Abstract
Objective
Modeling methods for busulfan-induced oligoasthenozoospermia are controversial. We aimed to systematically review the modeling method of busulfan-induced oligospermia and asthenozoospermia, and analyze changes in various evaluation indicators at different busulfan doses over time.
Methods
We searched the Cochrane Library, PubMed databases, Web of Science, the Chinese National Knowledge Infrastructure, and the Chinese Biomedical Literature Service System until April 9, 2022. Animal experiments of busulfan-induced spermatogenesis dysfunction were included and screened. The model mortality and parameters of the evaluation indicators were subjected to meta-analysis.
Results
Twenty-nine animal studies were included (control/model: 669/1829). The mortality of mice increased with busulfan dose. Significant spermatogenesis impairment occurred within 5 weeks, regardless of busulfan dose (10–40 mg/kg). Testicular weight (weighted mean difference [WMD]: − 0.04, 95% CI: − 0.05, − 0.03), testicular index (WMD: − 2.10, 95% CI: − 2.43, − 1.76), and Johnsen score (WMD: − 4.67, 95% CI: − 5.99, − 3.35) were significantly decreased. The pooled sperm counts of the model group were reduced by 32.8 × 106/ml (WMD: − 32.8, 95% CI: − 44.34, − 21.28), and sperm motility decreased by 37% (WMD: − 0.37, 95% CI: − 0.47, − 0.27). Sperm counts decreased slightly (WMD: − 3.03, 95% CI: − 3.42, − 2.64) in an intratesticular injection of low-dose busulfan (4 − 6 mg/kg), and the model almost returned to normal after one seminiferous cycle.
Conclusion
The model using low-dose busulfan (10 − 20 mg/kg) returned to normal after 10 − 15 weeks. However, in some spermatogenesis cycles, testicular weight reduction and testicular spermatogenic function damage were not proportional to busulfan dose. Sperm counts and motility results in different studies had significant heterogeneity. Standard protocols for sperm assessment in animal models were needed to reduce heterogeneity between studies.
Keywords: Busulfan, Oligospermia, Asthenospermia, Oligoasthenospermia, Spermatogenic dysfunction, Mouse, System evaluation
Introduction
Male infertility is a worldwide reproductive health issue affecting about 15% of reproductive-age couples worldwide [1]. Oligospermia and asthenozoospermia, characterized by reduced sperm and motile sperm concentrations, are common causes of male infertility. However, the exact pathogenesis of oligoasthenospermia has not been completely elucidated, and therapies are limited [2].
Animal models are powerful tools for studying male infertility mechanisms and developing preclinical therapeutics [3, 4]. Currently, modeling methods for spermatogenesis dysfunction are primarily divided into chemical and physical processes. The chemical method is the most commonly used because it is easy to operate and practical [5–8]. However, many kinds of chemical drugs are used for modeling, and each drug has shortcomings. For instance, the modeling cycle of Tripterygium wilfordii is long. It generally takes 4 weeks of continuous administration, and the model can return to normal in about 20–35 days after modeling [9]. Other commonly used chemicals, such as cyclophosphamide, adenine, and ornidazole, have long molding cycles and model recovery and high mortality [5].
Busulfan is a cytotoxic bifunctional alkylating agent that has significant testicular toxicity. It can lead to oligospermia, azoospermia, and testicular atrophy in men [10]. Therefore, busulfan is considered the most desirable agent for modeling oligospermia and azoospermia [3, 11]. The main mechanism by which busulfan causes male infertility is DNA alkylation, which inhibits cell division and kills spermatogenic cells at all levels, especially spermatogonial stem cells. Busulfan damages spermatogenic cells through oxidative stress and disrupts the Sertoli cells’ intercellular junctions [10, 12]. The typical dose of busulfan intraperitoneal injection in mice for temporary azoospermia is 30–40 mg/kg [13–15]. However, in some studies, the dose of busulfan-induced oligospermia and asthenospermia is 30–40 mg/kg [6, 16]. Essential differences between azoospermia and oligoasthenozoospermia have been reported. Many researchers believe that the extent of damage caused by busulfan to testicular cells increases with dose, and some of them induced partial damage to testicular cells by using small doses of busulfan (4–35 mg/kg) to establish oligoasthenozoospermia models [7, 13, 17]. Interestingly, some studies have shown that the degree of damage to fertility after intraperitoneal injection of busulfan is not fully proportional to dose [3, 4, 13]. Previous research indicated that after a single injection of 15, 30, or 45 mg/kg busulfan, the mice lost fertility at week 4, regardless of the busulfan dose [3]. However, no further explanation or systematic analysis were provided.
In most studies, the evaluation of modeling methods is not comprehensive enough for the systematic analysis of multiple influencing factors [3, 5], including model animal species, outcome observation time points, and evaluation indicators. Consequently, comparable and quantification outcomes are unavailable to readers. Therefore, we evaluated the quality of the included animal experimental studies and extracted relevant influencing factors in detail. We aimed to review the modeling method of busulfan-induced oligospermia and asthenozoospermia systematically. Moreover, changes in various evaluation indicators were analyzed at different busulfan doses over time.
Method
Search strategy
We searched the Cochrane Library, PubMed, China National Knowledge Infrastructure, and the Chinese Bio-Medical Literature Service System (Sino Med) without limits on language (the time limit was updated on April 9, 2022). Chinese was used for Chinese database retrieval. The search terms were busulphan, busulfan, myleran, male infertility, oligospermia, oligoasthenospermia, oligoasthenoteratospermia, spermatogenesis disorder, mice, and mouse.
