Inflammatory bowel disease (ulcerative colitis type) severity shows inverse correlation with semen parameters and testosterone levels

INTRODUCTION

Inflammatory bowel disease (IBD) is a chronic disease affecting the digestive tract. IBD is caused by factors such as the immune system, environmental factors, gut flora, and genetics.^1–3 IBD is divided into two subtypes: ulcerative colitis (UC) and Crohn’s disease (CD). The incidence of CD is approximately 2.1–6 per 100 000 people, while UC is seen in 2.2–4.5 per 100 000 people. The incidences of CD and UC in the group of 20–29 years are 16.6–18.7 per 100 000 people. Although data from developing countries are lacking, there is a rising incidence of UC in industrialized countries in Asia, the Middle East, and South America.⁴

Hypogonadism can affect approximately 40% of patients with IBD,^2,5 which may be caused by a direct effect of inflammatory mediators on the reproductive axis and testicular function, poor nutrition, decreased leptin levels, and long-term glucocorticoid treatment in these patients.⁶ The prevalence of male infertility in patients with IBD is higher than that in the general population. In a case–control study, Moody et al.⁷ found that the number of children born to fathers with CD was significantly lower than that of men with UC and the general population. The causes of male infertility in patients with IBD include surgery, medication, active disease, malnutrition, alcohol consumption, tobacco use, and psychological factors.⁸ Our previous research demonstrated that the degree of rectal bleeding in IBD had a significant impact on testosterone and free testosterone levels.¹ The study also found a significant correlation of semen parameters, particularly progressive motility, immotile spermatozoa, and normal spermatozoa morphology with the degree of rectal bleeding in IBD.¹ Grosen et al.⁹ stated that in patients with severe IBD, the progressive motility of spermatozoa was impaired, which increased significantly in the remission phase. During the active phase of the disease, testosterone levels decreased and returned to normal levels after the remission phase.⁹ The release of inflammatory mediators in active IBD increases reactive oxygen species (ROS) and oxidative stress. The presence of pro-inflammatory mediators such as tumor necrosis factor-α (TNF-α) and interleukin 1 (IL-1) can also increase the cytokine-mediated anti-fertility effect.⁹ This study aims to determine the profile of semen and sex steroids in patients with IBD and analyze how the Mayo score relates to the semen and sex steroid profiles in UC patients.

PATIENTS AND METHODS

This cross-sectional study was conducted on patients with UC-type IBD (UC-IBD) who visited the Gastroenterology Clinic, Adam Malik General Hospital, Medan, Indonesia, from December 2022 to March 2023. The study was approved by the Ethics Commission of the Adam Malik General Hospital/Faculty of Medicine, University of North Sumatra, Medan, Indonesia (Approval No. 441/KEPK/USU/2022). After the patients got the researcher’s explanation, the patients signed informed consent and willing to undergo an examination. Informed consent of patients was unsubmitted to the article due to patient’s data protection.

Samples’ and patients’ selection criteria

Patients diagnosed with UC-IBD were included in this study. This type was selected because the number of patients with UC-IBD is greater than that of patients with CD. The patients were examined by an intern with more than 3 years of experience in treating patients with IBD. The first author performed the diagnosis of IBD through anamnesis, physical examination, colonoscopy, and tissue histopathology. The patients recruited for this study were men aged 20–40 years, married, undergoing treatment for IBD, and willing to masturbate to provide a semen sample and donate a blood sample for hormonal analysis.

Laboratory tests

After the diagnosis of IBD was established, the intern calculated the Mayo score and then classified the patients as mild, moderate, and severe based on the criteria proposed by Schroeder.¹⁰ Subsequently, the patients were asked for their consent for semen and sex steroid examinations. The patients who agreed to be sampled were asked to abstain for 2–5 days and postcompletion of the abstinence period; they were directed to the masturbation room for collection of the semen sample. For sex steroid examinations, the patients were requested to fast for at least 8 h and then donate their blood samples in the morning at approximately 7 a.m.–9 a.m. Semen examination procedures were performed following the World Health Organization (WHO) 2010 standards,¹¹ while sex steroid examinations were performed using an enzyme-linked immunosorbent assay (ELISA) kit (Roche Diagnostics Ltd., Shanghai, China).

