Objective

To study the prevalence of spermatogonia in adult subjects with Klinefelter syndrome (KS) using MAGE-A4 and UCHL1 (PGP9.5) immunohistochemistry as markers for undifferentiated spermatogonial cells. We aimed to compare this method to the gold standard of hematoxylin and eosin (H & E) staining with histologic analysis in the largest reported cohort of adult subjects with KS.

Design

A retrospective cohort study.

Setting

Infertility Clinic and Institute for Regenerative Medicine.

Patient(s)

This study consisted of 79 adult subjects with KS and 12 adult control subjects.

Intervention(s)

The subjects with KS (n = 79) underwent bilateral testicular biopsy in an initial effort to recover spermatozoa for in vitro fertilization and intracytoplasmic sperm injection. The institutional review board approved the use of a portion of the archived diagnostic pathology paraffin blocks for the study. The samples were superimposed onto microscopic slides and labeled with the PGP9.5 and MAGE-A4 antibodies. Subjects (n = 12) who had previously consented to be organ donors via the National Disease Research Interchange were selected as controls. Dedicated genitourinary pathologists examined the H & E-, PGP9.5-, and MAGE-A4–stained tissue for presence of undifferentiated spermatogonia and spermatozoa with the use of a virtual microscopy software.

Main Outcome Measure(s)

The primary outcome was the presence of MAGE-A4–positive or UCHL1-positive tubules that indicate undifferentiated spermatogonia. Supportive outcomes include assessing the biopsy specimen for the following: total surface area; total seminiferous tubule surface area; total interstitium surface area; the total number of seminiferous tubules; and MAGE-A4– negative or UCHL1-negative tubules. Additionally, clinical information, such as age, karyotype, height, weight, mean testicle size, and hormonal panel (luteinizing hormone, follicle-stimulating hormone, and testosterone), was obtained and used in a single and multivariable analysis with linear regression to determine predictive factors for the number of UCHL1-positive tubules.

Result(s)

The mean age of the subjects in the KS group was 32.9 ± 0.7 years (range, 16–48). UCHL1 (PGP9.5) and MAGE-A4 staining showed that 74.7% (n = 59) and 40.5% (n = 32) of the subjects with KS, respectively, were positive for undifferentiated spermatogonia compared with 100% (n = 12) of the control subjects who were positive for both the markers. Hematoxylin and eosin with microscopic analysis showed that only 10.1% (n = 8) of the subjects were positive for spermatogonia. The mean number of positive tubules per subject with KS was 11.8 ± 1.8 for UCHL1 and 3.7 ± 1.0 for MAGE-A4. Secondary analysis showed 7 (8.9%) adult subjects with KS as positive for spermatozoa on biopsy. The population having negative testicular sperm extraction results (n = 72) showed a spermatogonia-positive rate of 1.4%, (n = 1), 72.2% (n = 52), and 34.7% (n = 25) using H & E, UCHL1, and MAGE-A4, respectively. Further analysis showed that 54 (75.0%) subjects were either positive for UCHL1 or MAGE-A4. Twenty (27.8%) subjects were positive for both UCHL1 and MAGE-A4. Multivariate analysis with linear regression showed no significant correlation between clinical variables and the number of UCHL1-positive tubules found on biopsy specimens.

Conclusion(s)

We report a cohort of adult subjects with KS undergoing analysis for the presence of undifferentiated spermatogonia. UCHL1 and MAGE-A4 immunostaining appear to be an effective way of identifying undifferentiated spermatogonia in testicular biopsy specimens of subjects with KS. Despite observing deterioration in the testicular architecture, many patients remain positive for undifferentiated spermatogonia, which could be harvested and potentially used for infertility therapy in a patient with KS who is azoospermic and has negative testicular sperm extraction results.

中文翻译：

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形态学和免疫组织化学分析作为一种方法来识别患有 Klinefelter 综合征的成年受试者未分化的精原细胞：一项队列研究

客观的

使用 MAGE-A4 和 UCHL1 (PGP9.5) 免疫组织化学作为未分化精原细胞的标记物，研究患有 Klinefelter 综合征 (KS) 的成人受试者中精原细胞的患病率。我们旨在将此方法与苏木精和伊红 (H & E) 染色的金标准进行比较，并在最大报告的 KS 成年受试者队列中进行组织学分析。

设计

一项回顾性队列研究。

环境

不孕症诊所和再生医学研究所。

患者）

该研究包括 79 名患有 KS 的成人受试者和 12 名成人对照受试者。

干预措施

患有 KS 的受试者 (n = 79) 接受了双侧睾丸活检，以初步努力恢复精子用于体外受精和胞浆内单精子注射。机构审查委员会批准使用部分存档的诊断病理石蜡块进行研究。将样品叠加到显微镜载玻片上并用 PGP9.5 和 MAGE-A4 抗体标记。先前通过国家疾病研究交流中心同意成为器官捐献者的受试者 (n = 12) 被选为对照。专门的泌尿生殖病理学家使用虚拟显微镜软件检查了 H & E、PGP9.5 和 MAGE-A4 染色的组织是否存在未分化的精原细胞和精子。

主要观察指标）

主要结果是存在表明未分化精原细胞的 MAGE-A4 阳性或 UCHL1 阳性小管。支持性结果包括评估活检标本的以下方面：总表面积；生精小管总表面积；间质总表面积；曲细精管总数；和 MAGE-A4– 阴性或 UCHL1 阴性小管。此外，还获得了临床信息，例如年龄、核型、身高、体重、平均睾丸大小和激素组（黄体生成素、促卵泡激素和睾酮），并用于单变量和多变量线性回归分析以确定UCHL1 阳性小管数量的预测因素。

结果）

KS 组受试者的平均年龄为 32.9 ± 0.7 岁（范围 16-48 岁）。UCHL1 (PGP9.5) 和 MAGE-A4 染色显示，分别有 74.7% (n = 59) 和 40.5% (n = 32) 的 KS 受试者未分化精原细胞呈阳性，而 100% (n = 12)是正面为标志的控制主题。苏木精和伊红显微镜分析表明，只有 10.1% (n = 8) 的受试者精原细胞呈阳性。每个 KS 受试者的平均阳性小管数对于 UCHL1 为 11.8 ± 1.8，对于 MAGE-A4 为 3.7 ± 1.0。二次分析显示 7 名 (8.9%) 成年 KS 受试者活检精子呈阳性。睾丸精子提取结果为阴性的人群 (n = 72) 使用 H & E 显示精原细胞阳性率为 1.4% (n = 1)、72.2% (n = 52) 和 34.7% (n = 25)，分别为 UCHL1 和 MAGE-A4。进一步分析表明，54 名 (75.0%) 受试者为 UCHL1 或 MAGE-A4 阳性。二十名 (27.8%) 受试者对 UCHL1 和 MAGE-A4 均呈阳性。线性回归的多变量分析显示临床变量与活检标本上发现的 UCHL1 阳性小管数量之间没有显着相关性。

结论

我们报告了一组患有 KS 的成年受试者正在接受未分化精原细胞存在的分析。UCHL1 和 MAGE-A4 免疫染色似乎是识别 KS 受试者睾丸活检标本中未分化精原细胞的有效方法。尽管观察到睾丸结构恶化，但许多患者仍对未分化精原细胞呈阳性，这些精原细胞可以被采集并可能用于无精子症且睾丸精子提取结果为阴性的 KS 患者的不育治疗。