The use of polarized light microscopy in IVF

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1 The use of polarized light microscopy in IVF Expert Rev. Obstet. Gynecol. 6(3), (2011) Suha Kilani 2 and Michael G Chapman 1 1 School of Women s and Children s Health, University of New South Wales, Sydney, NSW 2052, Australia 2 IVF Australia, 225 Maroubra Road, Maroubra, Sydney, NSW 2035, Australia Author for correspondence: skilani@ivf.com.au LC-PolScope technology, formally sold as Spindle View, is a microscopic imaging system that utilizes polarized light. It was initially designed for noninvasive imaging of structures within living cells, for example, the meiotic spindle, zona pellucida and sperm acrosome. The system has been developed as an adjustment to the standard light microscope with the addition of two polarizers and image analysis computer software. The unique feature of this device is the ability to capture live images of these biological structures, thus enabling quantitative analysis. As a noninvasive tool, this system has been used in IVF laboratories during intracytoplasmic sperm injection to avoid damaging the meiotic spindle while injecting individual spermatozoa into the oocyte. The latest version of this device (Oosight ) allows instant image analysis for quicker and easier use in IVF laboratories. This version also has better resolution, which allows a more detailed assessment of intracellular structures and may assist scientists in the selection of oocytes with the greatest potential to produce a pregnancy. Recent published data show higher pregnancy rates in embryos derived from oocytes where a normally shaped meiotic spindle was identified. Keywords: embryo implantation intracytoplasmic sperm injection IVF meiotic spindle polarized light microscopy Infertility is defined as the inability of a couple to conceive after 1 year of unprotected sexual intercourse. Approximately 15% of couples worldwide experience some form of infertility. The treatment of infertility with IVF has become an almost routine procedure for millions of couples experiencing infertility. The first IVF baby was conceived in By 2010 more than 4 million babies had been born as a result of assisted reproductive technology (ART). IVF involves stimulation of ovaries with FSH to produce multiple follicular development. When these follicles are appropriately developed, oocyte retrieval is undertaken under ultrasound control. The oocytes are then inseminated either by placing them with prepared semen in culture medium to allow fertilization to occur naturally (standard IVF) or by injection of individual oocytes with a single spermatozoa (intracytoplasmic sperm injection [ICSI]). The latter technique is used for significant male factor infertility, or for patients with previous poor fertilization rates or failed fertilization with standard IVF. Polarized light microscopy for viewing oocytes can only be used with ICSI since in this process the cumulus mass, which surrounds the oocyte, has been stripped away from the oocyte. Embryos are transferred transcervically to the uterus on day 2 or 3 after egg collection (four to eight cells) termed the cleavage stage or on day 5 (>100 cells) termed the blastocyst stage. A pregnancy test is performed 16 days after oocyte retrieval. The selection of embryos with the highest implantation potential for transfer is one of the major challenges in ART [1]. Historically, multiple embryo transfer was used to maximize pregnancy rates. However, with improved embryo quality, multiple pregnancy rates increased with their associated potential risks of prematurity and cerebral palsy [2]. This has led to the necessity for a reduction in the number of embryos replaced. Thus, selection of the best embryo has become essential, especially now that elective single embryo transfer is recommended or even mandated in a number of countries. Traditionally, embryo quality assessment has been synonymous with and restricted to morphological evaluation using white light microscopy. Highly sophisticated systems of assessment have been developed, such as oocyte grading, first polar body morphology [3], pronuclear stage grading [4], embryo grading and /EOG Expert Reviews Ltd ISSN

