The spindle observation and its relationship with fertilization after intracytoplasmic sperm injection in living human oocytes

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1 FERTILITY AND STERILITY VOL. 75, NO. 2, FEBRUARY 2001 Copyright 2001 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. The spindle observation and its relationship with fertilization after intracytoplasmic sperm injection in living human Wei-Hua Wang, Ph.D., a Li Meng, Ph.D., a Rickard J. Hackett, M.Sc., a Rudolf Odenbourg, Ph.D., b and David L. Keefe, M.D. a,b,c Women and Infants Hospital of Rhode Island, Brown University School of Medicine, Providence, Rhode Island Received April 25, 2000; accepted August 9, Supported by National Institutes of Health K and by the Women and Infants Hospital Faculty Research Fund. Presented at the 55th Conjoint Annual Meeting of the American Society for Reproductive Medicine and the Canadian Fertility and Andrology Society, Toronto, Ontario, Canada, September 25 30, Reprint requests: David L. Keefe, M.D., Division of Reproductive Endocrinology, Women and Infants Hospital of Rhode Island, 101 Dudley Street, Providence, Rhode Island (FAX: ; dkeefe@wihri.org) a Division of Reproductive Medicine and Infertility, Department of Obstetrics and Gynecology, Women and Infants Hospital of Rhode Island, Brown University School of Medicine. b Division of Reproductive Medicine and Infertility, Department of Obstetrics and Gynecology, New England Medical Center, Boston, Massachusetts. c Marine Biological Laboratory, Woods Hole, Massachusetts /01/$20.00 PII S (00) Objective: To image spindles in living human and to examine the relation between spindles and fertilization after ICSI. Design: The LC polscope was used to examine spindles in an observational study of living. Setting: Academic IVF clinic. Patient(s): Women being treated for infertility. Intervention(s): Oocytes retrieved from patients for infertility treatment were examined before ICSI. Aged, unfertilized after IVF or ICSI were examined with polscope and confocal microscopes to compare the two methods. Main Outcome Measure(s): Spindle structure in living and fertilization after ICSI. Result(s): Spindles could be imaged in 61.4% of. More with spindles than without spindles fertilized normally after ICSI (61.8% vs. 44.2%). Spindles in most aged were partially or completely disassembled, and only a few microtubules around the chromosomes or dispersed microtubules in the cytoplasm were observed. Confocal images of immunostained spindles were almost identical to polscope images of spindle birefringence. Conclusion(s): Spindles in living human can be imaged by using the polscope. A birefringent spindle in human may clinically predict the quality and age of. This method also can be used to monitor spindle position during ICSI. (Fertil Steril 2001;75: by American Society for Reproductive Medicine.) Key Words: Human, ICSI, spindle, polarization microscope In unfertilized metaphase 2, the meiotic spindle is crucial for normal chromosome alignment and separation of maternal chromosomes during meiosis (1, 2). Disruption of the meiotic spindle results in rearrangement or scatter of chromosomes throughout the cytoplasm (3 8) and may contribute to aneuploidy after fertilization. Aneuploidy is one of the most commonly observed patterns of abnormal fertilization in humans (9 11) and is the major reason for early embryo death, spontaneous abortion, and inherited disease (12). The dramatic decrease in clinical pregnancy rates in older women is closely related to the occurrence of aneuploidy (9, 10). Battaglia et al. (13) found that the proportion of with abnormal meiotic spindles was significantly higher in older women than in younger women and concluded that abnormal spindles may contribute to the high prevalence of aneuploidy in embryos from this population. Although the reasons for abnormal spindle structure in older women are not completely understood, it presumably contributes to their low success rate in achieving pregnancy after assisted fertilization. Other factors, such as altered temperature and ph (3 8) and oocyte age (2, 14, 15), also cause spindle disassembly in mammalian. All of these fac- 348

