{"paper_id":"ea20cf7e-414f-48dc-8dc7-973d98aa2f88","body_text":"Premature ovarian failure (POF) is a common endocrine disease causing female\ninfertility that it is characterized by high gonadotropin expression\n[follicle-stimulating hormone (FSH) ≥ 40 mIU/mL], low estradiol (E2)\nexpression, and follicular dysplasia in women aged less than 40 years ( Rudnicka  et al ., 2018 ). POF is\nassociated with impaired ovarian function leading to either hormonal disorders\n(hypoestrogenism) or loss of residual follicles that consequently increase the\npossibility of menstrual abnormalities, pregnancy failures, and impaired\nhealth-related quality of life. The POF is almost a rare outcome in a general\npopulation (prevalence 1%) ( Coulam  et\nal ., 1986 ). However, older ages (35-40 year), family\nhistory, cigarette smoking, cancer treatment (chemotherapy and radiation) ( Kenney  et al ., 2001 ) and\ngenetic agents are the POF risk factors ( Rudnicka\n et al ., 2018 ;  Franić-Ivanišević  et al ., 2016 ).\nPOF is a severe health condition with multiple negative outcomes deteriorating\npatients’ life ( Rudnicka  et al .,\n2018 ). Therefore, several therapeutic approaches like stem cell therapy\nare introduced to treat different modalities of POF ( Li  et al ., 2010 ;  Liu\n et al. , 2014a ). Mesenchymal stem cells (MSC) could\nbe extracted from different sources including adipose, human endometrium, human\namniotic, fluid cells, human placenta, human bone marrow, and menstrual blood ( Fu  et al ., 2021 ). Previous\nstudies reported promising outcomes for various types of MSCs in terms of\nimprovement of ovarian function in rats with POF. According to available evidence,\nMSCs play a critical role in the proliferation and repairing damaged tissues ( Manshadi  et al ., 2019 ;  Phinney & Prockop, 2007 ;  Liu  et al ., 2014a ). Local\nstimuli like inflammatory cytokines, ligands of Toll-like receptors, and hypoxia\nhave strong interaction with MSCs that lead to generating a large amount of growth\nfactors that are crucial in tissue regeneration ( Castro-Manrreza & Montesinos, 2015 ;  Dabrowska  et al ., 2019 ;  Wei  et al ., 2013 ). They can also modulate the\nexpression level of ovarian hormones to the normal level and also increase the\nnumber of follicles ( Manshadi  et\nal ., 2019 ). However, most of the available evidence is limited\nto animal studies and rat models and there is lack of data regarding the efficacy of\nMSCs in humans. Moreover, in most of the previous studies, MSCs harvested from the\nplacenta, bone marrow, and amniotic fluids and the MSCs extracted from peripheral\nblood were rarely investigated. The extraction of MSCs from peripheral blood is more\nconvenient than other types of MSC ( Borlongan\n et al ., 2010 ;  Mou\n et al ., 2013 ). Higher in-vitro survival and\nproliferation rate turn them into an appropriate and safe agent for cell therapy\n( Fu  et al ., 2021 ;  Manshadi  et al ., 2019 ). The\ncurrent study aimed to evaluate the therapeutic effect of MSCs derived from\nperipheral blood in the treatment of women with the POF background.\n\nThe current study was a pilot study carried-out on women younger than 40 years of\nage with premature ovarian failure who were referred to our infertility center\nin 2022. The current study was reviewed and approved by the Review Board and\nCommittee, and informed consent was completed for each study participant with\nage<40 and premature ovarian failure (POF) (with amenorrhea that occurred in\nat least four consecutive previous cycles and were confirmed with a high serum\nFSH level). Primary amenorrhea, history of severe drug allergy or autoimmune\ndisease, severe systemic disorders such as diseases of the cardiovascular\nsystem, liver, kidney and hematopoietic systems, family history of severe\ngenetic disease of benign or malignant ovarian tumors or endometrioma,\ninfectious diseases such as positive antibodies against HIV or syphilis and\nviral hepatitis were the exclusion criteria. We also excluded patients with\nabnormal liver function, abnormal prolactin and thyroid function.\nPatients with inclusion criteria received 5mg per kg body weight of subcutaneous\nGCS-F per day for five consecutive days. Then, all the participants were\nmonitored using a blood cell count test until the last day of injection before\nsampling. Three hours after the injection of the last dose of GCS-F, 120cc blood\nsamples were taken from each patient and placed under apheresis. The\nlaparoscopy-guided procedure was performed to inject 2cc into the navel of the\novary due to the small size of the ovaries and difficulty in vaginal access.\nMobilization of stem cells is currently most commonly achieved by the\nadministration of cytokines alone or preceded by chemotherapy. G-CSF (or\nfilgrastim, Neupogen, Amgen) is the most commonly used cytokine in this setting.\nG-CSF stimulates neutrophil production and maturation, and it induces the\nrelease of various proteases into the marrow, which disrupt the adhesion of CD34\nstem cells to the marrow stroma, facilitating their subsequent release into the\nPB. The default dose of G-CSF at our institution is 10 mg/kg/day for 5 days.