{"paper_id":"68887b0a-39d8-4929-911c-cf0d094636de","body_text":"1\nVol.:(0123456789)Scientific Reports |        (2020) 10:20911  | https://doi.org/10.1038/s41598-020-77781-w\nwww.nature.com/scientificreports\nEpinephrine minimizes \nthe use of bipolar coagulation \nand preserves ovarian reserve \nin laparoscopic ovarian cystectomy: \na randomized controlled trial\nEun Young Park1, Kyu‑Hee Hwang2, Ji‑Hee Kim2, San‑Hui Lee1, Kyu‑Sang Park2, \nSeong Jin Choi1* & Seung‑Kuy Cha2*\nWe propose a novel method, the epinephrine compression method (Epi‑pledget), as a hemostasis \nmethod for ovarian cystectomy. A total of 179 patients undergoing laparoscopic ovarian cystectomy \nwith stripping were randomly allocated into three groups: the bipolar coagulation group, the Epi‑\npledget group, and the coagulation after Epi‑pledget (Epi & Coagulation) group. Serum anti‑Müllerian \nhormone (AMH) levels and antral follicle count (AFC) by ultrasonography were measured to determine \nthe preservation of ovarian function. To evaluate the postoperative ovarian cellular proliferative \nactivity and tissue damage in a mouse model, we operated on the ovaries of mice with an artificial \nincision injury and applied two hemostatic methods: coagulation and Epi‑pledget. Eight weeks after \nsurgery, the AMH rate significantly decreased in the bipolar coagulation group compared with the \nEpi‑pledget group. The AFC decline rate was also significantly greater in the coagulation group than \nthe Epi‑pledget group. Specifically, patients with endometrioma had a significantly greater decline \nof serum AMH in the coagulation group than the Epi‑pledget group. In a histopathological analysis in \nmice, the Epi‑pledget group showed ameliorated fibrotic changes and necrotic findings in the injured \nlesion compared with the bipolar coagulation group. The Epi‑pledget method for ovarian stripping \nhas an additional benefit of maximizing the preservation of the ovarian reserve, especially for the \nendometriotic ovarian cyst type.\nSurgical laparoscopy is the most common technique for the treatment of benign ovarian cysts. Laparoscopic \novarian cystectomy has been demonstrated to improve post-operative fecundability and reduce the recurrence \nrate compared with fenestration and coagulation of the cyst  wall\n1. However, laparoscopic cystectomy is associated \nwith a risk of surgical injury to the remnant normal ovarian  tissue1. The radical laparoscopic surgical approach \nresulted in an improvement of several disease-related symptoms and quality of life  indices2. Balancing the surgical \nradicality between the success of treatment (reduction of pain and recurrence) and the ovarian reserve is a chal-\nlenging issue. Another crucial issue in laparoscopic ovarian cystectomy is how to reduce the damage and bleeding \nto preserve ovarian function. Many factors have been reported to affect the ovarian reserve, such as different \nsurgical techniques for the excision (traditional stripping and combined excisional/ablative techniques)\n3, mode \nof hemostasis (suture, compression, and cauterization)4, operational method (cystectomy vs. wedge resection), \nand cyst characteristics (size, location, and pathologic types)5,6. Notably, hemostasis by bipolar coagulation, which \nis commonly used, can result in thermal damage to healthy ovarian follicles and reduce the ovarian  reserve7–9.\nPharmacological intervention is an alternative hemostasis method for minimizing tissue damage. Vasopressin \nis commonly used for hemostasis that occurs from the stripping of ovarian cysts and  myomectomy10. However, \nintra-operative local injections of vasopressin frequently cause cardiopulmonary complications, such as cardiac \nOPEN\n1Department of Obstetrics and Gynecology, Yonsei University Wonju College of Medicine, 20 Ilsan-ro, \nWonju, Gangwondo 26426, Republic of Korea. 2Department of Physiology, Department of Global Medical \nScienceMitohormesis Research Center, Institute of Mitochondrial Medicine, Yonsei University Wonju College \nof Medicine, 20 Ilsan-ro, Wonju, Gangwondo 26426, Republic of Korea.  *email: choisj@yonsei.ac.kr ; skcha@\nyonsei.ac.kr\n\n2\nVol:.