Drug-free In Situ Activation of Follicle through Laparoscopic Ovarian Cortical Incisions in Patients with Premature Ovarian Insufficiency

Drug-free In Situ Activation of Follicle through Laparoscopic Ovarian Cortical Incisions in Patients with Premature Ovarian Insufficiency | 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 Research Article Drug-free In Situ Activation of Follicle through Laparoscopic Ovarian Cortical Incisions in Patients with Premature Ovarian Insufficiency Yuan Li, Xiaohui Wang, Kun Wang, Hongzhan Zhang, Jiaping Su, Rui Li, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7378665/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 Individuals with premature ovarian insufficiency (POI) face significant challenges in attaining favorable reproductive outcomes. To date, no effective interventions have been established to substantially improve this complex clinical scenario. Methods We conducted a clinical trial in a tertiary university-affiliated hospital to study a protocol of Drug-free In Situ Activation (Df-ISA) of ovarian follicles for treatment of POI patients in improving reproductive outcomes. Five women were enrolled and accepted the Df-ISA procedure through longitudinal and transverse mechanical cutting on one ovary. The contralateral ovary remained intact as control. Changes in antral follicle count (AFC), oocyte retrieval rates, embryo cryopreservation, clinical pregnancy and live birth outcomes were clinical observational indicators. Results Antral follicle development was observed in all five patients by ultrasound monitoring after Df-ISA. A total of 21 echo-free antral follicle-like structures were identified and one viable cleavage embryo were obtained in the 6-month follow-up. In the longer follow-up, a total of seven cleavage embryos were obtained from two patients, and one live birth was achieved. Paired t-test revealed a statistically significant difference of follicle-like structures observed between the Df-ISA ovaries and the contralateral control ovaries. Conclusions The "Longitudinal and Latitudinal cutting in situ" Df-ISA procedure represents a novel infertility treatment for patients with POI. This procedure promotes the growth of residual antral ovarian follicles through dense mechanical disruption, yielding encouraging results that warrant further investigation. The study adhered to the principles outlined in the Declaration of Helsinki and was granted ethical approval by the Ethics Research Committee of the University of Hong Kong-Shenzhen Hospital and has been registered in the National Medical Research Registration and Filing Information System, with the registration number ChiCTR2400086676.Every participant has provided informed consent. Drug-free In Situ Activation Premature ovarian insufficiency Mechanical disruption Reproductive outcome Laparoscopic Ovarian Cortical Incisions Figures Figure 1 Figure 2 Figure 3 INTRODUCTION Infertile women suffering from premature ovarian insufficiency (POI) encounter substantial difficulties in achieving favorable reproductive outcomes. POI is marked by diminished ovarian function resulting from a reduced quantity and/or compromised quality of the remaining oocytes [ 1 ] . As delineated by the guidelines of the European Society of Human Reproduction and Embryology (ESHRE), POI is defined as oligomenorrhea or amenorrhea lasting for at least four months, in conjunction with elevated serum follicle-stimulating hormone (FSH) levels exceeding 25 IU/L on two occasions separated by a time interval of more than 4 weeks, in women under the age of 40 [ 2 ] . Despite attempts to enhance in vitro fertilization (IVF) outcomes in these patients using diverse ovarian stimulation protocols and adjunctive therapies, success remains elusive [ 3 ] . The idea of using mechanical fragmentation to treat POI was initially implemented through the conception of in vitro activation (IVA) described by Dr. Kawamura et al. in 2013 [ 4 ] . This procedure entails fragmenting ovarian cortical tissue into 2×2×1 mm 3 cubes, followed by chemical activation with specific drugs before auto-transplantation. This method successfully promoted growth of residual follicles in the treated ovarian cortex after autografting to the POI patient and led to a healthy birth after oocyte retrieval and IVF-embryo transfer [ 4 ] . Further studies have optimized the procedure and achieved positive therapeutic effects [ 5 – 7 ] . Fabregues et al. reported the first pregnancy after drug-free in vitro activation (Df-IVA) of follicles and fresh tissue auto-transplantation in a POI patient in 2018 [ 8 ] . The procedure obviated the need for a 2-day drug incubation period and necessitated only a single surgery for follicle activation. In 2019, Dr. Kawamura's team employed the drug-free procedure in patients with poor ovarian response (POR) and diminished ovarian reserve (DOR) and resulted in one live birth, two ongoing pregnancies, and one miscarriage [ 9 ] . Zhang et al. developed an in vivo ovarian cortex slicing technique by scratching ovary with 3 superficial lesions. The procedure represented the first attempt at in vivo in situ ovarian cortex slicing, and successfully achieved follicle development, implying potential therapeutic benefits for patients with POI [ 10 ] . It has been demonstrated that mechanical shear can activate the phosphoinositide 3-kinase (PI3K)/Akt and Hippo signaling pathways which are crucial in folliculogenesis from the primordial follicle to the early antral follicle [ 5 , 11 – 14 ] and fragmentation of ovarian cortex facilitates the conversion of globular actin (G-actin) into filamentous actin (F-actin), leading to disruption of the Hippo signaling pathway thereby stimulating growth of the secondary follicles [ 15 ] . Based on these findings, we sorted to optimize the fragmentation procedure by performing in situ cutting of the ovarian cortex. Here, we refined the ovarian cutting technique of our drug-free in situ activation (Df-ISA) procedure by employing both longitudinal and transverse incisions. The objective of the study was to ascertain whether extensive in situ cortical incision of the ovary can induce follicular activation and improve reproductive outcome in patients with POI. MATERIALS AND METHODS Ethics statement This study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Research Committee of the University of Hong Kong-Shenzhen Hospital under a registry identification number hkuszh2020121 on June 2020. It has been registered in the National Medical Research Registration and Filing Information System, with the registration number ChiCTR2400086676. Study design This was a pilot clinical study involving five patients with POI between March 2021 and October 2023 in a tertiary university-affiliated hospital. The study aimed to evaluate the impact of our Df-ISA procedure on changes in antral follicle count (AFC), oocyte retrieval rates, embryo cryopreservation, clinical pregnancy and live birth of POI patients. Inclusion and Exclusion Criteria A total of five patients diagnosed with POI according to the ESHRE criteria were included in this study. To ensure population homogeneity and minimize potential confounding factors, the study excluded individuals with a history of prior surgical procedures involving at least one ovary, those with a personal history of ovarian malignancies, including borderline ovarian tumors, as well as those with contraindications for laparoscopic surgery or an abnormal karyotype. Hormonal measurements Concentrations of anti-Müllerian hormone (AMH), FSH, luteinizing hormone (LH), progesterone (P), and estradiol were measured using commercially available immunoassay kits (Beckman Coulter, Brea, CA, USA). The assay concentration ranges for FSH and estradiol were 0.1–250 mIU/ml and 15–2000 