Granulocyte Colony-Stimulating Factor Treatment in Women with Premature Ovarian Insufficiency: A Pilot Study of Biological Activity and Menstrual Resumption

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Premature ovarian insufficiency (POI) occurs when this decline happens before age 40, causing infertility. Bone marrow stem cells may help restore ovarian function, as some women conceive after bone marrow transplants. Studies suggest that mobilizing stem cells with Granulocyte Colony-Stimulating Factor (G-CSF) can improve ovarian response in women with diminished ovarian reserve, possibly without needing ovarian infusion. Our study aimed to evaluate if G-CSF injections alone could improve ovarian function in women with POI. Methods: This was a pilot, non-randomized, open-label clinical trial including 11 women aged 25–40 years with clinical POI and menopausal symptoms, defined by elevated follicle-stimulating hormone (FSH) on two occasions, low anti-Müllerian hormone (AMH), and reduced antral follicle count (AFC). Participants received up to three rounds of subcutaneous G-CSF administered daily for four days per month over 60 days. Ovarian reserve markers (FSH, AMH, AFC), menstruation resumption, and menopausal symptoms were assessed at baseline and multiple follow-up points over 12 months. Results: The mean age of participants was 34.1 ± 5.2 years (BMI 23.96 ± 4.0 kg/m²). GCS-F injections resulted in significant increases in white blood cells and mild elevation of liver enzymes which returned to baseline within one month. By four months, significant improvements in menopausal symptoms were reported. Mean FSH decreased from 54.3 ± 24.6 IU/L at baseline to 29.0 ± 8.1 IU/L at six months (p = 0.008). AMH and AFC rose modestly (0.55 ± 0.2 to 3.89 ± 0.5 pmol/L; 1.09 ± 1.0 to 1.2 ± 2.3), though not significantly. Menstruation resumed in 7 of 11 women (63.6%, p = 0.031). One participant showed marked response including retrieval of three mature oocytes. Conclusions: G-CSF injections were associated with menstrual resumption and symptom relief in most women with POI, suggesting biological activity. Although improvements in ovarian reserve markers were modest and disappointing in terms of the potential for assisted reproduction, these findings may support further evaluation of G-CSF in larger, controlled trials to clarify its clinical benefit and therapeutic potential. Trial registration number: NCT06117982 (https://clinicaltrials.gov/study/NCT06117982?cond=The%20Impact%20of%20Granulocyte%20Colony%20Stimulating%20Factor%20on%20Premature%20Ovarian%20Insufficiency&rank=1 Primary Ovarian Insufficiency Granulocyte Colony-Stimulating Factor Ovarian Reserve Follicle Stimulating Hormone Menstruation Figures Figure 1 INTRODUCTION It is generally accepted that women are born with all the eggs they will have in their lifetime, estimated to be about two million. There appears to be progressive depletion of oocytes such that by puberty, about four hundred thousand quiescent follicles are remaining, of which a small number initiate growth each month during a woman's reproductive life. Depletion of this primordial pool of follicles occurs over time, resulting in menopause at around age 50 years in most women (1). The rate at which the pool of resting follicles is lost may be accelerated because of genetic or chromosomal abnormalities (e.g. women with Turner’s syndrome or FMR-1 premutations), environmental factors (exposure to environmental toxicants or viral infection), iatrogenic causes (chemotherapy or radiation therapy for cancer) or in many cases from unknown causes (2). Over time, women with accelerated loss of follicles may develop premature ovarian insufficiency (POI), also known as premature ovarian failure, defined by elevated gonadotropins, hypoestrogenism and anovulatory cycles below 40 years of age and impacting about 1% of women [ 3 ]. Women with POI develop ovarian resistance to gonadotropic signals, disrupting ovulation and menstrual cycles and ultimately rendering fertility treatments such as IVF ineffective. Interestingly, patients who develop POI after chemotherapy have been observed to achieve spontaneous pregnancy after a bone marrow transplant [ 4 , 5 , 6 ]. This is thought to be related to bone marrow-derived stem cells, possibly leading to the recruitment of pre-existing quiescent follicles through various mechanisms. It is speculated that hematopoietic stem cells may differentiate into oocyte-support cells (granulosa/theca/cumulus or stromal cells) and sensitize the follicular environment to gonadotropic stimulation. Alternatively, paracrine factors from the stem cells, such as growth factors and angiogenic factors, could result in neovascularization and increased ovarian perfusion and facilitated delivery of gonadotropins to primordial follicles [ 7 , 8 ]. There is much evidence to suggest that BM hematopoietic stem cells can restore ovarian function in both mouse models and human studies [ 8 – 13 ]. In clinical studies, hematopoietic stem cells were either collected via direct aspiration from a patient's bone marrow (BM) or via a less invasive technique using granulocyte colony-stimulating factor (G-CSF) to mobilize stem cells from the BM into the peripheral blood. In the latter procedure, BM-derived stem cells (BMDSC) are then collected through peripheral blood plasmapheresis using flow cytometry. In both instances, BMDSCs are either infused in the ovarian stroma of women with POI via laparoscopy or alternatively infused into the ovarian artery. In one study from Spain, women who had diminished ovarian response (DOR) and were considered as poor responders to gonadotropins were treated with G-CSF for 5 days at a mean dose of 600 mcg/d (9). Peripheral blood was collected to isolate BMDSC, and these stem cells were subsequently infused into one ovarian artery, using the other ovary as a control. The authors demonstrated that autologous stem cell ovarian transplantation (ASCOT) following G-CSF treatment resulted in improvements in antral follicle count and AMH levels in 81% of patients (13/16). Overall, comparing pre- and post-ASCOT IVF cycles, they noted improvements in AFC, a reduced cycle cancellation rate, and better fertilization rates [ 9 ]. A total of 5 pregnancies occurred post-ASCOT treatment (3 spontaneous) [ 9 ]. Interestingly, although they only infused stem cells into one ovarian artery, they noted an effect on the control ovary as well, suggesting that perhaps these stem cells and/or growth factors released from them was able to travel through the bloodstream and exert their function elsewhere. Hence, this raises the question of whether ovarian artery infusion is required at all, and if simply mobilizing BMDSC from BM into peripheral blood is sufficient to see these positive outcomes. As a result, the same group performed a follow-up study in 10 women with POI undergoing ASCOT compared to 10 women receiving only GCS-F (mean dose 600 mcg/d for 5 days) without apheresis and intraovarian artery infusion of hematopoietic stem cells (10). Both groups yielded similar results, with 6 out of 10 women in each group exhibiting visible basal antral follicles. Additionally, two patients in each group underwent IVF, and one patient had a single embryo transferred in each group. This study suggests that intra-ovarian artery infusion of stem cells may not be required for folliculogenesis. Based on these promising results in women with DOR, the objective of the present study was to determine if hematopoietic stem cell mobilization could be beneficial in improving ovarian response in women with POI. Since both bone marrow aspiration and ASCOT are invasive procedures, we chose to use G-CSF injections and stem cell mobilization from the BM alone, similar to the previous study in DOR women (10). G-CSF treatment is safe and has been routinely used in healthy donors for decades to prepare them for allogeneic stem cell transplants. METHODS Study Design and Ethics This was a pilot, non-randomized, open-label clinical trial evaluating the effect of granulocyte colony-stimulating factor (G-CSF) on ovarian reserve in women with premature ovarian insufficiency (POI). The study was reviewed and approved by the Veritas Independent Review Board. Additionally, Health Canada authorized the trial under Part C, Division 5 of the Food and Drug Regulations, with no objection to its initiation. The study is also registered on ClinicalTrials.gov (NCT06117982) Eligibility Criteria Participants were women aged 25–40 years diagnosed with clinical POI and menopausal symptoms such as hot flashes, night sweats, insomnia, and vaginal dryness. The laboratory diagnosis of POI included two measurements of serum follicle-stimulating hormone (FSH) ≥ 30 IU/L at least one month apart, antral follicle count (AFC) < 5 and anti-Müllerian hormone (AMH) < 3 pmol/L (0.43 ng/ml). Exclusion criteria included autoimmune disorders, hematopoietic malignancies, sickle cell disease, or comorbidities that precluded infertility treatment or pregnancy (e.g., HIV/AIDS, hepatitis B/C, breast cancer, or BMI > 40 kg/m²). Concurrent use of other medical or fertility treatments was not permitted, except for natural estrogen therapy for vasomotor symptom management. Recruitment and Consent Eligible participants received both verbal and written study information and were given time to review the materials at home. Follow-up phone calls were made for those who agreed to be contacted. A total of 11 participants provided written informed consent and enrolled in the study. Intervention and Clinical Procedures Participants attended an in-person baseline visit for assessment and medication administration. Pre-treatment assessments included hormonal testing (FSH, AMH, AFC), a complete blood count (CBC), liver enzymes (ALT, ALP, LDH), renal function tests (creatinine, sodium, potassium, uric acid), and a spleen ultrasound. Each participant received a 0.5 mL subcutaneous injection of 300 µg granulocyte-colony stimulating factor (G-CSF) (Neupogen® prefilled syringes, Amgen, USA) for four consecutive days. The first injection was administered in the clinic and followed by a 60-minute observation period. Subsequent injections were self-administered at home after training. All baseline tests, except AMH and AFC, were repeated on the day after the fourth injection. Participants returned on Day 30 for reassessment, including all baseline tests, and a second four-day G-CSF cycle, with follow-up testing on Day 35. A third four-day G-CSF cycle was offered at Day 60 if there was no measurable improvement in FSH, AMH or AFC. Monthly follow-up visits were conducted for up to 12 months. Patients with a reduction in FSH to < 20 IU/L and an increase in basal antral follicle count were offered in vitro fertilization (IVF) to assess the possibility of oocyte retrieval and embryo development. Statistical Analysis All analyses were performed using IBM SPSS Statistics, Version 29.0 (IBM Corp., Armonk, NY). Continuous variables were tested for normality using the Shapiro-Wilk test. Normally distributed variables were reported as mean ± standard deviation. Repeated-measures ANOVA was used to assess longitudinal changes in FSH, AMH, and AFC across three time points: baseline, month 4, and month 6. Where the overall F-test was significant, Bonferroni-corrected pairwise comparisons were conducted as post hoc tests. Changes in menstruation status (yes/no) between baseline and month 4 were analyzed using McNemar’s test. Missing data were handled via listwise deletion. Additionally, one participant who demonstrated an exceptional clinical response (including successful oocyte retrieval) was described separately in the results. Statistical significance was defined as a two-tailed p -value < 0.05. RESULTS Patient Demographics A total of eleven patients diagnosed with primary ovarian insufficiency (POI) were enrolled in the study, with the first participant enrolled on October 13, 2023, and the final participant recruited on June 13, 2024. The mean age at baseline was 34.1 ± 5.2 years, and the mean body mass index (BMI) was 23.96 ± 4.0 kg/m². Enrollment in the study was stopped after these 11 patients, as we observed no robust clinical response in AFC following G-CSF treatment. Instead, we decided to follow the initial 11 subjects for up to 12 months to determine if there was any long-term response. Table 1 lists the demographic and clinical characteristics of the 11 patients included in this study with POI. In terms of