Polarized light microscopy of Meiotic Spindles in ICSI
2019年度 年次大会-講演抄録｜オーストラリア生殖医学会 交換講演 Exchange Lecture of The Fertility Society of Australia（FSA）
The meiotic spindle as a marker of embryo viability is gaining traction in the field of ART. The spindle is a highly dynamic cytoskeletal structure comprised of microfilaments and microtubules（Kim et al.1998）. Continual polymerisation and depolymerisation of its microtubule constituents render it morphologically fluctuant depending on the stage of meiosis. At metaphase II it is normally described as a barrel- shaped, symmetrical structure with pointed, anastral poles, radially oriented at the periphery of the oocyte）.
Ploidy is, to a large degree, contingent on the dynamic forces of the meiotic spindle. However, the assembly of tubulin units which constitute microtubules are highly sensitive to physical changes such as temperature and pH, as well as physiological changes such as ageing. These changes result in the disassembly of the spindle, corrupting normal spindle dynamics and chromosomal organisation. It is therefore unsurprising that oocyte meiosis is a highly error- prone process, with up to 20% of oocytes displaying aneuploidy due to chromosomal nondisjunction and premature sister chromatid separation Segregation errors give rise to aneuploid embryos, which in turn are predisposed to unsuccessful fertilisation, maturation arrest, spontaneous abortion, developmental anomalies, and genetic diseases. These findings have thus established the premise for the meiotic spindle as a marker for an oocyte’sdevelopmental potential.
The Spindle Under Polarised Light Microscopy
Conventional light microscopy, while non-invasive and capable of sustaining oocyte viability, fails to provide sufficient contrast to accurately differentiate the meiotic spindle from the translucent cytoplasm. Polarised light microscopy offers a non-invasive method of spindle visualisation by exploiting the refractive properties of its molecular architecture. The meiotic spindle is an anisotropic structure that, when subjected to polarised light, diverges the beam into two orthogonal planes. This occurs as the spindle microtubules, arranged in parallel arrays, cause the beam’s optical path to traverse at varying speeds, in turn shifting the plane of light. The specimen under scrutiny appears dark, except for its anisotropic structures, allowing them to be visualised . This intrinsic optical property is coined birefringence and can be quantified and known as retardance. Higher retardance values correlate with greater levels of macromolecular compactness and structural organisation, which in turn suggest higher order alignment and better organised structures. Evenly- distributed birefringence is also a positive indicator of spindle health.
The Meiotic Spindle as a Predictor of Fertility Outcomes
Many studies have supported the role of the meiotic spindle in appropriately distributing genetic material. As such, it follows that the visualisation of the meiotic spindle at the time of intracytoplasmic sperm injection （ICSI） correlates with improved fertility outcomes. A number of studies have demonstrated a significant increase in fertilisation rates amongst present-spindled oocytes relative to absent-spindled oocytes .
These results were in contradiction with other studies which found no statistically significant difference in fertilisation rates.
Of the reviewed studies investigating blastulation rates as an outcome, all purported a statistically significant correlation between meiotic spindle presence and increased blastocyst formation.
Two of the three studies comparing the effect of spindle presence on embryo quality found a significantly increased proportion of high-quality embryos derived from spindle present oocytes relative to those that were absent. Conversely, Others have found this increase in the formation of high-quality embryos to be statistically insignificant.
Three of the four reviewed studies found that a significantly higher proportion of oocytes with spindles achieved a clinical pregnancy, relative to those without spindles Moreover, we found that, amongst the oocytes which resulted in a clinical pregnancy, spindle density was significantly higher（30±1.23nm） compared to those that did not（2.5±0.7nm）（p=0.02）.
Few studies have investigated the difference in fertility outcomes between spindles based on morphology as opposed to presence. We developed a classification system and found that 90% of normal- spindled oocytes were successfully fertilised compared to 72% of oocytes with morphologically abnormal spindles（p<0.001）. Our more recent prospective cohort study also observed the ploidy of embryos with normal and abnormal spindles. The abnormal class was further stratified into dysmorphic, translucent, telophase and non-visible spindles. Translucent and non-visible spindles yielded significantly lower proportions of euploid embryos of 9.9%（OR 0.25, 95% CI 0.13-0.46）and 13.5% respectively（OR 0.35, 95% CI 0.19-0.63）relative to the other two groups（p£0.001）.
Although there exists no definitive consensus, current data suggests that the presence of the spindle apparatus with good morphological features is an indicator for more favourable fertility outcomes.
Our other area of interest has been the potential to improve fertilisation rates with ICSI. In our data fertilisation rates were improved by the use of PLM because it ensures spindle damage can be avoided at the time of sperm injection.