P-233 Novel, non-invasive imaging techniques in quality assessment of mammalian eggs
Authors: Fluks M , Rak A , Czajkowska K , Borecka K , Tamborski S , Szkulmowski M , Ajduk A 
Published: June 2023

Abstract

Study question
Can Optical Coherence Microscopy (OCM) imaging and Cytoplasmic Movement Velocity (CMV) analysis be useful in quality assessment of mature mammalian oocytes?

Summary answer
OCM allows for assessment of oocytes’ metaphase spindles, and CMV – the actomyosin cytoskeleton functionality, therefore they are potentially useful in oocyte selection for IVF.

What is known already
The success rate of IVF has significantly improved over the recent years, yet its efficiency plummets with the increasing age of female patients. Therefore, novel approaches to oocyte/embryo quality assessment are in high demand. Among the most promising new methods are OCM, a label-free, 3D tool for subcellular structure visualization, such as the meiotic spindle in mature oocytes or nuclear structure in immature oocytes, and the analysis of CMV, a time-lapse imaging-based method for studying properties of the actomyosin cytoskeleton, crucial for a plethora of cellular process. Both these methods provide unique insights into oocyte competence.

Study design, size, duration
MII spindle dimensions were measured in OCM-imaged freshly ovulated oocytes collected from young mice (FO-MII, n = 108) and correlated with the outcome of preimplantation development achieved after the oocytes’ activation. Spindles of FO-MII (n = 45) were also compared to spindles of oocytes collected from mice in advanced maternal age (AMA-MII, n = 44). CMV was compared in time-lapse imaged FO-MII (n = 24), postovulatory aged oocytes collected from young mice (PA-MII, n = 16), and AMA-MII (n = 19).

Participants/materials, setting, methods
Oocytes were collected from hormonally primed mice and imaged using a customized OCM protocol. They were then activated parthenogenetically and cultured for 5 days, followed by immunofluorescence staining to assess the total number of cells and number of cells in the first embryonic cell lineages. FO-MII, PA-MII, and AMA-MII were time-lapse imaged (6 frames per minute; 15 min). The images were subjected to particle image velocimetry analysis and the mean CMV was calculated.

Main results and the role of chance
Oocytes that achieved the blastocyst stage had smaller spindle volume (p &lt; 0.001) and width (p &lt; 0.05) as compared to those failing to do so. Univariate logistic regression analysis indicated that a 1 μm rise in the spindle width decreases the odds of blastocyst formation by 28% and the number of cells on average by 8, whereas a 100 μm3 rise in the volume – by almost 10% and 2.7 cells respectively. Moreover, the spindle volume is also related to the number of trophectodermal cells, and the spindle length - to the number of primitive endoderm cells in the blastocysts. Noteworthily, young oocytes’ spindles had significantly smaller volume (p &lt; 0.0001), as well as were narrower (p &lt; 0.0001) compared to their counterparts from aged females. A multivariate linear regression model combining spindle length with three morphokinetic parameters (t3, cc2b, tSB) explained 60% of the variability in the total number of cells in 5-day-old embryos (R2 = 0.604) while models based solely on a single morphokinetic parameters explained only up to approx. 54% (R2 = 0.537, for tSB). On the other hand, CMV analysis revealed that freshly ovulated eggs display faster cytoplasmic movements than those postovulatory-aged (p &lt; 0.0001). Reduced cytoplasmic speed was also observed in oocytes collected from aged mice (p &lt; 0.0001).

Limitations, reasons for caution
All studies presented here were performed on a mouse model, so the obtained data needs to be verified in the target species for validation of these methods as novel tools in oocyte selection protocols.

Wider implications of the findings
Our results suggest that OCM-based assessment of the MII spindle and short, 15-minute CMV imaging allow for the selection of higher quality oocytes and could potentially find application in assisted reproductive technologies, as they are non-invasive and may enhance the universally acclaimed oocyte/embryo quality assessment protocols.

Trial registration number
not applicable