{"paper_id":"4bea5b73-65ab-4b6a-8bb8-2bcad3303b48","body_text":"1\nVol.:(0123456789)Scientific Reports |         (2021) 11:8495  | https://doi.org/10.1038/s41598-021-87965-7\nwww.nature.com/scientificreports\nA randomized controlled trial \nof ovarian reserve preservation \nand hemostasis during ovarian \ncystectomy\nSoo Jin Park1, Aeran Seol1, Nara Lee2, Seungmee Lee3, Hee Seung Kim1* & PRAHA Study \nGroup*\nThe preservation of ovarian reserve during laparoendoscopic single-site (LESS) ovarian cystectomy is \ncrucial for reproductive-age women. This study was a single-blinded, single-center, and randomized \ncontrolled trial to evaluate the effect of hemostatic agents on the preservation of ovarian reserve and \nhemostasis during LESS ovarian cystectomy. Patients with unilateral ovarian cyst were randomized \nto the hemostatic agent and coagulation groups according to the hemostasis method. Afterwards, \nthe patients underwent LESS ovarian cystectomy, and hemostasis was performed after ovarian cyst \nexcision according to the assigned hemostasis method. If hemostasis was not completed within \n10 min. After discharge, the patients were followed until 3 months after surgery. We compared the \nhemoglobin, anti-Müllerian hormone (AMH) levels, and ovarian volumes before surgery, and 2 days, \n1 week, and 3 months after surgery (3 M-POST), and the decline ratio between the two groups. The \ndecline ratio of serum AMH levels was greater at 3 M-POST in the coagulation than in the hemostatic \nagent group (median intention-to-treat [ITT], − 36.7 vs. − 13.3%; per-protocol [PP], − 36.8 vs. \n− 13.3%; P < 0.05). Notably, the difference of the decline ratio of serum AMH levels was only shown \nin endometriosis patients (median; ITT, − 50.7 vs. − 14.4%; PP , − 50.7% vs. − 14.4%; P < 0.05), while \nthere was no difference in non-endometriosis patients. In conclusion, Hemostatic agents may be \nnon-inferior to bipolar coagulation for preserving ovarian reserve and hemostasis during LESS ovarian \ncystectomy, in particular, for endometriosis patients. (Trial registry: ClinicalTrials.gov Identifier \nNCT03374397).\nPreservation of ovarian reserve after ovarian cystectomy is a critical issue, especially in women of childbear -\ning age. After stripping ovarian cysts, hemorrhage often persists in the parenchymal bed of the ovary, and the \ndegree of ovarian reserve preservation is known to vary depending on the hemostatic methods  used1, 2. Even \nthough surgical suturing is the primary method for hemostasis during ovarian cystectomy, bipolar coagulation \nis frequently used since multi-port laparoscopic surgery has become more  common3, and LESS surgery has also \nemerged as a growing trend for treating benign gynecologic  diseases4, 5. Moreover, different types of hemostatic \nagents showing similar hemostatic effects have been developed and introduced in a clinical setting because \nbipolar coagulation leads to a decrease in ovarian reserve by tissue  damage6, 7.\nAlthough relevant randomized controlled trials (RCTs) have reported that hemostatic agents showed better \neffects for preserving ovarian reserve than bipolar coagulation with no difference in the hemostatic effect during \nlaparoscopic ovarian  cystectomy8, 9, it is challenging to estimate improvements in ovarian reserve preservation \nand hemostasis from hemostatic agents used during laparoscopic ovarian cystectomy because of the differ -\nent types of surgical approaches to hemostasis, and inconsistent assessment of the degree of ovarian reserve \npreservation.\nThus, we performed this study, called the “PRservation of ovArian reserve and Hemostasis during LESS \novArian cystectomy (PRAHA) trial” to compare the degree of ovarian reserve preservation and the hemostatic \nOPEN\n1Department of Obstetrics and Gynecology, Seoul National University College of Medicine, 101 Daehak -Ro \nJongno-Gu, Seoul 03080, Republic of Korea. 2Department of Obstetrics and Gynecology, CHA Gangnam Medical \nCenter, CHA University School of Medicine, Seoul 06135, Republic of Korea. 3Department of Obstetrics and \nGynecology, Keimyung University School of Medicine, Daegu 41931, Republic of Korea.  *A list of authors and their \naffiliations appears at the end of the paper. *email: bboddi0311@gmail.com\n\n2\nVol:.(1234567890)Scientific Reports |         (2021) 11:8495  | https://doi.org/10.1038/s41598-021-87965-7\nwww.nature.com/scientificreports/\neffects between bipolar coagulation and oxidized cellulose polymer as a hemostatic agent while minimizing the \nrelevant bias.