2 tors may disrupt subsequent fertilization, embryo development, implantation, and fetal development. Conventional methods to image the spindle rely on fixation (3 8, 13 15) and are therefore of limited value in clinical IVF and in studies of spindle dynamics. Currently, the only method of testing embryos for aneuploidy is preimplantation genetic selection, either by polar body (16) or blastomere biopsy (12). However, these techniques may not reflect mosaicism, and they require oocyte or embryo biopsy. Until now, no satisfactory method could distinguish morphologically normal for use in human IVF; thus, some transferred embryos presumably resulted from abnormal. It is well known that up to 80% of aneuploidy has maternal origin and that spindle integrity is closely related to distribution of chromosomes. Therefore, a method to detect spindle architecture may help predict the distribution of chromosomes and reduce the occurrence of aneuploid embryos transferred during IVF. A polarization microscope (polscope) was recently developed to study birefringence of living cells (17). The polscope uses novel electrooptical hardware and digital processing to image macromolecular structures in cells on the basis of their birefringence (18). Birefringence is an inherent physical property of highly ordered molecules, such as microtubules. Because meiotic spindles in are highly birefringent, their structures can be viewed by the polscope. The polscope has already been used to image spindles in hamster (19). Because it illuminates specimens with the same intensity of light as differential interference contrast (DIC) (unpublished observation) and because DIC (which also uses a form of polarized light) has been used safely during IVF for over 20 years, the polscope should be nontoxic to human. We found no detrimental effects on mouse oocyte or embryo development after exposure to the polscope (20); the polscope therefore has potential for application in human IVF. Intracytoplasmic sperm injection has been used in human IVF clinics for several years (21). To avoid damage to the spindle that may cause abnormal chromosomal separation, during ICSI, embryologists orient the oocyte on the holding pipette on the basis of the position of the first polar body (21). However, the effects of ICSI on chromosomes, especially the 30% 40% of that fail to fertilize, are unknown. It is possible that microinjection damages spindles during ICSI and thus disrupts chromosomal separation in some. The movement of the polar body in the perivitelline space or migration of the spindle deeper into the oocyte could be misleading as to the relation between the spindle and polar body. The position of the polar body is not an accurate predictor of spindle position in mammalian, as demonstrated by polscope imaging of hamster (19). In combination, a method of detecting spindle morphology and locating spindle position would probably increase the safety and efficiency of assisted fertilization. We sought to examine [1] whether spindles in living human could be imaged by using the polscope, [2] whether the polscope images of the spindle could be confirmed by immunocytochemical staining and confocal microscope imaging in in which fertilization failed, [3] whether the presence of birefringent spindles and fertilization of after ICSI are related, and [4] whether the presence of a birefringent spindle and oocyte age are related. The latter would suggest that the polscope may help select structurally normal for rescue ICSI. MATERIALS AND METHODS Sources of Oocytes Approval was obtained from the Institutional Review Board of Women and Infants Hospital to study unfertilized human and to study images of obtained during human IVF. We obtained from two sources. One source was the stimulated ovaries of consenting patients undergoing oocyte retrieval for ICSI. After retrieval, were cultured in P1 medium (Irvine Scientific, Santa Ana, CA) containing 6% synthesized serum substitute (SSS; Irvine Scientific) for 5 6 hours. Before examination with the polscope, the cumulus was removed by pipetting in modified human tubal fluid medium (Irvine Scientific) containing 80 IU/mL hyaluronidase (Sigma Chemical Co., St. Louis, MO). We also examined that had remained unfertilized 1 4 days after IVF or ICSI. Only without obvious morphologic degeneration were used. Spindle Examination and ICSI in Fresh Oocytes with the Polscope For imaging spindles and ICSI, each oocyte was placed in a5- L drop of modified human tubal fluid medium covered with warm paraffin oil (Gallard-Schleserger, Coral Place, NY) in a Bioptechs TC3 Culture Dish System (Bioptechs Inc., Butler, PA). The system consists of a temperature controller; a stage adapter; and the TC3 dish, which has a specially coated clear glass bottom that is 0.15 mm thick. The dishes were maintained at 37 C during examination and ICSI. Oocytes were examined under a Zeiss Axiovert 100 with a Neofluar 40 strainfree objective and LC Polscope optics and controller (CRI, Cambridge, MA), combined with a computerized image analysis system (MetaMorph Universal Imaging System, West Chester, PA). The images were saved on a JAZZ disk (Jazz Drive, Iomega Corp., Roy, UT) for subsequent analysis. After imaging, ICSI was conducted, and with or without spindles were cultured separately for hours in P1 medium (Irvine Scientific), supplemented with 10% synthesized serum substitute for examination of fertilization. For image analysis, images were reloaded and the Meta- Morph computerized imaging system was used to measure the pole-to-pole size of the spindle, the distance between FERTILITY & STERILITY 349

