Sexual intercourse with leukocytospermic men may be a possible booster of oxidative stress in female partners of infertile couples
Reet Mändar, Department of Microbiology, University of Tartu, Ravila 19, Tartu 50411, Estonia. E-mail: email@example.com
Human semen has undoubtedly significant influence on the organism of the female counterpart. At the same time there are no studies in English literature investigating the influence of sexual intercourse on oxidative stress level in women's organism. Seventeen infertile couples where male partners were with (n = 5) or without (n = 12) leukocytospermia were enrolled in the study. Systemic oxidative stress levels were measured, whereby twice in female partners – before and 8–12 h after sexual intercourse. The men with leukocytospermia were characterized by oxidative stress that was substantially transferred to their partners during sexual intercourse as revealed by increase in 8-isoprostanes level (median 32.7 vs. 70.4 ng/mmol creatinine, p = 0.006). Sexual intercourse with male partner having leukocytospermia increases the oxidative stress level in the women's organism that may interfere with fertilization.
Infertility represents an increasing medical problem affecting approximately 15% of couples around the world although its treatment is stressful, invasive and costly. The causes of infertility have not been completely elucidated, yet this condition is commonly believed to result from the synergistic negative influence of several factors. Although prostatitis is not widely accepted as a precursor or cause of infertility, there is some evidence that this condition may reduce semen quality. In addition to obstructive and immunologic mechanisms, prostatitis-associated oxidative stress (OxS) that is characterized by imbalance between production and detoxification of reactive oxygen species (ROS) may affect sperm function parameters, including decreased sperm motility, sperm number, and sperm–oocyte fusion (Potts & Pasqualotto, 2003; Wagenlehner et al., 2008; Agarwal & Sekhon, 2010; Gharagozloo & Aitken, 2011; Jungwirth et al., 2012; Lavranos et al., 2012).
At the same time the role of OxS in female infertility is significantly less studied, however, the influence of OxS on the maturation of oocytes and early pregnancy loss as well as the increased levels of ROS in infertile women have been noted (Agarwal et al., 2005, 2012; Combelles et al., 2009; Mansour et al., 2009; Ruder et al., 2009; Gupta et al., 2010).
Human semen has undoubtedly significant influence on the organism of the female counterpart. At the same time, according to our best knowledge there are no studies in English literature investigating the influence of sexual intercourse on OxS level in women's organism.
We hereby present the preliminary results about the influence of the sexual intercourse on partner's OxS level in infertile couples.
Materials and methods
The study was carried out at the Andrology Centre of Tartu University Hospital (Estonia) between April 2009 and May 2010. The study group included 17 consecutive couples (in total 34 subjects – 17 men and 17 women) who consulted a physician because of infertility of the couple and who wished to participate in the study (Table 1). Both partners had been investigated for common causes of infertility. In three men NIH IIIa category prostatitis (chronic prostatitis/chronic pelvic pain syndrome, inflammatory subtype) and in two men NIH IV category prostatitis (asymptomatic inflammatory prostatitis) was diagnosed (Krieger et al., 1999). As all these five men had significant inflammation in their semen [more than 1 million white blood cells (WBCs) per mL] and as WBCs are the main source of ROS in semen, we refer these men thereinafter as leukocytospermic men. Clinical, demographic and microbiological data of the subjects are presented elsewhere (Borovkova et al., 2011). None of the participants had received antimicrobial therapy within 3 months and anti-inflammatory medications for at least 1 month before evaluations.