2 Kilani & Chapman the degree of fragmentation [5]. These are influenced by human bias with significant variability and, ultimately, have reduced accuracy. Other noninvasive approaches have been studied to assess embryo quality. Measurement of either embryo excretion products in the culture medium, such as amino acids or pyruvate [6], or embryo uptake of nutrients such as glucose or oxygen have been correlated with pregnancy outcomes. Most of these techniques have not been rapid enough to be used in a clinical setting and have neither sensitivities nor specificities that make them particularly useful in practice. A recent automated approach measuring the profile of metabolic products of the embryo (termed metabolomics) is more rapid and appears to show promise in picking the embryos with the greatest pregnancy potential [7]. Genetic testing of single cells biopsied from the embryo can select those with a normal chromosomal makeup, but this is invasive and the subsequent pregnancy rates may be adversely affected by the procedure [8,9]. Noninvasive assessment of oocytes with the use of polarized light microscopy has allowed the assessment of their normality prior to fertilization [10]. In particular, the assessment of the meiotic spindle and zona pellucida has been a breakthrough [11,12]. It was initially applied in research in mice, but more recently it has been used on human oocytes [13,14]. It would seem that it is possible to predict the oocytes most likely to result in a pregnancy, and so assist in the selection of the best embryo for transfer. Introduction to the device The sample is illuminated with circularly polarized light. The standard (mechanically operated) compensator is replaced by a liquid crystal (LC) compensator. The LC universal compensator and grayscale charge coupled device camera are controlled by a host computer. Five images are taken, with the LC compensator changed to five different settings. The five images are then used to calculate the sample s birefringence at every pixel, based on patented polarimetric algorithms. Image analysis software Camera Polarizer 2 Polarizer 1 Figure 1. Inverted microscope with two polarizers and image ana lysis software. Image courtsey of CRi Oosight Imaging System on an Olympus inverted microscope. The LC-PolScope technology, sold as Oosight imaging system, is a commercial brand of orientation-independent polarized light microscopy. The device allows noninvasive detection and ana lysis of the birefringent objects in human oocytes, such as the meiotic spindle. Biological structures are often transparent or translucent and appear to have little or no detail when they are backilluminated by white light. However, when those same tissues are placed between a pair of crossed polarizers, the structures become visible (birefringence theory; Figure 1), and the greater the degree of order in the biological structure and the more organized the structure is, the greater the birefringence. This bending of the light path when it hits a structure compared with the path of a straight beam of light can be measured in nanometers (called retardance) and can be interpreted by the specific computer program, and is displayed on the screen as grayscale values. The physical length of the structures can also be measured and are expressed in µm. The design of the LC-PolScope technology developed by Cambridge Research and Instrumentation (CRi) is based on a conventional polarized light microscope with some critical adaptations. Retardance is measured in nanometers and is the difference between the path lengths of the polarized light waves (Figure 2). Structures that are imaged can be captured by the camera and be stored for later ana lysis. Depending on the number of oocytes to be injected, an experienced scientist using the LC-PolScope/Oosight takes 5 10 min longer than the conventional intracytoplasmic sperm injection. Preparation of the image system merely involves swapping the two polarizers with the standard light filters and booting up the Oosight software. The oocyte is placed in a glass-bottomed dish in culture medium. It is then imaged in four planes rotating with the injection needle to optimally view the meiotic spindle. The images are saved into an individual folder within the software program. The oocyte is then injected such that the needle avoids the meiotic spindle. The saved images can then be analyzed using the PolScope/Oosight software to establish quantitative parameters including retardance, length and width measurements of the zona pellucida and spindle. The initial capital cost of this equipment when compared with the standard light microscopy is an extra US$20,000 30,000, which is a small additional cost in the context of a fully functional IVF laboratory. There are extra costs in the disposable glass-bottomed dishes that are geometrically perfect to avoid distortion of the polarized light (US$3 10 compared with US$1 for a plastic dish for conventional ICSI). In addition, there is the expense of scientific time due to the slightly longer time to use the system. These amounts should add no more than min per case, even with a large number of oocytes. The use of polarized light microscopy is moving from being a research tool to application in the clinical setting. Early studies 242 Expert Rev. Obstet. Gynecol. 6(3), (2011)