3 FIGURE 1 Spindles in living human imaged by using the polscope just before ICSI. (A), An oocyte with a spindle located just under the plasma membrane, near the polar body. (B), An oocyte with a spindle forming at 43 to the polar body. (C), An oocyte with a spindle that migrated to a deeper position (arrow) in the cytoplasm and that is forming at 42.5 to the polar body. Original magnification, 300). pb polar body. Wang. Spindle observation in living human eggs. Fertil Steril polar body and spindle, and the angle between the spindle and the polar body. Spindle Examination in Aged Oocytes under the Polscope and Confocal Microscopes Unfertilized without obvious morphologic abnormalities were selected on days 1 4 after IVF or ICSI and examined under the polscope. After imaging, were fixed separately in 3.7% paraformaldehyde (Sigma Chemical Co.) in phosphate-buffered saline (PBS) and treated overnight with 0.1% Triton X-100 in PBS. Oocytes were incubated first in a blocking solution (PBS containing 2 mg/ml bovine serum albumin and 150 mm glycine) for 30 minutes, then in PBS containing anti -tubulin (1:300) (Sigma Chemical Co.) for 1 hour. After thorough washing in PBS containing 0.1% Tween 20, were incubated with fluorescein isothiocyanate conjugated goat antimouse IgG (1:32) (Sigma Chemical Co.) in PBS and Tween for 1 hour. Samples were then stained with propidium iodide, 10 g/ml, in PBS for examination of chromosomes. Finally, the were mounted on slides and examined by using laser confocal microscopy. Confocal microscopy was performed by using a Carl Zeiss LSM 410 laser confocal imaging system equipped with an argon laser and mounted on a Zeiss microscope. The images were compared with those obtained by using the polscope. Statistical Analysis Comparisons were made by using the 2 test. P.05 was considered statistically significant. RESULTS Spindles in Fresh Oocytes Five hundred thirty-three from 51 cycles were examined by using the polscope, and spindles were imaged in 61.4% of the (Fig. 1). The size of the spindles was m, and distance between the spindle and the first polar body was m. On the basis of the angle formed by the polar body, the center of oocyte, and the spindle, were divided into four groups. In group 1, had spindles on a line formed between the polar body and center of oocyte or located less than 5 relative the to polar body (Fig. 1A); 18.7% of were in this group. In group 2, had spindles located 6-45 relative to the polar body (Fig.1B, C); 69.1% of were in this group. In group 3, had spindles located relative to the polar body; 11.3% of were in this group. Finally, in group 4, had spindles located 90 relative to the polar body; 0.9% of were in this group. In most, spindles were close to the plasma membrane (Fig. 1A, B); however, in some, they appeared to have migrated to a position deep within the cytoplasm (Fig. 1C). Relation between Spindle and Fertilization after ICSI Because most (in groups 1 and 2) had a spindle located in a position close to the first polar body ( 45 ), ICSI was conducted after the were rotated to place the first polar body at 90 relative to the injection needle. Intracytoplasmic sperm injection in without spindles was also performed in this manner. In with spindles located at other positions ( 45 from the polar body or the spindle moved to a deeper position), ICSI was performed after were rotated to a position so that the injection needle avoided the spindles. As shown in Table 1, after ICSI, more with spindles than without spindles were fertilized normally, forming two pronuclei (61.8% vs. 44.2%; P.05). In contrast, more without a spindle 350 Wang et al. Spindle observation in living human eggs Vol. 75, No. 2, February 2001

4 TABLE 1 Relation between spindle and fertilization of human after ICSI. Presence of spindle examined with 3 PN with 2 PN with 1 PN 0PN degenerated Yes (61.4) b (4.6) (61.8) b (1.8) (23.9) b (8.0) No (38.6) c (1.0) (44.2) c (2.9) (39.8) c (12.1) Total (3.2) (55.0) (2.3) (30.0) (9.6) Note: Oocytes were collected from 51 cycles and examined by using the polscope just before ICSI. PN pronucleus or pronuclei. a Percentage of number of examined. b P.05 with values in the same column. c P.05. Wang. Spindle observation in living human eggs. Fertil Steril than with a spindle remained unfertilized (P.05). Oocytes without spindles tended to degenerate more frequently than those with intact spindles, but the difference was not statistically