pg/ml, respectively. The detection limit for the serum AMH assay were 0.02 ng/ml. Pre-Df-ISA hormonal treatment Because all patients presented with oligomenorrhea or amenorrhea, they were administered Hormone Replacement Therapy (HRT) drug Femoston (Abbott Biologicals B.V., Netherlands) to maintain a regular menstrual cycle prior to the Df-ISA procedure. They also received Femoston treatment after the procedure. Df-ISA procedure Prior to surgery, the target ovary for resection was determined using a random envelope method. A previously published protocol of ovarian cortex fragmentation was used with modification [ 8 ] . During the laparoscopic surgery, we first evaluated the macroscopic appearance of bilateral ovaries and ensured adequate exposure of the surgical field. To minimize thermal injury and reduce surgical trauma to the delicate ovarian tissue, we utilized sharp dissection with scissor and scalpel rather than electrocoagulation. Upon complete exposure of the ovary on the operative side, the ovarian cortex was meticulously incised with a scalpel. Initially, incisions were made perpendicular to the long axis of the ovary and encircling it, maintaining an interval of 1–2 mm and a depth of 1–2 mm. Subsequently, additional incisions were performed parallel to the long axis of the ovary and perpendicular to the initial incisions, with the same interval and depth of 1–2 mm. Incisional scratches encircle the entire ovarian surface, resembling a horizontal and vertical mango cut. All the procedures were performed by the same 2 surgeons and the appearance of the ovaries during surgery is depicted in Fig. 1 . Oocyte Retrieval, fertilization and Outcome Follow-up We chose to perform the Df-ISA procedure during the follicular phase. After surgery, patients continued to receive Femoston for HRT within the same cycle to maintain normal reproductive endocrine environment. The patients were followed-up for at least six months. Hormonal assessment and transvaginal ultrasound were conducted during the early follicular phase of each menstrual cycle. If echo-free antral follicle-like structure was observed, mild stimulation was initiated with the patient's consent. When the diameter of one follicle exceeded 15 mm, recombinant human chorionic gonadotropin (Ovitrelle® 500 µg, Merck, Israel) was administered. Oocyte retrieval was performed 34–36 hours post-HCG administration under transvaginal ultrasound guidance. Following retrieval of mature oocytes, standard IVF/ Intracytoplasmic Sperm Injection (ICSI) procedures were performed. Day-2 cleavage embryos were cryopreserved by vitrification and stored in liquid nitrogen. Blastocyst culture was not performed due to the limited number of embryos obtained. The vitrified embryos were warmed and transferred under a hormone replacement protocol. Pregnancy was confirmed by measuring serum hCG-β levels 2 weeks after embryo transfer. In patients who failed to achieve ovarian follicular response, Femoston was continued monthly. Statistical analysis Continuous variables were presented by medians and ranges. The IBM SPSS v23.0 software (IBM Corp, New York, USA) was used for all statistical analyses. The graphs were created using Prism 9 (GraphPad Software Inc., San Diego, CA, USA). For the comparison of antral follicle counts between the Df-IVA ovary and the contralateral control ovary in patients, a paired t-test was performed for statistical analysis with significance set at p < 0.05. RESULTS Patient characteristics A total of five patients with POI were enrolled in this study. The median age of the cohort was 33.20 years (31.00–37.00 years), and the median serum FSH level was 72.76 mIU/ml (33.19–136.22 mIU/ml) at the time of enrollment. During preoperative monitoring, no antral follicle-like structure was detected in the ovaries of all five patients. Comprehensive baseline characteristics and hormonal profiles of the patients are detailed in Table 1 . Table 1 Baseline characteristics of patients enrolled PatientID Age Years of diagnosis of POI Karyotype abnormality Baseline hormone profile AFC of the ovary before ISA Pharmaceutical supplement AMH(ng/mL) FSH(mIU/mL) E2(pg/mL) Left side Right side Patient 01 37 2 No < 0.02 33.19 42.46 0 0 Estradiol/Dydrogesterone Tablets Patient 02 31 5 No < 0.02 136.22 < 15.00 0 0 Estradiol/Dydrogesterone Tablets Patient 03 34 5 No < 0.02 73.35 < 15.00 0 0 Estradiol/Dydrogesterone Tablets Patient 04 32 3 No < 0.02 87.23 < 15.00 0 0 Estradiol/Dydrogesterone Tablets Patient 05 32 5 No 0.03 33.79 138.26 0 0 Estradiol/Dydrogesterone Tablets Follicle development, oocyte retrieval, fertilization and pregnancy outcome During the 6-month follow-up period after Df-IVA, follicular development was observed in all five patients. A total of 21 antral follicle-like structures were identified in 25 ultrasound monitoring sessions. In two patients, oocyte retrieval was performed, yielding a total of 2 MII oocytes (one viable cleavage embryos and one degenerated cleavage embryo obtained) (Table 2 ). In a longer follow-up period, a total of 11 oocyte retrieval procedures were performed, ultimately yielding 7 viable cleavage embryos (Fig. 2 ). Patient 3 underwent transfer of a single viable vitrified-warmed cleavage embryo but failed to achieve clinical pregnancy. Patient 5 underwent a vitrified-warmed embryo transfer of two viable cleavage embryos on August 2024 and achieved a live birth (Table 2 ). Table 2 Follicular development, cryopreserved embryos and pregnant outcome after surgery Oocyte retrieval and IVF/ICSI outcome during the 6-month follow-up period after Df-ISA Cryopreserved embryos and pregnant outcome in longer follow-up period after Df-ISA Number of patients recruited 5 Total number of cryopreserved embryos ready for transfer 7 Number of AFCs observed 21 Number of patients with cryopreserved embryos transferred 2 Cycle number with oocyte retrieval 2 Number of patients being pregnant after ET/FET 1 Oocyte obtained with oocyte retrieval 2 Number of live birth 1 Viable cleavage embryo number obtained via IVF/ICSI 1 Given that the surgical and control sides of the ovaries were randomly assigned pre-operatively, post-operative follow-up allowed for a comparative analysis of the number of antral follicle-like structures between the two sides to assess the surgical effect. In the ongoing follow-up, a total of 27 antral follicle-like structures were observed in the Df-ISA ovaries of the five patients in 35 ultrasound monitoring sessions, while only 6 antral follicle-like structures were identified in the control ovaries. A paired t-test revealed a statistically significant difference between the two groups (P = 0.0005) (Fig. 3 ). DISCUSSION Our innovative Df-ISA procedure achieved, for the first time, in-situ dense micro-incision of the entire ovarian cortex in a cohort of 5 POI patients, yielding encouraging outcomes. All participants exhibited follicular recruitment, with 2 patients producing viable embryos and ultimately achieving one live birth. Mechanobiology, an emerging field that researches the contribution of physical forces on changing to cellular biology, has perfectly inspired the concept of fragmenting the ovarian cortex for activation of primordial follicles. It is long known that ovarian wedge resection or laparoscopic ovarian laser drilling promote follicle growth in patients with polycystic ovary syndrome (PCOS) [ 11 , 16 ] . Since the first conception of IVA demonstrated by Dr. Kawamura et al. in 2013 [ 4 ] , the IVA protocol was gradually simplified. Initially, the protocol required 2 surgical operations for tissue collection and autologous transplantation and a 2-day drug treatment of the ovarian cortex [ 4 ] . Subsequently, the IVA procedure was optimized from requiring in vitro drug culture shortened to completing all steps in a single