Inclusion and exclusion criteria
The inclusion criteria were as follows: (i) busulfan-induced animal experiments about spermatogenesis dysfunction in mice; (ii) preclinical and mechanistic studies related to the treatment of oligospermia and asthenozoospermia; (iii) model evaluation experiment after busulfan modeling.
The following exclusion criteria were used: (i) model using other chemicals; (ii) animal experiments on rats and other species; (iii) lack of indicators for evaluating spermatogenesis; (iv) relevant data in the study could not be extracted; (v) review articles without original data.
Data collection and quality assessment
The quality of included studies was assessed with SYRCLE’s risk of bias tool. Two authors (RP and WZ) independently screened the literature according to the inclusion and exclusion criteria and resolved disagreements with a third review author (NC). SYRCLE’s risk of bias tool contains ten entries and 22 sub-entries for evaluating seven types of bias: selection, performance, detection, attrition, reporting bias, and other bias. The evaluation results were expressed by “Y,” “N,” and “UN,” which represent low-risk, high-risk, and uncertain risk bias, respectively [18, 19].
We designed a data extraction form in excel, and two review authors (RP and WZ) used it to extract data from eligible studies. Extracted data were compared, and any discrepancy was resolved through discussion. Data extracted included mouse strain, busulfan dose, modeling method, evaluation time point after modeling, study type, and model evaluation indicators. Model evaluation indicators were divided into 10 items, as shown in Table 1: ① histopathology (HE staining), ② organ weight and organ index (testis weight, testicular index, epididymal weight, and epidiymis index), ③ epididymal sperm count, ④ sperm motility, ⑤ sperm morphology, ⑥ female and male mice mated for the assessment of their fertility, ⑦ percentage of spermatogenic cells (flow cytometry), ⑧ Johnsen score and simplified version of evaluation in testicular seminiferous tubules, ⑨ protein markers assessed by immunohistochemistry or Western blot, and ⑩ Mouse mortality.
Table 1.
Basic characteristics of included studies
| Author | Year | Mice strains | Dose (mg/kg) and mode of administration‡ | Evaluation time point (weeks)† | N (control/model) | Evaluation indicators§ |
|---|---|---|---|---|---|---|
| Wang Yong-bin [20] | 2007 | Kunming | 30;40;50 (1 ip) | 3;4;6 | 11/59 | ②⑧⑩ |
| Zhang Shu-cheng [32] | 2009 | NIH | 40 (1 ip) | 17 | 20/11 | ①③④⑩ |
| Zhang Wei [21] | 2009 | ICR | 15;30;40 (1 ip) | 4;8;12 | 15/45 | ①⑧ |
| Zhang Ci [22] | 2009 | Kunming | 40 (1 i.p.) | 4;12 | 0/40 | ①⑩ |
| Wang De-zhi [13] | 2010 | BALB/c | 10;20;30;40;50 (1 ip) | 4;8;12;24;48 | 50/171 | ①⑥⑦⑩ |
| Luo Xiao-min [23] | 2012 | Kunming | 10;15 (2 ip, 24 days apart) | 4;8 | 18/36 | ①⑨ |
| Wei Gang [33] | 2012 | NIH | 40 (1 ip) | 17 | 14/10 | ③②④⑩ |
| Li Jing-ping [24] | 2013 | NIH | 30 (1 ip) | 2;4;8 | 15/15 | ①⑧⑩ |
| Zhang Sa [25] | 2014 | Kunming |
35;40;45 (1 ip) 10;15 (itt, bilateral injection) |
0.4;2;3;4;5;6 | 45/45 | ①⑧⑩ |
| Qin Yu-sheng [26] | 2014 | ICR |
40 (1 ip) 4;6 (itt, bilateral injection) |
2;3;4;5;10 | 60/180 | ①⑧⑩ |
| Wang, J [27] | 2014 | Kunming | 10;20;30 (1 ip) | 5;7;9;11;15 | 30/90 | ②⑩ |
| Bao Guo [28] | 2015 | ICR;NIH; Balb/c | 30;40;45 (1 ip) | 0.5;5;7.5;10 | 223/873 | ①④⑩ |
| Wang Ju-hua [29] | 2015 | Kunming | 10;20;30 (1 ip) | 5;7;9;11;13 | 26/78 | ①⑧⑩ |
| Sheng Wen-jie [30] | 2017 | C57BC/6 | 30 (1 ip) | 1;2;4 | 6/6 | ①② |
| Qu Ning [34] | 2018 | C57BC/6 | 40 (1 ip) | 9;17 | 20/20 | ①③⑥⑨ |
| Xie Yun [12] | 2019 | C57BC/6 | 20;30;40 (2 ip, 3 h apart) | 5;9 | 7/21 | ①⑨ |
| Ziaeipour, S [7] | 2019 | NMRI | 40 (1 ip) | 5;10 | 12/12 | ①③ |
| Meng Xue-dan [35] | 2020 | Balb/c | 20 (1 ip) | 2 | 6/6 | ①③④⑥ |
| Zhao Xi [36] | 2020 | ICR | 4 (itt, bilateral injection) | 4 | 6/6 | ②③④ |
| Wang Hui-hui [37] | 2020 | Kunming | 35 (1 ip) | 4 | 8/13 | ①③④⑦ |
| Li Hui-yun [31] | 2021 | BALB/c | 30;40 (1 ip) | 2;4;6;8;10 | 15/30 | ①⑧ |
| Ji Zhi-yong [38] | 2021 | C57BC/6 | 35 (1 ip) | 8 | 8/8 | ①⑧ |
| Moghadam, M. T [39] | 2021 | / | 30 (1 ip) | 7 | 6/6 | ②③④⑤⑧ |
| Yu.SH [40] | 2020 | ICR | 40 (1 ip) | 5 | 10/10 | ③④ |
| Chen Zhi-hong[41] | 2020 | C57BC/6 | 30 (1 ip) | 10 | 8/8 | ①③④ |
| Zhao Xi[42] | 2022 | ICR | 4 (itt, bilateral injection) | 6 | 5/5 | ①②③④⑨ |
| Ji, M [43] | 2007 | MCH-ICR | 35 (1 ip) | 5.7 | 7/7 | ①②③④ |
