Micronucleus counts correlating with male infertility: a clinical analysis of chromosomal abnormalities and reproductive parameters

Abstract

Investigating the correlation between micronucleus formation and male infertility has the potential to improve clinical diagnosis and deepen our understanding of pathological progression. Our study enrolled 2252 male patients whose semen was analyzed from March 2023 to July 2023. Their clinical data, including semen parameters and age, were also collected. Genetic analysis was used to determine whether the sex chromosome involved in male infertility was abnormal (including the increase, deletion, and translocation of the X and Y chromosomes), and subsequent semen analysis was conducted for clinical grouping purposes. The participants were categorized into five groups: normozoospermia, asthenozoospermia, oligozoospermia, oligoasthenozoospermia, and azoospermia. Patients were randomly selected for further study; 41 patients with normozoospermia were included in the control group and 117 patients with non-normozoospermia were included in the study group according to the proportions of all enrolled patients. Cytokinesis-block micronucleus (CBMN) screening was conducted through peripheral blood. Statistical analysis was used to determine the differences in micronuclei (MNi) among the groups and the relationships between MNi and clinical data. There was a significant increase in MNi in infertile men, including those with azoospermia, compared with normozoospermic patients, but there was no significant difference between the genetic and nongenetic groups in azoospermic men. The presence of MNi was associated with sperm concentration, progressive sperm motility, immotile spermatozoa, malformed spermatozoa, total sperm count, and total sperm motility. This study underscores the potential utility of MNi as a diagnostic tool and highlights the need for further research to elucidate the underlying mechanisms of male infertility.

INTRODUCTION

Infertility disorders in males account for 30%–50% of infertility cases,1 with approximately 7% of the global male population afflicted with this condition.2 The etiology of male infertility is multifaceted, encompassing genetic predispositions, and pathological factors. Male infertility can also serve as a proxy for assessing overall individual health,3 given its correlation with an increased risk of cardiovascular diseases, the incidence of cancer, the development of metabolic syndrome, and a variety of chronic maladies. The instability of germ cell DNA, a hallmark of male infertility, is closely tied to the compromised cellular environment within which spermatozoa are produced.4 The DNA damage sustained by spermatogonial stem cells directly impairs the generation of spermatids. Oxidative stress in sperm samples can be assessed by direct detection of reactive oxygen species (ROS) or by measuring the oxidoreduction potential of spermatozoa.5 DNA damage in human spermatozoa can be detected by nuclear staining (such as eosin) or Raman microspectroscopy.6 The whole pathological process of systemic DNA instability in individuals induces male germ cell DNA damage, resulting in male infertility. Evaluating the systemic DNA stability of individuals can facilitate the identification of potential causes of male infertility, augment our insights, and potentially provide information on the development of therapeutic interventions aimed at enhancing male fertility.

The generation of micronuclei (MNi) is an important characteristic of DNA instability. Cellular DNA instability primarily refers to DNA damage that perturbs the integrity of the chromosomal fabric, precipitating anomalous chromosomal segregation or erroneous repair mechanisms during mitosis. Such maladies result in the exclusion of entire chromosomes or chromosomal fragments from the daughter nuclei, and during mitosis, these dissociated DNA segments have the propensity to form MNi. The quantification of MNi frequency is a routine and extensively employed endpoint in the cytokinesis-block micronucleus (CBMN) assay in medical diagnosis.7 To prevent cell cycle progression into telophase, a chemical inhibitor, cytochalasin-B (Cyt-B), is used to curtail the formation of the microfilament ring, which is critical for mitotic spindle function.8 The CBMN assay, which detects MNi, is routinely employed in the assessment of DNA integrity and is indispensable for evaluating the deleterious effects of occupational exposures, such as in the annual surveillance of radiation-exposed personnel.9

MNi are associated with population characteristics7 and reproductive disease pathology.10 Many biological samples are assayed by CBMN, including oral mucosa epithelial cells and peripheral blood lymphocytes (PBLs), whereas the general MNi of individuals can be assayed on PBLs. An increase in MNi in PBL is associated with reproductive failure11,12 and idiopathic infertility.13 For male infertility, the MNi in somatic cells (such as PBL) is positively correlated with DNA damage in spermatozoa,14 and infertile males have an increased frequency of MNi in PBL;15 however, the correlation between the MNi in PBL and semen parameters is unknown.

