Karyotype of sperm from BT group.

The sperm of infertile men have higher rates of chromosomal abnormalities than those of fertile men. Miscarriage rate is also higher following testicular sperm extraction combined with intracytoplasmic sperm injection (TESE-ICSI). Sperm chromosomal abnormalities are assumed to be the cause of miscarriages. Previous testicular sperm karyotyping studies have only examined a few selected chromosomes using fluorescence in situ hybridization. The aim of this study was to provide a more detailed analysis of sperm karyotyping by analyzing all chromosomes using next-generation sequencing (NGS) in clinically usable testicular sperm. Sperm discarded after clinical use was collected for NGS. Additionally, sperm were individually collected by micromanipulation from patients with obstructive azoospermia (OA) and non-obstructive azoospermia (NOA) who underwent TESE-ICSI. For comparison, ejaculated sperm from control and balanced translocation (BT) carriers were examined. Karyotyping was performed on individual sperm cells using NGS. The number of normal and aberrant sperm was compared. Seventeen patients participated in this study: control (n = 4), BT (n = 3), OA (n = 5), and NOA (n = 5). Ten sperm samples per patient were analyzed. The total acquisition rate for single sperm karyotyping was 85% (145/170). Karyotyping of sperm from the BT group revealed sperm with unbalanced chromosomes derived from carrier translocations. Among the NOA group, 7/41 (17%) sperm samples exhibited aberrant karyotypes, whereas no aberrant sperm were identified in the control and OA groups. Individual differences were observed in the frequency of sperm chromosomal abnormalities among patients with NOA. In conclusion, sperm chromosomal abnormalities are frequently observed in patients with NOA even after sperm selection for clinical use. As the frequency of chromosomal abnormalities varies among patients with NOA, single sperm sequencing may help identify patients with NOA most likely to benefit from PGT-A.

不育男性的精子染色体异常发生率高于生育男性，且接受睾丸精子提取联合卵胞浆内单精子注射（TESE-ICSI）治疗后，流产率也更高。目前认为精子染色体异常是流产的诱因之一。既往睾丸精子核型分析研究仅通过荧光原位杂交（fluorescence in situ hybridization, FISH）技术，对少数选定的染色体进行检测。本研究旨在通过下一代测序（NGS）技术，对临床可用的睾丸精子的全部染色体开展分析，以实现更细致的精子核型研究。本研究收集了临床使用后废弃的精子用于NGS检测；此外，通过显微操作技术，从接受TESE-ICSI治疗的梗阻性无精子症（OA）与非梗阻性无精子症（NOA）患者中分别采集单个精子样本。为进行对照比较，同时对健康对照者及平衡易位（BT）携带者的射出精子开展检测。采用NGS技术对单个精子细胞进行核型分析，并对比正常与异常精子的数量。本研究共纳入17名受试者，其中健康对照组4例、平衡易位携带者组3例、梗阻性无精子症组5例、非梗阻性无精子症组5例，每名受试者分析10个精子样本。单个精子核型分析的总获取率为85%（145/170）。平衡易位携带者组的精子核型分析结果显示，存在由携带者易位引发的染色体不平衡的精子。在非梗阻性无精子症组中，7/41（17%）的精子样本呈现异常核型，而健康对照组与梗阻性无精子症组均未检测到异常精子。非梗阻性无精子症患者的精子染色体异常频率存在个体差异。综上，即便经过临床用精子筛选流程，非梗阻性无精子症患者的精子仍常存在染色体异常。由于非梗阻性无精子症患者的染色体异常频率存在个体差异，单个精子测序或可帮助甄别出最能从植入前非整倍体遗传学检测（PGT-A）中获益的NOA患者。