Early pregnancy loss (EPL) in the mare is defined as loss of pregnancy between initial detection and day 65 of gestation. It occurs in 710% of pregnancies and yet little is known about the underlying pathologies. A lack of suitable conceptus material has limited investigation into the role of chromosomal defects in EPL. The objective of this study was to develop a method to isolate and culture placental cells isolated from failed pregnancies to enable further genetic characterisation. Conceptus material was collected from thoroughbred mares suffering an EPL by sterile uterine lavage. Conceptuses were shipped in HBSS/10% horse serum/10 μg/ml amphotericin. Tissue was isolated from the allantochorion and chorion and cells cultured at 37 °C 8% CO2 in i) Chang D (Irvine Scientific) + amphotericin B + kanamycin sulphate, ii) AmnioChrome Plus (Lonza) + amphotericin B or iii) DMEM/10% FCS supplemented with penicillinstreptomycin and L-glutamine. Viability of cells was determined by trypan blue exclusion. Chromosome preparations were attained using colcemid and hypotonic solutions and methanol-acetic acid fixation. Twenty-nine conceptuses were submitted to the laboratory. The median gestation age EPL was detected was 42 days (range 1865) and mean gestational age at time of uterine flush was 45.5 days (range 2470). The median time between uterine lavage and arrival at the laboratory was 22 h (range 3.572). These factors did not influence the outcome of the cultures. Cells were successfully isolated and cultured from 62% of submitted conceptuses and 79% of conceptus cells selected for culture. Viable cells were more likely isolated with the use of AmniochromePlus compared to Chang D (P=0.003). Successful karyotyping from metaphase spreads identified from culture of one of the conceptuses showed a normal karyotype. In conclusion, we have identified a method to isolate, culture and subsequently karyotype cells derived from failed early equine pregnancies.
20 Jul 2015 - 22 Jul 2015