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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 13  |  Issue : 4  |  Page : 190-194

Significance of differentiation of round cell count in semen in infertile men: A cost-effective study


Department of Pathology, Bharati Vidyapeeth (Deemed to be University) Medical College and Hospital, Sangli, Maharashtra, India

Date of Web Publication16-Apr-2019

Correspondence Address:
Dr. Vishrabdha Rahul Pawar
Department of Pathology, Bharati Vidyapeeth Deemed University Medical College and Hospital, Sangli - 416 414, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jdmimsu.jdmimsu_32_17

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  Abstract 


Background: The significance of the presence of immature germ cells (IGCs) and leukocytes in semen have not very frequently been highlighted in the diagnostic criteria of male infertility. Round cell count differentiation aids in better clinical correlation. Aim and Objectives: To differentiate the round cells into IGCs and leukocytes in the semen and to find their correlation with the total sperm count and sperm motility. Materials and Methods: Semen samples from patients at Bharati hospital, Sangli for evaluation of infertility from July 2014 to January 2015 were analyzed as per the WHO criteria. After the consent and collection of the semen sample, routine semen analysis was done. Smears of semen were prepared and stained by Leishman stain. The round cells were counted and differentiated into IGCs and leukocytes. Results: We studied 103 cases. Semen parameters such as total sperm count, motility were noted. The smears were studied for differential counts of round cell count into IGC count and leukocyte. The cases were divided into four groups based on total sperm count. Maximum number of cases was found in group III. Our study shows that round cell count is 5%–10% of the total sperm count. Negative correlation was noted between the total sperm count and IGCs. The correlation of total sperm count and leukocytes was negative and significant. Conclusion: The present study shows that differentiation of round cells in the semen can be effectively differentiated into spermatogenic and nonspermatogenic cells with the use of simple and cost-effective stain like Leishman stain. The correlation of sperm count with the IGCs and leukocytes and sperm motility is of immense use to find out the possible cause of infertility.

Keywords: Immature germ cells, infertility, round cells, semen


How to cite this article:
Balasubramaniam S, Pawar VR. Significance of differentiation of round cell count in semen in infertile men: A cost-effective study. J Datta Meghe Inst Med Sci Univ 2018;13:190-4

How to cite this URL:
Balasubramaniam S, Pawar VR. Significance of differentiation of round cell count in semen in infertile men: A cost-effective study. J Datta Meghe Inst Med Sci Univ [serial online] 2018 [cited 2019 Jul 19];13:190-4. Available from: http://www.journaldmims.com/text.asp?2018/13/4/190/256206




  Introduction Top


The WHO defines infertility as the time of 1 year of unprotected intercourse without conception.[1] Approximately about 10%–15% of couples suffer from infertility all over the world. Female factor is responsible for 35% and male factor 45% of cases while the rest of the couples have either the combination or unexplained infertility.[2] In India, the infertility rate is 9% of the reproductive population, and among them, 50% are due to male factors.[3] Pathogenesis of male infertility is multi-factorial, and any alteration to normal physiology of reproductive organs may affect sperm functions resulting in oligozoospermia (low sperm count), asthenozoospermia (loss of motility), teratozoospermia (abnormal morphology), azoospermia (absence of sperms in the ejaculate), and in severe form, oligoasthenoteratozoospermia causes problem for a successful fertilization.[4] Thus, 15% of couples fail to achieve conception in a 1 year period and present to receive treatment for infertility.[5] Nearly 90% of cases male infertility is due to either low sperm count or poor semen quality or the combination of two. Recent studies have indicated that the decline in semen quality and quantity could be due to increased prevalence of sexually transmitted diseases or urogenital infections.[2]

While approaching an infertile couple in India, semen analysis proves to be a cost-effective method keeping in mind the socioeconomic factors in mind. Recommending a semen analysis as a first laboratory evaluation helps to identify the male patients who should be evaluated for treatable malefactors.[6] In addition, the analysis provides information on the functional status of the germ epithelium, epididymis, and accessory sex glands. The results of the semen analysis are taken as a surrogate measure of male fecundity and pregnancy risk.[7] Therefore, semen analysis is an indispensable diagnostic tool in the evaluation of fertility potential of the male partners of infertile couple.

