Abstract
Increased sperm DNA fragmentation index (DFI) despite normal semen analysis may be encountered in couples with unexplained recurrent pregnancy loss. Therefore, implementing strategies for selecting sperm cells with appropriate DNA integrity may improve the outcomes of assisted reproductive technology (ART). This study compared the efficacy of short abstinence, magnetic activated cell sorting (MACS), and zeta potential in obtaining sperm cells of improved quality. Accordingly, among couples with recurrent ART failures or recurrent abortions, 30 men with increased sperm DFI (sperm chromatin dispersion [SCD] more than 18 %) were enrolled. Subsequently, each participant provided a semen specimen after abstaining for 2 to 3 days. This specimen was divided into three parts. Sample 1 underwent semen analysis and DNA integrity/protamination evaluation using SCD/chromomycin A3 (CMA3) indices. Samples 2 and 3 underwent similar assessments after processing with zeta potential and the MACS techniques, respectively. Finally, Sample 4 was provided the following day after a short abstinence of 24 hr, and was analyzed as the previous samples. Based on the results, Sample 1 had the highest sperm count, and MACS preserved more sperm compared with zeta potential. By applying the three mentioned strategies, the percentage of sperm cells with progressive movement increased significantly, with MACS being more effective compared with the zeta potential (p < .001). MACS and zeta potential methods provided more sperm with normal morphology and fewer sperm with abnormal SCD/CMA3 tests (p < .001). These three strategies may provide sperm exhibiting better DNA integrity, with the MACS being the superior approach.
Introduction
Sperm function tests are gaining increased popularity in the era of assisted reproductive technology (ART). While routine assessment of male fertility potential relies on semen analysis, more advanced tests, such as sperm DNA fragmentation index (DFI), are proving to be more beneficial in certain cases. Couples with apparently normal semen analysis of the male partner but increased sperm DFI may experience recurrent spontaneous pregnancy loss (especially in the first trimester), ART failure, or unexplained infertility (Dai et al., 2021; Ghuman et al., 2023). Sperm DNA damage can stem from intrinsic factors, including abortive apoptosis, and extrinsic factors, such as immaturity of sperm or activated leukocytes (Muratori et al., 2019).
In ART, achieving a successful pregnancy often hinges on selecting a single sperm of the highest quality. Current strategies aimed at improving sperm DNA integrity include addressing underlying conditions, such as varicocele and male accessory gland infection, lifestyle modifications, such as weight loss and smoking cessation (Esteves et al., 2020), prescribing empirical antioxidants, practicing frequent ejaculation, or short abstinence periods (24 h or less) (Marinaro & Schlegel, 2023; Vahidi et al., 2021), employing advanced sperm processing techniques (Zhang et al., 2024), and considering testicular sperm extraction (Benchaib et al., 2024). These approaches are intended to enhance sperm quality and potentially improve the outcomes of ART.
There are various advanced techniques for sperm selection. Magnetic activated cell sorting (MACS), zeta potential, physiologic intracytoplasmic sperm injection (PICSI), intracytoplasmic morphologically selected sperm injection (IMSI), and microfluidics are some of the techniques implemented for selecting sperm (Vahidi et al., 2022).
MACS is effective for detecting apoptotic sperm and is based on the externalization of phosphatidylserine from the inner to the outer surface of the sperm cell membrane, which occurs in the early stages of apoptosis. Annexin-V protein has an affinity for binding to phosphatidylserine, and thus, Annexin-V conjugated with biodegradable magnetic microbeads in combination with a magnetic field can be used to separate defective sperm in the early stages of apoptosis. Some authors suggest that MACS may provide sperm with better DNA integrity (Pinto et al., 2021). Zeta potential exploits the negative charge (–16 to —20 mV) on mature sperm’s outer surface to bind to positively charged centrifuge tubes, thereby retaining mature sperm while immature sperm and debris wash away. This technique may also be associated with better DNA integrity and ART outcomes (Chan et al., 2006; Kheirollahi-Kouhestani et al., 2009).