3 The use of polarized light microscopy in IVF Device Profile have shown that meiotic spindle visualization can be an important tool for predicting better fertilization potential, embryo development and clinical outcomes [14,15]. Wang et al. studied the effect of cooling and warming on the meiotic spindle integrity using the LC-PolScope. They indicated that the LC-Polscope, can be used to image spindles in living oocytes. allowing ana lysis of spindle kinetics in the living state. The results of this study have shown that human meiotic spindles are exquisitely sensitive to alterations in temperature [16]. This is an important practical issue when handling oocytes in the clinical setting. Subsequent research has shown that injecting oocytes away from the spindle, localized by polarized light microscopy, resulted in higher fertilization rates and better embryo quality [16,17]. Traditionally when ICSI is performed, the oocyte is held and the first polar body (1PB) is placed either at the 12 or 6 o clock position to avoid disrupting the meiotic spindle, which had been believed to be universally beneath the 1PB. However, polarized light microscopy has shown that the meiotic spindle is not always located under the 1PB and can be anywhere in the hemisphere [17,18]. When oocytes are injected away from the meiotic spindle, the embryos generated have been shown to have better morphology and less fragmentation than control oocytes [17]. This viewing of the meiotic spindle with polarized light reduces the incidence of damaging the meiotic spindle during ICSI. Spindle integrity of frozen/thawed oocytes has been studied [19,20]. Chen et al. demonstrated that oocytes with good spindle morphology verified by the LC-PolScope before vitrification had a higher survival rate of intact oocytes after thawing compared with those with poor or undetected spindle images. They also showed a good agreement of spindle morphology by both the LC-PolScope and fluorescent staining [19]. There are studies in the literature that investigated the zona pellucida using the LC-PolScope. Madaschi et al. investigated possible factors contributing to the zona pellucida birefringence score and meiotic spindle visualization, and evaluated whether these structures may predict intracytoplasmic sperm injection outcomes. The results showed that selection of embryos based on zona pellucida and meiotic spindle imaging can significantly improve implantation and pregnancy rates [21]. A recent study used OCTAX polaraide to analyze whether a change in 3D structure of the zona pellucida could indicate suboptimal gamete quality. The results showed that in approximately a third of all gametes, the inner zona layer was not detected. This absence was a negative predictor of compaction, blastulation and pregnancy. In cases of successful zona imaging, the score based on the birefringence of the inner zona layer was a strong predictor of blastocyst formation, but not of embryo quality or pregnancy. The authors concluded that zona imaging was a helpful tool during oocyte selection [22]. Retardance Figure 2. Spindle retardance measured in nm. Clinical profile and post-marketing findings. Data taken from [26]. Micrometers micrometers Kilani et al. compared the zona pellucida thickness and density of oocytes from older women (>38 years) with those of younger women ( 38 years). The zona pellucida thickness and density was measured at four locations (3, 6, 9 and 12 o clock). For the zona pellucida thickness, the zona was measured from the outer layer to the inner layer and the average of the four measurements was used for the ana lysis. While for the zona pellucida density, the average measurements of the innermost birefringent layer of the whole zona pellucida was used for ana lysis. The results of the study showed that oocytes of older women had thicker and denser zonas than younger women. Blastocysts formed from these oocytes had thicker and denser zonas [23]. It is suggested that embryos with thicker zonas are less likely to hatch. Assisted hatching has been proposed for those oocytes, although no studies to date have tested this hypothesis. The optimal time for injecting oocytes is a controversial issue in the literature. Polarized light microscopy enables serial observation of oocytes over time from stripping of the cumulus cells until ICSI. Our group has shown the dynamic nature of the formation and dissolution of the meiotic spindle. Analysis of images of oocytes, captured at five time intervals using LC-PolScope showed that the largest percentage of oocytes had a visible spindle at h post-trigger. This time interval has been suggested to be the most suitable time for ICSI [24,25]. In an initial study using the LC-PolScope to assess markers within the egg which might assist in assessing the better quality oocytes, we compared oocytes resulting in a pregnancy to those that did not generate a pregnancy. In this small retrospective study the rate of spindle normality, defined as barrel shaped and complete spindle, was significantly higher in oocytes in the pregnant patients compared with oocytes in the nonpregnant patients (100 vs 33%; p < 0.001) [26]. Spindle density was also significantly higher in those oocytes resulting in pregnancy (3.0 ± 1.23 nm vs 2.5 ± 0.7 nm; p = 0.02). It was concluded that these oocyte markers might provide a useful noninvasive tool in the selection of the embryo most likely to produce a pregnancy. Wang et al. examined whether spindle morphologic features imaged with the LC-PolScope in living human oocytes matured in vitro can be used to predict chromosome configuration and 243

