Image of the meiotic spindle: its influence on human reproduction

Andréa Chemin; Vinicius Bonato da Rosa; José Augusto Lucca Neto; Alessandro Schuffner

JBRA Assisted Reproduction
REVIEW ARTICLE
doi: 10.5935/1518-0557.20130060

Image of the meiotic spindle: its influence on human reproduction

Andréa Chemin, Vinicius Bonato da Rosa, José Augusto Lucca Neto, Alessandro Schuffner

Conceber - Centro de Reprodução Humana – Curitiba – PR

Recebido em 28-05-2012
Aceito em 22-04-2013

Corresponding author:
Alessandro Schuffner
Clinica Conceber - Centro de reprodução humana
Av. Republica Argentina, 210, 17° andar
CEP: 80240-210
Curitiba – PR
alessandro@clinicaconceber.com.br

RESUMO
Nos últimos anos, a utilização clínica da imagem do fuso meiótico tem sido sugerida como um possível recurso para obtenção dos melhores embriões, melhores taxas de implantação embrionária e gravidez. O fuso meiótico controla os movimentos dos cromossomos durante as diferentes fases da meiose. A imagem do fuso meiótico em oócitos “in vivo”, através da microscopia de luz polarizada, permite aos embriologistas a avaliação morfológica do fuso meiótico de forma não invasiva. Portanto esta técnica pode ser utilizada com sucesso na escolha do melhor momento para ICSI. Segundo os autores, ainda é discutível os resultados desse parâmetro, assim sendo sugere-se a utilização em forma conjunta com outras técnicas com o objetivo de alcançar melhores resultados clínicos.
Palavras-chave: fuso meiótico, octax, desenvolvimento embrionário, ICSI, Seleção oocitária.

ABSTRACT
Recently, the clinical use of the image of the meiotic spindle has been suggested as a possible resource to achieve the best embryos, higher rates of embryo implantation and pregnancy. The meiotic spindle controls the movements of chromosomes during the different phases of meiosis. The image of the meiotic spindle in oocytes “in vivo” by means of polarized light microscopy, allows embryologists make the morphological evaluation of the meiotic spindle as a non-invasive technique . Therefore this technique can be successfully used in choosing the best moment of ICSI. According to the authors, the results of this parameter are still controversial, so it is suggested to use with other techniques in order to achieve better clinical results.
Keywords: meiotic spindle, octax, embryonic development, ICSI, oocyte selection.

INTRODUCTION
During oocyte maturation it is observed the formation of the 1st polar body which is indicative of the end of the first meiotic division and also the formation of the second polar body indicative of fertilization (Oliveira et al., 2009). The exact mechanisms that regulate the meiotic division blockage and reactivation are still unknown but some evidence suggest that the main control of the meiotic cycle is associated with the regulation and activation of the maturation promoting factor (Han, S. J.; Conti, M., 2006).
A number of studies confirmed that the quality of the oocyte is associated to factors related to nuclear and cytoplasmic competence. This cytoplasmic competence is responsible for the ability of the oocyte to undergo fertilization and first cleavage stages. The nuclear competency is characterized by the chromatin and meiotic spindle quality (Vieira et al., 2008).
The polarized light microscopy is a non invasive method that allows a real time estimation of oocyte quality by the assessment of the presence and position of the meiotic spindle. With the use of birefringence, it also possible to evaluate the oocyte maturation and to select the appropriate time for ICSI or cryopreservation. Therefore avoiding damage to the spindle during this procedures (Madaschi et al., 2006; Montang e Ven, 2008) and preserving cellular viability, fertilization and normal embryo development (Rienzi et al., 2003).
The goal of this review was to assess the issues related to the observation of the meiotic spindle, as well as its clinical importance in the selection of the best embryo, which should benefit couples in reproductive treatment.

MATERIALS AND METHODS
This review was conducted by searching different online database such as MEDLINE, EMBASE, SCIELO e LILAC’S, search websites such as PUbMed, GOOGLE SCHOLAR e SCIRUS and journals such as FERTILITY and STERILITY, HUMAN REPRODUCTION e REPRODUCTIVE BIOMEDICINE. The search comprised the period from 1999 until 2012. The keywords used were: meiotic spindle, embryonic development, ICSI and oocyte selection.

