Experimentally Induced Endometritis Impairs the Developmental Capacity of Bovine Oocytes†

Abstract

Uterine infection is associated with infertility in women and dairy cows, even after the resolution of infection. However, the mechanisms causing this persistent infertility are unclear. Here, we hypothesized that induced endometritis in non-lactating dairy cows would reduce the developmental competence of oocytes. Non-lactating Holstein cows received an intrauterine infusion of endometrial pathogenic bacteria (Escherichia coli and Trueperella pyogenes; n = 12) or vehicle control (n = 11) on day 2 of the estrous cycle. Bacterial infusion increased expression of endometrial inflammatory mediators, and a mucopurulent discharge in the vagina confirmed the establishment of endometritis. Oocytes were collected by transvaginal ultrasound-guided ovum pickup on days 2, 24, 45, and 66 following infusion and subjected to in vitro fertilization and embryo culture. Bacterial infusion resulted in fewer cleaved oocytes developing to morulae compared to vehicle-infused controls (30.7 versus 45.0%), with the greatest effect observed in oocytes collected on day 24. Development to morula was inversely correlated with endometrial expression of IL6 on day 6. The expression of genes associated with embryo quality did not differ significantly between morulae from bacteria-infused and control cows. Artificial insemination 130 days after intrauterine infusion resulted in normal, filamentous embryos that produced interferon tau 16 days after conception in both infusion groups. This model of experimentally induced uterine infection successfully resulted in endometritis and a reduction in the proportion of oocytes that developed to morulae following in vitro fertilization. In conclusion, endometritis reduced the capacity of oocytes to develop to morulae.

Keywords: embryo; endometritis; female infertility; inflammation; oocyte.

Graphical Abstract

Figure 1

Timeline of major experimental events. Estrous cycles were synchronized with gonadotrophin-releasing hormone and prostaglandin F_2α prior to intrauterine infusion of either LB broth vehicle medium (vehicle; n = 11) or pathogenic E. coli and T. pyogenes in LB broth (bacteria; n = 12) on experimental day 0. Major events include oocyte pickup (OPU, ), endometrial cytobrush () sampling, artificial insemination (AI), progesterone (P4) administration, and slaughter. Timeline is not drawn to scale.

Figure 2

Establishment and quantification of uterine disease. Vaginal mucus (A) was collected and graded on a scale of 0 to 4 based on the presence of mucopurulent discharge. Data are the mean grade ± SEM. The proportion of polymorphonuclear cells in cytological samples (B) was assessed in a total of 200 cells per cow. Each dot represents a cow and the solid line represents the mean. Rectal temperatures (C) are displayed as mean ± SEM. Plasma haptoglobin (D) was evaluated on day 5 relative to infusion, and each dot represents an individual cow and the solid line represents the mean.

Figure 3

Endometrial expression of inflammatory mediators following intrauterine infusion. Expression of AKR1C4 (A), CXCL8 (B), IL1B (C), IL6 (D), PTGES (E), PTGS2 (F), PTPRC (G), and TNF (H) in cytobrush samples was evaluated by real-time RT-PCR. Data are presented as expression relative to GAPDH. Each dot represents a single cow and the solid line indicates the mean. Comparisons between treatments at a given day are indicated by ^* when P ≤ 0.05.

Figure 4

Effect of intrauterine infusion on developmental capacity of oocytes following in vitro fertilization and embryo culture. Oocytes were collected via ultrasound-guided transvaginal oocyte pickup on days 2, 24, 45, and 66 relative to infusion of either LB broth vehicle medium (vehicle; n = 11) or pathogenic E. coli and T. pyogenes in LB broth (bacteria; n = 12) and subjected to in vitro fertilization and embryo culture. Pooled oocytes from each cow were maintained as an individual replicate throughout insemination and culture. Each dot represents an individual cow, and the solid line represents the mean of the treatment. The proportion of oocytes that cleaved 3.5 days post-insemination (A), the proportion of oocytes to develop to morulae 6 days post-insemination (B), and the proportion of cleaved oocytes to develop to morulae 6 days post-insemination (C) are shown. Comparisons between treatments on a specific day are indicated by ^* when P ≤ 0.05.

Figure 5

Effect of intrauterine infusion on gene expression of IVF derived morula stage embryos. Morula stage embryos derived by oocyte pickup, in vitro fertilization, and embryo culture from cows receiving intrauterine infusion of either LB broth vehicle medium (vehicle; n = 11) or pathogenic E. coli and T. pyogenes in LB broth (bacteria; n = 12) were probed for gene expression of BAX (A), BCL2 (B), DNMT3A (C), HSPA1A (D), IGF2R (E) and SLC2A1 (F) by real-time RT-PCR. Data are presented as expression relative to the geometric mean of the housekeeping genes GAPDH, SDHA, and RLP19. Each dot represents the average expression for an individual cow, and the solid line represents the mean of the treatment.

Figure 6

Association between morula development and endometrial inflammation. Endometrial expression of CXCL8 (A), IL1B (B), IL6 (C), PTGES (D), PTGS2 (E), and TNF (F) was determined by real-time RT-PCR on day 6 relative to infusion. Linear correlation was performed using the total proportion of cleaved oocytes to develop to morulae from all cows.

Figure 7

Effect of intrauterine infusion on embryo recovery, interferon tau concentration, serum progesterone, and anti-Müllerian hormone. Cows were synchronized and inseminated on day 130 post-infusion of either LB broth vehicle medium (vehicle; n = 11) or pathogenic E. coli and T. pyogenes in LB broth (bacteria; n = 12). Uterine content was collected ex vivo, 16 days post-insemination. All recovered embryos were filamentous in morphology (A). Interferon tau (IFNT) concentration (B) was quantified in uterine fluid by ELISA. Serum progesterone (C) was quantified 15 days after insemination. Each dot represents a cow and the solid line is the mean. Anti-Müllerian hormone (D) was quantified on day −2 prior to intrauterine infusion and day 145 after infusion. Each dot represents a cow.

Figure 8

Effect of intrauterine infusion on gene expression of trophectoderm from in vivo-derived embryos. Cows were synchronized and inseminated on day 130 post infusion of either LB broth vehicle medium (vehicle; n = 11) or pathogenic E. coli and T. pyogenes in LB broth (bacteria; n = 12). Total RNA was isolated from trophectoderm of day 16 in vivo-derived embryos, and expression of CDKN1C (A), IFNT2 (B), and PPARG (C) was evaluated by real-time RT-PCR. Data displayed are expression relative to the geometric mean of the housekeeping genes ACTB and GAPDH. Each dot represents an embryo and the solid line depicts the mean.