Associations of Ureaplasma urealyticum infection with male infertility and intrauterine insemination outcomes.

Approximately 15% of couples worldwide are currently experiencing infertility issues, 1 2 with male infertility accounting for approximately 50% of cases. 3 Numerous factors contribute to male infertility, including genital tract infection. 2 Ureaplasma urealyticum (UU) has been suggested as one of the predominant pathogens. These infections include urethritis, seminal infection, prostatitis, epididymitis, and orchitis, which can cause damage to the male reproductive system. 4 Although the adverse effects of genital tract infections on female infertile patients have been well studied, the impact of seminal UU infection in semen on male infertility and intrauterine insemination (IUI) outcomes remains a subject of debate. 5 Seminal UU infection may contribute to male infertility by affecting semen quality. 6 7 Additionally, it can also affect the outcomes of women’s natural pregnancies and assisted reproductive technology. UU infection can impair sperm quality directly by interacting with and damaging organs and spermatozoa, or indirectly by inducing inflammatory and oxidative stress reactions. 8 9 10 11 Specifically, it leads to increased levels of reactive oxygen species (ROS) in semen and disrupts the internal environment for spermatogenesis, resulting in decreased sperm motility as well as elevated levels of sperm DNA fragmentation index (DFI) and high DNA stainability (HDS). 12 However, other studies have reported that seminal UU infection may not have a substantial negative effect on semen quality, pregnancy rates, or assisted reproduction outcomes. 13 While studies have examined the effects of genital tract infections on fertility, 4 6 the specific impact of UU infection in the context of IUI remains relatively unexplored. IUI is often offered to infertile young couples with a low predicted chance of natural conception before more invasive in vitro fertilization (IVF) treatment is considered. 14 In light of these considerations, our study aimed to investigate whether seminal UU infection affects semen quality, pregnancy outcomes, and neonatal outcomes in patients undergoing IUI treatment. This study provides valuable insights into the management of male infertility, particularly that of men with UU infection, and its potential implications for treatment decisions, including the selection of IUI as a preferred approach for nonsevere infertility cases.

JH, XSZ and YYW designed the study. YYW, SYL, and XHJ collected the data. WJL, YYW, XC, and LMW performed the data parameter analysis. JH and XYS wrote the manuscript. XSZ, SB, and XHJ revised the manuscript. All authors read and approved the final manuscript.