Applying short abstinence (≤ 24 hr) is based on the stressor effect of the epididymis on vulnerable sperm, potentially attributed to high levels of reactive oxygen species produced by immature sperm, activated leukocytes, and epididymal epithelial cells (Vahidi et al., 2021). Since the epididymis poses stress on sperm cells during their transition, applying short abstinence could potentially decrease this exposure and result in obtaining sperm with improved DFI. While the World Health Organization (WHO) recommends an abstinence period of 2 TO 7 days for semen collection (World Health Organization, 2021), shorter abstinence may be beneficial for patients with increased sperm DFI (Hanson et al., 2018). Previous studies have discussed the advantages and disadvantages of advanced sperm selection techniques (Pinto et al., 2021). MACS, for example, is an advanced and expensive sperm processing technique that is not available in most andrology labs or ART centers. Moreover, a considerable sperm concentration is required for performing MACS. Zeta potential, in contrast, is less expensive than MACS but is operator-dependent and requires experienced technicians. To the best of our knowledge, short abstinence, while seemingly simple, has not yet had its efficacy compared with advanced sperm processing techniques. This study aimed to compare the efficacy of these three methods (MACS, zeta potential, and short abstinence) for obtaining sperm with improved DNA integrity in patients with increased sperm DNA fragmentation in their ejaculated semen samples.
Material and Methods
Patients
Thirty-eight male patients who were referred to our center were primarily enrolled in the study. Measurement of sperm DFI is mainly advocated in patients with unexplained infertility, recurrent pregnancy loss (RPL), recurrent abortion (more than two abortions), among others (Agarwal et al., 2016). For this reason, male partners of couples with increased sperm DFI (sperm chromatin dispersion [SCD] more than 18% in our lab) who had recurrent ART failures (more than two in vitro fertilization [IVF]/intracytoplasmic sperm injection [ICSI] failures) or recurrent abortions (two or more consecutive abortions) were recruited.
Cases with moderate to severe oligoasthenoteratozoospermia (sperm concentration less than 5 million cells per milliliter), pyospermia (more than 1 million white blood cells [WBCs] per milliliter of semen), known genetic defects (including karyotype or azoospermia factor c [AZFc] abnormalities), varicocele, history of smoking-alcohol consumption-undescended testis, and known hormonal derangement were excluded from the survey. A single urologist supervised all the patients.
Collecting the Semen Sample
The patients were instructed to provide a semen sample through masturbation after abstaining for 2 to 3 days. The specimen was divided into three parts for analysis. The first part (neat sample or Sample 1) was used for semen analysis and sperm DNA integrity/protamination evaluation based on the WHO 2021 criteria (World Health Organization, 2021) and SCD/chromomycin A3 (CMA3) indices, respectively. The second and third parts (Samples 2 and 3) underwent similar assessments after processing with zeta potential and the MACS techniques, respectively.
In addition, the patients were asked to provide another semen sample the following day after a short abstinence duration of 24 h (Sample 4). Semen analysis and DNA integrity/protamination assessment were also performed for this sample.
A brief description of the methods and indices used in this study is provided in Table 1.
A Brief Description of the Implemented Methods and Indices.
Note. DFI = DNA fragmentation index; SCD = sperm chromatin dispersion; CMA3 = chromomycin A3; MACS = magnetic activated cell sorting.
Conventional Semen Analysis
After semen liquefaction at room temperature, the samples were analyzed by light microscopy, following the instructions outlined in the WHO 2021 guideline (World Health Organization, 2021). Sperm morphology was assessed based on the strict criteria (Menkveld et al., 1990), and sperm motility was graded as progressive, nonprogressive, and immotile (World Health Organization, 2021).
SCD and CMA3 Tests
Assessment of sperm DNA damage and protamination was conducted using the SCD and CMA3 tests, respectively. Sperm DFI was assessed using the SCD technique and the Halosperm kit as previously described (Gill et al., 2019). The process involved preparing a mixture of melted agarose and sperm cells. Ten microliters of this mixture were placed at the center of a super-coated slide. After denaturation, lysis, and dehydration, the sperm cells were stained with eosin and thiazine and examined under light microscopy at 1,000× magnification. Evaluating more than 300 sperm cells for each sample, DFI was determined based on the size of the halo surrounding spermatozoa. Spermatozoa with large or medium-sized halos were categorized as having low DFI, whereas those with small or no halos were labeled as having high DFI. The percentage of sperm cells with abnormal DNA fragmentation was also recorded.
