Chlamydial induction of hydrosalpinx in 11 strains of mice reveals multiple host mechanisms for preventing upper genital tract pathology

Abstract

The female lower genital tract is constantly exposed to microbial infection, some of which can ascend to and cause pathology such as hydrosalpinx in the upper genital tract, which can affect fertility. To understand host mechanisms for preventing upper genital tract pathology, we screened 11 inbred strains of mice for hydrosalpinx induction by C. muridarum. When examined on days 60 to 80 after intravaginal infection, the 11 strains fell into 3 groups based on their hydrosalpinx severity: CBA/J and SJL/J mice were highly susceptible with a hydrosalpinx score of 5 or greater; Balb/c, C57BL/6J, C57BL/10J, C3H/HeJ and C3H/HeN were susceptible with a score between 1 and <5; NOD/ShiLtJ, DBA/1J, DBA/2J and A/J were resistant with a score of <1. The diverse range of mouse susceptibility to hydrosalpinx induction may reflect the varied clinical outcomes of C. trachomatis-infected women. When the 11 strains were infected via an intrauterine inoculation to bypass the requirement for ascension, higher incidence and more severe hydrosalpinges were induced in most mice, indicating that the interaction between chlamydial ascension and host control of ascension is critical for determining susceptibility to hydrosalpinx development in many mice. However, a few mouse strains resisted significant exacerbation of hydrosalpinx by intrauterine infection, indicating that these mice have evolved ascension-independent mechanisms for preventing upper genital tract pathology. Together, the above observations have demonstrated that different strains of mice can prevent upper genital tract pathology by using different mechanisms.

Figure 1. Hydrosalpinx development in 11 strains of mice following lower genital tract infection with C. muridarum.

Eleven different strains of female mice as indicated on the left of each representative image (with n = 5 to 23 mice in each strain as marked below each image) were intravaginally infected with C. muridarum. Sixty to 80 days after infection, all mice were sacrificed for observing hydrosalpinx. A representative image from each strain of mice is presented with the whole genital tract in the left and the magnified oviduct/ovary portion in the right. The number of mice with positive hydrosalpinx (as marked with red arrows in the magnified oviduct/ovary images) were counted and recorded as % of hydrosalpinx-positive mice. Furthermore, the severity of each hydrosalpinx was scored based on the criteria described in the Materials and Methods and marked with numbers in white in the corresponding images. The hydrosalpinx severity scores were compared between different strains of mice using Wilcoxon Rank Sum while the hydrosalpinx incidences were compared using Fisher's Exact. Note that the hydrosalpinx scores of NOD/ShiLtJ (panel h), DBA/1J (i), DBA/2J (j) and A/J (k) mice were <1 and significantly lower than those from other strains of mice (as noted with stars, p<0.05). These four strains were designated “resistant” to hydrosalpinx induction (right column) while the CBA/J (a) and SJL/J (b) mice with the highest rates and the most severe hydrosalpinx was designated “highly susceptible” (left column). The remaining strains all developed significant hydrosalpinx, thus were designated as “susceptible” (c–g in the central column).

Figure 2. Hydrosalpinx examined under microscopy.

The same genital tract tissues described in Fig. 1 legend were subjected to sectioning for H&E staining and microscopic observation. Both lumenal dilation and inflammatory infiltration were scored for oviduct tissues. Representative images from each group of mice taken under 10× (left columns, panels a–k) and 100× (right column, panels a1-k2) objective lens respectively are shown. The rectangles in each 10× image indicate where the two 100× images shown on the right were taken. Both the oviduct lumenal dilation (left) and inflammatory infiltration (right) scores (mean plus/minus standard deviation) from each group are listed under the corresponding images. Note that the lumenal dilation scores were significantly lower in the resistant strains (panels h–k) than highly susceptible (a & b) and 3 of 5 susceptible strains of mice (c–e). *p<0.05

Figure 3. Live C. muridarum organism recovery from vaginal swabs along the infection time course.

After the intravaginal infection (iv), the same eleven strains of mice described in Fig.1 legend were monitored for live chlamydial organism shedding from the lower genital tract. The number of live organisms recovered from the vagina/cervix swabs is expressed as log₁₀ IFUs per swab and displayed along the Y-axis. The eleven strains of mice were classified into 3 distinct groups (panels a and b for highly susceptible, c–g for susceptible and h–k for resistant) as described in Fig. 1 legend. The H-2 haplotype of each mouse strain is also listed in the corresponding bar graph. Note that the susceptibility to hydrosalpinx induction did not correlate with either the level or duration of live organism shedding or the H-2 haplotypes.

Figure 4. Hydrosalpinx development and live organism shedding following intrauterine infection with C. muridarum.

Each of the 11 strains of mice was inoculated with 2×10⁵ IFUs of C. muridarum via an intrauterine route (iu). Sixty days after inoculation, all mice were sacrificed for observing hydrosalpinx as described in Fig. 1 legend. Based on hydrosalpinx severity scores, the 11 strains were re-categorized into highly susceptible (with a median severity score of 5 or above, panels a-f for mouse strains CBA/J, SJL/J, Balb/cJ, C57BL/6J, C3H/HeJ & DBA/2J respectively), susceptible (<5 and >1, panels g-j for C57BL/10J, C3H/HeN, NOD/ShiLtJ & DBA/1J) or resistant (<1, panel k for A/J). The hydrosalpinx incidences (%) along with total number of mice per strain (in bracket) are listed under the corresponding hydrosalpinx scores. The live organism shedding was monitored post infection as described in Fig. 3 legend and expressed as Log10 IFUs (shown along the Y-axis of each panel). The H-2 haplotypes are listed after the corresponding mouse strain name. Note that the hydrosalpinx severity did not have any obvious correlations with the live organism shedding course or H-2 haplotypes except for H-2^d. Both Balb/cJ (H-2^d) and DBA/2J (H-2^d) were highly susceptible to hydrosalpinx induction.