\nResults\nParticipants. During this period, 66 patients were assessed for eligibility, and six, five, two, and one patient \nwere excluded due to low anti-Müllerian hormone (AMH) serum levels (< 0.05 ng/mL), refusal to participate, \nthe disappearance of a unilateral ovarian cyst, and the development of bilateral ovarian cysts. Finally, 52 patients \nwere randomly assigned to the coagulation and hemostatic agent groups, and each received planned treatment \nfor ITT analysis. However, four patients were lost to follow-up, and the database of the remaining 48 patients was \nused for PP analysis (Fig. 1). The clinicopathologic characteristics between the two groups were well-balanced, \nexcept for the direction of the ovarian cyst (Table 1).\nFigure 1.  Flowchart based on Consolidated Standards of Reporting Trials.\n\n3\nVol.:(0123456789)Scientific Reports |         (2021) 11:8495  | https://doi.org/10.1038/s41598-021-87965-7\nwww.nature.com/scientificreports/\nOperative outcomes. In terms of operative outcomes, there were no differences in operation time, esti-\nmated blood loss, transfusions, hospital stay lengths, complications, and reoperation between the two groups. \nMoreover, the time for hemostasis and the success rates of hemostasis at 4, 7, and 10 min after surgery did not \ndiffer between the two groups (Table 2). In the coagulation group, five patients received additional hemostasis \nby suturing after 10 min, and among them, four (80%) and one (20%) had endometriosis and mature cystic tera-\ntoma, respectively. In the hemostatic agent group, five patients failed to hemostasis within 10 min, and among \nthem, three (60%), one (20%), and one (20%) had endometriosis, a mature cystic teratoma, and a functional cyst, \nrespectively. Ovarian cyst recurrence did not occur during the 3 month follow-up period.\nHemostasis and preservation of the ovarian reserve. Table 3 depicts the comparison of Hb and \nserum AMH levels, and ovarian volumes at before surgery (PRE), and at 2 days (2D-POST), 1 week (1 W-POST), \nand 3  months after surgery (3  M-POST), which showed no differences. Figure  2 shows the time-dependent \nchanges in Hb and serum AMH levels and ovarian volumes in the two groups. Hb levels tended to decrease \nimmediately after surgery, and then gradually increased, whereas the serum AMH levels and ovarian volumes \ntended to decrease over time. However, there were no differences in these trends between the two groups. Fur-\nthermore, we evaluated the decline ratio in Hb and serum AMH levels and ovarian volumes between PRE and \nthe other time points. There were no differences in the decline ratio of Hb levels and ovarian volumes between \nthe two groups, whereas the decline ratio of in serum AMH levels was greater 3 M-POST in the coagulation \ngroup than in the hemostatic agent group. In particular, the decline ratio of serum AMH levels was greater in the \ncoagulation group than in the hemostatic agent group when only patients with endometriosis were included in \nTable 1.  Clinicopathologic characteristics. a Data were shown as mean ± standard deviation.\nCharacteristics Hemostatic agent (n = 26, %) Coagulation (n = 26, %) P value\nAge (years)a 32.5 ± 6.7 30.3 ± 6.7 0.23\nBody mass index (kg/m2)a 22.6 ± 2.9 21.1 ± 3.7 0.08\nPrevious abdominal surgery 5 (19.2) 6 (23.1) 1.00\nDirection of ovarian cyst < 0.05\nRight 17 (65.4) 9 (34.6)\nLeft 6 (23.1) 20 (76.9)\nHistology 0.45\nEndometriosis 12 (46.2) 14 (53.8)\nMature cystic teratoma 8 (30.8) 10 (38.5)\nSerous cystadenoma 2 (7.7) 0 (0)\nMucinous cystadenoma 1 (3.8) 0 (0)\nFunctional cyst 2 (7.7) 2 (7.7)\nBorderline tumor 1 (3.8) 0 (0)\nTable 2.  Operative outcomes. PCDS posterior cul de sac. a Data were shown as mean ± standard deviation. \nb Data were shown as median with range.\nParameter Hemostat agent (n = 26, %) Coagulation (n = 26, %) P value\nOperation time (min)a 24.2 ± 11.1 23.3 ± 16.1 .81\nEstimated blood loss (mL)b 25 (0, 100) 50 (7.5, 50) .78\nAdhesion\nNo adhesion 21 (80.8) 19 (73.1)\nPCDS 1 (3.8) 3 (11.5)\nUterus, PCDS 0 3 (11.5)\nRectum, uterus, PCDS 4 (15.4) 1 (3.8)\nTransfusion 1 (3.8) 0 (0) 1.00\nLength of hospital stay (d)b 3 (3, 3) 3 (3, 3) .32\nComplication 0 (0) 0 (0)\nReoperation 0 (0) 0 (0)\nHemostasis\nTime (min)a 6.7 ± 4.9 7.2 ± 6.1 .76\nFour-min success 11 (42.3) 12 (46.2) 1.00\nSeven-min success 15 (57.7) 14 (53.8) 1.00\nAdditional suturing for incomplete hemostasis after 10 min 5 (19.2) 5 (19.2) 1.00\n\n4\nVol:.