O-013 Intraovarian treatment with a specific combination of stem cell-secreted factors reactivates ovarian function in a premature ovarian insufficiency (POI) mouse model
A Buigues, C Rodríguez-Hernández, J Martínez, P Blázquez-Simón, A Pellicer, S HerraizAbstract
Study question
Does a combination of thrombospondin 1 (THBS1), kit ligand (KITLG), and fibroblast growth factor 2 (FGF2) promote ovarian function in a POI mouse model?
Summary answer
Two consecutive intraovarian injections of THBS1, KITLG, and FGF2 rescue the reproductive outcomes in POI mice by improving oocyte yield and early embryo development.
What is known already
Different stem cell-based strategies have been explored to recover ovarian function in patients with impaired ovarian reserves, suggesting that beneficial effects are mainly mediated by the secretion of stem cell-soluble factors. Our previous findings suggest that the specific growth factors (GFs) THBS1, KITLG and FGF2 play a crucial role in these regenerative ovarian effects, observed in both mice and humans. Specifically, THBS1 and FGF2 seem to be related to the promotion of follicle development and ovarian niche vascularization, while KITLG associates with ovulation and ovarian fibrosis. Further research is still required to validate their potential to recover ovarian function.
Study design, size, duration
This experimental animal study involved 24 eight-week-old NOD/SCID female mice randomized into four groups: Young-healthy (as reference), POI-Sham, POI-GFs-low, and POI-GFs-high. Thirteen days after POI induction by chemotherapy, mice received a 10-µl intraovarian injection of either saline (POI-Sham), low- or high-dose of GFs, followed by a second injection 13 days later. After two weeks, mice underwent ovarian hyperstimulation and euthanasia for ovary/oocyte collection. Retrieved MII-oocytes were subjected to in vitro fertilization and embryo culture.
Participants/materials, setting, methods
POI condition was induced by a single intraperitoneal injection of cyclophosphamide (120mg/kg) and busulfan (12mg/kg). Intraovarian injections were performed using 30-gauge needles. The low-dose GFs treatment consisted of 2µg/ml THBS1 +400pg/ml KITLG +40pg/ml FGF2, while the high dose included 10µg/ml THBS1 +400ng/ml KITLG +10 ng/ml FGF2. Ovarian hyperstimulation was induced with 10 IU of PMSG, followed by 10 IU of hCG 48 hours later. Oocytes were released by tearing the ampulla of the oviducts.
Main results and the role of chance
Ovarian mass (POI-Sham: 6.0±2.4 mg vs. Young-healthy: 17.4±2.9 mg; p < 0.001), fibrotic area (14.8±3.5% vs. 4.9±0.6%; p = 0.005) and oocyte yield (1.6±1.8 vs. 25.0±13.7; p = 0.008) were significantly impaired by chemotherapy. Interestingly, POI females receiving a double intraovarian injection of a high dose of GFs showed improved ovarian mass (POI-GFs high: 10.2±2.7 mg vs. POI-Sham: 6.0±2.4 mg; p = 0.010) and fibrosis (9.5±3.2% vs. 14.8±3.5%; p = 0.049), and a 5-fold increase in the number of retrieved oocytes when compared to POI-Sham (7.9±4.9 vs. 1.6±1.8, p = 0.023), suggesting a promotion of gonadotropin-responsive follicles. This treatment with high GFs dose also increased the percentage of oocytes with optimal meiotic spindle assembly (78.6% vs. 60%) and chromosome alignment (71.4% vs. 60%) when compared to POI-Sham mice. In vitro fertilization rates were similar between groups, but the POI-Sham group showed impaired embryo development compared to Young-healthy females, with decreases in blastocyst formation (81.8% vs. 98.3%, p = 0.002) and hatching rates (63.6% vs. 92.5%, p = 0.002). These impairments were mitigated in both the low- and the high-dose GFs-treated mice (POI-GFs-low: 88.9% and 83.3%, respectively; POI-GFs-high: 93.0% and 70.0%, respectively).
Limitations, reasons for caution
Further validation using human ovarian samples is required as this study was conducted using an animal model of POI. The magnitude of the observed effects may vary due to interspecies differences.
Wider implications of the findings
These results suggest that the combination of THBS1, KITLG, and FGF2 has the potential to recover ovarian function in POI females, increasing the number of retrieved mature-oocytes and improving embryo development. Upon validation in humans, this approach could represent a promising therapeutic alternative to be developed for women with POI.
Trial registration number
No