Scholarly article on topic 'Leukocytospermia: Overview of diagnosis, implications, and management of a controversial finding'

Leukocytospermia: Overview of diagnosis, implications, and management of a controversial finding Academic research paper on "Clinical medicine"

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Abstract of research paper on Clinical medicine, author of scientific article — Juan Sebastian Sandoval, Doug Raburn, Suheil Muasher

Abstract Background The progress in assisted reproductive techniques over the last 30years has raised interest in studying what in 1992 the World Health Organization (WHO) defined as abnormal: the presence of >1×106 leukocytes/mL in a semen sample. Leukocytospermia is found in both fertile and infertile men with and without evidence of infections of the genital tract. However, despite extensive research done in this subject, the clinical implications of this condition remain unclear. Results The accuracy that the cut-off value that defines leukocytospermia has in predicting clinical significance remains unclear. Furthermore, there are conflicting data supporting a physiologic and pathologic role of leukocytes in a semen sample through the generation of reactive oxygen species. Although some authors describe no effects on fertilization and pregnancy outcomes after in vitro fertilization with or without intra-cytoplasmic sperm injection, others suggest the exact opposite. Finally, research on treatment of leukocytospermia with vitamin E, COX-2 inhibitors and antibiotics has also shown contradicting data. Conclusions Thus, it seems reasonable to agree that there is no agreement on the diagnosis, implications, and management of leukocytospermia in patients without evidence of genital tract infection.

Academic research paper on topic "Leukocytospermia: Overview of diagnosis, implications, and management of a controversial finding"

Middle East Fertility Society Journal (2013) 18, 129-134

Middle East Fertility Society Middle East Fertility Society Journal

www.mefsjournal.org www.sciencedirect.com

REVIEW

Leukocytospermia: Overview of diagnosis, implications, and management of a controversial finding

Juan Sebastian Sandoval *, Doug Raburn, Suheil Muasher

Duke University, Division of Reproductive Endocrinology & Fertility, Duke Fertility Center, 5704 Fayetteville Rd., Durham, NC 27713, United States

Received 14 January 2013; accepted 27 February 2013 Available online 11 July 2013

KEYWORDS

Leukocytospermia; Reactive oxygen species; Peroxidase staining; In-vitro fertilization; Intra-cytoplasmic sperm injection

Abstract Background: The progress in assisted reproductive techniques over the last 30 years has raised interest in studying what in 1992 the World Health Organization (WHO) defined as abnormal: the presence of >1 x 106 leukocytes/mL in a semen sample. Leukocytospermia is found in both fertile and infertile men with and without evidence of infections of the genital tract. However, despite extensive research done in this subject, the clinical implications of this condition remain unclear.

Results: The accuracy that the cut-off value that defines leukocytospermia has in predicting clinical significance remains unclear. Furthermore, there are conflicting data supporting a physiologic and pathologic role of leukocytes in a semen sample through the generation of reactive oxygen species. Although some authors describe no effects on fertilization and pregnancy outcomes after in vitro fertilization with or without intra-cytoplasmic sperm injection, others suggest the exact opposite. Finally, research on treatment of leukocytospermia with vitamin E, COX-2 inhibitors and antibiotics has also shown contradicting data.

Conclusions: Thus, it seems reasonable to agree that there is no agreement on the diagnosis, implications, and management of leukocytospermia in patients without evidence of genital tract infection.

© 2013 Production and hosting by Elsevier B.V. on behalf of Middle East Fertility Society.

* Corresponding author. Tel.: +1 (919) 572 4673; fax: +1 (919) 484 0461.

E-mail address: juanse.s@duke.edu (J.S. Sandoval).

Peer review under responsibility of Middle East Fertility Society.

1110-5690 © 2013 Production and hosting by Elsevier B.V. on behalf of Middle East Fertility Society. http://dx.doi.org/10.1016/j.mefs.2013.02.004

Contents

1. Introduction......................................................................................................................................................130

2. Etiology............................................................................................................................................................130

3. Prevalence........................................................................................................................................................130

4. Diagnosis..........................................................................................................................................................131

5. Clinical implications..........................................................................................................................................131

6. Management......................................................................................................................................................133

7. Discussion........................................................................................................................................................133

References........................................................................................................................................................133