(1234567890)Scientific Reports |         (2021) 11:8495  | https://doi.org/10.1038/s41598-021-87965-7\nwww.nature.com/scientificreports/\nthe analysis. However, no difference was seen between the two groups when only those with non-endometriosis \nwere analyzed (Table 4).\nDiscussion\nWe evaluated the effect of oxidized cellulose polymer as one of the hemostatic agents on the preservation of \novarian reserve and hemostasis in patients who underwent LESS ovarian cystectomies. The hemostatic agent \nhad a hemostatic effect similar to bipolar coagulation and a better effect for preserving ovarian reserve than \nbipolar coagulation.\nTable 3.  Comparison of hemoglobin (Hb) levels and serum anti-Müllerian hormone (AMH) levels and \novarian volume, and the intention-to-treat (ITT) and the per-protocol populations (PP). PRE just before \nsurgery, 2D-POST at 2 days after surgery, 1 W-POST at 1 week after surgery, 3 M-POST at 3 months after \nsurgery. a Data were shown as mean ± standard deviation. b Data were shown as median with range.\nCharacteristics\nITT PP\nHemostatic agent \n(n = 26) Coagulation (n = 26) P value\nHemostatic agent \n(n = 25) Coagulation (n = 23) P value\nHb (mg/dL)a\nAll\n PRE 12.2 ± 1.7 12.4 ± 1.2 0.76 12.2 ± 1.7 12.4 ± 1.1 0.57\n 2D-POST 10.9 ± 1.5 11.2 ± 1.2 0.37 10.9 ± 1.5 11.3 ± 1.1 0.21\n 1 W-POST 12.3 ± 1.4 12.7 ± 1.2 0.25 12.3 ± 1.4 12.8 ± 1.1 0.13\n 3 M-POST 12.6 ± 1.1 12.9 ± 1.1 0.31 12.6 ± 1.1 12.9 ± 1.1 0.39\nEndometriosis\n PRE 11.4 ± 1.8 12.3 ± 1.2 0.15 11.4 ± 1.8 12.4 ± 1.2 0.12\n 2D-POST 10.5 ± 1.9 11.1 ± 1.2 0.31 10.5 ± 1.9 11.1 ± 1.2 0.30\n 1 W-POST 11.7 ± 1.6 12.7 ± 1.1 0.08 11.7 ± 1.6 12.8 ± 1 0.06\n 3 M-POST 12.2 ± 1.3 13 ± 1.1 0.10 12.2 ± 1.3 13 ± 1.1 0.10\nNon-endometriosis\n PRE 12.9 ± 1.3 12.6 ± 1.2 0.33 12.9 ± 1.3 12.5 ± 1.1 0.44\n 2D-POST 11.2 ± 0.9 11.4 ± 1.2 0.77 11.2 ± 0.9 11.6 ± 1 0.34\n 1 W-POST 12.9 ± 1 12.9 ± 1.3 0.99 12.8 ± 1.1 12.9 ± 1.1 0.86\n 3 M-POST 13 ± 0.8 12.9 ± 1.1 0.73 13 ± 0.8 12.8 ± 1.1 0.54\nAMH (ng/mL)a\nAll\n PRE 5.7 ± 5.6 4.6 ± 4.2 0.46 5.7 ± 5.7 4.5 ± 4.3 0.44\n 2D-POST 4.1 ± 4.3 4.3 ± 4.3 0.30 4.1 ± 4.4 3.0 ± 2.4 0.31\n 1 W-POST 4.3 ± 4.3 2.8 ± 2.2 0.13 4.2 ± 4.4 2.8 ± 2.4 0.19\n 3 M-POST 4.8 ± 2.9 6.3 ± 2.9 0.17 4.8 ± 6.3 2.9 ± 2.9 0.19\nEndometriosis\n PRE 3.8 ± 3.2 4.4 ± 3.5 0.63 3.8 ± 3.2 3.9 ± 3.1 0.92\n 2D-POST 2.5 ± 1.9 2.9 ± 2.1 0.64 2.5 ± 2 2.8 ± 2.1 0.78\n 1 W-POST 2.5 ± 1.9 2.4 ± 1.5 0.85 2.5 ± 2 2.3 ± 1.5 0.82\n 3 M-POST 2.6 ± 2 2.1 ± 2 0.51 2.6 ± 2 2.1 ± 2 0.51\nNon-endometriosis\n PRE 7.2 ± 6.8 4.9 ± 5.1 0.33 7.4 ± 7.1 5.3 ± 5.6 0.45\n 2D-POST 5.5 ± 5.4 3.3 ± 2.6 0.26 5.5 ± 5.6 3.3 ± 2.8 0.27\n 1 W-POST 5.8 ± 5.2 3.3 ± 2.9 0.15 5.8 ± 5.5 3.5 ± 3.1 0.24\n 3 M-POST 6.9 ± 8.1 3.8 ± 3.1 0.26 6.9 ± 8.1 4 ± 3.6 0.31\nOvarian volume (cm3)b\nAll\n PRE 25.3 (2.9, 911.7) 23.5 (5.4, 293.5) 0.55 25.1 (2.9, 911.7) 24.6 (5.4, 293.5) 0.89\n 2D-POST 13.1 (1.2, 217.1) 11.1 (0.6, 149.2) 0.49 12.6 (1.2, 217.1) 11.2 (0.6, 149.2) 0.77\n 1 W-POST 16.1 (2.1, 190.9) 13.4 (6.4, 258.6) 0.26 15.9 (2.1, 190.9) 13.6 (6.4, 258.6) 0.50\n 3 M-POST 11 (3.4, 66.4) 9.8 (1.2, 43.7) 0.07 11 (3.4, 66.4) 9.2 (1.2, 43.7) 0.06\nEndometriosis\n PRE 19.4 (8.2, 216.5) 16.9 (5.4, 293.5) 0.82 19.4 (8.2, 216.5) 15.3 (5.4, 293.5) 0.65\n 2D-POST 16.2 (1.2, 217.1) 11.4 (0.6, 149.2) 0.56 16.2 (1.2, 217.1) 11.2 (0.6, 149.2) 0.44\n 1 W-POST 17 (2.1, 190.9) 10.8 (6.4, 258.6) 0.35 17 (2.1, 190.9) 10.4 (6.4, 258.6) 0.38\n\n5\nVol.:(0123456789)Scientific Reports |         (2021) 11:8495  | https://doi.org/10.1038/s41598-021-87965-7\nwww.nature.com/scientificreports/\nRelevant RCTs have reported no difference in the hemostatic effect between bipolar coagulation and hemo-\nstatic  agents8–12. In these studies, the combination of gelatine granules and human thrombin (Floseal; Bax-\nter Healthcare Corporation Fremont, CA, USA) produced a better hemostatic effect than oxidized cellulose \n polymers13 which were mainly used for comparing the hemostatic effect to bipolar coagulation. However, few \nstudies to date have evaluated whether oxidized cellulose polymers, expected to have a less hemostatic effect \nthan the combination of gelatine granules and human thrombin, could have a hemostatic effect similar to bipolar \ncoagulation during laparoscopic ovarian cystectomy.