reproductive history, seven participants were nulligravid, while four had a history of previous pregnancies (gravidity range: 0–3; parity range: 0–1). A family history of POI was reported by four patients (36.3%). The average duration since POI diagnosis was 4.13 ± 4.3 years. Menopausal symptoms were assessed at baseline and again at month 4. At baseline, the most reported symptoms included hot flashes (6/11; 54.5%), irritability (5/11; 45.5%), poor concentration (5/11; 45.5%), vaginal dryness (7/11; 63.6%), insomnia (7/11; 63.6%), and night sweats (7/11; 63.6%). Less frequently reported symptoms included mood swings, weight gain, and memory difficulties. At 4 months post-treatment, several symptom domains demonstrated improvement. The prevalence of hot flashes decreased to 2/11 (16.6%), although this reduction did not reach statistical significance (McNemar’s test, p = 0.125). Statistically significant improvements were observed in the prevalence of vaginal dryness (0/11; p = 0.016), insomnia (1/11; p = 0.031), and night sweats (1/11; p = 0.031). Although reductions in irritability and poor concentration were noted, these changes did not achieve statistical significance. Safety Profile: Short-Term Side Effects and Laboratory Changes The treatment was well tolerated, with no serious adverse events reported throughout the study. The most observed side effect was lower back pain (6 out of 11 participants (54.5%)) and mild to moderate headache, experienced by 5 out of 11 participants (45.4%) within 24 hours following G-CSF administration. Other transient effects included fatigue (2 out of 11 participants (18.1%)) and injection site discomfort (9.0%). All symptoms were self-limited, resolving spontaneously within 1–2 days without the need for medical intervention. Routine laboratory monitoring, including complete blood count, liver enzymes, renal function markers (creatinine, sodium, potassium, uric acid), and spleen size via ultrasound, was performed at baseline, 1 day following each G-CSF treatment course and monthly thereafter. Significant transient increases were observed in white blood cell count a day after the final G-CSF injection. The mean white blood cell (WBC) count at baseline was 5.6 ± 0.26 ×10⁹/L, which increased to 29.0 ± 4.3 ×10⁹/L on Day 5. Repeated-measures ANOVA revealed that this increase in WBC on Day 5 compared to baseline was significant (F (2, 22) = 30.94, p 0.05), indicating that the increase was transient. This pattern of WBC increase was seen immediately after injections and returned to baseline after one month and was consistent across subsequent injections. Regarding liver function tests, alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) showed a significant increase from baseline to Day 5. The mean ALP at baseline was 67.2 ± 5.6 U/L, which increased to 179.2 ± 19.4 U/L on Day 5 ( p = 0.001). Similarly, LDH increased from a mean of 182.2 ± 5.7 U/L at baseline to 344.7 ± 26.2 U/L on Day 5 ( p = 0.001). However, these increases were transient, as both ALP and LDH levels returned to baseline after one month (mean ALP: 72.44 ± 7.0 U/L, LDH: 185.8 ± 10.2 U/L), with no significant difference between Day 30 and baseline ( p > 0.05). This trend was also observed after each subsequent treatment cycle, where ALP and LDH levels increased post-injection and returned to baseline after one month. Importantly, ALT levels remained stable throughout the study period, showing no significant changes from baseline to Day 30. Renal function was consistently normal throughout the study period. Tests for creatinine, sodium, potassium, and uric acid showed no significant changes from baseline to Day 30 or at any subsequent time points, confirming that no renal adverse effects were associated with G-CSF treatment. No evidence of clinically significant organ dysfunction, neutropenia or renal dysfunction. No splenomegaly was observed on ultrasound. All other laboratory markers remained stable throughout the study period. Hormonal and Ovarian Reserve Changes We assessed FSH, AMH, and AFC levels in patients over six months following the initial administration of G-CSF. Unfortunately, two of the eleven participants withdrew from the study after the third month. FSH Levels Regarding FSH levels over time, the mean FSH level decreased from a baseline of 54.26 ± 24.6 IU/L to 33.24 ± 28.01 IU/L four months post-treatment. A paired-samples t-test indicated a significant reduction in FSH levels from baseline to four months (t (9) = -2.511, p = 0.033). A repeated-measures ANOVA was conducted to evaluate changes in FSH levels over time at three time points: baseline, 4 months, and 6 months (Table 2). Mauchly’s Test was applied to assess the sphericity assumption. The analysis revealed a significant effect of time on FSH levels, F (2, 16) = 4.684, p = 0.025, partial η² = 0.36, indicating that FSH levels decreased significantly across the time points (Figure 1A and 1D). Post hoc pairwise comparisons, adjusted using the Bonferroni correction, showed a significant reduction in FSH levels at 6 months (29.00 ± 24.25) compared to baseline ( p = 0.008). However, no significant differences were observed between baseline and 4 months ( p = 0.181), or between 4 and 6 months ( p = 1.00), suggesting that the significant decrease in FSH levels occurred specifically between baseline and 6 months. AMH Levels Mean AMH levels increased from 0.21 ± 0.15 pmol/L at baseline to 0.49 ± 1.13 pmol/L at 4 months post-treatment (Figure 1B and 1E). Despite this apparent rise, the difference was not statistically significant on a paired sample t-test (t(9) = 0.927, p = 0.376). To assess changes over time, a repeated-measures ANOVA was conducted. Mauchly’s test indicated a violation of the sphericity assumption ( p = 0.001); therefore, Greenhouse-Geisser correction was applied. The corrected analysis revealed no statistically significant effect of time on AMH levels (F (2,12) = [value missing], p = 0.362, partial η² = 0.140), suggesting a small effect size. Bonferroni-adjusted post hoc comparisons further confirmed that changes from baseline to 4 months ( p = 0.879) and from baseline to 6 months ( p = 1.000) were not statistically significant. AFC (Antral Follicle Count) The mean antral follicle count (AFC) showed a slight increase from baseline to 6-month follow-up (Figure 1C and 1F). At baseline, the mean AFC was 1.09 ± 1.0. This increased to 2.1 ± 2.6 at 4 months and declined to 1.2 ± 2.3 at 6 months post-treatment. Despite the observed numerical increase at 4 months, the differences were not statistically significant based on paired sample t-tests (t (9) = 1.636, p = 0.133; and t(9) = 0.258, p = 0.803, respectively). To evaluate overall change over time, a repeated-measures ANOVA was conducted, which also demonstrated no significant effect of time on AFC (F (2,16) = 0.720, p = 0.502, partial η² = 0.083), indicating a small effect size. Post hoc comparisons using Bonferroni correction further confirmed the lack of statistically significant differences between baseline and either follow-up (baseline vs. 4 months: p = 0.908; baseline vs. 6 months: p = 1.000). These findings suggest that the observed variations in AFC over the study period may be due to random fluctuations rather than an actual treatment effect. Menstruation Resumption At baseline, none of the participants reported regular menstrual bleeding; one participant reported irregular bleeding. By 4 months post-treatment, 7 out of 11 participants (63.6%) reported resumption of menstruation. McNemar’s test indicated that this increase was statistically significant ( p = 0.031), suggesting a meaningful improvement in menstrual activity following treatment. Individual Patient Response Among the 11 patients enrolled, one individual demonstrated a particularly robust clinical and biochemical response following treatment. At baseline, her FSH was 30.1 IU/L, AMH was 0.55 pmol/L, and AFC was 1. By month 4, her FSH had declined to 6.38 IU/L, AMH had increased to 3.89 pmol/L, and AFC rose to 8. She resumed spontaneous menstruation starting the second month post-treatment. Notably, she proceeded to oocyte retrieval, which resulted in the collection of three MII oocytes, representing a significant improvement in ovarian function relative to her baseline status. Because she had no partner, she opted to cryopreserve the 3 mature oocytes. DISCUSSION The present study shares conceptual similarities with a previous trial by Pellicer et al. (10), which investigated G-CSF administration for hematopoietic stem cell mobilization in women with diminished ovarian reserve. Our study, however, focused specifically on women with premature ovarian insufficiency (POI) and employed a different dosing regimen including 300 µg of G-CSF administered daily for four consecutive days per month over three months, with follow-up for six months to one year. Our data confirm that this G-CSF regimen effectively and consistently mobilized hematopoietic stem cells, as demonstrated by significant and reproducible increases in white blood cell (WBC) counts following each injection cycle. This hematologic response suggests that the intervention is exhibiting successful biological activity. Despite this, no significant changes were observed in AMH or antral follicle count (AFC) in 9 of the 11 participants over the 6- to 12-month follow-up period. Serum FSH levels, however, declined significantly, from 54.3 ± 24.6 IU/L at baseline to 33.24 ± 28.01 IU/L at month 4 and 29.0 ± 24.25 IU/L at month 6, suggesting a partial improvement in the gonadotropic hormonal environment. Of particular interest, although 10 of 11 participants were amenorrheic at study entry (with one reporting irregular menses), seven women resumed menstrual cyclicity following G-CSF treatment. This occurred despite the absence of significant improvements in AMH or AFC. Although the return of menses may reflect ovarian responsiveness and possible ovulation, serum FSH levels remained above the threshold typically required for controlled ovarian stimulation and follicular recruitment. Thus, the clinical utility of G-CSF for fertility enhancement in this cohort remains limited. Nevertheless, these menstrual changes, along with symptom improvement, suggest that G-CSF may support ovarian or hypothalamic-pituitary-ovarian axis function in a biologically meaningful way, meriting further exploration. In contrast to the weak ovarian reserve changes observed in most participants, one patient demonstrated a markedly favourable response, including normalization of FSH, an increase in AFC, resumption of menses, and successful retrieval of three oocytes in an IVF cycle. While this outcome raises the possibility of a true treatment response, spontaneous fluctuations in ovarian function are well-documented in young women with POI. This makes it difficult to attribute such improvement definitively to G-CSF without a control group. Still, her outcome highlights the heterogeneity of POI and suggests that a subset of women might benefit from G-CSF therapy under specific biological conditions. Various other experimental therapies for POI have been described in the literature with mixed success. One of the first to be published involved in vitro activation by disrupting the Hippo signalling pathway to stimulate dormant primordial follicles (14). This procedure involved surgical resection of one ovary, mincing the ovarian cortex tissue and treating in vitro with Akt activators. The ovarian tissue fragments were then reimplanted in the pelvis with follicular development in 8/27 patients, and one live baby was born (14). This procedure involves two surgeries and has not been widely adopted in clinical practice. Another study in 19 women with POI used laparoscopic ovarian biopsy without chemically induced Akt activation. Resumption of ovarian function occurred in 10 women, and two pregnancies occurred following treatment (15). Patients with the highest ovarian cortex stiffness were most likely to respond to treatment. The authors speculated that the rigidity of the ovarian cortical extracellular matrix limits expansion of the follicle and oocyte maturation, resulting in quiescent follicles. Reduction of ovarian cortex stiffness by mechanical disruption may be the mechanism of action that allows follicle growth (15). Another treatment that has been more recently explored is platelet-rich plasma (PRP) injection into the ovaries of women with POI. In one study, 311 women (age 24–40) diagnosed with POI based on ESHRE