| Wang, C. N [44] | 2020 | ICR | 6 (itt, bilateral injection) | 2 | 10/10 | ①③ |
| Rezaei, F [15] | 2021 | NMRI | 40 (1 ip) | 3 | 8/8 | ①③④⑤⑦ |
†At various intervals post molding, the testes and epididymis were collected for evaluation (weeks)
‡Single intraperitoneal injection: 1 ip; bilateral intratesticular injections: itt, bilateral injection
§Evaluation indicators: ① histopathology (HE staining), ② organ weight and organ index, ③ epididymal sperm counts, ④ sperm motility, ⑤ sperm morphology, ⑥ female and male mice were mated to assess their fertility, ⑦ percentage of spermatogenic cells, ⑧ Johnsen score and simplified version of evaluation in testicular seminiferous tubules, ⑨ protein markers assessed by immunohistochemistry or western blot, ⑩ mouse mortality
Statistical analyses
In the study, the evaluation index units were uniformly converted: testicular weight, average weight of bilateral testicles (g); epididymis weight, average weight of bilateral epididymis (g); sperm count analyzed using computer-assisted sperm analysis (CASA) or hemocytometer counting chamber (106/ml); sperm motility (%); percentage of sperm showing forward movement.
The testicular index and mouse mortality were calculated according to the following formula:
- Testicular index (mg/g)
testicular weight (mg)/body weight (g).
- Mice mortality
number of dead mice/total number of mice in the molding group.
We conducted random-effects models to calculate pooled weighted mean differences (WMDs) and their 95% confidence intervals (CI) for continuous variables in STATA software 12.0. We performed a meta-analysis on mouse mortality by using random effects in R software 4.1.2. The heterogeneity of the studies was established by I2, and I2 < 50% indicated low heterogeneity, whereas I2 > 75% indicated high heterogeneity. Egger’s test was used in detecting publication bias.
The dose of busulfan and the time point of model observation were stratified for subgroup analysis. Five weeks is the period of the seminiferous epithelium, known as the seminiferous cycle [3, 4]. Therefore, observation time points were categorized into four groups: within one seminiferous cycle (≤ 5 weeks), within two seminiferous cycles (5–10 weeks), within three seminiferous cycles (10–15 weeks), and over three seminiferous cycles (≥ 15 weeks).
Results
Study selection
A total of 261 nonduplicated studies were obtained, and 173 studies were excluded at the title and abstract levels. Finally, 88 full-text articles were evaluated. Through further screening and evaluation of the bias risk of research, 59 full texts were excluded according to the exclusion criteria. Finally, 29 studies were included, composed of 15 experimental studies of methods of modeling and 14 animal pharmacodynamic experiments (Fig. 1). The reasons for exclusion were as follows: animal species did not meet the criteria (P), intervention measures were other chemicals (I), evaluation indicators were insufficient for a meta-analysis (C), outcome indicators were unavailable, and data could not be extracted (O). Another reason was that risk assessment showed a high risk.
Fig. 1.
PRISMA flow diagram detailing process of study selection for the meta-analysis. Key: CNKI, China National Knowledge Infrastructure. Sino Med, Chinese Biomedical Literature Service System
Characteristics of included studies
As shown in Table 1, all animal experiments included 2498 mice (669 controls and 1829 models). The modeling methods included intraperitoneal injection and low-dose intratesticular injection, and the minimum doses was 4 mg/kg, and the maximum was 50 mg/kg. After modeling, observation time varied between 3 days and 48 weeks. A total of 57% (15/27) of studies were experimental studies of modeling methods, including multiple modeling doses and assessment indicators at different time points [7, 12, 13, 20–31]. The remaining 14 studies were animal pharmacodynamic experiments, and only the data of control and model groups were extracted [15, 32–44]. Fourteen studies reported sperm counts [7, 15, 32–37, 39–44], and eleven reported sperm motility [15, 32, 33, 35–37, 39–43]. Six studies used assessment for damage to seminiferous tubules, including apparent disordered and hollow structures [12, 13, 20, 21, 25, 29]. However, the outcomes in these six studies were extremely different to quantify, and thus, they were not included in the meta-analysis of the Johnsen score.