\nIn terms of the preservation of ovarian reserve, three relevant RCTs emphasized the beneficial effect of \nthe combination of gelatine granules and human thrombin for preserving the remaining ovarian reserve after \nlaparoscopic ovarian  cystectomy8, 10, 11. In these studies, the preservation effect was calculated to measure the \nserum AMH levels PRE and 3 M-POST, as in this study. The decline ratio of serum AMH levels ranged from \n41.2 to 41.9% in patients treated with bipolar coagulation, whereas they were 15.4–16.1% in those treated with \nthe hemostatic agent. However, these studies could not evaluate the decline ratio in serum AMH levels after \nunilateral ovarian cystectomy because 13–35% of the patients had bilateral ovarian cysts, which requires further \ninvestigation in well-designed RCTs.\nAnother RCT evaluated the effect of oxidized regenerated cellulose (ORC) on ovarian reserve after laparo -\nscopic cystectomy of  endometriosis14. In the study, AMH decline ratio at 6 months decreased by 54.1% in the \ncystectomy-only group, and 45.4% in the cystectomy and ORC apply group. Besides, endometriosis recurrence \nwas significantly higher in the group that applied ORC during the follow-up period up to 30 months. Although \nthe AMH decline ratio was greater in the study than in our study, the hemostasis method was not mentioned \nin the group that did not apply ORC In our study, follow-up was only performed for up to 3 months, whereas \nin the study, follow-up was performed for up to 30 months, and it was confirmed that the effect of lowering the \nrecurrence of endometriosis as well as preserving ovarian reserve could be expected by using ORC, which might \nform a chemical ablation in the ovarian cortex after cystectomy.\nIn this study, we found that oxidized cellulose polymer showed a similar hemostatic effect as bipolar coagula-\ntion during LESS ovarian cystectomies. To the best of our knowledge, this is the first RCT to show a hemostatic \neffect of oxidized cellulose polymer comparable to bipolar coagulation during LESS ovarian cystectomy. When \nwe consider that the combination of gelatine granules and human thrombin is more expensive than oxidized \ncellulose polymer, and a diagnosis-related group (DRG) program has been adopted for most of the gynecologic \nsurgeries in our country since July  201315, this finding is significant because oxidized cellulose polymer can be \nused cost-effectively and safely for hemostasis during LESS ovarian cystectomy.\nMoreover, we compared the time-dependent changes in ovarian volumes between the two groups and found \nno difference in the decline ratio of ovarian volumes despite similar tendencies to decrease after surgery. However, \nwe found a beneficial effect of the hemostatic agent on the preservation of the remaining ovarian reserve com-\npared to bipolar coagulation. In this study, we found a decline in serum AMH levels of 13.3% in the hemostatic \nagent group, compared to a decline by bipolar coagulation of 36.7%. Above all, we should note that this effect \nwas shown only in patients with endometriosis, and serum AMH levels were declined by 50.7% after bipolar \ncoagulation during LESS ovarian cystectomy. In contrast, there was no difference in the decline ratio between \nthe two treatments in patients with non-endometriosis.\nFigure 2.  Repeated measure analysis of variance for comparing variables over time (a) hemoglobin (Hb) levels \n(b) anti-Müllerian hormone (AMH) levels (c) ovarian volume.\n\n6\nVol:.(1234567890)Scientific Reports |         (2021) 11:8495  | https://doi.org/10.1038/s41598-021-87965-7\nwww.nature.com/scientificreports/\nAlthough thermal damage by bipolar coagulation can further reduce ovarian function compared to hemo -\nstatic  agents16, the experienced gynecologist who performed the LESS ovarian cystectomies made an effort to \nminimize the removal of healthy ovarian tissue and the use of bipolar coagulation for preserving the remaining \novarian reserve in this study. No differences in the decline ratio of ovarian volume between the two groups sup-\nport the success of this effort.\nFurthermore, this effort could have contributed to the lack of difference in the decline ratio of serum AMH \nlevels between the hemostatic agent and coagulation groups when only patients with non-endometriosis were \nincluded in the analysis. The preservation effect by minimizing the use of bipolar coagulation can be supported by \nprevious studies where there was no difference in the decline ratio of serum AMH levels between the hemostatic \nTable 4.  