In this study, we evaluated the frequency of MNi within the PBL of normozoospermic and non-normozoospermic males. This study provides a new insight that an increase in MNi in PBL has occurred in non-normozoospermic males compared with normozoospermic males. Collectively, our study demonstrated a significant correlation between the increase in MNi in PBL and the severity of male infertility, irrespective of genetic or nongenetic factors. This observation suggests that a reduction in the frequency of MNi in PBL may benefit infertile male patients. Anti-genotoxic drugs that reduce the frequency of MNi in PBL, such as naringin,16 Ulva rigida crude extracts,17 Peltigera rufescens (Weis) Humb. extracts,18 chlorogenic acid,19 and curcumin,20 are expected to improve male infertility. Combinations of these agents with antioxidants that have long been used to treat male infertility, such as vitamin C, vitamin E, selenium, zinc, or glutathione,21 may be more effective. Whether the MNi in PBL can be used as an indicator of potential male infertility warrants further investigation and integration into medical treatment.

PATIENTS AND METHODS

Study population

Ethical approval for this study was obtained from the Academic Committee of the Third Affiliated Hospital of Guangzhou Medical University (Guangzhou, China; Approval No. YLKS2024-106), and all participants signed informed consent before the study started. From March 2023 to July 2023, a total of 2252 male patients, including infertile males, and fertile male partners of infertile females, who underwent semen analysis in the Third Affiliated Hospital of Guangzhou Medical University, were enrolled in this study. Cytogenetic analysis was performed to identify sex chromosome abnormalities in individuals. The semen analysis was interpreted as described in the 5^th version of the World Health Organization (WHO) human semen analysis guidelines published in 2010.22 When the total sperm count was less than 39 × 10⁶, the patients were assigned to the oligozoospermic group; asthenozoospermia referred to a total motility below 40% or PR below 32%; and azoospermia, defined as no spermatozoa being detected in three samples from three ejaculates. The premium principle of samples selected for the CBMN assay is representative of clinical characteristics. We collected peripheral blood from infertile male patients at 1 month for the CBMN assay, while the clinical data were collected at 5 months to save manpower and lower costs. One-quarter to one-third of the infertile male cases were assayed by CBMN (Figure 1). The control group included normozoospermic patients (n = 41), whose number of patients was similar to that of the asthenozoospermic group (n = 41), which was the largest subgroup of infertile males. The other study groups were set up according to the proportion of clinical patients, including oligozoospermic group (n = 9), oligoasthenozoospermic group (n = 21), and nongenetic azoospermic group (n = 34). All of the genetically azoospermic patients were enrolled in this study for the CBMN assay during the study period. Four of the genetically azoospermic patients were excluded because of blood clotting and a delay in genetic analysis. Fourty-six patients were diagnosed as azoospermia, with 12 in the genetic azoospermia group and 34 in the nongenetic azoospermia group (Figure 1).

Figure 1.

Flowchart of the cytogenetic analysis of male infertility. CBMN: cytokinesis-block micronucleus.

Genetic analysis of the Y chromosome

Azoospermia factor (AZF) microdeletions of the Y chromosome were analyzed via Y chromosome microdeletion kit (Cat#PG07001Y4; Tellgen, Shanghai, China) at 6 sites (sY84 and sY86 for AZFa; aY127 and sY134 for AZFb; and sY254 and sY255 for AZFc), with sex-determining region of Y-chromosome (SRY) and zinc finger protein X-linked gene (ZFX)/zinc finger protein Y-linked gene (ZFY) as the control.

G-banding analysis

Peripheral blood (400 µl) was seeded in lymphocyte culture medium (Cat#S.001-13; Guangzhou Dahui Biotech, Guangzhou, China). The activated cells were cultured for 72 h at 37°C and 5% CO₂, treated with 0.25 µg ml⁻¹ colchicine (Cat#S.003-10; Guangzhou Dahui Biotech) for 20 min, and then harvested. The lymphocytes were hypotonically treated with 0.075 mol l⁻¹ KCI at 37°C for 20 min and then fixed in methanol/acetic acid (3:1, v/v) for 1 h. The fixed cells were dropped onto cold slides and stained with Giemsa (Cat#S.003-9; Guangzhou Dahui Biotech). The G-banding images of mitosis were captured using a microscope (Axio-Imager, Carl Zeiss AG, Jena, Germany), and chromosomal karyotypes were analyzed with an autovision system (Hangzhou Diagens Biotechnology, Hangzhou, China).

