Micro-Trauma Induced Follicular Activation: The Role of Ovarian Pricking in Fertility Enhancement"

Micro-Trauma Induced Follicular Activation: The Role of Ovarian Pricking in Fertility Enhancement" | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Case Report Micro-Trauma Induced Follicular Activation: The Role of Ovarian Pricking in Fertility Enhancement" Dr. Jayesh Amin, Dr. Rudri Agrawal, Dr. Paresh Makwana This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8082011/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Women with diminished ovarian reserve (DOR) represent one of the most challenging groups in assisted reproductive technology (ART), often exhibiting poor response to conventional stimulation protocols and limited success in in vitro fertilization (IVF). Strategies aimed at activating dormant follicles have gained increasing attention, particularly approaches that modulate intra-ovarian signaling pathways such as the Hippo pathway. Ovarian pricking, a minimally invasive technique involving targeted disruption of the ovarian cortex under transvaginal ultrasound guidance, has been hypothesized to stimulate follicular activation and improve oocyte yield. This study aimed to evaluate whether ovarian pricking can enhance follicular recruitment and improve subsequent fertility outcomes in women with DOR who had previously failed IVF cycles. Results This prospective study included 24 infertile women aged 30–35 years with AMH levels < 0.5 ng/mL, reduced antral follicle counts, and ovarian volume of 1.5–2 cm³. Ovarian pricking was performed transvaginally under ultrasound guidance using a standard oocyte retrieval needle, followed by estradiol valerate administration and serial monitoring. Of the 24 patients, 22 (91.7%) demonstrated a follicular response, with a significant increase in the mean number of antral follicles from 0.42 ± 0.50 before pricking to 3.96 ± 1.30 post-procedure (t = − 12.274, p = 0.001). Among the 22 responders, 18 (81.8%) exhibited sustained follicular growth during controlled ovarian stimulation with recombinant FSH plus HMG under an antagonist protocol. Oocyte retrieval was successful in all 18, and 16 (88.9%) yielded mature (MII) oocytes. Blastocyst formation was achieved in 8 of these 16 patients (50%), demonstrating the feasibility of the approach in supporting downstream embryonic development. Four women showed an initial response but failed to progress to stimulation. Conclusions Ovarian pricking appears to be a novel and promising adjunct for the management of women with DOR, offering a minimally invasive means of activating dormant follicles and improving follicular recruitment, oocyte maturation, and blastocyst development. The high response rate observed in this study supports its potential clinical utility, possibly through modulation of the Hippo signalling pathway. However, outcomes varied among patients, highlighting the need for individualized treatment strategies. Larger, long-term prospective studies are warranted to validate efficacy, optimize protocols, and define predictors of success, as well as to assess the impact on live birth outcomes. Sexual & Reproductive Medicine Diminished Ovarian Reserve Follicular Activation Fertility Enhancement Ovarian Pricking Ovarian Stimulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Research Highlights Ovarian pricking triggered follicular response in 91.7% of DOR patients. Ovarian stimulation succeeded in 81.8% with 88.9% MII retrieval. Blastocyst formation occurred in 50% of MII cases. Technique may activate dormant follicles via Hippo pathway. Background Diminished ovarian reserve (DOR) presents a major challenge in assisted reproductive technology (ART), particularly for women desiring to conceive using their own gametes. Recent advances have shifted focus toward strategies that awaken dormant follicles within the ovary, such as mechanical disruption of the ovarian cortex to modulate intra-ovarian pathways—most notably, the Hippo signaling pathway. Ovarian pricking has emerged as a potential technique to activate primordial follicles and enhance folliculogenesis. This study evaluates the feasibility, efficacy, and safety of ovarian pricking in women with low ovarian reserve. The development of ovarian follicles is a highly regulated process that involves the coordinated action of various signaling pathways. At birth, a finite pool of primordial follicles is established, which remains dormant for years. However, only a small fraction of these follicles are activated to mature and ovulate, while the majority undergo atresia. During follicular activation, granulosa cells undergo a notable transformation from a flattened to a cuboidal shape, coinciding with an expansion in oocyte volume, signaling the onset of follicular growth. Recent studies have shed light on the critical role of mechanical signaling in regulating the activation of primordial follicles. Specifically, the Hippo and Akt signaling pathways have been identified as key regulators of follicular development. The Hippo pathway, known for its role in controlling organ size, is mechanoresponsive and plays a crucial role in maintaining the quiescence of primordial follicles. In contrast, the Akt pathway promotes follicular growth and survival. Mechanical factors, such as ovarian rigidity, also influence follicular development by modulating the activity of these pathways. Understanding the complex interplay between mechanical signaling pathways and follicular development can provide valuable insights into the mechanisms governing female reproductive health and may lead to the development of new therapeutic strategies for reproductive disorders. (Shah JS et al,2018). The quiescence of primordial follicles is regulated by a delicate balance between Hippo and Akt signaling pathways. The Hippo pathway acts as a brake on follicular activation by responding to mechanical cues from the microenvironment. When cells sense increased stiffness, it triggers a signaling cascade involving Rho GTPase and ROCK ( Rho-associated protein kinase) which phosphorylate Yes-associated protein ( YAP ) and transcriptional coactivator with PDZ-binding motif (TAZ), preventing them from entering the nucleus and promoting growth. Conversely, the Akt pathway promotes follicular activation, while phosphatase and tensin homolog (PTEN) counteracts this effect by dephosphorylating phosphatidylinositol-3,4,5-triphosphate (PIP3), allowing forkhead box O3 (FOXO3) to maintain follicular quiescence. However, when a follicle is subjected to mechanical forces, such as fragmentation or mechanical injury, this balance is disrupted, triggering a cascade of events that ultimately lead to its activation. The mechanical stress disrupts the Hippo pathway through Rho GTPase and ROCK, resulting in the dephosphorylation of YAP and TAZ, which then translocate to the nucleus. Concurrently, the Akt pathway is activated through the phosphorylation of phosphatidylinositol-4,5-bisphosphate (PIP2) to PIP3, allowing Akt (protein kinase B) to phosphorylate