(1234567890)Scientific Reports |        (2020) 10:20911  | https://doi.org/10.1038/s41598-020-77781-w\nwww.nature.com/scientificreports/\narrest, hypotension, and pulmonary  edema11–13. Therefore, we propose an alternative pharmacological interven-\ntion for hemostasis, epinephrine pledget compression (Epi-pledget). The Epi-pledget is widely used in endoscopic \nsinonasal surgery for local vasoconstriction and bleeding  control14, subsequently reducing blood leakage to \nthe maxillary sinus and subcutaneous tissue after canine fossa  puncture15. A recent study showed that topical \nepinephrine applied in prostate surgery facilitated post-operative  hemostasis16. Intravenous low-dose epineph-\nrine was used in total hip arthroplasty, resulting in a reduction of both bleeding and inflammatory  reaction17. \nIn addition, an epinephrine injection successfully produced hemostasis for bleeding of colonic diverticula in \n colonoscopy\n18. The present study evaluated the efficacy of the Epi-pledget on hemostasis to minimize subsequent \ncomplications including decreased ovarian reserve, tissue damage and bleeding in laparoscopic cystectomy.\nResults\nTable 1 shows the baseline characteristics of all participants in the three groups. The mean age, BMI, a maximum \ndiameter of ovarian cysts, and hemoglobin level did not differ among the three groups. Also, there was no dif -\nference in preoperative anti-Müllerian hormone (AMH) and antral follicle count (AFC) levels among the three \ngroups (Table 1, Supplementary Table S1).\nFigure  1 illustrates tissue changes in representative cases after hemostasis using different methods. We \nobserved thermal injury in the bipolar coagulation group (Fig.  1). The Epi-pledget compression successfully \nstopped bleeding without burn injury in the stripped lesion of laparoscopic ovariectomy (Fig. 1).\nWe measured the levels of AMH and AFC before and after surgery. Both the serum level of AMH and AFC \ncount significantly decreased in the bipolar coagulation group compared with the Epi-pledget compression group \nand the Epi & Coagulation group (Table 2). Of note, no significant difference was observed regarding AMH and \nAFC between the Epi-pledget compression group and the Epi & Coagulation group (Table  2). We investigated \nthe effect of hemostatic methods on AMH and AFC in patients stratified according to age. The group consisting \nof older subjects (age ≥  34) showed a significant difference in AMH according to different hemostatic methods \n(Table 2). Among the subgroups of the four types of ovarian cyst, the differences of AMH and AFC decline were \ninsignificant (Supplementary Table S2).\nHistopathological examination was conducted in all patient cases (n = 198), yielding the following diagnoses: \nbenign ovarian cyst, either mature cystic teratoma (n = 75, 42%) or endometrioma (n = 69, 39%); or serous and \nmucinous cystadenoma (n = 35, 20%). No significant differences were evident in the mean maximum diameter \nof each ovarian cyst type [6.34  ± 1.7 cm (mean ± standard deviation) in the endometrioma group, 6.90 ± 2.0 in \nthe teratoma group, and 6.72 ± 2.86 in the serous and mucinous cyst group]. Furthermore, the rates of decline \nin serum AMH and AFC did not differ among the subtypes of ovarian cyst (data not shown).\nNext, we performed a subgroup analysis based on the endometrioma and non-endometrioma groups (Sup-\nplementary Table S1, Fig. 2). In the case of endometrioma, the Epi-pledget compression group showed less AMH \ndecline than the coagulation group (Fig. 2A, Supplementary Table S1). When comparing the rate of serum AMH \ndecline in the coagulation group, patients with endometrioma showed a greater AMH decline than those with \nnon-endometrioma (Supplementary Table S2). The rate of AFC decline was not significantly different among \nthe three hemostatic intervention groups and the ovarian pathologic cyst types.\nIn hemoglobin changes and operative time based on the three hemostasis methods, we found that neither \ndiffered between the bipolar coagulation group and the Epi-pledget compression group (Fig. 3A–D). However, \nthe coagulation group and the Epi & Coagulation group showed differences in operation time (Fig. 3C,D).\nTable 1.  Preoperative patient baseline characteristics. Data are presented as mean ± standard deviation, \nmedian (interquartile ranges), or frequencies (percentages). Epi-pledget Epinephrine compression, Hb \nhemoglobin, AMH anti-Mullerian hormone, AFC antral follicle count.