significant because of the small sample. Spindles in Aged Oocytes On day 1 (16 18 hours after IVF or ICSI), spindles were seen in 8 of 11 (73%) that had been confirmed as unfertilized (Fig. 2A). Spindles in aged were shorter ( m) than those in fresh. Spindle structure seen with the polscope was similar to that seen by using confocal microscopy (Fig. 2A, insert). In 3 on day 1 (27%) and 27 on days 2 4 (100%), no birefringent spindles were seen by using the polscope. The images obtained by confocal microscopy indicated that the spindle was disassembled in these eggs (Fig. 2B, C). Chromosomes did not align on the center of spindles (Fig. 2A, insert) or were scattered throughout the cytoplasm (Fig. 2B, C). DISCUSSION Our results indicate that spindles in living human can be imaged by using the LC polscope. A higher fertili- FIGURE 2 Spindles in aged human as seen with the polarization microscope (polscope) and confocal microscope. (A), A day 1 oocyte with a small spindle. The confocal microscopy image of this oocyte was nearly identical to the polscope image (insert); (B), Confocal microscope image of aged on day 2. (C), Confocal microscope image of aged on day 3. In the latter two images, no spindle can be seen in the and chromosomes are scattered throughout the cytoplasm (arrows). The green portions represent microtubules, red represents chromatin, and yellow represents the overlay of the green and red images. Original magnification, 300). pb polar body. Wang. Spindle observation in living human eggs. Fertil Steril FERTILITY & STERILITY 351

5 zation rate was achieved in with birefringent spindles, indicating that the presence of spindles in human predicts oocyte quality. The polscope also can be used to monitor spindle position during oocyte micromanipulation, such as ICSI or nuclear transfer. An increased incidence of chromosomal aneuploidies is thought to contribute to decreased clinical pregnancy and implantation rates in older women (9, 10). In IVF clinics, such abnormalities have been detected in from young women as well, especially those with pathologic ovarian conditions (12). Such anomalies may arise from abnormal organization of microtubules or other cytoskeletal factors that lead to abnormal spindle formation. Because the cytoskeleton is sensitive to environmental changes, disruption of spindle architecture may also occur during oocyte maturation under the nonphysiologic conditions presented by the IVF laboratory. Battaglia et al. (13) recently reported that the proportion of with abnormal spindles was significantly higher in older than in young women, and abnormal spindles were associated with abnormal chromosomal distribution. We found a patient-dependent effect on the proportions of with or without birefringent spindles, but we did not find a relation between birefringent spindles and patient age. Other factors presumably influenced the presence of birefringent spindles in our study. The rate of abnormal spindle structure also increases as age in culture (2, 14, 15). Results of these studies provide strong evidence that abnormal chromosomal alignment in aged results from abnormal spindle structure. Although these studies suggest a mechanism to explain aneuploidy in humans, direct confirmation of the association between aneuploidy and spindle structure is needed. We found that spindles in living human can be imaged by using the polscope and that after ICSI, more with spindles than without spindles were fertilized. From these results, we conclude that the polscope can be useful in selecting on the basis of spindle birefringence before ICSI. On the basis of our results, we do not know whether without birefringent spindles develop to aneuploid embryos; further studies are necessary to directly test this hypothesis. We detected birefringent spindles in only 61.4% of living human. It was not clear why the other did not show birefringent spindles, but this finding was probably not related to technical limitations of the polscope; previously, we imaged spindles in more than 95% of metaphase II mouse (Wang et al. Unpublished data) and hamster (19) by using the same technique. Imaging of spindles in living with the polscope is based on birefringence, an inherent physical property of the microtubules. Thus, that do not show spindle birefringence were most likely injured during oocyte development, maturation, or in vitro manipulation. Indeed, meiotic spindles in mammalian are exquisitely sensitive to environmental changes, including ph and temperature (3 8). It seems that human are more sensitive to temperature fluctuations than are from other animals (3, 6, 8, 22), and spindle structure is disrupted even when are exposed briefly to temperature even slightly less than 37 C (4). Although we