Keywords
leucocytospermia - infertility - reactive oxygen species - semen
Introduction
The World Health Organization (WHO) defines leucocytospermia as the presence of peroxidase
positive leucocytes in concentrations of or greater than 106/mL of semen.[1] Leucocytospermia has been found in both fertile and infertile men with or without
evidence of genital tract infections. Significant leucocytospermia, however, has more
often been described in cases with underlying infection.[2] Earlier studies found leukocytes to have a positive effect on semen quality as these
cells were supposed to scavenge damaged sperms.[3]
[4] Recent work, however, suggests that an increase in leucocytes may negatively impact
semen quality and morphology due to reactive oxygen species (ROS)-mediated damage
and possibly defective spermiogenesis.[5]
[6]
[7]
[8] Leucocytospermia is a rare cause of infertility with a variable incidence of 2 to
35% in such cases.[9] As abnormal sperm morphology and leucocytospermia can affect the outcome of various
interventional procedures involved in infertility management, reporting of leucocytospermia
and associated morphological defects is important. Many centers across developing
nations still follow a Neubauer’s chamber/wet mount-based analysis of “pus cells.”
This work was done with the following objectives: (1) to examine the incidence of
leukocytospermia at a leading academic center examining a large number of infertility
patients; (2) to ascertain the role of performing cytomorphological analysis in the
work up of male infertility, with special reference to leukocytospermia and ascertaining
any additional utility, and (3) to examine the cytomorphological features of sperms
in cases of leucocytospermia.
Materials and Methods
The current work was a retrospective study conducted over a period of 3 months (November
2017 to January 2018) at the Clinical Pathology laboratory of our institute. Case
data of couples being worked up for primary infertility referred to our laboratory
for semen analysis was retrieved. All cases signed out as “leukocytospermia” in the
study period were included for analysis. This included cases in which the Neubauer’s
chamber estimation of pus cells was more than 106/mL and which was corroborated on smear examination. Cases in which a minimum of one
pus cell per five oil immersion fields but not fitting the WHO definition of leukocytospermia
were also included in the analysis .Cases in which pus cells were observed but in
which the possibility of contamination could not be ruled out (increased squamous
epithelial cells/presence of coccobacilli) were excluded from the study. Cases in
which a rare or occasional pus cell was observed in the entire smear with a normal
Neubauer’s chamber count were excluded from the analysis.
As per our laboratory protocol, semen analysis was performed within 1 hour after ejaculation
and evaluated as per the latest WHO guidelines for semen examination.[1] It was ensured that all samples were collected after a minimum of 3 days of abstinence.
Wet preparations were made for analysis of sperm count, motility, and other discernible
cells, if any. Leukocytes, if noted were reported per high power field (hpf). The
modified Neubauer’s chamber was used to validate sperm counts and to provide leukocyte
counts. Myeloperoxidase stain was done to confirm the white blood cells (WBC) counts.
Sperm motility was graded as progressive motility, nonprogressive motility, and immobility.
Viability of sperms was assessed on a 0.5% (w/v) Eosin Y-stained preparation. Samples
were also run on an automated sperm quality analyzer SQA-IIC-P (Medical Electronic
Systems, Caesarea, Israel) for obtaining functional and motile sperm concentrations
(MSCs). Papanicolaou (PAP)-stained smears were prepared for morphological analysis
and sperms were subsequently scored for head, midpiece, and tail defects by observation
in oil immersion at 1000×. As a single sperm may show multiple defects, overlap in
classifying sperms within these categories was noted. Based on the morphological findings,
the sperm deformity index (SDI) was calculated by dividing the total number of sperm
defects by the total number of sperms evaluated through a random selection. In addition,
the teratozoospermia index (TZI) was calculated by dividing the total number of sperm
defects by the total number of abnormal sperms. Pus cell evaluation was also done
on the cytosmear preparations with the number of pus cells documented per five hpf.
All procedures followed were in accordance with the ethical standards of the responsible
committee on human experimentation (institutional and national) and with the Helsinki
Declaration of 1975, as revised in 2008.
