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

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.

cell fusion of two adjacent oogonia or oocytes during oogenesis. 7 In addition, it is important for fertilization, reproduction, and development to examine chromosome numbers in mammalian gametes including GOs.
In general, cytogenetic analyses of chromosome numbers are needed to collect a lot of cells at metaphase and special staining. Therefore, it is difficult to assess the chromosome numbers in oocytes. Recently, next-generation sequencing (NGS) and the related technology have been developed with remarkable progress in DNA sequence technology, and then, we could apply it to chromosome analyses, aneuploid screening of embryos. 8 On the other hand, micromanipulation for oocytes/embryos in mammals has already been developed well, a lot of genetically modified and cloned animals was generated, 9 and tremendous babies were also born via micromanipulations such as intracytoplasmic sperm injection (ICSI) worldwide. In the present study, we applied these novel techniques to human GOs that are valuable and unique materials to clarify numerical chromosomal constitution during oocyte maturation.
The aim of the present study was to assess chromosome numbers in CSCs and polar bodies of human GOs by using micromanipulation for sampling and NGS for analyses of the chromosome numbers.

| Oocyte recovery
When patients agreed to provide GOs for research purposes by the informed consent form, recovered GOs were used in the present study. Oocyte recovery was performed at natural cycles or minimal ovarian stimulation (by clomiphene citrate or aromatase inhibitor) cycles in Yamashita Shonan Yume Clinic in Kanagawa, Japan, and recovered GOs at that time were then cryopreserved. The GOs were used for chromosome assessments in the present study.
Oocyte recovery in natural cycles was conducted as follows:

| GO collection and vitrification
Obtained oocyte-cumulus complexes were treated to remove cumulus cells while leaving a few layers and observed under an inverted microscope (IX-73; Olympus Corporation, Tokyo, Japan) to check oocyte maturation and measure its cytoplasm size in diameter.
Regarding the size of recovered oocytes, those with cytoplasm diameters exceeding 140 µm were judged as GOs. 4 The vitrified/warmed GOs were cultured until micromanipulation for sampling in incubators at 37.0°C in the atmosphere of 6% CO 2 , 5% O 2 , and 89% N 2 .

| GO micromanipulation for sampling polar bodies and CSCs
To avoid the effects of in vitro maturation culture, we only used mature GOs at oocyte recovery. Vitrified and warmed GOs were cultured for 10 minutes, as a pre-treatment for micro-manipulation for sampling. Quinn's Advantage TM Protein Plus Cleavage Medium added cytochalasin B (C6762-5MG; Sigma-Aldrich) to 5 µg/mL was used for the culture before the sampling micromanipulation for 10 minutes to improve cell viability after micro-manipulation. 12 As micro-manipulation medium ( Inc; Figure 1A). As shown in Figure 1B, the polar body 1 was aspirated with a glass pipette (PIN20-20FT; PRIME TECH LTD.) with an inner diameter of 20 µm and transferred to a PCR tube (K77301; BIOplastics) containing 2 µL of phosphate-buffered saline (PBS), and frozen and stored in a freezer at −30°C up to the chromosome analyses. As shown in Figure 1C, the CSC 1 corresponding to the polar body 1 was aspirated with the pipette, transferred to another PCR tube, and frozen and stored. One set of polar body 1 and CSC 1 derived from the same GO were collected as mentioned above, and the remaining polar body 2 and CSC 2 derived from the same GO were also collected each PCR tubes in the same procedure ( Figure 1D,E).
After collecting two sets of polar bodies and CSCs derived from the same one GO, the infrared diode laser was applied to the zona pellucida and a hole with a width of about 100 µm was made, and aspirated cytoplast remaining in the zona pellucida from the hole. The cytoplast was transferred to a PCR tube and frozen and stored up to the chromosome analyses ( Figure 1F). We sent collected genomic samples that were derived from polar bodies, CSCs, and cytoplasts to Igenomix Japan KK to examine the chromosome numbers of the GOs by NGS analyses.

| Chromosome analyses of GOs by NGS
The collected polar bodies, CSCs, and cytoplasts in the tubes were sent to Igenomix Japan KK for NGS analysis to examine the chromosome numbers. The protocol for this NGS analysis was as follows Scientific). Data analysis was performed with Ion Reporter software, which aligns the reads using the last human genome build (hg19; Thermo Fisher Scientific).

| Statistical analysis
GO cytoplast size was measured to two decimal places, and the mean was shown in mean ± SE. Statistical analysis of the GOs recovery rate between ovarian cycles was determined by one-way ANOVA followed by Tukey's test for multiple comparisons. Significance difference was set at P < .05. in GOs recovery rates between patient's ovarian treatment.

| GO collection and vitrification
The 16 mature GOs were vitrified and stored in liquid nitrogen. Four of the vitrified GOs were warmed, and all the 4 GOs were survived and used in the present study.

| GO micromanipulation for sampling polar bodies and CSCs
The successful rate of micromanipulation for sampling (Figure 3) from the GOs was 75% (3/4), and polar bodies and CSCs of the 3 GOs were analyzed by NGS method to examine the chromosome numbers.

| Chromosome analyses of GOs by NGS
As shown in Table 2 Figure S1). GO the chromosomal abnormality occurred during the first meiosis.
In the present study, we identified chromosomal abnormalities in the polar bodies of GOs and CSCs of GOs ( Table 2). The chromosomal abnormalities observed were monosomy and trisomy, and tetrasomy was not observed. The increase in miscarriage rates in older pregnancies is largely due to chromosomal aneuploidy that occurs during the first meiosis. 13 It has been reported that this is one of the causes of the decrease in cohesin in the oocyte chromosome with aging, 14 and it is thought that this is because the decrease in cohesin causes the early separation of bivalent chromosomes. 15 It is considered that the chromosomal abnormalities in GOs observed in the present study might be due to the early separation of bivalent chromosomes. The age of patients whom GOs was used in the present study was 46 for # 3, 43 for # 11, and 38 for # 16, respectively, and it is undeniable that the age affected chromosomal abnormalities of the GOs. In addition, it is considered that GOs are highly likely to be cells that are prone to chromosomal abnormalities due to their structural characteristics.
About the chromosome analysis by NGS method applied in the present study, there is concern that the genomic amount of the analysis sample is smaller than general samples. In PGT-A in human blastocysts, considering the possibility that abnormal cells are contained, a method to collect and analyze 5-10 cells at once is recommended. 16 Since the NGS analysis used in the present study was a   The results of the present study showed that CSCs and corresponding polar bodies of GOs could be collected as genomic samples by micromanipulation and that CSCs and the polar bodies could be analyzed by NGS method. Furthermore, it was revealed that human GOs should not be oocytes targeted for ART, as the number of chromosomes was abnormal. However, as the present study was limited to the analysis of 3 GOs, it is necessary to further increase the number of chromosome analysis of GOs.
In conclusion, we 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 including aneuploidy, GOs should be excluded from clinical purpose as gamete sources for embryo transfer in the human.