criteria underwent intraovarian PRP injection (16). PRP treatment resulted in increased antral follicle count (AFC) and serum anti-mullerian hormone (AMH), while serum follicle-stimulating hormone (FSH) did not change significantly. After PRP injection, 201 women who developed antral follicles attempted IVF, 82 (26.4% of total) developed embryos, and 57 underwent embryo transfer, resulting in 13 pregnancies (22.8% per transfer, 4% of total). In total, of the 311 women treated with PRP, 25 (8.0%) achieved livebirth/sustained implantation (spontaneously or after IVF), while another 25 (8.0%) had cryopreserved embryos. These findings suggest that in women with POI, intraovarian injection of autologous PRP is a potential treatment option. The mechanism of action of PRP in POI is unknown. Platelets play a central role in hemostasis. They also contribute to wound healing, mediated by the release of secretory proteins on platelet activation (17). The a granules of platelets contain numerous proteins that may provide an influence on wound healing, including platelet derived growth factor (PDGF- aa, bb, and ab isomers), transforming growth factor-b (TGF-b, b1 and b2 isomers), platelet factor 4, interleuken-1, platelet-derived angiogenesis factor, vascular endothelial growth factor, epidermal growth factor, platelet- derived endothelial growth factor, epithelial cell growth factor, insulin-like growth factor, osteocalcin, osteonectin, fibrinogen, vitronectin, fibronectin, and thrombospondin-1. These secretory proteins comprise growth factors, cytokines and chemokines. It is possible that one, or many of these factors together, could contribute to improvement in ovarian function in women with POI. Another speculation is that intraovarian injection of PRP may disrupt the Hippo pathway similar to mechanical disruption of the ovarian cortex with in vitro activation (2). However, none of the POI treatments, such as surgery to disrupt the Hippo pathway and PRP injections, have undergone controlled studies and pregnancy outcomes with IVF are very poor, less than 5%, consistent with potential spontaneous improvement rather than treatment efficacy. In the present study of G-CSF injections, aside from the single participant who showed a notable ovarian response, the remaining 10 patients did not demonstrate clinically meaningful improvements in fertility outcomes. Nevertheless, the resumption of menstrual cycles in 7 of the 11 participants, despite persistently low AMH and AFC, is an encouraging observation. The strength of this study is the enrollment of women with POI, rather than DOR as in most of the previous studies of G-CSF. Patients with POI and menopausal symptoms including elevated FSH are a more homogeneous group and less likely to resolve spontaneously whereas patients with DOR may often show fluctuations in ovarian response from month to month making interpretation of any treatment response less convincing. However, the weakness of our study is the small patient population undergoing treatment. We purposely stopped enrollment in the study since our aim was to achieve an IVF cycle for each patient and the results after 11 patients did not support this aim. It is also possible that there could be some spontaneous fluctuation in ovarian activity even in POI patients which can make interpretation of results problematic in the absence of a control group. In conclusion, this pilot study demonstrates that G-CSF may be biologically active in women with POI, as evidenced by a significant reduction in FSH levels and the resumption of menstruation in over 60% of participants. These findings suggest a potential modulatory effect on the hypothalamic-pituitary-ovarian axis. However, the lack of significant improvements in AMH, AFC, or fertility outcomes limit the strength of the conclusions. Nonetheless, the favourable safety profile and the robust individual response observed in one participant provide a rationale for larger, controlled studies to evaluate efficacy further and to identify subgroups most likely to benefit. Abbreviations GCSF Granulocyte Colony-Stimulating Factor POI Premature ovarian insufficiency FSH follicle-stimulating hormone AMH anti-Müllerian hormone AFC antral follicle count DOR diminished ovarian response BMDSC Bone Marrow-derived stem cells ASCOT autologous stem cell ovarian transplantation IVF In vitro Fertilization CBC complete blood count BMI body mass index PRP platelet-rich plasma Declarations Ethics Approval and Consent to Participate: The study was reviewed and approved by the Veritas Independent Review Board. Additionally, Health Canada authorized the trial under Part C, Division 5 of the Food and Drug Regulations, with no objection to its initiation. Consent for publication: Not Applicable Availability of data and materials: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests: The authors declare that they have no competing interests. Funding: This study was supported by TRIO Fertility. No external grants or specific funding agencies were involved. Authors’ Contribution: YS: Conceptualization, Methodology, Project administration, Data curation, Formal analysis, Project administration, Software, Visualization, Writing – original draft, Writing – review & editing ; LD: Writing – review & editing; MA: Investigation, Writing – review & editing; RC: Conceptualization, Investigation, Methodology, Funding acquisition, Supervision, Writing – original draft, Writing – review & editing. All authors read and approved the final manuscript. We thank the nursing, lab, and pharmacy teams at TRIO Fertility for their support, and especially Angela Lains, LBT, for her dedicated contributions to the laboratory component of this study. We are also deeply grateful to the patients who participated and made this research possible. References Hansen KR, Knowlton NS, Thyer AC, Charleston JS, Soules MR, Klein NA. A new model of reproductive aging: the decline in ovarian non-growing follicle number from birth to menopause. Hum Reprod 2008;23:699-708. Yahyavi Y, Kheradi N, Karimi A, Ebrahimi-Kalan A, Ramezani F, Yousefi S, et al. Novel Advances in Cell-Free Therapy for Premature Ovarian Failure (POF): A Comprehensive Review. Adv Pharm Bull 2024;14:543-57. The ESHRE Guideline Group on POI, L. Webber, M. Davies, R. Anderson, J. Bartlett, D. Braat, et al. Vermeulen, ESHRE Guideline: management of women with premature ovarian insufficiency, Hum Reprod 2016;31:926–37. Hershlag A, Schuster MW. Return of fertility after autologous stem cell transplantation. Fertil Steril 2002;77(2):419-21. Merloni A., Hawley J, Levy WC, Gooley T, Cd B, Deeg HJ, et al. Pregnancies following high-dose cyclophosphamide with or without high- dose busulfan or total-body irradiation and bone marrow transplantation. Blood 1996; 87 , 3045–52. Salooja N, Szydlo R, Socié G, Rio B, Chatterjee RM, Ljungman P, et al. Pregnancy outcomes after peripheral blood or bone marrow transplantation: a retrospective survey. Lancet. 2001;28;358:271-6 Tandulwadkar S, Karthick MS. Combined Use of Autologous Bone Marrow-derived Stem Cells and Platelet-rich Plasma for Ovarian Rejuvenation in Poor Responders. J Hum Reprod Sci 2020;13:184-190. Buigues A, Marchante M, de Miguel-Gómez L, Martinez J, Cervelló I, Pellicer A, et al. Stem cell-secreted factor therapy regenerates the ovarian niche and rescues follicles. Am J Obstet Gynecol . 2021;225(1):65.e1-14. Herraiz S, Romeu M, Buigues A, Martínez S, Díaz-García C, Gómez-Seguí I, et al. Autologous stem cell ovarian transplantation to increase reproductive potential in patients who are poor responders. Fertil Steril . 2018;110:496-505.e1. Herraiz S, Pellicer N, Romeu M, Pellicer A. Treatment potential of bone marrow-derived stem cells in women with diminished ovarian reserves and premature ovarian failure. Curr Opin Obstet Gynecol . 2019;31:156-62. El Andaloussi A, Igboeli P, Amer A, Al-Hendy A. Intravenous Infusion of Nucleated Peripheral Blood Cells Restores Fertility in Mice with Chemotherapy-Induced Premature Ovarian Failure. Biomedicines . 2018;6:93. Edessy, M., Hosni, H. N., Shady, Y. A., Waf, Y., Bakr, S., & Kamel, M. M. (2015). Autologous stem cells therapy, The first baby of idiopathic premature ovarian failure. Acta Medica International , 3 (1), 19. Gupta S, Lodha P, Karthick MS, Tandulwadkar SR. Role of Autologous Bone Marrow-Derived Stem Cell Therapy for Follicular Recruitment in Premature Ovarian Insufficiency: Review of Literature and a Case Report of World's First Baby with Ovarian Autologous Stem Cell Therapy in a Perimenopausal Woman of Age 45 Year. J Hum Reprod Sci . 2018;11(2):125-30. Kawamura K, Cheng Y, Suzuki N, Deguchi M, Sato Y, Takae S, Ho CH, Kawamura N, Tamura M, Hashimoto S, Sugishita Y, Morimoto Y, Hosoi Y, Yoshioka N, Ishizuka B, Hsueh AJ. Hippo signaling disruption and Akt stimulation of ovarian follicles for infertility treatment. Proc Natl Acad Sci U S A. 2013;110:17474-9. Méndez M, Fabregues F, Ferreri J, Calafell JM, Villarino A, Otero J, Farre R, Carmona F. Biomechanical characteristics of the ovarian cortex in POI patients and functional outcomes after drug-free IVA. J Assist Reprod Genet . 2022;39(8):1759-67. Cakiroglu Y, Saltik A, Yuceturk A, Karaosmanoglu O, Kopuk SY, Scott RT, et al. Effects of intraovarian injection of autologous platelet rich plasma on ovarian reserve and IVF outcome parameters in women with primary ovarian insufficiency. Aging (Albany NY) . 2020;12(11):10211-22. Pietrzak WS, Eppley BL. Platelet rich plasma: biology and new technology. J Craniofac Surg . 2005;16(6):1043-54. Tables Table 1: Demographic and clinical characteristics of individual patients. Case Age at Baseline BMI Family History of POI FSH a (IU/L) FSH b (IU/L) AMH a (pmol/L) AMH b (pmol/L) AFC a AFC b Menstruation a Menstruation b 1 34.00 22.9 No 32.5 28.10 0.20 0.12 2.00 2.00 No No 2 34.00 26.6 No 50.0 40.40 0.19 0.07 0.00 0.00 No Yes 3 31.00 21.8 No 48.1 5.97 0.11 0.33 3.00 5.00 No Yes 4 40.00 22.1 Yes 84.9 2.55 0.30 0.36 1.00 1.00 Yes Yes 5 38.00 20.8 Yes 100.0 74.50 0.12 0.09 1.00 3.00 No No 6 38.00 23.7 Yes 81.4 76.00 0.06 0.09 0.00 0.00 No Yes 7 38.00 20.5 No 41.5 40.20 0.02 0.08 0.00 0.00 No Yes 8 25.00 21.3 No 30.0 6.38 0.55 3.89 1.00 8.00 No Yes 9 32.00 30.6 No 63.2 63.90 0.40 0.08 2.00 1.00 No No 10 40.00 21.3 Yes 35.3 7.03 0.26 0.25 2.00 4.00 No Yes 11 26.00 32.0 No 30.00 20.70 0.12 0.11 0.00 0.00 No No BMI: Body Mass Index; FSH: Follicle Stimulating Factor; AMH: Anti-Mullerian Hormone; AFC: Antral Follicle Count a: Values at Baseline; b: Values at the 4 th month post treatment Table 2. Hormonal and Ovarian Reserve Markers Over Time. Values are reported in mean ± SD. Time Point FSH (IU/L) AMH (pmol/L) AFC (n) Baseline 54.26 ± 24.65 0.21 ± 0.15 1.09 ± 1.04 Month 2 42.96 ± 31.03 0.35 ± 0.38 1.09 ± 1.22 Month 4 33.24 ± 28.01 0.49 ± 1.13 2.18 ± 2.60 Month 6 29.0 ± 24.25 0.37 ± 0.73 1.22 ± 2.33 Month 12 25.50 ± 12.88 0.09 ± 0.05 1.75 ± 0.95 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 07 Jan, 2026 Read the published version in Reproductive Biology and Endocrinology → Version 1 posted Editorial decision: Revision requested 06 Nov, 2025 Reviews received at journal 05 Nov, 2025 Reviewers agreed at journal 04 Nov, 2025 Reviewers agreed at journal 03 Nov, 2025 Reviewers agreed at journal 01 Nov, 2025 Reviewers agreed at journal 01 Nov, 2025 Reviewers agreed at journal 01 Nov, 2025 Reviewers agreed at journal 01 Nov, 2025 Reviewers agreed at journal 01 Nov, 2025 Reviews received at journal 31 Oct, 2025 Reviewers agreed at journal 30 Oct, 2025 Reviewers agreed at journal 30 Oct, 2025 Reviewers agreed at journal 30 Oct, 2025 Reviewers invited by journal 30 Oct, 2025 Editor assigned by journal 23 Oct, 2025 Submission checks completed at journal 23 Oct, 2025 First submitted to journal 21 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7917847","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":541130827,"identity":"09cc3ae2-b607-4c27-8ac2-fb8d150c4296","order_by":0,"name":"Yasaman Sadeghi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDElEQVRIiWNgGAWjYDADfiA+wGADJJmZGw4QpUWyDaQljYGBh5mx4QBRegyOgUiQFgbGBgZ8WgyOH3/46UbNHXvj+70PD3xIsMm3Z2dsPPyxjUGeXwy7RoMzOcbSOceeJW47xm5wcEZCmmUPyGEH2xgMZ85OwK7lQA6DdA7b4QSzY2wMh3l/HDbggWpJMLiNQ8v5549/5/w7bG/cBtTCk/CfCC03Esykc9sOM25gA2s5QFiL5I03Zta5fYcTZxxLYwD6JdmA5zBQy5lzEjj9wnc+/fHtnG+H7fmbjzF/+JBgZ8Def/jwh4oyG3l+aexaFA5gFWZkk8AqDgLyDdjF/+DUMQpGwSgYBSMPAAAnC2eaiKohfgAAAABJRU5ErkJggg==","orcid":"","institution":"IVIRMA Global Research Alliance, TRIO Fertility","correspondingAuthor":true,"prefix":"","firstName":"Yasaman","middleName":"","lastName":"Sadeghi","suffix":""},{"id":541130829,"identity":"af8534e7-b416-4af3-af39-07d8c73ba0d1","order_by":1,"name":"Livia Deda","email":"","orcid":"","institution":"IVIRMA Global Research Alliance, TRIO Fertility","correspondingAuthor":false,"prefix":"","firstName":"Livia","middleName":"","lastName":"Deda","suffix":""},{"id":541130830,"identity":"44edeb72-bb79-4b7e-b0aa-68737f775997","order_by":2,"name":"Mohammad Albar","email":"","orcid":"","institution":"King Abdulaziz University","correspondingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"","lastName":"Albar","suffix":""},{"id":541130831,"identity":"31622844-69c9-4bdc-b19e-a8f162de35d3","order_by":3,"name":"Robert Casper","email":"","orcid":"","institution":"IVIRMA Global Research Alliance, TRIO Fertility","correspondingAuthor":false,"prefix":"","firstName":"Robert","middleName":"","lastName":"Casper","suffix":""}],"badges":[],"createdAt":"2025-10-22 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09:28:16","extension":"html","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":96146,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7917847/v1/25743f838426bcb09f34ad93.html"},{"id":95529674,"identity":"ecb60a2e-5461-4441-9628-5b6bddcfc9f5","added_by":"auto","created_at":"2025-11-10 10:17:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":93424,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges in Ovarian Reserve Markers Over Time\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A–C) \u003c/strong\u003eLine graphs showing mean ± standard deviation of \u003cstrong\u003e(A)\u003c/strong\u003eserum FSH (IU/L), \u003cstrong\u003e(B)\u003c/strong\u003e antral follicle count (AFC), and \u003cstrong\u003e(C)\u003c/strong\u003eanti-Müllerian hormone (AMH, pmol/L) at baseline and follow-up time points.