Risk of bias assessment
As shown in Table 2, one study was excluded for potential high risk through SYRCLE tool evaluation [45]. Nearly 40% of studies reported on the generation or application of sequence, baseline characteristics, investigator and animal keeper blinding, and randomness in outcome assessment. More than 90% of the articles reported randomization of animal placement, and no bias in reporting of results was found. However, 71% of studies (10/14) did not state the blinding method of sperm counts and sperm motility [7, 15, 32, 33, 36, 37, 39–41, 43]. The result of Egger’s test suggested no obvious publication bias (P >|t|= 0.206).
Table 2.
Assessment of risk of bias of the included studies using SYRCLE’s risk of bias tool
| Study | ① | ② | ③ | ④ | ⑤ | ⑥ | ⑦ | ⑧ | ⑨ | ⑩ |
|---|---|---|---|---|---|---|---|---|---|---|
| Wang, Y. B. (2007) | U | Y | U | Y | U | U | U | U | Y | Y |
| Zhang, S. C. (2009) | U | N | U | Y | U | U | N | Y | Y | Y |
| Zhang, W. (2009) | U | U | U | Y | U | U | U | Y | Y | Y |
| Zhang, C (2003) | U | Y | U | Y | U | U | U | Y | Y | Y |
| Wang, D. Z. (2010) | Y | Y | U | Y | Y | Y | U | Y | Y | Y |
| Luo, X. M. (2010) | Y | U | U | U | U | U | U | U | Y | Y |
| Wei, G. (2012) | U | U | U | Y | U | U | U | Y | Y | Y |
| Li J., P. (2013) | U | U | U | Y | U | U | U | Y | Y | Y |
| Zhang, S. (2014) | Y | Y | Y | Y | Y | Y | U | Y | Y | Y |
| Qin, Y. S. (2014) | Y | U | U | Y | U | Y | U | Y | Y | Y |
| Bao, G. (2015) | Y | U | U | Y | U | U | U | Y | Y | Y |
| Wang, J. H. (2015) | Y | U | U | Y | U | U | U | Y | Y | Y |
| Sheng, W. J. (2017) | Y | U | U | Y | U | U | U | U | Y | Y |
| Meng, X. D. (2020) | U | U | U | Y | Y | U | Y | Y | Y | Y |
| Wang, H. H. (2020) | Y | Y | U | Y | U | U | U | Y | Y | Y |
| Li, H. Y. (2021) | U | N | U | Y | U | U | U | Y | Y | Y |
| Ji, Z. Y. (2021) | Y | U | U | Y | U | U | U | Y | Y | Y |
| Wang, J. (2014) | Y | U | U | Y | U | U | U | Y | Y | Y |
| Qu, N. (2018) | Y | Y | U | Y | Y | Y | Y | Y | Y | Y |
| Xie, Y. (2020) | Y | Y | U | Y | U | Y | U | Y | Y | Y |
| Ziaeipour, S. (2019) | Y | Y | U | Y | Y | Y | U | Y | Y | Y |
| Zhao, X. (2020) | U | Y | U | Y | U | Y | U | Y | Y | Y |
| Moghadam, M. T. (2021) | Y | U | U | Y | Y | U | U | Y | Y | Y |
| Zhao, X. (2022) | Y | Y | U | Y | Y | Y | Y | Y | Y | Y |
| Chen, Z. (2021) | U | U | U | Y | Y | Y | U | Y | Y | Y |
| Yu, S. (2020) | U | U | U | Y | Y | U | U | Y | Y | Y |
| Ji, M. (2007) | U | Y | U | Y | Y | U | U | Y | Y | Y |
| Rezaei, F. (2021) | U | Y | U | Y | U | U | U | Y | Y | Y |
| Wang, C. N. (2020) | Y | Y | U | Y | Y | Y | Y | Y | Y | Y |
Key: ① generation or application of sequence, ② baseline characteristics, ③ concealed allocation, ④ randomization of animal placement, ⑤ blinded (investigator and animal keeper blinding), ⑥ randomness in outcomes assessment, ⑦ blinded (outcome evaluators); ⑧ incomplete data report, ⑨ Selective reporting of results, ⑩ other sources of bias, Y, yes; N: no; U, uncertain
Systematic review of modeling methods
The meta-analysis results showed that the most commonly used modeling method for busulfan-induced oligoasthenozoospermia models was intraperitoneal injection (dose: 10–50 mg/kg). As shown in Fig. 2, the mortality rate of mice increased with busulfan dose. The overall mouse mortality rate was 23% (WMD: 0.23, 95% CI: 0.13, 0.34). The overall mortality rate of mice in the model group was less than 11% when the busulfan dose was less than 35 mg/kg. In addition, no death due to low-dose (< 6 mg/kg) intratesticular injection of busulfan was reported in mice.
Fig. 2.
Forest plot and meta-analysis of mouse mortality
Meta-analysis of evaluation indicators
Testicular weight and testicular index
The meta-analysis demonstrated a significant reduction in testicular weight (WMD − 0.04, 95% CI: − 0.05, − 0.03) in the busulfan-induced mouse model, as shown in Fig. 3. The testicular weight of the low-dose busulfan (10 mg/kg) group slightly decreased (WMD, − 0.01; 95% CI: − 0.04, 0.01). However, reduction in testicular weight in the high-dose groups was not entirely proportional busulfan dosage. Owing to the self-recovery of spermatogenic function after modeling with busulfan, performing a subgroup analysis on the basis of observation time point is necessary.
Fig. 3.