surgery, yielding gratifying clinical outcome [ 6 – 8 , 10 , 17 ] . All the modifications restored ovarian function and fertility to a certain extent. These clinical trial phenomena are consistent with the observations that disruption of the Hippo signaling pathway through ovarian tissue fragmentation can activate the dormant primordial follicles [ 12 , 15 ] . In our study, all five patients had small ovarian volumes with a more solid texture. Only two of them had one antral follicle-like structure each at enrollment. After Df-ISA, antral follicle development were observed in all five patients. The observation is consistent with previous report showing an increase in AFC counts in POR patients following drug-free IVA [ 9 ] . Other studies reported that mechanical stimulation of the ovaries could activate dormant follicles in most patients with Extremely Poor Ovarian Response (EPOR), restore hormonal responsiveness, and lead to multiple live births [ 18 ] . The mechanisms underlying the use of different mechanical stimulation methods for improving ovarian function may vary slightly: The IVA procedure may primarily act on activation of dormant primordial follicles, whereas the Whole Ovary Laparoscopic Incision (WOLI) procedure may mainly rescue the developing secondary follicles [ 19 ] . In our study cohort following the Df-ISA procedure, 2 patients underwent oocyte retrieval with a retrieval rate of 40%, consistent with a reported retrieval rate of 35.71% for POI patients [ 20 ] . Interestingly, the oocyte retrieval rate was 70% in DOR patients undergoing cortical tissue fragmentation and autologous-transplantation [ 17 ] . This suggests that the success of Df-ISA depends on the presence of residual ovarian follicles to some extent. The relatively higher success rate in DOR patients highlights the importance of baseline ovarian reserve in determining the efficacy of Df-ISA. In this study, a total of 11 oocyte retrievals were performed in patients with POI in longer follow-up. Among these, two retrievals failed to obtain oocytes. All the retrieved oocytes were subjected to IVF or ICSI. Two oocytes exhibited abnormal fertilization, the remaining 7 oocytes were normally fertilized and yielded 7 viable cleavage embryos. The fertilization rate was 77.8%, which is slightly higher than that reported by Grin (59.0%/72.0%) in POI/POR patients [ 21 ] and Kawamura (68.7%) in POR patients with DOR using in vitro activation [ 9 ] . These data support the effectiveness of mechanical scratch in activating and maturing oocytes for fertilization. Among the five patients enrolled in our study, two had embryo cryopreservation. Ferreri et al. [ 20 ] and Fabregues [ 8 ] et al. reported that approximately 28–30% of POI patients undergoing drug-free IVA achieved embryo cryopreservation. In POR patients, Grin et al. reported that all studied patients (7/7) had at least one embryo cryopreserved, compared to 35% of patients in the POI group [ 21 ] . This comparison highlights that severe follicle depletion remains a significant factor in the reproductive outcomes of IVA. Among the five patients enrolled, two patients had 1 and 6 utilzable cleavage embryos respectively. The patient with only one embryo underwent HRT embryo transfer but failed to get pregnant. The other patient underwent HRT embryo transfer of two cleavage embryos and successfully had a live birth. Another study reported that among 14 patients with POI who underwent Df-IVA, only one clinical pregnancy occurred, which unfortunately ended in an early miscarriage [ 21 ] . These observations suggest that the pregnancy and live birth rates for POI patients undergoing IVF treatment remain at a relatively low level. We observed a significantly higher number of antral follicles in the operated ovary compared with the untouched control ovary. Among 5 patients, antral follicle development was all observed post-Df-ISA and most follicular recruitment occurred in the operated ovary with a paired t-test P value < 0.001. This suggested Df-ISA can lead to rapid induction of residual secondary follicles growth into antral follicles. However, another research found no difference in the number of mature follicles after ovarian stimulation 10 weeks after Df-IVA versus the control ovaries (1.0 vs.0.7 follicles, P = 0.35) in POI patients [ 17 ] . Biomechanics shows that fragmentation of human and mice ovarian cortex into small cubes promotes conversion of G-actin into F-actin [ 15 ] , which disrupts the Hippo signaling pathway and induces nuclear localization of the Yes-associated protein and connective tissue growth factors to activate secondary follicle growth in vitro and in vivo [ 4 , 11 , 12 , 15 , 22 – 24 ] . Theoretically, the more cuts on the ovarian cortex mean more activation and ample evidence underscores the essential role of mechanical signaling in the development of the ovary, follicle, and oocyte during gametogenesis [ 4 , 23 ] . In our research, we conducted both longitudinal and transverse cut in situ on the ovary to increase density of the incisions so as to have more effect on the actin and therefor stronger action on the Hippo pathway and downstream factors. The limitations of our study are that it is a single-center pilot study, and the sample size is small. We plan to continue to recruit patients in the hope of obtaining higher levels of positive evidence. Given that IVA/ISA efficacy is likely contingent upon the presence of residual ovarian follicles, there is a pressing need for future studies to devise more sensitive assays capable of accurately predicting both the quantity of growing follicles and the residual primordial follicle pool in POI patients. This study underscores the therapeutic potential of Df-ISA for patients with POI, highlighting its capacity to induce follicular activation, enhance oocyte retrieval rates, and improve reproductive outcomes. Future research endeavors should prioritize head-to-head comparisons between drug-free and chemically activated IVA/ISA protocols, as well as delve into angiogenic mechanisms, to elucidate optimal treatment strategies. Declarations Author Contribution Author contributionsStudy design: TW, YL, XW, KW; Conduct of the clinical study: YL, XW, HZ, RL, JS, PZ; Quality assessment of the clinical study: RHWL, EHYN, WSBY, J.V.Z, XCW, TW; Data analysis: YL, XW; Manuscript preparation: YL. All authors approved the final version of the manuscript. Acknowledgement Yuan Li#, Xiaohui Wang#, Kun Wang#, Hongzhan Zhang, Jiaping Su, Rui Li, Peikun Zhao, Raymond H W Li, Ernest H Y Ng, William S B Yeung, J.V. Zhang, Xiangcai Wei*, Tianren Wang*#Co-first author*Corresponding author Support Funding ： This work was supported by Shenzhen Fundamental Research Program, China (No: JCYJ20200109150429414); Shenzhen Science and Technology Program, China (No. RCYX20200714114705073, No. KQTD20190929172749226); the National Natural Science Foundation of China (No: 81971453); General Program Sponsored by Basic Research Fund in Shenzhen Natural Science Foundation (JCYJ20240813113030040); National Key R&D Program of China 2024YFA1803001;Shenzhen Medical Research Fund B2404004; Guangdong Basic and Applied Basic Research Foundation 2024A1515010059; Shenzhen Science and Technology Program JCYJ20220818101218040; Shenzhen Key Laboratory of Metabolic Health (Grant No. ZDSYS20210427152400001) References Steiner A Z, Pritchard D, Stanczyk F Z, et al. Association Between Biomarkers of Ovarian Reserve and Infertility Among Older Women of Reproductive Age[J]. JAMA, 2017, 318(14): 1367-1376. 