RESULTS
SPINDLE DETECTION: PRESENCE, LOCALIZATION AND ITS APPLICATION ON IVF/ICSI

A study about the spindle localization in relation to the first polar body was conducted by Rienzi et al. (2003). The results showed a decrease in fertilization rates in oocytes with more than 90° spindle deviation in relation to the polar body. However, the position of the spindle had no effect on embryo development.
A meta-analysis from nine studies was published by Petersen et al., 2009. Fertilization rate was higher (75,6%) for oocytes in which the meiotic spindle was visualized compared to the group of oocytes in which the spindle were not observed (61,5%) (P<0,0001) (Table 1.).
The percentage of embryos with good pronuclear morphology was significantly higher in embryos developed from oocytes in which the spindle was observed (25,1%) compared to embryos from oocytes which spindle was not (12,7%) (P = 0,003; OR 1,71, IC 95%).
Cleavage rate was statistically higher in embryos that came from oocytes which the meiotic spindle was observed (63,1%) compared to embryos which spindle was not observed in the oocyte (43,9%) (P < 0,0001). Also, the observation of the meiotic spindle in the oocytes was positively related to better quality embryos on day 3 (37,3% vs. 28,0%; P < 0,003). These results combined suggested the importance of the meiotic spindle observation on embryo development.

Tabela 1. Meta análise investigando a influência da visualização do fuso meótico em oócitos humanos submetidos a ICSI (PETERSEN et al., 2009).

BLASTOCIST DAY +5
The percentage of embryos reaching the blastocyst stage was significantly higher (P < 0,0001) among those originated from oocytes in which the meiotic spindle was observed (50,7%) comparing to blastocysts that came from oocytes with the absence of the spindle (28,6%). The blastocysts rate was higher for oocytes in which the spindle was observed (50,7%) compared to those not observed (28,6%) by microscopy.

IMPLANTATION AND PREGNANCY RATE
Despite the differences in the previous parameters of fertilization and embryo development, the observation of the meiotic spindle was not associated with higher rates of implantation or with higher pregnancy rates. The implantation and pregnancy rates were similar among embryos that originated from oocytes with the spindle observed (15,4% and 28,5%, respectively) compared with embryos in which the spindle was not observed (9,8% and 17,6%, respectively). The presence of meiotic spindle has been associated with higher birefringence of the zona pellucida (Madaschi et al, 2009) and higher fertilization rates (Fang et al., 2007; Rama Raju et al., 2007; Braga et al., 2008, Madaschi et al., 2008). However, there are conflicting results for the association of spindle observation and early cleavage with lack of association (Cohen et al., 2004), or positive association (Moon et al., 2003; Rama Raju et al., 2007; Braga et al., 2008, Madaschi et al., 2008). Other results indicate that higher pregnancy rates were observed with oocytes in which the meiotic spindle was observed (Madaschi et al., 2008, 2009).
Another important issue is the correlation between the position of the meiotic spindle and the polar body with fertilization and cleavage rates (Fang et al., 2007) and early embryonic development (Cooke et al., 2003). However, Moon et al., 2003 did not observe the same correlation. Pregnancy rate and normal spindle morphology (barrel shape with high birefringence) were correlated (Kilani et al. 2009).

