Visualization of the metaphase II meiotic spindle in living human oocytes using the Polscope enables the prediction of embryonic developmental competence after ICSI

Jeong-Hee Moon1, Chang-Seop Hyun, Seok-Won Lee, Weon-Young Son, San-Hyun Yoon and Jin-Ho Lim

Maria Infertility Hospital, Seoul, Korea

1 To whom correspondence should be addressed at: In Vitro Fertilization Laboratory, Maria Infertility Hospital, 103–11, Sinseol-dong, Dongdaemun-gu, Seoul, Korea e-mail: mjhee{at}yahoo.co.kr


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Meiotic spindles in living human oocytes can be visualized by the Polscope. This study investigated the relationship between the presence/location of the spindle in metaphase II (MII) oocytes and developmental competence of embryos in vitro. METHODS: The spindles in 626 MII oocytes were examined by the Polscope and divided into six groups (A–F) based on the presence or absence of the spindles and the angle between the spindle and the first polar body. After ICSI, the fertilization and embryo development were evaluated. RESULTS: Meiotic spindles were imaged in 523 oocytes (83.5%), while 103 (16.5%) did not have a visible spindle (group F). The majority of oocytes (68.8%) had the spindle directly beneath or adjacent to the first polar body (groups A and B: 48.2 and 20.6%). Oocytes in group C (11.2%) had the spindle located between 60 and 120° angle away from the first polar body, those in group D (2.4%) had the spindle located between 120 and 180° angle and those in group E (1.1%) had the spindle located at 180° angle to the first polar body. The fertilization and embryonic development were similar in the oocytes with spindles regardless of spindle position. However, the rate of high quality embryos was significantly higher in the oocytes (64.2%) with visible spindles than in the oocytes (35.9%) without spindle and multipronuclear proportion showed a slight tendency to increase in oocytes without spindles. (10.7 versus 5.9%, P = 0.12; NS). CONCLUSIONS: the presence of a bi-refringent meiotic spindle in human oocytes by using the Polscope can predict a higher embryonic developmental competence. However, the relative position of the spindle within the oocyte doesn’t appear to influence the developmental potential of embryos.

Key words: embryo development/ICSI/MII oocytes/Polscope/spindle


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The meiotic spindle plays an important role in the oocyte during chromosome alignment and separation at meiosis. It has been reported that spindle abnormalities in oocytes during meiosis increase with age in women over 40 years, which is a contributing factor to the aneuploidy in embryos after fertilization (Battaglia et al., 1996Go) and poor embryo development (Janny et al., 1996Go; Volarcik et al., 1998Go; Chang et al., 2002Go).

The Polscope was recently developed to study spindles in living cells non-invasively (Liu et al., 2000Go) including in human oocytes (Wang et al., 2001a,b,cGo). The Polscope, which combines innovations in polarization optics with novel image-processing software, enables us to image bi-refringent spindles regardless of their orientation (Oldenbourg et al., 1995Go). It had no detrimental effects on oocyte maturation and subsequent embryonic development when the Polscope was used to image mouse oocytes (Liu et al., 2000Go); so this technique has been successfully used in human assisted reproduction technology (Wang et al., 2001aGo,bGo,cGo). During ICSI, the location of the metaphase II (MII) spindle is commonly assessed in relation to the location of the first polar body. The ICSI needle must avoid the metaphase spindle during sperm injection. However, recent reports showed that the location of the first polar body does not predict accurately the location of the MII meiotic spindle in mammalian oocytes (Silva et al., 1999Go) including human oocytes (Wang et al., 2001aGo,b,c). Damage to the spindles may happen in some oocytes if the spindles are away from the first polar body. Therefore, the use of a Polscope may be an alternative approach for decreasing spindle damage during ICSI in human IVF.