1. Introduction

Although research about the presence of white blood cells in a semen sample has been done for more than 20 years, the lack of consensus is a recurring theme when it comes to definitions, implications and management of this condition in an individual without a reproductive tract infection. Defined in 1992 by the World Health Organization (WHO) as the presence of at least 1 x 106 leukocytes/mL in a semen sample (1), leukocy-tospermia has been found to be present in both fertile and infertile men with and without evidence of infections of the genital tract. Simultaneous progress over the last 30 years in assisted reproductive techniques (ART) has motivated numerous authors to investigate this subject. Unfortunately, the diagnosis of leukocytospermia in asymptomatic patients has shown contradicting effects in pregnancy rates of spontaneous cycles, intra uterine insemination (IUI) and in vitro fertilization (IVF) with and without intra-cytoplasmic sperm injection (ICSI). Consequently, the methods used for its diagnosis, prevalence, clinical significance, accuracy of this defined concentration to predict effects on the semen quality, and the role of leukocytes in normal sperm function remain controversial.

2. Etiology

In order to define the etiology of leukocytospermia, people who present with this finding can be divided in two groups:

those without and those with a genital tract infection (GTI).

As it will be discussed later, the etiology of leukocytospermia

in people without a GTI remains unclear. On the other hand,

genital tract infections can affect the urethra, epididymis, testicles, and prostate. Asymptomatic infectious urethritis is a rare condition occurring in only 5-10% of the cases (2). While epididymitis is always symptomatic, chronic orchitis can occasionally be a silent condition. On the other hand, the clinical manifestations of prostatitis can vary significantly from one person to another. This is exemplified by the 1998 NIH classification of the prostatitis syndromes (Table 1).

The lack of symptoms is not indicative of the absence of an infectious or inflammatory process in the urogenital tract. However, it has been described that there is an association between significant leukocytospermia and an underlying infection (2). Thus it is important to clarify that for the purpose of this review the discussion will focus on leukocytospermia in patients without evidence of urogenital tract infection.

3. Prevalence

There is a wide variation of the prevalence of leukocytosper-mia depending on which author is being quoted. A review of the available literature supports a prevalence anywhere from 16.1% to 60.7% (8). Its correlation with bacteriospermia is controversial, particularly in asymptomatic men, where its presence should raise the question of probable contamination (8). Conversely, it seems to be that in symptomatic men, with evidence of accessory gland infection, leukocytospermia has a better association with the presence of bacteriospermia. Consequently, it has been stated that most cases of leukocytospermia are not the result of a bacterial infection or bacteriospermia (8). Interestingly, in 2008 Pasqualotto showed a positive correlation between smoking and the presence of leukocytospermia

Table 1 NIH classification of prostatitis syndrome (3).

Category Manifestations Findings

I. Acute bacterial prostatitis Urinary symptoms with or Bacteriuria and pyuria

without systemic signs/symptoms

II. Chronic bacterial prostatitis Recurrent bacterial UTls by Lower urinary tract cultures grow bacteria between

the same organism symptomatic episodes

III. Chronic prostatitis/chronic Urological pain* Leukocytes in expressed prostatic secretions,

pelvic pain syndrome: Urological pain post-prostate massage urine or semen

A. Inflammatory No evidence of inflammation

B. Non-inflammatory

IV. Asymptomatic Inflammatory prostatitis No history of genitourinary tract pain - Elevated PSA fi prostate biopsy shows prostatitis.

- Infertility fi significant leukocytospermia

* Without: active urethritis, urogenital cancer •, urinary tract disease, functionally significant urethral stricture, neurological disease affecting the

bladder.

Table 2 Diagnostic tests used to detect leukocytospermia in patients without evidence of genital tract infection (4,9,20-22).

Test Method Pros Cons

Direct counting of round cells Immunocytology Peroxidase staining Visually with microscope Cell type-specific monoclonal antibodies Granulocyte peroxidases oxidizes a benzidine derivate which precipitates in the form of a brown color Inexpensive Highly specific Considered the gold standard Recognizes PMN which are the predominant WBCs in semen Highly inaccurate No standardization on exact immunohistological staining method No standardization on exact monoclonal antibodies Expensive Time-consuming Cannot identify non-peroxidase rich WBCs Uses toxic solutions Poor performance compared to the gold standard

(r = 0.311, P = 0.009) (9). Given the wide range of prevalence of this condition in both fertile and infertile men, the key issues to address are the clinical implications of leukocytospermia and levels above which it becomes clinically significant.

