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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 10  |  Issue : 3  |  Page : 126-130

Aniline blue staining method for sperm DNA fragmentation index: A guide to sperm quality and infertility management – An observational study in males from South Maharashtra


1 Professor, Department of Anatomy, Bharati Vidyapeeth (Deemed to be University) Medical College and Hospital, Sangli, Maharashtra, India
2 Professor and Head, Department of Anatomy, Bharati Vidyapeeth (Deemed to be University) Medical College and Hospital, Sangli, Maharashtra, India
3 Professor, Department of Pathology, Bharati Vidyapeeth (Deemed to be University) Medical College and Hospital, Sangli, Maharashtra, India

Date of Submission06-May-2021
Date of Decision09-Jun-2021
Date of Acceptance20-Jul-2021
Date of Web Publication30-Jul-2021

Correspondence Address:
Priya Santosh Patil
Department of Anatomy, Bharati Vidyapeeth (Deemed to be University) Medical College and Hospital, Sangli, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/NJCA.NJCA_31_21

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  Abstract 


Background: Infertility rate all over the globe is on the rise and semen analysis forms the only basic investigation for male infertility. The success of infertility treatment depends on many semen parameters. In this study, we focus on sperm DNA fragmentation (SDF) by the aniline blue staining method. The DNA fragmentation index (DFI) was correlated with other semen parameters. The semen parameters in low DFI and high DFI (HDFI) groups were compared to highlight the importance of DFI in infertility management. Methodology: The sample size was 60. The semen parameters were studied. The sperm chromatin integrity and DFI were calculated. Statistical analysis was performed by calculating mean, standard deviation, Spearman's correlation coefficient, and unpaired t-test. Results: DFI showed a significant negative correlation with sperm count, progressive motility of sperms, sperm vitality, and a positive correlation with sperm deformity index (SDI). The cases were grouped according to DFI and showed statistically significant differences in the two groups with respect to sperm count, motility, sperm vitality, and SDI indicating poor sperm quality. HDFI group underwent prolonged infertility treatment. Conclusion: Acidic aniline blue staining method is a feasible screening test for sperm DFI which correlates well with semen analysis parameters such as low sperm counts, low progressive motility, low sperm vitality, and high SDI. A high SDF has a negative impact on infertility management.

Keywords: Aniline blue, chromatin, DNA fragmentation, infertility, semen


How to cite this article:
Patil PS, Mudiraj NR, Mane VP. Aniline blue staining method for sperm DNA fragmentation index: A guide to sperm quality and infertility management – An observational study in males from South Maharashtra. Natl J Clin Anat 2021;10:126-30

How to cite this URL:
Patil PS, Mudiraj NR, Mane VP. Aniline blue staining method for sperm DNA fragmentation index: A guide to sperm quality and infertility management – An observational study in males from South Maharashtra. Natl J Clin Anat [serial online] 2021 [cited 2021 Dec 8];10:126-30. Available from: http://www.njca.info/text.asp?2021/10/3/126/322803




  Introduction Top


The infertility rate all over the globe is on a rise and 40%–50% is due to the male factor.[1] Semen analysis forms the basic, noninvasive investigation for male infertility. Although none of the currently used parameters accurately predict sperm quality, the diagnosis and treatment rely greatly on the semen reports.[2] The threshold values of routine semen parameters are decreasing.[3] As infertility is considered a social taboo in developing countries, an infertile couple has to go through a lot of social, psychological, and financial stress during treatment.[4] Scientific advances give an infertile couple many options of assisted reproduction techniques (ARTs). The success of ART depends on many factors, semen being equally important where recently sperm DNA has gained crucial value. There are various DNA fragmentation index (DFI) assessment techniques which are recommended for use with different principles underlying their use. Some of them require sophisticated laboratory equipment including fluorescent microscopes or expertise which may not be available at all places. However, some of the tests are easy to perform as well as interpret and can provide valuable information. Many of these options are still very costly and out of reach of many clinicians as well as patients.[5],[6]

It is essential to try to reduce the burden on the infertile couple when offering the best possible investigations and treatment options. In a developing country like ours, it is not always possible to perform all high-end and costly investigations in every couple. Hence to alleviate the above problem, we aim to focus on the utility of the acidic aniline blue staining method for sperm DFI. This cost-effective method can be easily adopted by all laboratories even in the peripheral rural areas. In this study, we also observe the correlation between DFI and other semen parameters as well as compare the semen parameters in the high DFI group (HDFI) with that of the low DFI (LDFI) group.