4 Kilani & Chapman select oocytes with normal chromosomes. His study demonstrated that the spindle images obtained with the LC-PolScope in living human oocytes correlate with those in fixed oocytes as imaged by confocal microscopy. Wang concluded that spindle images with the LC-PolScope can be applied to human IVF in order to help predict chromosomally normal oocytes for insemination [27]. Subsequently, using the more recent Oosight system, 100 women producing more than 900 oocytes and undergoing ICSI were prospectively followed up. Oocytes were individually cultured and followed up until embryo transfer. Scientists blinded to the Oosight results selected blastocysts based on morphological criteria. Only single embryo transfers were performed. Analysis of outcomes showed a 60% pregnancy rate in those derived from normally spindled oocytes. There were no pregnancies in those derived from oocytes with abnormal spindles [Kilani S, Cooke S, Tilia L, Chapman MG. Does meiotic spindle normality predict improved blastocyst development, implantation and live birth rates?; Manuscript submitted]. We therefore believe that polarized light microscopy has a significant role to play in the selection of the best embryo [28]. On the basis of this study we are now using Oosight in routine practice to select the blastocyst for transfer when there is a cohort of morphologically similar blastocysts. It is also being used in patients with repeated implantation failure to attempt to define a possible oocyte issue in their failure to conceive. Other groups have published work with LC-PolScope in which they have addressed issues that are very important to the daily life of an IVF scientist. Similar to our findings, the detection of the meiotic spindle in in vitro maturated oocytes and frozen thawed oocytes [29,30] suggested better quality oocytes. Polarized light microscopy was found to be a helpful tool to decide the timing of ICSI for oocytes matured in vitro [31]. Rama Raju et al. demonstrated that the quantitative measurement of the length and retardance of the meiotic spindle and zona pellucida has a positive predictive value in relation to embryonic development [32]. A recent meta-ana lysis has investigated the outcome of ICSI when the spindle was visualized. The results of the ana lysis showed significantly higher fertilization rates when the spindle was viewed. Embryos derived from oocytes with visible spindles cleaved better and formed more blastocysts. However, the clinical pregnancies and implantation rates was not different between the two groups. This meta-ana lysis looked only at the presence or absence of the meiotic spindle [33]. Recent studies involving more precise assessment of normal and abnormal meiotic spindle morphology seem to be more discriminatory [34]. Another study has investigated the ability of the LC-PolScope to detect spindle chromosomal configurations in MII oocytes [35]. In this study, images of frozen thawed oocytes were captured and analyzed by LC-PolScope and then these oocytes were fixed and analyzed by confocal microscopy. The measurement of the spindle axis using PolScope was significantly smaller than the measurement using confocal microscopy. The conclusion of the study was that the spindle retardance measurement has limited predictive value. Therefore, the authors suggested that PolScope is not an efficient method for assessing the spindle. This technology is increasingly being utilized by scientists around the world to improve outcomes for assisted reproductive techniques. Alternative devices To our knowledge, there are no alternative approaches that can noninvasively detect the meiotic spindle and the zona pellucida other than the polarized light microscope. There are different companies producing polarized light microscopy systems for ART; Reseach Instruments with Oosight; and MTG with OCTAX polaraide. There are a number of publications using the two systems and generally producing similar conclusions. We only have experience with Oosight. To our knowledge, Oosight is the only commercial device that provides quantitative measurement of retardance, although the OCTAX system has an automated oocyte grading system based upon birefringence. The development of the Polscope to Oosight with the introduction of more user-friendly software and better optics allows this technique to be feasibly utilized in the clinical setting. Conclusion The goal of all IVF cycles is accepted to be a healthy singleton pregnancy derived from a single embryo transfer. The pressure is on the