4. Diagnosis

Direct counting of round cells in a semen sample is a highly inaccurate method of diagnosing leukocytospermia because white blood cells cannot be distinguished from immature germ cells using light microscopy (4). Although immunocytology using monoclonal antibodies is recognized as the gold standard for diagnosis of leukocytospermia (3-5), this method is not feasible for the daily medical practice (Table 2). The lack of standardization on the exact immunohistological staining method with the specific monoclonal antibodies to be used is one of the major limiting factors (4). Additionally, these techniques are time consuming and expensive. Consequently, the WHO recommends peroxidase staining as the best next option to diagnose leukocytospermia (3). Peroxidases are enzymes that break down H2O2, liberating O2. This molecule oxidizes a benzidine derivate present in the staining solution, which precipitates in the form of a brown color, allowing these cells to be recognized under light microscopy. In contrast to immunohis-tological staining (which can recognize any leukocyte depending on the monoclonal antibody that is being used) this method only identifies cells rich in peroxidase such as polymor-phonuclear (PMN) granulocytes and macrophages. Although they constitute the predominant white blood cell types seen in semen (50-60% and 20-30%, respectively), T-lymphocytes, which represent 2-5% of semen leukocytes, are not recognized by this staining (5,6).

The main limiting factors of the peroxidase staining are lack of identification of non-peroxidase rich white blood cells, its reliance on the use of toxic solutions, and the poor performance of this test when compared to the gold standard. Ricci et al. used flow cytometry with anti-CD45 and anti-CD53 monoclonal antibodies to contrast these two methods. When compared to flow cytometry using anti-CD45, the sensitivity and specificity of peroxidase staining was 58.8% and 92.8%, respectively. When compared to flow cytometry using anti-CD53, its sensitivity and specificity was 55.5% and 94.1%, respectively (4). Years later, Villegas et al. used peroxidase staining in a group of 46 patients to establish a prevalence of leukocytospermia of 19.6%. When analyzing the same samples

using indirect immunofluorescence with anti-CD45, anti-CD15, anti-CD68, anti-CD2/T, and anti-CD22 monoclonal antibodies, its prevalence increased to 41.3% (5). It has been postulated that a possible explanation for the poor performance of this test when compared to immunocytology techniques could be that an inflammatory process induces the release of myeloperoxidase (5). The consequent loss of activity of this enzyme in granulocytes would make them undetectable by this staining. Another major concern of peroxidase staining is the use of benzidine products which are toxic substances that are considered carcinogens (bladder cancer) and have raised concerns about possible teratogenicity (7). It seems counterintuitive to use these types of compounds in andrology laboratories where ART procedures are being done. Because of these reasons, new, reliable, cost-effective and safe methods to detect leukocytes in semen samples need to be developed.

5. Clinical implications

Early research dating back to the late 1980s and early 1990s, described a positive influence of leukocytes in the sperm analysis through phagocytosis of damaged sperm. In contrast, more recent research has consistently regarded leukocytosper-mia as a negative factor for semen quality (8,10). In 1997, Thomas described a negative correlation between PMN and normal sperm (r = —0.276, P < 0.01) and by unknown mechanisms, a positive correlation with midpiece abnormalities (r = 0.225; P < 0.04) (11). Years later, Aziz et al. reported a positive correlation between leukocytospermia and higher sperm deformity index (SDI) scores (r = 0.32; P = 0.008), acrosomal damage (r = 0.36; P = 0.003), tail deformities (r = 0.33; P = 0.005), cytoplasmic droplet (r = 0.31; P = 0.01), and midpiece defects (r = 0.27; P = 0.026) (10). Pasqualotto also described a negative correlation between leu-kocytospermia and sperm motility (r = —0.211; P = 0.042) (9). In 2011, Sing saw leukocytospermia in 64.5% of 138 cases with abnormal morphologic features in comparison with 17% of 63 cases with normal morphologic features (P < 0.001), however, none of these variables were associated with specific morphologic defects (13).

Some conflicting results were described in 2008 by Lackner, who stated that non-leukocytospermia in ICSI had the greatest impact on fertilization by having the best chance of achieving a pregnancy (OR = 0.952) (14). These findings contradicted the results from Yilmaz, who found that even though leukocyto-

Table 3 Summary of conclusions by various authors (1,5,6,10,13,15,16,18,18,23,24).

