141 Ovarian tissue injury resulted by puncture alters follicular activation in mice

Demand for assisted reproductive techniques has increased considerably in human and veterinary medicine. Currently, ovarian puncture is being widely used but its effect on ovarian follicular mobilisation and fertility is still not fully determined. In this scenario, the aims of the present study were to: (i) standardise a murine experimental model for ovarian puncture, and (ii) investigate the effects of tissue injury caused by the procedure on ovarian follicular mobilisation and mice fertility. Female mice were randomly distributed into three groups (n = 60 divided into Control, Sham, and Puncture groups). The Puncture group was submitted to an experimental puncture procedure, the Sham group underwent the surgical procedure without puncture. This study was performed in two parts. The first experiments were related to follicular dynamic and the second to reproductive aspects. Oestrous cycle was monitored before and after the procedure. 96 h after puncture, the animals were killed and the ovaries were recovered for morphofunctional analysis (histopathological analysis, follicle quantification per section, morphometry, immunohistochemistry, qPCR). For the superovulation analysis and fertility assay, new animals were submitted to the same procedures (n = 60). Data were analysed for normality using the D’Agostino Pearson followed by analysis of variance. The experimental puncture procedure was successfully standardised. The oestrous cycle duration was not altered by the procedure (P > 0.05). Besides the inflammatory events that occurred in both Sham and Puncture groups, the second one showed a higher (P < 0.0001) volumetric density of pathological elements (presence of polymorphonuclear cells, inflammatory exudate, and haemorrhagic focus). The number of primordial follicles was reduced by 72.7% in Puncture group (P = 0.008) compared with the Control, and this procedure also increased the number of atretic follicles (P = 0.002, higher in Puncture group) follicles. Immunohistochemistry analysis showed a higher proliferation index (BrdU) on granulosa cells of primordial follicles (P < 0.001) caused by puncture and that the atresia, by Caspase3 activation, occurred predominantly in secondary follicles (P < 0.001). qPCR showed an upregulation of genes related to collagen deposition and inflammatory response (IL1A, COL1A1, and COL4A1; P < 0.001) in punctured ovaries. The superovulation assay showed a reduced number of oocytes collected from Puncture animals (P = 0.044); their morphological quality was not affected (P > 0.05). No significant differences regarding the fertility aspects of the animals were observed (P > 0.05). Our findings indicate that the ovarian puncture affects the follicular activation, and inflammation is triggered by this procedure. Punctured ovaries were able to overcome the inflammation, maintaining female fertility. The present study brings a new approach to ovarian puncture studies and shows unprecedented results related to the inflammatory kinetics resulting from the puncture and its effects on follicle activation.

This research was supported by CAPES, FAPEMIG, and CNPq.