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D–F)\u003c/strong\u003e Boxplots of \u003cstrong\u003e(D)\u003c/strong\u003e FSH, \u003cstrong\u003e(E)\u003c/strong\u003e AFC, and \u003cstrong\u003e(F)\u003c/strong\u003eAMH comparing distributions at baseline, month 4, and month 12. Each plot shows the interquartile range, median, and outliers. FSH declined significantly, while AFC and AMH increased significantly post-treatment, with sustained improvements at month 4.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7917847/v1/93bdc6a3dbc0d2b786fb4da5.png"},{"id":100069121,"identity":"41283342-0a02-4ffa-81f6-a393dea76145","added_by":"auto","created_at":"2026-01-12 16:09:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":954161,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7917847/v1/0a6260a4-9c87-4580-9beb-07c918ba658e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Granulocyte Colony-Stimulating Factor Treatment in Women with Premature Ovarian Insufficiency: A Pilot Study of Biological Activity and Menstrual Resumption","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eIt is generally accepted that women are born with all the eggs they will have in their lifetime, estimated to be about two million. There appears to be progressive depletion of oocytes such that by puberty, about four hundred thousand quiescent follicles are remaining, of which a small number initiate growth each month during a woman's reproductive life. Depletion of this primordial pool of follicles occurs over time, resulting in menopause at around age 50 years in most women (1). The rate at which the pool of resting follicles is lost may be accelerated because of genetic or chromosomal abnormalities (e.g. women with Turner\u0026rsquo;s syndrome or FMR-1 premutations), environmental factors (exposure to environmental toxicants or viral infection), iatrogenic causes (chemotherapy or radiation therapy for cancer) or in many cases from unknown causes (2). Over time, women with accelerated loss of follicles may develop premature ovarian insufficiency (POI), also known as premature ovarian failure, defined by elevated gonadotropins, hypoestrogenism and anovulatory cycles below 40 years of age and impacting about 1% of women [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Women with POI develop ovarian resistance to gonadotropic signals, disrupting ovulation and menstrual cycles and ultimately rendering fertility treatments such as IVF ineffective.\u003c/p\u003e\u003cp\u003eInterestingly, patients who develop POI after chemotherapy have been observed to achieve spontaneous pregnancy after a bone marrow transplant [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. This is thought to be related to bone marrow-derived stem cells, possibly leading to the recruitment of pre-existing quiescent follicles through various mechanisms. It is speculated that hematopoietic stem cells may differentiate into oocyte-support cells (granulosa/theca/cumulus or stromal cells) and sensitize the follicular environment to gonadotropic stimulation. Alternatively, paracrine factors from the stem cells, such as growth factors and angiogenic factors, could result in neovascularization and increased ovarian perfusion and facilitated delivery of gonadotropins to primordial follicles [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThere is much evidence to suggest that BM hematopoietic stem cells can restore ovarian function in both mouse models and human studies [\u003cspan additionalcitationids=\"CR9 CR10 CR11 CR12\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In clinical studies, hematopoietic stem cells were either collected via direct aspiration from a patient's bone marrow (BM) or via a less invasive technique using granulocyte colony-stimulating factor (G-CSF) to mobilize stem cells from the BM into the peripheral blood. In the latter procedure, BM-derived stem cells (BMDSC) are then collected through peripheral blood plasmapheresis using flow cytometry. In both instances, BMDSCs are either infused in the ovarian stroma of women with POI via laparoscopy or alternatively infused into the ovarian artery.\u003c/p\u003e\u003cp\u003eIn one study from Spain, women who had diminished ovarian response (DOR) and were considered as poor responders to gonadotropins were treated with G-CSF for 5 days at a mean dose of 600 mcg/d (9). Peripheral blood was collected to isolate BMDSC, and these stem cells were subsequently infused into one ovarian artery, using the other ovary as a control. The authors demonstrated that autologous stem cell ovarian transplantation (ASCOT) following G-CSF treatment resulted in improvements in antral follicle count and AMH levels in 81% of patients (13/16). Overall, comparing pre- and post-ASCOT IVF cycles, they noted improvements in AFC, a reduced cycle cancellation rate, and better fertilization rates [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. A total of 5 pregnancies occurred post-ASCOT treatment (3 spontaneous) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Interestingly, although they only infused stem cells into one ovarian artery, they noted an effect on the control ovary as well, suggesting that perhaps these stem cells and/or growth factors released from them was able to travel through the bloodstream and exert their function elsewhere. Hence, this raises the question of whether ovarian artery infusion is required at all, and if simply mobilizing BMDSC from BM into peripheral blood is sufficient to see these positive outcomes.\u003c/p\u003e\u003cp\u003eAs a result, the same group performed a follow-up study in 10 women with POI undergoing ASCOT compared to 10 women receiving only GCS-F (mean dose 600 mcg/d for 5 days) without apheresis and intraovarian artery infusion of hematopoietic stem cells (10). Both groups yielded similar results, with 6 out of 10 women in each group exhibiting visible basal antral follicles. Additionally, two patients in each group underwent IVF, and one patient had a single embryo transferred in each group. This study suggests that intra-ovarian artery infusion of stem cells may not be required for folliculogenesis.\u003c/p\u003e\u003cp\u003eBased on these promising results in women with DOR, the objective of the present study was to determine if hematopoietic stem cell mobilization could be beneficial in improving ovarian response in women with POI. Since both bone marrow aspiration and ASCOT are invasive procedures, we chose to use G-CSF injections and stem cell mobilization from the BM alone, similar to the previous study in DOR women (10). G-CSF treatment is safe and has been routinely used in healthy donors for decades to prepare them for allogeneic stem cell transplants.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStudy Design and Ethics\u003c/h2\u003e\u003cp\u003eThis was a pilot, non-randomized, open-label clinical trial evaluating the effect of granulocyte colony-stimulating factor (G-CSF) on ovarian reserve in women with premature ovarian insufficiency (POI). The study was reviewed and approved by the Veritas Independent Review Board. Additionally, Health Canada authorized the trial under Part C, Division 5 of the Food and Drug Regulations, with no objection to its initiation. The study is also registered on ClinicalTrials.gov (NCT06117982)\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eEligibility Criteria\u003c/h3\u003e\n\u003cp\u003eParticipants were women aged 25\u0026ndash;40 years diagnosed with clinical POI and menopausal symptoms such as hot flashes, night sweats, insomnia, and vaginal dryness. The laboratory diagnosis of POI included two measurements of serum follicle-stimulating hormone (FSH)\u0026thinsp;\u0026ge;\u0026thinsp;30 IU/L at least one month apart, antral follicle count (AFC)\u0026thinsp;\u0026lt;\u0026thinsp;5 and anti-M\u0026uuml;llerian hormone (AMH)\u0026thinsp;\u0026lt;\u0026thinsp;3 pmol/L (0.43 ng/ml).\u003c/p\u003e\u003cp\u003eExclusion criteria included autoimmune disorders, hematopoietic malignancies, sickle cell disease, or comorbidities that precluded infertility treatment or pregnancy (e.g., HIV/AIDS, hepatitis B/C, breast cancer, or BMI\u0026thinsp;\u0026gt;\u0026thinsp;40 kg/m\u0026sup2;). Concurrent use of other medical or fertility treatments was not permitted, except for natural estrogen therapy for vasomotor symptom management.\u003c/p\u003e\n\u003ch3\u003eRecruitment and Consent\u003c/h3\u003e\n\u003cp\u003eEligible participants received both verbal and written study information and were given time to review the materials at home. Follow-up phone calls were made for those who agreed to be contacted. A total of 11 participants provided written informed consent and enrolled in the study.\u003c/p\u003e\n\u003ch3\u003eIntervention and Clinical Procedures\u003c/h3\u003e\n\u003cp\u003eParticipants attended an in-person baseline visit for assessment and medication administration. Pre-treatment assessments included hormonal testing (FSH, AMH, AFC), a complete blood count (CBC), liver enzymes (ALT, ALP, LDH), renal function tests (creatinine, sodium, potassium, uric acid), and a spleen ultrasound.\u003c/p\u003e\u003cp\u003eEach participant received a 0.5 mL subcutaneous injection of 300 \u0026micro;g granulocyte-colony stimulating factor (G-CSF) (Neupogen\u0026reg; prefilled syringes, Amgen, USA) for four consecutive days. The first injection was administered in the clinic and followed by a 60-minute observation period. Subsequent injections were self-administered at home after training. All baseline tests, except AMH and AFC, were repeated on the day after the fourth injection.\u003c/p\u003e\u003cp\u003eParticipants returned on Day 30 for reassessment, including all baseline tests, and a second four-day G-CSF cycle, with follow-up testing on Day 35. A third four-day G-CSF cycle was offered at Day 60 if there was no measurable improvement in FSH, AMH or AFC. Monthly follow-up visits were conducted for up to 12 months. Patients with a reduction in FSH to \u0026lt;\u0026thinsp;20 IU/L and an increase in basal antral follicle count were offered in vitro fertilization (IVF) to assess the possibility of oocyte retrieval and embryo development.