and export FOXO3 from the nucleus to the cytoplasm. With FOXO3 no longer inhibiting growth, YAP/TAZ bind to transcriptional enhanced associate domain (TEAD), promoting the expression of growth factors like CCN [cysteine-rich protein 61 (CYR61/CCN1), connective tissue growth factor (CTGF/CCN2), and nephroblastoma overexpressed (NOV/CCN3)] and baculoviral inhibitors of apoptosis repeat containing (BIRC) proteins, which drive follicular activation and cell growth. Research suggests that mechanical stress can activate follicle growth by disrupting the ovarian Hippo signaling pathway. The process begins with mechanical stress but is sustained by localized biochemical changes that drive follicular development ( Fig. 1 and Fig. 2 ) Method Study Design and Participants - This was a prospective study conducted at a tertiary fertility center from January 2025 to July 2025. The case series included twenty-four women aged 30–35 years with infertility, anti-Müllerian hormone (AMH) levels < 0.5 ng/mL, and low ovarian volume (1.5–2 cm³). All participants had one or no visible follicles on transvaginal ultrasound (TVS). In addition, all patients had a history of at least three previous failed IVF attempts or demonstrated hyporesponse/no response in prior IVF cycles attempted elsewhere. ( Fig. 3) Procedure Under total intravenous anesthesia and TVS guidance, ovarian pricking was performed via the vaginal route using an oocyte retrieval needle. Post-procedure, patients were prescribed estradiol valerate 2 mg once or twice daily. Monitoring and Stimulation TVS was repeated every 10 days for a maximum of 30 days. Response was defined as an increase in visible follicles compared to baseline. Responders underwent controlled ovarian stimulation using recombinant FSH (rFSH) and human menopausal gonadotropin (HMG), followed by antagonist protocol and dual trigger. Outcomes Primary outcomes included follicular response, number of oocytes retrieved, and number of MII oocytes. Secondary outcome included blastocyst formation. Results Of the 24 patients, 22 (91.7%) exhibited a positive response to pricking and underwent ovarian stimulation. Among these 22, 18 (81.8%) patients responded to the ovarian stimulation, and remaining 4 (18.2%) showed initial response to stimulation but follicular growth was not seen during subsequent stimulation. Oocyte retrieval was done in all 18 patients who responded to ovarian stimulation. Mature (MII) oocytes were retrieved in 16 of these 18 patients (88.9%). Among16 patients in whom mature oocytes were retrieved, blastocysts were formed in 8 (50%) patients. (Table 1 ) ( Fig. 4) The mean number of follicles increased markedly from 0.42 ± 0.50 pre-pricking to 3.96 ± 1.30 post-pricking. This difference was statistically significant ( t = − 12.274, df = 23, p = 0.001* ), indicating that pricking led to a significant increase in the number of follicles. (Table 2) Table 1 Patient outcomes following ovarian pricking and subsequent ovarian stimulation, oocyte retrieval, and blastocyst formation. Patient Outcome Number of Patients Percentage (%) Total patients 24 100.0 No response to pricking 2 8.3% Responded to pricking/ ovarian stimulation started 22 91.7 Initial response to stimulation but no further growth 4 18.2 Responded to stimulation and OPU was done 18 81.8% Mature oocytes retrieved 16 88.9 Blastocysts formed 8 50.0 (Data are presented as number of patients (n) and percentage (%). Percentages are calculated relative to the corresponding denominator at each step.) Table-2, Comparison of number of follicles before and after pricking (Paired ‘t’ test applied) Parameter Mean ± SD ‘t’ value, df P value CI (95%) Pre-pricking 0.42 ± 0.50 -12.274, df = 23 0.001* -4.14 to -2.94 Post-pricking 3.96 ± 1.30 Data are expressed as mean ± standard deviation (SD). A paired t-test was applied to compare pre- and post-pricking follicle counts. Paired t-test demonstrated a significant increase in follicle numbers post-pricking compared with pre-pricking (t = -12.274, df = 23, p = 0.001, 95% CI: -4.14 to -2.94). CI = Confidence Interval; df = degrees of freedom. Discussion This prospective study explored the utility of ovarian pricking as a minimally invasive technique to activate dormant ovarian follicles in women with diminished ovarian reserve (DOR). With 81.8% (18/22) follicular response rate and 88.9% (16/18) mature oocyte retrieval rate among responders, our results demonstrate that ovarian pricking may enhance folliculogenesis in a clinically significant subset of low reserve patients. This aligns with evolving concepts in ovarian biology, particularly the mechanical disruption of the Hippo signaling pathway to promote follicular activation. Historically, ovarian stimulation strategies have primarily focused on exogenous gonadotropin administration or adjuvants such as DHEA, CoQ10, or GH to enhance ovarian response. However, these options often show limited benefit in women with advanced follicular depletion. The idea of mechanical disruption to activate quiescent follicles originates from animal studies. Early murine models demonstrated that ovarian drilling or fragmentation could upregulate the PI3K/Akt pathway, facilitating the activation of dormant primordial follicles (Li et al., 2010). Subsequently, built on this concept; Kawamura et al. (2013) introduced the concept of in vitro activation (IVA) using ovarian tissue fragmentation and AKT stimulation. While effective, the need for surgery and in vitro manipulation made it less feasible. Siristatidis et al in 2014 , hypothesised that transvaginal oocyte trauma may be a promising approach to enhance IVF outcomes in poor responders. As this innovative technique remains unexplored in humans, further investigation is necessary to determine its efficacy and safety. Key considerations, such as ovarian volume requirements, threshold values for biomarkers like antral follicle count (AFC), anti-Müllerian hormone (AMH), and follicle-stimulating hormone (FSH), number of prior IVF attempts, and patient age, need to be established to guide the application of this modality. kawamura et al. (2016) observed that mechanical injury, such as ovarian drilling, alters local growth factor expression, PTEN and FOXO3a, potentially facilitating follicular activation. Historically, ovarian drilling via laparoscopy was employed to treat anovulatory PCOS, leveraging stromal destruction to reduce androgen production and promote follicular recruitment. Though effective, this approach was invasive and not tailored to DOR patients. Rather it was associated with complications like decreased ovarian reserve and other complications inherent to nature of surgery. Unlike ovarian drilling, ovarian pricking via transvaginal route under ultrasound guidance, as performed in our study, is less invasive, non-damaging to ovary and allows for real-time assessment, associated with lower morbidity, and allows repeatability. Mechanistic Insights and Molecular Basis The scientific foundation of our approach parallels that of Kawamura et al. (2013), who