第一部分 血清抗苗勒管激素与睾酮比值对先天性低促性腺激素性性腺功能减退症患者的生精预测

背景: 成年先天性低促性腺激素性性腺功能减退症（congenital hypogonadotropichypogonadism，CHH）患者普遍面临生精功能障碍导致的不孕症，这一病理状态不仅造成家庭关系紧张、心理健康受损及社会经济负担的"三重压力"，更因促性腺激素治疗方案的效果的冗长和不确定性而影响生精结局。临床上迫切需要准确且客观的指标来预测生精结局。抗苗勒管激素（AMH）作为睾丸支持细胞功能的标志物，在男性不育疾病中发挥着重要作用。睾酮（T）由间质细胞分泌，参与精子的生成过程，二者在整个生命周期相互作用、相互影响。AMH 和 T 在男性不育疾病中的研究尚不明确，目前尚无研究表明，血清 AMH 或 AMH/T 能否作为早期预测 CHH生精结局的预测指标。
目的:本研究旨在通过分析CHH患者治疗前后激素水平的变化，探究AMH和AMH/T能否作为预测 CHH 患者促性腺激素治疗成功的标志物。
对象和方法:本研究为回顾性、单中心队列研究，纳入 2020 年 2 月至 2024 年 12 月北京协和医院就诊的且有生育需求的男性 CHH 患者。患者均规律接受促性腺激素治疗，根据疗效（精液中能否检测到精子）分为生精组和未生精组。收集促性腺激素治疗前后的激素水平变化，通过建立单变量和多变量回归分析模型，比较治疗前和治疗后血清 AMH 和 AMH/T 预测效能。同时对年龄分层进行亚组分析。
结果: 本研究最终纳入的 32 例 CHH 患者中，成功生精组 12 例，未生精组 20 例。中位治疗时间 8.5 个月。生精组的 AMH 低于未生精组，分别为 5.03（9.74,15.51）ng/mL和 15.3（8.35,23.05）ng/mL，血清 AMH 不能作为 CHH 患者生精结局的独立预测因子（P＞0.05）。血清 AMH/T 在生精组和未生精组中有显著差异（P=0.003），调整年龄、睾丸体积和血清 FSH 等协变量后，AMH/T（OR 0.95，95% CI 0.67-1.00，P =0.039）和△AMH/T（OR 0.95，95% CI 0.91-1.00，P = 0.040）是 CHH 患者促性腺激素治疗生精成功独立预测因子，其中 AMH/T 的预测效能优于△AMH/T（AUC 0.84vs 0.82）。在 18-25 岁 CHH 患者中 AMH/T（OR 0.94，95% CI 0.92-1.00）和△AMH/T（OR 0.95，95% CI 0.93-1.00）均可预测生精结局（P＜0.05）。
结论: 1. 血清 AMH/T 是男性 CHH 患者促性腺激素治疗生精结局的独立预测因子，血清AMH/T＜10.71 时，预测成功生精敏感性 94%，特异性为 65%。2. 相比于△AMH/T，AMH/T 在对生精结局的预测性更高，在临床治疗的过程中，△AMH/T 可以作为辅助指标来预测生精 3. 血清 AMH/T 和△AMH/T 在 18-25 岁 CHH 患者中预测效果更好。