\u003c/p\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eAll analyses were performed using IBM SPSS Statistics, Version 29.0 (IBM Corp., Armonk, NY). Continuous variables were tested for normality using the Shapiro-Wilk test. Normally distributed variables were reported as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Repeated-measures ANOVA was used to assess longitudinal changes in FSH, AMH, and AFC across three time points: baseline, month 4, and month 6. Where the overall F-test was significant, Bonferroni-corrected pairwise comparisons were conducted as post hoc tests. Changes in menstruation status (yes/no) between baseline and month 4 were analyzed using McNemar\u0026rsquo;s test. Missing data were handled via listwise deletion. Additionally, one participant who demonstrated an exceptional clinical response (including successful oocyte retrieval) was described separately in the results. Statistical significance was defined as a two-tailed \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePatient Demographics\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of eleven patients diagnosed with primary ovarian insufficiency (POI) were enrolled in the study, with the first participant enrolled on October 13, 2023, and the final participant recruited on June 13, 2024. The mean age at baseline was 34.1 ± 5.2 years, and the mean body mass index (BMI) was 23.96 ± 4.0 kg/m². Enrollment in the study was stopped after these 11 patients, as we observed no robust clinical response in AFC following G-CSF treatment. Instead, we decided to follow the initial 11 subjects for up to 12 months to determine if there was any long-term response.\u003c/p\u003e\n\u003cp\u003eTable 1 lists the demographic and clinical characteristics of the 11 patients included in this study with POI. In terms of reproductive history, seven participants were nulligravid, while four had a history of previous pregnancies (gravidity range: 0–3; parity range: 0–1). A family history of POI was reported by four patients (36.3%). The average duration since POI diagnosis was 4.13 ± 4.3 years.\u003c/p\u003e\n\u003cp\u003eMenopausal symptoms were assessed at baseline and again at month 4. At baseline, the most reported symptoms included hot flashes (6/11; 54.5%), irritability (5/11; 45.5%), poor concentration (5/11; 45.5%), vaginal dryness (7/11; 63.6%), insomnia (7/11; 63.6%), and night sweats (7/11; 63.6%). Less frequently reported symptoms included mood swings, weight gain, and memory difficulties.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAt 4 months post-treatment, several symptom domains demonstrated improvement. The prevalence of hot flashes decreased to 2/11 (16.6%), although this reduction did not reach statistical significance (McNemar’s test, \u003cem\u003ep\u003c/em\u003e = 0.125). Statistically significant improvements were observed in the prevalence of vaginal dryness (0/11; \u003cem\u003ep\u003c/em\u003e = 0.016), insomnia (1/11; \u003cem\u003ep\u003c/em\u003e = 0.031), and night sweats (1/11; \u003cem\u003ep\u003c/em\u003e = 0.031). Although reductions in irritability and poor concentration were noted, these changes did not achieve statistical significance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSafety Profile: Short-Term Side Effects and Laboratory Changes\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe treatment was well tolerated, with no serious adverse events reported throughout the study. The most observed side effect was lower back pain (6 out of 11 participants (54.5%)) and mild to moderate headache, experienced by 5 out of 11 participants (45.4%) within 24 hours following G-CSF administration. Other transient effects included fatigue (2 out of 11 participants (18.1%)) and injection site discomfort (9.0%). All symptoms were self-limited, resolving spontaneously within 1–2 days without the need for medical intervention.\u003c/p\u003e\n\u003cp\u003eRoutine laboratory monitoring, including complete blood count, liver enzymes, renal function markers (creatinine, sodium, potassium, uric acid), and spleen size via ultrasound, was performed at baseline, 1 day following each G-CSF treatment course and monthly thereafter. Significant transient increases were observed in white blood cell count a day after the final G-CSF injection. The mean white blood cell (WBC) count at baseline was 5.6 ± 0.26 ×10⁹/L, which increased to 29.0 ± 4.3 ×10⁹/L on Day 5. Repeated-measures ANOVA revealed that this increase in WBC on Day 5 compared to baseline was significant (F (2, 22) = 30.94, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). However, by Day 30, WBC levels returned to baseline values (mean 5.4 ± 0.37 ×10⁹/L), with no significant difference between Day 30 and baseline (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05), indicating that the increase was transient. This pattern of WBC increase was seen immediately after injections and returned to baseline after one month and was consistent across subsequent injections.\u003c/p\u003e\n\u003cp\u003eRegarding liver function tests, alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) showed a significant increase from baseline to Day 5. The mean ALP at baseline was 67.2 ± 5.6 U/L, which increased to 179.2 ± 19.4 U/L on Day 5 (\u003cem\u003ep\u003c/em\u003e = 0.001). Similarly, LDH increased from a mean of 182.2 ± 5.7 U/L at baseline to 344.7 ± 26.2 U/L on Day 5 (\u003cem\u003ep\u003c/em\u003e = 0.001). However, these increases were transient, as both ALP and LDH levels returned to baseline after one month (mean ALP: 72.44 ± 7.0 U/L, LDH: 185.8 ± 10.2 U/L), with no significant difference between Day 30 and baseline (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05). This trend was also observed after each subsequent treatment cycle, where ALP and LDH levels increased post-injection and returned to baseline after one month. Importantly, ALT levels remained stable throughout the study period, showing no significant changes from baseline to Day 30.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRenal function was consistently normal throughout the study period. Tests for creatinine, sodium, potassium, and uric acid showed no significant changes from baseline to Day 30 or at any subsequent time points, confirming that no renal adverse effects were associated with G-CSF treatment. No evidence of clinically significant organ dysfunction, neutropenia or renal dysfunction. No splenomegaly was observed on ultrasound. All other laboratory markers remained stable throughout the study period.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHormonal and Ovarian Reserve Changes\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe assessed FSH, AMH, and AFC levels in patients over six months following the initial administration of G-CSF. Unfortunately, two of the eleven participants withdrew from the study after the third month.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFSH Levels\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRegarding FSH levels over time, the mean FSH level decreased from a baseline of 54.26 ± 24.6 IU/L to 33.24 ± 28.01 IU/L four months post-treatment. A paired-samples t-test indicated a significant reduction in FSH levels from baseline to four months (t (9) = -2.511, \u003cem\u003ep\u003c/em\u003e = 0.033).\u003c/p\u003e\n\u003cp\u003eA repeated-measures ANOVA was conducted to evaluate changes in FSH levels over time at three time points: baseline, 4 months, and 6 months (Table 2). Mauchly’s Test was applied to assess the sphericity assumption. The analysis revealed a significant effect of time on FSH levels, F (2, 16) = 4.684, p = 0.025, partial η² = 0.36, indicating that FSH levels decreased significantly across the time points (Figure 1A and 1D).\u003c/p\u003e\n\u003cp\u003ePost hoc pairwise comparisons, adjusted using the Bonferroni correction, showed a significant reduction in FSH levels at 6 months (29.00 ± 24.25) compared to baseline (\u003cem\u003ep\u003c/em\u003e = 0.008). However, no significant differences were observed between baseline and 4 months (\u003cem\u003ep\u003c/em\u003e = 0.181), or between 4 and 6 months (\u003cem\u003ep\u003c/em\u003e = 1.00), suggesting that the significant decrease in FSH levels occurred specifically between baseline and 6 months.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAMH Levels\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMean AMH levels increased from 0.21 ± 0.15 pmol/L at baseline to 0.49 ± 1.13 pmol/L at 4 months post-treatment (Figure 1B and 1E). Despite this apparent rise, the difference was not statistically significant on a paired sample t-test (t(9) = 0.927, \u003cem\u003ep\u003c/em\u003e = 0.376).\u003c/p\u003e\n\u003cp\u003eTo assess changes over time, a repeated-measures ANOVA was conducted. Mauchly’s test indicated a violation of the sphericity assumption (\u003cem\u003ep\u003c/em\u003e = 0.001); therefore, Greenhouse-Geisser correction was applied. The corrected analysis revealed no statistically significant effect of time on AMH levels (F (2,12) = [value missing], \u003cem\u003ep\u003c/em\u003e = 0.362, partial η² = 0.140), suggesting a small effect size.\u003c/p\u003e\n\u003cp\u003eBonferroni-adjusted post hoc comparisons further confirmed that changes from baseline to 4 months (\u003cem\u003ep\u003c/em\u003e = 0.879) and from baseline to 6 months (\u003cem\u003ep\u003c/em\u003e = 1.000) were not statistically significant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAFC (Antral Follicle Count)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mean antral follicle count (AFC) showed a slight increase from baseline to 6-month follow-up (Figure 1C and 1F). At baseline, the mean AFC was 1.09 ± 1.0. This increased to 2.1 ± 2.6 at 4 months and declined to 1.2 ± 2.3 at 6 months post-treatment. Despite the observed numerical increase at 4 months, the differences were not statistically significant based on paired sample t-tests (t (9) = 1.636, \u003cem\u003ep\u003c/em\u003e = 0.133; and t(9) = 0.258, \u003cem\u003ep\u003c/em\u003e = 0.803, respectively).\u003c/p\u003e\n\u003cp\u003eTo evaluate overall change over time, a repeated-measures ANOVA was conducted, which also demonstrated no significant effect of time on AFC (F (2,16) = 0.720, \u003cem\u003ep\u003c/em\u003e = 0.502, partial η² = 0.083), indicating a small effect size. Post hoc comparisons using Bonferroni correction further confirmed the lack of statistically significant differences between baseline and either follow-up (baseline vs. 4 months: \u003cem\u003ep\u003c/em\u003e = 0.908; baseline vs. 6 months: \u003cem\u003ep\u003c/em\u003e = 1.000).\u003c/p\u003e\n\u003cp\u003eThese findings suggest that the observed variations in AFC over the study period may be due to random fluctuations rather than an actual treatment effect.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMenstruation Resumption\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt baseline, none of the participants reported regular menstrual bleeding; one participant reported irregular bleeding. By 4 months post-treatment, 7 out of 11 participants (63.6%) reported resumption of menstruation. McNemar’s test indicated that this increase was statistically significant (\u003cem\u003ep\u003c/em\u003e = 0.031), suggesting a meaningful improvement in menstrual activity following treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIndividual Patient Response\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmong the 11 patients enrolled, one individual demonstrated a particularly robust clinical and biochemical response following treatment. At baseline, her FSH was 30.1 IU/L, AMH was 0.55 pmol/L, and AFC was 1. By month 4, her FSH had declined to 6.38 IU/L, AMH had increased to 3.89 pmol/L, and AFC rose to 8. She resumed spontaneous menstruation starting the second month post-treatment. Notably, she proceeded to oocyte retrieval, which resulted in the collection of three MII oocytes, representing a significant improvement in ovarian function relative to her baseline status. Because she had no partner, she opted to cryopreserve the 3 mature oocytes.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe present study shares conceptual similarities with a previous trial by Pellicer et al. (10), which investigated G-CSF administration for hematopoietic stem cell mobilization in women with diminished ovarian reserve. Our study, however, focused specifically on women with premature ovarian insufficiency (POI) and employed a different dosing regimen including 300 \u0026micro;g of G-CSF administered daily for four consecutive days per month over three months, with follow-up for six months to one year.