\nPegaGen® is the brand name of Pegfilgrasti (cinnagen company, Iran).\nPegaGen ®  is supplied in 6 mg/0.6 mL prefilled syringes and\nautoinjectors (Physioject™). This drug is available in packs containing 1\nsterile pre-filled syringe or Physioject™ and a patient information\nleaflet. The dose given once a day, enables stem cell mobilization after the\nlast dose of cbc is rechecked, and if there is no side effect, with no problem\nconcerning the isolation.\nFor each patient, 120 mL of peripheral blood was collected in a 4 centrifugable\nsyringe device (Rooyagen PBSCs Kit, Arya Mabna company, Iran), each device\ncontaining 5 ml of 3.8% (wt/vol) sodium citrate. Subsequently, the blood was\ncentrifuged for 12 minutes, in the first stage,1600 RPM (450 G). In the next\nstep, the supernatant plasma part was drawn using another 20 ml syringe and\ntransferred to a number eight, 10 ml VBCT sterile tube and centrifuged at 3,500\nRPM (2,000 G) in the second round. After finishing the centrifuge, the upper\nsoup was discarded and only 2 cc of plasma remained, then the buffy coat\nresuspended to the remaining plasma. However, it should be noted that the buffy\ncoat pellet part contained leukocytes, platelets and CD34 positive PBSCs.\nWe collected data on age, body mass index (BMI), type and duration of infertility\nas baseline characteristics of the study participants. Serum level of\nFollicle-stimulating hormone (FSH), Estradiol (E2), and Anti-mullerian hormone\n(AMH) were the main investigated outcomes that were assessed at baseline (prior\nthe intervention), month two and four of the very small stem cell intervention.\nWe also measured the number of Antral follicle count (AFC) at baseline,\n2 nd  and 4 th  month of intervention. Menstrual\nrestoration and pregnancy were the secondary outcomes that were measured during\nthe follow-up examinations in the second and fourth months.\nWe described the data using mean and standard deviation. We also provided number\nand proportion for dichotomous variables. We checked the normality distribution\nusing the Shapiro-Wilk test. We assessed the Whitin-group variability over the\nstudy period using repeated ANOVA measurements. All statistical analyses were\nperformed using the Stata software (Ver 17.0, StataCorp, College Station, Texas,\nUSA) and a  p -value<0.05 was considered significant.\n\nThe current study was performed on 20 patients with premature ovarian failure\nbackground who underwent one-month treatment with very small steam cells from\nperipheral blood. Average age of the study participants was 36.2 (1.6) years.\nAverage BMI was 26.6 (3.0) kg/m 2  and 60.0% of the study participants had\nprimary infertility. Moreover, average duration of the infertility was 4.0 (1.7)\nyears ( Table 1 ).\nStudy participants baseline characteristics.\nWe assessed serum level of three hormones including FSH, AMH, and E2 at baseline and\nin two follow-up time in 2 nd  month and 4 th  month of\ntherapeutic course, as well. Average serum level of FSH was 45.0 mIU/mL (12.1) at\nbaseline and continually decreased in the second month and in month four, and\nreached 44.3 (17.4) mIU/mL, and 33.2 (12.4) mIU/mL, respectively. The within-group\nvariability was statistically significant ( p -value<0.001). There\nwas an upward trend in serum level of AMH where the average AMH level was 0.10 prior\nto the intervention and increased to 0.13 in the 2 nd  month; and 0.15 in\nthe fourth month. The observed trend was statistically significant\n( p -value<0.001). We also depicted a decreasing pattern in\nthe serum level of E2. The E2 level was 85.7 (23.6) pg/ml on average at baseline;\nhowever, after two months it reached 81.2 (22.7) pg/ml, and the average E2 was 77.2\n(25.6) pg/ml in the fourth month. The observed change was statistically significant\n( p -value=0.001) ( Table\n2 ).\nThe trend of FSH, AMH, and E2 hormone serum levels over the one-month\ntreatment period with very small stem cells from peripheral blood in\npatients with premature ovarian failure.\nData were provided as mean and standard deviation.\nAverage number of AFC was 2.0 (0.8) at baseline. There was a gradual increase in the\nsecond month (Mean AFC=2.2), and after four months it increased to 3.1 (1.8) as the\nhighest. The increase we found was statistically significant\n( p -value<0.001) ( Figure\n1 ).\nFigure 1 Change in the average number of AFC during the four-month treatment with\nvery small stem cells from the peripheral blood in patients with\npremature ovarian failure.\nChange in the average number of AFC during the four-month treatment with\nvery small stem cells from the peripheral blood in patients with\npremature ovarian failure.\nThere was also menstrual restoration in 10% of our study participants after a 4-month\nMSC intervention ( Figure 2 ).\nFigure 2 Ratio of patients with and without menstrual restoration after the\nfour-month treatment with very small stem cells from peripheral blood in\npatients with premature ovarian failure.