proposed that mechanical fragmentation of ovarian tissue could disrupt the Hippo signaling cascade, leading to upregulation of growth factors (YAP, CCN2, and BIRC5) and follicular activation. Their in vitro activation (IVA) protocol, though promising, required laparoscopic removal and reimplantation of ovarian cortex, making it less accessible. In contrast, our technique relies on in situ activation, avoiding surgical excision. Similarly, Zhao et al. (2018) demonstrated that mechanical ovarian disruption in mice led to primordial follicle activation and progression to the antral stage. Translating this concept into clinical practice, ovarian pricking offers a non-excisional approach that could induce a comparable effect. Ernst et al. (2017) performed transcriptomic profiling of human oocytes from primordial and primary follicles and revealed distinct molecular signatures associated with dormancy and activation. Dormant oocytes exhibited enrichment of genes involved in transcriptional repression, chromatin remodeling, and cell cycle inhibition, whereas primary follicle oocytes demonstrated upregulation of genes related to metabolic activity, RNA processing, and translational machinery. Crucially, pathways such as PI3K-AKT, TGF-β, and FOXO3 were implicated in the transition from dormancy to growth, underscoring a tightly regulated oocyte-intrinsic mechanism for follicular activation. These findings highlight the biological significance of awakening dormant follicles, a process that holds potential for therapeutic manipulation in infertility treatment. In line with this concept, our study explored the mechanical disruption of follicular dormancy via ovarian pricking, aiming to stimulate the intrinsic activation pathways described by Ernst et al. The parallel between their molecular insights and our interventional approach supports the rationale for mechanically-induced follicular recruitment as a viable fertility-enhancing strategy in women with diminished ovarian reserve. In line with Hsueh (2020) , our study underscores the pivotal role of Hippo signaling disruption via mechanical stress in activating dormant follicles. The review details how ovarian fragmentation enhances actin polymerization, fostering YAP nuclear translocation, and upregulating CCN and BIRC growth and survival factors, thereby facilitating follicle growth. Clinical protocols leveraging this mechanism—such as drug-free IVA, combination IVA with Akt stimulators, and in vivo mechanical incision techniques—have shown promising outcomes in POI, DOR, and resistant ovary patients. Hsueh further highlights translational opportunities, suggesting refinement of mechanical approaches and localized actin-enhancing interventions to improve efficacy while mitigating invasiveness and tissue damage While promising, these biochemical activators are not widely approved or available. In contrast, our approach uses mechanical intervention alone, making it more accessible, safer, and cost-effective in routine practice. Zhao et al. (2021) comprehensively reviewed the molecular circuitry controlling mammalian primordial follicle activation, identifying the PI3K/PTEN/Akt/FOXO3 axis as the core regulatory pathway within oocytes. Under physiologic conditions, PTEN maintains quiescence by restricting PIP3 accumulation; activation of PI3K initiates downstream Akt phosphorylation and FOXO3 nuclear export, committing follicles to growth. In pre-granulosa cells, mTORC1 signaling functions upstream of PI3K/Akt, with crosstalk from MAPK3/1 and potential modulation by DNA damage response pathways. These insights have informed the development of in vitro activation (IVA) protocols, which combine mechanical disruption (e.g. ovarian fragmentation) with pharmacologic stimulation of PI3K/mTOR to awaken dormant follicles. The authors note that while such approaches have yielded promising results in premature ovarian insufficiency and poor ovarian responders, outcomes vary and excessive PTEN inhibition may paradoxically reduce efficacy, underscoring the need for individualized modulation. Mechanistically, these findings support the rationale for ovarian pricking as a potential in vivo analogue of IVA, wherein local stromal disruption could relieve Hippo pathway constraints and secondarily engage the PI3K/mTOR signaling cascade to promote follicular recruitment. Pellicer et al ( 2023) concluded that In cases of irreversible damage, such as premature ovarian insufficiency (POI) or diminished ovarian reserve (DOR), follicle reactivation has shown encouraging results in experimental settings. However, robust clinical evidence is limited, although ongoing research is yielding more promising data. Interpretation of non-responders- Despite overall favorable results in our study, a few patients did not develop mature oocytes or blastocysts, suggesting that not all dormant follicles are functionally competent. Factors such as fibrosis, vascular compromise, or intrinsic oocyte quality may influence this outcome. Additionally, genetic and epigenetic programming of early follicles may limit response to mechanical signals. This highlights the need for personalized protocols and predictive biomarkers, such as ovarian tissue transcriptomics or real-time follicular fluid profiling. Clinical Implications and Future Directions This study reinforces the concept that ovarian tissue retains latent regenerative potential, even in women classified as having DOR. Given the non-invasive nature of ovarian pricking, it could be considered prior to stimulation in patients with low AMH or antral follicle count, especially those seeking to attempt IVF with autologous oocytes. To strengthen clinical application, future trials should address: Comparative effectiveness versus conventional stimulation alone. Live birth outcomes, perinatal safety, and long-term offspring health. Molecular assessment of pricking-induced gene expression changes. Integration with adjunct therapies such as PRP or stem cell infusion. Limitations This study has limitations including a small sample size and absence of a control group. Pregnancy and live birth rates were not measured. Furthermore, no molecular assessments were conducted to confirm mechanistic pathways such as Hippo signaling modulation. Variability in individual ovarian biology could have influenced outcomes. Larger randomized controlled trials are needed to validate these preliminary findings. Conclusion Ovarian pricking appears to be a promising, low-cost, and minimally invasive method to activate dormant follicles in women with diminished ovarian reserve. The technique yielded favorable outcomes in terms of mature oocyte retrieval and blastocyst formation. With further validation, ovarian pricking may become a valuable adjunct in the management of poor responders in IVF cycles. Declarations Ethics approval and consent to participate This study was conducted in accordance with the ethical principles outlined in the Declaration of Helsinki (2013 revision). The study protocol was reviewed and approved by