LAUSR

Can multiple intraovarian injections of platelet-rich plasma (PRP) enhance ovarian function and fertility outcomes in a mouse model of premature ovarian insufficiency (POI)? A double administration of PRP ameliorates the deleterious effects of chemotherapy on ovarian fibrosis and embryo development in mice. PRP has emerged as an alternative approach to restore fertility in women with POI, a condition characterized by the loss of ovarian function before the age of 40. Previous studies suggest that PRP therapy has the potential to enhance ovarian tissue regeneration and follicular activation in damaged ovaries. However, the clinical efficacy and optimal protocols for PRP preparation and administration remain uncertain. Further research is needed to establish effective treatment regimens. Therefore, this study aims to evaluate the potential benefits of repeated PRP injections, providing sustained stimulation across multiple menstrual cycles. POI condition was induced in thirteen 8-week-old NOD/SCID female mice, while 4 untreated mice were used as a reference (Young-Healthy). Thirteen days after POI induction, mice were randomized to receive 1 or 2 intraovarian injections of PRP (POI PRP-1X, n = 4 and POI PRP-2X, n = 5 respectively) or 2 injections of PBS (POI-Sham, n = 4), with a 13-day interval between injections. Twelve days after treatments, mice underwent controlled ovarian stimulation (COS) and euthanasia to evaluate ovarian function. POI was induced by intraperitoneal administration of cyclophosphamide and busulphan (120 mg/kg, 12 mg/kg). For intraovarian injections, 10 µl of either PBS or activated PRP from normoresponder patients (1801-FIVI-003-AP) was injected into each ovary. Following COS and euthanasia, ovaries were collected and fixed to evaluate ovarian fibrosis (Picrosirius Red staining) and vascularization (Isolectin-B4 inmunostaining). Retrieved oocytes underwent in vitro fertilization (IVF) with C57BL/6 sperm, and embryos were cultured to assess fertilization rate and embryo development. Chemotherapy administration successfully reproduced the POI condition in mice as evidenced by the signs of ovarian damage observed in the POI-Sham group. Specifically, the ovarian/body mass ratio (POI-Sham: 0.3±0.1, Young-Healthy: 0.7±0.1; p < 0.001) and the total number of ovulated oocytes (POI-Sham= 1.6±1.8, Young-Healthy: 25.0±13.7; p = 0.001) were significantly reduced in POI-Sham mice compared with Young-Healthy females. The POI-Sham group also exhibited higher levels of ovarian fibrosis (POI-Sham: 14.8±3.5%, Young-Healthy: 4.9±0.6%; p = 0.005), although no differences were found in ovarian vascularization. Single and double PRP intraovarian injections did not improve ovarian mass or oocyte yield in POI mice. However, both PRP regimens were able to reduce ovarian fibrosis, being this decrease statistically significant in the 1PRP dose group (POI PRP-1X: 7.2±0.9%, POI PRP-2X: 10.9±3.7% vs. POI-Sham: 14.8±3.5%; p = 0.008 and p = 0.061 respectively). Chemotherapy did not affect fertilization rates, but the POI-Sham group showed impaired embryo development, with reduced blastocyst formation (POI-Sham: 81.8%, Young-Healthy: 98.3%; p = 0.002) and hatching rates (POI-Sham: 63.6%, Young-Healthy: 92.5%, p = 0.035) compared to controls. Notably, these negative effects were not observed in those POI mice treated with two PRP injections (POI PRP-2X: 91.3%, 91.3% respectively; p=NS vs. Young-Healthy). This study was conducted in a mouse model of POI, requiring additional studies with human samples to confirm this results and assess their clinical application. PRP treatment mitigates some of the deleterious effects induced by chemotherapy in mice, and a double PRP injection seems to improve the benefits of this treatment in embryo development. However, further research is still needed to clarify the therapeutic potential of PRP to promote ovarian function and restore female fertility. No