 2 in air with 100% humidity. Both immature and mature eggs were imaged in the study. Embryos resulting from both ICSI (n 19) and IVF (n 64) were cultured in P1 media through postretrieval day 3 with SSS (Irvine Scientific). TABLE 1 and variance in thickness and retardance as measured around individual eggs and embryos. Immature eggs (n 33) Zona Pellucida Imaging in Living Oocytes and Embryos with the PolScope To image zonae pellucida, each egg and embryo was placed in a 5- L drop of modified N-2-hydroxyethylpiperazine-N -2-ethanesulfonic acid-buffered human tubule fluid covered with warm paraffin oil (Gallard-Schleserger, Coral Place, NY) in a glass-bottomed (0.17 mm thick) Petri dish (50 mm diameter) (WilCo; World Precision Instruments, Sarasota, FL). Dishes were maintained at 37 C 0.5 C during examination with a thermal plate (Tokai Hit Thermo Plate; Zander Medical, Vero Beach, FL) and objective heater (Cambridge Research and Instrumentation), and eggs were examined with a microscope (Zeiss Axiovert S100TC; Carl Zeiss International, Jena, Germany) affixed with a 0.55 condenser and LD Acroplan 40 objective Numerical Appeture (NA) 0.60 with correction collar) equipped with LC Pol- Scope filters, analog video camera (Kohu; Cambridge Research & Instrumentation), and a personal computer running image analysis software (Spindle View 3.9; Cambridge Research and Instrumentation). Experimental Design Zonae pellucida were imaged by focusing at an equatorial plane of each egg and embryo to ensure the accuracy of image representation of thickness of zona layers. Digital images were saved to a compact disk for subsequent analysis. A computerized imaging analysis system (Spindle View 3.9) was used to measure the thickness and retardance of zona layers in eight octants by drawing chords extending from the inner boundary of the zona outward (Fig. 1D). Retardance magnitude was collected at the midpoint of the chord for each layer. All measurements were collected by the same observer. The observer was blinded to the source of the eggs, and seven eggs were imaged twice to calculate R 2 values for thickness and retardance. R 2 were 0.85 and 0.95, respectively, for the corresponding images, thus confirming the reproducibility and reliability of the image capture and analysis system. Statistical Analysis Variation in thickness and retardance were analyzed with the F test, and differences in means with Student s one-tailed t-test. Percentage composition comparisons of layers between eggs and embryos were made by a root-squared, arc-sine transformation of percents and analyzed by analysis of variance. Patient-specific data collection was restricted to cycles in which six or more eggs or embryos were imaged. Differences were considered statistically significant at P.05. RESULTS Mature eggs (n 92) Day 3 embryos (n 83) Zona layer 1 Thickness ( m) b Retardance (nm) Zona layer 2 Thickness ( m) Retardance (nm) a c Zona layer 3 Thickness ( m) b Retardance (nm) b Zona total thickness ( m) b Note: Values are mean SD (one-tailed t-test; P 0.05). a Statistically significant difference between immature and mature eggs. b Statistically significant decrease between mature eggs and day 3 embryos. c Statistically significant increase between mature eggs and day 3 embryos. Although differential interference contrast can be used to noninvasively observe the overall zona properties, only polarized light optics allows the multilaminar structure of the zona to be imaged and quantified in living eggs and embryos (Fig. 1). In both eggs and embryos the innermost layer (layer 1) is the thickest of the three zona layers and also exhibits the 852 Pelletier et al. PolScope views laminar zona Vol. 81, Suppl 1, March 2004

4 FIGURE 2 Embryo zona are thicker, primarily owing to thinning of layer 3. Comparisons of overall zona pellucida thickness (A), individual layer thickness (B), and individual layer retardance (C). Small dots are averages of eight measurements from individual eggs and embryos; large dots and bars represent averages and SDs. highest retardance, indicated by brightness in the PolScope image (Figs. 1, 2, Table 1). The outermost layer is thinner than layer 1, and exhibits lower retardance, whereas the middle layer is the thinnest of the three zona layers and exhibits little to no retardance. No differences in individual layer thickness or overall zona thicknesses were observed between immature (germinal vesicle, meiosis I) and mature (meiosis II) eggs (Table 1). The zonae of embryos differed in thickness and retardance compared with mature eggs. Each zona layer was thinner in embryos than in eggs, thus contributing to an overall thinning of the zona in embryos (Figs. 1, 2, Table 1). In embryos, layer 3 contributes less to the overall zona thickness than it does in eggs, thus permitting layers 1 and 2 to contribute more to the embryo zona thickness (Fig. 1E). The retardance of layer 1 did not differ between eggs and embryos. The retardance of layer 2 increased in embryos, and the retardance of layer 3 decreased in embryos compared with eggs. For layers 1 and 3, which exhibit significant retardance, thickness correlates positively with retardance (Fig. 3, Table 2). For example, in day 3 embryos, the retardance increases 0.38 nm with each increasing thickness micron. This relationship holds for immature eggs, mature eggs, and embryos (Fig. 3), as well as in the cohort of embryos from individual FERTILITY & STERILITY 853

