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The use of polarized light microscopy in IVF Expert Rev. Obstet. Gynecol. 6(3), 241–246 (2011)
Suha Kilani†2 and Michael G Chapman1 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:
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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 1978. 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. www.expert-reviews.com
10.1586/EOG.11.17
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
© 2011 Expert Reviews Ltd
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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.
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 analysis 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 analysis. 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. Introduction to the device The oocyte is placed in a glass-bottomed dish in culture The sample is illuminated with circularly polarized light. The standard (mechanically operated) compensator is replaced by a medium. It is then imaged in four planes rotating with the injecliquid crystal (LC) compensator. The LC universal compensator tion needle to optimally view the meiotic spindle. The images and grayscale charge coupled device camera are controlled by a are saved into an individual folder within the software program. The oocyte is then injected such that the needle avoids the meihost computer. Five images are taken, with the LC compensator changed to five different settings. The five images are then used otic spindle. The saved images can then be analyzed using the to calculate the sample’s birefringence at every pixel, based on PolScope/Oosight software to establish quantitative parameters including retardance, length and width measurements of the zona patented polarimetric algorithms. pellucida and spindle. The initial capital cost of this equipment when compared with the standard Image analysis software light microscopy is an extra US$20,000– 30,000, which is a small additional cost in the context of a fully functional IVF laboPolarizer 1 Camera ratory. 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 Polarizer 2 should add no more than 10–15 min per case, even with a large number of oocytes. The use of polarized light microscopy is Figure 1. Inverted microscope with two polarizers and image analysis software. moving from being a research tool to appliImage courtsey of CRi Oosight Imaging System on an Olympus inverted microscope. cation in the clinical setting. Early studies 242
Expert Rev. Obstet. Gynecol. 6(3), (2011)
The use of polarized light microscopy in IVF
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Retardance
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 analysis of spindle kinetics in the living state. The Micrometers results of this study have shown that human 24.25153 micrometers meiotic spindles are exquisitely sensitive to alterations in temperature [16] . This is an Figure 2. Spindle retardance measured in nm. important practical issue when handling Clinical profile and post-marketing findings. oocytes in the clinical setting. Data taken from [26] . Subsequent research has shown that injecting oocytes away from the spindle, localized by polarized Kilani et al. compared the zona pellucida thickness and density light microscopy, resulted in higher fertilization rates and better of oocytes from older women (>38 years) with those of younger embryo quality [16,17] . women (≤38 years). The zona pellucida thickness and density Traditionally when ICSI is performed, the oocyte is held and was measured at four locations (3, 6, 9 and 12 o’clock). For the the first polar body (1PB) is placed either at the 12 or 6 o’clock zona pellucida thickness, the zona was measured from the outer position to avoid disrupting the meiotic spindle, which had been layer to the inner layer and the average of the four measurements believed to be universally beneath the 1PB. However, polar- was used for the analysis. While for the zona pellucida density, ized light microscopy has shown that the meiotic spindle is not the average measurements of the innermost birefringent layer of always located under the 1PB and can be anywhere in the hemi- the whole zona pellucida was used for analysis. The results of sphere [17,18] . When oocytes are injected away from the meiotic the study showed that oocytes of older women had thicker and spindle, the embryos generated have been shown to have better denser zonas than younger women. Blastocysts formed from these morphology and less fragmentation than control oocytes [17] . This oocytes had thicker and denser zonas [23] . viewing of the meiotic spindle with polarized light reduces the It is suggested that embryos with thicker zonas are less likely incidence of damaging the meiotic spindle during ICSI. to hatch. Assisted hatching has been proposed for those oocytes, Spindle integrity of frozen/thawed oocytes has been stud- although no studies to date have tested this hypothesis. ied [19,20] . Chen et al. demonstrated that oocytes with good spindle The optimal time for injecting oocytes is a controversial issue morphology verified by the LC-PolScope before vitrification had a in the literature. Polarized light microscopy enables serial observahigher survival rate of intact oocytes after thawing compared with tion of oocytes over time from stripping of the cumulus cells until those with poor or undetected spindle images. They also showed a ICSI. Our group has shown the dynamic nature of the formagood agreement of spindle morphology by both the LC-PolScope tion and dissolution of the meiotic spindle. Analysis of images and fluorescent staining [19] . of oocytes, captured at five time intervals using LC-PolScope There are studies in the literature that investigated the zona showed that the largest percentage of oocytes had a visible spindle pellucida using the LC-PolScope. Madaschi et al. investigated at 39–41 h post-trigger. This time interval has been suggested to possible factors contributing to the zona pellucida birefringence be the most suitable time for ICSI [24,25] . score and meiotic spindle visualization, and evaluated whether In an initial study using the LC-PolScope to assess markers these structures may predict intracytoplasmic sperm injection within the egg which might assist in assessing the better quality outcomes. The results showed that selection of embryos based oocytes, we compared oocytes resulting in a pregnancy to those on zona pellucida and meiotic spindle imaging can significantly that did not generate a pregnancy. In this small retrospective improve implantation and pregnancy rates [21] . study the rate of spindle normality, defined as barrel shaped and A recent study used OCTAX polarAide™ to analyze whether complete spindle, was significantly higher in oocytes in the prega change in 3D structure of the zona pellucida could indicate sub- nant patients compared with oocytes in the nonpregnant patients optimal gamete quality. The results showed that in approximately (100 vs 33%; p