the Institutional Ethics Committee of WINGS IVF Academy with approval number 2025/006/34D. Written informed consent was obtained from all participants prior to enrollment. Consent for publication All participants provided written consent for publication of anonymized clinical data. Availability of data and materials The datasets generated and analyzed 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 did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Authors’ contributions Dr. Jayesh Amin- Conceptualization, Methodology, Supervision, Validation, Visualization, Supervision, Validation, Visualization. Dr. Rudri Agrawal- Formal analysis, Investigation, Project administration, Writing – original draft, Writing – review & editing, Data curation Dr. Paresh Makwana- Data curation, Investigation All authors read and approved the final manuscript. Acknowledgements The authors thank the clinical and embryology team of NOVA WINGS IVF for their assistance in patient care and laboratory support. The authors are also grateful to the participating women for their cooperation. References Shah JS, Sabouni R, Cayton Vaught KC, Owen CM, Albertini DF, Segars JH (2018) Biomechanics and mechanical signaling in the ovary: a systematic review. J Assist Reprod Genet 35:1135–1148. https://doi.org/10.1007/s10815-018-1180-y Li J, Kawamura K, Cheng Y, Liu S, Klein C, Liu S, Duan EK, Hsueh AJ (2010) Activation of dormant ovarian follicles to generate mature eggs. Proc Natl Acad Sci U S A 107:10280–10284. https://doi.org/10.1073/pnas.1001198107 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 (2013) Hippo signaling disruption and Akt stimulation of ovarian follicles for infertility treatment. Proc Natl Acad Sci U S A 110:17474–17479. https://doi.org/10.1073/pnas.1312830110 Siristatidis C, Vogiatzi P, Bettocchi S, Basios G, Mastorakos G, Vrachnis N (2014) Transvaginal ovarian trauma, poor responders and improvement of success rates in IVF: Anecdotal data and a hypothesis. Med Hypotheses 83:227–231. https://doi.org/10.1016/j.mehy.2014.04.022 Kawamura K, Kawamura N, Hsueh AJ (2016) Activation of dormant follicles: a new treatment for premature ovarian failure? Curr Opin Obstet Gynecol 28:217–222. https://doi.org/10.1097/GCO.0000000000000268 Zhao Y, Zhang Y, Li J, Zheng N, Xu X, Yang J, Xia G, Zhang M (2018) MAPK3/1 participates in the activation of primordial follicles through mTORC1-KITL signaling. J Cell Physiol 233:226–237. https://doi.org/10.1002/jcp.25868 Ernst EH, Grøndahl ML, Grund S, Hardy K, Heuck A, Sunde L, Franks S, Andersen CY, Villesen P, Lykke-Hartmann K (2017) Dormancy and activation of human oocytes from primordial and primary follicles: molecular clues to oocyte regulation. Hum Reprod 32:1684–1700. https://doi.org/10.1093/humrep/dex238 Hsueh AJW, Kawamura K (2020) Hippo signaling disruption and ovarian follicle activation in infertile patients. Fertil Steril 114:458–464. https://doi.org/10.1016/j.fertnstert.2020.07.031 Zhao Y, Feng H, Zhang Y, Zhang JV, Wang X, Liu D, Wang T, Li RHW, Ng EHY, Yeung WSB et al (2021) Current understandings of core pathways for the activation of mammalian primordial follicles. Cells 10:1491. https://doi.org/10.3390/cells10061491 Pellicer N, Cozzolino M, Diaz-García C, Galliano D, Cobo A, Pellicer A, Herraiz S (2023) Ovarian rescue in women with premature ovarian insufficiency: facts and fiction. Reprod Biomed Online 46:543–565. https://doi.org/10.1016/j.rbmo.2022.12.011 Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 1 posted 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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10:21:03","extension":"html","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":65834,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8082011/v1/c24ab21661f32f9f3e081571.html"},{"id":95795680,"identity":"a845e598-14a4-406f-8de8-872c54b55265","added_by":"auto","created_at":"2025-11-13 07:44:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":161282,"visible":true,"origin":"","legend":"\u003cp\u003eDormant Follicle\u003c/p\u003e\n\u003cp\u003eFIG.1 - Regulation of Quiescent phase of primordial follicle.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8082011/v1/294470c5c1afa294aeae28cd.png"},{"id":95795681,"identity":"cf44c583-93e6-45c9-a724-8fe72e7c7db4","added_by":"auto","created_at":"2025-11-13 07:44:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":168943,"visible":true,"origin":"","legend":"\u003cp\u003eActivated Follicle\u003c/p\u003e\n\u003cp\u003eFIG-2- Pathway depicting the signalling for Follicular activation\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8082011/v1/47d584369a111335b34b9244.png"},{"id":95818652,"identity":"74fb3606-9312-422d-b4e1-3ec560f020ac","added_by":"auto","created_at":"2025-11-13 10:21:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":178078,"visible":true,"origin":"","legend":"\u003cp\u003eSelection of study participants\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8082011/v1/7cc7ffffc11cc1a7b957e31b.png"},{"id":95818649,"identity":"2744d106-f068-4d13-bcb8-0a0f4ee5b515","added_by":"auto","created_at":"2025-11-13 10:21:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":452568,"visible":true,"origin":"","legend":"\u003cp\u003eResponse to ovarian pricking\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8082011/v1/577e8804e5cbf5afc99d0ed0.png"},{"id":96253689,"identity":"b2e98d0f-c942-41f4-84ac-92eb1e2cf5ad","added_by":"auto","created_at":"2025-11-19 07:45:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1524130,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8082011/v1/b89e87b0-bce4-433a-9325-6b23cc321d6c.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eMicro-Trauma Induced Follicular Activation: The Role of Ovarian Pricking in Fertility Enhancement\"\u003c/p\u003e","fulltext":[{"header":"Research Highlights","content":"\u003cul\u003e\n \u003cli\u003eOvarian pricking triggered follicular response in 91.7% of DOR patients.\u003c/li\u003e\n \u003cli\u003eOvarian stimulation succeeded in 81.8% with 88.9% MII retrieval.\u003c/li\u003e\n \u003cli\u003eBlastocyst formation occurred in 50% of MII cases.\u003c/li\u003e\n \u003cli\u003eTechnique may activate dormant follicles via Hippo pathway.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Background","content":"\u003cp\u003eDiminished ovarian reserve (DOR) presents a major challenge in assisted reproductive technology (ART), particularly for women desiring to conceive using their own gametes. Recent advances have shifted focus toward strategies that awaken dormant follicles within the ovary, such as mechanical disruption of the ovarian cortex to modulate intra-ovarian pathways\u0026mdash;most notably, the Hippo signaling pathway.