从先前接受过化疗的儿童癌症患者身上获得的人类青春期前和青春期周围的睾丸细胞能否形成睾丸类器官 (TO)？从儿童癌症患者身上收集的睾丸组织中获得的类器官的形成与 SRY-Box 转录因子 9 (SOX9) 呈正相关支持细胞中的表达，这又与之前接受的烷基化化疗呈负相关。接受高性腺毒性治疗的青春期前和青春期男孩只能通过睾丸组织冷冻保存来保护其生育潜力。如今，还没有成熟的临床工具可以使用这些睾丸样本恢复生育能力。类器官有望为创建此类平台提供基本的早期见解。然而，生成与先天睾丸非常相似的 TO，以便能够彻底监测生殖细胞分化和体细胞功能的必要步骤，仍然是一个挑战。我们使用基于基质胶的三层梯度培养系统来生成人类 TO并揭示化疗暴露是否影响 TO 形成能力以及青春期前和围青春期睾丸体细胞的功能。对 11 名男孩（年龄 7.7±±4.1（平均值±标准差）岁）的睾丸细胞进行了评估，以了解与之前化疗暴露相关的 TO 形成以及活检当天收集的石蜡包埋睾丸组织样本的组织学切片中 SOX9 的表达，并与睾丸组织样本取自 28 名连续患者（年龄 6.9±±3.8（平均值±标准差）岁）。所有 39 名患者均参与了生育力保存项目 NORDFERTIL；使用内部生物库收集的另外 10 个样本（来自 5.5±3.5（平均值±SD）岁的男孩，没有潜在的病理学）作为对照。我们从 0.8-13.4 岁的男孩那里获得了 49 个睾丸组织样本。将新鲜样品（n = 11）解离成单细胞悬浮液，并应用于三层梯度培养系统中以形成类器官。作为生育力保存项目 NORDFERTIL 的一部分获得的另外 28 个样本的组织学切片以及来自病理生物库样本库的 10 个样本用于评估先前接触烷化剂对睾丸样本的影响。睾丸类器官的形成是根据形态特征来定义的，例如显示索状形成的区室结构，并通过免疫组织化学染色评估生殖细胞和体细胞的睾丸细胞特异性标记物的蛋白质表达。通过特定的酶联免疫吸附测定来分析睾酮和抗苗勒氏管激素 (AMH) 的产生。我们的结果显示，11 个青春期前睾丸样本中有 4 个形成了 TO，其显示出被间质样区域包围的分隔的索状结构和在 7 天的培养期内，睾酮和 AMH 水平不断增加。我们观察到 SOX9 表达与 TO 形成呈正相关。此外，活检前接触烷化剂与SOX9表达呈负相关（P = 0.006）。N/A。由于可用材料数量有限，39个青春期前和围青春期睾丸组织样本中只有11个可以被检测到。用于类器官形成实验。从卡罗林斯卡大学医院病理科内部生物库样本采集中获得的睾丸组织样本，如果没有睾丸病理报告，则被认为是正常的，并纳入研究。然而，有关该生物库中包含的患者既往医疗和/或睾丸体积的详细信息尚不可用。我们的观察表明，SOX9 表达可能作为 TO 形成的假定指标，表明支持细胞在促进类器官中的关键作用青春期前和围青春期睾丸组织的形成、曲细精管完整性和睾丸功能。这项研究得到了瑞典儿童癌症基金会 (PR2019-0123; PR2022-0115; TJ2020-0023) (J.-B.S.) 的资助。 、芬兰癌症协会 (K.J.)、芬兰儿科研究基金会 (K.J.)、瑞典研究理事会 (2018-03094; 2021-02107) (J.-B.S.) 以及 Birgitta 和 Carl-Axel Rydbeck 儿科研究研究补助金 (2020 年) -00348；2020-00335；2021-00317）（J.-B.S.和K.J.）。 Y.C.和Y.Y。获得国家留学基金委奖学金。 J.P.A-L。得到了瑞典研究委员会的医学与健康启动补助金 (2022-01467) 的支持。 R.T.M.得到 UKRI 未来领袖奖学金 (MR/S017151/1) 的支持。 MRC 生殖健康中心得到了 MRC 中心拨款 (MR/N022556/1) 的支持。作者声明不存在竞争利益。© 作者 2024。由牛津大学出版社代表欧洲人类生殖和胚胎学学会出版。

Can human pre- and peri-pubertal testicular cells obtained from childhood cancer patients, previously treated with chemotherapy, form testicular organoids (TOs)?Organoid formation from testicular tissue collected from childhood cancer patients positively correlates with SRY-Box transcription factor 9 (SOX9) expression in Sertoli cells, which in turn negatively correlates with previous exposure to alkylating chemotherapy.Pre- and peri-pubertal boys exposed to highly gonadotoxic therapies can only safeguard their fertility potential through testicular tissue cryopreservation. Today, there is no established clinical tool to restore fertility using these testicular samples. Organoids hold promise in providing fundamental early insights in creating such platforms. However, the generation of TOs that closely resemble the innate testis, to enable a thorough monitoring of the necessary steps for germ cell differentiation and somatic functionalities, remains a challenge.We used a Matrigel-based three-layer gradient culture system to generate human TOs and to reveal whether chemotherapy exposure affects TO formation capacity and the functionality of pre- and peri-pubertal testicular somatic cells. Testicular cells of 11 boys (aged 7.7 ± 4.1 (mean ± SD) years) were assessed for TO formation in relation to previous chemotherapy exposure and SOX9 expression in histological sections of paraffin-embedded testicular tissue samples collected on the day of biopsy and compared with testicular tissue samples obtained from 28 consecutive patients (aged 6.9 ± 3.8 (mean ± SD) years). All 39 patients were