Forest plot and meta-analysis of testicular weight
We performed a subgroup analysis of testicular weight by stratifying busulfan dose and observation time according to the mouse seminiferous cycle. The main results are presented in Fig. 4. The testicular weights of mice in the 20 mg/kg busulfan group returned to normal after three seminiferous cycles (15 weeks). In the first seminiferous cycle of the 30 mg/kg busulfan group, the testicular weight significantly decreased compared with that in the control (the overall decrease was 0.07 g) but subsequently gradually increased. However, it considerably decreased after three spermatogenic cycles. The testicular weight of the 40 mg/kg busulfan group continued to decline and did not recover in three spermatogenesis cycles.
Fig. 4.
Subgroup analysis of testicular weight
As shown in Fig. 5, the testicular index significantly decreased in almost all busulfan dose groups compared with that in the control. The testicular index of mice in the 10 mg/kg busulfan group returned to normal within 15 weeks. The testicular index of the 20 mg/kg group decreased significantly in the second seminiferous cycle (WMD − 5.18, 95% CI: − 5.64, − 4.73), and recovery started in the third seminiferous cycle. The testicular index of the 40 mg/kg group showed a trend of recovery within three seminiferous cycles, but considerable heterogeneity was observed among the subgroups (I2 > 75%).
Fig. 5.
Subgroup analysis of testicular index
Johnsen score
The state of spermatogenic cells in testicular tissues was quantitatively evaluated by the Johnsen score [46, 47]. As shown in Fig. 6, the pooled Johnsen score significantly decreased in all groups compared with the control. The testicular Johnsen score of the 30 mg/kg busulfan group had no recovery trend within two spermatogenic cycles (WMD: − 4.59, 95% CI: − 7.26, − 1.92). The Johnsen score of 40 mg/kg busulfan decreased continuously in two spermatogenic cycles (WMD: − 6.23, 95% CI: − 7.15, − 5.31).
Fig. 6.
Subgroup analysis of Johnsen score
Sperm counts and motility
Figure 7A shows that epididymal sperm count decreased in almost all dose busulfan groups compared with the control. The pooled sperm counts decreased by 32.84 × 106/ml (WMD: − 32.84, 95% CI: − 44.34, − 21.28). We performed a subgroup analysis of sperm counts by stratifying the observation time and busulfan dose. The results showed that heterogeneity between studies did not decrease in subgroup analysis, as shown in Fig. 7B
Fig. 7.
Epididymal sperm count forest plot (A) and subgroup of sperm count (B)
Sperm motility decreased in all dose busulfan groups compared with the control. The pooled sperm count deceased by 37% (WMD: − 0.37, 95% CI: − 0.47, − 0.27). The 40 mg/kg busulfan group had the most significant decrease in sperm motility, with a low heterogeneity (I2 = 12.7%), as shown in Fig. 8.
Fig. 8.
Epididymal sperm motility forest plot
Discussion
Present meta-analysis results indicate that the mouse mortality rate increased with busulfan dose after modeling. The pooled mortality of the 30 mg/kg busulfan group was 10%, and significant spermatogenesis disorder lasted for 10 − 15 weeks [12, 48]. Therefore, modeling doses within 30 mg/kg can achieve low mortality and incomplete spermatogenesis damage (within 10 weeks) in mice.
In our analysis, the commonly used evaluation indicators were organ weight or index (22/27), histopathological HE staining (23/27), and sperm count (15/27). Only 7% (2/27) of studies reported in vivo fertility assay (female and male mice were mated to assess their fertility), and 19% (5/27) reported the percentage of spermatogenic cells. Examining sperm morphology is essential to assessing male infertility [49–51]. However, a standardized assessment of sperm morphology is rarely reported, and the number of studies was insufficient for meta-analysis.
Testicular weight and testicular index were most sensitive to changes and decreased significantly in the first spermatogenic cycle (Fig. 4). Testicular weight gradually recovered to normal in 3 spermatogenic cycles after low-dose busulfan modeling. However, testicular weight and Johnsen score continuously decreased after the administration of a high dose (> 30 mg/kg) of busulfan. These observations were consistent with the previous experimental studies [52, 53]. Moreover, we found an interesting phenomenon. Decrease in the testicular index in the 20 mg/kg busulfan group was more pronounced than in the 40 mg/kg dose group in the second spermatogenic cycle after modeling. This finding indicated that reduction in testicular weight was not entirely proportional to busulfan dose.
The results of sperm count in different studies showed significant heterogeneity. Although the heterogeneity of the analysis results was reduced by subgroup analysis, some results showed high heterogeneity (I2 > 50%), which may be related to inevitable laboratory heterogeneity among studies [49, 54]. Busulfan has different effects on the different strains of mice. In contrast to ICR mice, NIH mice have more significant spermatogenic damage and higher mortality [28]. Balb/C and Swiss mice showed testicular degeneration after a single intraperitoneal injection of busulfan (40 mg/kg). However, testicular function and fertility recovered spontaneously at 90 days in Swiss mice, but not in Balb/C mice [52]. We tried to stratify the busulfan dose and observation time point and conducted subgroup analysis on mouse strains. However, owing to the limited number of studies, subgroup analysis cannot be conducted. The subgroup analysis of mouse strains is inappropriate without controlling the dosage of busulfan and the spermatogenic cycle. Therefore, subsequent meta-analysis is needed to verify difference in sperm counts in the models of different mouse strains.