第二部分 基于精浆代谢组学预测先天性低促性腺激素性性腺功能减退症患者生精疗效
背景: 先天性低促性腺激素性性腺功能减退症（congenital hypogonadotropic hypogo- nadism，CHH）是以发育延迟和嗅觉缺失为特征的疾病，主要影响男性的生育能力。该疾病通常由于下丘脑分泌促性腺激素缺乏，导致睾酮水平减低和生精功能障碍。促性腺激素（HCG/HMG）治疗作为一种有效的干预手段，被应用于 CHH 患者的生育治疗中。然而，促性腺激素治疗的反应存在个体差异，如何精确、客观的预测并优化治疗效果仍然是临床研究的难点。近年来随着高通量技术的发展，代谢组学技术逐渐兴起，在男性不育方面成为研究热点，但目前尚无研究表明是否能通过精浆代谢组学中的代谢物预测 CHH 患者促性腺激素治疗预后（生精结局）。
目的: 本研究旨在通过谢组学对 CHH 患者促性腺激素治疗后的精浆进行检测，探寻CHH 患者促性腺激素治疗成功的潜在预测生物标志物。
方法: 招募于 2022 年 11 月至 2023 年 8 月在北京协和医院内分泌科就诊且有生育需求的男性 CHH 患者。根据严格的纳排标准，最终纳入 39 名患者。所有患者均接受了为期 24 个月的促性腺激素治疗，并根据治疗反应（精子生成）分为生精组和未生精组。在随访的第一阶段收集精浆样本，并使用超高效液相色谱-串联质谱法（UHPLC-MS/MS）进行代谢组学分析。通过正交偏最小二乘判别分析（OPLS-DA）和单变量统计分析评估两组之间代谢物的差异。最后，探讨了差异表达代谢物与精液参数之间的关联。
结果: 本研究纳入 39 名患者，其中生精组 26 名和未生精组 13 名。通过代谢组学分析揭示生精组和未生精组精浆代谢产物差异，发现 42 种差异表达的代谢物，主要涉及脂质、氨基酸、核苷和有机氧化合物。其中，硫苷脂（SHexCer (d34:1)）和溶血磷脂酰胆碱（LPC (22:6)）显示出对治疗反应的高敏感性和特异。SHexCer (d34:1)与精子密度（r=0.69,P＜0.0001）、精子总数（r=0.62,P＜0.0001）和精子总活力（r=0.55,P＜0.0001）相关性最密切；LPC (22:6)与精子密度（r=0.64,P＜0.0001）、精子总数（r=0.56,P＜0.0001）正相关关系。受试者工作特征（ROC）分析进一步证实，这两种代谢物在区分生精组与未生精组方面具有良好的诊断能力，其曲线下面积（AUC）分别为 0.93 和 0.89。
结论: SHexCer (d34:1)和 LPC (22:6)的显著升高可能反映了生精过程中脂质代谢的活跃程度，这一发现为预测 CHH 患者对促性腺激素治疗的反应提供了潜在的生物标志物。

Part 1: The Predictive Value of the Serum Anti-Müllerian Hormone to Testosterone Ratio for Spermatogenesis in Patients with Congenital Hypogonadotropic Hypogonadism

Context: Spermatogenic dysfunction, as a direct consequence of gonadotropin deficiency, represents the major etiology of infertility in CHH-affected adult males. This pathological state imposes a "triple burden" of strained familial relationships, psychological distress, and socioeconomic costs, exacerbated by the prolonged and uncertain therapeutic efficacy of gonadotropin regimens in achieving spermatogenic success. There is an urgent clinical need for reliable and objective biomarkers to predict spermatogenic outcomes. Anti-Müllerian hormone (AMH), a biomarker of Sertoli cell functionality, plays a pivotal role in male infertility disorders. Testosterone (T), secreted by Leydig cells, is integral to spermatogenesis. Although AMH and T exhibit lifelong bidirectional regulatory interactions,their roles in predicting CHH-related spermatogenic outcomes remain undefined. To date, no studies have established serum AMH or the AMH/T ratio as early predictive indicators for spermatogenic success in CHH. 
Objective: This study aimed to investigate whether AMH and AMH/T could serve as biomarkers for predicting the success of gonadotropin therapy in CHH patients by analyzing changes in hormone levels before and after treatment. 
Methods: This retrospective single-center cohort study systematically evaluated the predictive value of AMH/T ratio in male congenital hypogonadotropic hypogonadism (CHH) patients undergoing fertility treatment. The study population comprised CHH patients with documented fertility demands who received standardized gonadotropin therapy at Peking Union Medical College Hospital between February 2020 and December 2024. Following a comprehensive treatment protocol, participants were categorized into spermatogenesis (sperm-positive) and non-spermatogenesis (sperm-negative) groups based on standardized post-treatment semen analysis results. Serum AMH and testosterone (T) levels were measured pre- and post-treatment. Univariate and multivariate regression models were constructed to evaluate the predictive performance of baseline and post-treatment AMH/T ratios. Age-stratified subgroup analyses were further conducted
Results: Among the 32 congenital hypogonadotropic hypogonadism (CHH) patients included in this study, 12 achieved successful spermatogenesis (spermatogenesis group), while 20 did not (non-spermatogenesis group), with a median treatment duration of 8.5 months. Serum anti-Müllerian hormone (AMH) levels were significantly lower in the spermatogenesis group compared to the non-spermatogenesis group [5.03 (9.74–15.51) ng/mL vs. 15.3 (8.35–23.05) ng/mL], though AMH alone was not an independent predictor of spermatogenic outcomes (P>0.05). The AMH/testosterone (AMH/T) ratio exhibited significant intergroup disparity (P=0.003). After adjusting for age, testicular volume, and serum FSH levels, both baseline AMH/T (OR 0.95, 95% CI 0.67–1.00, P=0.039) and ΔAMH/T (OR 0.95, 95% CI 0.91–1.00, P=0.040) emerged as independent predictors of successful spermatogenesis following gonadotropin therapy, with baseline AMH/T demonstrating superior predictive performance (AUC 0.84 vs. 0.82 for ΔAMH/T). In CHH patients aged 18–25 years, both AMH/T (OR 0.94, 95% CI 0.92–1.00) and ΔAMH/T (OR 0.95, 95% CI 0.93–1.00) were significant predictors of spermatogenesis (P<0.05). 
Conclusions: 1. Serum AMH/T as an Independent Predictor: The serum anti-Müllerian hormone-to-testosterone ratio (AMH/T) serves as an independent predictor of spermatogenic outcomes in male CHH patients undergoing gonadotropin therapy. A threshold of AMH/T <10.71 predicts successful  permatogenesis with 94% sensitivity and 65% specificity. 
2. Superior Predictive Performance of AMH/T: Compared to ΔAMH/T (post-treatment change in AMH/T), baseline AMH/T demonstrates higher predictive accuracy (AUC 0.84 vs. 0.82). While ΔAMH/T may function as an adjunct biomarker, baseline AMH/T remains the primary indicator for clinical decision-making. 
3. Age-Stratified Efficacy: Both AMH/T and ΔAMH/T exhibit enhanced predictive efficacy in CHH patients aged 18–25 years, underscoring the importance of early intervention in younger cohorts