Cytokinesis-block micronucleus assay

The experimental procedure followed the protocol of G-banding analysis with some variation. Cultured lymphocytes were treated with 3 µg ml⁻¹ cytochalasin B (Cat#HY-16928; MedChemExpress, Shanghai, China) for 48 h and harvested at 72 h without colchicine treatment. The duration of hypotonia was 5 min. The MNi score reflects the incidence of MNi in binuclear cells (Figure 2), and 1000 cells were analyzed per sample. Permission had been granted from the Academic Committee of the Third Affiliated Hospital of Guangzhou Medical University to publish the data involved in this study.

Figure 2.

CBMN assay of MNi in PBL culture. (a) Binuclear cells without MNi and (b) binuclear cells with MNi (black arrow) were detected in the PBL culture. CBMN: cytokinesis-block micronucleus; MNi: micronuclei; PBL: peripheral blood lymphocyte.

Statistical analyses

Statistical analysis was performed with SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). The Mann–Whitney U test was used to examine the differences among the infertile groups. Spearman correlation analysis and linear regression were used to describe the correlations between age, semen volume, semen pH, and frequency of MNi in PBL, as well as correlations between sperm quality and MNi frequency. The significance level in all the tests was set to P < 0.05.

RESULTS

The MNi score in the azoospermia group with abnormal sex chromosomes

All participants in the study were karyotyped, with two cases of sex chromosome anomalies being selected for additional investigation. These included 11 cases of 47,XXY and one instance of a mosaic karyotype of 45,X[14]/46,X,del(Y)(q11.2)[⁸⁶], as shown in Figure 3. The mosaicism patients were tested for microdeletions of the Y chromosome, and four sites (aY127, sY134, sY254, and sY255) were not detected, indicating a loss of AZFb and AZFc. The patients with abnormal sex chromosomes were all azoospermic (Table 1). There were no significant differences between genetic and nongenetic azoospermia in terms of semen volume, semen pH or MNi (all P > 0.05; Table 1), and the azoospermic group was formed by genetic and nongenetic azoospermia in the following part of this study.

Figure 3.

Karyotype analysis of the male infertility population. There are three karyotypes of these infertile male populations: (a) normal, (b) Klinefelter syndrome, and mosaic karyotype of 45,X[14]/46,X,del(Y)(q11.2)[⁸⁶], while karyotypes of (c) 45,X and (d) 46,X,del(Y)(q11.2) are shown.

Table 1.

Comparison of genetic and nongenetic azoospermic patients

Characteristic	Genetic (n=12)	Nongenetic (n=34)	P
Age (year), mean±s.d.	34.2±4.4	32.6±4.7	0.238
Semen volume (ml), mean±s.d.	2.63±1.66	2.84±1.92	0.900
Semen pH (mean±s.d.)	6.28±2.94	7.08±1.86	0.345
MNi (‰), mean±s.d.	13.42±7.24	11.18±5.88	0.380

MNi: micronuclei; s.d.: standard deviation

MNi scores in the infertile male groups

The study population was randomly selected according to the clinical proportion of male infertility, and the clinical characteristics of the groups were compared (Table 2). The groups were similar in terms of age, semen volume, and semen pH. By definition, the azoospermic group had no spermatozoa. The other groups presented significant increases in IM and sperm malformation but significant decreases in the sperm concentration, PR, total sperm count, and total motility (all P < 0.05). The total sperm count of the asthenozoospermic group was not different from that of the normozoospermic group (P = 0.097). The clinical characteristics were then matched across the groups, and further studies compared the MNi within the groups (Table 2). There was a significant increase in MNi in the infertile groups compared with the normozoospermic groups (all P < 0.05), but there was no significant difference in MNi among the male infertility groups (all P > 0.05), as shown in Table 2.

Table 2.

