Cytobrush: a tool for molecular evaluation of the bovine endometrium

In cattle, molecular understanding of uterine biology in vivo requires probing of the uterus using invasive techniques, such as biopsies. Repeated sampling may cause inflammation, physical damage and impact fertility. Therefore, validation of less traumatic sampling techniques that are repeatable and allow the collection of representative and reliable samples are necessary. In this context, this study aimed to: (1) compare the structural and functional aspect of endometrial samples collected by transcervical biopsy and cytobrush and (2) characterize the expression profile of genes involved in the luteolytic mechanism in bovine endometrium using the cytobrush technique. In Experiment 1, estrus of five Nelore cows was synchronized using a protocol based on progesterone and estradiol injections. Ovulations were detected by ultrasonography. Ten days after ovulation, endometrial samples were taken transcervically from the uterus, both by a cervical brush adapted to the tip of an artificial insemination gun and, subsequently, using a biopsy apparatus. Samples were submerged in Trizol reagent and stored at -80C until RNA extraction. The abundance of transcripts characteristic of epithelium (KRT18), stroma (VIM), immune cells (CD3D) and endothelial cells (FLT1) and transcripts involved in uterine function during the estrous cycle (PGR, PTGES, AKR1C4) was measured by RT-qPCR and compared between probing techniques. The fixed effect of technique was analyzed by one-way ANOVA using the PROC MIXED procedure (SAS version 9.2). Abundance of VIM and FLT1 was 9.2 and 275 fold greater in biopsy samples, respectively (P 0.05). In Experiment 2, estrus of five Nelore cows was synchronized and the day of ovulation (D0) estimated by ultrasonography. On days 10, 13, 16 and 19 endometrial samples were collected using the cytobrush method. OXTR, ESR1 and PGR2 transcript abundance was measured by qPCR and analyzed by repeated-measures ANOVA using the PROC MIXED procedure (SAS version 9.2). The least significant difference test was used for mean comparisons among days. There was an effect of day on the abundance of all transcripts (PGR2 and OXTR, P u003c 0.05; ESR1, P u003c 0.1). Abundance of ESR1 decreased gradually from D10 to D16 then increased again on D19. There was a gradual increase in the abundance of OXTR over time, and its greatest abundance was noted on day 19. In contrast, abundance of PGR2 mRNA was maximum on day 10 and then gradually decreased (P u003c 0.05). In summary, tissues obtained using the cytobrush technique provide representative samples, enriched in epithelial and immune cells, compared with biopsies. Temporal dynamics of expression of select transcripts were consistent with uterine function at the end of the estrous cycle. In conclusion, cytobrush sampling can conveniently substitute biopsy sampling, providing a safe, repeatable and less traumatic method to study bovine uterine biology in vivo.