Fig. 1: Follicles develop an asymmetric morphology prior to ovulation both in vivo and in vitro, which can be used to predict the site of follicle rupture.

A During follicle development, follicles grow from a symmetric morphology where the oocyte (asterisk) is surrounded by even layers of granulosa cells, to an asymmetric morphology where the oocyte (asterisk) is surrounded by cumulus cells and a fluid-filled antral cavity forms (arrow). The antral cavity continues to expand and will eventually rupture open to release the cumulus oocyte complex. B During ex vivo follicle growth, follicles begin exhibiting signs of asymmetry at around day 4 of culture and become entirely asymmetric by day 8 of culture. n = 51 follicles. C The site of rupture of follicles grown ex vivo can be reliably predicted based on morphology of the follicle. Rupture occurs in the region of the follicle wall that is expanded due to the antral cavity and is optically lighter in color on brightfield microscopy. D Antral stage follicles (i) can be subdivided into distinct regions (ii) by an object detection model’s prediction of the ruptured (in yellow) follicle region and unruptured (in red) follicle region. (iii) The CAM indicates that the model prediction concentrated on differences in cellular density to distinguish between the ruptured and unruptured regions of the follicle. n = 92 follicles. E Our experimental paradigm refers to these distinct regions as the putative unruptured side and putative ruptured side in the no-hCG condition and as the unruptured and ruptured side in the post-hCG condition. F Based on morphologic hallmarks of the putative ruptured and unruptured sides and guidance from the machine learning model, follicles in the no-hCG condition can be microdissected. The COC was removed and excluded so that only the contribution of the follicle was analyzed. Images are from the same follicle, before and after microdissection. The pink arrowhead denotes the putative ruptured side and the light blue arrowhead denotes the putative unruptured side. G 12 h post-hCG, follicles were microdissected to separate the unruptured region from the region that has ruptured open during ovulation with the COC removed. Images are from the same follicle, before and after microdissection. The dark pink arrowhead denotes the ruptured side and the dark blue arrowhead denotes the unruptured side.