Spindle examination in unfertilized eggs using the polarization microscope can assist rescue ICSI

Reproductive BioMedicine Online (2013) 26, 280 285 www.sciencedirect.com www.rbmonline.com ARTICLE Spindle examination in unfertilized eggs using the polarization microscope can assist rescue ICSI Jeong Hee Moon *, Weon-Young Son, Sara Henderson, Alina Mahfoudh, Michael Dahan, Hananel Holzer MUHC Reproductive Center, Department of Obstetrics and Gynecology, McGill University, Montreal, Quebec, Canada * Corresponding author. E-mail address: MJHEE@yahoo.co.kr (JH Moon). Dr. Moon is currently working as an embryologist at the McGill Reproductive Center, Department of Obstetrics and Gynecology, Royal Victoria Hospital, McGill University, Quebec, Canada. She obtained a MSc in developmental biology in Seoul Women s University, Seoul, Korea in 1996 and her PhD at the School of Medicine, Seoul National University in 2008. She started her work as a clinical embryologist at Maria Infertility Center, Seoul, Korea in 1996, and she was an IVF laboratory director in the Department of Obstetrics and Gynecology, Bundang Seoul National University Hospital, in Kyungkido, Korea during 2002 2008. She joined the McGill Reproductive Center in 2009. Abstract Rescue ICSI can induce a high rate of 3 pronuclei (PN) formation from double insemination in eggs already fertilized by IVF but lacking signs of normal pronuclear formation. This study was performed to determine whether the number of 3PN embryos could be reduced by using the polarization microscope for rescue intracytoplasmic sperm injection (ICSI). As a study group, after conventional insemination, 81 unfertilized mature oocytes from 11 couples were checked for the number of spindles using the polarization microscope. One spindle (82.7%) or two spindles (17.3%) were observed in this group. Rescue ICSI was only performed on the unfertilized oocytes showing one spindle. In the control group, 87 mature oocytes which lacked visualization of any fertilization signs were selected for rescue ICSI and none of them underwent observation of the spindle. After rescue ICSI, the normal fertilization rate in the study group was significantly higher than in the control (68.7% versus 43.7%; P = 0.0032). The rate of 3PN or 4PN embryos was significantly decreased in the study group with one spindle compared with the group without observation of the spindle (4.5% versus 26.4%; P = 0.0004). RBMOnline ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: polarization microscope, rescue ICSI, spindle Introduction One of the major disappointments that an infertile couple may face during an IVF cycle is low fertilization rate or total fertilization failure. Various reasons for failed fertilization after insemination have been reported, including defects of the spermatozoon or oocyte of the interaction between them (Van Blerkom et al., 1994; Kruger et al., 1986; Lui et al., 2000). Failed fertilization following IVF or intracytoplasmic sperm injection (ICSI) can be emotionally difficult and add to the financial burden for the couple. Performing rescue ICSI on unfertilized eggs, 1-day-old, may be a method to prevent cycle cancellation even though it may result in poor embryos with genetic abnormalities (Morton et al., 1997; Pehlivan et al., 2004), poor pregnancy rate (Kuczynski et al., 2002; Chen and Kattera, 2003) and 1472-6483/$ - see front matter ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rbmo.2012.10.019

Spindle examination using the polarization microscope prior to rescue ICSI 281 higher formation of 3 pronuclei (PN) (Nagy et al., 1995; Lundin et al., 1996). Currently, some studies have reported a successful live birth following a cycle which required rescue ICSI (Lundin et al., 1996; Yuzpe et al., 2000; Shalom-PAZ et al., 2011). It has also been reported that reducing the time between egg retrieval and rescue ICSI on aged eggs can provide a higher rate of normal fertilization (Chen and Kattera, 2003; Nagy et al., 2006). The timing of rescue ICSI is crucial because as the oocyte ages, the success rate decreases due to a decrease in normal fertilization rates and increase in 3PN formation (Nagy et al., 1995; Pehlivan et al., 2004). Also, one of the possible causes of 3PN formation is that the rescue ICSI could induce double insemination of oocytes that were already fertilized prior to rescue ICSI in embryos where signs of fertilization had already disappeared or their formation were delayed, resulting in a second spermatozoon inseminating the oocyte (Pehlivan et al., 2004). Fertilization is a process that culminates when the male and female