Between January 2021 and January 2023, a total of 877 infertile couples who underwent IUI at The First Affiliated Hospital of USTC (Hefei, China) were included in this study. Unexplained infertility, mild male factor infertility (sperm concentration: 10 × 10 6 –15 × 10 6 ml −1 ; progressive motility [PR]: 15%–32%; malformation rate ≤97%; semen volume: 1–1.5 ml; or semen liquefaction time > 60 min), mild endometriosis, ovulation disorders, and sexual dysfunction are common indications for IUI. On the basis of the UU detection results, the infertile couples were divided into two groups: the UU-positive (UU+) group and the UU-negative (UU−) group. After three rounds of exclusion, only 245 infertile couples remained for inclusion in the final analysis ( Figure 1 ). The exclusion criteria were as follows: (1) females with pelvic endometriosis, adenomyosis, diminished ovarian reserve, bilateral fallopian tube obstruction, or complications such as pelvic effusion; (2) males with azoospermia, severe oligoasthenoteratozoospermia, varicocele, orchitis, cryptorchidism, urological trauma, or diseases caused by genetic defects (including single gene diseases and abnormal chromosome number and structure); and (3) patients who tested negative for UU after 1 week of antibiotic treatment. The majority of infected male patients are asymptomatic, whereas some patients may experience pruritus, discomfort, or tingling at the meatus or urethra. Additionally, they may also present with testicular and perineal pain accompanied by varying degrees of urgency, dysuria, and other urinary symptoms. Flow diagram from the original cohort recruitment to the final comprehensive evaluation study. UU: Ureaplasma urealyticum ; IUI: intrauterine insemination; UU+: UU-positive; UU−: UU-negative. This study was approved by the Ethics Committee of the First Affiliated Hospital of USTC (Approval No. 2022-re-417), and written informed consent was obtained from all participating patients. All semen samples were subjected to UU culture and drug sensitivity tests in accordance with the manufacturer’s instructions (Zhong Ai Sheng Hebei Biotechnology Co., Ltd., Xingtai, China). The semen was placed in liquid medium, which was then dispensed into wells containing indicators that include urea and arginine to reflect the growth of potential agents. The plates were subsequently incubated in a 36°C ± 1°C incubator. The presence of UU was indicated by a color change (UU decomposes urea in the culture medium), resulting in an increase in pH. The color of the medium changed from orange-yellow to red after 24 h of incubation. In the second step, identification and colony-forming units were performed in similar wells for positive specimens. Subsequently, the detection of 1 × 10 4 color changing units (CCUs) per ml growth or greater was considered a positive result. Co-infection with other microorganisms including Mycoplasma hominis ( M. hominis ), Chlamydia trachomatis ( C. trachomatis ), Neisseria gonorrhoeae ( N. gonorrhoeae ), and fungi was determined via routine laboratory methods. Latex immunochromatography tests (Abogen Biosciences Co., Ltd., Hangzhou, China) were used for C. trachomatis testing. Standard culturing methods (Autobio Biosciences Co., Ltd., Zhengzhou, China) were employed to detect N. gonorrhoeae . Fungi were identified via the fungal slide culture method (Tianda Diagnostic Reagents Co., Ltd., Hefei, China). Semen samples were collected from patients following a period of 2–7 days of abstinence by masturbation into sterile containers. The standard semen parameters including semen volume, sperm concentration, percentage of spermatozoa with progressive motility (PR), percentage of spermatozoa with nonprogressive motility (NP), total motility (PR+NP), and percentage of spermatozoa with inactive motility (IM) were assessed in a specialized seminal laboratory using computer-assisted sperm analysis (CASA; SAS Medical, Beijing, China). Straight-line velocity (VSL) was measured to classify sperm movement: VSL ≥15 μm s −1 for the forward motion (PR), 5 μm s −1 ≤ VSL<15 μm s −1 as a non-forward movement (NP), and VSL < 5 μm s −1 was defined as immobility (IM). The assessment of sperm concentration and sperm motility was carried out by Saes SAS-II sperm quality analyzer (SAS Medical). Sperm morphology, leukocyte count, and anti-sperm antibodies were determined through Diff-Quick staining, benzidine peroxidase staining, and mixed antiglobulin reaction, respectively (Anke Biotechnology, Hefei, China). The morphological parameters of sperm and peroxidase-positive cells were analyzed under a microscope (Leica DM2500, Wetzlar, Germany). All analyses were conducted and evaluated following the 5 th edition of World Health Organization (WHO) manual. 15 Fluorescence signals from spermatozoa stained with acridine orange were analyzed using the BD Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA). The resulting data were processed using DFI viewer software (Cellpro, Ningbo, China), yielding values for HDS and sperm DFI. The HDS value reflects the percentage of immature nuclear spermatozoa, while the DFI represents the extent of damage to sperm nuclear chromatin. Ovulation was monitored on the basis of the woman’s natural or artificially induced menstrual cycle. Once the dominant follicle diameter exceeded 14 mm, B-ultrasound monitoring was conducted daily in conjunction with urinary luteinizing hormone (LH) detection. When a dominant follicle reached a diameter of >18 mm and a urinary LH peak was present, human chorionic gonadotropin (hCG) at a dosage of 8000–10 000 IU (Lizhu Pharm, Zhuhai, China) was injected into the muscle in the morning, followed by insemination within 24 h. Clinical pregnancy was confirmed through blood testing and urinary hCG assessment 17 days after the procedure. If the test was positive, an ultrasound examination was performed after 2–3 weeks to check for the presence of a gestational sac and fetal heartbeat during biochemical pregnancy. Continued follow-up included early, middle, and late pregnancy information as well as delivery outcomes and neonatal conditions. Statistical analyses of the data were performed via R version 3.5.3 (R Core Team, Vienna, Austria). In general, continuous variables with a normal distribution are expressed as the mean ± standard deviations (s.d.), whereas nonnormally distributed variables are presented as median (interquartile range [IQR]). Categorical variables are represented as frequencies (percentages). Student’s t -test was applied for normally distributed continuous variables, whereas the Mann–Whitney U test was employed for nonnormally distributed variables. Pearson’s Chi-square test or Fisher’s exact test was used for qualitative data parameter analyses. Univariate and multivariate logistic regression analyses were used to adjust for confounding factors. P < 0.05 was considered statistically significant.

