Morphological and biomechanical characterisation of the ovine cervix

Due to the anatomy of the ovine cervix, transcervical artificial insemination is not routinely possible in ewes, meaning sperm must be deposited at the external osmium of the cervix. To better understand how sperm traverse the cervix, there is a need to develop more physiologically relevant ex-vivo models. One of the first steps in this is to assess the functionality of the cervix to establish if differences exist along the length of the cervix and characterise the remodelling that may occur across the duration of the cycle. This project aimed to evaluate the cervical micro-environment and biomechanical properties to elucidate the underlying mechanisms orchestrating sperm transport across the cervix. To histologically evaluate the cervix, crossbred ewes were synchronised and slaughtered at either the follicular (n = 6) or luteal (n = 7) phase of their cycle. The reproductive tract was isolated, and the cervix was sectioned circularly (up to 10 segments along the length of the cervix), formalin-fixed, wax embedded, sectioned, and stained with haematoxylin and eosin stain. The types of epithelial cells (squamous vs columnar), their heights, cervical crypt surface area, depth and tortuosity were quantified. Biomechanical studies using uniaxial testing investigated the macro?biomechanical effects on cervical tissue during the follicular (n = 14 ewes) and luteal phase (n = 14 ewes) of the reproductive cycle and compared two cryopreservation methods (vitrification or controlled rate freezing) to fresh tissue. Each cervix was sectioned into 3 longitudinal strips which were split across the 3 treatments. To evaluate the effects of reproductive phase and effects of cryopreservation, the tissue was biomechanically characterised using uniaxial cyclic loading tension. Samples were subjected to 10 cycles of uniaxial loading at a rate of 10 mm/min, ranging from 0% to 20% engineering strain, with elastic modulus of the linear region of the stress-strain curve used to evaluate samples. Histologically, the cervix presented with a squamocolumnar junction containing columnar and squamous epithelium at the external ostium. The remainer of the cervix was primarily composed of columnar epithelium. Columnar epithelial cells were exclusively found through segments 2 through 10 (1 = External cervical ostium; 10 = Internal cervical ostium) in both reproductive phases. The height of the columnar epithelium was greater in the follicular (33.3 ± 1.31 µm) compared to the luteal (26.7 ± 1.21 µm) phase (mean ± s.e.m; p < 0.001). There was an increase in cervical microgroove surface area during the follicular phase (p < 0.05). There was also an increase in cervical microgroove surface area, depth and tortuosity in the segments nearest internal cervical ostium compared to those nearest the external ostium (p < 0.01). Biomechanical analysis revealed neither of the two cryopreservation treatments had any significant effects on the macro-structure of the cervix when compared to fresh tissue. This study provides a detailed characterization of the ovine cervical canal, revealing hormonally driven cellular changes during the reproductive cycle, with a significant epithelial height increase and microgroove structural changes including increased surface area, depth and tortuosity during the follicular phase. Biomechanically revealed no structural differences between reproductive phases and demonstrated that cryopreservation preserves the biomechanical integrity of cervical tissue, supporting its potential for future reproductive research and biobanking applications.