STUDY QUESTION Does RXFP2 disruption impair male fertility? SUMMARY ANSWER We identified biallelic variants in RXFP2 in patients with male infertility due to spermatogenic arrest at the spermatid stage, supporting a role of RXFP2 in human spermatogenesis, specifically in germ cell maturation. WHAT IS KNOWN ALREADY Since RXFP2, the receptor for INSL3, plays a crucial role in testicular descent during prenatal development, biallelic variants lead to bilateral cryptorchidism, as described in four families to date. While animal models have also suggested a function in spermatogenesis, the postnatal functions of RXFP2 and its ligand INSL3, produced in large amounts by the testes from puberty throughout adulthood, are largely unknown. STUDY DESIGN, SIZE, DURATION A family with two male members affected by impaired fertility due to spermatogenic maturation arrest and a history of bilateral cryptorchidism underwent clinical, endocrinological, histological, genomic, in vitro cellular, and in silico investigations. PARTICIPANTS/MATERIALS, SETTING, METHODS The endocrinological and histological findings were correlated with publicly available single-cell RNA sequencing (scRNA-seq) data. The genomic defects have been characterized using long-read sequencing and validated with in silico modeling and an in vitro cyclic AMP reporter gene assay. MAIN RESULTS AND THE ROLE OF CHANCE An intragenic deletion of exon 1-5 of RXFP2 (NM_130806.5) was detected in trans with a hemizygous missense variant c.229G>A, p.(Glu77Lys). The p.(Glu77Lys) variant caused no clear change in cell surface expression or ability to bind INSL3, but displayed absence of a cAMP signal in response to INSL3, indicating a loss-of-function. Testicular biopsy in the proband showed a maturation arrest at the spermatid stage, corresponding to the highest level of RXFP2 expression in scRNA-seq data, thereby providing a potential explanation for the impaired fertility. LIMITATIONS, REASONS FOR CAUTION Although this is so far the only study of human cases that supports the role of RXFP2 in spermatogenic maturation, this is corroborated by several animal studies that have already demonstrated a postnatal function of INSL3 and RXFP2 in spermatogenesis. WIDER IMPLICATIONS OF THE FINDINGS This study corroborates RXFP2 as gene implicated in autosomal recessive congenital bilateral cryptorchidism due to biallelic variants, rather than autosomal-dominant cryptorchidism due to monoallelic RXFP2 variants. Our findings also support that RXFP2 is essential in human spermatogenesis, specifically in germ cell maturation, and that biallelic disruption can cause male infertility through spermatogenic arrest at the spermatid stage. STUDY FUNDING/COMPETING INTEREST(S) Funding was provided by the Bellux Society for Pediatric Endocrinology and Diabetology (BELSPEED) and supported by a Research Foundation Flanders (FWO) senior clinical investigator grant (E.D.B., 1802220N) and a Ghent University Hospital Special Research Fund grant (M.C., FIKO-IV institutional fund). The authors declare no conflict of interest. TRIAL REGISTRATION NUMBER N/A.

中文翻译：

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双等位基因 RXFP2 变异导致先天性双侧隐睾症和男性不育症，支持 RXFP2 在精子发生中的作用。


研究问题 RXFP2 破坏会损害男性生育能力吗？总结答案 我们在男性不育症患者中发现了 RXFP2 中的双等位基因变异，这是由于精子细胞期生精停滞而引起的，这支持 RXFP2 在人类精子发生中的作用，特别是在生殖细胞成熟中的作用。已知的内容由于 INSL3 受体 RXFP2 在产前发育过程中的睾丸下降中起着至关重要的作用，因此双等位基因变异会导致双侧隐睾症，如迄今为止的四个家庭所述。虽然动物模型也表明了精子发生的功能，但 RXFP2 及其配体 INSL3 的出生后功能在很大程度上是未知的，这些配体从青春期到成年期由睾丸大量产生。研究设计、规模、持续时间 一个有两名男性成员的家庭因生精成熟停滞和双侧隐睾病史而受生育能力受损的影响，接受了临床、内分泌、组织学、基因组学、体外细胞学和计算机调查。参与者/材料、环境、方法 内分泌学和组织学发现与公开可用的单细胞 RNA 测序 （scRNA-seq） 数据相关。已使用长读长测序对基因组缺陷进行了表征，并通过计算机建模和体外环状 AMP 报告基因测定进行了验证。主要结果和机会的作用 在具有半合子错义变异 c.229G>A， p.（Glu77Lys） 的反式中检测到 RXFP2 外显子 1-5 的基因内缺失 （NM_130806.5）。p.（Glu77Lys） 变体未引起细胞表面表达或结合 INSL3 的能力发生明显变化，但显示响应 INSL3 的 cAMP 信号缺失，表明功能丧失。 先证者的睾丸活检显示精子细胞阶段成熟停滞，对应于 scRNA-seq 数据中 RXFP2 表达的最高水平，从而为生育能力受损提供了潜在的解释。局限性，谨慎的原因尽管这是迄今为止唯一支持 RXFP2 在精子发生成熟中的作用的人类病例研究，但几项动物研究证实了这一点，这些研究已经证明了 INSL3 和 RXFP2 在精子发生中的出生后功能。研究结果的更广泛意义 本研究证实 RXFP2 是与双等位基因变异引起的常染色体隐性先天性双侧隐睾症有关的基因，而不是由于单等位基因 RXFP2 变异引起的常染色体显性遗传隐睾症。我们的研究结果还支持 RXFP2 在人类精子发生中是必不可少的，特别是在生殖细胞成熟中，并且双等位基因破坏可以通过在精子细胞阶段的生精停滞导致男性不育。研究资金/利益争夺资金由 Bellux 儿科内分泌学和糖尿病学协会 （BELSPEED） 提供，并得到佛兰德斯研究基金会 （FWO） 高级临床研究者资助 （E.D.B.， 1802220N） 和根特大学医院特殊研究基金资助 （M.C.， FIKO-IV 机构基金）。作者声明没有利益冲突。试验注册号 N/A。