5 FIGURE 3 Zona retardance increases proportionally with increasing layer thickness. A relationship exists between zona layer 1 thickness and layer 1 retardance for immature eggs, mature eggs, and embryos. Inset demonstrates that this positive correlation holds for a cohort of embryos from a single patient. patients (inset, Fig. 3). Therefore, thicker zona layers display greater retardance. Human zona layers display variation between eggs and embryos of a cohort and between patients (Fig. 4). The cohort of eggs and embryos from some patients has similar layer thicknesses and retardances. For example, patient 14 (Fig. 4A and C) had eight day 3 embryos all having comparable layer 1 thickness as well as retardance levels. By contrast, thickness and retardance differed in the cohort of eggs and embryos from other patients (Fig. 4A and C). In addition to variation in zona properties between members of a cohort, we found variation in layer thicknesses and retardances of zona around individual oocytes (Fig. 4B and D). For example, layer 1 thickness is very consistent at each position measured around all eggs from patient 6, thus leading to low values in thickness variance (Fig. 4B). Only some eggs from patient 7 showed consistent layer 1 thicknesses (low values in thickness variance), whereas other eggs from this patient had very inconsistent zonae (high value in thickness variance). A similar phenomenon of variance in retardance around individual eggs was observed (Fig. 4D). Human zona layers display great variation in retardance and thickness. DISCUSSION This study quantitatively characterized zona layers in immature oocytes, mature oocytes, and cleavage-stage embryos. Specifically, the zona is thinner in embryos than in eggs, and this thinning primarily results from thinning in the outermost layer. Thicker zonae of oocytes and embryos also have greater retardance. Lastly, the zona can be irregular around individual eggs and embryos, as well as between eggs and embryos of a cohort and between patients. Recently, it has been proposed that overall zona thickness could serve as a clinical diagnostic for embryo selection, and during the last few years much interest in this subject has been generated (25). Lacking from this debate has been detailed knowledge of the variation in zona properties between eggs and embryos. Here we report the first quantitative characterization of zona properties from living human eggs and embryos and demonstrate that not only does overall zona thickness vary, but the thickness and molecular organization of subzonal layers vary as well. The zona pellucida of cleavage-stage embryos is thinner than that of eggs. It is well known that zonae thin by the blastocyst stage (23, 24, 26 31), and here we show this phenomenon already has begun by day 3. All three layers constituting the zona pellucida are thinner in embryos than in eggs. However, proportionally, layers 1 and 2 contribute less and layer 3 more to the thinning process, thus indicating a possible stretching of the zona as the embryo increases in diameter (32, 33). Alternatively, zona thinning is unlikely to TABLE 2 Thicker zonas have a higher retardance. Thickness 10 m Thickness 10 m Retardance comparison thickness ( m) retardance (nm) thickness ( m) retardance (nm) (one-tailed t-test) Immature eggs P.05 Mature eggs P.05 Day 3 embryos P.05 Note: Values are mean SD. 854 Pelletier et al. PolScope views laminar zona Vol. 81, Suppl 1, March 2004

6 FIGURE 4 Zona layers exhibit variation within a cohort of eggs or embryos, between patients, and around individual eggs. Comparisons of zona pellucida layer thickness (A), variance in layer thickness (B), layer retardance (C), and variance in layer retardance (D) from cohorts of embryos grouped by patient. The zona of eggs and embryos from the same patient vary considerably (A and C). The thickness and retardance of the zona varies around individual eggs and embryos (B and D). Small dots are averages of eight measurements from individual eggs and embryos; large dots and bars represent averages and SDs. result from protease digestion because thinning occurs most dramatically in the outermost layer. Retardance increases proportionally with increases in the inner and outermost layers thickness. The increased retardance could reflect greater molecular organization of zona pellucida filaments making up the zona (5, 34). Alternatively, the elevated retardance of thicker zona layers might be caused by a longer light path. Human zona layers display variance between oocytes and embryos from a given cohort. The zona matrix is formed during follicular development in the ovary, and epitope tagging of zona proteins demonstrated that zona layers are laid down in temporal sequence, with the inner layer laid down last (1). The oocytes and resulting embryos we examined were harvested after gonadotropin stimulation, which alters follicular recruitment, and oocytes were harvested from follicles of different sizes and maturation. This variation in oocyte origin might underlie the variance we observed in the zona of oocytes and embryos and might serve as a proxy of egg/embryo quality. FERTILITY & STERILITY 855

7 Subzonal layers vary around individual eggs. Spatial heterogeneity in cytoplasmic maturation could account for variation in the zona around eggs. Eggs are relatively large cells, and regional or spatial differences in enzymatic or protein production within the egg cytoplasm or the surrounding granulosa cells could alter zona protein deposition. Alternatively, the degree of attachment of granulose cells to the egg might vary and could regionally restrict zona formation and result in the variance in zona parameters around individual eggs. The variation in subzonal layers observed in this study might provide a noninvasive marker for egg and embryo quality. Imaging the human zona with the PolScope might prove useful in clinical embryo morphology grading and has the potential to resolve the controversy over the clinical utility of assisted hatching. Future studies relating variance in zona parameters to stimulation regimes, embryo quality assessment, and clinical outcome are underway. 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