Data analysis

Parametric tests were performed on the numerical data. After the data were collected, they were analyzed using GraphPad Prism (version 9.5.0, GraphPad Software, San Diego, CA, USA). The distribution of the frequency data (mean and median) was determined for each variable. If a parameter was normally distributed, the data were displayed as mean ± standard deviation (s.d.); if not, the parameters were presented as median and interquartile range (IQR).

The analysis of differences in each parameter of semen and sex steroids based on the Mayo score was performed using the one-way analysis of variance (ANOVA) test if the data were normally distributed, and the Kruskal–Wallis test if not. Post hoc analysis was performed after this test. To assess the correlation (r) between the Mayo score and semen and sex steroid parameters, a Spearman correlation test was performed if the data were normally distributed, and a Pearson correlation test was performed if the data were not normally distributed. P < 0.05 indicates statistically significant.

RESULTS

Patient characteristics

A total of 120 patients were analyzed in this study. The mean ± s.d. age of the patients was 32.9 ± 4.2 years. In addition, the patients’ average body mass index (BMI; mean ± s.d.: 22.68 ± 3.43 kg m⁻²) was within the normal range according to the BMI criteria for Asians. The distribution of the IBD samples according to the Mayo score was as follows: mild (26.7%), moderate (55.0%), and severe (18.3%). Among all the patients, 106 (88.3%) had not received any treatment, nine patients had taken treatment with mesalazine and methylprednisolone, and five patients had used mesalazine alone. One hundred and eleven (92.5%) of the patients were married and had children. The patients were examined for semen and sex steroids after fasting for at least 8 h. Detailed characteristics of the patients are listed in Table 1.

Semen parameters differ among IBD groups

Semen parameters were compared among three patient groups classified according to the Mayo disease score. The Kruskal–Wallis test showed a significant difference in sperm concentration among the groups (P = 0.0007). The post hoc test revealed significant differences between the mild and moderate groups (P = 0.045) and between the mild and severe groups (P = 0.0005), but the differences between the moderate and severe groups were not significant (P = 0.100). The mild group presented with a higher sperm concentration (median [IQR]: 47.7 [38.0–54.6] ×10⁶ ml⁻¹) than that in the moderate (median [IQR]: 38.0 [13.0–54.4] ×10⁶ ml⁻¹) and severe (median [IQR]: 14.0 [12.0–44.0] ×10⁶ ml⁻¹) groups. In the one-way ANOVA, significant differences in sperm motility were observed among the three groups (P < 0.0001). The post hoc test showed significant differences between the mild and moderate groups (P < 0.0001), between mild and severe groups (P < 0.0001), and between moderate and severe groups (P < 0.0001). The mild group had higher total sperm motility (mean ± s.d.: 80.1% ± 8.2%) in comparison with the moderate (mean ± s.d.: 68.8% ± 13.1%) and severe (mean ± s.d.: 55.5% ± 7.1%) groups.

Similar to sperm concentration and motility, sperm morphology differed significantly among the three patient groups (P < 0.0001). This was followed by a post hoc test, which showed significant differences between the mild and moderate groups (P < 0.0001), between the mild and severe groups (P < 0.0001), and between the moderate and severe groups (P = 0.0001). The mild group had fewer percentage of abnormal sperm counts, with a median of 37.5% (IQR: 25.3%–44.8%) compared to the moderate group with 84.5% (IQR: 70.0%–97.0%) and the severe group with 98.0% (IQR: 98.0%–99.3%). In comparison, Kruskal–Wallis analysis showed no significant differences in leukocyte count (P = 0.160), number of immature germ cells (P = 0.745), and semen volume (P = 0.898) among the three groups. The results of this analysis are shown in Figure 1.