Comparison of decline ratio of hemoglobin (Hb) levels and serum anti-Müllerian hormone (AMH) \nlevels, and ovarian volume in the intention-to-treat (ITT) and the per-protocol populations (PP). a Data were \nshown as mean ± standard deviation. b Defined as (the value at 2 days after surgery—the value just before \nsurgery)/the value just before surgery × 100. c Defined as (the value at 1 week after surgery—the value just \nbefore surgery)/the value just before surgery × 100. d Defined as (the value at 3 months after surgery − the value \njust before surgery)/the value just before surgery × 100. e Data were shown as median with range.\nCharacteristics\nITT PP\nHemostatic agent \n(n = 26) Coagulation (n = 26) P value\nHemostatic agent \n(n = 25) Coagulation (n = 23) P value\nHb (%)a\nAll\n Decline ratio  2Db − 10.8 ± 7.6 − 8.6 ± 7.7 0.30 − 10.6 ± 7.7 − 8.5 ± 7.8 0.34\n Decline ratio  1Wc 1.2 ± 7.1 3.3 ± 7.0 0.29 1.3 ± 7.2 3.7 ± 7.3 0.27\n Decline ratio  3Md 5 ± 13.9 3.9 ± 6.6 0.73 5 ± 13.9 4.1 ± 6.7 0.78\nEndometriosis\n Decline ratio  2Db − 8.6 ± 6.7 − 9.2 ± 8.8 0.83 − 8.6 ± 6.7 − 9.7 ± 9 0.73\n Decline ratio  1Wc 2.8 ± 6.4 3.2 ± 6.5 0.87 2.8 ± 6.4 3.7 ± 6.6 0.74\n Decline ratio  3Md 7.9 ± 16.4 5.1 ± 6.7 0.57 7.9 ± 16.4 5.1 ± 6.7 0.57\nNon-endometriosis\n Decline ratio  2Db − 12.7 ± 8 − 7.7 ± 6.3 0.10 − 12.7 ± 8 − 7.7 ± 6.3 0.09\n Decline ratio  1Wc − 0.1 ± 7.6 3.5 ± 7.8 0.25 − 0.1 ± 7.6 3.5 ± 7.8 0.28\n Decline ratio  3Md 2.3 ± 11.1 2.5 ± 6.6 0.95 2.3 ± 11.1 2.5 ± 6.6 0.88\nAMH (%)a\nAll\n Decline ratio  2Db − 27.6 ± 16.3 − 28 ± 25.8 0.94 − 28 ± 16.5 − 28.4 ± 25 0.95\n Decline ratio  1Wc − 20.5 ± 28.1 − 33.1 ± 29.2 0.12 − 21.1 ± 28.5 − 32.3 ± 29.5 0.19\n Decline ratio  3Md − 13.3 ± 34.1 − 36.7 ± 34.3 0.02 − 13.3 ± 34.2 − 36.8 ± 35 0.02\nEndometriosis\n Decline ratio  2Db − 27.8 ± 15.2 − 27.1 ± 26.4 0.94 − 27.8 ± 15.2 − 24.9 ± 26.1 0.74\n Decline ratio  1Wc − 24.6 ± 29.8 − 34.7 ± 32 0.42 − 24.6 ± 29.8 − 31.6 ± 31.1 0.57\n Decline ratio  3Md − 14.4 ± 40.5 − 50.7 ± 32.8 0.02 − 14.4 ± 40.5 − 50.7 ± 32.8 0.02\nNon-endometriosis\n Decline ratio  2Db − 27.4 ± 17.8 − 29.2 ± 26.2 0.84 − 28.2 ± 18.2 − 33 ± 24.1 0.59\n Decline ratio  1Wc − 16.9 ± 27.1 − 31.2 ± 26.7 0.19 − 17.9 ± 28 − 33.2 ± 29 0.21\n Decline ratio  3Md − 12.2 ± 28.8 − 20.2 ± 29.2 0.51 − 12.2 ± 28.9 − 18.6 ± 30.3 0.61\nOvarian volume (%)e\nAll\n Decline ratio  2Db − 45.2 (− 99.3, 154.8) − 53.7 (− 96.3, 189.5) 0.77 − 41.8 (− 99.3, 154.8) − 52.9 (− 96.3, 189.5) 0.75\n Decline ratio  1Wc − 26.9 (− 91.4, 138.1) − 48.4 (− 97.6, 401.8) 0.74 − 26.6 (− 91.4, 138.1) − 50.9 (− 97.6, 401.8) 0.91\n Decline ratio  3Md − 56.5 (− 98.3, 223.3) − 51.1 (− 99.6, 123.4) 0.90 − 56.5 (− 98.3, 223.3) − 52.4 (− 99.6, 123.4) 0.73\nEndometriosis\n Decline ratio  2Db − 38.5 (− 85, 40.4) − 49.5 (− − 96.3, 189.5) 0.84 − 38.5 (− 85, 40.4) − 46.2 (− 96.3, 189.5) 0.89\n Decline ratio  1Wc − 24.4 (− 82.6, 17.2) − 48.5 (− 97.6, 401.8) 1.00 − 24.4 (− 82.6, 17.2) − 46.1 (− 97.6, 401.8) 0.81\n Decline ratio  3Md − 47.7 (− 83.1, 217) − 47 (− 100. .123.4) 0.85 − 47.7 (− 83.1, 217) − 47 (− 100, 123.4) 0.85\nNon-endometriosis\n Decline ratio  2Db − 63 (− 99.3, 154.8) − 61.7 (− 87, 56.5) 1.00 − 57.9 (− 99.3, 154.8) − 61.7 (− 87, 56.5) 0.83\n Decline ratio  1Wc − 42.1 (− 91.4, 138.1) − 42.6 (− 78.5, 103) 0.63 − 27.2 (− 91.4, 138.1) − 58.5 (− 78.5, 103) 0.98\n Decline ratio  3Md − 63.1 (− 98.3, 223.3) − 68.8 (− 96.1, 40) 0.87 − 63.1 (− 98.3, 223.3) − 73.7 (− 96.1, 39.9) 0.61\n\n7\nVol.:(0123456789)Scientific Reports |         (2021) 11:8495  | https://doi.org/10.1038/s41598-021-87965-7\nwww.nature.com/scientificreports/\nagent and bipolar coagulation groups, (23 vs. 19%; P  = 0.47)9, and there was no clear association between the \nremaining ovarian reserve and the number of follicles removed in the  specimens17.\nHowever, patients with endometriosis showed further decreases in AMH 3 M-POST in terms of decline ratio \nin the coagulation group. Despite relevant evidence, this finding can be explained by the following hypothesis. \nSince most patients with endometriosis show pelvic adhesion, we commonly perform adhesiolysis sufficiently \nfrom tissues surrounding the lesion for complete cystectomy. During the procedure, the vascular system within \nthe ovarian cortex or surrounding the ovary can be injured, which can result in lower serum AMH levels caused \nby inadequate blood  supply17. This hypothesis can be supported by a previous study that showed a lower peak \nsystolic velocity in the ovary subjected to laparoscopic  surgery18. Furthermore, endometriosis itself or surgery \nmight decrease ovarian reserve than non-endometriotic cyst because the formation of the endometriotic cyst \nwall is formed by invagination of ovarian cortex comprising ovarian  reserve19.