part of the fertility preservation project NORDFERTIL; an additional 10 samples (from boys aged 5.5 ± 3.5 (mean ± SD) years, without an underlying pathology) in an internal biobank collection were used as controls.We obtained 49 testicular tissue samples from boys aged 0.8-13.4 years. Fresh samples (n = 11) were dissociated into single-cell suspensions and applied to a three-layer gradient culture system for organoid formation. Histological sections of another 28 samples obtained as part of the fertility preservation project NORDFERTIL, and 10 samples from a sample collection of a pathology biobank were used to evaluate the effects of prior exposure to alkylating agents on testicular samples. Testicular organoid formation was defined based on morphological features, such as compartmentalized structures showing cord formation, and protein expression of testicular cell-specific markers for germ and somatic cells was evaluated via immunohistochemical staining. Hormone secretion was analysed by specific enzyme-linked immunosorbent assays for testosterone and anti-Müllerian hormone (AMH) production.Our results revealed that 4 out of 11 prepubertal testicular samples formed TOs that showed compartmentalized cord-like structures surrounded by interstitial-like areas and increasing levels of both testosterone as well as AMH over a 7-day culture period. We observed that SOX9 expression was correlated positively with TO formation. Moreover, exposure to alkylating agents before biopsy was inversely correlated with SOX9 expression (P = 0.006).N/A.Due to the limited amount of material available, only 11 out of the 39 pre- and peri-pubertal testicular tissue samples could be used for the organoid formation experiments. The testicular tissue samples obtained from a sample collection of the internal biobank of Department of Pathology, Karolinska University Hospital were considered normal and included in the study if no testicular pathology was reported. However, detailed information regarding previous medical treatments and/or testicular volumes of the patients included in this biobank was not available.Our observations suggest that SOX9 expression may serve as a putative indicator of TO formation, indicating a critical role of Sertoli cells in promoting organoid formation, seminiferous tubule integrity, and testicular function in pre- and peri-pubertal testicular tissue.This study was supported by grants from the Swedish Childhood Cancer Foundation (PR2019-0123; PR2022-0115; TJ2020-0023) (J.-B.S.), Finnish Cancer Society (K.J.), Finnish Foundation for Paediatric Research (K.J.), Swedish Research Council (2018-03094; 2021-02107) (J.-B.S.), and Birgitta and Carl-Axel Rydbeck's Research Grant for Paediatric Research (2020-00348; 2020-00335; 2021-00073; 2022-00317) (J.-B.S. and K.J.). Y.C. and Y.Y. received a scholarship from the Chinese Scholarship Council. J.P.A-L. was supported by a Starting Grant in Medicine and Health (2022-01467) from the Swedish Research Council. R.T.M. was supported by a UKRI Future Leaders Fellowship (MR/S017151/1). The MRC Centre for Reproductive Health was supported by an MRC Centre Grant (MR/N022556/1). The authors declare no competing interests.© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.