pronuclei fuse in activated oocytes, which is mediated by the sperm centrosome with regards to microtubule organization into the first mitotic spindle (Asch et al., 1995; Terada et al., 2010). Abnormal cytoskeletal organization on aged unfertilized eggs was visualized using fluorescence microscopy by Pickering et al. (1988). Also, Schatten (1994) demonstrated using immunostaining some sperm aster enlargement as the male pronucleus forms during the developmental process of fertilization. Currently, the use of a modified polarizing microscope (LC-Polscope) has made it possible to identify spindles in living oocytes without fixing, staining or creating any detrimental effects to the eggs (Wang et al., 2001; Moon et al., 2003; Rienzi et al., 2003). This study postulated that if the spermatozoon is already in the oocyte, the organization of microtubules, which is necessary for the fertilization procedure, could be examined with the polarization microscope which allow the avoidance of double sperm injection through rescue ICSI. Therefore, this study was done to determine if the polarization microscope can help select unfertilized mature oocytes without sperm entry from IVF in order to prevent double insemination by rescue ICSI on already fertilized eggs. Materials and methods Patients and experimental design This retrospective study was approved by the Research and Ethics Board of McGill University Health Centre on 28 October 2010. A low fertilization rate (<20%) occurred in 10 IVF cycles (female age, mean ± SD, 34 ± 4 years) between June 2009 and May 2010. Fertilization rate was calculated as the number of 2PN embryos from the number of mature eggs. All couples in these IVF groups met standard criteria for IVF treatment, including normal semen motility, concentration and strict morphology as assessed by World Health Organization (WHO, 1999), i.e. concentration >20 10 6 /ml, sperm motility >50% (grades a + b) or >25% (grade a) and morphologically normal spermatozoa >15%. During this period, 87 unfertilized mature eggs had rescue ICSI performed (19 22 h after insemination) without examination of the spindle by polarization microscope (LC-Polscope; Cri, USA). The embryological data were used as a control to compare to those which had the spindle checked prior to rescue ICSI. From June 2010 to May 2011, 11 women (female age 34 ± 4.5 years) had low fertilization rates (<20%) following conventional IVF and were treated with rescue ICSI. A total of 81 unfertilized eggs was examined for spindle presence by polarization microscopy prior to rescue ICSI. Eggs with only one spindle and one PB (Figure 1A), had rescue ICSI performed. Eggs with two spindle positions (Figure 1B) did not undergo rescue ICSI. Stimulation and insemination Stimulation protocol, follow up and oocyte retrieval were performed as previously described (Buckett et al., 2007). Three hours after egg retrieval, oocyte cumulus corona complexes were inseminated by conventional insemination with 125,000 150,000/ml motile spermatozoa per insemination dish, containing 3 5 eggs. Ejaculated spermatozoa from male partners were liquefied for 30 min. The concentration and motility of the sperm specimens were verified prior to insemination and had normal parameters by WHO standards (WHO, 1999). Sperm samples were separated on three layer gradients of 95%, 70% and 50% PureSperm (Global, USA) by centrifugation at 1500 g for 20 min. Sperm pellets were washed twice in gamete buffer (Cook, Australia) by centrifugation at 800 g for 10 min. After processing, concentration and progressive motility were re-determined for insemination. Sperm suspension prepared on the day of oocyte retrieval was kept at room temperature over night. Spermatozoa with progressive motility and normal morphology were chosen for rescue ICSI on day 1 after collection. Assessment of fertilization and spindle examination Cumulus cells surrounding each oocyte were removed 16 18 h after insemination to assess fertilization, which was determined by checking for the presence of 2 PN and two PB. Eggs without signs for fertilization (exclusively 0 PN), were selected for spindle observation using the Polscope spindle view system (LC-Polscope) with a computerized image analysis system (Spindleview; Cri). Each oocyte was put into individual 10 ll drops of gamete buffer (Cook, Australia) and covered with mineral oil (Cook) in a glass-bottom culture dish (Fluorodish; World Precision Instruments, USA). The drops were kept at 37 C by maintaining the heated stages at 38.5 C. The oocytes were rotated with an injection pipette until the spindle position was visible. Unfertilized eggs with only one spindle and one PB observed with the polarization microscope had rescue ICSI performed soon after checking the spindle in the same glass bottom dish (17 19 h after insemination). Microinjection, re-assessment of fertilization and development of embryos Unfertilized metaphase II eggs with only one spindle and one PB identified by polarization microscopy had rescue ICSI performed within 1 h of checking the spindle. Eggs with two spindles examined with the polarization microscope were excluded from rescue ICSI. The spermatozoa produced