Results
Out of a total of 348 samples received during this period, 6 samples (1.72%) were
diagnosed as having “leukocytospermia.” The couples evaluated in this work presented
to the gynecology outpatient clinic of our institute with the complaint of an inability
to conceive for an average (median) period of 1.5 years. The median age of the male
subjects under evaluation was 32.0 years. The respective female partners had no previous
conceptions and, on extensive evaluation for hormonal/structural dysfunction as a
possible cause for infertility, had unremarkable test results. Five cases, in addition,
were analyzed which, though failing to reach the WHO cut off for leukocytospermia,
displayed the presence of pus cells (≥1/5 hpf) on cytosmear preparations (1.43%).
One case in this subset also had post-treatment semen analysis. The results of the
semen evaluation of all samples are summarized in [Table 1]. Six out of the total cases evaluated (6/11 = 54.5%) were those in which the Neubauer’s
chamber WBC count was more than the WHO defined cut off of leukocytospermia (1 × 10
6/mL). All cases of leukocytospermia had pus cells on cytosmear evaluation also, albeit
to varying degrees as illustrated in [Table 1]. Out of the five additional cases examined, four had available automated counts.
Three (3/5 = 60%) cases in this subset showed oligospermia. Overall, six cases (54.5%)
showed oligospermia, four of which in addition had a low Neubauer’s chamber white
blood cell count (< 1 × 10 6/mL). Decreased motility and viability was noted in 70% of analyzed cases. Nine cases
(81.8%) showed reduced MSC, functional sperm concentration (FSC), and sperm motility
index. Automated analyzer generated values showed more than 4% morphologically normal
spermatozoa in all cases. However, analysis of the PAP-stained smears showed mid-piece
defects in two cases (18.1%) in the form of asymmetrical insertion of the midpiece
into the head, thick or irregular neck (
[Fig. 1b]
), sharply bent neck (
[Fig. 2b]
) abnormally thin, or a combination of these. All cases diagnosed with leukocytospermia
had an SDI and TZI which were in normal range (≤ 1.6).
Table 1
Semen parameters obtained on automated semen analysis, Neubauer chamber analysis,
and morphological evaluation
|
Case no.
|
Duration of inability to conceive (years)
|
Age
(years)
|
TSC
|
MT (%)
|
Morphology (%)
|
FSC (million/mL)
|
MSC (Million/mL)
|
SMI
|
Viability
(%)
|
Pus cell count
(×106)
|
Number of pus cells/5 hpf
|
Morphological evaluation
|
TZI
|
SDI
|
|
|
|
SQA-IIC-P Automated analyzer
|
|
|
|
H
|
M
|
T
|
N
|
ABN
|
|
|
|
Abbreviations: ABN, number of abnormal sperms; FSC, functional sperm concentration;
H, head; hpf, high power field; M, mid-piece; MSC, motile sperm concentration; MT,
motility; N, number of normal sperms; SDI, sperm deformity index; SMI, sperm motility index;
T, tail; TSC, total sperm concentration; TZI, teratoz teratozoospermia index; U/A,
unavailable.
