Single-cell analysis of pathology in Klinefelter syndrome and idiopathic male infertility

Klinefelter syndrome (KS), also known as 47,XXY, is characterized by a distinct set of physiological abnormalities, commonly including infertility. The molecular basis for Klinefelter-related infertility is still unclear, largely due to the cellular complexity of the testis and the intricate endocrine and paracrine signaling that regulates spermatogenesis. Here, we demonstrate an analysis framework for dissecting human testis pathology that uses comparative analysis of single-cell RNA-sequencing data from the biopsies of 13 human donors. By comparing donors from a range of ages and forms of infertility, we generate gene expression signatures that characterize normal testicular function and distinguish clinically distinct forms of male infertility. Unexpectedly, we identified a subpopulation of Sertoli cells within multiple cases of KS that lack transcription from the XIST locus, with the consequence of increased X-linked gene expression compared to all other KS cell populations. By systematic assessment of known signaling pathways, we identify 72 pathways potentially active in testis, dozens of which appear upregulated in KS. Altogether our data support a model of pathogenic changes in interstitial cells cascading from loss of X-inactivation in pubertal Sertoli cells, and nominate testicular GNRH1 as a dosage-sensitive factor secreted by Sertoli cells that may contribute to the process. Our findings demonstrate the value of comparative patient analysis in mapping genetic mechanisms of disease, and identify an epigenetic phenomenon in KS Sertoli cells that may prove important for understanding causes of infertility and sex chromosome evolution. Overall design: To dissect the pathological biology in Klinefelter syndrome (KS) and idiopathic male infertility, we combined and analyzed 26300 single cell transcriptomes derived from the testis of 12 carefully selected donors, chosen to allow us to isolate expression changes that are attributes of the specific defects of KS, versus changes that are general hallmarks of defects in spermatogenesis, and endocrine dysfunction. Our primary analysis framework is called Sparse Decomposition of Arrays (SDA), a matrix factorization method that we find very useful for summarizing large, multi-donor scRNA-seq data into manageable sets of co-regulated genes, known as SDA components. We have recently shown the value of SDA in interpreting testis pathology in mice13 and herein we expand on its application by showing utility in humans.

克兰费尔特综合征（Klinefelter syndrome, KS），又称47,XXY综合征，以一系列特征性生理异常为主要临床表现，其中不育症最为常见。目前克氏综合征相关不育的分子机制仍未阐明，这在很大程度上源于睾丸组织的细胞复杂性，以及调控精子发生的复杂内分泌与旁分泌信号网络。
本研究构建了一套可用于解析人类睾丸病理的分析框架：该框架通过对13名人类受试者睾丸活检样本的单细胞RNA测序（single-cell RNA-sequencing, scRNA-seq）数据开展比较分析得以实现。通过对比不同年龄、不同不育亚型的受试者，我们构建了可表征正常睾丸功能的基因表达特征谱，并可区分临床分型各异的男性不育症。
出乎意料的是，我们在多例克氏综合征患者样本中鉴定出一群XIST基因座转录缺失的塞尔托利细胞（Sertoli cells），与其余所有克氏综合征细胞群相比，这群细胞的X连锁基因表达水平显著升高。通过对已知信号通路的系统性评估，我们共鉴定出72条可能在睾丸中发挥活性的信号通路，其中数十条在克氏综合征样本中呈现上调表达。
综合本研究数据，我们提出如下致病模型：青春期塞尔托利细胞的X染色体失活缺失，会引发间质细胞的级联病理改变；同时我们提出，睾丸来源的促性腺激素释放激素1（GNRH1）作为一种由塞尔托利细胞分泌的剂量敏感因子，可能参与了这一病理进程。
本研究结果证实了比较患者队列分析在解析疾病遗传机制中的重要价值，并在克氏综合征患者的塞尔托利细胞中发现了一种表观遗传现象，该现象或可为理解不育症的病因及性染色体进化提供关键线索。
【整体实验设计】
为解析克氏综合征与特发性男性不育（idiopathic male infertility）的病理生物学特征，我们整合并分析了12名经过严格筛选的受试者的睾丸组织单细胞转录组数据，共计26300个细胞。受试对象的筛选标准旨在帮助我们分离出仅与克氏综合征特有缺陷相关的表达变化，以及与精子发生缺陷、内分泌功能紊乱通用特征相关的表达变化。我们采用的核心分析框架名为矩阵稀疏分解法（Sparse Decomposition of Arrays, SDA），这是一种矩阵分解方法，可将大规模多受试者的单细胞RNA测序数据汇总为一系列共调控基因集合（即SDA组分），便于后续分析。我们此前已证实SDA在解析小鼠睾丸病理中的应用价值13，本研究则进一步拓展了其应用范围，验证了该方法在人类样本中的实用性。