Gsl120

An intact nucleus, clear borders, and strong fluorescence signals were the criteria for sperm nuclei scoring. The definitions of various numerical abnormalities were as follows: 1) nullisomy, no signal for the given probe and one signal for the other control probe; 2) disomy, two signals for the given probe and one signal for the other control probe; 3) diploidy, two signals for both the given probe and the other control probe; and 4) “others” consisting of trisomy (three signals for the given probe and one signal for the other control probe), triploidy (three signals for both the given probe and the other control probe), tetraploidy (four signals for both the given probe and the other control probe), and multiple aneuploidies (in which the signals for the given probe and the other control probe differ from those described above). The signal patterns for the various numerical chromosomal abnormalities are shown in Supplementary Figure S1.
The scoring system was developed using an automated system (GSL-120; Leica Biosystems Richmond Inc.), with provision for automated slide loading, cell finding, image capturing, signal scanning, and sorting. The system captured approximately 1,000 sperm cells and sorted the different signal patterns. An experienced technician double-checked the signal from each cell, using the scoring criteria described above, to ensure classification accuracy.

Karyotyping was performed following the standard procedures, and karyotypes were scanned on Leica GSL120. At least 20 metaphases were counted, and five metaphases were analyzed. Abnormal karyotypes were named basing on ISCN 2020.

Karyotyping was performed following the standard cytogenetic protocol^{5 (link)}, and karyotypes were scanned on Leica GSL120. At least 20 metaphases were counted, and five metaphases were analyzed. Karyotype analysis and description were based on ISCN 2020^{6 (link)}.

Amniotic fluid (20 mL) was collected by amniocentesis under ultrasonic guidance and then centrifuged at 800×g/min for 10 min. After removing the supernatant, the remaining 1.5 mL of liquid was mixed and inoculated in two 3.5-mL culture bottles containing AmnioMAX™ II Complete culture medium (BIOAMF™-2, Biological Industries, Inc.). The culture medium was changed 6–7 days after inoculation. The cells were collected, and sections were prepared for Giemsa banding when more than 10 cell colonies were present. Images were scanned and collected by using a Leica Biosystems GSL-120 and Lecia CytoVisin analysis system (California Richmond, USA). We analyzed 5 mitotic figures and 30 counts for each sample.

The karyotypes of hiPSC lines were analyzed by G-banding method (GTG staining) at Nuwacell Biotechnology Co., Ltd. Using proliferating iPSCs, 30 metaphase spreads were counted at minimal resolution of 400 bands. Briefly, iPSCs at passage 31 or 36 were treated with 50 ng/mL colchica at 37°C for 2 hours, trypsinized into single cells, resuspended, and washed once in DPBS. The cells were resuspended in low permeability of KCl at 37°C for 20 to 40 min, fixed with methanol and acetic acid (3:1) overnight. The supernatant was discarded and the cells were dropped onto the precooled slides immediately and dried for 1 to 2 hours at 75°C. The cells on slides were treated with the Giemsa staining and dried at RT. The cells were scanned by Leica scanner (GSL120) and then subjected to chromosome karyotype analysis.