In addition, whether sperm analysis methods are reported in detail is of great significance for tracing the source of heterogeneity and is a basis for analyzing the comparability of research results in different studies. F12 medium containing 0.1% bovine serum albumin can facilitate the transmigration of sperm from the epididymis [41]. In contrast to Qu et al. using PBS to disperse epididymal sperm [34], Chen et al. used a nutrient-rich medium [41] and significantly higher sperm concentration and vitality. Both studies were conducted on C57 mice, the dosage of busulfan was the same, and the observation time was 9–10 weeks after modeling. What is more, compared with the results of CASA, the results of sperm concentration counting with a hemocytometer (100 μm) had lower uncertainties [54].
Our research has many advantages but faces significant challenges. First, owing to the limited number of studies, the subgroup analyses of some specific observation times were not performed. Second, we found that standard detailed protocols for sperm count and sperm motility practices should be considered. The exact sperm analysis method and blinding method of evaluators for manual counts were essential to reduce the heterogeneity of results. In addition, we only included relevant studies in mice to minimize differences among different model animals. As for whether these results apply to rats and other animals, more research is needed. To the best of our knowledge, our study is the first to comprehensively analyze the modeling method of oligoasthenozoospermia, which has a good reference value for follow-up research.
Conclusion
Intraperitoneal injection of low-dose busulfan (< 30 mg/kg) has low mortality in the mouse model of oligoasthenospermia. Evaluation indicators in the model group significantly decreased within 10 weeks. However, the decrease may not be directly proportional to busulfan dose. Moreover, sperm counts have significant heterogeneity in different studies. Therefore, standardized protocols for sperm count and motility evaluation in the animal model are urgently needed. Ensuring the comparability of different research results is essential for evaluating infertility models.
Author contribution
Conceptualization: NC, RP; methodology: RP, JL, AZ; software: RP, JL, DS; validation: AZ, XR, YL, WZ; formal analysis: HH, XL, LL, YW; investigation and data curation: RP, WZ; writing-original draft: RP, JY, JL; writing-review and editing: RP, JY, NC; supervision: YB, NC.
Funding
This work is supported by the Natural Science Foundation of China (No. 81673248).
Data Availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Declarations
Conflict of interest
The authors declare no competing interests.
Footnotes
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Ruiyang Pu and Jing Liu are the co-first authors of this article.
References
- 1.Vander Borght M, Wyns C. Fertility and infertility: definition and epidemiology. Clin Biochem. 2018;62:2–10. doi: 10.1016/j.clinbiochem.2018.03.012. [DOI] [PubMed] [Google Scholar]
- 2.Agarwal A, Baskaran S, Parekh N, et al. Male infertility. Lancet. 2021;397:319–333. doi: 10.1016/s0140-6736(20)32667-2. [DOI] [PubMed] [Google Scholar]
- 3.Zohni K, Zhang X, Tan SL, et al. The efficiency of male fertility restoration is dependent on the recovery kinetics of spermatogonial stem cells after cytotoxic treatment with busulfan in mice. Hum Reprod. 2012;27:44–53. doi: 10.1093/humrep/der357. [DOI] [PubMed] [Google Scholar]
- 4.Borg CL, Wolski KM, Gibbs GM, et al. Phenotyping male infertility in the mouse: how to get the most out of a ‘non-performer’. Hum Reprod Update. 2010;16:205–224. doi: 10.1093/humupd/dmp032. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Wang H. Chen L [A brief review of animal model of spermatogenesis dysfunction] Chin J Hum Sex. 2017;26:102–104. [Google Scholar]
- 6.Liu W, Zhang L, Gao A, et al. Food-derived high arginine peptides promote spermatogenesis recovery in busulfan treated mice. Front Cell Dev Biol. 2021;9:791471. doi: 10.3389/fcell.2021.791471. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Ziaeipour S, Rezaei F, Piryaei A, et al. Hyperthermia versus busulfan: finding the effective method in animal model of azoospermia induction. Andrologia. 2019;51:e13438. doi: 10.1111/and.13438. [DOI] [PubMed] [Google Scholar]
- 8.Boekelheide K. Mechanisms of toxic damage to spermatogenesis. J Natl Cancer Inst Monogr. 2005;34:6–8. doi: 10.1093/jncimonographs/lgi006. [DOI] [PubMed] [Google Scholar]