\u003c/p\u003e\u003cp\u003eOvarian pricking has emerged as a potential technique to activate primordial follicles and enhance folliculogenesis. This study evaluates the feasibility, efficacy, and safety of ovarian pricking in women with low ovarian reserve. The development of ovarian follicles is a highly regulated process that involves the coordinated action of various signaling pathways. At birth, a finite pool of primordial follicles is established, which remains dormant for years. However, only a small fraction of these follicles are activated to mature and ovulate, while the majority undergo atresia. During follicular activation, granulosa cells undergo a notable transformation from a flattened to a cuboidal shape, coinciding with an expansion in oocyte volume, signaling the onset of follicular growth.\u003c/p\u003e\u003cp\u003eRecent studies have shed light on the critical role of mechanical signaling in regulating the activation of primordial follicles. Specifically, the Hippo and Akt signaling pathways have been identified as key regulators of follicular development. The Hippo pathway, known for its role in controlling organ size, is mechanoresponsive and plays a crucial role in maintaining the quiescence of primordial follicles. In contrast, the Akt pathway promotes follicular growth and survival. Mechanical factors, such as ovarian rigidity, also influence follicular development by modulating the activity of these pathways. Understanding the complex interplay between mechanical signaling pathways and follicular development can provide valuable insights into the mechanisms governing female reproductive health and may lead to the development of new therapeutic strategies for reproductive disorders. \u003cb\u003e(Shah JS et al,2018).\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe quiescence of primordial follicles is regulated by a delicate balance between Hippo and Akt signaling pathways. The Hippo pathway acts as a brake on follicular activation by responding to mechanical cues from the microenvironment. When cells sense increased stiffness, it triggers a signaling cascade involving Rho GTPase and ROCK ( Rho-associated protein kinase) which phosphorylate Yes-associated protein ( YAP ) and transcriptional coactivator with PDZ-binding motif (TAZ), preventing them from entering the nucleus and promoting growth. Conversely, the Akt pathway promotes follicular activation, while phosphatase and tensin homolog (PTEN) counteracts this effect by dephosphorylating phosphatidylinositol-3,4,5-triphosphate (PIP3), allowing forkhead box O3 (FOXO3) to maintain follicular quiescence.\u003c/p\u003e\u003cp\u003eHowever, when a follicle is subjected to mechanical forces, such as fragmentation or mechanical injury, this balance is disrupted, triggering a cascade of events that ultimately lead to its activation. The mechanical stress disrupts the Hippo pathway through Rho GTPase and ROCK, resulting in the dephosphorylation of YAP and TAZ, which then translocate to the nucleus. Concurrently, the Akt pathway is activated through the phosphorylation of phosphatidylinositol-4,5-bisphosphate (PIP2) to PIP3, allowing Akt (protein kinase B) to phosphorylate and export FOXO3 from the nucleus to the cytoplasm. With FOXO3 no longer inhibiting growth, YAP/TAZ bind to transcriptional enhanced associate domain (TEAD), promoting the expression of growth factors like CCN [cysteine-rich protein 61 (CYR61/CCN1), connective tissue growth factor (CTGF/CCN2), and nephroblastoma overexpressed (NOV/CCN3)] and baculoviral inhibitors of apoptosis repeat containing (BIRC) proteins, which drive follicular activation and cell growth. Research suggests that mechanical stress can activate follicle growth by disrupting the ovarian Hippo signaling pathway. The process begins with mechanical stress but is sustained by localized biochemical changes that drive follicular development ( Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e"},{"header":"Method","content":"\u003cp\u003e\u003cstrong\u003eStudy Design and Participants\u003c/strong\u003e-\u003c/p\u003e\n\u003cp\u003eThis was a prospective study conducted at a tertiary fertility center from January 2025 to July 2025. The case series included twenty-four women aged 30\u0026ndash;35 years with infertility, anti-M\u0026uuml;llerian hormone (AMH) levels\u0026thinsp;\u0026lt;\u0026thinsp;0.5 ng/mL, and low ovarian volume (1.5\u0026ndash;2 cm\u0026sup3;). All participants had one or no visible follicles on transvaginal ultrasound (TVS). In addition, all patients had a history of at least three previous failed IVF attempts or demonstrated hyporesponse/no response in prior IVF cycles attempted elsewhere. ( Fig.\u0026nbsp;3)\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eProcedure\u003c/h2\u003e\n\u003cp\u003eUnder total intravenous anesthesia and TVS guidance, ovarian pricking was performed via the vaginal route using an oocyte retrieval needle. Post-procedure, patients were prescribed estradiol valerate 2 mg once or twice daily.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eMonitoring and Stimulation\u003c/h3\u003e\n\u003cp\u003eTVS was repeated every 10 days for a maximum of 30 days. Response was defined as an increase in visible follicles compared to baseline. Responders underwent controlled ovarian stimulation using recombinant FSH (rFSH) and human menopausal gonadotropin (HMG), followed by antagonist protocol and dual trigger.\u003c/p\u003e\n\u003ch3\u003eOutcomes\u003c/h3\u003e\n\u003cp\u003ePrimary outcomes included follicular response, number of oocytes retrieved, and number of MII oocytes. Secondary outcome included blastocyst formation.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eOf the 24 patients, 22 (91.7%) exhibited a positive response to pricking and underwent ovarian stimulation. Among these 22, 18 (81.8%) patients responded to the ovarian stimulation, and remaining 4 (18.2%) showed initial response to stimulation but follicular growth was not seen during subsequent stimulation. Oocyte retrieval was done in all 18 patients who responded to ovarian stimulation. Mature (MII) oocytes were retrieved in 16 of these 18 patients (88.9%). Among16 patients in whom mature oocytes were retrieved, blastocysts were formed in 8 (50%) patients. (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) ( Fig.\u0026nbsp;4)\u003c/p\u003e\n\u003cp\u003eThe mean number of follicles increased markedly from \u003cstrong\u003e0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003c/strong\u003e pre-pricking to \u003cstrong\u003e3.96\u0026thinsp;\u0026plusmn;\u0026thinsp;1.30\u003c/strong\u003e post-pricking. This difference was statistically significant (\u003cstrong\u003et\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;12.274, df\u0026thinsp;=\u0026thinsp;23, p\u0026thinsp;=\u0026thinsp;0.001*\u003c/strong\u003e), indicating that pricking led to a significant increase in the number of follicles. (Table\u0026nbsp;2)\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003ePatient outcomes following ovarian pricking and subsequent ovarian stimulation, oocyte retrieval, and blastocyst formation.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ePatient Outcome\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eNumber of Patients\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ePercentage (%)\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTotal patients\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e24\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e100.