\nBipolar Cauterization group \n(n = 65) Epi-pledget group (n = 62)\nEpi-pledget and Cauterization \ngroup (n = 52) P value\nAge (years) 32.6 ± 9.1 32.8 ± 8.0 32 ± 9.6 0.911\nBody mass index (kg/m\n2) 21.5 ± 3.7 21.9 ± 5.1 23.6 ± 6.2 0.074\nParity (n (%))\nNulliparous 49 (75%) 51 (82%) 34 (65%)\nParous 16 (15%) 11 (18%) 18 (35%)\nMaximum diameter of ovarian \ncyst (cm) 6.3 ± 2.3 6.9 ± 1.7 6.8 ± 2.2 0.304\nPreoperative Hb level (mg/dL) 12.13 ± 1.1 12.31 ± 1.2 12.62 ± 2.2 0.427\nPreoperative AMH level (ng/\ndL) 5.16 (2.54–7.26) 5.47 (2.6–7.66) 3.90 (2.89–5.9) 0.057\nPreoperative AFC (n) 2.52 ± 1.08 2.21 ± 0.94 2.25 ± 1.04 0.211\nType of ovarian cyst (n)\nEndometriotic cyst 20 26 13\nMature cystic teratoma 3 2 3\nSerous cystadenoma 11 11 2\nMucinous cystadenoma 31 23 34\n\n3\nVol.:(0123456789)Scientific Reports |        (2020) 10:20911  | https://doi.org/10.1038/s41598-020-77781-w\nwww.nature.com/scientificreports/\nTogether these results indicate that Epi-pledget compression is a favorable hemostasis intervention for the fol-\nlicle reserve in patients. To examine pathological changes after applying hemostatic interventions, we employed a \nmouse model and evaluated acute and chronic changes in the injured ovary tissues after hemostatic interventions \n(Fig. 4A). No cardiovascular or other complications were observed in the mice, although the same concentrations \nof epinephrine (0.1%) were used in human patients. Electrocoagulation intervention exaggerated blood vessel \ncollapse and fibrotic changes in mouse ovaries more so than epinephrine intervention (Fig. 4B,C). Moreover, elec-\ntrocauterization caused a wide range of tissue injuries with eosinophilic granulomatous tissue formation around \nthe necrotic area and a decrease in the number of growing follicles, while Epi-pledget intervention ameliorated \nthe tissue and follicle injuries (Fig.  4D,E). Next, we examined the proliferative potential after hemostasis inter-\nventions using Ki-67, a proliferation marker. Ki-67 immunoreactivity in the granulosa cell layer of the growing \nFigure 1.  An operative finding of hemostasis on a laparoscopic ovarian cystectomy lesion. Hemostasis with \nepinephrine-soaked pledget compression (Epi-pledget compression) and bipolar coagulation before and after \nlaparoscopic stripping of ovarian cysts. Y ellow and blue arrows indicate bleeding of the laparoscopic cystectomy \nlesion and Epi-pledget compression on the stripping lesion, respectively.\nTable 2.  Comparison of AMH and AFC decline rates between hemostatic groups. Data are presented \nas median (interquartile ranges). Decline rate was defined as 100 x [preoperative level (AMH or AFC) – \npostoperative level (AMH or AFC)] / preoperative level (AMH or AFC). AMH anti-Mullerian hormone, AFC \nantral follicle count, Tukey HSD Tukey honest significant differences. \na p < 0.05 vs. Bipolar coagulation group \nby Tukey HSD post hoc comparison. b p < 0.05 vs. Epi-pledget compression group by Tukey HSD post hoc \ncomparison.\nTypes of hemostasis\nP-valueBipolar Cauterization group Epi-pledget group Epi-pledget & Cauterization group\nAll subjects (n = 65) (n = 62) (n = 52)\nRate of AMH decline (%) 24.5 (8.1–40.4) 0.8 (–18.7–39.2) a 10.34 (–17–30.0) a 0.005\nRate of AFC decline (%) 33.3 (– 41.7–60.0) b 25.0 (–100–50) 25.0 (–50–50) 0.026\nAge < 34 years (n = 42) (n = 34) (n = 31)\nRate of AMH decline (%) 24.4 (7.5–40.6) 4.0 (–24.8–35.6) 20.5 (–11.1–43.3) 0.052\nRate of AFC decline (%) 33.3 (– 25.0–50.0) 29.2 (–50–45.8) 0 (–100–50) 0.125\nAge ≥ 34 years (n = 23) (n = 28) (n = 21)\nRate of AMH decline (%) 27.1 (14.9–39.22) – 2.4 (–8.5–40.5) – 3.7 (–37.5–18.5) a 0.003\nRate of AFC decline (%) 50.0 (–41.7–63.3) 0 (–100–50) 33.3 (–50.0–50.0) 0.09\n\n4\nVol:.(1234567890)Scientific Reports |        (2020) 10:20911  | https://doi.org/10.1038/s41598-020-77781-w\nwww.nature.com/scientificreports/\nFigure 2.  Comparison of AMH decline rates between hemostatic groups according to ovarian pathologic \nsubtypes. AMH decline rates (%) in endometrioma (A) and non-endometrioma (B) groups. Decline rate was \ndefined as 100 × [preoperative AMH level – postoperative AMH level] / preoperative AMH level. *p < 0.05. \nCaut cauterization, Epi epinephrine-pledget compression, Epi + Caut cauterization and epinephrine-pledget \ncompression.\nFigure 3.  