282 JH Moon et al. Figure 1 Unfertilized egg with one spindle (A) and two spindles (B) after failure of fertilization ( 400 magnification); arrows indicate spindles; arrow heads indicate polar bodies. Bars = 50 lm. and used for the original insemination were reused for the rescue ICSI procedure. On the day of rescue ICSI (6 8 h after rescue ICSI), examination for evidence of PN for fertilization was performed. A comparison of the normal fertilization rate of the study group with that of the control group (rescue ICSI performed for unfertilized eggs without checking spindle) was then performed. Embryo quality was classified as good, fair or poor depending on the number and the size of blastomeres and percentage of anucleate fragments on day 2 after rescue ICSI (Reinblatt et al., 2011). Good-quality embryos were defined as those with 4 cells, grade 2 (defined as embryo with 0 20% fragmentation). Fair-quality embryos were those with 2 or 4 cells, grade 3 (embryo fragmentation 20 50%). Finally, poor-quality embryos had 1 4 cells, grade 4 (embryo fragmentation >50% or arrested development). Statistics Statistical evaluation was performed using Fisher s Exact test. Differences were considered significant at P < 0.05. Results The control group had 141 eggs retrieved from 10 women (14.1 ± 6.0 oocytes per oocyte retrieval), of which 107 (75.9%) were in metaphase II. After insemination by IVF, 17 of 107 eggs (15.9%) were fertilized and 87 unfertilized eggs had rescue ICSI performed (Table 1). The study group had a total of 151 eggs collected from 11 women (13.7 ± 5.4 oocytes per oocyte retrieval) of which 112 (74.2%) were in metaphase II. The fertilization rate after IVF was 18.8% (21 out of 112 matured eggs) (Table 1). Five oocytes (4.5%) were abnormally shaped or ruptured (Table 1) and therefore did not have the spindle checked with the polarization microscope. Of the 81 unfertilized eggs following IVF examined with the polarization microscope, 67 (82.7%) showing one spindle had rescue ICSI performed (Table 2, Figure 1A) and 14 (17.3%) oocytes showed two spindles and did not undergo rescue ICSI (Table 2, Figure 1B). As shown in Table 2, in the study group, after rescue ICSI, 46 out of 67 eggs (68.7%) showed evidence of normal fertilization (2PN). In the control group, 38 out of 87 eggs (43.7%) showed signs of normal fertilization following rescue ICSI (P = 0.0032). This is significantly Table 1 Outcome of IVF and rescue intracytoplasmic sperm injection in the study (polarization microscopy) and control patients. higher than the normal fertilization rate obtained in the control group. However, the percentage of good embryos was not statistically different between the two groups (37% versus 36.8%; Table 2). In the study group, three out of 67 eggs (4.5%) had 3 or 4 PN, which is significantly less (P = 0.0004) than in the control group (23 out of 87, 26.4%; Table 2). Discussion Study group (n = 11) Control (n = 10) Patient s age (years) 34 ± 4.5 34 ± 4.0 Retrieved eggs 151 141 Mature oocytes at fertilization 112 (74.2) 107 (75.9) assessment Normally (2PN) fertilized 21 (18.8) 17 (15.9) oocytes Abnormally (1PN or 3PN) 5 (4.5) 3 (2.8) fertilized oocytes Oocytes with abnormality 5 (4.5) 0 (0) Oocytes with failed fertilization 81 (72.3) 87 (81.3) Values are mean ± SD, n or n (%). Complete fertilization failure, or poor fertilization, occurs frequently in unexplained infertile couples after conventional insemination of oocytes (Barlow et al., 1990; Lipitz et al., 1994). Rescue ICSI has been performed in this unexpected situation to avoid cycle cancellation and increase the number of available embryos even though it may result in low pregnancy rates (Lundin et al., 1996; Morton et al., 1997), high 3PN formation (Nagy et al., 1995) or genetically aberrant embryos (Pehlivan et al., 2004). Rescue ICSI is usually performed 1 day after failed insemination. Therefore, the poor results obtained could be due to the effect of the oocyte ageing. Chen and Kattera (2003) have shown that higher pregnancy and fertilization rates are obtained when rescue ICSI was performed 6 h as opposed to 22 h after failed IVF