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Drug-free In Vitro Activation of Ovarian Follicles and Fresh Tissue Autotransplantation in Patients with Poor ovarian response and Premature Ovarian Insufficiency[J]. F S Sci, 2025. Hendriks M L, Ket J C, Hompes P G, et al. Why does ovarian surgery in PCOS help? Insight into the endocrine implications of ovarian surgery for ovulation induction in polycystic ovary syndrome[J]. Hum Reprod Update, 2007, 13(3): 249-64. Shah J S, Sabouni R, Cayton Vaught K C, et al. Biomechanics and mechanical signaling in the ovary: a systematic review[J]. J Assist Reprod Genet, 2018, 35(7): 1135-1148. Cheng Y, Feng Y, Jansson L, et al. Actin polymerization-enhancing drugs promote ovarian follicle growth mediated by the Hippo signaling effector YAP[J]. FASEB J, 2015, 29(6): 2423-30. Additional Declarations No competing interests reported. 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Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYPACmwQJEMXYQKyGAwxppGs5TIIWg+NnD7/+uOd8nuSMBOaPP3cwyJn3LyCg5UxemsWBZ7eLpSUS2CQkzzAYy9x4gF+L2YEcM4MDB24nzpPI/8Zg2MaQOEPiAAEt59+AtJwDaklg/pBIlJYbOcYPDhw4kDhbIoFB4iBIC38Dfi32N96YMZw5kJw4s+cBm2Rjm4SxhAR+HQyS/TnGHyoO2CXOOA4KsTYbOQl+Ag4DAjZkY4FsoAsJAeYPqHwibBkFo2AUjIKRBQDWlUo29x0RigAAAABJRU5ErkJggg==","orcid":"","institution":"The University of Hong Kong-Shenzhen Hospital","correspondingAuthor":true,"prefix":"","firstName":"Tianren","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-08-15 05:53:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7378665/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7378665/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":96556773,"identity":"4e402de5-28c7-424e-aaea-39b6a401ba61","added_by":"auto","created_at":"2025-11-23 11:50:00","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":862921,"visible":true,"origin":"","legend":"","description":"","filename":"TechnicalCheckISAmanuscript.docx","url":"https://assets-eu.researchsquare.com/files/rs-7378665/v1/fcb85d9203d96a13398b0400.docx"},{"id":96556769,"identity":"2b94e433-e644-4ead-8c61-70dd81f3b8c2","added_by":"auto","created_at":"2025-11-23 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09:17:11","extension":"xml","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":78134,"visible":true,"origin":"","legend":"","description":"","filename":"bed8f43ffd234a0dbb05c24544181a131structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7378665/v1/2da64462ae4518b10cea04db.xml"},{"id":96556782,"identity":"c80f156f-373f-4948-b08a-04f539738670","added_by":"auto","created_at":"2025-11-23 11:50:01","extension":"html","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":90558,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7378665/v1/68ecaeb4e903fac4f84e7648.html"},{"id":96556771,"identity":"49f764ea-c4a4-4078-bcc6-902fd321e77a","added_by":"auto","created_at":"2025-11-23 11:50:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":547018,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe appearance of the ovary that was transected both transversely and longitudinally by the surgical blade during laparoscopy\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7378665/v1/bf492f7ac2d01dad3ac4fc07.png"},{"id":96556772,"identity":"edc76454-fd4f-4c9a-8096-0f815780585b","added_by":"auto","created_at":"2025-11-23 11:50:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":199038,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFollicular development, cryopreserved embryos and pregnant outcome after Df-ISA\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7378665/v1/e3b88f7a3b3bbf9bb38183c9.png"},{"id":96604755,"identity":"25d41a9b-0733-4ec6-a943-6af8894d68f4","added_by":"auto","created_at":"2025-11-24 09:14:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":52677,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of AFCs observed between surgical side and control side of ovaries\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7378665/v1/c0c7c0fa175c74d4724aba15.png"},{"id":100359887,"identity":"1d820c1a-c01e-4560-970b-bd5f0922fbbd","added_by":"auto","created_at":"2026-01-16 07:27:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2292404,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7378665/v1/4cd505e5-9cf2-4f05-a96f-3e3057893a4f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Drug-free In Situ Activation of Follicle through Laparoscopic Ovarian Cortical Incisions in Patients with Premature Ovarian Insufficiency","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eInfertile women suffering from premature ovarian insufficiency (POI) encounter substantial difficulties in achieving favorable reproductive outcomes. POI is marked by diminished ovarian function resulting from a reduced quantity and/or compromised quality of the remaining oocytes \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. As delineated by the guidelines of the European Society of Human Reproduction and Embryology (ESHRE), POI is defined as oligomenorrhea or amenorrhea lasting for at least four months, in conjunction with elevated serum follicle-stimulating hormone (FSH) levels exceeding 25 IU/L on two occasions separated by a time interval of more than 4 weeks, in women under the age of 40 \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Despite attempts to enhance in vitro fertilization (IVF) outcomes in these patients using diverse ovarian stimulation protocols and adjunctive therapies, success remains elusive \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe idea of using mechanical fragmentation to treat POI was initially implemented through the conception of in vitro activation (IVA) described by Dr. Kawamura et al. in 2013\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. This procedure entails fragmenting ovarian cortical tissue into 2\u0026times;2\u0026times;1 mm\u003csup\u003e3\u003c/sup\u003e cubes, followed by chemical activation with specific drugs before auto-transplantation. This method successfully promoted growth of residual follicles in the treated ovarian cortex after autografting to the POI patient and led to a healthy birth after oocyte retrieval and IVF-embryo transfer \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Further studies have optimized the procedure and achieved positive therapeutic effects \u003csup\u003e[\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Fabregues et al. reported the first pregnancy after drug-free in vitro activation (Df-IVA) of follicles and fresh tissue auto-transplantation in a POI patient in 2018 \u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. The procedure obviated the need for a 2-day drug incubation period and necessitated only a single surgery for follicle activation. In 2019, Dr. Kawamura's team employed the drug-free procedure in patients with poor ovarian response (POR) and diminished ovarian reserve (DOR) and resulted in one live birth, two ongoing pregnancies, and one miscarriage \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Zhang et al. developed an in vivo ovarian cortex slicing technique by scratching ovary with 3 superficial lesions. The procedure represented the first attempt at in vivo in situ ovarian cortex slicing, and successfully achieved follicle development, implying potential therapeutic benefits for patients with POI \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIt has been demonstrated that mechanical shear can activate the phosphoinositide 3-kinase (PI3K)/Akt and Hippo signaling pathways which are crucial in folliculogenesis from the primordial follicle to the early antral follicle \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e and fragmentation of ovarian cortex facilitates the conversion of globular actin (G-actin) into filamentous actin (F-actin), leading to disruption of the Hippo signaling pathway thereby stimulating growth of the secondary follicles \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eBased on these findings, we sorted to optimize the fragmentation procedure by performing in situ cutting of the ovarian cortex. Here, we refined the ovarian cutting technique of our drug-free in situ activation (Df-ISA) procedure by employing both longitudinal and transverse incisions. The objective of the study was to ascertain whether extensive in situ cortical incision of the ovary can induce follicular activation and improve reproductive outcome in patients with POI.