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Abstract
Objectives The association between Mycoplasma genitalium (M. genitalium) serum antibodies and infertility in women and men, as well as infertility subtypes, was investigated.
Methods Stored serum was obtained from two patient cohorts: infertile couples (239 women and 243 men) attending a gynaecological outpatient clinic between October 1997 and February 2001 and 244 age-matched spontaneously pregnant women. An enzyme immunoassay was used to detect serum immunoglobulin G (IgG) antibodies to M. genitalium in these samples. Patient's Chlamydia trachomatis seropositivity had been previously determined. Risks were calculated using multivariate logistic regression.
Results M. genitalium serum IgG was more common among women of infertile couples (5.4%) than among fertile controls (1.6%) (OR (95%CI) 3.45 (1.10 to 10.75)), adjusting for C. trachomatis IgG (adjusted OR=3.00 (0.95 to 9.47)). Of the women with tubal factor infertility (TFI) 9.1% had M. genitalium IgG compared with 4.6% of women without TFI (OR=2.07 (0.60 to 7.05)); (AOR=1.20 (0.32 to 74.40)). In patients IgG positive to both microorganisms the OR for having TFI was increased (OR=4.86 (1.22 to 19.36)) compared with those positive to C. trachomatis IgG only (AOR=3.14 (1.58 to 6.20)). No associations were found with other infertility diagnoses. Only two men of the infertile couples were M. genitalium IgG positive (0.8%).
Conclusions M. genitalium serum IgG was associated with infertility in women, however insignificant after adjustment for C. trachomatis IgG, but not with infertility subtypes within this study. M. genitalium IgG seroprevalence among men was very low and not associated with male factor infertility.
- M GENITALIUM
- INFERTILITY
- SEROLOGY
- ANTIBODIES
- CHLAMYDIA TRACHOMATIS
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Introduction
Mycoplasma genitalium (M. genitalium) is an established cause of non-gonococcal urethritis, but the evidence for an association with infertility is inconclusive.1 M. genitalium infection has been associated with pelvic inflammatory disease (PID) and tubal factor infertility (TFI), as reviewed by Daley et al;1 however, the clinical and epidemiological evidence is limited. The bacterium is reported to establish long-term infection of human endocervical epithelial cells, resulting in chronic inflammatory cytokine secretion in vitro,2 and to cause PID and cervicitis in primates inoculated with the bacterium.3
Since M. genitalium has been shown to adhere to spermatozoa4 and affect sperm motility in vitro, it is also suggested to affect male fertility.
The aim of the present study was to further elucidate the relationship between M. genitalium and infertility in women and men, by analysing M. genitalium immunoglobulin G (IgG) antibodies in stored sera, previously analysed for Chlamydia trachomatis antibodies, from a well-characterised cohort of infertile couples and their fertile controls.5 A secondary aim was to evaluate if there were any associations with TFI, other infertility diagnoses, pregnancy rates and pregnancy outcomes.
Methods
This is an extended analysis of stored sera from a previously published cross-sectional case–control study.5 The Human Ethics Committee of the Medical Faculty, Umeå University, approved of the study.
Study population
The study population has been described in detail previously.5 In brief, all consecutive couples (244 women, 244 men) attending the gynaecological outpatient clinic at the University Hospital of Northern Sweden, Umeå, due to infertility during the period of October 1997 through February 2001, were included. Blood was drawn from both partners at their first hospital visit and analysed for C. trachomatis IgG antibodies. Serum was stored at −20°C, and was retrievable from 239 and 243 of the women and men respectively for M. genitalium IgG analysis.
Infertility assessment was conducted according to routine infertility work-up including mid-luteal progesterone, semen analysis according to the WHO as modified by the Nordic Association for Andrology/European Society of Human Reproduction and Embryology, clinical examination and gynaecological ultrasound. Thereafter, the couples were advised about treatment according to the findings. Tubal assessment was carried out in 129 women using hysterosalpingosonography (HSS) (n=64), laparoscopy (n=43) or both (n=20). One hundred and fifteen women either became spontaneously pregnant before tubal assessment was performed (n=58), which was considered a surrogate marker for tubal patency, decided to end investigation (n=15) or had other infertility factors that indicated the need for assisted reproductive techniques (n=42). TFI was defined as one or both tubes being occluded or dilated at HSS or laparoscopic chromopertubation. The follow-up period was 14–54 months (mean 37 months), where after infertility diagnosis, treatments, pregnancies and pregnancy outcomes were retrieved from the medical records. For all women where a pregnancy was not registered, a structured telephone interview was conducted (AI) and responses regarding fertility treatments, pregnancies and pregnancy outcome were recorded.
As fertile controls, women attending the Antenatal Care programme in the same region, reporting spontaneous pregnancies without infertility (according to the medical records), were included (n=244). Controls were matched with respect to age (birth year) and year of blood sampling. Serum samples were stored at −20°C.
The primary outcome was the prevalence of M. genitalium serum IgG antibodies among women and men of infertile couples and fertile women, and the association with infertility. Secondary outcomes were association of M. genitalium IgG with TFI, other infertility diagnoses, pregnancy rates and outcomes, as well as association with C. trachomatis IgG antibodies. An additional outcome was the association of M. genitalium IgG combined with C. trachomatis IgG with infertility and TFI.
M. genitalium antibody analysis
M. genitalium IgG antibodies were detected using an M. genitalium lipid-associated membrane protein-enzyme immunoassay as previously described.6 Briefly, 100 µL of plasma samples diluted 1/50 in blocking solution were used. Serum from a patient with an M. genitalium PCR-positive result in the urogenital tract specimen was used as a positive control, and pooled sera from blood donors were used as a negative control in each run. The cut-off level was set to 0.3 optical density and was determined as 3 SDs above the negative control mean.