While evaluating a semen sample, we rely on the reference threshold values for semen analysis given by the WHO since 1987. The most recent publication of the 2010 criteria has included lower reference values than the 1999 manual based on a population study of fertile men across 14 countries. The development of the new WHO criteria and application of clear reference values should help to reduce the incidence of misdiagnosis of fertility problems and improve clinical care.[8]

The findings of the semen analysis influence medical management of an infertile couple. The ejaculate invariably contains cells other than spermatozoa, termed as round cells. These round cells indicate forms other than spermatozoa; thus grouping together both leukocytes and immature germ cells (IGCs).[9] The presence of leukocytes may be suggestive of genital infection while others suggest that the enzymes of leukocytes in semen may impair semen quality.[10] Increase in the IGCs in semen is reflected as “stress pattern” in semen.[10] Thus, it is important to differentiate leukocytes from IGCs which are also often observed in semen that are difficult to be distinguished visually. This calls for differentiation of round cells into IGCs and leukocytes.[9] This differentiation ensures proper grouping of the round cells, which efficiently points to the underlying deficient factor. These findings make it easier for the clinician to proceed with the treatment of the infertile men.[9] The round cells observed in semen sample could be either spermatogenic origin or nonspermatogenic origin such as epithelial cells, neutrophils or lymphocytes. According to the WHO manual, ejaculate should contain <5 × 106 round cells/ml while the number of leukocytes should be <1 × 106/ml.[1]

For appreciation and further differentiation of these cells different staining techniques have been used. It is observed that the size of leukocytes in blood smear and semen samples appears the same after using the Papanicolaou stain. Hence, the Papanicolaou stain is usually preferred to stain the smears of semen samples.[2],[9] However, in this study, we have used a more simple and cost-effective Leishman stain. Other leukocytes such as monocytes, lymphocytes, and epithelial cells are differentiated on routine stain by their morphological features. The spermatids and spermatocytes can appear similar to leukocytes mainly neutrophils with the segmented nuclei.[9],[11]

For clinical evaluation, it is of particular importance to distinguish between spermatogenic and nonspermatogenic cells in semen. Complete absence of spermatogenic cells suggests an obstructive cause to the azoospermia that may be corrected by surgery. The presence of spermatogenic cells in semen samples having few if any mature spermatozoa suggest a testicular malfunction where the impaired spermatogenesis could be of endocrinological origin.[9]

The presence of leukocytes in semen is an important factor interfering with the fertilizing potential of spermatozoa. It has been seen that the antibiotic therapy was unable to decrease the presence of white blood cells (WBC) in infertile patients with leukocytes, thus making the management of leukocytes controversial. It has to be evaluated with semen culture and possibly antisperm antibody tests.[6] The prevalence of leukocytospermia (>106 WBC/ml semen) among male infertile patients is approximately 10%–20%. It is associated with decreased sperm number and impaired sperm motility.[1],[12]

The 2010 WHO recommendation is to evaluate progressive motility with a simplified binary categorization in which sperms are classified as either forward progressive (rapid or sluggish) or nonprogressive.[6] The state of having a total motility <40% or progressive motility <32% is referred to as asthenozoospermia.[1] Impaired motility can occur because of abnormal sperm production, varicocele, infection, antisperm antibodies, heat, or other toxic factors.[7]

The significance of round cell differentiation has not commonly been highlighted. In our study, the correlations between IGCs and leukocytes are analyzed with respect to other semen parameters.


  Materials and Methods Top


Inclusion criteria

Semen samples from all male patients who had come to Bharati Hospital, Sangli for evaluation of infertility and who fulfilled the WHO criteria of Infertility, i.e., the period of 1 year of unprotected intercourse without conception, were analyzed.

Exclusion criteria

The patients who did not fulfill the criteria of 1 year of unprotected intercourse without conception were excluded from the study.