Postoperative change of hemoglobin and operation time. (A) Hemoglobin (Hb) decline rate in \nindividual patients between hemostatic groups. Decline rate (%) was defined as 100 × [preoperative Hb \nlevel – postoperative Hb level] / preoperative Hb level. (B) Summary of panel A. (C) Operation time (min) of \npatients between hemostatic groups. (D) Summary of panel C. *p < 0.05. Data are presented as mean ± standard \ndeviation. Caut cauterization, Epi epinephrine-pledget compression, Epi + Caut cauterization and epinephrine-\npledget compression, Hb hemoglobin.\n\n5\nVol.:(0123456789)Scientific Reports |        (2020) 10:20911  | https://doi.org/10.1038/s41598-020-77781-w\nwww.nature.com/scientificreports/\nfollicle was markedly reduced in the electrocoagulation group, while the Epi-pledget intervention maintained \nKi-67-positive cells (Fig.  5A). This was further confirmed by the Ki-67 index, which was significantly lower in \nthe electrocoagulation group compared with the epinephrine group (Fig. 5B).\nDiscussion\nThe present study demonstrates the beneficial effects of epinephrine pledget compression on an ovarian cystec-\ntomy lesion preserving ovarian reserve. Additionally, we also provide evidence that the Epi-pledget causes less \nthermal damage to the ovarian parenchyma than bipolar coagulation in an animal model. Electrocoagulation \nwas shown to cause a greater decline in serum AMH and/or AFC after surgery than  suture\n19,20. The hemostatic \nmethod in group 3 included both Epi-pledget and bipolar cauterization. In detail, the major hemostatic method \nin group 3 was the Epi-pledget and the bipolar cauterization was the assistant hemostatic method for oozing \nsite after the application of the Epi-pledget. The Epi-pledget reduced the time of application of the bipolar cau-\nterization and might alleviate the ovarian damage by electronic power. In hemostatic interventions according to \nthe pathologic subtype of ovarian cyst, the rate of serum AMH decline was lower in the endometrioma group \nwith bipolar coagulation than in the endometrioma group with Epi-pledget compression or Epi & coagulation. \nAnimal experiments supported the notion that bipolar coagulation aggravated the reduction of ovarian reserve \nafter surgery and caused irreversible ovarian tissue damage compared with that of the Epi-pledget. The primary \npathophysiological mechanisms in irreversible ovarian tissue injury were inflammation and fibrosis that were \ninduced by the thermal damage by electrocauterization rather than vasoconstriction and ischemic change by \npharmacological intervention.\nThe suture had fewer adverse effects than bipolar coagulation in bleeding ovarian tissue after  stripping\n19,20. \nMaterials of gel or film type, such as elastin-thrombin matrix sealant, have also been proposed for  hemostasis21. \nFigure 4.  Quality of fibrosis and ovarian tissue reservation by hemostasis interventions in an injured ovary \nmice model. (A) Transverse incision at the lower back of the mouse and entry into the retroperitoneal space \nto approach the ovary. (B) Masson’s trichrome stain of mouse ovaries of each group (magnification, 200×). \nNecrosis region (*) and fibrotic tissue region (arrow, magnification, 400×) were found in the electrocauterization \ngroup. (C) Quantification of fibrotic area (%). *p < 0.05 (D) Histological evaluation of injured ovarian \ntissues after hemostasis with Epi-pledget compression and bipolar coagulation. The tissue of the Epi-pledget \ncompression was almost similar to the sham group, whereas necrosis and fibrotic tissue change were observed in \nthe coagulation group. An asterisk indicates necrotic regions. Magnification, 200×. (E) The follicles of each slide \nwere counted to obtain the mean value. **p < 0.01. Sham normal ovary, Caut electrocauterization group, Epi \nepinephrine-soaked cotton pledget compression.\n\n6\nVol:.(1234567890)Scientific Reports |        (2020) 10:20911  | https://doi.org/10.1038/s41598-020-77781-w\nwww.nature.com/scientificreports/\nHowever, in patients with a past medical history of using hemostatic sealant during gynecologic surgery, adhe-\nsion to the adjacent adnexal organ or bowel obstruction has been  reported22.