Effects Of Low-intensity Pulsed Ultrasound Combined With Human Amnion-derived Mesenchymal Stem Cell Transplantation On Premature Ovarian Insufficiency

Backgroud and Objectives:POI is pathologically characterized by oligomenorrhea or amenorrhea,low levels of gonadal hormones,and high levels of FSH（>25 mIU/ml）in females before the age of 40 years.POI affects about 1%of women under the age of 40 years old and 0.1%of women under the age of 30 years old,and can induce multiple health risks.Chemotherapy is a commonly used treatment method for women bearing tumors,which could induce ovarian failure,including GC and oocyte apoptosis,follicle loss,vascular damage,and tissue fibrosis,especially in young females.Today,there is no effective therapy for POI.Therefore,it is of great importance to develop and/or improve the treatment strategies for the irreversible pathogenesis of POI.Regenerative medicine research suggests that MSC transplantation may partly restore the ovarian structure and function in animal models of POI,which may provide an effective treatment method.hAD-MSCs have the features of MSCs.Self-renewal capacity,multipotency,low immunogenicity,and noninvasive application without controversy make hAD-MSCs promising and useful source of stem cells for transplantation and regenerative medicine.However,whether hAD-MSC transplantation can restore the ovarian function in chemotherapy-induced POI is still unknown.Thus,the effects of hAD-MSC transplantation on chemotherapy-induced POI in rats will be explored in our study.LIPUS is a form of mechanical vibration energy transmission.The acoustic pressure wave produced by LIPUS is able to transmit into and through living cells,which may result in a series of biochemical events at the cellular level.LIPUS is pulsed emission with low-intensity and low thermal effect,with minimal or no adverse effects on cells.LIPUS can promote expression of various growth factors and anti-inflammatory molecules in cells,including FGF2,IGF-1,and VEGF,which are necessary to keep the follicle growing and to reduce GC apoptosis in ovary.MSCs have been demonstrated to have the ability to sense and respond to physical stimuli.Thus,we speculate that LIPUS might promote the expression and secretion of those growth factors in hAD-MSCs.If the speculation is certificated,whether LIPUS-pretreated hAD-MSC transplantation could be more efficient to treat patients with POI will be explored.This study is to explore the effects and mechanism of h AD-MSC and LIPUS-pretreated hAD-MSC transplantation on chemotherapy-induced POI in rats.Methods:（1）hAD-MSCs were isolated from the amnion of term placentas and identified by flow cytometry and differentiation culture.（2）Effects of LIPUS on the viability and proliferation of h AD-MSCs were investigated by Cell Counting Kit-8,cell cycle and EdU assays,through which the optimal condition was determined and used in the following experiments.Western blotting was used to determine the protein expression levels.Expression and secretion of growth factors promoted by LIPUS in hAD-MSCs were detected by RT-qPCR and ELISA assay in vitro.（3）The animals were randomly divided into the control,POI,hAD-MSC treatment（hAD-MSCs）,and LIPUS-pretreated hAD-MSC treatment（LIPUS+hAD-MSCs）groups（n=40 each group）.To establish the POI model,the rats from the POI,hAD-MSCs,and LIPUS+hAD-MSCs groups were first sterilized by intraperitoneal injection of CTX,with the dose of 50mg/kg on the first day and the daily dose of 8 mg/kg for the next14 consecutive days,while the rats from the control group were only injected with normal saline.The hAD-MSCs isolated from amnion were exposed to LIPUS or sham irradiation for five consecutive days and then labeled with PKH26.At 24h after chemotherapy,the rats from the hAD-MSCs and LIPUS+hAD-MSCs groups were injected with 0.6ml PBS containing 4×10⁶ hAD-MSCs and LUPUS-pretreated hAD-MSCs labeled with PKH-26,respectively,via the tail vein,while the rats from the control and POI groups were only injected with 0.6ml PBS.At 0,2,4,6,8,and 10w after cell transplantation,the rats were sacrificed.The location and homing of the transplanted hAD-MSCs in the ovarian tissues were tracked,and estrous cycle,serum sex hormone levels,follicle counts,ovarian pathological changes,GC apoptosis,Bcl-2 and Bax expression,and pro-inflammatory cytokine levels in ovaries were examined.