Table 1. Clinical and demographic overview of study subjects
|Age1||31.6 (25–40)||28.8 (25–33)||32.8 (25–40)|
|White blood cells in semen (M/mL)1||1.6 (0–13.3)||5.1 (1.3–13.3)*||0.2 (0–0.9)*|
|Semen volume (mL)1||3.6 (2–6.6)||3.22 (2–4.6)||3.8 (2.5–6.6)|
|Sperm concentration (M/mL)1||60.4 (2.5–270)||49.4 (9–128)||64.9 (2.5–270)|
|Total sperm count (M)1||203.1 (8.5–918)||134 (27–285.6)||231.9 (8.5–918)|
|A+B sperm motility (%)1||33.2 (0–56)||35.4 (15–54)||32.3 (0–56)|
|Smoking (no, yes)||13, 4||3, 2||10, 2|
|Trying to conceive (years)1||1.9 (0.5–5.5)||2.4 (0.5–5)||1.8 (0.5–5.5)|
|Age1||29.9 (21–39)||29.4 (26–34)||30.2 (21–39)|
|Menarche (age)1||13.5 (11–15)||14.7 (14–15)||13.2 (11–15)|
|No. of pregnancies1||1.2 (0–4)||0.6 (0–3)||1.4 (0–4)|
|No. of deliveries1||0.7 (0–2)||0.2 (0–1)||0.9 (0–2)|
|No. of artificial abortions1||0.3 (0–2)||0.4 (0–2)||0.3 (0–1)|
|No. of spontaneous abortions1||0.2 (0–2)||0 (0–0)||0.3 (0–2)|
|Gynecological diseases in history2||16/17||5/5 ||11/12|
|Gynecological surgery in history3||6/17||2/5||4/12|
|Smoking (no, yes)||14, 3||3, 2||11, 1|
|Trying to conceive (years)1||1.9 (0.5–5.5)||2.4 (0.5–5)||1.8 (0.5–5.5)|
Participation in the study was voluntary. All subjects were at least 18 years old. Informed consent was obtained from all of the patients. The study was approved by the Ethics Review Committee on Human Research of the University of Tartu.
Semen and urine samples of male partners were collected during menstruation of the female counterpart. The samples were self-collected into a sterile collection tubes in a private room near laboratories after washing the glans penis with warm water. After ejaculation, the semen was incubated at 37 °C for 25–45 min for liquefaction. For biochemical analyses, urine and seminal plasma were frozen at −20 °C.
The two urine samples from women were taken 3–5 days later (on the 6th to 8th day of the cycle) – before intercourse, and 8–12 h after intercourse. Mid-stream urine samples from women were self-collected at home after washing the vulva with warm water and stored at 4 °C until transporting to laboratory just after collecting the second sample.
In both partners, 8-isoprostanes (8-EPI) were measured in urine. Diene conjugates (DC) and total antioxidant capacity (TAC) were measured in seminal plasma. ROS-TAC score was calculated as ratio of DC and TAC.
The analysis of semen was performed according to WHO guidelines (World Health Organization, 1999). Semen volume was estimated by weighing the collection tube with the semen sample and subsequently subtracting the pre-determined weight of the empty tube assuming 1 g = 1 mL. Motility was assessed to report the number of motile spermatozoa (WHO motility classes A + B). Sperm concentration was assessed using the improved Neubauer haemocytometers. Total sperm count was calculated by multiplying semen volume by sperm concentration.
For cytological analysis, Bryan–Leishman stained semen smears were made and examined with the use of oil immersion microscopy (magnification: ×1000) by an experienced microscopist. The WBC concentration in semen was calculated by using the known sperm concentration. One hundred round cells were counted twice, and their mean value was registered.
Diene conjugates were measured according to the method previously described (Recknagel & Glende, 1984) with minor modifications (Starkopf et al., 1995). Briefly, samples (0.15 mL) + 0.15 mL 0.9% NaCl (reagent blank contains only isotonic saline) were incubated at 37 °C for 30 min, 0.25% butylated hydroxytoluene (0.015 mL) was added and the lipids were extracted by heptane/isopropanol (1 : 1, total volume 1.8 mL). Then the samples were acidified by 5 m hydrochloric acid (0.5 mL). After extraction by cold heptane (1.6 mL), samples were centrifuged (for 5 min at 300 g) and absorbance of heptane fraction was measured spectrophotometrically at absorbance maximum at 234 nm.
Total antioxidant capacity (TAC)
A novel automated colorimetric measurement method was employed (Erel, 2004). By this method, a colourless molecule, reduced 2,2′-azinobis(3-ethylbenzthiazoline-6-sulphonate) (ABTS), is oxidized to a blue-green ABTS*+, using hydrogen peroxide in acidic medium (the acetate buffer 30 ml/L pH 3,6). When the coloured ABTS*+ is mixed with any substance, that can be oxidized, it is reduced to its original colourless ABTS form again. The ABTS*+ is decolorized by antioxidants according to their concentrations and antioxidant capacities. This change of colour is measured as a change in absorbance at 660 nm.