\nVasoconstriction agents are widely used for hemostasis by the tourniquet effect on blood  vessels23. Agents \nare injected directly into the bleeding lesion to constrict blood vessels, but these methods could cause a harmful \nreaction to the systemic circulation. Epi-pledget compression has been applied to the bleeding lesion in vari-\nous other clinical  settings\n15 and used as a reasonable choice to assist in securing the operative field. However, \nepinephrine itself could have cardiovascular  effects23. The mechanism of cardiac muscle susceptibility to hyper-\nsensitivity is not well understood, but increases in arterial blood pressure, changes in heart rate, and increased \ncardiac muscle autonomy are factors affecting  arrhythmia\n24. In this study, we conducted hemostasis by the 0.1% \nepinephrine-soaked pledget compression method, not an injection. Our results showed that during the operation \nof 114 patients with the Epi-pledget, no systemic cardiovascular problems occurred. Furthermore, there were \nno cardiovascular complications in animal experiments.\nThere were no significant differences in pre-postoperative hemoglobin changes and operation time between \nthe bipolar coagulation and epinephrine intervention groups. Indeed, the advantages of electro-cauterization \nin laparoscopic ovarian stripping are a reduction in hemorrhage and a shorter operation  time\n23. In the present \nstudy, we showed that simple Epi-pledget compression alone or assisted application of Epi-pledget compression \ncould be comparable to the cauterization method. The Epi & Coagulation group had a significantly longer opera-\ntion time than the coagulation group, but this did not affect the decline in AMH and hemoglobin levels. These \nresults suggest that surgeons should consider the use of Epi-pledget compression alone or with minimal assis -\ntance of coagulation to preserve the ovarian reserve after laparoscopic ovarian cystectomy. Similar to our study, \nthe Epi-pledget or epinephrine-involved intervention was applied in other fields of surgery or  procedures\n14–18. \nThe effectiveness of hemostasis and the preservation of normal tissue function of the Epi-pledget indicated the \npotential application at the surgery for other hormone-secreting  tissues25.\nThe challenging issue in the surgical treatment of ovarian cysts is the possibility of encountering an unex-\npected ovarian malignancy. Approximately 0.9% and 3.0% of benign-appearing ovarian cysts were reported as \nunsuspected ovarian cysts in pre- and post-menopausal patients,  respectively\n26. As another study  recommended26, \nFigure 5.  Proliferative capability by hemostasis interventions. (A) Proliferation capability was evaluated using \nKi-67 immunoreactivity in mouse ovary tissue. Magnification, 200×. Stained nuclei (arrow) of the cauterization \ngroup ovarian tissue with Ki-67; the stained nuclei of normal growing follicular cells with Ki-67 (arrow) are \neasily identified. Magnification of inset, 400×. (B) Ki-67 index was calculated as the percentage of the number of \nstained nuclei at 1 and 4 days after the operation. **p < 0.01 vs. Sham group.\n\n7\nVol.:(0123456789)Scientific Reports |        (2020) 10:20911  | https://doi.org/10.1038/s41598-020-77781-w\nwww.nature.com/scientificreports/\nwe strictly selected subjects via medical history, physical examination, serum CA125, and the transvaginal US. \nAs a result, we did not report any patients diagnosed with ovarian malignancy.\nThere were several limitations to our study. First, the sample size of the endometrioma group was relatively \nsmall for showing the statistical significance of epinephrine effects on ovarian reserve levels. Further studies \nshould increase the sample size and evaluate the quality of tissue healing to clarify the tissue changes according to \nvarious hemostatic methods, such as atraumatic suturing or various hemostatic sealants. Second, outcomes were \nmeasured over a relatively short 6–8-week follow-up period to determine the postoperative change in AMH levels \nor in tissue damages. Finally, our study compared the effects of coagulation with epinephrine. Further studies \nare needed to examine the hemostatic effects with ovarian reserve by various hemostatic sealants and vasocon-\ntraction agents such as vasopressin. Also, further comparison analyses between pharmacological interventions \nand suturing methods are required. Despite these limitations, our study provides unique strengths: a prospec -\ntive design, inclusion of a variety of cyst types, and comparison of a new hemostatic method with established \nhemostatic methods concerning the preservation of ovarian reserve and pathologic subtype. Moreover, we used \nanimal experiments to examine the pathologic changes in tissue after hemostatic surgery.