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. 2021 Mar 18;20(3):260-266.
doi: 10.1002/rmb2.12378. eCollection 2021 Jul.

Chromosomal analyses of human giant diploid oocytes by next-generation sequencing

Hiroomi Kawano  1   2 , Naoki Yamashita  2 , Junya Ito  1 , Naomi Kashiwazaki  1
Affiliations

Chromosomal analyses of human giant diploid oocytes by next-generation sequencing

Hiroomi Kawano et al. Reprod Med Biol. .

Abstract

Purpose: Although giant oocytes (GOs) having about twice cytoplasmic volume compared with general oocytes in mammals including the human are rarely recovered, it is thought that GOs have potentially chromosomal abnormalities. The aim of the present study was to assess chromosome numbers in chromosome-spindle complexes (CSCs) and polar bodies of human GOs by using micromanipulation for sampling and next-generation sequencing (NGS) for analyses of the chromosome numbers.

Methods: When recovered oocytes whose cytoplasm has lager than 140 µm or above, the oocytes were defined as GOs, and recovered GOs were vitrified. After warming, the CSCs, polar bodies, and enucleated cytoplasm were collected by micromanipulation from 3 GOs. The collected samples were analyzed by NGS.

Results: Chromosomal aneuploidy in the GOs was confirmed in all the three GOs. Comparing the CSCs with the chromosomes from polar bodies, the deletion and overlapping chromosome numbers were complementary in each GO.

Conclusions: The authors could collect the CSCs and the polar bodies from human GOs by micromanipulation, and then could analyze the chromosome numbers of the GOs by NGS method. As our data suggest that human GOs have chromosomal abnormalities, GOs should be excluded from clinical purpose as gamete sources for embryo transfer in the human.

Keywords: cell size; cytodiagnosis; high‐throughput nucleotide sequencing; micromanipulation; oocytes.

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Conflict of interest statement

Conflict of interest: The authors declare no conflict of interest in association with this manuscript. Human rights statement and informed consent: The present study was approved by the institutional ethics review board of Yamashita Shonan Yume Clinic held in October 2015 (Reference number: YSYC‐07) and National Center for Global Health and Medicine Ethics Review Board held in February 2020 (Reference number: 3370). All patients signed an informed written consent form before entering the study, and they were informed that they could terminate their cooperation with us whenever they wanted without any consequences.

Figures

FIGURE 1
FIGURE 1
Micromanipulation procedure for sampling polar bodies and chromosome‐spindle complexes (CSCs) from a mature giant oocyte in the human. A, Laser perforation of the zone pellucida. B, Tubing polar body 1. C, Tubing CSC 1. D, Tubing polar body 2. E, Tubing CSC 2. F, Tubing cytoplast. CSC(s), Chromosome‐spindle complex(es)
FIGURE 2
FIGURE 2
A giant oocyte with two polar bodies and two chromosome‐spindle complexes (CSCs). A, A giant oocyte with two polar bodies (arrows). B & C, A giant oocyte with two CSCs in the cytoplasm. The CSCs (arrows) were observed below the polar bodies by polarized microscopy. D, A normal metaphase Ⅱ oocyte with one polar body (arrow). E, A normal metaphase Ⅱ oocyte with one CSC (arrow)
FIGURE 3
FIGURE 3
Micromanipulation for sampling of polar bodies, chromosome‐spindle complexes (CSCs), and remained cytoplast from a mature giant oocyte in the human. A, A giant oocyte retained with a holding pipette. The two polar bodies (arrows) are clearly visible by polarized light microscopy. B & C, Confirmation of the two CSCs in the cytoplasm by polarized light microscopy. The CSCs (arrows) were directly observed below the polar bodies. D, Tubing the polar body (arrow). E, Tubing the CSC (arrow) which was observed in the glass pipette. F, A cytoplast (arrow) taken out of the zona pellucida
See this image and copyright information in PMC

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