Marilen Benner

CHAPTER 7 196 poorly understood - but possibly far-reaching - consequences underscore the need for careful assessment and restraint use of antibiotics during pregnancy. Pregnant women are generally excluded from clinical trials, and, other than what can be deduced from retrospective studies, few approaches take into account the unique adaptations occurring to maintain a healthy pregnancy. These present results highlight the importance of in vivo studies on medication used during gestation to employ pregnancy models, taking into account the unique immunological properties, and possible tissue-specific effects, of this period. MATERIALS AND METHODS Animals 8-week-old, specific pathogen-free, male C57Bl/6 mice and 8-week-old nulliparous female Balb/c, purchased from Envigo (Horst, The Netherlands) were housed at the animal facility of the Utrecht University (Utrecht, The Netherlands) on a reversed 12 h light/dark cycle with unlimited access to water and semi ‐ purified AIN ‐ 93G soy protein ‐ based rodent diet (Ssniff Spezialdiäten GmbH, Soest, Germany). Upon arrival, mice were habituated to the laboratory conditions for two weeks prior to the start of the study. The male mice were mated with a separate set of Balb/c females (Envigo, Horst, The Netherlands) prior to the experiment, and males with proven fertility were selected to mate with the experimental females. Males were housed individually before and after mating, and female mice were housed 2 per cage. Animal procedures were approved by the Ethical Committee for Animal Research of the Utrecht University and conducted according to the European Directive 2010/63/EU on the protection of animals used for scientific purposes (AVD108002016597). Experimental design After 14 days of acclimatization, bedding from the cages of assigned breeder males was added to the cages of experimental female mice to facilitate synchronization of the females’ cycle. After three days, males and females were housed together for 72 hours in a 1:2 ratio. Vaginal plugs were scored to assess the time of mating. After mating, the females were randomly assigned into the control group or the antibiotic treatment group. Antibiotic treatment was carried out by adding a mix of 2.5 mg/ml neomycin (Sigma Aldrich, Zwijndrecht, The Netherlands), 0.5 mg/ml metronidazole (Sigma Aldrich), and 0.09 mg/ml polymyxin (Sigma Aldrich) in the drinking water. The treatment consisted of two courses, starting with 4 days antibiotic treatment with followed by 10 days without antibiotics and another 4 days with antibiotics (Figure 1A). The animals were weighed before mating and at the end of experiment to evaluate the weight gain. The pregnant mice were killed by cervical dislocation on day 17 after mating, after which the number of fetuses and resorptions were assessed and tissue samples were collected for further analysis. During the sectioning, which was carried out in a laminar flowhood, sterile surgical instruments were