  Methodology Top


Study design

This cohort, observational study was conducted at the Department of Anatomy, at a Tertiary Care Hospital and Medical College located in South Maharashtra.

Sample size

As per previous study[7] the mean DFI in the infertile group was 32.88 ± 7.41 and in fertile group was 22.5 ± 2.81. Taking effect size 1.85, α (level of significance) as 1%, and power 95%, the minimum sample size obtained from statistical software G power 3.1 was n = 26, hence we included 60 cases considering 30 in each group having LDFI and HDFI.

Sixty male subjects coming to the laboratory for semen analysis as a part of infertility treatment during the study period of 2 years were included. Informed consent was taken and the subjects were asked to collect semen samples by masturbation after 3–4 days of abstinence. Male subjects who came for semen analysis but had some medical or surgical problems like fever, hydrocele, varicocele which may alter semen parameters were excluded. Required Institutional Ethical Committee clearance was obtained (Letter no.BVDUMCandH/Sangli/IEC/149/15 Dated: October 8 and 9, 2015).

Sample collection and semen analysis

The samples were collected in sterile containers, allowed to liquefy and then observed as a wet mount under the microscope for sperm motility and sperm count as per procedure given in WHO Manual.[3]

Other sperm parameters studied were sperm morphology, sperm vitality, and sperm chromatin integrity.

Sperm morphology

To determine the sperm morphology and Sperm deformity index (SDI), the slides were stained by Papanicolaou stain [Figure 1]. Two hundred sperms were observed under ×100 and categorized into normal and abnormal. The percentage of abnormal sperms and SDI was then calculated.[3],[8]
Figure 1: Papanicolaou stained slide under ×100 for sperm morphology. Arrows show abnormal spermatozoa

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Sperm vitality

The sperm vitality was observed after mixing 10 ul of semen with 50 ul Eosin-Nigrosin stain and then preparing the smears after 30 s. The slides were observed under ×100, the unstained sperms were counted as vital and pink colored as dead/nonvital [Figure 2].
Figure 2: Eosin - Nigrosin stained slide under ×100 for sperm vitality. Arrows show dead spermatozoa

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Sperm chromatin

To determine sperm chromatin integrity, semen slides were prepared by the feathering method. They were fixed in 3% glutaraldehyde for 30 min, followed by staining with Acidic Aniline blue for 5 min.[9],[10] The stained slides were seen under ×100. The sperms with unstained or lightly stained nuclei were counted as normal, while the dark blue stained sperms were counted as abnormal or having fragmented DNA [Figure 3]. A total of 200 sperms were counted and the DFI was recorded as percentage, i.e. the number of fragmented sperms per 100 sperms counted.
Figure 3: Acidic aniline blue stained slide under ×100 for sperm chromatin. Arrow shows spermatozoa with fragmented DNA (with darkly stained head as compared to those with unstained or lightly stained adjacent sperm heads)

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Statistical analysis

All the readings of variables were entered in Excel sheet and a master chart was prepared. Statistical analysis was done by calculating mean, standard deviation, and unpaired “t” test to check the significant difference in the means of various sperm parameters. Analysis was done using Microsoft excel and SPSS 22.0 statistical analysis software (IBM Corp, Armonk, New York, USA).