\nOur study reveals that hemostasis by Epi-pledget compression significantly increased ovarian reserve preser-\nvation compared with cauterization during laparoscopic benign ovarian cystectomy. Experiments using a mouse \ncystectomy model support the findings that the Epi-pledget intervention effectively ameliorates irreversible tissue \ndamage causing the reduction of ovarian reserve. Based on the effectiveness and safety of bleeding control, the \nEpi-pledget showed similar effectiveness as bipolar coagulation. Our study suggests that Epi-pledget compression \nincreases the preservation of the ovarian reserve by minimizing the use of coagulation.\nMethods\nParticipants. According to the Consolidated Standards of Reporting Trials (CONSORT), we conducted a \nrandomized controlled trial of patients who underwent laparoscopic ovarian cystectomy between January 2019 \nand September 2019 in the Department of Obstetrics and Gynecology at the Y onsei Wonju Severance Chris-\ntian Hospital. The study was approved by the Institutional Review Board (IRB) of the Y onsei Wonju Severance \nChristian Hospital (CR118035). All procedures were conducted in adherence to the Declaration of Helsinki and \nCONSORT.\nInclusion criteria were as follows: unilateral ovarian cyst diagnosed by ultrasound examination; diameter \nof the ovarian cyst between 3 and 15 cm; and appropriate medical status for laparoscopic surgery (American \nSociety of Anesthesiologists Physical Status classification 1 or 2). In detail, we included operative subjects with \nsymptomatic huge size of ovarian cysts. We operated on patients with pathologic ovarian cysts (endometriotic \ncyst, dermoid cyst, serous cyst, mucinous cyst) that had multi-septated and heterogeneous features diagnosed by \ntransvaginal ultrasonogram (US). Exclusion criteria were as follows: evidence of any other endocrine disorder \nsuch as diabetes mellitus, thyroid dysfunction, hyperprolactinemia, and Cushing’s syndrome; postmenopausal \nstatus; pregnancy; the use of any hormonal treatment (i.e., use of oral contraceptive pills in the three months \nbefore surgery); previous surgery due to adnexal pathology; any complication during operation; conversion to \nlaparotomy; and loss of follow-up. We initially included 198 participants in this study, of which 179 participants \ncompleted the follow-up check. All patients provided written informed consent before participation (Fig. 6).\nWe named the epinephrine soaked pledget (hemostatic method) as Epi-pledget. We annotated the hemostatic \nmethod using the Epi-pledget and bipolar coagulation as Epi & Coagulation. Patients were randomly assigned \nto one of three groups: (1) bipolar coagulation, (2) Epi-pledget, and (3) epinephrine with coagulation (Epi & \nCoagulation), at a 1:1:1 ratio using a random permuted-block algorithm and a comprehensive internet-based \nrandomization service for clinical trials available at http://www.rando mize.net. The surgeon was not involved in \ngroup allocation. The patients were blinded to the hemostatic method to be used in their operation.\nStudy protocol. The primary outcome of the present study was the reserve of ovary tissue and the function \nafter laparoscopic ovarian cyst removal. The ovarian reserve was mainly checked by AFC and AMH. US exami-\nnation was performed with the 5–6-MHz transvaginal transducer (SSD-α10, Aloka, Japan) to determine the uni-\nlateral ovarian cyst size and antral follicle count (AFC) of both ovaries. All subjects underwent transvaginal and/\nor transrectal US examination twice, once preoperatively and once postoperatively. The preoperative examina-\ntion was done in the early follicular phase of the menstrual cycle (days 3–7). The postoperative examination was \ndone on a similar day during the second menstrual cycle at 6–8 weeks. Using the US, the AFC was determined \nas the total number of follicles with a diameter smaller than 10 mm, and both ovaries were counted using the \nlargest cross-sectional sagittal view of the ovary.\nIn all study groups, blood samples were taken within two weeks prior to and within 24 h after surgery. AMH \nlevels were checked on the same day as US examinations. The decline rate of the serum AMH/AFC level (%) \nwas calculated as follows: 100 × ([preoperative AMH/AFC level—postoperative AMH/AFC level] / preoperative \nAMH/AFC level).