Briefly, 200 μL of reagent 1 (R1: acetate buffer 0,4 mol/L, pH 5,8) was carefully mixed with 5 μL of test sample in cuvette and incubated for 4 min at 37 °C in the spectrophotometer. The first absorbance was taken before the mixing with 20 μL R2 [R2: the ABTS*+ in acetate buffer 30 mmol/L, pH 3,6 (as sample blank)]. The last absorbance was taken at the end of incubation (5 min). The bleaching rate is inversely related with the TAC of sample. The reaction rate was calibrated with Trolox, which is used as a traditional standard for TAC measurement assays. The results are expressed in mmol Trolox equivalent/L. Within- and between-batch precision data obtained by TAC method were 2.5 and 2.9% respectively.
8-Isoprostanes (8-EPI) in urine
Competitive enzyme-linked immunoassay for determining levels of 8-EPI in biological samples (BIOXYTECH 8-Isoprostane Assay, Cat. No.21019; Oxis International, Inc., Portland, OR, USA) was used as described earlier (Kullisaar et al., 2003). Briefly, 8-EPI in the samples or standards competes for binding (to the antibody coated on the plate) with 8-EPI conjugated to horseradish peroxidase (HRP). The peroxidase activity results in colour development when the substrate is added. The intensity of the colour is proportional to the amount of 8-EPI–HRP bound and inversely proportional to the amount of 8-EPI in the samples or standards. The urinary concentrations of isoprostanes were corrected by urinary creatinine concentrations to account for the differences in renal excretory function.
The study tested the null hypothesis that there is no difference in systemic OxS level in women's organism before and after sexual intercourse. Statistical analyses were performed with the use of SigmaStat (Systat Software, Chicago, IL, USA). The study groups were compared with Student's t-test (in case of normal distribution) and Mann–Whitney rank sum test (in case of non-parametric distribution). Pearson product-moment correlation was used to find out correlations between different markers. Statistical significance was assumed at p < 0.05 level for all parameters.
The leukocytospermic men were characterized by OxS as revealed by elevated levels of OxS markers (DC, 8-isoprostanes) and decreased antioxidant levels (TAC), although because of small groups these differences were above significance level. However, the ROS-TAC score (DC/TAC ratio) was significantly higher in leukocytospermic men than the rest of men (Table 2).
Table 2. Markers of oxidative stress in infertile couples
|DC (μm)||13.8 (11.0–17.1)||8.0 (6.7–12.6)||0.167|
|TAC (mmol/l)||1.5 (1.3–1.8)||1.9 (1.8–1.9)||0.052|
|ROS-TAC score||9.1 (6.9–10.8)||4.4 (3.6–6.9)||0.024|
|8-Isoprostanes (ng/mmol creatinine)||51.9 (49.6–70.3)||48.7 (39.9–83.6)||ns|
|8-Isoprostanes in all women (ng/mmol creatinine)||45.2 (32.3–66.1)||59.7 (45.3–77.6)||0.164|
|8-Isoprostanes in partners of leukocytospermic men (ng/mmol creatinine)||32.7 (31.1–38.6)||70.4 (56.5–77.2)||0.006|
|8-Isoprostanes in partners of non-leukocytospermic men (ng/mmol creatinine)||51.2 (34.5–66.2)||54.8 (39.8–76.8)||ns|
Sexual intercourse increased systemic OxS level in women's organism as revealed by the levels of 8-isoprostanes in urine (Table 2). This shift was slightly over the significance level for the whole group, but it was remarkably prominent in women whose partner had leukocytospermia. In addition, there was no correlation in 8-isoprostanes' levels between the women's pre- and post-intercourse samples as well as the women's pre-intercourse samples and that of their men, but there was significant correlation between the women's post-intercourse samples with that of their men (R = 0.59, p = 0.015) that confirms influence of male OxS on their female partners. Therefore, the null hypothesis was rejected for women whose partner had leukocytospermia.
We have noted for the first time that sexual intercourse increases the OxS level in the women's organism when their male partner has leukocytospermia.
Oxidative stress is a consequence of an imbalance between the production of ROS and the body's antioxidant defence mechanisms. It has been implicated in the pathogenesis of many human diseases including atherosclerosis, cancer, diabetes, liver damage, rheumatoid arthritis, cataracts, AIDS, inflammatory bowel disease and central nervous system disorders. In case of inflammation in male genital tract, the high-grade OxS is associated with alterations in metabolism, motility and DNA damage of spermatozoa. Excessive ROS levels cause peroxidative damage of the unsaturated fatty acids in the sperm membrane, and lead to incompetence of sperm–oocyte fusion (Aitken et al., 1992; Erenpreiss et al., 2006; Makker et al., 2009). Leukocytospermic prostatitis patients are characterized by both local and systemic OxS that was revealed also by our previous study (Kullisaar et al., 2008). Local OxS appears because of increased numbers of peroxidase-positive leukocytes in semen that produce large amounts of ROS, whereas systemic OxS that is evident as increased levels of urinary 8-isoprostanes and reduced levels of blood glutathione arises because of local inflammation in the prostate (Türk & Kullisaar, 2011; Kullisaar et al., 2012). In addition, prostatitis is associated with genetic polymorphisms related directly to antioxidative defence, or immune cross-reactivity against proteins that repair oxidative damage (Arisan et al., 2006; Mazzoli, 2010).
