Infertility is a severe problem that affects 10%–15% of couples worldwide, among which male infertility is a factor in ~50% of the cases [1]. Azoospermia, which is defined as the absence of sperm in at least two ejaculates, can be divided into two types: obstructive azoospermia (OA) and non-OA (NOA) [1]. OA patients have normal spermatogenesis and mature sperm in testes and can father a biological child through testicular sperm retrieval and intracytoplasmic sperm injection (ICSI) [2]. By contrast, NOA patients usually have various degrees of spermatogenesis impairment and can be faced with complicated assisted reproductive technology (ART) strategies to father a biological child [2]. The precise diagnosis of spermatogenesis in NOA patients is crucial for clinical decision making regarding ART.
Analysis of the specificity and efficiency of a new RT-qPCR panel (A) Specificity of the RT-qPCR primers in the new panel as indicated by the dissociation curve. (B) Amplification efficiency of the primers determined by serial dilution. E means efficiency.
Spermatogenesis is an orchestrated series of developmental events involving mitosis, meiosis, and spermiogenesis [3,4]. Histological staining is a conventional method to evaluate spermatogenesis, but divergent histological reporting systems and limitations in biopsy sites decrease its value for the assessment of testicular biopsy specimens [5]. Thus, molecular diagnosis might be a better option.
Recently, the single-cell RNA sequencing technique identified specific markers of human germ cells and somatic cells. For example, UTF1 [6] and HMGA1 [7] are specific for spermatogonial stem cells (SSCs); SOHLH2 [6] and PRAME [7] for differentiating spermatogonia; SCML1 [7], DPH7 [7], and DSG3 [7] for L1, L2, and L3, respectively (the three stages of leptotene spermatocytes); TDRG1 [7] for zygotene; CCDC112 [7] for pachytene; AURKA [7] for diplotene; C9orf116 [7] for spermatocyte 7; TEX29 [7], NFKBIB [7], IQCF3 [7], and LELP1 [7] for S1, S2, S3, and S4 (the four stages of spermatids), respectively; and AMH [7] for Sertoli cells. Based on these specific markers, we aimed to precisely assess spermatogenesis by analyzing the expression levels of specific markers in testis biopsy samples.
In the present study, we developed a new real-time quantitative PCR (RT-qPCR) panel for the evaluation of spermatogenesis in NOA men. This panel consists of primers for marker genes of Sertoli cells (AMH), SSCs (HMGA1), differentiating spermatogonia (PRAME), the pachytene stage (CCDC112), the S2 stage (NFKBIB), the S3 stage (IQCF3), and the S4 stage (LELP1), as well as GAPDH (Supplementary TableS1). Our results showed that the primers were specific (Fig.1A), with high amplification efficiencies from 0.9–1.03 (Fig.1B).
Evaluation of spermatogenesis using the new RT-qPCR panel (A–J) Histological staining of testicular biopsy specimens from OA and SCOS patients. Spermatogenic cells were present in the OA group but not in the SCOS group, as indicated by histology. Representative results of five OA (A–E) and five SCOS (F, J) specimens are shown. Bar = 40 μm. (K) Relative expression levels of the new panel in five OA and five SCOS patients as determined by RT-qPCR. **P < 0.01, ***P < 0.001.
To test this new panel, testicular biopsy specimens were collected from patients who underwent testicular sperm extraction (TESE) for ICSI in Shanghai Ninth People’s Hospital affiliated to JiaoTong University School of Medicine with their consent. Testicular biopsy specimens from 10 infertile male patients were assessed by histological staining. Five OA and five Sertoli cell-only syndrome (SCOS) cases were identified. In the OA group, spermatogenic cells and somatic cells were observed in the biopsy specimens (Fig.2A–E). In the SCOS group, no tubule-containing spermatogenic cells were found (Fig.2F,J). Clinical information (Supplementary Table S2), including age and the levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), and testosterone (T), showed no significant differences between the OA and SCOS groups. FSH and PRL levels were higher in the SCOS group than those in the OA group (Supplementary TableS2). Expectedly, the expressions of all these genes were detected in the OA group (Fig.2K). However, no spermatogenesis markers (Supplementary TableS1) were detected in the SCOS group (Fig.2K). In addition, the expression level of the Sertoli cell-specific marker (AMH) was significantly lower in the SCOS group than that in the OA group (Fig.2K).
Assessment of spermatogenesis in a PA case using the new RT-qPCR panel (A) Histological staining of a PA case. Bar = 40 μm. The red arrow indicates pachytene spermatocytes. (B) Relative expression level of the new panel in the PA case compared with the OA group. ***P < 0.001.
Patients with NOA often undergo multiple rounds of TESE or micro-TESE with the hope of harvesting sperm to father a biological child through ICSI. However, spermatozoa are successfully obtained in only 40%–60% of such patients [8]. To avoid unnecessary operations, numerous biomarkers have been reported to predict the success rate of sperm retrieval, but none of these markers is completely credible [8]. AMH is a protein secreted by Sertoli cells that has been considered a direct marker of the function of Sertoli cells, which supports the highly ordered spermatogenesis process [9]. Our results showed that AMH expression was significantly lower in the SCOS group than in the OA group (Fig.2K), indicating the impairment of Sertoli cell function. Moreover, the spermatogenesis markers were completely undetectable in complete SCOS patients (Fig.2K), which was consistent with the histological staining results, suggesting that a second procedure may not be feasible.
To further validate the diagnostic effectiveness of this new panel, a pachytene stage arrest (PA) case was included. The age and the levels of FSH, LH, PRL, and T in the PA case were 34 years, 9.38 mIU/ml, 11.95 mIU/ml, 3.39 ng/ml, and 13.49 ng/ml, respectively. Histological staining showed the clear arrest of spermatocytes at the pachytene stage (Fig.3A). The expression levels of the panel of genes (Supplementary TableS1) in whole PA specimens were determined. Compared with OA specimens (Fig.2A–E and Supplementary TableS2), the PA specimens showed comparable expression levels of the Sertoli cell marker AMH and the SSC marker HMGA1 but significantly lower expression levels of the differentiating spermatogonia marker PRAME and the pachytene stage marker CCDC112 (Fig.3B). The expression levels of the S2 stage marker NFKBIB, S3 stage marker IQCF3, and S4 stage marker LELP1 in the PA case were too low to be detected (Fig.3B). In this PA case diagnosed by histological staining, the presence of SSCs was confirmed by RT-qPCR analysis (Fig.3). Thus, if sperm retrieval is unsuccessful, the PA patient has a chance to preserve SSC-enriched cells and undergo SSC therapy in the future [3].
In conclusion, this study provided a robust new RT-qPCR panel for the assessment of spermatogenesis in testis biopsy specimens from NOA men. The high sensitivity of the RT-qPCR assay and simple interpretation of the results provide a fast and precise diagnostic method for clinical decision making during ART. This method is worthy of further validation by tracking the outcomes of micro-TESE in larger cohorts.
Acknowledgment
We appreciate Prof. Yiping Li (Shanghai Institute of Biochemistry and Cell Biology, Shanghai, China) for the generous help during specimen preparation.
Funding
This work was supported by the grants from the National Natural Science Foundation of China (No. 81571486) and the Open Project of Shanghai Key Laboratory of Molecular Andrology (No. SLMA-09).
References
Author notes
These authors contributed equally to this work.