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNo response to pricking\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e8.3%\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eResponded to pricking/ ovarian stimulation started\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e22\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e91.7\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eInitial response to stimulation but no further growth\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e18.2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eResponded to stimulation and OPU was done\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e81.8%\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMature oocytes retrieved\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e88.9\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBlastocysts formed\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e50.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e(Data are presented as number of patients (n) and percentage (%). Percentages are calculated relative to the corresponding denominator at each step.)\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u003cstrong\u003eTable-2, Comparison of number of follicles before and after pricking\u003c/strong\u003e \u003cstrong\u003e(Paired \u0026lsquo;t\u0026rsquo; test applied)\u003c/strong\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Taba\" border=\"1\"\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eParameter\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u0026lsquo;t\u0026rsquo; value, df\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eP value\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCI (95%)\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePre-pricking\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e-12.274, df\u0026thinsp;=\u0026thinsp;23\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.001*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e-4.14 to -2.94\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePost-pricking\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e3.96\u0026thinsp;\u0026plusmn;\u0026thinsp;1.30\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eData are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). A paired t-test was applied to compare pre- and post-pricking follicle counts. Paired t-test demonstrated a significant increase in follicle numbers post-pricking compared with pre-pricking (t = -12.274, df\u0026thinsp;=\u0026thinsp;23, p\u0026thinsp;=\u0026thinsp;0.001, 95% CI: -4.14 to -2.94). CI\u0026thinsp;=\u0026thinsp;Confidence Interval; df\u0026thinsp;=\u0026thinsp;degrees of freedom.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis prospective study explored the utility of ovarian pricking as a minimally invasive technique to activate dormant ovarian follicles in women with diminished ovarian reserve (DOR). With 81.8% (18/22) follicular response rate and 88.9% (16/18) mature oocyte retrieval rate among responders, our results demonstrate that ovarian pricking may enhance folliculogenesis in a clinically significant subset of low reserve patients. This aligns with evolving concepts in ovarian biology, particularly the mechanical disruption of the Hippo signaling pathway to promote follicular activation.\u003c/p\u003e\n\u003cp\u003eHistorically, ovarian stimulation strategies have primarily focused on exogenous gonadotropin administration or adjuvants such as DHEA, CoQ10, or GH to enhance ovarian response. However, these options often show limited benefit in women with advanced follicular depletion.\u003c/p\u003e\n\u003cp\u003eThe idea of mechanical disruption to activate quiescent follicles originates from animal studies. Early murine models demonstrated that ovarian drilling or fragmentation could upregulate the PI3K/Akt pathway, facilitating the activation of dormant primordial follicles \u003cstrong\u003e(Li et al., 2010).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSubsequently, built on this concept; \u003cstrong\u003eKawamura et al. (2013)\u003c/strong\u003e introduced the concept of in vitro activation (IVA) using ovarian tissue fragmentation and AKT stimulation. While effective, the need for surgery and in vitro manipulation made it less feasible.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSiristatidis et al in 2014\u003c/strong\u003e, hypothesised that transvaginal oocyte trauma may be a promising approach to enhance IVF outcomes in poor responders. As this innovative technique remains unexplored in humans, further investigation is necessary to determine its efficacy and safety. Key considerations, such as ovarian volume requirements, threshold values for biomarkers like antral follicle count (AFC), anti-M\u0026uuml;llerian hormone (AMH), and follicle-stimulating hormone (FSH), number of prior IVF attempts, and patient age, need to be established to guide the application of this modality.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ekawamura et al. (2016)\u003c/strong\u003e observed that mechanical injury, such as ovarian drilling, alters local growth factor expression, PTEN and FOXO3a, potentially facilitating follicular activation.\u003c/p\u003e\n\u003cp\u003eHistorically, ovarian drilling via laparoscopy was employed to treat anovulatory PCOS, leveraging stromal destruction to reduce androgen production and promote follicular recruitment. Though effective, this approach was invasive and not tailored to DOR patients. Rather it was associated with complications like decreased ovarian reserve and other complications inherent to nature of surgery. Unlike ovarian drilling, ovarian pricking via transvaginal route under ultrasound guidance, as performed in our study, is less invasive, non-damaging to ovary and allows for real-time assessment, associated with lower morbidity, and allows repeatability.\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003ch2\u003eMechanistic Insights and Molecular Basis\u003c/h2\u003e\n\u003cp\u003eThe scientific foundation of our approach parallels that of Kawamura et al. (2013), who proposed that mechanical fragmentation of ovarian tissue could disrupt the Hippo signaling cascade, leading to upregulation of growth factors (YAP, CCN2, and BIRC5) and follicular activation. Their in vitro activation (IVA) protocol, though promising, required laparoscopic removal and reimplantation of ovarian cortex, making it less accessible. In contrast, our technique relies on in situ activation, avoiding surgical excision.\u003c/p\u003e\n\u003cp\u003eSimilarly, \u003cstrong\u003eZhao et al. (2018)\u003c/strong\u003e demonstrated that mechanical ovarian disruption in mice led to primordial follicle activation and progression to the antral stage. Translating this concept into clinical practice, ovarian pricking offers a non-excisional approach that could induce a comparable effect.