Testosterone and SHBG differ among IBD groups

Four steroids were evaluated in this study. Analysis of total testosterone levels among IBD groups showed significant differences (P < 0.0001). The post hoc test results showed significant differences between the mild and moderate groups (P = 0.0007), between the mild and severe groups (P < 0.0001), and between the moderate and severe groups (P < 0.0001). The mild group had higher median testosterone levels of 12.8 (IQR: 9.9–13.5) nmol l^-1 in comparison to the moderate group (median [IQR]: 9.7 (9.0–10.8) nmol l^-1), and the severe group (median [IQR]: 8.3 [7.9–8.8] nmol l^-1). Comparison of free testosterone levels using post hoc test showed significant differences between the mild and moderate groups (P = 0.0006), between the mild and severe groups (P < 0.0001), and between the moderate and severe groups (P < 0.0001). The mild group had higher free testosterone levels (median [IQR]: 6.7 [5.1–7.3] nmol l^-1) than the moderate and severe groups.

One-way ANOVA for estrogen levels showed significant differences among the three groups (P = 0.043); however, the post hoc analysis showed no significant differences among the three groups.

A comparison of SHBG levels using the Kruskal–Wallis test showed significant differences among the three groups (P = 0.024). A post hoc comparison showed a significant difference between the mild and severe groups (P = 0.034). The mild group had lower SHBG levels (median [IQR]: 16.5 [3.7–17.6] nmol l^-1) than the moderate group (median [IQR]: 16.9 [15.8–18.0] nmol l^-1), and the severe group (median [IQR]: 17.6 [15.3–18.5] nmol l^-1). The overall results of this test are shown in Figure 2.

Correlation between IBD Mayo score and semen parameters and sex steroids

The Mayo score is an accepted method for classifying IBD according to severity. As there were significant differences in the number of parameters among the IBD groups based on the Mayo score, we performed a correlation analysis between the Mayo score and various study parameters (semen volume, sperm concentrations, rapid progressive, immotility, abnormal morphology, estradiol, SHBG, free testosterone, and total testosterone; Table 2). The Mayo score showed negative correlations with sperm concentration (r = −0.375, P < 0.0001), rapid progressive (r = −0.660, P < 0.0001), free testosterone (r = −0.732, P < 0.0001), and total testosterone (r = −0.721, P < 0.0001), and positive correlations with immotile sperm (r = 0.660, P < 0.0001), abnormal morphology (r = 0.657, P < 0.0001), and SHBG (r = 0.278, P = 0.002); but showed no significant correlation with estradiol. Because some of these parameters could be affected by the treatment that the patients were receiving, we also performed a correlation test after excluding patients who had received treatment (mesalazine and methylprednisolone) and had an excessive BMI (>22.9 kg m⁻²). These two drugs (mesalazine and methylprednisolone) independently affect sperm quality and sex steroids. The adjusted results showed a significant correlation between Mayo score and sperm concentration (P = 0.0127), rapid progressive motility (P < 0.0001), immotile sperm (P < 0.0001), sperm morphology (P < 0.0001), free testosterone levels (P < 0.0001), and total testosterone levels (P < 0.0001), as shown in Table 2.

DISCUSSION

The Mayo score is an objective assessment method that has long been used to assess the progression and severity of IBD. The Mayo score reflects IBD progression. IBD is thought to affect semen quality and male fertility; however, data on the effects of IBD severity on semen parameters are scarce. In this study, we attempted to classify patients with IBD according to disease severity based on the Mayo score and understand the impact of disease severity on semen parameters and hormone levels. We observed an inverse correlation between IBD severity and sperm concentration, sperm motility, and normal morphology. Similarly, a significant inverse correlation was observed between disease severity and total testosterone and free testosterone levels. In contrast, SHBG levels were directly correlated with IBD severity. The results of this study indicated that the severity of IBD is one of the possible factors that can cause disturbances in hormone levels, including testosterone, and affect semen quality in these patients. We reported similar findings in our previous study.¹ This study made a comprehensive comparison between the IBD severity and semen parameters.