（4）The animals were divided into the control,POI,Control-CM and LIPUS-CM groups（n=12 each group）.The rats from POI,hAD-MSCs,and LIPUS+hAD-MSCs groups were chosen to establish the POI model.The hAD-MSCs isolated from amnion were exposed to sham irradiation or LIPUS for five consecutive days,and the conditioned media（CM）were collected,which were named Control-CM or LIPUS-CM.50μL of Control-CM and LIPUS-CM were injected into the ovaries of POI rats in Control-CM and LIPUS-CM groups,respectively,while 50μL of Cell free-CM（CF-CM）were injected into the ovaries of normal and POI rats in the control and POI groups,respectively.At 2 and 4 w after CM injection,the rats were sacrificed.Estrous cycle,serum sex hormone levels,follicle counts,ovarian pathological changes,GC apoptosis and VEGF expression level in ovaries were examined in the four groups.Results:（1）hAD-MSCs were successfully isolated from the amnion and identified as multipotent mesenchymal stem cells.（2）LIPUS promoted the viability and proliferation of hAD-MSCs.Cell cycle analysis showed that LIPUS promoted cells to enter S and G2/M phases from G0/G1 phase.Western blot results showed that LIPUS significantly upregulated the expression of cyclin D1,cyclin E1,cyclin A2and cyclin B1.LIPUS at I_SATA=30mW/cm² and ET=30min was determined to be the optimal condition and was used in the following experiments.LIPUS at I_SATA=30mW/cm² and ET=30min promoted the expression and secretion of growth factors,including IGF-1,HGF,and VEGF,in hAD-MSCs in vitro.（3）PKH26-labeled hAD-MSCs mainly homed to ovaries in POI rats after cell transplantation in both hAD-MSCs and LIPUS+hAD-MSCs groups,and amounts of red fluorescent signal was found in ovaries.Both hAD-MSC and LIPUS-pretreated hAD-MSC transplantation increased the body and reproductive organ weights,improved ovarian function,and reduced reproductive organ injuries in POI rats.Transplantation of hAD-MSCs increased the Bcl-2/Bax ratio,and reduced GC apoptosis and ovarian inflammation induced by chemotherapy in ovaries.These effects could be improved by the pretreatment of LIPUS on hAD-MSCs.（4）PKH26-labeled hAD-MSCs were only located in the interstitium of ovaries,rather than in follicles,after transplantation in both hAD-MSCs and LIPUS+hAD-MSCs groups.hAD-MSCs did not express ZP3 or FSHR,which was the typical marker of oocyte or GC,at 8 w after cell transplantation,and these demonstrated that hAD-MSCs did not differentiate into oocyte or GC.（5）Both Control-CM and LIPUS-CM injecton into ovaries partially improved ovarian function,reduced ovarian injuries and GC apoptosis,and promoted VEGF expression level in ovaries in POI rats.However,there was no significant difference between Control-CM and LIPUS-CM groups.Conclusions:（1）LIPUS can promote the viability and proliferation of hAD-MSCs.LIPUS promotes expression and secretion of growth factors in hAD-MSCs.（2）Both hAD-MSC and LIPUS-pretreated h AD-MSC transplantation can repair ovarian injury and improve ovarian function in rats with chemotherapy-induced POI.LIPUS-pretreated h AD-MSC transplantation is more advantageous to reduce inflammation,improve local microenvironment,and inhibit GC apoptosis induced by chemotherapy in ovarian tissue of POI rats.（3）Both Control-CM and LIPUS-CM injecton into ovaries can partially improve ovarian function and reduce ovarian injuries.（4）The efficacy of h AD-MSC and LIPUS-pretreated hAD-MSC transplantation on chemotherapy induced POI is more likely to be partially mediated by hAD-MSCs through the paracrine pathway.