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eErnst et al. (2017)\u003c/strong\u003e performed transcriptomic profiling of human oocytes from primordial and primary follicles and revealed distinct molecular signatures associated with dormancy and activation. Dormant oocytes exhibited enrichment of genes involved in transcriptional repression, chromatin remodeling, and cell cycle inhibition, whereas primary follicle oocytes demonstrated upregulation of genes related to metabolic activity, RNA processing, and translational machinery. Crucially, pathways such as PI3K-AKT, TGF-\u0026beta;, and FOXO3 were implicated in the transition from dormancy to growth, underscoring a tightly regulated oocyte-intrinsic mechanism for follicular activation.\u003c/p\u003e\n\u003cp\u003eThese findings highlight the biological significance of awakening dormant follicles, a process that holds potential for therapeutic manipulation in infertility treatment. In line with this concept, our study explored the mechanical disruption of follicular dormancy via ovarian pricking, aiming to stimulate the intrinsic activation pathways described by Ernst et al. The parallel between their molecular insights and our interventional approach supports the rationale for mechanically-induced follicular recruitment as a viable fertility-enhancing strategy in women with diminished ovarian reserve.\u003c/p\u003e\n\u003cp\u003eIn line with \u003cstrong\u003eHsueh (2020)\u003c/strong\u003e, our study underscores the pivotal role of Hippo signaling disruption via mechanical stress in activating dormant follicles. The review details how ovarian fragmentation enhances actin polymerization, fostering YAP nuclear translocation, and upregulating CCN and BIRC growth and survival factors, thereby facilitating follicle growth. Clinical protocols leveraging this mechanism\u0026mdash;such as drug-free IVA, combination IVA with Akt stimulators, and in vivo mechanical incision techniques\u0026mdash;have shown promising outcomes in POI, DOR, and resistant ovary patients.\u003c/p\u003e\n\u003cp\u003eHsueh further highlights translational opportunities, suggesting refinement of mechanical approaches and localized actin-enhancing interventions to improve efficacy while mitigating invasiveness and tissue damage\u003c/p\u003e\n\u003cp\u003eWhile promising, these biochemical activators are not widely approved or available. In contrast, our approach uses mechanical intervention alone, making it more accessible, safer, and cost-effective in routine practice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eZhao\u003c/strong\u003e \u003cstrong\u003eet al.\u003c/strong\u003e \u003cstrong\u003e(2021)\u003c/strong\u003e comprehensively reviewed the molecular circuitry controlling mammalian primordial follicle activation, identifying the PI3K/PTEN/Akt/FOXO3 axis as the core regulatory pathway within oocytes. Under physiologic conditions, PTEN maintains quiescence by restricting PIP3 accumulation; activation of PI3K initiates downstream Akt phosphorylation and FOXO3 nuclear export, committing follicles to growth. In pre-granulosa cells, mTORC1 signaling functions upstream of PI3K/Akt, with crosstalk from MAPK3/1 and potential modulation by DNA damage response pathways. These insights have informed the development of in vitro activation (IVA) protocols, which combine mechanical disruption (e.g. ovarian fragmentation) with pharmacologic stimulation of PI3K/mTOR to awaken dormant follicles. The authors note that while such approaches have yielded promising results in premature ovarian insufficiency and poor ovarian responders, outcomes vary and excessive PTEN inhibition may paradoxically reduce efficacy, underscoring the need for individualized modulation. Mechanistically, these findings support the rationale for ovarian pricking as a potential in vivo analogue of IVA, wherein local stromal disruption could relieve Hippo pathway constraints and secondarily engage the PI3K/mTOR signaling cascade to promote follicular recruitment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePellicer et al ( 2023)\u003c/strong\u003e concluded that In cases of irreversible damage, such as premature ovarian insufficiency (POI) or diminished ovarian reserve (DOR), follicle reactivation has shown encouraging results in experimental settings. However, robust clinical evidence is limited, although ongoing research is yielding more promising data.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eInterpretation of non-responders-\u003c/h3\u003e\n\u003cp\u003eDespite overall favorable results in our study, a few patients did not develop mature oocytes or blastocysts, suggesting that not all dormant follicles are functionally competent. Factors such as fibrosis, vascular compromise, or intrinsic oocyte quality may influence this outcome. Additionally, genetic and epigenetic programming of early follicles may limit response to mechanical signals. This highlights the need for personalized protocols and predictive biomarkers, such as ovarian tissue transcriptomics or real-time follicular fluid profiling.\u003c/p\u003e\n\u003ch3\u003eClinical Implications and Future Directions\u003c/h3\u003e\n\u003cp\u003eThis study reinforces the concept that ovarian tissue retains latent regenerative potential, even in women classified as having DOR. Given the non-invasive nature of ovarian pricking, it could be considered prior to stimulation in patients with low AMH or antral follicle count, especially those seeking to attempt IVF with autologous oocytes.\u003c/p\u003e\n\u003cp\u003eTo strengthen clinical application, future trials should address:\u003c/p\u003e\n\u003cul\u003e\n\u003cli\u003e\n\u003cp\u003eComparative effectiveness versus conventional stimulation alone.\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003eLive birth outcomes, perinatal safety, and long-term offspring health.\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003eMolecular assessment of pricking-induced gene expression changes.\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003eIntegration with adjunct therapies such as PRP or stem cell infusion.\u003c/p\u003e\n\u003c/li\u003e\n\u003c/ul\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003ch2\u003eLimitations\u003c/h2\u003e\n\u003cp\u003eThis study has limitations including a small sample size and absence of a control group. Pregnancy and live birth rates were not measured. Furthermore, no molecular assessments were conducted to confirm mechanistic pathways such as Hippo signaling modulation. Variability in individual ovarian biology could have influenced outcomes. Larger randomized controlled trials are needed to validate these preliminary findings.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOvarian pricking appears to be a promising, low-cost, and minimally invasive method to activate dormant follicles in women with diminished ovarian reserve. The technique yielded favorable outcomes in terms of mature oocyte retrieval and blastocyst formation. With further validation, ovarian pricking may become a valuable adjunct in the management of poor responders in IVF cycles.