  Results Top


The current study was done on 60 male subjects undergoing treatment for male infertility and who had visited the laboratory for a semen analysis. The semen parameters such as sperm count, motility, morphology, sperm vitality, and sperm chromatin were recorded. The SDI and DFI were calculated. The DFI was correlated with other semen parameters as shown in [Table 1]. It was observed that the DFI showed a significant negative correlation with sperm count, progressive motility of sperms, sperm vitality, and a positive correlation with SDI. This indicates that DFI was high in samples having a low sperm count, progressive motility, and sperm vitality.
Table 1: Correlation of DNA fragmentation index with other semen parameters

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The cases were further grouped according to DFI into two groups as shown in [Table 2].
Table 2: Comparison of semen parameters in low DNA fragmentation index and high DNA fragmentation index groups

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Group 1 had subjects with a LDFI of <20– LDFI, while Group 2 included subjects with HDFI, equal to or more than 20. The mean DFI in Group 1 was 11.8%, while in Group 2 was 34.5%. Out of the total 60 males who underwent semen analysis as a part of infertility work-up, 43.3% (26) had a LDFI (<20), while 56.6% (34) had a HDFI (≥20).

The various semen parameters were correlated in these two groups. The age and weight of subjects were recorded. The mean age and mean weight were compared in both the groups as shown in [Table 2]. There is no statistically significant difference in the mean age and weight of subjects with LDFI and HDFI.

The sperm count was compared in the two groups as shown in [Table 2]. The mean of total sperm count in the HDFI was less; 40.9 million/ml as compared to the LDFI group. There is statistically significant difference in the total sperm count in both groups. The mean of the percentage of sperms showing progressive motility in the HDFI group is lower (27.2%) than in the LDFI group. There is a statistically significant difference in the progressive sperm motility among LDFI and HDFI.

The sperm vitality and sperm morphology in the form of SDI were compared in the two groups as per [Table 2]. The mean of the percentage of vital sperms in the HDFI group was slightly lower (56.8%) than in the LDFI group. The mean of SDI in the HDFI group is also higher (1.19) than in the LDFI group. There is statistically significant difference in sperm vitality and SDI among HDFI and LDFI.

The follow-up and feedback of the cases to know the infertility outcome, we tried to take a phone interview of all the participants in this study after 6 months and 1 year of semen analysis. The response from the participants was very poor and they were reluctant to talk. Some of the subjects refused to share any information even though they had given written consent initially. There were many of them who gave a history that they have changed their previous doctor or the laboratory. Out of 60 participants, 35 responded but did not agree to share their reports or treatments. Out of 35, those having LDFI were 19 and HDFI were 16. In the LDFI group, only 1 subject was still undergoing treatment for infertility after 1 year. In the HDFI group, 14 of them were still undergoing infertility treatment, only 2 had a female child aged 10 months and 4 months, respectively, during the 1 year of follow-up [Table 3].
Table 3: Follow-up of cases

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  Discussion Top


A routine semen analysis forms an important aspect of the infertility workup of a couple coming for treatment of infertility. In this study, the DFI showed negative correlation with other semen parameters such as sperm count, progressive motility of sperms, sperm vitality, and a positive correlation with SDI, hence proving that DFI reflects the quality of semen. Recent studies have highlighted the clinical significance of DFI as well as its correlation with various sperm parameters.[11],[12]

Out of the 60 semen samples examined, 56.6% showed a mean DFI of 34.5% which is quite high. The fact that there are chances of more than 57% of participants having such a HDFI highlights the need to include the sperm chromatin assessment and DFI in all semen analyses particularly done for assessment of male infertility when female parameters are normal. In this study, it was seen that the age or weight of the subjects showed no statistically significant difference when compared with the DNA fragmentation in LDFI and HDFI groups, though the mean age and weight were higher in Group 2 with HDFI. This may be due to the small sample size of the current study. In the study by Sheikh et al., there was no correlation of DFI and age,[6] while in another study by Al Omrani et al. in 2018, the authors found a positive correlation between HDFI and age as well as body mass index.[11] When the other sperm parameters viz: sperm count, motility, vitality, and morphology were compared, they showed a statistically significant difference in both groups. DFI had a negative correlation with sperm motility, vitality, and morphology.