\nRatio of patients with and without menstrual restoration after the\nfour-month treatment with very small stem cells from peripheral blood in\npatients with premature ovarian failure.\n\nThe present study aimed to investigate the therapeutic effect of very small stem\ncells from the peripheral blood on women with POF. We assessed the effect of very\nsmall stem cells on serum levels of E2, FSH, and AMH; and also, the effect of stem\ncell treatment on the number of AFC.\nOur data showed that the therapeutic approach with very small stem cells extracted\nfrom the peripheral blood could significantly increase the serum level of AMH; while\nits effect on FSH and E2 was inverse and led to a significant reduction in the serum\nlevel of E2 and FSH. Moreover, the stem cell intervention was associated with an\nincreased average AFC. Proportion of menstrual restoration and pregnancy was 10% and\nit was observed in 2 patients. Similar findings were reported by previous studies.\nSeveral studies have shown promising outcomes for different types of mesenchymal\nstem cells (MSC) regardless of their source of extraction in terms of recovering\novarian function ( Fu  et al .,\n2021 ;  Liu  et al .,\n2014a ;  Manshadi  et al. ,\n2019 ). Upregulation of AMH and increasing expression levels of FSH\nreceptor is the proposed mechanism for this improvement, which causes granulosa cell\napoptosis and follicular atresia ( Yoon,\n2019 ). A rat model study confirming our findings showed that a 30-day stem\ncell treatment could increase the levels of AMH, and AFC. They also showed that such\ntreatment could reduce FSH to near normal levels ( Liu  et al ., 2014b ;  Besikcioglu  et al ., 2019 ). Similarly, we observed that\nafter four months of stem cell treatment, the FSH level came down to less than 40\nmIU/mL. It is argued that stem cells might repair ovarian function in rats with POF\nthrough the overexpression of miR-21 and targeting phosphatase, and tension homolog\ndeleted on chromosome ten (PTEN) and recombinant human programmed cell death 4\n(PDCD4) ( Fu  et al ., 2017 ;\n Yang  et al ., 2020 ).\nBlood-derived stem cells were rarely used to improve ovarian function and there is\nlimited evidence regarding their effects on POF. However, recent studies have shown\nthat blood-derived stem cells could be used for tissue repair as they could impose a\nhigher proliferation rate ( Liu  et\nal ., 2014a ). In the study conducted by  Manshadi  et al . (2019) , the application of\nblood-derived stem cells in rats with POF led to improved hormone secretion as they\ncould be localized in the GC layer of immature follicles. According to their\nfindings, the transplantation of MSC increased the expression level of ovarian\ngranulose cell-specific including AMH, FST, and FSHR up to the normal level leading\nto an increase in the number of follicles. They also showed improvements in ovarian\nfollicular structure upon the injection of blood-derived MSCs ( Manshadi  et al ., 2019 ). These results were in\nline with of our findings and those of other previous studies ( Liu  et al ., 2014a ;  Sheikhansari  et al ., 2018 ). There is also some\nevidence regarding the positive effect of MSC on the repair of damaged tissues as\nMSCs could secrete cytokines and induce the secretion of bioactive molecules ( Lai  et al ., 2014 ;  2015 ). They also play a critical role in the\nproliferation and regeneration of damaged tissues, as they contain fibroblast growth\nfactor-2 which is a crucial factor in these regards ( Wang  et al ., 2017 ). MSCs have shown great potential in\nregenerative medicine due to their advantages, such as abundant sources, easy\naccess, multidirectional differentiation, and low immunogenicity and are safe. There\nare no side effects associated with stem cell therapy ( Gao  et al ., 2022 ). In very rare cases: a slight\nincrease in temperature after the procedure may happen; slight redness at the\ninjection site has been reported. In this study only one patient had redness at the\ninjection site, and after two hours it was resolved without any intervention.\nThe current study is one of the first attempts to evaluate the efficacy of\nblood-derived MSCs on women with POF as a pilot study. Our study was unique, because\nthe study samples were human and we evaluated the critical hormones representing the\noverall ovarian function. However, our findings must be interpreted in light of our\nlimitations, such as a small study group. In conclusion, our study indicated that\nblood-derived MSCs could significantly improve hormone secretion in women with POF.\nImplantation of MSCs in women with POF background was associated with an increase in\nAMH and AFC; while it reduced the serum level of E2 and FSH. MSCs could also cause\nmenstrual restoration and pregnancy in women with POF. Further studies are\nrequired.\nThe main limitations include the small sample size.","source_license":"CC0","license_restricted":false}