\u003c/p\u003e\u003cp\u003eOur data confirm that this G-CSF regimen effectively and consistently mobilized hematopoietic stem cells, as demonstrated by significant and reproducible increases in white blood cell (WBC) counts following each injection cycle. This hematologic response suggests that the intervention is exhibiting successful biological activity. Despite this, no significant changes were observed in AMH or antral follicle count (AFC) in 9 of the 11 participants over the 6- to 12-month follow-up period. Serum FSH levels, however, declined significantly, from 54.3\u0026thinsp;\u0026plusmn;\u0026thinsp;24.6 IU/L at baseline to 33.24\u0026thinsp;\u0026plusmn;\u0026thinsp;28.01 IU/L at month 4 and 29.0\u0026thinsp;\u0026plusmn;\u0026thinsp;24.25 IU/L at month 6, suggesting a partial improvement in the gonadotropic hormonal environment.\u003c/p\u003e\u003cp\u003eOf particular interest, although 10 of 11 participants were amenorrheic at study entry (with one reporting irregular menses), seven women resumed menstrual cyclicity following G-CSF treatment. This occurred despite the absence of significant improvements in AMH or AFC. Although the return of menses may reflect ovarian responsiveness and possible ovulation, serum FSH levels remained above the threshold typically required for controlled ovarian stimulation and follicular recruitment. Thus, the clinical utility of G-CSF for fertility enhancement in this cohort remains limited. Nevertheless, these menstrual changes, along with symptom improvement, suggest that G-CSF may support ovarian or hypothalamic-pituitary-ovarian axis function in a biologically meaningful way, meriting further exploration.\u003c/p\u003e\u003cp\u003eIn contrast to the weak ovarian reserve changes observed in most participants, one patient demonstrated a markedly favourable response, including normalization of FSH, an increase in AFC, resumption of menses, and successful retrieval of three oocytes in an IVF cycle. While this outcome raises the possibility of a true treatment response, spontaneous fluctuations in ovarian function are well-documented in young women with POI. This makes it difficult to attribute such improvement definitively to G-CSF without a control group. Still, her outcome highlights the heterogeneity of POI and suggests that a subset of women might benefit from G-CSF therapy under specific biological conditions.\u003c/p\u003e\u003cp\u003eVarious other experimental therapies for POI have been described in the literature with mixed success. One of the first to be published involved in vitro activation by disrupting the Hippo signalling pathway to stimulate dormant primordial follicles (14). This procedure involved surgical resection of one ovary, mincing the ovarian cortex tissue and treating in vitro with Akt activators. The ovarian tissue fragments were then reimplanted in the pelvis with follicular development in 8/27 patients, and one live baby was born (14). This procedure involves two surgeries and has not been widely adopted in clinical practice.\u003c/p\u003e\u003cp\u003eAnother study in 19 women with POI used laparoscopic ovarian biopsy without chemically induced Akt activation. Resumption of ovarian function occurred in 10 women, and two pregnancies occurred following treatment (15). Patients with the highest ovarian cortex stiffness were most likely to respond to treatment. The authors speculated that the rigidity of the ovarian cortical extracellular matrix limits expansion of the follicle and oocyte maturation, resulting in quiescent follicles. Reduction of ovarian cortex stiffness by mechanical disruption may be the mechanism of action that allows follicle growth (15).\u003c/p\u003e\u003cp\u003eAnother treatment that has been more recently explored is platelet-rich plasma (PRP) injection into the ovaries of women with POI. In one study, 311 women (age 24\u0026ndash;40) diagnosed with POI based on ESHRE criteria underwent intraovarian PRP injection (16). PRP treatment resulted in increased antral follicle count (AFC) and serum anti-mullerian hormone (AMH), while serum follicle-stimulating hormone (FSH) did not change significantly. After PRP injection, 201 women who developed antral follicles attempted IVF, 82 (26.4% of total) developed embryos, and 57 underwent embryo transfer, resulting in 13 pregnancies (22.8% per transfer, 4% of total). In total, of the 311 women treated with PRP, 25 (8.0%) achieved livebirth/sustained implantation (spontaneously or after IVF), while another 25 (8.0%) had cryopreserved embryos. These findings suggest that in women with POI, intraovarian injection of autologous PRP is a potential treatment option.\u003c/p\u003e\u003cp\u003eThe mechanism of action of PRP in POI is unknown. Platelets play a central role in hemostasis. They also contribute to wound healing, mediated by the release of secretory proteins on platelet activation (17). The a granules of platelets contain numerous proteins that may provide an influence on wound healing, including platelet derived growth factor (PDGF- aa, bb, and ab isomers), transforming growth factor-b (TGF-b, b1 and b2 isomers), platelet factor 4, interleuken-1, platelet-derived angiogenesis factor, vascular endothelial growth factor, epidermal growth factor, platelet- derived endothelial growth factor, epithelial cell growth factor, insulin-like growth factor, osteocalcin, osteonectin, fibrinogen, vitronectin, fibronectin, and thrombospondin-1. These secretory proteins comprise growth factors, cytokines and chemokines. It is possible that one, or many of these factors together, could contribute to improvement in ovarian function in women with POI. Another speculation is that intraovarian injection of PRP may disrupt the Hippo pathway similar to mechanical disruption of the ovarian cortex with in vitro activation (2). However, none of the POI treatments, such as surgery to disrupt the Hippo pathway and PRP injections, have undergone controlled studies and pregnancy outcomes with IVF are very poor, less than 5%, consistent with potential spontaneous improvement rather than treatment efficacy.\u003c/p\u003e\u003cp\u003eIn the present study of G-CSF injections, aside from the single participant who showed a notable ovarian response, the remaining 10 patients did not demonstrate clinically meaningful improvements in fertility outcomes. Nevertheless, the resumption of menstrual cycles in 7 of the 11 participants, despite persistently low AMH and AFC, is an encouraging observation.\u003c/p\u003e\u003cp\u003eThe strength of this study is the enrollment of women with POI, rather than DOR as in most of the previous studies of G-CSF. Patients with POI and menopausal symptoms including elevated FSH are a more homogeneous group and less likely to resolve spontaneously whereas patients with DOR may often show fluctuations in ovarian response from month to month making interpretation of any treatment response less convincing. However, the weakness of our study is the small patient population undergoing treatment. We purposely stopped enrollment in the study since our aim was to achieve an IVF cycle for each patient and the results after 11 patients did not support this aim. It is also possible that there could be some spontaneous fluctuation in ovarian activity even in POI patients which can make interpretation of results problematic in the absence of a control group.\u003c/p\u003e\u003cp\u003eIn conclusion, this pilot study demonstrates that G-CSF may be biologically active in women with POI, as evidenced by a significant reduction in FSH levels and the resumption of menstruation in over 60% of participants. These findings suggest a potential modulatory effect on the hypothalamic-pituitary-ovarian axis. However, the lack of significant improvements in AMH, AFC, or fertility outcomes limit the strength of the conclusions. Nonetheless, the favourable safety profile and the robust individual response observed in one participant provide a rationale for larger, controlled studies to evaluate efficacy further and to identify subgroups most likely to benefit.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGCSF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGranulocyte Colony-Stimulating Factor\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePOI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePremature ovarian insufficiency\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eFSH\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003efollicle-stimulating hormone\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAMH\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eanti-M\u0026uuml;llerian hormone\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAFC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eantral follicle count\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDOR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ediminished ovarian response\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eBMDSC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eBone Marrow-derived stem cells\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eASCOT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eautologous stem cell ovarian transplantation\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIVF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eIn vitro Fertilization\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCBC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ecomplete blood count\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eBMI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ebody mass index\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePRP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eplatelet-rich plasma\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cu\u003eEthics Approval and Consent to Participate:\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was reviewed and approved by the Veritas Independent Review Board. Additionally, Health Canada authorized the trial under Part C, Division 5 of the Food and Drug Regulations, with no objection to its initiation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eConsent for publication:\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eAvailability of data and materials:\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003cstrong\u003e\u003cu\u003e\u0026nbsp;\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eCompeting interests:\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eFunding:\u0026nbsp;\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by TRIO Fertility. No external grants or specific funding agencies were involved.\u003c/p\u003e\n\u003cp\u003eAuthors\u0026rsquo; Contribution:\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eYS:\u003c/em\u003e Conceptualization, Methodology, Project administration, Data curation, Formal analysis, Project administration, Software, Visualization, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing\u003cstrong\u003e;\u0026nbsp;\u003c/strong\u003e\u003cem\u003eLD:\u003c/em\u003e Writing \u0026ndash; review \u0026amp; editing; \u003cem\u003eMA:\u003c/em\u003e Investigation, Writing \u0026ndash; review \u0026amp; editing; \u003cem\u003eRC:\u003c/em\u003e Conceptualization, Investigation, Methodology, Funding acquisition, Supervision, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003eWe thank the nursing, lab, and pharmacy teams at TRIO Fertility for their support, and especially Angela Lains, LBT, for her dedicated contributions to the laboratory component of this study. We are also deeply grateful to the patients who participated and made this research possible.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eHansen KR, Knowlton NS, Thyer AC, Charleston JS, Soules MR, Klein NA. A new model of reproductive aging: the decline in ovarian non-growing follicle number from birth to menopause. \u003cem\u003eHum Reprod\u003c/em\u003e 2008;23:699-708.\u003c/li\u003e\n \u003cli\u003eYahyavi Y, Kheradi N, Karimi A, Ebrahimi-Kalan A, Ramezani F, Yousefi S, et al. Novel Advances in Cell-Free Therapy for Premature Ovarian Failure (POF): A Comprehensive Review. \u003cem\u003eAdv Pharm Bull\u003c/em\u003e 2024;14:543-57.\u003c/li\u003e\n \u003cli\u003eThe ESHRE Guideline Group on POI, L. Webber, M. Davies, R. Anderson, J. Bartlett, D. Braat, et al. Vermeulen, ESHRE Guideline: management of women with premature ovarian insufficiency, \u003cem\u003eHum Reprod\u003c/em\u003e 2016;31:926\u0026ndash;37.\u003c/li\u003e\n \u003cli\u003eHershlag A, Schuster MW. Return of fertility after autologous stem cell transplantation. \u003cem\u003eFertil Steril\u0026nbsp;\u003c/em\u003e2002;77(2):419-21.