Similar findings were observed by authors when they compared sperm parameters in fertile and nonfertile groups.[6],[13] In a recent study in 2020, it was shown that sperm DFI can be used as a measure of sperm quality in cryopreserved semen samples of mice.[14] A review in February 2020 also discussed the role of protamination in sperm chromatin integrity and causes of fragmentation.[15] The mean DFI in infertile males was quite higher; 48.7% in their study as compared to the mean in fertile males which was 12.9%. This finding adds to our finding that if we group patients according to the DFI, it can serve as a guide for the choice of fertility treatment options like a normal conception to intra-uterine insemination, in vitro fertilization or intra-cytoplasmic sperm injection.

In this study, during the follow up it was seen that 94.7% cases in the LDFI group had conceived during a period of 1 year while 87.5% cases in the HDFI group were still under infertility treatment. In a study where authors compared the ART outcome in males having HDFI, they saw that fertilization, cleavage or blastulation was not affected by DNA fragmentation or showed no significant statistical correlation, but they had a problem in implantation and continuation of pregnancy. They showed that all patients who achieved pregnancy had DFI <27% and no pregnancy was achieved in patients with DFI >27%.[5]

In this study, we used the acidic aniline blue staining method based on the affinity of aniline blue to histones, which is also recommended by many others.[9],[10],[16] In a review article by Agarwal et al., it has been clearly mentioned as to when to use DFI and how it can be a guide to the clinicians.[16] In another article by Mathur, in 2017, the author suggests that sperm DNA fragmentation (SDF) can be used as a very useful routine diagnostic tool which cannot be taken lightly.[17] In the comment by Pandiyan et al. regarding perspective on SDF, in 2017 the authors suggest that there are always going to be sperms with fragmented DNA in a routine semen sample. They are of the opinion that routine assessment of sperm DNA may not be recommended.[18] This perspective may be justified, as there are many different costly tests available and may not be affordable or accessible to all. However, the aniline blue staining method used here is easy to perform, cost-effective yet gives a clear differentiation between normal (light stained or unstained) and fragmented (darkly stained) sperms seen under a routine microscope having an oil immersion lens. This method can be easily followed in all laboratories having basic infrastructure, so can be utilized to include sperm chromatin assessment and DFI in every routine semen report at an affordable cost.

In developing countries like ours, fertility is a boon and infertility a curse, which comes with a social stigma, emotional and financial strain on the couple. In such a scenario, the availability of such a cost-effective test, which will provide valuable insight and clear-cut direction for further treatment options will be a real savior.

After the initial assessment of sperm chromatin by acidic aniline blue staining, the samples which show a HDFI that correlates with a history of infertility, bad obstetric history or ART failures can be segregated and assessed with more sophisticated and costly tests at higher centers. This would help in a focussed analysis of male infertility as well as reduce the financial burden on the couple.

Limitations of the study

A small sample size and poor follow-up due to the lack of patient compliance in our area was the main limitation of this study.


  Conclusion Top


The acidic aniline blue staining method is a feasible screening test for sperm DFI and correlates well with semen analysis parameters such as low sperm counts, low progressive motility, low sperm vitality, and high SDI. A high DFI has a negative impact on infertility management.

Acknowledgments

The authors would like to thank Bharati Vidyapeeth (Deemed to be University), Pune for the funding and BV (DU) MCH, Sangli for their support during this study. A heartfelt gratitude to the Dean of the Institute, HOD, faculty and nonteaching staff of the Department of Anatomy, Bharati Hospital Laboratory and outside laboratories for their co-operation. The authors pay respect towards all the subjects who have participated in this study. Sincere gratitude towards all the authors and researchers whose books and research work facilitated this study.