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eEthics statement\u003c/h2\u003e\u003cp\u003e This study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Research Committee of the University of Hong Kong-Shenzhen Hospital under a registry identification number hkuszh2020121 on June 2020. It has been registered in the National Medical Research Registration and Filing Information System, with the registration number ChiCTR2400086676.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eStudy design\u003c/h3\u003e\n\u003cp\u003eThis was a pilot clinical study involving five patients with POI between March 2021 and October 2023 in a tertiary university-affiliated hospital. The study aimed to evaluate the impact of our Df-ISA procedure on changes in antral follicle count (AFC), oocyte retrieval rates, embryo cryopreservation, clinical pregnancy and live birth of POI patients.\u003c/p\u003e\n\u003ch3\u003eInclusion and Exclusion Criteria\u003c/h3\u003e\n\u003cp\u003eA total of five patients diagnosed with POI according to the ESHRE criteria were included in this study. To ensure population homogeneity and minimize potential confounding factors, the study excluded individuals with a history of prior surgical procedures involving at least one ovary, those with a personal history of ovarian malignancies, including borderline ovarian tumors, as well as those with contraindications for laparoscopic surgery or an abnormal karyotype.\u003c/p\u003e\n\u003ch3\u003eHormonal measurements\u003c/h3\u003e\n\u003cp\u003eConcentrations of anti-M\u0026uuml;llerian hormone (AMH), FSH, luteinizing hormone (LH), progesterone (P), and estradiol were measured using commercially available immunoassay kits (Beckman Coulter, Brea, CA, USA). The assay concentration ranges for FSH and estradiol were 0.1\u0026ndash;250 mIU/ml and 15\u0026ndash;2000 pg/ml, respectively. The detection limit for the serum AMH assay were 0.02 ng/ml.\u003c/p\u003e\n\u003ch3\u003ePre-Df-ISA hormonal treatment\u003c/h3\u003e\n\u003cp\u003eBecause all patients presented with oligomenorrhea or amenorrhea, they were administered Hormone Replacement Therapy (HRT) drug Femoston (Abbott Biologicals B.V., Netherlands) to maintain a regular menstrual cycle prior to the Df-ISA procedure. They also received Femoston treatment after the procedure.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eDf-ISA procedure\u003c/h2\u003e\u003cp\u003ePrior to surgery, the target ovary for resection was determined using a random envelope method. A previously published protocol of ovarian cortex fragmentation was used with modification \u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. During the laparoscopic surgery, we first evaluated the macroscopic appearance of bilateral ovaries and ensured adequate exposure of the surgical field. To minimize thermal injury and reduce surgical trauma to the delicate ovarian tissue, we utilized sharp dissection with scissor and scalpel rather than electrocoagulation. Upon complete exposure of the ovary on the operative side, the ovarian cortex was meticulously incised with a scalpel. Initially, incisions were made perpendicular to the long axis of the ovary and encircling it, maintaining an interval of 1\u0026ndash;2 mm and a depth of 1\u0026ndash;2 mm. Subsequently, additional incisions were performed parallel to the long axis of the ovary and perpendicular to the initial incisions, with the same interval and depth of 1\u0026ndash;2 mm. Incisional scratches encircle the entire ovarian surface, resembling a horizontal and vertical mango cut. All the procedures were performed by the same 2 surgeons and the appearance of the ovaries during surgery is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eOocyte Retrieval, fertilization and Outcome Follow-up\u003c/h3\u003e\n\u003cp\u003eWe chose to perform the Df-ISA procedure during the follicular phase. After surgery, patients continued to receive Femoston for HRT within the same cycle to maintain normal reproductive endocrine environment. The patients were followed-up for at least six months. Hormonal assessment and transvaginal ultrasound were conducted during the early follicular phase of each menstrual cycle. If echo-free antral follicle-like structure was observed, mild stimulation was initiated with the patient's consent. When the diameter of one follicle exceeded 15 mm, recombinant human chorionic gonadotropin (Ovitrelle\u0026reg; 500 \u0026micro;g, Merck, Israel) was administered. Oocyte retrieval was performed 34\u0026ndash;36 hours post-HCG administration under transvaginal ultrasound guidance. Following retrieval of mature oocytes, standard IVF/ Intracytoplasmic Sperm Injection (ICSI) procedures were performed. Day-2 cleavage embryos were cryopreserved by vitrification and stored in liquid nitrogen. Blastocyst culture was not performed due to the limited number of embryos obtained. The vitrified embryos were warmed and transferred under a hormone replacement protocol. Pregnancy was confirmed by measuring serum hCG-β levels 2 weeks after embryo transfer. In patients who failed to achieve ovarian follicular response, Femoston was continued monthly.\u003c/p\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eContinuous variables were presented by medians and ranges. The IBM SPSS v23.0 software (IBM Corp, New York, USA) was used for all statistical analyses. The graphs were created using Prism 9 (GraphPad Software Inc., San Diego, CA, USA). For the comparison of antral follicle counts between the Df-IVA ovary and the contralateral control ovary in patients, a paired t-test was performed for statistical analysis with significance set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003ePatient characteristics\u003c/h2\u003e\u003cp\u003eA total of five patients with POI were enrolled in this study. The median age of the cohort was 33.20 years (31.00\u0026ndash;37.00 years), and the median serum FSH level was 72.76 mIU/ml (33.19\u0026ndash;136.22 mIU/ml) at the time of enrollment. During preoperative monitoring, no antral follicle-like structure was detected in the ovaries of all five patients. Comprehensive baseline characteristics and hormonal profiles of the patients are detailed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eBaseline characteristics of patients enrolled\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"10\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003ePatientID\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eAge\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eYears of diagnosis of POI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eKaryotype abnormality\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e\u003cp\u003eBaseline hormone profile\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003eAFC of the ovary before ISA\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003ePharmaceutical supplement\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAMH(ng/mL)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFSH(mIU/mL)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eE2(pg/mL)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eLeft side\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eRight side\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePatient 01\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.02\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e33.19\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e42.46\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003eEstradiol/Dydrogesterone