Spindle examination using the polarization microscope prior to rescue ICSI 283 Table 2 Result of rescue ICSI between the study (polarization microscopy) and control failed fertilization oocytes. Study group (n = 81) Control (n = 87) P- value Oocytes with one 67 (82.7) ND spindle Oocytes with two 14 (17.3) ND spindle Oocytes for rescue 67 87 ICSI 2PN after rescue 46 (68.7) 38 (43.7) 0.0032 ICSI Day-2 embryo quality Good 17 (37.0) 14 (36.8) NS Fair 14 (30.4) 11 (28.9) Poor 15 (32.6) 13 (34.2) 3PN or 4PN 3 (4.5) 23 (26.4) 0.0004 Values are n or n (%). ND = not determined; NS = not statistically significant. insemination. They suggested that a shortened time lapse after failed IVF might be better for fertilization, pregnancy and implantation rates. Nagy et al. (2006) also showed that rescue ICSI of unfertilized eggs 6 h after insemination can provide a normal fertilization rate. They identified the second polar body, which can be the earliest indication for egg activation following fertilization. However, it can be difficult to identify the polar bodies, because they often show fragmentation after insemination. Nagy et al. (1993) suggested that a high frequency of more than 2 PN after rescue ICSI for 1-day-old mature oocytes can result from non-extrusion of the second PB or duplication of the remaining maternal genetic material. However, the current study suggests that one of the reasons for the 3PN formation in the control group (26.4%) is that rescue ICSI might be performed on fertilized eggs which had already had sperm penetration following IVF. Machtinger et al. (2012) investigated two spindles in unfertilized eggs using immunostaining, and one of the spindles was attached to a sperm tail considered as paternal origin. So it is possible the origin of the third pronucleus can be from an additional spermatozoon; however, further research is needed. Traditionally in this study laboratory, rescue ICSI is performed on unfertilized eggs 3 h after checking fertilization (19 21 h later after insemination) to reduce the possibility of double insemination in eggs which may have experienced a problem with timing of the visualization of the pronuclei either they have already disappeared or the formation is retarded. Consequently, another reason for poor outcome with rescue ICSI can be due to the egg ageing. Therefore, this study suggests that rescue ICSI after analysis by polarization microscopy reduces the risk of double sperm injection and also oocyte ageing, thereby increasing selection of normal oocytes and overall rates of normal development after rescue ICSI. In the present study, rescue ICSI for 11 cycles was done following examination of the maternally derived spindle in the 67 unfertilized eggs with the polarization microscope on day 1 after collection. All of the oocytes had an insufficient number of binding spermatozoa (less than five) around zona pellucida. So when a birefringent spindle is present in this situation, with only one PB, a failure of sperm penetration was suggested. After examination of the spindle with the polarization microscope, 67 out of 81 eggs (82.7%) that had one spindle were selected, rescue ICSI was performed and then 14 out of 81 eggs that showed two spindles (17.3%) were excluded from rescue ICSI. Four out of the 14 unfertilized eggs with two spindles appeared to have a PN or proceeded to cleave the next day following evaluation (data not shown). Machtinger et al. (2012) suggested that failure of normal fertilization which is dependent on the cytoplasmic programming may exhibit the formation of a paternal, as well as a maternal, spindle. Such immature cytoplasmic conditions in eggs may give rise to abnormal processing and formation of premature chromosome condensation (Nasr-Esfahani et al., 2007). It has been proposed that a proportion of mature eggs will have no spindle visualized on egg collection day in stimulated IVF cycles (Wang et al., 2001; Moon et al., 2003). However, in the current study, a spindle could be visualized in all unfertilized eggs. Although no eggs were found which did not show spindle birefringence by the polarization microscope in the current study, this may be due to the small sample size. This is the first study to examine not only the maternally derived meiotic spindles but also the presence of higher-order alignment of microtubule bundles from spermatozoa, which occurred in apparently unfertilized inseminated eggs with the modified polarization light microscope, which uses novel electro-optical hardware and digital