Chlamydia trachomatis, Ureaplasma urealyticum and Mycoplasma hominis urogenital infections in patients with infertility. Results from a large cross-sectional study.

Female and male infertility have been associated to Chlamydia trachomatis, Ureaplasma urealyticum and Mycoplasma hominis urogenital infections. However, evidence from large studies assessing their prevalence and putative associations in patients with infertility is still needed. The study design was a cross-sectional study including 1554 female and 3610 male patients with infertility in Cordoba, Argentina. In women, the prevalence of C. trachomatis, U. urealyticum and M. hominis urogenital infection was 4.3%, 31.1% and 12.1% whereas in men it was 5.8%, 19.2% and 5.3%, respectively. C. trachomatis infection was signicantly more prevalent in men whereas U. urealyticum and M. hominis infections were more prevalent in women. Besides, C. trachomatis and U. urealyticum infections were signicantly higher patients younger than 25 years. Moreover, U. urealyticum and M. hominis infections were associated to each other in either infertile women or men. Our data revealed that C. trachomatis, U. urealyticum and M. hominis are prevalent uropathogens in infertile patients. Of clinical importance, C. trachomatis and U. urealyticum infections were more prevalent in young patients whereas U. urealyticum and M. hominis are reciprocal risk factors of their co-infection. These results highlight the importance of including the screening of urogenital infections in the diagnostic workup of infertility. C. trachomatis, U. urealyticum and M. hominis urogenital infections are associated with patient sex and age. In fact, individuals younger than 25 years at the highest risk of C. trachomatis and U. urealyticum infection. Moreover, our data show that men are at higher risk of C. trachomatis infection and, conversely, women are at higher risk of U. urealyticum and M. hominis infection.


Introduction
Urogenital infections are known causes of infertility 1 . Currently, infertility affects 15-20% of reproductive-aged couples worldwide and women and men equally contribute to infertility cases 1,2 . Sexually transmitted infections can impair fertility by different mechanisms: by directly damaging organs and gametes and/or, indirectly, by the induced in ammation and associated tissue damage, scarring and obstruction 1,3 . Moreover, infection-induced genital in ammation may alter the normal immunomodulation process that naturally occurs in the female genital tract after mating to facilitate fertilization, embryo implantation and promote embryo growth for a successful pregnancy 4 . Besides being the most frequent sexually transmitted bacterial infection worldwide, Chlamydia trachomatis is a common infection associated to infertility 5 . In women, C. trachomatis is a known cause of different urogenital pathologies such as acute urethritis, cervicitis and salpingitis that may lead to severe reproductive complications including pelvic in ammatory disease, chronic pelvic pain, ectopic pregnancy, miscarriage and tubal infertility 6,7 . In men, C. trachomatis is considered the most common agent of non-gonococcal urethritis and may cause epididymitis-orchitis, prostatitis, sperm tract obstructions and alterations in sperm quality 8,9 . On the other hand, Mycoplasma hominis and Ureaplasma urealyticum have also been recognized as sexually transmitted infections that could impair human fertility 1 . Although they are both known to colonize the female and male reproductive tracts as commensals, cumulative growing evidence has shown they are emerging sexually transmitted opportunistic pathogens able to cause asymptomatic chronic disorders affecting female and male fertility [10][11][12][13][14][15] . In men, U. urealyticum and M. hominis are causes of nongonococcal urethritis contaminating semen during ejaculation. Moreover, U. urealyticum has been proposed to cause prostatitis, epididymitis and infertility 1 .
In addition, reported data have shown that both U. urealyticum and M. hominis could impair sperm quality 11,16,17 . In women, U. urealyticum and M. hominis may cause different pathologies including acute urethritis, bacterial vaginosis, pelvic in ammatory disease and tubal infertility 14,18,19 . Moreover, the asymptomatic colonization by mycoplasmas or ureaplasmas could induce pro-in ammatory immune responses in the endometrium that may impair pregnancy outcomes 2,9,14,20 .
The detection rates of C. trachomatis, U. urealyticum and M. hominis in the urogenital tract form infertile women and men has shown striking variations across regions and countries and in different groups when individuals were classi ed according to age, ethnicity and socioeconomic status 5,21−23 . In that regard, a growing number of studies have been reported during the last decade. However, compelling available data from large cross-sectional studies is scarce [21][22][23] . Moreover, reported data about the association of these infections in either infertile women or men is limited [21][22][23] . Since these infections may play a signi cant role in the etiology of infertility, we herein conducted a large observational investigation into urogenital C. trachomatis, U. urealyticum and M. hominis infections in infertile women and men seeking care for infertility. Moreover, we analyzed the associations among infections and with demographic parameters such as patient sex and age.
Interestingly, a signi cant association between M. hominis and U. urealyticum infection was found in women (OR: 33 (Figure 1c, Table 3). As detailed in Table 3 (Table 3). Multivariate regression analysis further con rmed these tight associations, indicating that U. urealyticum and M. hominis act as mutual risk factors of infection either in women or men (Supplementary Table S1).
Noteworthy, only 2.0% of infected women (11 out of 539) and 1.5% of infected men (13 out of 871) were co-infected with the three uropathogens analyzed (Supplementary Figure S1).
Demographic parameters associated to C. trachomatis, U. urealyticum and M. hominis infection Remarkably, C. trachomatis, U. urealyticum and M. hominis infections were signi cantly associated with patient sex and age (Table 2). In fact, univariate regression analysis revealed that C. trachomatis infection was more likely to be detected in infertile men than in women with an odds ratio of 1.36 (95% CI: 1.02-1.80, p=0.034) ( Table 2). Moreover, a signi cant association was particularly found between C. trachomatis infection and men younger than 25 years (OR: 2.51, 95% CI: 1.40-4.48, p=0.002, Table 2) indicating that men, and especially those younger than 25 years, are at higher risk of infection than women (Table 2). Multivariate analysis further con rmed these associations (Supplementary Table S1).
These higher in infertile women than in infertile men 17 . Furthermore, our results also showed that C. trachomatis and U. urealyticum infections were signi cantly more prevalent in young patients, particularly in those younger than 25, indicating that age as a risk factor and in agreement with previously reported data 26,27 . In fact, it is known that sexually transmitted infections are directly related to sexual experience, having young people more frequent sexual intercourses, less consistency of condom use and one or multiple sexual partners 26,27 . On the other hand, when assessing co-infections, only 2.0% of women and 1.5% of men were co-infected with the three uropathogens analyzed.
Our results support previously reported data. In a large observational study, Chen  and 0.9%, respectively) 6 . In addition, considerably lower prevalence rates of C. trachomatis, U. urealyticum and M. hominis infection were reported in infertile women from the USA and Iran 36,37 . Several factors could underlie these differences such as disparities in population under study (geographical location, age, ethnicity, religion, socio-economic status, access to medical care, etc.) or study design (prospective versus retrospective, patient population size, methods used for diagnosis, etc.). However, our results are in line with cumulative reported data and supported by the large patient population analyzed, including 5164 infertile individuals (1554 women and 3610 men).
Conversely, and supporting recently reported data 38-40 , U. urealyticum or M. hominis were signi cantly associated to each other in either infertile women or men, indicating that U. urealyticum or M. hominis urogenital infection increased the risk of M. hominis or U. urealyticum co-infection. This tight infection association could be due to shared infection routes and/or pathophysiologic mechanisms 1,10 .
To our knowledge, this is the rst cross-sectional study to investigate the prevalence and association of C. trachomatis, U. urealyticum and M. hominis in infertile women and men from Argentina. Being one of the few cross-sectional study performed in Latin American countries 21,23 and the large number of patients analyzed are the main strengths of our study. However, our study has some limitations such as it did not include control groups of fertile women and men, in whom the prevalence of the infections under analysis could have been different from their infertile counterparts. Moreover, we did not have the information of the partner couple of every infertile women or men enrolled, which could have provided important information about infection concordance. In