scientists to select the best embryo for transfer. Scientists face a dilemma when there is more than one apparently good quality embryo available for transfer. The decision can be vital to achieving a pregnancy. Thus, there is a high demand for quantitative assessment that can be helpful in the selection process. There is growing evidence that the use of polarized light microscopy allows noninvasive early evaluation of human oocytes that can enable the detection of markers, particularly the meiotic spindle, which may assist scientists in selecting embryos later on. These markers are also giving better insight and understanding of the physiology and pathology of human oocyte and embryo development. Ultimately, the true value of the application of polarized light microscopy will be determined by well-conducted randomized controlled trials in the clinical arena. Expert commentary & five-year view In addition to oocytes and embryos, the sperm acrosome can be studied using polarized light microscopy. There has been very little application to this area, but it may be shown that morphological markers in the various structures of individual sperm for ICSI may indicate the optimum sperm for selection [36]. There is increasing demand for fertility preservation, in particular, as couples delay child bearing to an older age. Oocyte freezing at a younger age is an option as insurance against later deterioration of oocyte quantity and quality. As the spindle is the major structure that is affected by the freezing process, the use of the polarized light microscopy may assist in detecting its presence and, thus, their normality post-thawing. Technologic advances with polarized light microscopy are providing a higher resolution of images allowing finer detail to be visualized. Differentiation of normal from abnormal structures has 244 Expert Rev. Obstet. Gynecol. 6(3), (2011)

5 The use of polarized light microscopy in IVF Device Profile become more feasible. Newer versions of polarized light microscopy provide faster image ana lysis with less potentially detrimental temperature fluctuation. We would expect this technique to advance further over the next 5 years. Information resource Cambridge Research and Instrumentation, Inc.: Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript. Key issues Polarized light microscopy: Utilizes the capacity of polarized light to image intracellular structures not visible with white light microscopy. Allows noninvasive imaging of human oocytes, embryos and sperm to assess intracellular structure. Allows assessment of meiotic spindle normality as a new dimension to embryo selection to maximize pregnancy rates in IVF. Is a relatively inexpensive supplementation to a standard light microscope. Is moving from a research instrument to a useful clinical tool. References Papers of special note have been highlighted as: of interest of considerable interest 1 Scott L, Finn A, O Leary T, McLellan S, Hill J. Morphologic parameters of early cleavage-stage embryos that correlate with fetal development and delivery: prospective and applied data for increased pregnancy rates. Hum. Reprod. 22(1), (2007). 2 Ingram Cooke RW. Does neonatal and infant neurodevelopmental morbidity of multiples and singletons differ? Semin. Fetal Neonatal Med. 15(6), (2010). 3 Verlinsky Y, Lerner S, Illkevitch N et al. Is there any predictive value of first polar body morphology for embryo genotype or developmental potential? Reprod. Biomed. Online 7(3), (2003). 4 Tesarik J, Junca AM, Hazout A et al. Embryos with high implantation potential after intracytoplasmic sperm injection can be recognized by a simple, non-invasive examination of pronuclear morphology. Hum. Reprod. 15(6), (2000). 5 De Santis L, Cino I, Rabellotti E et al. Polar body morphology and spindle imaging as predictors of oocyte quality. Reprod. Biomed. Online 11(1), (2005). 6 Houghton FD, Hawkhead JA, Humpherson PG et al. Non-invasive amino acid turnover predicts human embryo developmental capacity. Hum. Reprod. 17(4), (2002). 7 Botros L, Sakkas D, Seli E. Metabolomics and its application for non-invasive embryo assessment in IVF. Mol. Hum. Reprod. 14(12), (2008). 8 Rius M, Obradors A, Daina G et al. Reliability of short comparative genomic hybridization in fibroblasts and blastomeres for a comprehensive aneuploidy screening: first clinical application. Hum. Reprod. 25(7), (2010). 9 Fishel S, Gordon A, Lynch C et al. Live birth after polar body array comparative genomic hybridization prediction of embryo ploidy-the future of IVF? Fertil. Steril. 93(3), 1006.e e10 (2010). 10 Shen Y, Stalf T, Mehnert C, Eichenlaub- Ritter U, Tinneberg HR. High magnitude of light retardation by the zona pellucida is associated with conception cycles. Hum. Reprod. 20(6), (2005). 11 Keefe D, Liu L, Wang W et al. Imaging meiotic spindles by polarization light microscopy: principles and applications to IVF. Reprod. Biomed. Online 7(1), (2003). 