processing to build images of birefringent macromolecular structures, including the meiotic spindle in human oocytes (Lui et al., 2000). Birefringence intensity is an indicator of a high-order paracrystalline alignment of microtubules that generate a contrast between the spindle and the rest of cell (Oldenbourg and Mei, 1995). Terada et al. (2010) reported on the aster organization from the centrosome of the spermatozoon after insemination with fixing and staining. In some cases after sperm entry, microtubule organization may have started, but failed to proceed to the next step to activate the eggs. This results in failed fertilization including PN formation (Flaherty et al., 1998). Also, Rawe et al. (2000) used the immunofluorescence analysis for sperm microtubules and chromatin in eggs after fertilization failure which showed abortive activation after sperm penetration. In other words, the presence of a spermatozoon within the eggs does not guarantee complete fertilization success, because some failures arise due to specific defects within microtubule organization including sperm astral microtubules for pronuclear apposition (Asch et al., 1995). Indeed, even though the commercially available polarization microscope did not permit detection of the type and intensity of birefringence associated with sperm cells (Baccetti, 2004), the current study suggests that there is considerable potential for the observation of spindles and that once the sperm astral microtubules assemble around the sperm head after apposition of the spermatozoon within the egg for fertilization (Schatten, 1994; Simerly et al., 1999), it might be visualized using polarization microscope.

284 JH Moon et al. This study postulates that the two-spindle stage is the initial stage of fertilization before PN formation. However, when checking the spindle every 2 h with the polarization microscope until two pronuclei appeared, two-spindle stages in some in-vitro matured eggs after sperm injection could not be observed in the process of normal fertilization (unpublished data). Two PN suddenly appeared without any evidence of two spindle positions developing. Therefore, the two spindles that appear in unfertilized eggs might be an abnormal stage which occurs when there is no true activation, resulting in egg arresting and remaining in metaphase II. Finally, sperm chromatin may become a condensed chromosome that organizes a second spindle for their metaphase stage. In this study, the control group, which did not have the spindles checked, had a higher percentage of 3PN embryos following rescue ICSI than those injected with only one spindle (26.4% versus 4.5%). The second injection of a spermatozoon by rescue ICSI in unfertilized eggs might induce another sign to activate the process for fertilization, consequently, resulting in 3 PN. In the study group, after checking the spindles with the polarization microscope following evaluation of fertilization, the normal fertilization rate (2PN) was also significantly higher after rescue ICSI than in the control group. Also, the reason for a comparatively higher 2PN rate in the study group compared with the control group might be the earlier attempt of rescue ICSI (17 19 h after insemination) after spindle examination with the Polscope (control group, 19 22 h after insemination). It might reduce the effect of egg ageing. This finding is likely to help selection of oocytes with only one spindle for rescue ICSI. However, there was no difference of developmental capacity between the two groups (control and study group) when comparing quality. Also, this study could not compare the pregnancy rate between the two groups after embryo transfer, because most of the cycles had a transfer of embryos produced from both IVF and rescue ICSI on day 3 after oocyte retrieval. Therefore, the embryo quality could only be evaluated 2 days after rescue ICSI. Day-2 embryo development was shown to be a poor predictor of continued development compared with the embryo on day 3 or blastocyst on day 5. Therefore it is difficult to draw conclusions from the embryo developmental data to determine whether early rescue ICSI could produce better quality embryos. In conclusion, these results suggest by examining the spindle of unfertilized eggs after fertilization failure with the polarization microscope can be helpful for the selection of eggs for rescue ICSI to decrease the risk of double insemination, lower the 3PN rate and increase the normal fertilization rate. References Asch, R., Simerly, C., Ord, T., Ord, V.A., Schatten, G., 1995. 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