Statistical methods
Pearson χ2 and, where applicable, Fisher’s exact test were used to determine differences in antibody prevalence between groups with dichotomous data. A two-tailed p value of <0.05 was considered significant. ORs and 95% CIs were calculated with binary logistic regression; multivariate analysis was performed to control for possible confounding. All analyses were done using the IBM SPSS Statistics, V.22.0.0.0.
Results
The prevalence of M. genitalium serum IgG antibodies among the infertile couples and the fertile women is presented in table 1. The prevalence of C. trachomatis IgG antibodies has been previously published;5 however, the values are included for comparative reasons and the possibility to present multivariate analyses.
Mycoplasma genitalium and Chlamydia trachomatis* serum IgG antibodies in women and men of infertile couples and spontaneously pregnant women
Concordance of M. genitalium IgG seropositivity (n=19, 2.6%) and C. trachomatis IgG seropositivity (n=143, 19.8%) was observed, with an OR of being M. genitalium IgG positive of 3.83 (1.52 to 9.61) if C. trachomatis IgG positive (n=9, 1.2%) in all women and men included in the study. M. genitalium IgG seropositivity was associated with infertility in the women (table 1), but the association was insignificant after adjusting for C. trachomatis IgG seropositivity. The same was true for C. trachomatis IgG seropositivity, which was also, unadjusted, associated with infertility. When adjusting for M. genitalium IgG seropositivity the association was no longer significant (table 1).
M. genitalium IgG antibodies were not significantly associated with TFI (table 1) and restricting the analysis regarding TFI to women assessed with laparoscopy and/or HSS changed the risk estimates for M. genitalium IgG-positive women minimally (OR=2.47 (0.58 to 10.45)).
There were no associations of M. genitalium antibodies with unexplained infertility (1/26, 3.8%) (OR=0.67 (0.08 to 5.38)), pregnancy rates among infertile women (6/88, 6.8%) (OR=1.44 (0.46 to 4.43)), miscarriages (1/27, 3.7%) (OR=0.72 (0.08 to 6.23)) or extrauterine pregnancies (0/6, 0.0%) (OR=0.00 (0.00 to ∞)).
Two men, with normal semen analyses, were found M. genitalium IgG positive and the female partner in the respective couples conceived before full infertility assessment was completed.
Discussion
In this study, M. genitalium serum IgG antibodies were shown to be more prevalent in women of infertile couples compared with spontaneously pregnant women, and the prevalence among men of infertile couples was very low. M. genitalium IgG and C. trachomatis IgG antibodies in the women showed strong concordance, but the prevalence of M. genitalium IgG was several times lower than the prevalence of C. trachomatis IgG antibodies.
This is, to our knowledge, the first study analysing M. genitalium serum IgG antibodies in both women and men of infertile couples, comparing also the prevalence in these women with that in proven fertile women. The long follow-up period regarding pregnancies with very few couples lost to follow-up is another strength. The method for M. genitalium IgG analysis has previously been validated and presents no cross-reactivity with M. pneumoniae. One limitation is that not all women were assessed by laparoscopy or HSS for tubal damage, resulting in reduced power to the analysis regarding TFI. The power to detect pregnancy outcome variables was limited with few cases with miscarriages and extrauterine pregnancies.
Although M. genitalium and C. trachomatis seropositivity rates were elevated in infertile women in this study, neither was by itself significantly associated with infertility in a multivariate analysis. M. genitalium has been suggested to reduce fertility by causing inflammatory changes to the fallopian tubes followed by TFI and has, in some studies, but not all, been associated with PID and infertility in women.1 ,6 ,7 No cervical M. genitalium DNA, and only one C. trachomatis DNA-positive case, was detected in a similar sized cohort study despite an association of serum antibodies with TFI.7 In the present study, M. genitalium IgG was not associated with TFI, possibly due to lack of power to detect such an association. Being seropositive to both M. genitalium and C. trachomatis is in this study associated with an increased OR for both TFI and infertility.
The low prevalence of M. genitalium serum IgG antibodies in the men of the infertile couples corroborates previous studies showing a low prevalence of M. genitalium bacteria in semen or first void urine specimen of infertile men, and a lack of significant associations with semen parameters or male factor infertility.1 ,8–10
In conclusion, the presence of M. genitalium serum IgG antibodies, indicating a past or present infection, was raised in the cohort of infertile women compared with fertile controls; however, the association was not significant after adjustment for C. trachomatis. Future studies with larger patient cohorts are warranted to verify the association between M. genitalium and female infertility. The finding of a very low frequency of M. genitalium IgG seropositivity in the men of the infertile couples renders it unlikely that M. genitalium is a causal factor in male factor infertility.
References
Supplementary materials
Abstract in Swedish
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Footnotes
▸ Additional material is published online. To view please visit the journal (http://dx.doi.org/10.1136/sextrans-2015-052011).
Handling editor Jackie Cassell
Contributors AI and JIO contributed to the design and data acquisition of the present study. MJ and HF developed the LAMP-EIA methodology and supervised the laboratory work. AI conducted the analyses and wrote the initial draft of the manuscript. All authors contributed to the interpretation of the results, manuscript revisions and approved the final manuscript.
Funding This work was supported by grants from the Örebro County Council Research Committee, the Swedish Medical Research Council and the Medical Faculty, Umeå University.
Competing interests None declared.
Ethics approval Human Ethics Committee of the Medical Faculty, Umeå University, Umeå (#09-059M).
Provenance and peer review Not commissioned; externally peer reviewed.