Study period

From July 2014 to January 2015 Infertility clinic at Bharati Hospital, Sangli.

A total of 103 males who complained of infertility for the past 1 year voluntarily agreed to participate in the study. The written informed consent was taken in local language The Institutional Ethics Committee guidelines were followed. The patients were asked to abstain from intercourse for 4 days. The samples were collected by masturbation into a clean wide-mouthed plastic semen container. Semen parameters such as total sperm count, motility, and round cells were recorded as per the dilution. These parameters taken were on the basis of the standard values given by the WHO manual. The routine semen analysis includes the macroscopic and microscopic examination. In the macroscopic examination, the following properties were noted.

Physical characteristics of semen

Volume, liquefaction, viscosity, pH, color, and odor.

Microscopic examination including the sperm count sperm motility and progression, sperm morphology. Leukocyte quantification is another part of the routine semen analysis. Although multiple reports point to sperm morphology as the parameter with the most discriminatory power, others indicate that sperm concentration and/or motility are the most valuable ones.

Using a drop of semen, smears were prepared by feathering technique as given in the 5th edition of the WHO manual of semen analysis.

Smears were stained by Leishman stain. The round cells were counted and differentiated into IGCs such as spermatocytes, spermatids, and leukocytes.

The round cells other than the leukocytes include the cells in various stages development of spermatids. They invariably include spermatogonia, spermatocytes, spermatids, and exfoliated epithelial cells. The number of nongerm cells in semen can be estimated in fixed wet preparation by the use of hemocytometer. The prevalence of round cells relative to spermatozoa can be assessed from slides.

Multinuclear spermatids are commonly found in the semen samples and need to be carefully distinguished from neutrophils and lymphocytes. Spermatids that have failed to complete development can be found in semen as multinucleated syncytium like structures containing three or four nuclei that are located in the periphery. These cells can be confused with the lobes of the multilobated nuclei of neutrophils. Keeping these morphological features in mind, the cells were identified, and their count was noted.

The data of various parameters obtained were analyzed using an SPSS software version 20 (SPSS Inc. 233 South Wacker Drive, 11th Floor Chicago, IL 60606-6412). P < 0.005 was considered statistically significant. Student's t-test was used to compare the data.


  Results Top


We studied 103 cases. After taking the consent and collecting the semen sample, the routine semen analysis was done. The semen parameters such as total sperm count, motility was noted and recorded. Smears were prepared and stained with Leishman stain. The smears were then screened under ×40 and then under ×100 and the differential counts of round cell count into IGC count and leukocyte were done. Cells were identified according to their size.

The IGC predominantly noted was the primary spermatocytes.

The cells were identified as:

Spermatogenic cells [Figure 1] and [Figure 2]
Figure 1: Spermatocyte

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Figure 2: Spermatid

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Nonspermatogenic cells appearing were [Figure 3], [Figure 4], [Figure 5] and [Table 1], [Table 2]
Figure 3: Macrophage

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Figure 4: Decreased number of round cells over the background of numerous sperm cells

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Figure 5: Increased number of round cells over the background of relatively less number of sperm cells

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Table 1: Groups of sperm count

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Table 2: Groups of sperm count and motility

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  • On correlating average sperm count with average immature germ cell count, a negative correlation was observed with a Pearsons coefficient of −0.21and Chi-square test showed P = 0.0262 which is statistically significant
  • The Pearson's coefficient between sperm count and motility was −0.35 and Chi-square test showed a P = 0.0002 which is extremely significant.