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eMerloni A., Hawley J, Levy WC, Gooley T, Cd B, Deeg HJ, et al. Pregnancies following high-dose cyclophosphamide with or without high- dose busulfan or total-body irradiation and bone marrow transplantation. \u003cem\u003eBlood\u003c/em\u003e 1996;\u003cem\u003e87\u003c/em\u003e, 3045\u0026ndash;52.\u003c/li\u003e\n \u003cli\u003eSalooja N, Szydlo R, Soci\u0026eacute; G, Rio B, Chatterjee RM, Ljungman P, et al. Pregnancy outcomes after peripheral blood or bone marrow transplantation: a retrospective survey. \u003cem\u003eLancet.\u0026nbsp;\u003c/em\u003e2001;28;358:271-6\u003c/li\u003e\n \u003cli\u003eTandulwadkar S, Karthick MS. Combined Use of Autologous Bone Marrow-derived Stem Cells and Platelet-rich Plasma for Ovarian Rejuvenation in Poor Responders. \u003cem\u003eJ Hum Reprod Sci\u003c/em\u003e 2020;13:184-190.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eBuigues A, Marchante M, de Miguel-G\u0026oacute;mez L, Martinez J, Cervell\u0026oacute; I, Pellicer A, et al. Stem cell-secreted factor therapy regenerates the ovarian niche and rescues follicles. \u003cem\u003eAm J Obstet Gynecol\u003c/em\u003e. 2021;225(1):65.e1-14.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eHerraiz S, Romeu M, Buigues A, Mart\u0026iacute;nez S, D\u0026iacute;az-Garc\u0026iacute;a C, G\u0026oacute;mez-Segu\u0026iacute; I, et al. Autologous stem cell ovarian transplantation to increase reproductive potential in patients who are poor responders. \u003cem\u003eFertil Steril\u003c/em\u003e. 2018;110:496-505.e1.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eHerraiz S, Pellicer N, Romeu M, Pellicer A. Treatment potential of bone marrow-derived stem cells in women with diminished ovarian reserves and premature ovarian failure. \u003cem\u003eCurr Opin Obstet Gynecol\u003c/em\u003e. 2019;31:156-62.\u003c/li\u003e\n \u003cli\u003eEl Andaloussi A, Igboeli P, Amer A, Al-Hendy A. Intravenous Infusion of Nucleated Peripheral Blood Cells Restores Fertility in Mice with Chemotherapy-Induced Premature Ovarian Failure. \u003cem\u003eBiomedicines\u003c/em\u003e. 2018;6:93.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eEdessy, M., Hosni, H. N., Shady, Y. A., Waf, Y., Bakr, S., \u0026amp; Kamel, M. M. (2015). Autologous stem cells therapy, The first baby of idiopathic premature ovarian failure. \u003cem\u003eActa Medica International\u003c/em\u003e, \u003cem\u003e3\u003c/em\u003e(1), 19.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eGupta S, Lodha P, Karthick MS, Tandulwadkar SR. Role of Autologous Bone Marrow-Derived Stem Cell Therapy for Follicular Recruitment in Premature Ovarian Insufficiency: Review of Literature and a Case Report of World\u0026apos;s First Baby with Ovarian Autologous Stem Cell Therapy in a Perimenopausal Woman of Age 45 Year. \u003cem\u003eJ Hum Reprod Sci\u003c/em\u003e. 2018;11(2):125-30.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eKawamura K, Cheng Y, Suzuki N, Deguchi M, Sato Y, Takae S, Ho CH, Kawamura N, Tamura M, Hashimoto S, Sugishita Y, Morimoto Y, Hosoi Y, Yoshioka N, Ishizuka B, Hsueh AJ. Hippo signaling disruption and Akt stimulation of ovarian follicles for infertility treatment. Proc Natl Acad Sci U S A. 2013;110:17474-9.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eM\u0026eacute;ndez M, Fabregues F, Ferreri J, Calafell JM, Villarino A, Otero J, Farre R, Carmona F. Biomechanical characteristics of the ovarian cortex in POI patients and functional outcomes after drug-free IVA. \u003cem\u003eJ Assist Reprod Genet\u003c/em\u003e. 2022;39(8):1759-67.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eCakiroglu Y, Saltik A, Yuceturk A, Karaosmanoglu O, Kopuk SY, Scott RT, et al. Effects of intraovarian injection of autologous platelet rich plasma on ovarian reserve and IVF outcome parameters in women with primary ovarian insufficiency. \u003cem\u003eAging (Albany NY)\u003c/em\u003e. 2020;12(11):10211-22.\u003c/li\u003e\n \u003cli\u003ePietrzak WS, Eppley BL. Platelet rich plasma: biology and new technology. \u003cem\u003eJ Craniofac Surg\u003c/em\u003e. 2005;16(6):1043-54.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1: Demographic and clinical characteristics of individual patients.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"784\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.84694%;\"\u003e\n \u003cp\u003eCase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.41837%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge at Baseline\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.9949%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBMI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.41837%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFamily History of POI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.27041%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFSH \u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e (IU/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.27041%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFSH \u003csup\u003eb\u003c/sup\u003e\u003c/strong\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003e(IU/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.69388%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAMH \u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003e(pmol/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.69388%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAMH\u0026nbsp;\u003c/strong\u003e\u003csup\u003eb\u0026nbsp;\u003c/sup\u003e(pmol/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.9949%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAFC \u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.9949%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAFC \u003csup\u003eb\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.1378%;\"\u003e\n \u003cp\u003eMenstruation \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.2653%;\"\u003e\n \u003cp\u003eMenstruation \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.84694%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.41837%;\"\u003e\n \u003cp\u003e34.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.9949%;\"\u003e\n \u003cp\u003e22.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.41837%;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.27041%;\"\u003e\n \u003cp\u003e32.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.27041%;\"\u003e\n \u003cp\u003e28.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.69388%;\"\u003e\n \u003cp\u003e0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.69388%;\"\u003e\n \u003cp\u003e0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.9949%;\"\u003e\n \u003cp\u003e2.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.9949%;\"\u003e\n \u003cp\u003e2.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.1378%;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.2653%;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.84694%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.41837%;\"\u003e\n \u003cp\u003e34.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.9949%;\"\u003e\n \u003cp\u003e26.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.41837%;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.27041%;\"\u003e\n \u003cp\u003e50.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.27041%;\"\u003e\n \u003cp\u003e40.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.69388%;\"\u003e\n \u003cp\u003e0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.69388%;\"\u003e\n \u003cp\u003e0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.9949%;\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.9949%;\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.1378%;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.2653%;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.84694%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.41837%;\"\u003e\n \u003cp\u003e31.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.9949%;\"\u003e\n \u003cp\u003e21.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.41837%;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.27041%;\"\u003e\n \u003cp\u003e48.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.27041%;\"\u003e\n \u003cp\u003e5.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.69388%;\"\u003e\n \u003cp\u003e0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.69388%;\"\u003e\n \u003cp\u003e0.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.9949%;\"\u003e\n \u003cp\u003e3.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.9949%;\"\u003e\n \u003cp\u003e5.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.1378%;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.2653%;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.84694%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.41837%;\"\u003e\n \u003cp\u003e40.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.9949%;\"\u003e\n \u003cp\u003e22.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.41837%;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.27041%;\"\u003e\n \u003cp\u003e84.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.27041%;\"\u003e\n \u003cp\u003e2.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.69388%;\"\u003e\n \u003cp\u003e0.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.69388%;\"\u003e\n \u003cp\u003e0.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.9949%;\"\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.9949%;\"\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.1378%;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.2653%;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.84694%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.41837%;\"\u003e\n \u003cp\u003e38.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.9949%;\"\u003e\n \u003cp\u003e20.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.41837%;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.27041%;\"\u003e\n \u003cp\u003e100.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.27041%;\"\u003e\n \u003cp\u003e74.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.69388%;\"\u003e\n \u003cp\u003e0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.69388%;\"\u003e\n \u003cp\u003e0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.9949%;\"\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.9949%;\"\u003e\n \u003cp\u003e3.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.1378%;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.2653%;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.84694%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.41837%;\"\u003e\n \u003cp\u003e38.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.9949%;\"\u003e\n \u003cp\u003e23.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.41837%;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.27041%;\"\u003e\n \u003cp\u003e81.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.27041%;\"\u003e\n \u003cp\u003e76.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.69388%;\"\u003e\n \u003cp\u003e0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.69388%;\"\u003e\n \u003cp\u003e0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.9949%;\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.9949%;\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.1378%;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.2653%;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.84694%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.41837%;\"\u003e\n \u003cp\u003e38.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.9949%;\"\u003e\n \u003cp\u003e20.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.41837%;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.27041%;\"\u003e\n \u003cp\u003e41.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.27041%;\"\u003e\n \u003cp\u003e40.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.69388%;\"\u003e\n \u003cp\u003e0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.69388%;\"\u003e\n \u003cp\u003e0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.9949%;\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.9949%;\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.1378%;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.2653%;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.84694%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.41837%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e25.00\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.9949%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e21.3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.41837%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.27041%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e30.