Part 2: Seminal Plasma Metabolomics Predicts Gonadotropin Therapy Outcomes in Congenital Hypogonadotropic Hypogonadism (CHH) Patients

Context: Congenital hypogonadotropic hypogonadism (CHH), characterized by delayed development and anosmia, primarily impairs male fertility due to deficient hypothalamic secretion of gonadotropin-releasing hormone (GnRH), resulting in hypogonadism and spermatogenic failure. Gonadotropin therapy (hCG/hMG) has been increasingly utilized to restore fertility in CHH patients. However, marked interindividual variability in therapeutic response underscores the need for objective biomarkers to predict spermatogenic outcomes. While metabolomics has emerged as a powerful tool in male infertility research, no studies have explored seminal plasma metabolomic profiles for predicting gonadotropin therapy efficacy in CHH. 
Objective:  This study aimed to identify seminal plasma metabolites predictive of successful spermatogenesis following gonadotropin therapy in CHH patients using metabolomics. 
Methods: From November 2022 to August 2023, male CHH patients seeking fertility treatment were recruited at the Department of Endocrinology, Peking Union Medical College Hospital. Following strict inclusion/exclusion criteria, 39 patients were included. All subjects received 24-month gonadotropin therapy and were stratified into spermatogenesis-achieving (n=26) and non-achieving (n=13) groups based on post-treatment semen analysis. Seminal plasma samples collected during the first follow-up phase were analyzed via ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Orthogonal partial least squares-discriminant analysis (OPLS-DA) and univariate statistical methods identified differential metabolites, which were subsequently correlated with semen parameters. 
Results: Metabolomic profiling revealed 42 differentially expressed metabolites between groups, predominantly enriched in lipids, amino acids, nucleosides, and organic oxygen compounds. Sulfatide (SHexCer (d34:1)) and lysophosphatidylcholine (LPC (22:6)) demonstrated high sensitivity and specificity for predicting treatment response. SHexCer (d34:1) exhibited strong positive correlations with sperm density (r=0.69, P<0.0001), total sperm count (r=0.62, P<0.0001), and motility (r=0.55, P<0.0001), while LPC (22:6) correlated significantly with sperm density (r=0.64, P<0.0001) and total count (r=0.56, P<0.0001). ROC analysis confirmed their robust discriminative capacity, with area under the curve (AUC) values of 0.93 and 0.89, respectively. 
Conclusion: Elevated SHexCer (d34:1) and LPC (22:6) levels may reflect active lipid metabolism during spermatogenesis, identifying these metabolites as potential biomarkers for predicting gonadotropin therapy responsiveness in CHH patients.