The association between semen quality and IBD is an interesting observation without an obvious answer. There are several possible explanations for this association. Increased oxidation-reduction potential (ORP) has been reported in IBD cases,¹² which may affect sperm concentration, total motility, and morphology.^12,13 In patients with IBD, ulcerative colitis and inflammation are generally linked to intestinal mucosal injury. This injury increases the activity of enzymes such as myeloperoxidase (MPO), which would result in an increase in ROS in these patients.³ Further, inadequate antioxidant activity in IBD has been reported to be associated with disease activity or low consumption of nutrients.¹⁴ This imbalance between ROS and antioxidant activity impacts various health parameters, resulting in poor semen quality. Although we did not examine the ORP in this study, we assumed that IBD patients with higher Mayo scores would present with higher ORP than those with lower Mayo scores.

Apart from the ORP factor, another possible cause of decreased semen quality in the IBD group of severe degree could be decreased testosterone levels. Testosterone is essential for spermatogenesis. We found an inverse correlation between the Mayo score and free testosterone levels. While the reasons behind decreased free testosterone levels in patients with IBD remain obscure, increased oxidation, poor nutrient absorption, and inflammatory disease might contribute to poor testosterone production in these patients. The relationship between testosterone levels and IBD is also indicated by the relatively higher incidence of autoimmune diseases in women than in men.¹⁵ Anti-inflammatory properties of testosterone are well-known,¹⁶ due to which, testosterone protects against several inflammatory diseases. The cause-and-effect relationship between testosterone level and IBD remains an interesting topic for future research. In this study, we attempted to rule out the possible effects of mesalazine treatment on spermatogenesis. A previous study has reported that this drug affects fertility potential.⁶ We found that the correlation between IBD and poor semen quality remained statistically significant even after excluding patients using medications for IBD management. This finding suggests that IBD is associated with reduced testosterone levels and fertility.

Another possible mechanism linking IBD with reduced fertility is inflammation and immunological reactions triggered by IBD. Chronic inflammation triggers the formation of pro-inflammatory cytokines, such as TNF-α and IL-1, in the male reproductive tract. This causes an increase in pro-infertility cytokines.^1,17 These pro-inflammatory cytokines have also been shown to increase aromatase expression, causing higher conversion of androgens into estrogens.¹⁸ In addition, these immune-triggering factors may also induce anti-sperm antibodies (ASA).¹⁹ Immune processes due to IBD may also be related to the existence and diversity of the gut microbiota. The effect of the gut microbiota on testosterone metabolism is well known.^20–22 Inflammatory processes in IBD may affect the gut microbiome,^21,22 resulting in altered nutritional status, inflammatory signaling, and eventual disturbance in body homeostasis, testosterone production, and spermatogenesis. These mechanisms are not yet precisely understood but appear to be the most convincing explanation for the association between IBD and compromised spermatogenesis. Unfortunately, we could not perform ASA, cytokine, or gut microbiome analyses.

Although IBD is one of the most challenging global health problems, it is now becoming clear that it affects organ systems beyond the gut. This study can form a basis for holistic IBD treatment, especially for patients with complaints of infertility or sexual dysfunction. A limitation of this study is that we did not measure the levels of ROS, DNA fragmentation index, mixed agglutination reaction, or gut microbiota, which could provide further insights into the possible mechanisms underlying our observations.

CONCLUSION

The IBD Mayo score showed a negative correlation with sperm concentration, rapid progressive motility, free testosterone, and total testosterone, and a positive correlation with immotile sperm, abnormal morphology, and SHBG, but showed no significant correlation with estradiol. The mechanisms underlying this observation are poorly understood, but increased ROS and cytokine production, poor nutrient absorption, and gut microbiome dysbiosis in IBD chronic inflammation could be chiefly responsible for this effect on semen parameters. Despite some inherent limitations, our findings suggest that treating IBD has far deeper implications than simply managing the gut problems in these patients.

AUTHOR CONTRIBUTIONS

DD and CP were involved in conceptualization, data curation, data analysis, visualization, and project administration. DD, CP, WSP, and HH prepared the original draft. DD, CP, RS, AS, and AR contributed to the writing and editing of the article. All authors read and approved the final manuscript.

COMPETING INTERESTS

All authors declare no competing interests.