- 9.Chang Z, Qin W, Zheng H, et al. Triptonide is a reversible non-hormonal male contraceptive agent in mice and non-human primates. Nat Commun. 2021;12:1253. doi: 10.1038/s41467-021-21517-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Chen X, Liang M, Wang D. Progress on the study of the mechanism of busulfan cytotoxicity. Cytotechnology. 2018;70:497–502. doi: 10.1007/s10616-018-0189-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Huang WL, Ding L, Yao JH, et al. Testicular quantitative ultrasound: a noninvasive monitoring method for evaluating spermatogenic function in busulfan-induced testicular injury mouse models. Andrologia. 2020;53:e13927 . doi: 10.1111/and.13927. [DOI] [PubMed] [Google Scholar]
- 12.Xie Y, Deng CC, Ouyang B, et al. Establishing a nonlethal and efficient mouse model of male gonadotoxicity by intraperitoneal busulfan injection. Asian J Androl. 2020;22:184–191. doi: 10.4103/aja.aja_41_19. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Wang DZ. The effect of busulfan on production of testicular germ cells, sexual behaviour and sterility of male mice [Doctor thesis] Wuhan University; 2010. [Google Scholar]
- 14.Ibrahim HF, Safwat SH, Zeitoun TM, et al. The therapeutic potential of amniotic fluid-derived stem cells on busulfan-induced azoospermia in Adult Rats. Tissue Eng Regen Med. 2021;18:279–295. doi: 10.1007/s13770-020-00309-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Rezaei F, Bayat M, Nazarian H, et al. Photobiomodulation therapy improves spermatogenesis in busulfan-induced infertile mouse. Reprod Sci. 2021;28:2789–2798. doi: 10.1007/s43032-021-00557-8. [DOI] [PubMed] [Google Scholar]
- 16.Zhao J, Wang M, Wang Y, et al. Endoplasmic reticulum stress promotes blood-testis barrier impairment in mice with busulfan-induced oligospermia through PERK-eIF2α signaling pathway. Toxicology. 2022;473:153193 . doi: 10.1016/j.tox.2022.153193. [DOI] [PubMed] [Google Scholar]
- 17.Luo J, Yang Y, Ji X, et al. NGF rescues spermatogenesis in azoospermic mice. Reprod Sci. 2021;28:2780–2788. doi: 10.1007/s43032-021-00511-8. [DOI] [PubMed] [Google Scholar]
- 18.Kraeuter AK, Phillips R, Sarnyai Z. The gut microbiome in psychosis from mice to men: a systematic review of preclinical and clinical studies. Front Psychiatry. 2020;11:799. doi: 10.3389/fpsyt.2020.00799. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Hooijmans CR, Rovers MM, de Vries RB, et al. SYRCLE's risk of bias tool for animal studies. BMC Med Res Methodol. 2014;14:43. doi: 10.1186/1471-2288-14-43. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Wang YB, Liu GS, Zhu SE, et al. Model establishment of transplantation of spermatogonial stem cells in mice. Chinese Journal of Comparative Medicine. 2007;17:676–680+701.
- 21.Zhang W, Xia XY, Ding H, et al. Establishment of recipient mouse model of stem cell transplantation into testicular seminiferous tubules and improvement of transplantation techniques. Zhonghua nan ke xue. 2009;15:703–707. [PubMed] [Google Scholar]
- 22.Zhang C, Wang LL, Jin HM, et al. Induction of azoospermia model in mice by busulfan. J New Med. 2003;13:201–202. [Google Scholar]
- 23.Luo XM, Zhang C, Yang SX, et al. Murine model of busulfan-induced spermatogenesis regeneration: a quantitative evaluation. Zhonghua nan ke xue. 2010;16:395–399. [PubMed] [Google Scholar]
- 24.Li JP, Guo WB, He JC, et al. Establishing a mouse model of Sertoli-cell-only syndrome by administration of busulfan. Zhonghua nan ke xue. 2013;19:300–5. [PubMed]
- 25.Zhang S. Preliminary establishment of mouse model for spermatogonial stem cell transplants [Master thesis] Guangxi University; 2014. [Google Scholar]
- 26.Qin YS. Testicular injection of busulfan for recipient preparation in transplantation of spermatogonial stem cells in mice [Master thesis] Jilin Agricultural University; 2014. [DOI] [PubMed] [Google Scholar]
- 27.Wang J, Xue X, Fan C, et al. Establishment of recipient model for spermatogonial stem cells transplantation in Kunming mice. Tissue Cell. 2014;46:249–254. doi: 10.1016/j.tice.2014.05.005. [DOI] [PubMed] [Google Scholar]
- 28.Bao G, Wang SM, Zhang CY, et al. Establishing azoospermia model with busulfan in three strains of mice. J Reprod Med. 2015;24:7. [Google Scholar]
- 29.Wang JH, Xie QQ, Fan CY, et al. Establishment of transplantation model for spermatogonial stem cells in Kunming mice with busulfan treatment. J Northwest A & F Univ-Nat Sci Ed. 2015;43:51–56. [Google Scholar]
- 30.Sheng WJ, Lei B. Effects of single-dose busulfan injection on the function of major organs in mice. Guangdong Med J. 2017;38:1684–1685+1690.
- 31.Li HY, Wang HF, Meng XL, et al. Establishment of NOA model in BALB/c mice induced by busulfan. Chinese Journal of Reprodctive Health. 2021;32:247–251+302.
- 32.Zhang SC, He B, Wang SM, et al. Comparative study on the effects of Wuziyanzong Pills and Jinkuishenqi Pills on spermatogenesis function. Chinese Journal of Family Planning. 2009;17:401–404. [Google Scholar]
- 33.Wei G, Chen XH, Zhang SC, et al. Study on gene expression profiling of Wuzi Yanzong Wan in regaining spermatogenic ability in mouse with azoospermia. Journal of Hebei TCM and Pharmacology. 2012;27:4–7+2.