Tablets\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePatient 02\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e31\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003eNo\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.02\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e136.22\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;15.00\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003eEstradiol/Dydrogesterone Tablets\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePatient 03\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e34\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003eNo\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.02\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e73.35\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;15.00\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003eEstradiol/Dydrogesterone Tablets\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePatient 04\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e32\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003eNo\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.02\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e87.23\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;15.00\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003eEstradiol/Dydrogesterone Tablets\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePatient 05\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e32\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003eNo\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e0.03\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e33.79\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e138.26\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003eEstradiol/Dydrogesterone Tablets\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eFollicle development, oocyte retrieval, fertilization and pregnancy outcome\u003c/h2\u003e\u003cp\u003eDuring the 6-month follow-up period after Df-IVA, follicular development was observed in all five patients. A total of 21 antral follicle-like structures were identified in 25 ultrasound monitoring sessions. In two patients, oocyte retrieval was performed, yielding a total of 2 MII oocytes (one viable cleavage embryos and one degenerated cleavage embryo obtained) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In a longer follow-up period, a total of 11 oocyte retrieval procedures were performed, ultimately yielding 7 viable cleavage embryos (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Patient 3 underwent transfer of a single viable vitrified-warmed cleavage embryo but failed to achieve clinical pregnancy. Patient 5 underwent a vitrified-warmed embryo transfer of two viable cleavage embryos on August 2024 and achieved a live birth (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eFollicular development, cryopreserved embryos and pregnant outcome after surgery\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOocyte retrieval and IVF/ICSI outcome during the 6-month follow-up period after Df-ISA\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCryopreserved embryos and pregnant outcome in longer follow-up period after Df-ISA\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber of patients recruited\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTotal number of cryopreserved embryos ready for transfer\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber of AFCs observed\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNumber of patients with cryopreserved embryos transferred\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCycle number with oocyte retrieval\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eNumber of patients being pregnant after ET/FET\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eOocyte obtained with oocyte retrieval\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eNumber of live birth\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eViable cleavage embryo number obtained via IVF/ICSI\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eGiven that the surgical and control sides of the ovaries were randomly assigned pre-operatively, post-operative follow-up allowed for a comparative analysis of the number of antral follicle-like structures between the two sides to assess the surgical effect. In the ongoing follow-up, a total of 27 antral follicle-like structures were observed in the Df-ISA ovaries of the five patients in 35 ultrasound monitoring sessions, while only 6 antral follicle-like structures were identified in the control ovaries. A paired t-test revealed a statistically significant difference between the two groups (P\u0026thinsp;=\u0026thinsp;0.0005) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eOur innovative Df-ISA procedure achieved, for the first time, in-situ dense micro-incision of the entire ovarian cortex in a cohort of 5 POI patients, yielding encouraging outcomes. All participants exhibited follicular recruitment, with 2 patients producing viable embryos and ultimately achieving one live birth.\u003c/p\u003e\u003cp\u003eMechanobiology, an emerging field that researches the contribution of physical forces on changing to cellular biology, has perfectly inspired the concept of fragmenting the ovarian cortex for activation of primordial follicles. It is long known that ovarian wedge resection or laparoscopic ovarian laser drilling promote follicle growth in patients with polycystic ovary syndrome (PCOS) \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Since the first conception of IVA demonstrated by Dr. Kawamura et al. in 2013\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e, the IVA protocol was gradually simplified. Initially, the protocol required 2 surgical operations for tissue collection and autologous transplantation and a 2-day drug treatment of the ovarian cortex\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Subsequently, the IVA procedure was optimized from requiring in vitro drug culture shortened to completing all steps in a single surgery, yielding gratifying clinical outcome\u003csup\u003e[\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. All the modifications restored ovarian function and fertility to a certain extent. These clinical trial phenomena are consistent with the observations that disruption of the Hippo signaling pathway through ovarian tissue fragmentation can activate the dormant primordial follicles\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn our study, all five patients had small ovarian volumes with a more solid texture. Only two of them had one antral follicle-like structure each at enrollment. After Df-ISA, antral follicle development were observed in all five patients. The observation is consistent with previous report showing an increase in AFC counts in POR patients following drug-free IVA \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Other studies reported that mechanical stimulation of the ovaries could activate dormant follicles in most patients with Extremely Poor Ovarian Response (EPOR), restore hormonal responsiveness, and lead to multiple live births \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. The mechanisms underlying the use of different mechanical stimulation methods for improving ovarian function may vary slightly: The IVA procedure may primarily act on activation of dormant primordial follicles, whereas the Whole Ovary Laparoscopic Incision (WOLI) procedure may mainly rescue the developing secondary follicles \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn our study cohort following the Df-ISA procedure, 2 patients underwent oocyte retrieval with a retrieval rate of 40%, consistent with a reported retrieval rate of 35.71% for POI patients \u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Interestingly, the oocyte retrieval rate was 70% in DOR patients undergoing cortical tissue fragmentation and autologous-transplantation \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. This suggests that the success of Df-ISA depends on the presence of residual ovarian follicles to some extent. The relatively higher success rate in DOR patients highlights the importance of baseline ovarian reserve in determining the efficacy of Df-ISA.