DISCUSSION
In human IVF practice, there is a daily need to deal with a limited number of oocytes and this situation leads to a continuous search for non invasive methods of oocyte assessment. Polarized light microscopy has been used as a tool to assess oocyte quality by analyzing the meiotic spindle birefringence and organization of the zona pellucida. Polarized light microscopy makes it possible to observer the birefringence in live cells without the need for fixation or staining. This allows the use of this technique in oocytes for IVF (Wang et al., 2001; Moon et al., 2003; Petersen et al., 2009). Published data suggests that oocyte assessment by this technique could be useful as a screening for potential abnormalities in the meiotic spindle. The technique could also be used to assess indirectly the chromosomes alignment, because in most cases these two situations are present simultaneously (Navarro et al., 2006).
Better selection of gametes is an important and potential area of study to improve the techniques used in reproductive medicine (Araújo et al., 2007). As a part of the continuous search for better biomarkers which could more precisely assess oocyte maturation and quality, evidence in the literature suggests that the image of the meiotic spindle can supply valuable information (Montang et al., 2011).
The absence of spindle birefringence led to lower fertilization rates and could be due to oocyte immaturity. On the other hand, a considerable number of oocytes were fertilized (fertilization rate of 61,5% for all studies included in the meta-analysis from Petersen et al., 2009). Possible explanations for this number include technical failure during spindle visualization (inadequate period for visualization of all oocytes) or the absence of the spindle did not prevent fertilization in all cases. However, Petersen et al., 2009 suggest that the absence of a meiotic spindle could have a posterior effect during embryonic development.
The meiotic spindle has an important role in the alignment and segregation of chromosomes during cell division. The integrity and viability of the spindle is necessary in order to prepare the mature oocyte for fertilization (Vieira et al., 2008). The spindle morphology and kinetics are affected by variables such as age, temperature and oocyte handling. Temperature is one of the variables that can lead to the formation of embryos with aneuplody due to nondisjunction, unbalanced junction or premature chromatid disjunction and chromosome segregation. The pH of the culture media is another important factor (Montang et al., 2008).
The incidence of spindle abnormalities was significantly higher in women with advanced age (Battaglia et., al. 1996). Later, it was confirmed by Volarcik et al., 1999, that aging is associated with the meiotic process and also to the in vitro maturation of human oocytes from non stimulated ovaries.
A study conducted by MOON et.al., 2003 showed that fertilization and embryonic development were similar in oocytes with spindle despite its position in the cell. However, the rate of embryo of good quality was higher in oocytes in which the spindle was observed (64,2%) than in the ones the spindle was not (35,9%). In another study, the fertilization rate in oocytes with spindle observed was statistically higher compared to oocytes in which the spindle was not detected (70,4% versus 62,2%, P = 0,035). There was no relationship between the spindle image and embryo cleavage on day 3 (Cohen et al., 2004). The identification of the meiotic spindle position in relation to the polar body could prevent a potential damage caused during ICSI. However, recent reports showed that the position of the first polar body is not a precise indicative for the spindle position in human oocytes (Wang et al., 2001). A possible reason could be a physical dislocation of the polar body during denudation of the cumulus cells (Rama Raju et al., 2007).
When oocytes were compared in relation to time from hCG injection, it was observed that the formation of the meiotic spindle was significantly lower on 36-37 hours post HCG (61,6%) compared to 38-39 hours (81,5%) and post 40-42 hours (78,1%). Therefore the best time to observe the spindle is before 38 hours post hCG.
Studies were conducted to assess the relation between the angle formed between the polar body and the spindle with fertilization rates. Angles lower than 90° did not affect this rate (Rienzi et al., 2003; Fang et al., 2007).
The disappearance of the meiotic spindle during observation could be a marker of oocyte maturation by meiotic stages. During the transition from metaphase I to metaphase II, the spindle disappears completely for about 40-60 min. Therefore, a simple observation of the spindle seems not to be enough to indentify oocytes without a viable spindle, abnormal (without spindle) or that just had a late entry on telophase. In this last case, there is a high probability of spindle formation two hours later (Montang et al., 2006).
Some authors (Hyun et al., 2007) showed that in vitro matured oocytes, although apparently matured (presence of the first polar body) were in fact at telophase I, following polarized light microscopy analysis. They also showed a lower fertilization rate compared to oocytes that finished the first meiotic division. Chamayou et al., 2006 analyzed 697 oocytes in metaphase II. It was not observed a relation between spindle presence or absence and clinical pregnancy (20,8% versus 17,5%; P> 0,05) and implantation rate following ICSI (11,9% versus 9,7%, P> 0,05).
Besides the meiotic spindle, the polarized light microscopy also allows the assessment of other characteristics from oocytes such as zona pellucida birefringence (Shen et al., 2005). There is a correlation between the meiotic spindle and characteristics of the zona pellucida that may influence embryonic development. Factors that may affect oocyte maturation, may also affect this two structures which are closely associated.
One of the main challenges in reproductive medicine is the search for better selection methods for gametes and embryos. The major goal is to achieve better embryos, increased fertilization, implantation rates, clinical results and reduce multiple gestations. Therefore, it is essential the development of new tools and improvement of the available ones such as the non invasive polarized light microscopy.

CONCLUSION
This review suggests that the assessment of presence and position of the meiotic spindle should help the achievement of better clinical results and predict rates of fertilization and normal embryonic development.
From this perspective, it is possible to indentify couples that could benefit from this method, in special for cases with recurrence of failures and therefore have an alternative individual therapy of each patient.
The importance of meiotic spindle visualization is still controversial and a critical review about this method deservers a more intense and coordinated work to reach a consensus and provide results of positive pregnancy and hope of having a health child.

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