Author Conclusions

Thomas, 1997 Negative correlation between PMN and normal sperm

(r = -0.276,

P < 0.01)

Positive correlation with midpiece abnormalities

(r = 0.225; P < 0.04)

Aziz, 2004 Positive correlation between:

Leukocytospermia and higher SDI scores (r = 0.32;

P = 0.008)

Acrosomal damage (r = 0.36; P = 0.003)

Tail deformities (r = 0.33; P = 0.005)

Cytoplasmic droplet (r = 0.31; P = 0.01)

Midpiece defects (r = 0.27; P = 0.026)

Yilmaz, 2005 Lower:

Fertilization rates (82% vs 87%)

Embryo development rates (79% vs 86%)

No statistical difference between:

Good embryo quality (88% vs 92%; P > 0.05),

Normal embryo cleavage (both 50%)

Pregnancy rates (30.3% vs 30.6%; P > 0.05)

Pasqualotto, 2008 Negative correlation between leukocytospermia and sperm

motility (r = -0.211; P = 0.042)

Lackner, 2008 Non-leukocytospermia in ICSI have the best chance of

becoming pregnant (OR = 0.952)

Sing, 2011 Leukocytospermia in 64.5% of 138 cases with abnormal

morphologic features vs 17% of 63 cases with normal

morphologic features (P < 0.001)

spermia had lower fertilization rates (82% vs 87%) and lower embryo development rates (79% vs 86%), there were no statistically significant differences in regard to good embryo quality (88% vs 92%; P > 0.05), normal embryo cleavage (both 50%), and pregnancy rates (30.3% vs 30.6%; P > 0.05) (15).

The lack of consistency in the findings from research done in this field (Table 3) raised the question of whether there is something else affecting the sperm quality that could be associated with the presence of seminal leukocytes but somehow disturbs the semen of some individuals more than others. The common denominator in this discussion seems to be reactive O2 species (ROS). Although the primary sources of ROS are WBCs, a portion of these is produced by sperm. A membrane bound NADPH oxidase produces superoxide anion, which by the action of superoxide dismutase gets converted into hydrogen peroxide. The combination of superoxide anion and hydrogen peroxide initiates the Haber-Weiss reaction to generate the hy-droxyl radical (16). Although this free radical will affect the unsaturated fatty acids-rich sperm membrane due to its powerful initiation of lipid peroxidation, when produced in controlled amounts, it fulfills a physiologic role in capacitation and fertilization (17-19). In fact, Attaran et al. measured the ROS levels in the follicular fluid of 53 women who underwent IVF, and

found that women who became pregnant had significantly higher ROS levels than those who did not(14). Thus it seems logical to think that the impact of WBCs and ROS in fertility depends on the degree rather than on their absolute presence or absence (14). Superoxide dismutase and catalase are two antioxidant enzymes. It has been seen that the levels of superoxide dismutase are positively correlated with sperm concentration (r = 0.204; P = 0.034) and negatively correlated with leukocytospermia (r = -0.228; P = 0.021) (9). When the ROS overwhelm the body's antioxidant defense mechanisms, oxidative stress occurs. This could be exemplified by the fact that there is a negative correlation between smoking and semen quality, which could be attributed, in part, to the effects that tobacco has in increasing levels of oxidants while decreasing levels of antioxidants in seminal plasma (9). Hence, a possible explanation for the conflicting results of the impact that leukocytosper-mia has on male fertility and overall sperm quality for ART could be the fact that there is a complex interaction with a fine balance between oxidant and antioxidant substances in semen.

It has been described that the loss of the sperm functional integrity secondary to ROS is mediated by multiple mechanisms (Table 4) including decreased mitochondrial activity, alteration in the sperm-egg binding, and loss of DNA integrity

Table 4 Summary of proposed effects of ROS in sperm function (1,5,6,10,13,15,16,18,18,23,24).

Location Mechanism Consequence

Cell membrane Mitochondria DNA Lipid peroxidation of unsaturated fatty acids Disruption of membrane selectivity inhibits oxidative phosphorylation decreasing ATP production • Oxidation of DNA bases • Single & double strand breaks Disruption of cell & organelle membranes Affected sperm motility DNA damage

Table 5 Summary of studied interventions for leukocytospermia in patients without evidence of genital tract infection

(3,8,12,26,27,28).