Wang et al. (2001bGo,cGo) also suggested that the presence of a bi-refringent spindle in the human oocyte predicts higher fertilization rate as well as embryo developmental compe tence after ICSI. They explained the possibility that oocytes without bi-refringent spindles may have abundant chromosomal abnormalities, which in turn might induce cell cycle arrest. Also, they suggested that changes in spindle structure might reflect cytoplasmic dysfunction or other damage to the oocytes (Wang et al., 2001aGo,b,c). Although Wang et al., (2001aGo,b,c) reported that the spindles were located in various areas during MII, it has not reported whether fertilization and embryo development vary depending on the location of meiotic spindle. In the present study, we examined the embryo developmental competence in the oocytes with various spindle positions and in the oocytes without visible spindles after ICSI.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The oocytes were collected from ovaries of 77 patients (mean age ± SD = 32 ± 4 years) with their consent. Superovulation was induced by GnRH agonist and hMG in either a long- or a short-treatment protocol. When two follicles reached 18 mm in diameter, 10 000 IU hCG (IVF-C; LG, Geneva, Switzerland) was administered. Oocytes were retrieved transvaginally 36–38 h after hCG injection. After oocyte– cumulus complex (OCC) retrieval, the OCC were denuded of their surrounding cumulus and corona cells by incubation in 0.0001% hyaluronidase (Sigma) in YS media (Yoon et al., 2001Go) with 10% human follicular fluid (hFF) for 1min and by repeated pipetting of the OCC in and out of a hand-drawn fine Pasteur pipette. After incubation in YS medium for 3–4 h, 626 metaphase II (MII) stage oocytes which have the extrusion of the first polar body were examined by Polscope. ICSI was performed taking care to avoid the spindles irrespective of polar body orientation, whereas oocytes without visible spindle were generally injected with their polar body at the 6 or 12 o’clock position during ICSI. The temperature was maintained at 37°C during imaging and ICSI. According to the angle formed between the spindle and the first polar body, oocytes were divided into six groups (Figure 1). Group A: oocytes had the spindle located exactly under the first polar body (Figure 1A); group B: oocytes had the spindle forming 0 to 60° angles (Figure 1B): group C: oocytes had the spindle forming 60 and 120° angles (Figure 1C); group D: oocytes had the spindle forming 120 and 180° angles (Figure 1D); group E: oocytes had the spindle forming a 180° angle and group F: oocytes had no visible spindle (Figures 1E and F).



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Figure 1. Bi-refringent spindles in living human oocytes imaged at metaphase II-stage with the Polscope just before ICSI. (A) Oocyte had the spindle located under the first polar body. (B) Oocyte had the spindle located between 0° and 60° relative to the polar body. (C) Oocytes had the spindle located between 60° and 120° relative to the polar body. (D) Oocytes had the spindle located between 120° and 180° relative to the polar body. (E) Oocytes had the spindle located exactly at 180° relative to the polar body. (F) Oocytes had no visible spindle. Original magnification, x 200. PB = Polar body; arrow = spindle; bar = 50 µm.

 
Fertilization was evaluated 18 h after ICSI. Zygotes with two pronuclei (PN) and 2nd polar body were co-cultured with cumulus cells in YS medium containing 10% hFF up to day 3. Embryonic development was assessed on day 3 of culture according to the regularity of blastomeres, the percentage and pattern of anucleate fragments, and all dysmorphic characteristic of the embryos. For this study, we defined embryos as high quality embryos if they had at least six blastomeres and <20% anucleate fragments and no apparent morphologic abnormalities. Embryos showing blastomere multi-nucleation, poor cell adhesion, uneven cell division and cytoplasmic abnormalities were defined as low quality.

Statistics
Statistical analysis was performed by the {chi}2-test.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Figure 1 shows the spindle images of MII oocytes in six groups by the Polscope before ICSI. MII oocytes had spindles at various locations relative to the first polar body. As shown in Figure 2, spindle was present in 83.5% of oocytes and was not visible in 16.5% of oocytes. Also, Figure 2 shows percentage of oocytes in each group with visible spindle. A total of 48.2, 20.6, 11.2, 2.4 and 1.1%, of oocytes were in group A, B, C, D, and E respectively. After ICSI, there was no difference in the proportions of oocytes forming 2PN between oocytes with (82.2%) and without (75.7%) a visible spindle (Figure 3). However, proportion of multipronuclear formation (3PN and 4PN) showed a slight but non-significant tendency to increase in oocytes without spindle compared to oocytes with spindle (10.7 versus 5.9%: P = 0.1219) (Figure 3). We examined fertilization rates of oocytes with different locations of spindles in groups A to E. There was no significant difference in the fertilization rates among the different groups, which were 84.8, 81.4, 77.1, 73 and 85.7% respectively. The rate of high quality embryos was 64.2% in oocytes with spindles, which was significantly higher than that (35.9%) in the oocytes without spindle (P < 0.01) (Figure 4). Again, there was no statistical difference in rates of high quality embryos among the groups of oocytes with a visible spindle (group A = 62.5%; group B = 63.8%; group C = 70.4%; group D = 77.8%; and group E = 66.7%).



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Figure 2. Relationship between meiotic spindle and the first polar body in living human oocytes imaged with the Polscope. Spindle was present in 83.5% (523/626) of oocytes and was not visible in 16.5% (103/626) of oocytes. Distribution of oocytes in groups A, B, C, D and E was 48.2% (302/626), 20.6% (129/626), 11.2% (70/626), 2.4% (15/626) and 1.1% (7/626) respectively.