maintained at 37 C during all manipulations, some unexpected environmental changes, including transient fluctuations in temperature or ph, may have caused spindle disassembly. Furthermore, oocyte age, maternal age, and other patient-dependent factors may disrupt spindles. Maternal age has been found to play an important role in abnormal spindle dynamics (13). We did not find a close relationship between maternal age and spindle architecture (data not shown); however, all were retrieved from women undergoing infertility treatment, who presumably exhibit variable degrees of reproductive aging that do not always correlate with chronological age. Future studies must examine the relation between spindle structure and maternal age. When we compared images of the same spindles obtained by polscope and by confocal microscope, we found that the two images were nearly superimposible. As in previous reports (2, 14), the spindles in aged (16 18 hours after IVF or ICSI) were shorter than those in fresh under the polscope. By analyzing images obtained by confocal microscopy, we confirmed that aged had deformed spindles and abnormal chromosomal alignment. As did previous investigators (2, 14, 15), we found that aged had small or absent spindles. These results suggest that abnormal spindle structure and scatter of chromosomes may explain the low efficiency of ICSI performed on in vitro aged. Although we did not attempt rescue ICSI on day 1, it is very likely that abnormal embryos would have been produced from such aged. Therefore, the polscope may help determine whether can be used for rescue ICSI based on the birefringence, size, and position of the spindles. We also found that polar body position could predict the exact spindle position only in a small proportion (about 20%) of. Thus, monitoring spindle position during ICSI may reduce spindle damage and chromosome breaks, especially because most embryologists aspirate the egg cytoplasm before injecting the sperm. The observation of spindles in living also is important for basic and applied research. Traditionally, to examine spindle dynamics or to study the effect of environmental changes on spindles, only fixed samples have been used; it was therefore difficult to examine the dynamic behavior of individual spindles. Moreover, fixation introduces artifacts that alter the architecture of the spindles. However, examination of spindles in living with the polscope overcomes such disadvantages because spindles can be observed in their living state continuously over time. Studies using fluorescently labeled tubulin also are limited by the need to excite the fluorescent dyes and by the tending 352 Wang et al. Spindle observation in living human eggs Vol. 75, No. 2, February 2001

6 of all fluorescent dyes to quench over time. Quantitative analysis of spindle fiber density is thus severely limited. Nuclear transfer also has been widely used in animal breeding and other biomedical research (23, 24). A key step during nuclear transfer is enucleation of the recipient oocyte. Currently, most laboratories remove the nucleus after fluorescent staining of chromatin. However, fluorescence imaging is detrimental to, because it excites the cell with high-frequency light and involves artificial fluorescent dyes. The low developmental ability of reconstructed embryos may result in part from the detrimental effects of fluorescent dyes and fluorescent light. In addition, because chromosomes are usually associated with spindle fibers, removal of the spindles in living may be required for complete removal of all the chromosomes in recipient before nuclear transfer. So far, we have found that spindles in living hamster (19), mouse (20), and human are visible under the polscope. The examination of spindles in other mammals will extend the practical uses of the polscope. In conclusion, our results indicate that the polscope is useful for selection of spindle-positive and monitoring of spindle position during human ICSI. We also found that in vitro aging disrupts spindle structure. Use of the polscope is not detrimental to ; because the are imaged in seconds, the polscope is suitable for everyday general use in the laboratory. Because we did not separately culture the fertilized eggs (embryos), we do not know whether the quality of embryos and pregnancies differs between with or those without spindles. Further studies must culture embryos individually after polscope imaging and ICSI to test the hypotheses that spindle birefringence and architecture can predict the developmental potential of the resulting embryos. References 1. Zamboni L, Fine morphology of mammalian fertilization. New York: Harper & Row, Pickering SJ, Johnson MH, Braude PR. 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