Sperm protamination status was assessed by CMA3 staining as described by others (Vahidi et al., 2018). Briefly, Carnoy’s solution was added to air-dried smears and fixed at 4°C for 10 min. The smears were then stained with CMA3 solution for 10 min, washed, and evaluated under a fluorescent microscope (Olympus BX51, Japan) at 1,000× magnification using a specific filter (464–470 nm). Spermatozoa appearing bright yellow or dull yellow were classified as CMA3-positive and CMA3-negative, respectively. The percentage of abnormal spermatozoa (CMA3-positive) was reported accordingly.
MACS Technique
For sperm selection using the MACS approach, the semen sample was first washed and then mixed with a combination of Annexin V microbeads and binding buffer from the MACS kit (Miltenyi Biotec, Germany). After an additional wash and a 15-min incubation at 4°C, another 1 ml of binding buffer was added to the sample. Subsequently, the mixture was loaded into a MiniMACS column (Miltenyi Biotec, Germany) under a magnetic field. Annexin V-positive cells were retained within the column, while Annexin V-negative (nonapoptotic) cells passed through.
Zeta Potential Technique
The technique was performed similarly to the protocol previously described (Nasr Esfahani et al., 2016). After undergoing density gradient centrifugation (DGC), the yielded pellets were washed with albumin-free Ham’s F-10 medium. The pellets were then diluted with 4 ml of Ham’s F-10 and resuspended in 5 ml centrifuge tubes. Subsequently, the centrifuge tube, enclosed in a latex glove, was twisted two or three turns and swiftly removed from the glove to expose spermatozoa to a positive electrical charge. The tube was then left at room temperature for 1 min, allowing the adherence of the high-quality sperm cells to the charged tube wall. The medium containing nonadhering spermatozoa was then removed from the tube. To neutralize the positive charge and recover adhered spermatozoa, the inner surface of the tube was washed with 4 ml of Ham’s F-10 containing albumin (making a total volume of 5 ml). Following this step, the tube was centrifuged at 300 g for 5 min. The pellet of spermatozoa was then carefully resuspended in 1 ml of Ham’s F-10 supplemented with albumin, providing cells appropriate for ICSI.
Short Abstinence
For assessing the effects of short abstinence on sperm quality, the patients were instructed to provide a second semen specimen with an interval shorter than 24 h from the first one, as this cut-off has been associated with low rates of DNA fragmentation in some studies (Hanson et al., 2018).
Ethical Considerations
This study was approved by the Institutional Review Board and Ethics Committee of our institution. Informed written consent was obtained from all the participants, and all of the experiments were performed based on the guidelines outlined by the Declaration of Helsinki.
Statistical Analysis
The data were summarized using mean and standard deviation (SD), as well as median and interquartile range (IQR). The Shapiro–Wilk test was used to evaluate the normality of data distribution. The Friedman test was employed to compare the parameters among the four samples. To identify each pair with significant differences, we conducted post hoc analyses. Bonferroni correction was applied to control for multiple comparisons. All statistical analyses were performed using IBM SPSS version 26 for Windows (IBM Corp., Armonk, NY, USA). The p-value < .05 was considered statistically significant.
Results
Applying the exclusion criteria, 30 men were finally enrolled for the assessment of sperm quality parameters.
Table 2 summarizes the results of semen analysis, SCD, and CMA3 tests conducted on the neat sample, as well as samples provided using each technique (i.e., MACS, zeta potential, and short abstinence). Sperm concentration, motility, and morphology were the parameters investigated during semen analysis.
Comparative Analysis of Various Sperm Parameters Across Four Different Samples.
Note. Values with similar uppercase letters had statistically significant differences in pairwise comparisons. a <.001; b <.001; c .002; d <.001; e <.001; f.019; g.016; h.007; i.031; j.012; k.027; l.001. MACS = magnetic activated cell sorting; SD = standard deviation; IQR = interquartile range; SCD = sperm chromatin dispersion; CMA3 = chromomycin A3.
The sperm count was significantly higher in Sample 1 than in Samples 2, 3, and 4. In addition, Sample 2 had significantly more sperm cells compared with Sample 3. Finally, sperm count in Samples 3 and 4 showed a significant difference.
On the contrary, Sample 1 exhibited the lowest progressive motility, implying that applying the mentioned techniques may aid in selecting sperm cells with better motility. Moreover, sperm cells provided with the MACS technique were significantly more motile than those found in specimens processed with zeta potential. Short abstinence also yielded more progressive sperm; however, these cells did not show significantly different motility compared with cells selected using other techniques. It should be noted that the nonprogressive motility was not significantly different among the four samples, indicating that none of the methods had a significant effect on the proportion of sperm that moved in a nonlinear fashion.
