Until recently, the bladder was considered a sterile environment, and the finding of bacteria in the urine an indication of a bladder infection. However, the culture-independent method, 16s rRNA gene sequencing, has revealed the presence of a permanent bacteria colonization (microbiota) in the bladder, without signs of actually infection. The composition and variation of the urinary microbiota in healthy women is poorly described. Moreover, the effects of hormonal changes during menopause is not well understood. 
Studies have shown that the urinary microbiota differs  in women with bladder dysfunctions for example urgency urinary incontinence in comparison to healthy controls by a decrease in the relative abundance of Lactobacillus. Several of the studies however, do not take into account the menopausal status of the included women. 
Previous studies have demonstrated a decrease in the relative abundance of Lactobacillus in the vagina after menopause.  We have therefore hypothized that the same could be true for the bladder and the urine. We aimed to compare urine samples from pre- and postmenopausal women without any bladder symptoms to evaluate the role of hormonal changes in  menopause  status on the composition of the  urinary microbiota.

A total of 100 women (50 premenopausal aged 18 to 50 years and 50 postmenopausal aged more than 55 years) were planned for inclusion in the study. Exclusion criteria for the premenopausal women  was  current pregnancy, whereas vaginal bleeding within the last year, or estrogen replacement therapy (oral or vaginal) were the exclusion criteria for the postmenopausal women. For both group, exclusion criteria were antibiotic treatment within the last three months, recurrent urinary tract infections (defined by us as two or more infections in the last year) and incontinence or irritative bladder symptoms (evaluated by the questionnaires ICIQ-UI-SF and ICIQ-OAB). Moreover, women with a positive routine culture of the collected urine sample were also excluded. 
Urine samples were collected by placement of a urethral catheter and stored at -80°C until further analyses. Bacterial DNA was isolated from 10 ml of urine using the QIAamp Viral RNA Mini Kit (Qiagen). Negative urine controls were designed by performing DNA extraction on Nuclease-free water. Bacterial composition of urine samples was  determined by 16S rRNA gene sequencing targeting the V4 region. Both richness, alpha-diversity (Shannon Index) and abundance were assessed.

Overall, 41 premenopausal (37 ± 9.5 years) and 42 postmenopausal (63 ± 6.9 years) women were included in this study. Participant characteristics such as BMI and smoking status were comparable between the two groups.
Samples with first PCR amplicon concentrations below levels of the negative control were excluded. This resulted in urine samples from 21 women being excluded, leaving 36 (88%) women in the premenopausal group and 26 (62%) women in the postmenopausal group for subsequent analyses. 
A total of 2,763 unique Operational Taxonomic Units (OTUs) were identified. Urine from postmenopausal women showed a significant higher number of observed OTUs (higher richness) and a significant higher alpha- diversity compared to that of premenopausal women. Although Lactobacillus was the most highly represented bacteria genus in both groups, the relative abundance of Lactobacillus accounts for 78% of the bacteria in premenopausal women as opposed to 38% in postmenopausal women. The postmenopausal women appeared to have a broader representation of different bacteria, as supported by the higher alpha-diversity, including presence of Gardnerella, Streptococcus, Prevotella, and E. Shigella.

Our study shows that the urinary microbiota differs significantly between healthy pre- and postmenopausal women . A decrease in the abundance of Lactobacillus was observed in the latter group together with a parallel  increase in other unique bacteria. Furthermore, the urine from postmenopausal women is characterized by  higher alpha diversity, meaning that the abundance of the individual unique bacteria are more evenly distributed. In future urinary microbiota research it is therefore very important to take into account the age and menopausal status of the included women in conjunction with interpretation of the results. Further studies are needed to investigate which other factors may influence the urinary microbiota besides menopausal status, and the potential role of altered urinary microbiota on the development of bladder dysfunction and recurrent urinary tract infection.

The urinary microbiota of healthy pre- and postmenopausal women differs significantly. Age and menopausal status are important factors to consider when evaluating the urinary microbiota in women with bladder dysfunction in controlled studies. This is important knowledge for future research in the area of urinary microbiota and bladder diseases.