Clinical characteristics of the study population and micronuclei comparison

Characteristic	Normozoospermia (n=41)	Asthenozoospermia (n=41)		Oligozoospermia (n=9)		Oligoasthenozoospermia (n=21)		Azoospermia (n=46)		Total (n=158)
		Value	P	Value	P	Value	P	Value	P
Age (year), mean±s.d.	33.2±5.6	36.5±6.5	0.020*	31.9±5.8	0.732	36.8±8.1	0.071	33.0±4.6	0.800	34.4±6.2
Semen volume (ml), mean±s.d.	3.20±1.32	3.61±1.61	0.307	2.87±1.57	0.441	2.92±1.34	0.430	2.79±1.84	0.236	3.13±1.59
Semen pH (mean±s.d.)	7.61±0.32	7.57±0.35	0.582	7.89±0.32	0.026*	7.55±0.38	0.851	6.87±2.19	0.289	7.39±1.25
Sperm concentration (×106 ml−1), mean±s.d.	67.16±43.81	49.62±37.48	0.031*	11.14±6.61	0.001*	7.98±4.77	0.001*	-	-	32.00±40.72
PR (%), mean±s.d.	57.37±9.90	21.71±10.12	0.001*	45.44±9.12	0.004*	15.95±11.09	0.001*	-	-	25.23±23.78
IM (%), mean±s.d.	38.88±10.33	76.27±10.89	0.001*	51.33±9.07	0.004*	81.38±12.29	0.001*	-	-	43.62±33.42
Malformation rate (%), mean±s.d.	96.56±2.26	97.68±2.36	0.005*	98.89±0.33	0.001*	98.19±1.78	0.002*	-	-	69.09±44.46
Total sperm count (×106), mean±s.d.	207.20±144.18	175.90±162.67	0.097	24.83±8.10	0.001*	20.42±10.14	0.001*	-	-	103.53±143.70
Total motility (%), mean±s.d.	61.34±10.63	23.73±10.89	0.001*	48.67±9.07	0.004*	18.62±12.29	0.001*	-	-	27.32±25.36
MNi (‰), mean±s.d.	3.98±3.11	11.80±6.21	0.001*	11.33±4.12	0.001*	10.52±4.65	0.001*	11.76±6.26	0.001*	9.56±6.19

*P, a significant difference versus normozoospermia. For MNi, there is no significant difference among the four study groups (all P>0.05). -: not available; s.d.: standard deviation; PR: progressive sperm motility; IM: immotile spermatozoa; MNi: micronuclei

MNi score and semen quality

The correlations between MNi and semen quality values for all of the data from individuals with non-normozoospermia and normozoospermia were analyzed. The results are presented as scatter plots and linear regressions (Figure 4). The MNi score was not associated with age, semen volume, or semen pH (all P > 0.05) but was negatively associated with the sperm concentration (P < 0.001, r_s = −0.358), total sperm count (P < 0.001, r_s = −0.340), total motility (P < 0.001, r_s = −0.491), and PR (P < 0.001, r_s = −0.495) and positively associated with the IM (P < 0.001, r_s = 0.489) and malformation rates (P = 0.002, r_s = 0.291).

Figure 4.

Correlation analysis of the MNi and clinical parameters. The MNi score was not associated with (a) age, (b) semen volume or (c) semen pH but was associated with (d) sperm concentration, (e) PR, (f) IM, (g) malformation rate, (h) total sperm count, and (i) total motility. r_s is coefficient of rank correlation. MNi: micronuclei; PR: progressive sperm motility; IM: immotile spermatozoa.

DISCUSSION

The core manifestation of male infertility is defective sperm structure and function, with oxidative stress-induced DNA damage being regarded as a leading cause of abnormal spermatozoa.23 Some lifestyles that induce oxidative stress (OS) in the cellular environment, such as smoking, alcohol intake, and radiation, also stimulate factors affecting the integrity of sperm chromatin, resulting in male infertility.24 OS-induced DNA damage is a complex biochemical process that occurs during spermatogenesis,25 and the sperm DNA fragmentation (SDF) assay is used clinically to test sperm DNA fragmentation, which is considered the final stage of DNA damage.26 Systemic DNA damage analysis is performed on peripheral blood lymphocytes, which suggests that exogenous sources increase cellular OS or that endogenous sources lack antioxidants. The study of the correlation between male infertility and DNA damage would benefit the understanding of the pathology and the search for a cure.