\u003c/p\u003e\u003cp\u003eIn this study, a total of 11 oocyte retrievals were performed in patients with POI in longer follow-up. Among these, two retrievals failed to obtain oocytes. All the retrieved oocytes were subjected to IVF or ICSI. Two oocytes exhibited abnormal fertilization, the remaining 7 oocytes were normally fertilized and yielded 7 viable cleavage embryos. The fertilization rate was 77.8%, which is slightly higher than that reported by Grin (59.0%/72.0%) in POI/POR patients\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e and Kawamura (68.7%) in POR patients with DOR using in vitro activation \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. These data support the effectiveness of mechanical scratch in activating and maturing oocytes for fertilization. Among the five patients enrolled in our study, two had embryo cryopreservation. Ferreri et al.\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e and Fabregues\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e et al. reported that approximately 28\u0026ndash;30% of POI patients undergoing drug-free IVA achieved embryo cryopreservation. In POR patients, Grin et al. reported that all studied patients (7/7) had at least one embryo cryopreserved, compared to 35% of patients in the POI group \u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. This comparison highlights that severe follicle depletion remains a significant factor in the reproductive outcomes of IVA.\u003c/p\u003e\u003cp\u003eAmong the five patients enrolled, two patients had 1 and 6 utilzable cleavage embryos respectively. The patient with only one embryo underwent HRT embryo transfer but failed to get pregnant. The other patient underwent HRT embryo transfer of two cleavage embryos and successfully had a live birth. Another study reported that among 14 patients with POI who underwent Df-IVA, only one clinical pregnancy occurred, which unfortunately ended in an early miscarriage \u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. These observations suggest that the pregnancy and live birth rates for POI patients undergoing IVF treatment remain at a relatively low level.\u003c/p\u003e\u003cp\u003eWe observed a significantly higher number of antral follicles in the operated ovary compared with the untouched control ovary. Among 5 patients, antral follicle development was all observed post-Df-ISA and most follicular recruitment occurred in the operated ovary with a paired t-test P value\u0026thinsp;\u0026lt;\u0026thinsp;0.001. This suggested Df-ISA can lead to rapid induction of residual secondary follicles growth into antral follicles. However, another research found no difference in the number of mature follicles after ovarian stimulation 10 weeks after Df-IVA versus the control ovaries (1.0 vs.0.7 follicles, P\u0026thinsp;=\u0026thinsp;0.35) in POI patients \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eBiomechanics shows that fragmentation of human and mice ovarian cortex into small cubes promotes conversion of G-actin into F-actin \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e, which disrupts the Hippo signaling pathway and induces nuclear localization of the Yes-associated protein and connective tissue growth factors to activate secondary follicle growth in vitro and in vivo \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Theoretically, the more cuts on the ovarian cortex mean more activation and ample evidence underscores the essential role of mechanical signaling in the development of the ovary, follicle, and oocyte during gametogenesis\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. In our research, we conducted both longitudinal and transverse cut in situ on the ovary to increase density of the incisions so as to have more effect on the actin and therefor stronger action on the Hippo pathway and downstream factors.\u003c/p\u003e\u003cp\u003eThe limitations of our study are that it is a single-center pilot study, and the sample size is small. We plan to continue to recruit patients in the hope of obtaining higher levels of positive evidence. Given that IVA/ISA efficacy is likely contingent upon the presence of residual ovarian follicles, there is a pressing need for future studies to devise more sensitive assays capable of accurately predicting both the quantity of growing follicles and the residual primordial follicle pool in POI patients.\u003c/p\u003e\u003cp\u003eThis study underscores the therapeutic potential of Df-ISA for patients with POI, highlighting its capacity to induce follicular activation, enhance oocyte retrieval rates, and improve reproductive outcomes. Future research endeavors should prioritize head-to-head comparisons between drug-free and chemically activated IVA/ISA protocols, as well as delve into angiogenic mechanisms, to elucidate optimal treatment strategies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAuthor contributionsStudy design: TW, YL, XW, KW; Conduct of the clinical study: YL, XW, HZ, RL, JS, PZ; Quality assessment of the clinical study: RHWL, EHYN, WSBY, J.V.Z, XCW, TW; Data analysis: YL, XW; Manuscript preparation: YL. All authors approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eYuan Li#, Xiaohui Wang#, Kun Wang#, Hongzhan Zhang, Jiaping Su, Rui Li, Peikun Zhao, Raymond H W Li, Ernest H Y Ng, William S B Yeung, J.V. Zhang, Xiangcai Wei*, Tianren Wang*#Co-first author*Corresponding author\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eSupport Funding\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003eThis work was supported by Shenzhen Fundamental Research Program, China (No: JCYJ20200109150429414); Shenzhen Science and Technology Program, China (No. RCYX20200714114705073, No. KQTD20190929172749226); the National Natural Science Foundation of China (No: 81971453); General Program Sponsored by Basic Research Fund in Shenzhen Natural Science Foundation (JCYJ20240813113030040); National Key R\u0026amp;D Program of China 2024YFA1803001;Shenzhen Medical Research Fund B2404004; Guangdong Basic and Applied Basic Research Foundation 2024A1515010059; Shenzhen Science and Technology Program JCYJ20220818101218040; Shenzhen Key Laboratory of Metabolic Health (Grant No. ZDSYS20210427152400001)\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSteiner A Z, Pritchard D, Stanczyk F Z, et al. Association Between Biomarkers of Ovarian Reserve and Infertility Among Older Women of Reproductive Age[J]. JAMA, 2017, 318(14): 1367-1376.\u003c/li\u003e\n\u003cli\u003eEuropean Society for Human R, Embryology Guideline Group On P O I, Webber L, et al. ESHRE Guideline: management of women with premature ovarian insufficiency[J]. Hum Reprod, 2016, 31(5): 926-37.\u003c/li\u003e\n\u003cli\u003eDatta A K, Maheshwari A, Felix N, et al. Mild versus conventional ovarian stimulation for IVF in poor, normal and hyper-responders: a systematic review and meta-analysis[J]. Hum Reprod Update, 2021, 27(2): 229-253.\u003c/li\u003e\n\u003cli\u003eKawamura K, Cheng Y, Suzuki N, et al. Hippo signaling disruption and Akt stimulation of ovarian follicles for infertility treatment[J]. Proc Natl Acad Sci U S A, 2013, 110(43): 17474-9.\u003c/li\u003e\n\u003cli\u003eVo K C T, Kawamura K. In Vitro Activation Early Follicles: From the Basic Science to the Clinical Perspectives[J]. Int J Mol Sci, 2021, 22(7).