In addition, the control sample contained the highest proportion of immotile cells. The sperm cells provided by the MACS technique showed the lowest rate of immobility, with a significantly different rate compared with Sample 3.
Regarding sperm morphology, the neat sample contained the lowest rate of sperm with normal morphology, compared with Samples 2 and 3. However, the rate of sperm cells with acceptable morphology did not differ significantly while short abstinence was applied.
Using the SCD test, it was revealed that Sample 1 had the highest rate of abnormal cells, which was significantly higher than those of Samples 2 and 3, demonstrating that both the MACS and zeta potential techniques reduced the percentage of sperm with DNA damage. While the DFI of sperm cells analyzed after short abstinence was higher than cells provided after processing with MACS and zeta potential techniques, no significant differences regarding DFI were evident when comparing Samples 1 and 4.
Similar to the pattern revealed by the SCD assay, Sample 1 had the highest cells with abnormal protamination in the CMA3 test. This may indicate that the mentioned techniques could be effective in selecting the sperm cells with improved protamination. Furthermore, it should be noted that short abstinence provided a significantly higher rate of abnormally protaminated cells compared with the MACS approach.
Discussion
Factors, such as sperm morphology, sperm motility, sperm concentration, and DFI, play a significant role in analyzing semen quality and male fertility (Tanga et al., 2021). In this study, these variables were measured accordingly, providing valuable insights into the condition of sperm provided using different approaches.
To our knowledge, this is the first study to compare the results of three commonly used sperm selection techniques (MACS, zeta potential, and short abstinence) in obtaining sperm cells of improved quality. ART imposes a tremendous economic and psychological burden on infertile couples and insurance companies (Njagi et al., 2023). Besides the direct financial burden of the conventional ART processes, using the sperm processing techniques in patients with increased sperm DFI may bring additional costs. In this regard, choosing a more affordable method with moderate to high efficacy seems rational. Since andrologists are on the front lines of managing patients with increased sperm DFI, they have an undeniable role in addressing this issue before proceeding to ART. Therefore, choosing the appropriate technique remains a challenge for these specialists. For this purpose, the current study aimed to compare the effectiveness of different common sperm selection techniques of various expenditures (in descending order: MACS, zeta potential, and short abstinence). The results showed that more advanced approaches, such as MACS, may be more effective than the other aforementioned methods in obtaining sperm cells with better gross quality (progressive motility and normal morphology) and DNA integrity (lower sperm DFI and acceptable chromatin packaging). However, it was noted that sperm concentration may decrease as a result of the processing technique.
It has been demonstrated that infertile men may have a higher proportion of spermatozoa undergoing pro-apoptotic processes (initiated programmed cell death), despite having normal motility and morphology (Garrido & Gil Juliá, 2024). Consequently, embryos derived from such spermatozoa are more likely to experience developmental arrest. Since conventional selection of sperm based on motility and morphology (as typically done in ICSI) cannot identify these pro-apoptotic sperm, MACS sperm selection, which is based on the externalization of phosphatidylserine, can address this issue (Garrido & Gil Juliá, 2024; Salehi Novin et al., 2023).
The result also demonstrated that in men with increased sperm DFI, a neat semen sample (without any processing) exhibits lower quality and decreased DNA integrity (higher DFI and incomplete protamination) compared with other methods. Therefore, it appears crucial to implement sperm selection techniques of any type, whether simple or advanced, for such patients. In other words, limited access to advanced sperm processing techniques should not discourage the application of alternative methods.
The current study is in line with that of Bucar et al. and Salehi Novin et al., in which processing of 100 (randomly assigned to five groups of 20 samples) and 60 semen samples, respectively, showed that MACS alone or in combination with DGC or swim up (SU) techniques can result in selecting sperm cells with better DNA integrity (Bucar et al., 2015; Salehi Novin et al., 2023).
In contrast, Cakar et al. evaluated 20 males (10 normozoospermic samples and 10 oligozoospermic samples) and showed that MACS in combination with SU or DGC, compared with SU or DGC alone, improved DNA integrity and protamination. However, this improvement was not statistically significant. This may be due to either their small sample size or the lack of data regarding the neat semen sample DFI (before processing) (Cakar et al., 2016).
