Analysis of optical properties of paracrystalline ultrastructures in human oocytes by PolScope microscopy correlated to embryo quality and viability
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Medizinisches Zentrum f├╝r Frauenheilkunde und Geburtshilfe
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The spindle apparatus and the zona pellucida are two essential organelles in oocytes, which can be viewed by PolScope microscopy non-invasively, due to their composition of molecularly ordered, paracrystalline fibres. The spindle apparatus is responsible for the high fidelity of chromosome segregation during oogenesis. Disturbances in spindle assembly increase the risk of chromosome mal-segregation in oocytes. Accordingly, oocytes possessing a birefringent spindle in PolScope microscopy tend to have a better developmental potential compared to those without a spindle. The zona pellucida is an extracellular matrix surrounding mammalian oocytes and pre-implantation embryos with an essential role in oocyte development, fertilization and implantation. Failures in expression of zona proteins correlate to subfertility or infertility in animals. Low expression of the zona proteins by the growing human oocyte may indicate reduced developmental potential. Retardance of light and magnitude of birefringence is linearly correlated to number and alignment of microtubules. Therefore, high mean retardance magnitude may reflect oocytes with healthy, robust spindle and/or zona pellucida that have a high developmental potential. Low birefringence might be indicative of structures with few and unaligned fibres and oocytes with lower developmental capacity. To test this notion, the current study employed PolScope microscopy in the IVF centre of Giessen University to non-invasively and quantitatively assess spindle and zona morphology in living human oocytes before they were subjected to intracytoplasmic sperm injection (ICSI). Quality and viability of the embryos after insemination of oocytes with high or low birefringence was compared retrospectively.
In total, oocytes from 182 stimulated ICSI cycles were screened by PolScope after informed consent of patients. For the assessment of spindle morphology and texture, 676 oocytes (mean age of patients was 32.5 ┬▒ 4.4 years) were analysed that developed into pre-embryos and were assessed for PN scores to select embryos for transfer, blindly with respect to spindle birefringence. Mean magnitude of retardance of spindles and pole-to-pole length was retrospectively compared between all oocytes giving rise to high or low PN score embryos, and was also analysed with respect to maternal age. Mean retardance was also compared between oocytes developing into non-transfer embryos and those selected for transfer after ICSI, respectively.
For the assessment of the zona morphology and texture, retardance magnitude and thickness of the inner, middle and outer layers of the ZP were quantitatively analysed by PolScope in 166 oocytes selected for transfer after ICSI (63 of the 103 cycles; mean age 32.8 ┬▒ 4.4 years) on the basis of pronuclear score at day 1. Blastomere number was determined at day 2. Data were compared between oocytes giving rise to conception cycles (CC; 65 oocytes/23 cycles) and non-conception cycles (NCC; 101 oocytes/40 cycles) and with respect to maternal age.
Results of the spindle analysis showed that magnitude of light retardance by the oocyte spindle was positively correlated to embryo quality after ICSI according to PN score in the IVF centre in Giessen. Good PN score pre-embryos were from oocytes with significantly higher birefringent spindles compared to lower PN score pre-embryos (P < 0.001). In addition, oocytes developing to pre-embryos with very poor quality had a significantly shorter spindle compared to those forming embryos with highest PN score (P < 0.001). Mean retardance was significantly higher in spindles of oocytes selected for transfer compared to non-transfer oocytes (P < 0.001). There was no clear relationship between maternal age and mean retardance in the cohort containing few patients ≥ 36 years.
Zona pellucida scoring revealed three distinctly different layers of the extracellular coat surrounding the human oocyte, whereas the zona appeared comparatively translucent, when viewed by a conventional light microscope equipped with Hoffmann interference optics. Embryos in the CC group tended to develop faster. The thickness of the inner zona layer was significantly different (P < 0.001), and the mean magnitude of light retardance was nearly 30% higher (P < 0.001) in the inner layer of the zona pellucida of oocytes contributing to a CC compared to a NCC. Nearly 90% percent of the oocytes containing a ZP with a mean retardance magnitude over 3.0 nm of the inner ZP layer contributed to a CC, significantly more compared with the NCC (c┬▓-test, P < 0.001).
In conclusion, the study shows that oocytes with a high mean retardance of light by the spindle develop into pre-embryos with good PN score. This finding has been confirmed by a small cohort of 59 oocytes from an IVF centre in Milano, Italy. PN-scoring was originally developed to identify presumably high quality embryos and may be predictive for aneuploidy. Since PN-scoring cannot be used for selection in some counties due to ethical and legal considerations, analysis of mean spindle retardance by PolScope microscopy may be as useful as PN-scoring to identify oocytes with higher or lower developmental potential, and combining with PN-scoring may provide additional information on oocyte health and quality.
A high birefringent and thick ZP is related to the presence of a dense coat of highly ordered ZP filaments and thus appears to reflect health and developmental potential of an oocyte. The mean magnitude of light retardance by the ZP inner layer presents a new, unique, non-invasive marker for embryo viability after ICSI. Combining screening for birefringence of the spindle apparatus and the zona pellucida together with other methods like PN-scoring, analysis of oocytes by quantitative PolScope microscopy may contribute significantly to identify the highest quality oocytes in ICSI cycles, to reduce multiple embryo transfers, and to improve oocyte handling and treatment of patients.
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