12 Konc J, Kanyo K, Cseh S. Visualization and examination of the meiotic spindle in human oocytes with polscope. J. Assist. Reprod. Genet. 21(10), (2004). 13 Liu L, Keefe DL. Ageing-associated aberration in meiosis of oocytes from senescence-accelerated mice. Hum. Reprod. 17(10), (2002). 14 Keefe D, Liu L, Wang W, Silva C. Imaging meiotic spindles by polarization light microscopy: principles and applications to IVF. Reprod. Biomed. Online 7(1), (2003). 15 Madaschi C, de Souza Bonetti TC, de Almeida Ferreira Braga DP, Pasqualotto FF, Iaconelli A Jr, Borges E Jr. Spindle imaging: a marker for embryo development and implantation. Fertil. Steril. 90(1), (2008). 16 Wang WH, Meng L, Hackett RJ, Odenbourg R, Keefe DL. Limited recovery of meiotic spindles in living human oocytes after cooling-rewarming observed using polarized light microscopy. Hum. Reprod. 16(11), (2001). 17 Cooke S, Tyler JP, Driscoll GL. Meiotic spindle location and identification and its effect on embryonic cleavage plane and early development. Hum. Reprod. 18(11), (2003). 18 Rienzi L, Ubaldi F, Martinez F et al. Relationship between meiotic spindle location with regard to the polar body position and oocyte developmental potential after ICSI. Hum. Reprod. 18(6), (2003). 19 Chen CK, Wang CW, Tsai WJ, Hsieh LL, Wang HS, Soong YK. Evaluation of meiotic spindles in thawed oocytes after vitrification using polarized light microscopy. Fertil. Steril. 82(3), (2004). 20 Rienzi L, Martinez F, Ubaldi F et al. Polscope analysis of meiotic spindle changes in living metaphase II human oocytes during the freezing and thawing procedures. Hum. Reprod. 19(3), (2004). 21 Madaschi C, Aoki T, de Almeida Ferreira Braga DP et al. Zona pellucida birefringence score and meiotic spindle visualization in relation to embryo development and ICSI outcomes. Reprod. Biomed. Online 18(5), (2009). 22 Ebner T, Balaban B, Moser M et al. Automatic user-independent zona pellucida imaging at the oocyte stage allows for the prediction of preimplantation development. Fertil. Steril. 94(3), (2010)

6 Kilani & Chapman 23 Kilani S, Cooke S, Kan A, Chapman MG. Do age and extended culture affect the architecture of the zona pellucida of human oocytes and embryos? Zygote 14(1), (2006). 24 Kilani S, Cooke S, Chapman MG. The change of the meiotic spindle over time a possible marker to optimise ICSI? ANZJOG 46(s2), A14 (2006). 25 Kilani S, Cooke S, Chapman MG. Time course of meiotic spindle development in MII oocytes. Zygote 1, 8 (2011). The first attempt investigating the meiotic spindle changes in mature oocytes from retrieval until intracytoplasmic sperm injection time. 26 Kilani S, Cooke S, Kan A, Chapman MG. Are there non-invasive markers in human oocytes that can predict pregnancy outcome? Reprod. Biomed. Online 18(5), (2009). Describes our initial experience with meiotic spindle normality and it s relationship to pregnancy outcome. 27 Wang WH, Keefe DL. Prediction of chromosome misalignment among in vitro matured human oocytes by spindle imaging with the PolScope. Fertil. Steril. 78(5), (2002). First paper to describe the relationship of spindle structure to chromosome abnormality. 28 Kilani S, Cooke S, Chapman MG. Can polarised light microscopy of oocyte ultrastructure improve pregnancy rates? ANZJOG 50(S1), 44 (2010). Inspects meiotic spindle structure using polarized light microscopy in frozen oocytes. 29 Molinari E, Revelli A, Racca C et al. Slow-freezing-induced changes of birefringent structures in human oocytes are related to responsiveness to ovulation induction. Reprod. Biomed. Online 20(5), (2010). 30 Fang C, Tang M, Li T, Peng W et al. Visualization of meiotic spindle and subsequent embryonic development in in vitro and in vivo matured human oocytes. J. Assist. Reprod. Genet. 24(11), (2007). 31 Hyun C, Cha J, Son W et al. Optimal ICSI timing after the first polar body extrusion in in vitro matured human oocytes. Hum. Reprod. 22(7), (2007). 32 Rama Raju GA, Prakash GJ, Krishna KM, Madan K. Meiotic spindle and zona pellucida characteristics as predictors of embryonic development: a preliminary study using PolScope imaging. Reprod. Biomed. Online 14(2), (2007). 33 Petersen CG, Oliveira JB, Mauri AL et al. Relationship between visualization of meiotic spindle in human oocytes and ICSI outcomes: a meta-analysis. Reprod. Biomed. Online 18(2), (2009). 34 Kilani S, Cooke S, Chapman MG. Does meiotic spindle normality predict blastocyst development and implantation? Reprod. Med. Endocrin. 7(4), (2010). 35 Coticchio G, Sciajno R, Hutt K, Bromfield J, Borini A, Albertini DF. Comparative analysis of the metaphase II spindle of human oocytes through polarized light and high-performance confocal microscopy. Fertil. Steril. 93(6), (2010). 36 Gianaroli L, Magli MC, Collodel G, Moretti E, Ferraretti AP, Baccetti B. Sperm head s birefringence: a new criterion for sperm selection. Fertil. Steril. 90(1), (2008). 246 Expert Rev. Obstet. Gynecol. 6(3), (2011)