Despite the informative results of the current study, several limitations still exist. The number of recruited participants is quite limited, which could be partly due to the high cost of the MACS kit. Accordingly, the current findings should be further examined in larger prospective studies. In terms of obtaining sperm with better DNA integrity, although it has been shown that MACS is superior to other strategies, such as zeta potential and short abstinence, whether this superiority is of clinical importance remains to be elucidated. Therefore, further studies can focus on assessing the rate of successful fertilization following each of the three techniques. Moreover, sperm DNA integrity/protamination was evaluated using SCD and CMA3 techniques due to the lack of access to flow cytometric system. Hence, it would be reasonable to further confirm the current results by more advanced techniques, such as sperm chromatin structure assay (SCSA) in upcoming experiments.
Proposed stepwise approach for patients with high sperm DFI
In our center, a stepwise approach is implemented for patients with increased sperm DFI (Figure 1). Initially, couples with RPL or those with unexplained infertility facing recurrent ART failures should be evaluated regarding maternal (e.g., anatomical abnormalities) or embryonal (e.g., chromosomal abnormalities) factors (Ma et al., 2022). After ruling out these etiologies and confirming the presence of high sperm DFI in the male partner, modifying the underlying conditions (that can lead to increased sperm DNA injury) is advocated. These conditions may include various diseases (such as varicocele and prostatitis), lifestyle-related factors (including obesity, sedentary lifestyle, smoking, and opium addiction), and occupational contact (heat and chemical exposure, radiation, etc.) (Farkouh et al., 2023). If this strategy fails or if there is no known factor for increased sperm DFI, as the next step, a trial of empirical antioxidant therapy for 3 to 6 months, based on previous published recommendations (Agarwal et al., 2023), may be recommended. If sperm DFI remained high despite medical therapy, obtaining a sample by applying short abstinence (24 h or less) may be beneficial (Vahidi et al., 2021). In nonresponders to previous strategies, advanced sperm processing techniques (such as zeta potential, MACS, IMSI, PICSI, and microfluidics) may be implemented during ART cycles based on semen characteristics (Ribas-Maynou et al., 2022). If the sperm DFI remained high despite using advanced sperm selection techniques, either a simple ICSI procedure or testicular sperm extraction plus ICSI may be proposed as the last option. Testicular sperm cells have not been exposed to the oxidative stress exhibited during transportation in the genital tract, justifying their application as cells with lower DFI (Esteves et al., 2015). Some published studies have indicated that ICSI may result in better pregnancy outcomes in patients with high sperm DFI (Chi et al., 2017), while there are controversies about the benefit of testicular sperm compared with their ejaculated counterparts in terms of ART outcomes (Ambar et al., 2021). Generally, guidelines remain cautious regarding testicular sperm extraction in patients with high sperm DFI and suggest this approach when other available strategies fail (Marinaro & Schlegel, 2023). Based on a recent meta-analysis, testicular sperm may lead to better pregnancy outcomes in this group of patients (Zhao et al., 2023); however, this analysis included studies with questionable quality. Notably, the surgical complications associated with this invasive procedure, such as hematoma, infection, and potential damage to testicular arteries (total testis loss), can further limit its widespread application (Ambar et al., 2021). Therefore, patients should be carefully selected, and extensive consultation should be performed before the procedure.
A Step-Wise Approach for Managing Patients with High Sperm DFI.
Conclusion
Sperm selection techniques may provide sperm cells with better DNA integrity compared with neat semen samples. The MACS technique may be superior to zeta potential and short abstinence in this regard. However, the clinical significance of this finding in addressing RPL or unexplained infertility should be further evaluated in future studies.
Footnotes
Acknowledgements
None
Ethical Consideration
The current study was approved by the Institutional Review Board and Ethics Committee of Iran University of Medical Sciences (Approval ID: IR.IUMS.FMD.REC.1399.533).
Consent to Participate
Informed written consent was obtained from all the participants, and all of the experiments were performed based on the guidelines outlined by the Declaration of Helsinki.
Consent for Publication
Not applicable.
Funding
The authors received no financial support for the research, authorship, and/or publication of this article.
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Data Availability Statement
The data supporting the current study’s findings are available from the corresponding author upon reasonable request.