Author Intervention Results

Suleiman, 1996 Vitamin E Improved pregnancy rates

Kessopoulou, 1995 Vitamin E No effect on pregnancy rates

Gambera, 2007 Cox-2 Decrease in leukocyte concentration

Lackner, 2006 Cox-2 Decrease in leukocyte concentration

Skau, 2008 Doxycycline Erythromycin, Considerable improvement in morphology

Trimethoprim/Sulfamethoxazole Minor improvement in motility and

concentration

Hamada, 2011 Doxycycline Improvement in leukocytospermia but no

difference in semen parameters

(20). The high plasma membrane concentration of polyunsaturated fatty acids makes sperm particularly susceptible to ROS. The mitochondrial membranes need to be selectively permeable to maintain an electrolytical gradient between the inner and outer compartments. When ROS are present in excess, the phospholipid membranes are altered, thus disrupting the membrane selectivity. This inhibits the oxidative phosphor-ylation which decreases ATP production, affecting the sperm motility (20). Studies from the 1990s and early 2000s have postulated multiple mechanisms of DNA damage such as lipid degradation by-products that bind to DNA, oxidation of DNA bases (mainly Guanosine), and direct interaction with DNA strand leading to non-specific single and double strand breaks (20). Zorn et al. compared IVF and ICSI outcomes in correlation with ROS. Their data support the deleterious effects of ROS in sperm quality by showing a negative association between seminal ROS and IVF results but no effects on ICSI outcome (21).

6. Management

There have been multiple attempts to establish if leukocyto-spermia needs to be treated, and if so, what the best management for this condition is (Table 5). The description of spontaneous downward variation of leukocytospermia seen in 40% of infertile men over a period of ±3 months (8) affects the reliability of studies that have investigated the effects of treating patients with this condition. In 1988 Aitken proposed the use of vitamin E (an antioxidant) in the culture media for IVF to enhance sperm-oocyte fusion by reducing ROS levels (16). However, while Kessopoulou, in 1995, showed no effect on pregnancy rates after treatment with vitamin E (22), Suleiman et al., in 1996, did find improvement with this intervention. Years later, Gambera and Lackner described the use of COX-2 inhibitors in patients with leukocytospermia. The former treated 47 infertile men and found an improvement in leukocyte concentration (decreased from 1.6 ± 1.1 million/mL to 0.4 ± 0.2 million/mL) (22). The latter, reported the treatment of 56 men, with a decrease in leukocyte concentration from 5.5 million/mL to 1.1 million/mL (P < 0.001) (23).

In 2003, a meta-analysis of 12 studies that used different broad-spectrum antibiotics (doxycycline, erythromycin, and trimethoprim/sulfamethaxazole) to treat patients with leuko-cytospermia, showed a considerable improvement in sperm morphology and a minor improvement in sperm motility and concentration (24). The main criticism that the authors of this

study had to other studies that failed to show improvement was the short duration of follow-up (<3 months). Most recently, Hamada compared natural pregnancy outcomes in subjects treated versus non-treated with doxycycline. This antibiotic interferes with the release of ROS from seminal leukocytes and nonviable sperm. In this study the cutoff for leu-kocytospermia was decreased to from 0.2 to 1 x 106. With this definition the study population consisted of 61 out of the original 223 subjects. Of these, 34 were treated and 27 were not. Resolution of leukocytospermia was seen in 56% of the subjects from the treated group vs 25% in the non-treated. In the treated group, 47% of subjects achieved pregnancy (OR: 3.7, CI: 1.1-11.7; P = 0.04). In the non-treated group only 20% achieved pregnancy. Despite these results, there was no difference in the semen parameters (25).

7. Discussion

From the early research on this subject done in the late 1970s, until now, there have been multiple investigators that have tried to better define and interpret the implications of the presence of leukocytes in a semen sample. The oldness of the original data used by the WHO to define leukocytospermia, the lack of a reliable, cost-effective method to detect and differentiate white blood cells (WBC) accurately, the significant variation between semen samples of same individuals and the conflicting results of research done in this subjects are hurdles that the scientific community has failed to overcome. Most of the studies published up to this date have been done in small samples and without taking into consideration known and probably unknown confounding variables. Thus, in order to be able to define the influence of leukocytospermia on semen quality and ART outcomes better-designed and larger trials should be done. Only then we will be able to establish accurate cutoff concentrations at which the presence of leukocytes may affect sperm and from this, clarify the clinical significance and best management of leukocytospermia.

References

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(26) Kaleli S, Ocer F, Irez T, Budak E, Aksu F. Does leukocytosper-mia ssociate with poor semen parameters and sperm functions in male infertility? The role of diferente seminal leukocyte concentrations. Eur J Obstet Gynecol Reprod Biol 2000;89:185-91.

(27) Aziz M, Agarwal A, Lewis-Jones I, et al. Novel associations between specific sperm morphological defects and leukocytosper-mia. Fertil Steril 2004;82:621-7.

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