\nAlthough this study showed the beneficial effect of the hemostatic agent on the preservation of ovarian reserve \nwith a hemostatic effect similar to bipolar coagulation during LESS ovarian cystectomy, the standard method of \nhemostasis is still suturing, even though there is a relatively long learning curve for laparoscopic suturing. RCTs \nthat compare the hemostatic and preservation efficacy between hemostatic agents and suturing during LESS \novarian cystectomy are needed and performed by experienced gynecologists with surgical proficiency. Moreover, \nRCTs evaluating the decline ratio of serum AMH levels according to the two hemostatic methods are required \nfor patients with unilateral ovarian endometriosis because the sample size in this study was calculated based on \nthe results of a previous study where various types of ovarian cysts were  included8.\nThe strengths of this study are that we enrolled only patients with a unilateral ovarian cyst, and researchers \nperformed supervise for consistency of surgical procedures during the LESS ovarian cystectomies using the same \nsurgical protocol, which minimized the use of bipolar coagulation for preserving ovarian reserve. Moreover, we \nevaluated ovarian function in terms of serum AMH levels and ovarian volume. As a result, we found a prominent \ndecrease in serum AMH levels using bipolar coagulation in patients with endometriosis. In contrast, the decline \nratio of the ovarian volume was similar between the two treatments, suggesting that ovarian volume could not \nreflect the remaining ovarian reserve after ovarian cystectomy.\nThe limitation is that we did not evaluate all parameters for more than 3 months after surgery. Regarding that \nprimordial follicle growth may take up to 180 days, at list 6 months follow up after surgery might be sufficient \nto evaluate ovarian reserve. The relatively small sample size could also act as a bias for interpreting these results \ndespite the well-designed trial. Additionally, the severity of adhesion or impaired vascularity in the enucleated \novary was not evaluated and could have reduced the serum AMH levels, especially in patients with endometriosis. \nFurthermore, more relevant studies should be conducted for validating these results because a single institution \nin this study performed the LESS ovarian cystectomies.\nMaterials and methods\nStudy design. This study was an investigator-initiated, single-blinded, randomized controlled trial con-\nducted at Seoul National University Hospital. The Institutional Review Board of Seoul National University \nHospital approved the study (No. 1707-079-869) before study initiation and registered at ClinicalTrials.gov on \nDecember 15, 2017 (No. NCT03374397). We obtained informed consent from all participants, and study was \nperformed according to the relevant guidelines and regulations the Institutional Review Board of Seoul National \nUniversity Hospital.\nParticipants. We enrolled patients with benign ovarian diseases consecutively from December 2017 to Feb-\nruary 2019 based on the following eligibility criteria: age ≥ 18 and ≤ 45 years; unilateral benign ovarian cysts \nconfirmed by pelvic ultrasonography; a regular menstrual period between 21 and 45 days; preoperative serum \nAMH levels of ≥ 0.50 ng/mL; planned LESS ovarian cystectomy; American Society of Anesthesiologists (ASA) \nphysical status classification 1–2; and written informed consent. The exclusion criteria were as follows: suspi-\ncious ovarian malignancy shown by imaging studies; bilateral ovarian disease; preoperative serum AMH levels \nof < 0.50 ng/mL; planned multi-port laparoscopic ovarian cystectomy; pregnancy or breastfeeding; comorbid \nendocrine disease, such as thyroid function abnormality, hyperprolactinemia, or Cushing’s disease; and history \nof hormonal therapy within 3 months. A simple ultrasound-based rule was used for evaluation of the ovarian \n cyst20. Benign rules included unilocular, size of solid component less than 7 mm, acoustic shadows, smooth \nmultilocular cyst less than 10 cm, and no Doppler flow, and malignant rules included irregular margin, ascites, \nmore than three papillary structure, and presence of Doppler flow.