P–186 Volumetric imaging provides insight into the 3D ultrastructural organization of maturing human oocytes
Authors: Trebichalská Z , Javůrek J , Kyjovská D , Tatíčková M , Kloudová S , Otevřel P , Hampl A , Holubcova Z 
Published: August 2021

Abstract

Study question
Is volume electron microscopy suitable for high-resolution oocyte imaging in three dimensions (3D)?

Summary answer
Focused Ion Bean Scanning Electron Microscopy (FIB-SEM) allows 3D visualization and quantitative analysis of ultrastructural features in the large human oocyte volume.

What is known already
Transmission electron microscopy (TEM) has been traditionally used to study the fine morphology of female gametes. However, 2D micrographs provide only limited information about the topology of subcellular structures. Volumetric studies are needed to elucidate the 3D organization of complex oocyte cytoplasm.

Study design, size, duration
An academic study conducted in collaboration with an IVF clinic. Advanced 3D - ultrastructural analysis was performed on 9 human oocytes representing 3 stages of maturation (3 germinal vesicle (GV), 3 metaphase I (MI), 3 metaphase (MII) oocytes) collected from February 2018 to November 2019.

Participants/materials, setting, methods
Spare IVF oocytes, donated by 9 young egg donors (22–29 years), were cultured in vitro until they reached a defined developmental stage. Each oocytés meiotic status was determined based on the presence/absence of a prophase nucleus, a polar body, and a non-invasively detectable MI/MII spindle. Following standard TEM preparation, individual oocyte-containing resin blocks were coated with a thin carbon layer[JJ1] , mounted on the microscope stage, and subjected to FIB-SEM imaging.

Main results and the role of chance
FIB-SEM tomography provided an unprecedented view of the oocytés intracellular morphology. Automated serial scanning of newly exposed sample surface generated large stacks (120–1294 slices) of ultrastructural images with 40–100 nm z-resolution. The tomographic reconstruction of acquired datasets revealed the spatial arrangement of inner oocyteś structures. The imaging protocol was optimized to ensure sufficient image detail, minimal noisiness, and time-efficiency of large volume scanning. Comparison of oocytes fixed at different maturation stages confirmed previous TEM observations that the cortical region of GV oocytes is deprived of membraneous structures, and major organelle redistribution occurs during the MI phase. Semi-automated 3D image segmentation was employed to distinguish distinct organelle populations and evaluated their abundance. Subsequent quantitative analysis of volumetric data showed that the mitochondrion occupies ∼ 5.27% of MII oocyte volume. In conclusion, the volumetric imaging, followed by advanced image analysis, maximizes the amount of morphological data obtained from a single human oocyte.

Limitations, reasons for caution
The imaging procedure was pioneered on a small number of hormonally-primed oocytes, which failed to complete development in vivo. There is a trade-off between resolution, the size of the 3D volume, and imaging time. Block-face ion milling during FIB-SEM imaging inevitably results in sample destruction.
Wider implications of the findings: This proof-of-concept study opens up new possibilities to study the delicate architecture of scarce human oocytes. Enhancing our knowledge of the spatial organization of ooplasm is pivotal for developing experimental and therapeutical strategies involving oocyte microsurgery.

Trial registration number
Not applicable