Financial support and sponsorship

The authors would like to thank Bharati Vidyapeeth (Deemed to be University), Pune for the funding, and BV (DU) MCH, Sangli for their support during this study.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Ganiga P, Pujeri S. Study on semen analysis in the evaluation of male infertility in coastal Karnataka, India. Int J Reprod Contracept Obstet Gynecol 2014;7:2603-7.  Back to cited text no. 1
    
2.
Komiya A, Kato T, Kawauchi Y, Watanabe A, Fuse H. Clinical factors associated with sperm DNA fragmentation in male patients with infertility. ScientificWorldJournal 2014;2014:868303.  Back to cited text no. 2
    
3.
World Health Organization. WHO Laboratory Manual for the Examination and Processing of Human Semen. 5th ed. Geneva, Switzerland: WHO Press; 2010.  Back to cited text no. 3
    
4.
Joshi P, Gopal N, Bhat V. Study of semen analysis patterns in infertile males. Int J Pharm Bio Sci 2011;1:44-9.  Back to cited text no. 4
    
5.
Larson-Cook KL, Brannian JD, Hansen KA, Kasperson KM, Aamold ET, Evenson DP. Relationship between the outcomes of assisted reproductive techniques and sperm DNA fragmentation as measured by the sperm chromatin structure assay. Fertil Steril 2003;80:895-902.  Back to cited text no. 5
    
6.
Sheikh N, Amiri I, Farimani M, Najafi R, Hadeie J. Correlation between sperm parameters and sperm DNA fragmentation in fertile and infertile men. Iran J Reprod Med 2008;6:13-8.  Back to cited text no. 6
    
7.
Venkatesh S, Shamsi MB, Deka D, Saxena V, Kumar R, Dada R. Clinical implications of oxidative stress and sperm DNA damage in normozoospermic infertile men. Indian J Med Res 2011;134:396-8.  Back to cited text no. 7
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8.
Aziz N, Buchan I, Taylor C, Kingsland CR, Lewis-Jones I. The sperm deformity index: A reliable predictor of the outcome of oocyte fertilization in vitro. Fertil Steril 1996;66:1000-8.  Back to cited text no. 8
    
9.
Patil P, Bambulkar S, Ajgaonkar S, Patil R, Patil A, Nikam V. DNA fragmentation index [DFI] of human semen by modified aniline blue method. Cibtech J Bioprotocols 2013;2:1-5.  Back to cited text no. 9
    
10.
Agarwal A, Tsarev I, Erenpreiss J, Said TM. Sperm chromatin assessment. In: Gardner DK, Weissman A, Howles CM, Zeev S, editors. Textbook of Assisted Reproductive Techniques Fourth Edition: Volume 1: Laboratory Perspectives. CRC Press; Boca Raton, FL, USA: 2012.  Back to cited text no. 10
    
11.
Al Omrani B, Al Eisa N, Javed M, Al Ghedan M, Al Matrafi H, Al Sufyan H. Associations of sperm DNA fragmentation with lifestyle factors and semen parameters of Saudi men and its impact on ICSI outcome. Reprod Biol Endocrinol 2018;16:49.  Back to cited text no. 11
    
12.
Basar MM, Kahraman S. Clinical utility of sperm DNA fragmentation testing: Practice recommendations based on clinical scenarios. Transl Androl Urol 2017;6:S574-6.  Back to cited text no. 12
    
13.
Patil PS, Patki SM, Patki SS, Patki US. DNA fragmentation index and its correlation with other sperm parameters in 4833 infertile males. Int J Reprod Contracept Obstet Gynecol 2018;7:3718-23.  Back to cited text no. 13
    
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Li MW, Lloyd KC. DNA fragmentation index (DFI) as a measure of sperm quality and fertility in mice. Sci Rep 2020;10:3833.  Back to cited text no. 14
    
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Hamilton TR, Assumpção ME. Sperm DNA fragmentation: Causes and identification. Zygote 2020;28:1-8.  Back to cited text no. 15
    
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Agarwal A, Majzoub A, Esteves SC, Ko E, Ramasamy R, Zini A. Clinical utility of sperm DNA fragmentation testing: Practice recommendations based on clinical scenarios. Transl Androl Urol 2016;5:935-50.  Back to cited text no. 16
    
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Mathur PP. Recognizing sperm DNA fragmentation testing in clinical evaluation of male fertility. Transl Androl Urol 2017;6:S681-2.  Back to cited text no. 17
    
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Pandiyan N, Pandiyan R, Raja DR. A perspective on sperm DNA fragmentation. Transl Androl Urol 2017;6:S661-4.  Back to cited text no. 18
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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