A total of 245 male patients from infertile groups were included in this study ( Figure 1 ). Among these patients, 53 patients were UU positive (the UU+ group) and 192 patients were UU negative (the UU− group). There were no significant differences between the two groups in terms of age, body mass index (BMI), duration of infertility, and infertility type (all P > 0.05). The reproductive hormonal parameters of the female spouses such as follicle-stimulating hormone (FSH), estradiol (E2), progesterone (P), prolactin (PRL), LH, testosterone (T), endometrial thickness on the day of IUI, and the type of ovulation induction modality used for IUI were not significantly different between the UU− and UU+ groups (all P > 0.05). In summary, there were no statistically significant differences between the two groups in terms of general clinical data parameters ( Table 1 ). General clinical data characteristics of the UU+ and UU− groups BMI: body mass index; FSH: follicle-stimulating hormone; E2: estradiol; P: progestational hormone; PRL: prolactin; LH: luteinizing hormone; UU+: Ureaplasma urealyticum -positive; UU−: Ureaplasma urealyticum -negative; s.d.: standard deviation; IQR: interquartile range Variables associated with semen quality were examined and are summarized in Table 2 . The proportion of patients with an abnormal sperm volume (<1.5 ml) was not significantly different between the UU+ group and the UU− group (15.1% vs 10.9%, P = 0.41). The sperm concentration was normal in most patients, with only a few patients in both groups showing abnormalities (<15 × 10 6 ml −1 ; 5.7% vs 2.6%, P = 0.38). The ratio of abnormal sperm PR (<32%) in UU+ semen samples was nearly the same as that in UU− semen samples ( n = 21 [39.6%] vs n = 72 [37.5%]; P = 0.79). More than 40% of the patients in both groups had abnormal total sperm motility (< 40%; n = 23 [43.4%] vs n = 81 [42.2%]; P = 0.88). Next, we evaluated several parameters, including the semen leukocyte count, anti-sperm antibody level, the proportion of abnormal anti-sperm antibody level (AsA > 10%) , abnormal sperm morphology, and the proportion of abnormal sperm DFI (DFI > 15%). There were no significant differences in these semen parameters between the UU+ and UU− group ( P > 0.05). The sperm HDS status was also compared between the UU+ and UU− groups. We detected a statistically significant difference in the sperm HDS status between the UU+ and UU− group (median [IQR]: 8.0 [6.1–11.3] vs 6.8 [5.0–9.5], P = 0.04), indicating that the sperm nuclear maturity of the UU+ group was lower than that of the UU− group. Characteristics of semen parameters in the UU+ and UU− groups of male infertile patients a Fisher’s exact test. AsA: antisperm antibody; DFI: DNA fragmentation index; HDS: high DNA stainability; UU+: Ureaplasma urealyticum -positive; UU−: Ureaplasma urealyticum -negative; IQR: interquartile range Comparisons between the UU+ and UU− groups regarding IUI pregnancy and neonatal outcomes revealed no statistically significant differences ( Table 3 ). The mean gestational age at birth (38.2 weeks vs 38.6 weeks, P = 0.39), biochemical pregnancy rate (35.9% vs 25.5%, P = 0.15), clinical pregnancy rate (32.1% vs 22.4%, P = 0.15), live birth rate (20.8% vs 17.7%, P = 0.61), and miscarriage rate (35.3% vs 20.9%, P = 0.32) were comparable between the two groups. Similarly, there were no significant differences in the rates of premature rupture of membranes (17.7% vs 2.3%, P = 0.07), premature birth (18.2% vs 8.8%, P = 0.58), and cesarean section (63.6% vs 47.1%, P = 0.34) between the UU+ and UU− groups. Additionally, there were no significant differences in the proportions of male and female neonates ( P = 0.55), birth weight ( P = 0.41), birth height ( P = 0.17), or Apgar score ( P = 0.40). In summary, the study revealed no significant differences in pregnancy and neonatal outcomes between the UU+ and UU− groups, suggesting that seminal UU infection did not adversely affect these outcomes following IUI procedures. Pregnancy outcomes and neonatal characteristics of the UU+ and UU− groups a Fisher’s exact test. UU+: Ureaplasma urealyticum -positive; UU−: Ureaplasma urealyticum -negative; s.d.: standard deviation After controlling for other variables, we examined the associations between UU infection status and outcomes. Univariate analysis showed that the odds ratio (OR) for clinical pregnancy rate in the UU+ group compared with the UU− group was 1.76 (95% confidence interval [CI]: 0.83–3.75, P = 0.14) after adjusting for potential confounders ( Table 4 ). Additionally, after adjustment, the OR for the miscarriage rate in the UU+ group compared with the UU− group was 1.93 (95% CI: 0.38–9.75, P = 0.43; Table 4 ), indicating that there was no clear association between UU infection and the miscarriage rate. For IUI outcomes, no significant associations were found between UU infection and the rates of normal birth weight or being appropriate for gestational age status. Overall, the results shown in Table 4 suggest that the UU infection status did not significantly influence most of the examined pregnancy and neonatal outcomes in this analysis. Multivariate logistic regression analysis of pregnancy and neonatal outcomes in UU+ group (with the UU− group as the reference group) UU−: Ureaplasma urealyticum -negative; UU+: Ureaplasma urealyticum -positive; OR: odds ratio; CI: confidence interval; -: the corresponding confidence intervals range from 0 to infinity