Ethical approval
The study was carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) standards and the Argentinian legislation for protection of personal data (Law 25326). The experimental protocol was approved by the Institutional Ethics Committee from the Hospital Nacional de Clinicas, Universidad Nacional de Cordoba (RePIS #3512). All patients provided a signed written informed consent form agreeing to share their own anonymous information prior to enrollment.
DNA extraction and C. trachomatis detection Total DNA was extracted from vaginal or semen samples and C. trachomatis infection detected by polymerase chain reaction (PCR) using the Chlamydia trachomatis 330/740 IC PCR kit (Sacace Biotechnologies Srl, Como, Italy) and within 4 h of sample collection.
M. hominis and U. urealyticum detection M. hominis or U. urealyticum infections were assessed by culture using the commercially available colorimetric kit Mycofast RevolutioN (ELITech Group, Puteaux, France). Brie y, 100 μl of vaginal swab seeded transport medium or semen were added to the M. hominis and U. urealyticum wells, sealed and incubated at 37°C ± 1°C for 24 h. After incubation, orange or red color changes indicated the presence of M. hominis and/or U. urealyticum, respectively.

Statistics
Demographic characteristics (age, sex) and co-infections were compared between infected and non-infected patients using the chi-square test and the odds ratios (OR) and 95% con dence intervals (CI) were calculated. Univariate and multivariate logistic regression analyses for all variables were performed to determine associations or risk factors for infections. The SPSS statistical software package (version 17.0; SPSS Inc., Chicago, USA) and GraphPad Prism 8.0.1 were used for analyses. A p<0.05 was considered statistically signi cant.

Ethical approval
The study was carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) standards and the Argentinian legislation for protection of personal data (Law 25326). The experimental protocol was approved by the Institutional Ethics Committee from the Hospital