Bench to Bedside
Session: Poster Session A
Cecily V. Bishop, PhD
Assistant Professor
Oregon State University
Corvallis, Oregon, United States
Cecily Bishop1, Sydney Bowne1, Tamar Weiss1, Carol Hanna2, Charles Estill1,3, Alison Ting4
1Department of Animal and Rangeland Sciences, College of Agricultural Sciences, Oregon State University, Corvallis, OR, USA.
2Division of Reproductive and Developmental Sciences, Oregon National Primate Research Center, Oregon Health & Science University, Beaverton, OR, USA.
3Department of Clinical Sciences, Carlson College of Veterinary Medicine, Oregon State University, Corvallis, OR, USA.
4Expanse Bio LLC, North Charleston, SC, USA.
Abstract Text: Prior research by our group and others suggest re-establishment of function is possible in ewes following ovary removal, vitrification, and re-transplantation to orthotopic site following re-anastomosis of vessels. The present study was to determine if oocyte retrieval and fertilization potential of vitrified ovaries. Ewes underwent bi-lateral ovariectomy followed by vitrification, re-warming and transplantation of 1 ovary back to the orthotopic site via re-anastomosis (vitrified; n=4), or transplantation of 1 ovary without vitrification (sham; n=1), or remained intact (n=5, non-surgical control). Vitrified, sham and 4/5 control ewes were allowed to recover for 30 days before analysis of cyclicity by evaluation of bi-weekly serum progesterone levels (P4) for 2 months, encompassing at least 2.5 estrous cycles, along with analysis of anti-mullerian hormone (AMH) mid-way through cycle evaluation period. These ewes were also exposed to a ram of proven fertility during this time. All control ewes (n=4) had P4 >0.5 ng/ml, and samples measured assayed at end of this period for pregnancy specific protein B (BioPRYN) indicated all 4 achieved pregnancy. All control ewes had measurable AMH (average concentration 2.49 ng/ml). Sham control ewe had P4 >0.5 ng/ml in most samples assayed. This female had measurable AMH, but 3-fold lower than average for cycle control females. One (of 4) vitrified female had consistent bi-weekly samples with P4 >0.5 ng/ml. While 2/4 vitrified ewes had sporadic samples with P4 >0.5 ng/ml, the remaining vitrified ewe never achieved a sample with P4 >0.5 ng/ml - this ewe tested positive for Ovine Progressive Pneumonia and was humanely euthanized. No vitrified ewes had AMH above detectable limits of the assay (0.025 ng/ml). All remaining vitrified (n=3) and sham (n=1) ewes, along with an additional, non-pregnant control ewe (n=1) underwent estrus synchronization and were stimulated to ovulate with gonadotropins (PG-600), followed by ovariectomy for follicular aspiration and histology. Prior to synchronization, the control ewe had AMH levels within the 95th percentile of control ewes. AMH levels in sham and vitrified ewes remained similar to those detected during cycle analyses. 5 follicles were aspirated from control ewe, with cumulus oocyte complexes (COCs) successfully retrieved from all follicles. Of these 5 COCs, all underwent successful IVM, and 4/5 fertilized. Similar to control, the sham ewe also had 5 follicles aspirated, with 5 COCs retrieved and all successfully matured, and 4/5 fertilized. In vitrified ewes, the 2 ewes with sporadic P4 levels had follicles present on ovary post-synchronization, but no oocytes were recovered. The remaining ewe with consistently elevated P4 in bi-weekly samples had 4 follicles aspirated with 2 COCs recovered. Both of these oocytes successfully matured and fertilized. Serum levels of Inhibin A were analyzed at the time of ovariectomy/follicle aspiration. Ewes in which COCs were successfully retrieved from follicles had Inhibin A levels in serum above detectable limits of the assay (range 6.9-3.6 ng/ml, lower limit of ELISA 2.3 ng/ml). These data provide further evidence indicating that whole ovary vitrification is successful in achieving resumption of some follicular activity in most ewes exposed to these protocols. In addition, while natural pregnancy was not achieved in this experiment group, COCs capable of fertilization were obtained in ewes with high levels of ovarian function. This provides further evidence that these protocols are a viable option for fertility preservation.
Funding: NIH SBIR 5R44HD104531 (A. Ting), and P51 OD 011092 (DPCPSI, ORIP, NIH).