The influence of male OxS on the woman's organism has not been investigated so far. Our study group included the subjects with and without leukocytospermia. We observed minimal increase in OxS level in women's organism when their partners did not have leukocytospermia, but we noted significant increase in OxS levels in the partners of leukocytospermic men that was statistically significant despite of very small study groups. This change may add an additional component to the couple's infertility that is already affected by prostatitis. As infertility is commonly a result of several synergistic factors, the subsidiary effect of OxS may be the ultimate component resulting this malady. In addition, OxS has been associated with termination of early pregnancy, too, as ROS or their products, lipid peroxides, stimulate synthesis of PGF2α that causes uterine contraction (Sugino et al., 2000).
The mechanism of OxS transfer during sexual intercourse needs to be elucidated. As it was evident in these women in systemic level, the transfer of not OxS markers, but rather the inducer(s) of OxS could be suspected. It is likely that pro-inflammatory cytokines and ROS released by the (transferred to woman) activated macrophages may influence the redox status and OxS level of the vagina and so increase the production of locally produced 8-isoprostanes in vagina which may have possible feed-back mechanism to influence the systemic OxS level in female partner (Türk & Kullisaar, 2011; Kullisaar et al., 2012). Moreover, it has been shown that motor activity of lower urinary tract and bladder obstruction result in elevation of urinary OxS markers (Tarcan et al., 2000; Matsumoto et al., 2010). Similar mechanical mechanism may somewhat contribute to the after-intercourse increase in OxS markers in our study. In addition, the oxidative damage of sperm cell membranes may influence redox status of vagina and may activate macrophages during the sexual intercourse. Pandya & Cohen (1985) have even considered some leukocytosis in cervix after insemination as physiological response. However, in our study significant differences between the groups were observed. We also noted somewhat lower (although statistically insignificant, p = 0.13) baseline 8-EPI levels in female partners of leukocytospermic men that may be associated with adaptation mechanism to chronic stressor from male side.
Measurement of 8-isoprostanes (the products of lipid peroxidation) in urine is a reliable approach to assess systemic OxS in vivo, providing an important tool for exploring its role in the pathogenesis of diseases. Isoprostanes are stereoisomers of prostaglandins that are formed primarily through the non-enzymatic peroxidation of arachidonic acid by ROS. Isoprostanes' pathway products exert potent bioactivity via both receptor-dependent and receptor-independent mechanisms and therefore may contribute to disease (Montuschi et al., 2007). Stability of 8-isoprostanes in different specimens and at different regimens has been investigated in several studies. It has been shown that they are very stable at −80 °C, but two cycles of freezing and thawing may decrease their levels by 30%. Isoprostane levels in urine are stable also at 4 °C or room temperature for up to 10 days, and no artifactual formation of the isoprostanes have been detected during the sampling and processing (Pratico et al., 1998; Kitano et al., 2006; Samitas et al., 2009). In our experiment two self-collected specimens were investigated and we asked the women to store the before-intercourse specimens at 4 °C overnight to avoid repeated freezing and thawing.
The weakness of our study is the absence of fertile control group, and also the study groups are very small. This kind of study is quite complicated and not many couples are motivated to participate. As concerns smoking status that may affect OxS levels, then there were some smokers in both women's group (Table 1). We noted that the baseline levels of 8-isoprostanes tended to be slightly higher in smoking women, but because of very small groups this difference was not significant.
In conclusion, sexual intercourse with male partner having leukocytospermia increases the OxS level in the women's organism that may interfere with fertilization.
The authors would like to thank Dr. Paul Korrovits, Dr. Kristo Ausmees and Ms. Kadri Poolak for excellent technical help. This study was supported by Estonian Science Foundation (grant no. 5701), Estonian Ministry of Education and Research (target financing no. SF0180132s08) and Enterprise Estonia (grant no. EU30200).