\nSample size calculation. The sample size was calculated based on a relevant RCT 8, where the decline ratio \nof AMH at 3 months after surgery was 41.2% (interquartile range 17.2–54.5%) and 16.1% (interquartile range \n8.3–44.7%) in the bipolar coagulation and hemostatic agent groups, respectively. The mean value of the differ -\nence between the two groups was 25.1%, and the standard deviation estimated from the assumption of normal \ndistribution was 27.7% and 27% for each group. When the sample size was based on a non-inferiority margin \nof 30%, reflecting an acceptable difference between the two groups, the estimated sample size was 52 patients \nto obtain 80% power and the two-sided significance levels of 5%, considering that 10% of the target number of \npatients would be eliminated.\nRandomization. All patients were randomly assigned to the coagulation and hemostatic agent groups at \na 1: 1 ratio after signing the informed consent form. A third-party gynecologist (AS) created and managed the \nrandomization table using a web-based program. Randomization table was created by simple calculation with-\n\n8\nVol:.(1234567890)Scientific Reports |         (2021) 11:8495  | https://doi.org/10.1038/s41598-021-87965-7\nwww.nature.com/scientificreports/\nout blockings. Then, AS managed the sequence without the other gynecologists knowing. The randomization \ntable was implemented with silver paper, so that the next assignment cannot be recognized. The randomization \nresults were released to investigators immediately before surgery, and blinding of patients was maintained until \nthe end of the study.\nSurgical procedure. All patients underwent LESS ovarian cystectomy by the single gynecologist (HSK) \nwith more than 100 cases per year of 10 years of experience, and all surgical procedures were monitored by \nauthors SJP and NL for consistency. The patient was placed in the Trendelenburg position after general anesthe-\nsia, and a Glove Port A (Medtech Inframed Corp., Seoul, Republic of Korea) was inserted through the umbilicus. \nIf unilateral ovarian cysts adhered to the surrounding tissues, they were mobilized from the surrounding tissues, \nand then the ovarian cortex was incised with monopolar scissor on the opposite side of the mesovarium longitu-\ndinally. The cleavage plane between the ovarian cortex and the cyst wall was identified, and cyst enucleation was \nperformed by pulling the cyst wall in a direction opposite to the ovarian cortex. After completing cyst enuclea-\ntion, bipolar coagulation was applied for patients in the coagulation group. In contrast, one piece of non-woven \nSurgiGuard, 10.2 × 10.2 cm (Hanmi Pharm. Co., Ltd., Seoul, Republic of Korea) was applied in the inner bleeding \narea of the ovarian cortex for those in the hemostatic agent group. If hemostasis failed within 10 min, suturing \nwas performed in the coagulation group, whereas bipolar coagulation, and suturing if needed, were conducted \nin the hemostatic agent group. After confirming the lack of bleeding, the peritoneal cavity was irrigated with \nnormal saline. All patients were discharged 2 days after surgery.\nEndpoints. In previous relevant studies, serum AMH levels, follicular stimulating hormone, the antral folli-\ncular count, and ovarian volume were used as markers for evaluating the remaining ovarian reserve after ovarian \ncystectomy. Among them, serum AMH levels are known to be stable during regular menstrual cycles and decline \nwith advanced age, reflecting the remaining ovarian  reserve21–23. In this study, we considered the decline ratio of \nserum AMH levels 3 months after surgery as the primary endpoint, which was defined as 100 × (the serum AMH \nlevel 3 months after surgery minus the serum AMH level just before surgery)/the serum level of AMH just before \nsurgery (%), based on a relevant  trial8.\nThe secondary endpoints were operative outcomes including operation time, the estimated blood loss, transfu-\nsions, length of hospital stay, complications, reoperation, time for hemostasis, and the success rates of hemostasis \nat 4, 7, and 10 min. Moreover, we compared the Hb levels (mg/dL) and the serum AMH levels (ng/mL), and \novarian volumes  (cm3) just before surgery (PRE), and at 2 days (2D-POST), 1 week (1 W-POST), and 3 months \nafter surgery (3 M-POST), and the decline ratio between the two groups. The ovarian volumes were estimated as \nfollows: volume  (cm3) = maximal sagittal diameter (length, cm) × maximal coronal diameter (width, cm) × maxi-\nmal transverse diameter (depth, cm) × 0.5233 on pelvic  ultrasonography24.