  Discussion Top


While approaching an infertile couple, semen analysis appears to be one of the most essential initial laboratory tests to be done for evaluating the male-related factors. No doubt the routine analysis mentions the “round cells” in the results, the specification of the round cells and their percentage composition is not mentioned. In our study, apart from the routine semen analysis we have gone further with the differentiation of round cells and have taken their percentage count. According to the WHO, a normal ejaculate of semen should contain sperms <5 million/mL.[9] This study has shown the round cell count to be 5%–10% of the total sperm count [Table 3]. On comparing the findings with that of Fedder et al.[13] and Patil et al.,[3] we have found out that our results were comparable.
Table 3: Groupwise distribution of cases showing average sperm count, average round cell count, average immature germ cell count, and average leucocyte count

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For the further differentiation of round cells, we made the smears of the liquefied semen and stained them with Leishman stain. Papanicolaou stain can also be used since it does not interfere with the size of the leukocytes in blood smears and semen sample.[9] However, keeping the cost-effectiveness in mind, we prefer Leishman to Papanicolaou since the latter happens to be expensive. Furthermore, most of the pathologists happen to be well versed with the appreciation of the leukocytes in a blood smear stained with the routine Leishman stain thus making it a more preferable stain. While staining with Papanicolaou, the multinucleated spermatids appearing in the semen samples are sometimes difficult to differentiate from neutrophils having a segmented nucleus, this problem is resolved using the peroxidase reaction.[9]

For clinical evaluation, it is necessary to differentiate between the cells of spermatogenic and nonspermatogenic origin. The differential counts of round cells showed 60%–70% of IGCs and 30%–40% leukocytes. These results were then correlated with the studies of Patil et al.,[3] Gandini et al.,[11] Fedder et al.[13] Highest concentrations of IGC can be found in the patients with genital tract infection.[7],[9] On correlating the total sperm count with IGCs, a negative correlation was found [Table 4]. This went in hand with Fedder et al.,[13] and Patil et al.[3] The separation of round cells into IGCs and leukocytes can be done better using Modified discontinuous Percoll gradient technique which achieves good separation of IGC with scanty leukocyte contamination.[11] Other techniques include the use of immunofluorescence microscopy and flow cytometry analysis. Our study demonstrates that the fractions from 30 to 45% showed the highest concentration of immature germ cells and the lowest leukocyte contamination. These data are supported by the use of permanent staining techniques that give a morphological definition even if they require a high level of experience from the analyst. In fact, spermatids and spermatocytes can seem, in some cases, very similar to leukocytes and, due to the presence of more than one nucleus, they can seem like polymorphonuclear neutrophils with a multilobulated nucleus. To overcome these possible mistakes, we also used immunofluorescence microscopy and flow cytometry analysis: rapid and simple methods that were particularly useful to discriminate the various subpopulations of leukocytes in the Percoll fractions.
Table 4: Correlation of sperm count with immature germ cell count (IGC)

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The inclusion of IGCs helps to find out the underlying cause of infertility. It indicates testicular malfunction with impaired spermatogenesis can be of endocrinal origin.[9] In normal subjects, IGCs represent about 90% of nonsperm cells present in semen.[12],[13] The concentration is 5%–10% of overall sperm concentration and is sometimes inversely correlated. The concentration of IGCs in azoospermia ranged from 0 to 5.67 × 106/ml in oligospermia. The values were found to be slightly higher than the results of Gandini et al.,[11] who had used the modified Percoll gradient technique for the separation of IGCs.

The WHO manual considers 1 × 106 m/ml as a threshold value for leukocytes in semen, and this is termed as leukocytospermia and it is associated with poor semen parameters. The prevalence of leukocytospermia among male infertility is approximately 10%–20%. However, it can also be an indicator to the clinician to give a trial by starting antibiotic treatment to improve the fertility of the patient. Leukocytes presence can result in the loss of motility[3],[8] increased number of WBC damage the sperm functions.[12] The results in our study indicate that the increased leukocyte count was responsible for infertility, despite the better total sperm count. The increased concentration of leukocytes in our study can be suggestive of inflammatory conditions of male genital tract.

The correlation between the total sperm count and motility was significantly negative. In addition, leukocytospermia is associated with impaired sperm motility.[10],[12] These favor our findings of negative correlation between sperm count and leukocytes and negative correlation with sperm motility.