\nOperative procedure. Operative laparoscopy was performed in the usual manner described in several \nstudies. After the entire cystic portion was separated from the ovarian cortex without vasopressin, a hemostatic \nmaterial, bipolar coagulation, Epi-pledget compression, or Epi & Coagulation was applied for hemostasis. In the \nepinephrine groups, diluted epinephrine (0.5 mg/mL of epinephrine) in 50 mL of saline solution was used. In the \nEpi-pledget compression group, the bleeding sites were covered with the Epi-pledget and compressed directly \nwith a laparoscopic applicator for 2 min to allow hemostasis (Fig. 1). In the Epi & Coagulation group, minimal \nelectrical bipolar coagulation was performed after compression with the Epi-pledget for 1 min. All operations \nin this study were performed by one surgeon experienced in laparoscopic ovarian cystectomy (Dr. E. Y . Park). \n\n8\nVol:.(1234567890)Scientific Reports |        (2020) 10:20911  | https://doi.org/10.1038/s41598-020-77781-w\nwww.nature.com/scientificreports/\nAn electrocardiogram, used to detect arrhythmia, was performed before and after applying compression of the \nEpi-pledget. During compression of the Epi-pledget, systolic and diastolic blood pressure and heart rate were \nmeasured by an anesthesiologist. The subsequent procedures included careful inspection of the pelvic and peri-\ntoneal cavity by peritoneal washings, adhesiolysis for release, mobilization of the ovaries from the surrounding \nstructures, and aspiration of the cyst material by a suction needle. A negative pressure drainage bag (Hemovac) \nwas inserted into the cul-de-sac for enucleation of large-sized (> 10 cm) ovarian cysts and large volume irriga-\ntion (> 1000 cc). Hemostasis, on the ovarian cyst, stripped the oozing bed, was performed either by a 35–40 W \ncurrent bipolar coagulation or through the application of an epinephrine-soaked cotton pledget. Total operative \ntime was calculated from skin incision to skin closure. To confirm the diagnosis and exclusion of malignancy, \nthe cyst wall was sent for histological assessment. None of the operated ovaries were sutured. The subjects were \ndischarged from the hospital postoperative 2 or 3 days without postoperative fever and narcotic analgesics. All \nsubjects were seen for follow-up at 6 to 8 weeks after surgery.\nA complicated case was defined as when a patient experienced acute or delayed bleeding or epinephrine-\nrelated cardiopulmonary problems (dysrhythmias, hypotension, and pulmonary edema). We checked the degree \nof bleeding by comparing hemoglobin (Hb) levels from the blood test at the pre-operative assessment with those \nat 1 day and 6 to 8 weeks after surgery. There was no complicated case during the follow-up and we did not \nobserve any anesthesiology problems. Furthermore, no patients experienced post-operative fever or infection \nof port site lesions.\nAnimal model. Thirty female mice (C57B6/J) aged 12 weeks and around 21–23 g were randomly divided \ninto three groups according to hemostasis techniques: the sham (no artificial incision injury and treatment to the \novary) group, the bipolar coagulation group, and the Epi-pledget compression group (n = 10 per group). After an \noperation was performed to create an artificial incision injury to the ovary, bipolar coagulation, and Epi-pledget \ncompression techniques were applied to the injured ovary. Five mice in each group were sacrificed at postopera-\ntive day 1. The remaining five mice were sacrificed at postoperative day 4. Except for the sham group, the ovary \nwounds in all other groups were treated with hemostasis. The hair on the backs of the mice was shaved 2 days \nbefore surgery. Mice were anesthetized via inhalation followed by a transverse incision at the lower back of the \nmouse (Fig. 4A) and entry into the retroperitoneal space to approach the ovary. A partial incision of the ovaries \nresulted in wound production and bleeding of the ovary. To stop the bleeding, bipolar coagulation and diluted \nepinephrine (0.5 mg/mL of epinephrine in 50 mL of saline solution)-soaked cotton pledget compression were \nintroduced. When the stoppage of bleeding was confirmed in the damaged lesion, the skin was closed by suture \nand the mice were checked for vital signs and complications at 1 and 4 days. All animal research protocols were \napproved by the Institutional Animal Care and Use Committee, Y onsei University Wonju College of Medicine, \nKorea (YWC-170622–1).\nFigure 6.  