\u003c/li\u003e\n\u003cli\u003eTanaka Y, Hsueh A J, Kawamura K. Surgical approaches of drug-free in vitro activation and laparoscopic ovarian incision to treat patients with ovarian infertility[J]. Fertil Steril, 2020, 114(6): 1355-1357.\u003c/li\u003e\n\u003cli\u003eSuzuki N, Yoshioka N, Takae S, et al. Successful fertility preservation following ovarian tissue vitrification in patients with primary ovarian insufficiency[J]. Hum Reprod, 2015, 30(3): 608-15.\u003c/li\u003e\n\u003cli\u003eFabregues F, Ferreri J, Calafell J M, et al. Pregnancy after drug-free in vitro activation of follicles and fresh tissue autotransplantation in primary ovarian insufficiency patient: a case report and literature review[J]. J Ovarian Res, 2018, 11(1): 76.\u003c/li\u003e\n\u003cli\u003eKawamura K, Ishizuka B, Hsueh A J W. Drug-free in-vitro activation of follicles for infertility treatment in poor ovarian response patients with decreased ovarian reserve[J]. Reprod Biomed Online, 2020, 40(2): 245-253.\u003c/li\u003e\n\u003cli\u003eZhang X, Han T, Yan L, et al. Resumption of Ovarian Function After Ovarian Biopsy/Scratch in Patients With Premature Ovarian Insufficiency[J]. Reprod Sci, 2019, 26(2): 207-213.\u003c/li\u003e\n\u003cli\u003eFarquhar C, Vandekerckhove P, Lilford R. Laparoscopic \u0026quot;drilling\u0026quot; by diathermy or laser for ovulation induction in anovulatory polycystic ovary syndrome[J]. Cochrane Database Syst Rev, 2001(4): CD001122.\u003c/li\u003e\n\u003cli\u003eVo K C T, Kawamura K. Ovarian Fragmentation and AKT Stimulation for Expansion of Fertile Lifespan[J]. Front Reprod Health, 2021, 3: 636771.\u003c/li\u003e\n\u003cli\u003eLi J, Kawamura K, Cheng Y, et al. Activation of dormant ovarian follicles to generate mature eggs[J]. Proc Natl Acad Sci U S A, 2010, 107(22): 10280-4.\u003c/li\u003e\n\u003cli\u003eHsueh A J, Kawamura K, Cheng Y, et al. Intraovarian control of early folliculogenesis[J]. Endocr Rev, 2015, 36(1): 1-24.\u003c/li\u003e\n\u003cli\u003eHsueh A J W, Kawamura K. Hippo signaling disruption and ovarian follicle activation in infertile patients[J]. Fertil Steril, 2020, 114(3): 458-464.\u003c/li\u003e\n\u003cli\u003eBayram N, Van Wely M, Kaaijk E M, et al. Using an electrocautery strategy or recombinant follicle stimulating hormone to induce ovulation in polycystic ovary syndrome: randomised controlled trial[J]. BMJ, 2004, 328(7433): 192.\u003c/li\u003e\n\u003cli\u003eLunding S A, Pors S E, Kristensen S G, et al. Biopsying, fragmentation and autotransplantation of fresh ovarian cortical tissue in infertile women with diminished ovarian reserve[J]. Hum Reprod, 2019, 34(10): 1924-1936.\u003c/li\u003e\n\u003cli\u003eChang C L, Chin T H, Hsu Y C, et al. Whole-Ovary Laparoscopic Incisions Improve Hormonal Response and Fertility in Patients with Extremely Poor Ovarian Response[J]. J Minim Invasive Gynecol, 2022, 29(7): 905-914.\u003c/li\u003e\n\u003cli\u003eChang C L. Facilitation of Ovarian Response by Mechanical Force-Latest Insight on Fertility Improvement in Women with Poor Ovarian Response or Primary Ovarian Insufficiency[J]. Int J Mol Sci, 2023, 24(19).\u003c/li\u003e\n\u003cli\u003eFerreri J, Fabregues F, Calafell J M, et al. Drug-free in-vitro activation of follicles and fresh tissue autotransplantation as a therapeutic option in patients with primary ovarian insufficiency[J]. Reprod Biomed Online, 2020, 40(2): 254-260.\u003c/li\u003e\n\u003cli\u003eGrin L, Berkovitz-Shperling R, Goldshtein G, et al. Drug-free In Vitro Activation of Ovarian Follicles and Fresh Tissue Autotransplantation in Patients with Poor ovarian response and Premature Ovarian Insufficiency[J]. F S Sci, 2025.\u003c/li\u003e\n\u003cli\u003eHendriks M L, Ket J C, Hompes P G, et al. Why does ovarian surgery in PCOS help? Insight into the endocrine implications of ovarian surgery for ovulation induction in polycystic ovary syndrome[J]. Hum Reprod Update, 2007, 13(3): 249-64.\u003c/li\u003e\n\u003cli\u003eShah J S, Sabouni R, Cayton Vaught K C, et al. Biomechanics and mechanical signaling in the ovary: a systematic review[J]. J Assist Reprod Genet, 2018, 35(7): 1135-1148.\u003c/li\u003e\n\u003cli\u003eCheng Y, Feng Y, Jansson L, et al. Actin polymerization-enhancing drugs promote ovarian follicle growth mediated by the Hippo signaling effector YAP[J]. FASEB J, 2015, 29(6): 2423-30.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","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":"Drug-free In Situ Activation, Premature ovarian insufficiency, Mechanical disruption, Reproductive outcome, Laparoscopic Ovarian Cortical Incisions","lastPublishedDoi":"10.21203/rs.3.rs-7378665/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7378665/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\u003eIndividuals with premature ovarian insufficiency (POI) face significant challenges in attaining favorable reproductive outcomes. To date, no effective interventions have been established to substantially improve this complex clinical scenario.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe conducted a clinical trial in a tertiary university-affiliated hospital to study a protocol of Drug-free In Situ Activation (Df-ISA) of ovarian follicles for treatment of POI patients in improving reproductive outcomes. Five women were enrolled and accepted the Df-ISA procedure through longitudinal and transverse mechanical cutting on one ovary. The contralateral ovary remained intact as control. Changes in antral follicle count (AFC), oocyte retrieval rates, embryo cryopreservation, clinical pregnancy and live birth outcomes were clinical observational indicators.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAntral follicle development was observed in all five patients by ultrasound monitoring after Df-ISA. A total of 21 echo-free antral follicle-like structures were identified and one viable cleavage embryo were obtained in the 6-month follow-up. In the longer follow-up, a total of seven cleavage embryos were obtained from two patients, and one live birth was achieved. Paired t-test revealed a statistically significant difference of follicle-like structures observed between the Df-ISA ovaries and the contralateral control ovaries.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \"Longitudinal and Latitudinal cutting in situ\" Df-ISA procedure represents a novel infertility treatment for patients with POI. This procedure promotes the growth of residual antral ovarian follicles through dense mechanical disruption, yielding encouraging results that warrant further investigation.\u003c/p\u003e\n\u003cp\u003eThe study adhered to the principles outlined in the Declaration of Helsinki and was granted ethical approval by the Ethics Research Committee of the University of Hong Kong-Shenzhen Hospital and has been registered in the National Medical Research Registration and Filing Information System, with the registration number ChiCTR2400086676.Every participant has provided informed consent.\u003c/p\u003e","manuscriptTitle":"Drug-free In Situ Activation of Follicle through Laparoscopic Ovarian Cortical Incisions in Patients with Premature Ovarian Insufficiency","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-23 11:49:56","doi":"10.21203/rs.3.rs-7378665/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":"c53e1567-c9cb-4583-aa0a-bb0c8cf60a81","owner":[],"postedDate":"November 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-10T04:08:51+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-23 11:49:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7378665","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7378665","identity":"rs-7378665","version":["v1"]},"buildId":"WvIrzKhiLBfengagbw6Ux","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}