The patients having leukocytospermia can be further investigated by reactive oxygen species or immunofluroscence microscopy. Some patients can be segregated and further analyzed for cytogenetic studies for sperm deoxyribonucleic acid (DNA) damage or DNA fragmentation index which may be the cause of subfertility. The common available tests are (Sperm Chromatin Structure Assay and the terminal deoxynucleotidyl transferase dUTP Nick End Labeling Assay.[1],[14]


  Conclusion Top


Our study shows that differentiation of round cells in the semen can be effectively differentiated into spermatogenic and nonspermatogenic cells with the use of simple and cost-effective stain like Leishman stain. The correlation of sperm count with the IGCs and leukocytes and sperm motility is of immense use to find out the possible cause of infertility.

Acknowledgment

We would like to express our gratitude to STS ICMR 2014, for giving encouraging research at the grass root level and the Department of Obstetrics and Gynecology, Bharati Vidyapeeth Medical College and Hospital, Sangli for lending us their support. Furthermore, we would like to thank all the staff members of Central Clinical Laboratory for their invaluable assistance and support.

Financial support and sponsorship

STS-ICMR 2014 scholarship.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
World Health Organization. WHO Laboratory Manual for the Examination and Processing of Human Sperm. 5th ed. Geneva: WHO Press; 2010.  Back to cited text no. 1
    
2.
Butt F, Akram N. Semen analysis parameters: Experiences and insight into male infertility at a tertiary care hospital in Punjab. J Pak Med Assoc 2013;63:558-62.  Back to cited text no. 2
    
3.
Patil PS, Humbarwadi RS, Patil AD, Gune AR. Immature germ cells in semen-correlation with total sperm count and sperm motility. J Cytol 2013;30:185-9.  Back to cited text no. 3
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4.
Sunanda P, Panda B, Dash C, Padhy RN, Routray P. Effect of age and abstinence on semen quality: A retrospective study in a teaching hospital. Asian Pac J Reprod 2014;3:134-41.  Back to cited text no. 4
    
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Tapisiz OL, Kiykac Altinbas S, Abike F, Goktolga U. Semen analysis from a point of view of gynecologist and recent developments. J Turk Soci Obstet Gynecol 2012;9:25-31.  Back to cited text no. 5
    
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Stahl PJ, Stember DS, Schlegel PN. Interpretation of the semen analysis and initial male factor management. Clin Obstet Gynecol 2011;54:656-65.  Back to cited text no. 6
    
7.
Sandro CE, Aggarwal A. Impact of the new WHO guidelines on diagnosis and practice of male infertility. Open Reprod Sci J 2011;3:7-15.  Back to cited text no. 7
    
8.
Murray KS, James A, McGeady JB, Reed ML, Kuang WW, Nangia AK, et al. The effect of the new 2010 World Health Organization criteria for semen analyses on male infertility. Fertil Steril 2012;98:1428-31.  Back to cited text no. 8
    
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Johanisson E, Campana A, Luthi R, de Agostini A. Evaluation of 'round cells' in semen analysis: A comparative study. Hum Reprod Update 2000;6:404-12.  Back to cited text no. 9
    
10.
Caşkurlu T, Tasci AI, Samasti M, Bayraktar Z, Cek M, Sevin G, et al. Immature germ cells in semen and their correlations with other semen parameters. Int Urol Nephrol 1999;31:389-93.  Back to cited text no. 10
    
11.
Gandini L, Lenzi A, Lombardo F, Pacifici R, Dondero F. Immature germ cell separation using a modified discontinuous percoll gradient technique in human semen. Hum Reprod 1999;14:1022-7.  Back to cited text no. 11
    
12.
Wolff H. The biologic significance of white blood cells in semen. Fertil Steril 1995;63:1143-57.  Back to cited text no. 12
    
13.
Fedder J, Askjaer SA, Hjort T. Nonspermatozoal cells in semen: Relationship to other semen parameters and fertility status of the couple. Arch Androl 1993;31:95-103.  Back to cited text no. 13
    
14.
Agarwal A, Said TM. Role of sperm chromatin abnormalities and DNA damage in male infertility. Hum Reprod Update 2003;9:331-45.  Back to cited text no. 14
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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