Study flow diagram showing the study protocol and sequence of events in this study.\n\n9\nVol.:(0123456789)Scientific Reports |        (2020) 10:20911  | https://doi.org/10.1038/s41598-020-77781-w\nwww.nature.com/scientificreports/\nHistological evaluations of ovaries and determination of ovarian reserve. Ovaries were harvested and included \nnormal and wounded areas. All tissues were fixed in 4% paraformaldehyde for 48 h at room temperature. After \nfixation, ovaries underwent dehydration in graded ethanol and xylene and then were embedded in paraffin. Sec-\ntions were cut with a thickness of 5 μm and attached onto slides. The sections were deparaffinized in xylene and \nrehydrated in graded alcohol. After hydrating with deionized water, the sections were stained with hematoxylin \nand eosin (H&E), Masson’s trichrome (MTC) for detecting fibrosis, or Ki-67 for detecting proliferation. Stained \nsections were examined in a bright field with an Olympus microscope and photographed with a DP73 camera; \nthe images were exported to CellSens Software. Fibrotic areas were measured and analyzed using Image J software \n(http://image j.nih.gov/ij/imdex /html).\nStatistical analysis. Data were analyzed using Prism software (version 6, GraphPad Software, San Diego, \nCA, USA). Baseline clinical characteristics and study outcomes were compared among the three groups using a \none-way analysis of variance (ANOV A) with multiple comparisons followed by Tukey’s multiple comparisons. \nP-values less than 0.05 were considered significant.\nEthics approval and consent to participate. All procedures involving human participants were in \naccordance with the ethical standards of the institutional and/or national research committee and the 1964 Dec-\nlaration of Helsinki and its later amendments or comparable ethical standards. Informed consent was obtained \nfrom all participants included in the study.\nReceived: 25 December 2019; Accepted: 12 November 2020\nReferences\n 1. Kitajima, M. et al. Changes in serum anti-Müllerian hormone levels may predict damage to residual normal ovarian tissue after \nlaparoscopic surgery for women with ovarian endometrioma. Fertil. Steril. 95, 2589-2591.e2581. https ://doi.org/10.1016/j.fertn \nstert .2011.01.036 (2011).\n 2. Busacca, M. et al. Determinants of long-term clinically detected recurrence rates of deep, ovarian, and pelvic endometriosis. Am. \nJ. Obstet. Gynecol. 195, 426–432. https ://doi.org/10.1016/j.ajog.2006.01.078 (2006).\n 3. Muzii, L. et al. Comparison between the stripping technique and the combined excisional/ablative technique for the treatment of \nbilateral ovarian endometriomas: A multicentre RCT. Hum. Reprod. 31, 339–344. https ://doi.org/10.1093/humre p/dev31 3 (2016).\n 4. Sugita, A. et al. 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S.J.C. and S.K.C. \nsupervised the entire study. All authors contributed to the study design and approved the final version of the \nmanuscript for submission.\nFunding\nThis study was supported by the Medical Research Center Program (2017R1A5A2015369) through the National \nResearch Foundation of Korea and funded by the Ministry of Health & Welfare, Republic of Korea (HI18C2196).\nCompeting interests \nThe authors declare no competing interests.\nAdditional information\nSupplementary information is available for this paper at https ://doi.org/10.1038/s4159 8-020-77781 -w.\nCorrespondence and requests for materials should be addressed to S.J.C. or S.-K.C.\nReprints and permissions information is available at www.nature.com/reprints.\nPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and \ninstitutional affiliations.\nOpen Access  This article is licensed under a Creative Commons Attribution 4.0 International \nLicense, which permits use, sharing, adaptation, distribution and reproduction in any medium or \nformat, as long as you give appropriate credit to the original author(s) and the source, provide a link to the \nCreative Commons licence, and indicate if changes were made. The images or other third party material in this \narticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the \nmaterial. If material is not included in the article’s Creative Commons licence and your intended use is not \npermitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from \nthe copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.\n© The Author(s) 2020","source_license":"CC0","license_restricted":false}