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch3\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThis study was conducted in accordance with the ethical principles outlined in the \u003cem\u003eDeclaration of Helsinki\u003c/em\u003e (2013 revision). The study protocol was reviewed and approved by the Institutional Ethics Committee of WINGS IVF Academy with approval number 2025/006/34D. Written informed consent was obtained from all participants prior to enrollment.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eAll participants provided written consent for publication of anonymized clinical data.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThe datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThis study did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/h3\u003e\n\u003col\u003e\n \u003cli\u003eDr. Jayesh Amin- Conceptualization, Methodology, Supervision, Validation, Visualization, Supervision, Validation, Visualization.\u003c/li\u003e\n \u003cli\u003eDr. Rudri Agrawal- Formal analysis, Investigation, Project administration, Writing – original draft, Writing – review \u0026amp; editing, Data curation\u003c/li\u003e\n \u003cli\u003eDr. Paresh Makwana- \u0026nbsp;Data curation, Investigation\u003c/li\u003e\n\u003c/ol\u003e\n\u003cul\u003e\n \u003cli\u003e\u003cstrong\u003eAll authors\u003c/strong\u003e read and approved the final manuscript.\u003c/li\u003e\n\u003c/ul\u003e\n\u003ch3\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThe authors thank the clinical and embryology team of NOVA WINGS IVF for their assistance in patient care and laboratory support. The authors are also grateful to the participating women for their cooperation.\u003cbr\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eShah JS, Sabouni R, Cayton Vaught KC, Owen CM, Albertini DF, Segars JH (2018) Biomechanics and mechanical signaling in the ovary: a systematic review. J Assist Reprod Genet 35:1135\u0026ndash;1148. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10815-018-1180-y\u003c/span\u003e\u003cspan address=\"10.1007/s10815-018-1180-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi J, Kawamura K, Cheng Y, Liu S, Klein C, Liu S, Duan EK, Hsueh AJ (2010) Activation of dormant ovarian follicles to generate mature eggs. 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Cells 10:1491. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/cells10061491\u003c/span\u003e\u003cspan address=\"10.3390/cells10061491\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePellicer N, Cozzolino M, Diaz-Garc\u0026iacute;a C, Galliano D, Cobo A, Pellicer A, Herraiz S (2023) Ovarian rescue in women with premature ovarian insufficiency: facts and fiction. Reprod Biomed Online 46:543\u0026ndash;565. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.rbmo.2022.12.011\u003c/span\u003e\u003cspan address=\"10.1016/j.rbmo.2022.12.011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"NOVA WINGS IVF ","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Diminished Ovarian Reserve, Follicular Activation, Fertility Enhancement, Ovarian Pricking, Ovarian Stimulation","lastPublishedDoi":"10.21203/rs.3.rs-8082011/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8082011/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eWomen with diminished ovarian reserve (DOR) represent one of the most challenging groups in assisted reproductive technology (ART), often exhibiting poor response to conventional stimulation protocols and limited success in in vitro fertilization (IVF). Strategies aimed at activating dormant follicles have gained increasing attention, particularly approaches that modulate intra-ovarian signaling pathways such as the Hippo pathway. Ovarian pricking, a minimally invasive technique involving targeted disruption of the ovarian cortex under transvaginal ultrasound guidance, has been hypothesized to stimulate follicular activation and improve oocyte yield. This study aimed to evaluate whether ovarian pricking can enhance follicular recruitment and improve subsequent fertility outcomes in women with DOR who had previously failed IVF cycles.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eThis prospective study included 24 infertile women aged 30\u0026ndash;35 years with AMH levels\u0026thinsp;\u0026lt;\u0026thinsp;0.5 ng/mL, reduced antral follicle counts, and ovarian volume of 1.5\u0026ndash;2 cm\u0026sup3;. Ovarian pricking was performed transvaginally under ultrasound guidance using a standard oocyte retrieval needle, followed by estradiol valerate administration and serial monitoring. Of the 24 patients, 22 (91.7%) demonstrated a follicular response, with a significant increase in the mean number of antral follicles from 0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50 before pricking to 3.96\u0026thinsp;\u0026plusmn;\u0026thinsp;1.30 post-procedure (t\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;12.274, p\u0026thinsp;=\u0026thinsp;0.001). Among the 22 responders, 18 (81.8%) exhibited sustained follicular growth during controlled ovarian stimulation with recombinant FSH plus HMG under an antagonist protocol. Oocyte retrieval was successful in all 18, and 16 (88.9%) yielded mature (MII) oocytes. Blastocyst formation was achieved in 8 of these 16 patients (50%), demonstrating the feasibility of the approach in supporting downstream embryonic development. Four women showed an initial response but failed to progress to stimulation.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eOvarian pricking appears to be a novel and promising adjunct for the management of women with DOR, offering a minimally invasive means of activating dormant follicles and improving follicular recruitment, oocyte maturation, and blastocyst development. The high response rate observed in this study supports its potential clinical utility, possibly through modulation of the Hippo signalling pathway. However, outcomes varied among patients, highlighting the need for individualized treatment strategies. Larger, long-term prospective studies are warranted to validate efficacy, optimize protocols, and define predictors of success, as well as to assess the impact on live birth outcomes.\u003c/p\u003e","manuscriptTitle":"Micro-Trauma Induced Follicular Activation: The Role of Ovarian Pricking in Fertility Enhancement\"","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-13 07:44:12","doi":"10.21203/rs.3.rs-8082011/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"df504b6b-62d2-4771-8a37-d48470f8cef1","owner":[],"postedDate":"November 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":57768418,"name":"Sexual \u0026 Reproductive Medicine"}],"tags":[],"updatedAt":"2025-11-13T07:44:13+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-13 07:44:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8082011","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8082011","identity":"rs-8082011","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}