 


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Figure 3. Fertilization rates of oocytes with or without visible spindle after ICSI according to the number of pronuclei (PN). (Poly-PNs = three or four pronuclei).

 


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Figure 4. Comparison of high quality embryo developmental rates for oocytes with or without a visible spindle.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study indicates that the location of the MII spindle in living human oocytes can be imaged noninvasively with the Polscope and that this method helps predict the embryonic developmental competence after ICSI.

We confirm that there was no significance difference in the rate of fertilization, multipronuclear formation and embryonic development between total MII oocytes injected post imaging with the Polscope and controls which were routinely injected with polar body held at 12 o’clock or 6 o’clock without Polscope. It indicated no detrimental effects of imaging with the Polscope in human.

The rates of fertilization and embryonic development were significantly lower in oocytes without visible spindle than oocytes with visible spindle. Wang et al., (2001aGo,b,c) suggested that certain environmental changes or stimuli that induce microtubule depolymerization cause spindle damage. For example, a change in ambient temperature can induce temporary disappearance of the spindle and this could also result in poor quality of embryos due to chromosomal abnormalities. They also reported limited spindle recovery after cooling–rewarming (Wang et al., 2001aGo,b,c). In this study, we examined the bi-refringent spindles in MII oocytes while maintaining the temperature at 37°C for 3–4 hr. This implies that other factors other than temperature have influenced the presence of spindle birefringence in MII oocytes without spindles.

In this study, we observed that the rate of multipronuclear formation appeared to be higher in oocytes without a bi-refringent spindle than in oocytes with bi-refringent spindle although this failed to be statistically significant. This result suggests that the high rate of multipronuclear formation in oocytes without a spindle may be the result of dispersion of chromosomes into clusters (e.g. in the absence of a spindle) followed by the formation of multipronuclei, each with haploid set of chromosomes. However, the relationship between absence of a spindle and multipronuclear formation after ICSI requires further investigation.

Furthermore, as Silva et al. (1999Go) and Hewitson et al. (1999Go) proposed, it seems that location of the first polar body does not accurately predict the position of meiotic spindle in the hamster oocytes. Wang et al. (2001aGo,b,c) also found that the first polar body does not predict the exact spindle position in living human oocytes. As in previous reports, in the present study, we found that 48.2% of oocytes had spindles directly beneath or adjacent to the first polar body, but 11.2% of oocytes had their spindles located in the path of the injection needle if the first polar body was at the 6 or 12 o’clock position during conventional ICSI and other oocytes (24.1%) had spindles in different locations. One possible explanation for the various spindle positions is that the first polar body does not remain attached to the oocyte due to removal of cumulus cells and this can cause free rotation of the polar body. Another possibility is that spindle positioning changes due to the fluidity of the membrane area to which the spindle is attached. Further study is required to confirm this suggestion.

There is no difference in fertilization rates and quality of embryos among groups (A–E) of oocytes with spindle. This might be due to minimizing the potential for the spindle damage during ICSI at the moment of injection.

In this study, the rate of high quality embryos was higher in oocytes with visible spindle than that in oocytes without visible spindle (P < 0.01). Some explanations are suggested for the lower developmental ability of oocytes without a bi-refringent spindle. It is possible that oocytes without bi-refringent spindles might have abundant chromosomal abnormalities, which in turn might induce cell cycle arrest, although direct confirmation of the association between aneuploidy and spindle structure is needed (Wang et al., 2001aGo,b,c). It also was reported that in a telophase I or an early prometaphase I stage of meiosis the spindle is still unordered and therefore not bi-refringent and hence cannot be imaged by using the Polscope (Eichenlaub-Ritter et al., 2002Go). Furthermore the damage to the spindle caused by ICSI might possibly be one reason for the poor quality of oocytes without bi-refringent spindle compared with those with bi-refringent spindles (Wang et al., 2001aGo,b,c), although due to the narrow gauge of the ICSI needle, the chance of damaging the spindle is probably very small. In conclusion, our results indicate oocytes in which a bi-refringent spindle can be visualized may have a higher embryonic developmental competence than oocytes without a bi-refringent spindle and that spindle position does not influence the rate of fertilization or embryonic development. Our results also indicate that the use of a Polscope in combination with ICSI may minimize the mechanical damage to the spindle caused during the ICSI procedure and thus reduce multipronuclear formation.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on September 9, 2002; accepted on December 20, 2002.