- 34.Qu N, Kuramasu M, Hirayanagi Y, et al. Gosha-Jinki-Gan recovers spermatogenesis in mice with busulfan-induced aspermatogenesis. Int J Mol Sci. 2018;19:2606. doi: 10.3390/ijms19092606. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.Meng XD. Study on spermatogenesis and molecular mechanism of Wuziyanzong pill [thesis of master] Beijing University of Chinese Medicine; 2020. [Google Scholar]
- 36.Zhao X, Liu Z, Gao J, et al. Inhibition of ferroptosis attenuates busulfan-induced oligospermia in mice. Toxicology. 2020;440:152489 . doi: 10.1016/j.tox.2020.152489. [DOI] [PubMed] [Google Scholar]
- 37.Wang HH, Zhang L, Xu RH, et al. Effect of Duzhong Butiansu capsule on busulfan-induced spermatogenic impairment in mice. Tradis Chin Drug Res Clin Pharmacol. 2020;31:69–78. [Google Scholar]
- 38.Ji ZY, Yao CC, Zhao JP, et al. Protective effect of L-carnitine on spermatogenic dysfunction mouse model. Shanghai Medical Journal. 2021;44:316–320. [Google Scholar]
- 39.Moghadam MT, Dadfar R, Khorsandi L. The effects of ozone and melatonin on busulfan-induced testicular damage in mice. JBRA Assist Reprod. 2021;25:176–184. doi: 10.5935/1518-0557.20200081. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40.Yu S, Zhao Y, Zhang FL, et al. Chestnut polysaccharides benefit spermatogenesis through improvement in the expression of important genes. Aging (Albany NY) 2020;12:11431–11445 . doi: 10.18632/aging.103205. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 41.Chen Z, Liu M, Hu JH, et al. Substance P restores spermatogenesis in busulfan-treated mice: a new strategy for male infertility therapy. Biomed Pharmacother. 2021;133:110868 . doi: 10.1016/j.biopha.2020.110868. [DOI] [PubMed] [Google Scholar]
- 42.Zhao X, Sang M, Han P, et al. Peptides from the croceine croaker (Larimichthys crocea) swim bladder attenuate busulfan-induced oligoasthenospermia in mice. Pharm Biol. 2022;60:319–325. doi: 10.1080/13880209.2022.2034895. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 43.Ji M, Minami N, Yamada M, et al. Effect of protopanaxatriol saponin on spermatogenic stem cell survival in busulfan-treated male mice. Reprod Med Biol. 2007;6:99–108. doi: 10.1111/j.1447-0578.2007.00172.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 44.Wang CN, Sang MM, Gong SN, et al. Two resveratrol analogs, pinosylvin and 4,4'-dihydroxystilbene, improve oligoasthenospermia in a mouse model by attenuating oxidative stress via the Nrf2-ARE pathway. Bioorg Chem. 2020;104:104295. doi: 10.1016/j.bioorg.2020.104295. [DOI] [PubMed] [Google Scholar]
- 45.Wu C, Zhang Y, Shen Q, et al. Resveratrol changes spermatogonial stem cells (SSCs) activity and ameliorates their loss in busulfan-induced infertile mouse. Oncotarget. 2016;7:82085–82096 . doi: 10.18632/oncotarget.12990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 46.Johnsen SG. Testicular biopsy score count–a method for registration of spermatogenesis in human testes: normal values and results in 335 hypogonadal males. Hormones. 1970;1:2–25. doi: 10.1159/000178170. [DOI] [PubMed] [Google Scholar]
- 47.Ito Y, Unagami M, Yamabe F, et al. A method for utilizing automated machine learning for histopathological classification of testis based on Johnsen scores. Sci Rep. 2021;11:9962. doi: 10.1038/s41598-021-89369-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 48.Moisan AE, Foster RA, Betteridge KJ, et al. Dose-response of RAG2-/-/gammac-/- mice to busulfan in preparation for spermatogonial transplantation. Reproduction. 2003;126:205–216. doi: 10.1530/rep.0.1260205. [DOI] [PubMed] [Google Scholar]
- 49.Björndahl L, Kirkman Brown J. The sixth edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen: ensuring quality and standardization in basic examination of human ejaculates. Fertil Steril. 2022;117:246–251. doi: 10.1016/j.fertnstert.2021.12.012. [DOI] [PubMed] [Google Scholar]
- 50.Sáez-Espinosa P, Robles-Gómez L, Ortega-López L, Aizpurua J, Gómez-Torres MJ. Immunofluorescence and high-resolution microscopy reveal new insights in human globozoospermia. Int J Mol Sci. 2022;23(3):1729. doi: 10.3390/ijms23031729. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 51.Shang Y, Zhu F, Wang L, Ouyang YC, Dong MZ, Liu C, et al. Essential role for SUN5 in anchoring sperm head to the tail. eLife. 2017;6:e28199. doi: 10.7554/eLife.28199. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 52.Gutierrez K, Glanzner WG, Chemeris RO, Rigo ML, Comim FV, Bordignon V, et al. Gonadotoxic effects of busulfan in two strains of mice. Reprod Toxicol (Elmsford, NY) 2016;59:31–39. doi: 10.1016/j.reprotox.2015.09.002. [DOI] [PubMed] [Google Scholar]
- 53.Bucci LR, Meistrich ML. Effects of busulfan on murine spermatogenesis: cytotoxicity, sterility, sperm abnormalities, and dominant lethal mutations. Mutat Res. 1987;176(2):259–268 . doi: 10.1016/0027-5107(87)90057-1. [DOI] [PubMed] [Google Scholar]
- 54.Gutierrez K, Glanzner WG, Chemeris RO, et al. Gonadotoxic effects of busulfan in two strains of mice. Reprod Toxicol. 2016;59:31–39. doi: 10.1016/j.reprotox.2015.09.002. [DOI] [PubMed] [Google Scholar]
Associated Data
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Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.