It is crucial to consider the limitations of this study and the potential impact of unmeasured confounders on these results. First, the relatively small sample size of the UU+ group, after excluding patients with infections and co-infections with other common uropathogens, may limit the generalizability of our findings. Second, our study focused primarily on infertility patients without comparing them to fertile couples in terms of relevant measures. Third, as a single-center retrospective case–control study, there is a possibility of selection bias. Therefore, larger studies across different centers and cohorts are needed to validate our findings. Fourth, the present study lacked patients data collected before and after antibiotic treatment, and the effect of antibiotic treatment could not be ruled out. Fifth, the culture method failed to achieve an absolute quantitative analysis of UU, and the amount of UU may be related to the outcome of the infection. Finally, we only included common pathogens, while many other pathogens that may affect IUI pregnancy outcomes, such as Escherichia coli , Proteus mirabilis , and Enterococcus faecalis , etc . were not included. In conclusion, the data from our study revealed no correlation between UU infection and IUI outcomes. Therefore, this suggests that IUI remains a viable and cost-effective option for infertile couples with UU infection who are facing infertility issues.

Approximately 15% of male infertility patients present with concurrent genital tract infections. 16 Colonization by Mycoplasma in the urogenital tract is common, with a significant number of asymptomatic carriers, 17 especially for UU in the male reproductive system. 18 However, there is disagreement concerning the impact of male genital tract infections, specifically seminal UU infections, on semen parameters and pregnancy outcomes. 19 20 Several studies have suggested that genital tract infections can lead to oligoasthenozoospermia as well as abnormal sperm DFI and HDS. 5 21 22 Some studies have suggested that concurrent UU infection of the genital tract may be secondary to an increase in seminal leukocytes, leading to an oxidative stress reaction in the semen and creating an abnormal seminiferous environment for sperm development. 9 11 This can negatively impact embryo development and decrease female pregnancy rates as indicated by elevated DFI and HDS levels. 23 However, other studies have reported no apparent correlation between genital tract infection, male semen parameters, and adverse pregnancy outcomes. 24 25 In the present study, an in-depth comparison and analysis were conducted between infertile males in the UU+ and UU− groups to examine the effects of male seminal UU infection on semen parameters, pregnancy outcomes, and neonates outcomes after IUI. Our study revealed that there were no statistically significant differences in semen volume, sperm concentration, sperm motility, seminal leukocyte count, anti-sperm antibody levels, normal sperm morphology, and sperm DFI. However, when the means of the two groups were compared, the effect of seminal UU infection on HDS was statistically significant. The sperm HDS is an additional parameter utilized in the detection of sperm DNA fragmentation via the sperm chromatin structure assay (SCSA) method and indicates the immaturity of the sperm nucleus. Previous studies have shown that UU can directly interact with sperm cells, causing sperm deformities. These deformities are likely to impair DNA repair and affect sperm HDS levels. 10 11 26 The influence of the DFI and HDS on pregnancy outcomes has been controversial in previous studies. It has been reported that men with a DFI < 27% and HDS level <10% have a significantly higher success rate of pregnancy following assisted reproductive technology (ART) treatment. 27 Similarly, reports suggest that the sperm DFI and HDS level can predict men’s fertility capacity, assist in therapeutic decisions, and assess the risk of congenital diseases in newborns. 28 In contrast, some scholars have reported that the sperm DFI does not affect the ART outcomes. 29 Best et al . 30 noted that neither the DFI nor the HDS level could predict the pregnancy rate as assessed by SCSA. In the present study, we found that there were no statistically significant differences in pregnancy outcomes between the UU+ and UU− groups. This discrepancy may be due to several reasons. First, although a significant difference in HDS was observed between the UU+ and UU− groups, the proportion of patients with HDS in the UU-positive group remained lower than the 15% that is generally believed to affect embryo quality. 31 Additionally, studies conducted on mice and humans revealed that oocytes might repair damaged DNA. 32 To control for potential confounding factors arising from other possible co-infections, we screened several common uropathogens in addition to UU, including M. hominis , C. Trachomatis , N. gonorrhoeae , and fungi. Only patients with a sole UU infection were subsequently included in our experimental group. In addition, we performed a semiquantitative analysis, and only patients with >1 × 10 4 CCUs ml −1 were included in the UU+ group. In this study, we found no significant difference in the prevalence of pregnancies conceived through IUI or neonatal outcomes between the two groups, suggesting that couples infected with UU can still achieve comparable outcomes to those of their UU-negative counterparts. This is particularly reassuring for couples undergoing fertility treatments, as it suggests that UU infection may not pose a significant barrier to successful conception through IUI. Clinicians can use this evidence to counsel couples infected with UU about their fertility treatment options, including IUI, without undue concern about compromised outcomes. This may help to improve patient satisfaction and adherence to treatment recommendations. Moreover, these results emphasize the necessity for ongoing research to clarify the complex interactions between UU infection and fertility outcomes, as well as the potential mechanisms underlying these associations. Notably, these microorganisms can potentially cause urethritis, prostatitis, epididymitis, and orchitis. Therefore, male individuals who test positive for UU should consider undergoing eradication therapy with antibiotics.

All authors declare no competing interests.