\nStatistical analysis. We performed a statistical analysis of both the intention-to-treat (ITT) and the per-\nprotocol populations (PP). The continuous variables between the coagulation and hemostatic agent groups were \ncompared by Student’s t or the Mann–Whitney U test, and the categorical variables were analyzed using Fish-\ner’s exact or the χ2 test. Repeated measure analysis of variance (ANOV A), including Pillai’s Trace, and Wilks’ \nLambda were used to analyze the outcomes according to the time points. All P-values of < 0.05 were considered \nstatistically significant. The statistical analyses were conducted using SPSS 20.0 (SPSS, Inc., Chicago, IL, USA).\nConclusions\nOxidized cellulose polymer as a hemostatic agent may be effective for hemostasis and preserving ovarian reserve \nduring LESS ovarian cystectomy compared to bipolar coagulation. Moreover, the use of hemostatic agents in \npatients of reproductive age suspected to have ovarian endometriosis should be considered to preserve ovarian \nreserve instead of bipolar coagulation.\nData availability\nThe datasets generated during and/or analyzed during the current study are not publicly available because the \ninformed consent did not include an explanation about public data sharing plan, but are available from the cor-\nresponding author on reasonable request.\nReceived: 15 August 2020; Accepted: 5 April 2021\nReferences\n 1. Ata, B., Turkgeldi, E., Seyhan, A. & Urman, B. Effect of hemostatic method on ovarian reserve following laparoscopic endometrioma \nexcision; comparison of suture, hemostatic sealant, and bipolar dessication. A systematic review and meta-analysis. J. Minim. \nInvasive Gynecol. 22, 363–372 (2015).\n 2. Peters, A., Rindos, N. B. & Lee, T. 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Serum anti-Mullerian hormone is more strongly related to ovarian follicular status than serum inhibin B, estradiol, \nFSH and LH on day 3. Hum Reprod 18, 323–327. https:// doi. org/ 10. 1093/ humrep/ deg042 (2003).\n 22. Iwase, A. et al. Serum anti-Mullerian hormone level is a useful marker for evaluating the impact of laparoscopic cystectomy on \novarian reserve. Fertil. Steril. 94, 2846–2849. https:// doi. org/ 10. 1016/j. fertn stert. 2010. 06. 010 (2010).\n 23. Dewailly, D. et al. The physiology and clinical utility of anti-Mullerian hormone in women. Hum. Reprod. Update  20, 370–385. \nhttps:// doi. org/ 10. 1093/ humupd/ dmt062 (2014).\n 24. Orsini, L. F ., Salardi, S., Pilu, G., Bovicelli, L. & Cacciari, E. Pelvic organs in premenarcheal girls: Real-time ultrasonography. \nRadiology 153, 113–116. https:// doi. org/ 10. 1148/ radio logy. 153.1. 64737 71 (1984).\nAcknowledgements\nWe sincerely appreciate Professor Jae-Weon Kim from Seoul National University College of Medicine for his \nassistance with managing the study fund, and the Medical Research Collaborating Centre (MRCC) from Seoul \nNational University Hospital for calculating the sample size for this trial.\nAuthor contributions\nStudy concepts: S.J.P ., A.S., S.L., N.L., and H.S.K. Study design: S.J.P ., S.L., and H.S.K. Data acquisition: S.J.P ., A.S., \nN.L., and H.S.K. Quality control of data and algorithms: A.S., S.L., and N.L. Data analysis and interpretation: \nS.J.P ., A.S., S.L., N.L., and H.S.K. Statistical analysis: S.J.P ., and H.S.K. Manuscript preparation: S.J.P ., and H.S.K. \nManuscript editing: S.J.P ., and H.S.K. Manuscript review: S.J.P ., A.S., S.L., N.L., and H.S.K.\nCompeting interests \nSJP , AS, SL, and NL declare no competing interests. HSK received funding for this study from Hanmi Pharm. \nCo., Ltd. Hanmi Pharm. Co., Ltd supported this research without any role in the study design, data collection, \ndata analysis, data interpretation, or writing of the report.\nAdditional information\nCorrespondence and requests for materials should be addressed to H.S.K.\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.\n\n10\nVol:.(1234567890)Scientific Reports |         (2021) 11:8495  | https://doi.org/10.1038/s41598-021-87965-7\nwww.nature.com/scientificreports/\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) 2021\n \nPRAHA Study Group\nAeran Seol1, Eunji Lee1, Ga Won Yim4, Haerin Paik1, Hee Seung Kim1, Hyunji Lim1, Jae-Weon \nKim1, Jaehee Mun1, Junhwan Kim1, Nara Lee2, Seungmee Lee3 & Soo Jin Park1\n4Department of Obstetrics and Gynecology, Dongguk University Ilsan Hospital, Goyang, Republic of Korea.","source_license":"CC0","license_restricted":false}