|
|
1
|
1.0
|
32
|
10
|
15
|
16
|
1
|
1.5
|
40
|
25
|
2.4
|
6
|
60
|
34
|
11
|
130
|
70
|
1.5
|
0.525
|
|
2
|
1.5
|
42
|
83
|
54
|
37
|
27.5
|
44.8
|
230
|
60
|
2.2
|
5
|
30
|
59
|
7
|
124
|
76
|
1.26
|
0.48
|
|
3
|
1.5
|
28
|
80
|
53
|
36
|
25
|
42.4
|
220
|
64
|
1.1
|
1
|
71
|
26
|
1
|
124
|
76
|
1.28
|
0.49
|
|
4
|
1.0
|
32
|
54
|
44
|
54
|
1.5
|
23.7
|
150
|
60
|
4.5
|
8
|
52
|
16
|
02
|
140
|
60
|
0.35
|
1.16
|
|
5
|
2.0
|
25
|
37
|
36
|
23
|
4.7
|
13.3
|
97
|
50
|
1.3
|
2
|
60
|
41
|
2
|
138
|
62
|
1.6
|
0.515
|
|
6
|
2.0
|
35
|
14
|
19
|
17
|
1.4
|
2.6
|
50
|
35
|
1.6
|
3
|
45
|
51
|
2
|
122
|
78
|
1.25
|
0.49
|
|
7
|
1.0
|
30
|
12
|
17
|
16
|
1.2
|
2
|
44
|
12
|
0.3
|
1
|
28
|
5
|
17
|
150
|
50
|
1
|
0.25
|
|
8
|
1.0
|
29
|
10
|
20
|
17
|
1.2
|
2
|
45
|
15
|
0.8
|
2
|
30
|
10
|
10
|
50
|
50
|
1
|
0.5
|
|
9 (pretreatment)
|
1.5
|
32
|
U/A
|
U/A
|
U/A
|
0.02
|
0.02
|
0.7
|
U/A
|
0.9
|
2
|
U/A
|
|
10 (post-treatment)
|
1.5
|
32
|
11
|
16
|
16
|
1.1
|
1.7
|
42
|
50
|
0.8
|
2
|
50
|
46
|
23
|
14
|
86
|
1.38
|
1.19
|
|
11
|
1.0
|
28
|
33
|
35
|
21
|
3.9
|
11.5
|
89
|
52
|
0.4
|
4
|
20
|
40
|
28
|
80
|
120
|
0.73
|
0.44
|
|
Median, Range
|
1.25
(1.0–2.0)
|
32
(25–42)
|
23.5
(10–83)
|
27.5
(15–55)
|
19
(16–54)
|
1.45
(0.02–27.5)
|
2.6
(0.02–44.8)
|
50
(0.7–230)
|
50
(12–65)
|
1
(0.3–4.5)
|
|
|
|
|
|
|
1.27 (0.35–1.6)
|
(0.25–1.19)
|
Fig. 1 Papanicolaou-stained semen cytosmears show (A) tapered heads, bent neck pieces, and polymorphonuclear cells (Papanicolaou stain,
100×); (B) Mid piece defect—thick midpiece inserting into the sperm head, inset shows a higher
magnification highlighting the defective sperm neck (Papanicolaou stain, 100× and
1000×).
Fig. 2 Semen smears show (A) tapered spermatozoa heads in a background of polymorphonuclear cells; (B) Bent neck piece displaying an angle of 249.5 degrees (Papanicolaou stain, 200× and
1000×).
In the two cases in which pre and post treatment samples were evaluated, the first
case showed a mild improvement in semen parameters whereas in the other, the derangements
in counts and morphology persisted. Follow-up data of other cases was unavailable.
Discussion
Leucocytospermia is commonly associated with infection or inflammation of male accessory
glands and urogenital tract.[2] There is a wide variation of the prevalence of leucocytospermia, ranging from 16.1
to 60.7%.[10] Increased leucocytes lead to increased ROS and sperm dysfunction by lipid peroxidation
(
[Fig. 3]
). There are various causes of leucocytospermia which may be classified as infectious
and noninfectious. Infectious causes include bacterial and viral infections of the
genitourinary tract, commonly prostatitis and epididymitis. Commonly implicated organisms
include Chlamydia trachomatis, Ureaplasma urealyticum, and Escherichia coli. Viruses such as herpes simplex, cytomegalovirus, hepatitis B may also be causative.
Noninfectious causes include consumption of nicotine, marijuana, and alcohol.[11] Other factors such as prolonged abstinence and anal intercourse may also lead to
an increased number of white blood cells in the semen.[12] Lipid peroxidation impacts the polyunsaturated fatty acids in sperm cell membranes
which lead to DNA damage. This coupled with impaired repair mechanisms and maturation
lead to decreased sperm motility, abnormal morphology, and overall decrease in sperm
fertilization capacity and viability which explains the possible cause of infertility
in our cases, in addition to oligospermia seen in around half of our cases.[13]
Fig. 3 Schematic diagram illustrating the role of leukocytospermia in male infertility.