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.27041%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e6.38\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.69388%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.55\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.69388%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e3.89\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.9949%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.00\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.9949%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e8.00\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.1378%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.2653%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eYes\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.84694%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.41837%;\"\u003e\n \u003cp\u003e32.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.9949%;\"\u003e\n \u003cp\u003e30.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.41837%;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.27041%;\"\u003e\n \u003cp\u003e63.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.27041%;\"\u003e\n \u003cp\u003e63.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.69388%;\"\u003e\n \u003cp\u003e0.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.69388%;\"\u003e\n \u003cp\u003e0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.9949%;\"\u003e\n \u003cp\u003e2.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.9949%;\"\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.1378%;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.2653%;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.84694%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e10\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.41837%;\"\u003e\n \u003cp\u003e40.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.9949%;\"\u003e\n \u003cp\u003e21.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.41837%;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.27041%;\"\u003e\n \u003cp\u003e35.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.27041%;\"\u003e\n \u003cp\u003e7.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.69388%;\"\u003e\n \u003cp\u003e0.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.69388%;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.9949%;\"\u003e\n \u003cp\u003e2.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.9949%;\"\u003e\n \u003cp\u003e4.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.1378%;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.2653%;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.84694%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e11\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.41837%;\"\u003e\n \u003cp\u003e26.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.9949%;\"\u003e\n \u003cp\u003e32.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.41837%;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.27041%;\"\u003e\n \u003cp\u003e30.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7.27041%;\"\u003e\n \u003cp\u003e20.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.69388%;\"\u003e\n \u003cp\u003e0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.69388%;\"\u003e\n \u003cp\u003e0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.9949%;\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.9949%;\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.1378%;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.2653%;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eBMI: Body Mass Index; FSH: Follicle Stimulating Factor; AMH: Anti-Mullerian Hormone; AFC: Antral Follicle Count\u003c/p\u003e\n\u003cp\u003ea: Values at Baseline; b: Values at the 4\u003csup\u003eth\u003c/sup\u003e month post treatment\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. Hormonal and Ovarian Reserve Markers Over Time. Values are reported in mean\u0026nbsp;\u003c/strong\u003e\u0026plusmn; \u003cstrong\u003eSD.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTime Point\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eFSH (IU/L)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAMH (pmol/L)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAFC (n)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eBaseline\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e54.26 \u0026plusmn; 24.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.21 \u0026plusmn; 0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.09 \u0026plusmn; 1.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMonth 2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e42.96 \u0026plusmn; 31.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.35 \u0026plusmn; 0.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.09 \u0026plusmn; 1.22\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMonth 4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e33.24 \u0026plusmn; 28.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.49 \u0026plusmn; 1.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2.18 \u0026plusmn; 2.60\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMonth 6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e29.0 \u0026plusmn; 24.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.37 \u0026plusmn; 0.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.22 \u0026plusmn; 2.33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMonth 12\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e25.50 \u0026plusmn; 12.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.09 \u0026plusmn; 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.75 \u0026plusmn; 0.95\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"reproductive-biology-and-endocrinology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbej","sideBox":"Learn more about [Reproductive Biology and Endocrinology](http://rbej.biomedcentral.com)","snPcode":"12958","submissionUrl":"https://submission.nature.com/new-submission/12958/3","title":"Reproductive Biology and Endocrinology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Primary Ovarian Insufficiency, Granulocyte Colony-Stimulating Factor, Ovarian Reserve, Follicle Stimulating Hormone, Menstruation","lastPublishedDoi":"10.21203/rs.3.rs-7917847/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7917847/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWomen are born with a limited number of eggs, which decline over time. Premature ovarian insufficiency (POI) occurs when this decline happens before age 40, causing infertility. Bone marrow stem cells may help restore ovarian function, as some women conceive after bone marrow transplants. Studies suggest that mobilizing stem cells with Granulocyte Colony-Stimulating Factor (G-CSF) can improve ovarian response in women with diminished ovarian reserve, possibly without needing ovarian infusion. Our study aimed to evaluate if G-CSF injections alone could improve ovarian function in women with POI.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis was a pilot, non-randomized, open-label clinical trial including 11 women aged 25–40 years with clinical POI and menopausal symptoms, defined by elevated follicle-stimulating hormone (FSH) on two occasions, low anti-Müllerian hormone (AMH), and reduced antral follicle count (AFC). Participants received up to three rounds of subcutaneous G-CSF administered daily for four days per month over 60 days. Ovarian reserve markers (FSH, AMH, AFC), menstruation resumption, and menopausal symptoms were assessed at baseline and multiple follow-up points over 12 months.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mean age of participants was 34.1 ± 5.2 years (BMI 23.96 ± 4.0 kg/m²). GCS-F injections resulted in significant increases in white blood cells and mild elevation of liver enzymes which returned to baseline within one month. By four months, significant improvements in menopausal symptoms were reported. Mean FSH decreased from 54.3 ± 24.6 IU/L at baseline to 29.0 ± 8.1 IU/L at six months (p = 0.008). AMH and AFC rose modestly (0.55 ± 0.2 to 3.89 ± 0.5 pmol/L; 1.09 ± 1.0 to 1.2 ± 2.3), though not significantly. Menstruation resumed in 7 of 11 women (63.6%, p = 0.031). One participant showed marked response including retrieval of three mature oocytes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eG-CSF injections were associated with menstrual resumption and symptom relief in most women with POI, suggesting biological activity. Although improvements in ovarian reserve markers were modest and disappointing in terms of the potential for assisted reproduction, these findings may support further evaluation of G-CSF in larger, controlled trials to clarify its clinical benefit and therapeutic potential.\u003c/p\u003e\n\u003cp\u003eTrial registration number: NCT06117982\u003c/p\u003e\n\u003cp\u003e(https://clinicaltrials.gov/study/NCT06117982?cond=The%20Impact%20of%20Granulocyte%20Colony%20Stimulating%20Factor%20on%20Premature%20Ovarian%20Insufficiency\u0026amp;rank=1\u003c/p\u003e","manuscriptTitle":"Granulocyte Colony-Stimulating Factor Treatment in Women with Premature Ovarian Insufficiency: A Pilot Study of Biological Activity and Menstrual Resumption","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-10 09:28:11","doi":"10.21203/rs.3.rs-7917847/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-06T16:39:03+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-05T12:13:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"6582604423134843649213175289700591884","date":"2025-11-04T14:26:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"53832838862794084641105553303939820949","date":"2025-11-03T14:15:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"161081399846673522629113817642350036586","date":"2025-11-02T03:01:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"94224495326516270507754057471244865876","date":"2025-11-01T19:44:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"217920228450683465346225138222481155544","date":"2025-11-01T15:50:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"296284508643137668882032903827702781481","date":"2025-11-01T12:32:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"164613201762328084916150129733783367569","date":"2025-11-01T11:48:52+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-01T01:10:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"133589401493474890374705739195341237319","date":"2025-10-30T16:21:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"105342439048540087000507992132740584409","date":"2025-10-30T15:53:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"209941104799308816590249060153555378313","date":"2025-10-30T14:06:18+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-30T11:12:41+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-23T09:47:58+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-23T09:47:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"Reproductive Biology and Endocrinology","date":"2025-10-21T15:07:04+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"reproductive-biology-and-endocrinology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbej","sideBox":"Learn more about [Reproductive Biology and Endocrinology](http://rbej.biomedcentral.com)","snPcode":"12958","submissionUrl":"https://submission.nature.com/new-submission/12958/3","title":"Reproductive Biology and Endocrinology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"de72a988-b13a-401a-9bce-2578316687b3","owner":[],"postedDate":"November 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-12T16:01:22+00:00","versionOfRecord":{"articleIdentity":"rs-7917847","link":"https://doi.org/10.1186/s12958-025-01510-z","journal":{"identity":"reproductive-biology-and-endocrinology","isVorOnly":false,"title":"Reproductive Biology and Endocrinology"},"publishedOn":"2026-01-07 15:57:28","publishedOnDateReadable":"January 7th, 2026"},"versionCreatedAt":"2025-11-10 09:28:11","video":"","vorDoi":"10.1186/s12958-025-01510-z","vorDoiUrl":"https://doi.org/10.1186/s12958-025-01510-z","workflowStages":[]},"version":"v1","identity":"rs-7917847","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7917847","identity":"rs-7917847","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}