The CBMN assay is commonly used for detecting DNA damage by cell morphology, as it is specific for once-divided binucleated (BN) cells.8 The nuclear division index (NDI) indicates the rate of cellular division, and other scores are used to monitor DNA damage. During the BN cell phase, nucleoplasmic bridges (NPBs) are formed upon DNA misrepair or telomere end-fusions, and nuclear buds (NBUDs) indicate the elimination of amplified DNA or DNA repair complexes. Therefore, MNi is a general indicator of chromosomal breakage or whole chromosome loss. All the CBMN parameters (NDI, NPBs, NBUDs, and MNi) are related.8 For peripheral blood lymphocytes of the general population, the MNi is 5.06 ± 3.11 (mean ± standard deviation [s.d.])7 or 6.27 ± 2.66 (mean ± s.d.)27 per 1000 cells, which is consistent with our detection of 3.98 ± 3.11 (mean ± s.d.) per 1000 cells.

An increase in MNi occurred in cells with chromosomal abnormalities. Compared with those of healthy controls, the lymphocytes of patients with Down syndrome also exhibit an increased MNi frequency; moreover, patients with potential complications of Down syndrome, such as congenital anomalies and aplastic anemia, also demonstrate a Down syndrome-like MNi frequency increase.28 Direct evidence that chromosomal abnormalities induce an increase in MNi has also been provided in a study of chromosomal instability in mosaic Down syndrome, in which trisomic leukocytes exhibit increased MNi compared with disomic leukocytes.29 In this study, we showed that, compared with normozoospermic individuals, patients with Klinefelter syndrome (the genetic azoospermic group) presented a similar increase in MNi frequency as nongenetic azoospermic patients did, and our results are consistent with the findings in Down syndrome patients.28,29 However, the mechanism of chromosome abnormality-induced DNA damage and pathological abnormalities remains unclear.

As a monitor of DNA damage, MNi increases in some neurodegenerative diseases, such as Alzheimer’s disease (AD) and Parkinson’s disease (PD).30 An increase in system MNi occurs in infertile couples,12,13 and in males with reproductive failure, an increase in MNi in PBL has been detected;11 however, those males display normal sperm quality. In this study, we randomly selected different infertile males from our clinic and reported a similar increase in the MNi in PBL among patients with different degrees of male infertility.

All of our enrolled patients had reproductive needs, and the ages of most of them were between 28 years and 40 years. We used semen parameter values to group the population. While some parameter values reflected the clinical characteristics of the study population, there were no significant differences in semen volume or semen pH. The regression analysis demonstrated that the frequency of MNi was significantly correlated with the clinical grouping but not the semen volume or pH. Our study had a retrospective design where we selected the study population; thus, potential bias for arbitrariness exists, and more prospective studies are needed to determine the clinical significance of MNi in male infertility diagnosis and treatment. Multiple factors affect semen quality, such as increased obesity, poor diet, and exposure to environmental toxins.31 Anti-genotoxic drugs are the most convenient way to influence an individual’s frequency of MNi in PBL.16,17,18,19,20 Our study provides evidence that MNi is an independent indicator associated with semen quality and suggests that decreasing the frequency of MNi in the PBL could improve male semen parameters.

CONCLUSION

Our research revealed that MNi in PBL is a potential indicator of male infertility; a higher frequency of MNi in PBL indicates worse semen quality in males, and both genetic and nongenetic forms of azoospermia are associated with an increase in the frequency of MNi in PBL. An increase in this frequency was observed within male infertility cohorts and was significantly correlated with various semen parameter values. Elucidating the molecular underpinnings of this phenomenon is essential for elucidating the intricate interplay between the frequency of MNi in PBL and attendant semen quality. Exploring the potential of reducing the frequency of MNi in PBL as a therapeutic way of enhancing male fertility appears to be a promising direction in future, warranting further investigations in the biomedical domain.

AUTHOR CONTRIBUTIONS

XFS, DW, and SHZ conceived and designed the study. SHZ, WJQ, QYP, LHC, JFW, and SQH contributed to the collection and management of the clinical data. SHZ and DW performed CBMN assay. XFS and YJX contributed to genetic analysis and G-banding analysis. SHZ analyzed the data, and DW, XFS, and YJX drafted the manuscript. All authors read and approved the final manuscript.

COMPETING INTERESTS

All authors declare no competing interests.