The present work emphasizes the role of a detailed morphological analysis of semen
in the clinical pathology laboratory. With the advent of automated semen analysis,
parameters such as total FSC, MSC, sperm motility index, percentage of progressively
motile spermatozoa, and percentage of morphologically normal spermatozoa are readily
available. In our study, however, despite more than 4% morphologically normal sperms
in all our cases, two cases (18.1%) showed midpiece defects on cytological evaluation,
an observation also highlighted in patients with leucocytospermia by Thomas et al
in 1997.[14] This work described 79 cases of semen analyses finding a positive correlation between
polymorphonuclear cells and midpiece abnormalities and a negative correlation with
normal sperms. Both these cases had an increased number of pus cells both on Neubauer’s
chamber as well as on PAP-stained smears.
All our cases (6/11) displaying more than 1 × 10 6/mL pus cells, i.e., leukocytospermia, displayed pus cells on cytosmear evaluation.
In addition, five (45.4%) cases showed pus cells (one and more) on evaluation of five
hpfs. These all showed pus cell counts less than 1 × 10 6/mL on modified Neubauer chamber estimation. This could possibly be explained by the
pus cells inducing an altered microenvironment for the spermatozoa leading to nonspecific
microaggregates, as described by Bhardwaj et al.[15] These being heavier tend to settle downward and despite adequate mixing of the semen,
may escape being sampled into the Neubauer’s chamber resulting in false low counts.
Previous work by Moretti et al has also suggested a hampered semen interpretation
in the presence of pus cells.[16] However, the authors concluded that pus cells may have no pathogenic role which
is contrary to our work, wherein all cases having autoanalyzer and smear documentation
of pus cells presented with infertility. Our findings hence may suggest a greater
need to validate automated values with cytological evaluation as part of standard
practice.
Our cases showed a normal SDI in both the patients which was in contrast to findings
by Aziz et al who reported a positive correlation between leucocytospermia and higher
mean SDI score which was 1.9 in their study.[17] This may be due to the fact that leukocytes scavenge damaged sperms resulting in
a possibly normal SDI. All the remaining cases also showed normal SDI and TZI scores.
Menkveld and Kruger observed many tapered heads in cases of leucocytospermia as seen
in our cases in addition to other features of poor sperm morphology.[18] Tapered heads were also seen in the cases which had mid piece defects.
All cases described herein highlight a significant correlation between leucocytospermia
and semen parameters like SMI, MSC, FSC, motility, and viability suggesting an association
with poor sperm quality. The morphologist’s report assumes critical importance, as
a recent review of management of leucocytospermia has suggested, antibiotics and antioxidants
may improve sperm quality and subsequent chances of fertility.[2]
The current work is limited by the absence of immunocytological evaluation of the
white blood cells on the samples and microbiological correlation including reflexive
semen culture. to document any association with bacteriospermia in our cases. As the
compliance of subjects to give a second sample for culture in our sociocultural setting
is low, this was not performed on our study cohort. In addition, adequate follow-up
clinical data was not available despite the best of our efforts. Further prospective
studies on larger cohorts with multidisciplinary correlation, are required to validate
a practical and user-friendly morphological criteria of diagnosing leukocytospermia
in challenging conditions, as well as the actual clinical import of this observation
in indigenous study populations.
To conclude, leukocytospermia is a diagnostic and clinical enigma. Challenges remain
in the diagnosis in a routine laboratory setting with resource constraints being a
real issue. In this scenario, a thorough cytomorphological analysis on simple and
inexpensive PAP-stained preparations of semen smears can possibly improve the detection
of leukocytospermia as well as morphological indicators of sperm quality, even in
settings where automated semen analyzers may not be available. It could double up
as a useful tool not only in arriving at the correct diagnosis in cases of male infertility
but also as a quality control measure to screen out cases where the sample may appear
contaminated or unsatisfactory for analysis. As seen herein, it may also aid to negate
the vagaries seen in Neubauer chamber analysis of cell counts, still being practiced
in many laboratories.
