Patient selection
From June 2012 to September 2015, infertile women and women who wish to preserve ovarian function under the age of 40 with suspected endometriomas, which diagnosed by vaginal ultrasonography and MRI had been allocated. The subjects were counselled to choose three-step surgical management or one-step surgery, and prospectively followed up at least one year after surgery. Women who selected three step management should be infertile and should not receive any hormonal treatment before allocation. Among the subjects who selected one-step surgery, women received hormonal therapy within three months before the surgery were excluded. This study was accomplished at Nagasaki University Hospital. Institutional review board had been approved this study. Written informed consent were obtained from all subjects.
Surgical procedures
Three-step surgical management
First look laparoscopy (FLL) was performed under general anesthesia with standard four ports placement. Peritoneal fluids were collected at the beginning of surgery. Portion of peritoneal fluids was sent to cytological assessment and rest of them were centrifuged and aliquots were stored at -20˚C. Endometriomas were fenestrated , then chocolate-like fluids were aspirated and inner side of the cyst was irrigated. Small fragment of cyst wall was biopsied for pathological examination. Adhesiolysis was performed as much as possible. Staging of endometriosis by ASRM scoring system was recorded[10].
After the first look laparoscopy, oral progestin, dienogest 2mg daily (b.i.d.) were prescribed and continued for three months until second look surgery.
Second look laparoscopy (SLL) was performed in similar way to first look surgery. Peritoneal fluids were collected at the beginning of surgery. Portion of peritoneal fluids was sent to cytological assessment and rest of them were centrifuged and aliquots were stored at -20˚C. After the adhesiolysis, cystectomy with the care to conserve normal ovarian tissue was performed according to the procedures reported previously[11-13]. In the case with bilateral lesions, hemi-lateral cyst wall ablation with bipolar coagulator with reduced power setting was performed.
One-step surgical management
Women who did not receive three-step procedure, cystectomy was performed as similar to second look surgery. Accordingly, in the case with bilateral lesions, cyst wall ablation with bipolar coagulator was performed instead of cystectomy after the complete adhesiolysis.
AMH measurement and post-operative follow up
Patient’s blood was collected before and after the surgery and serum AMH levels were determined by ELISA (AMH genII, Beckman-Coulter, Tokyo, Japan) at out-sourcing laboratory (SRL, Tokyo, Japan) irrespective of menstrual cycles. The subjects were followed up 1-3month intervals and blood was collected at every their visits. In women had three-step management, serum AMH levels were determined at one month after FLL, three months after dienogest treatment just before SLL, and one month after SLL. For group comparison, serum AMH levels after surgery were categorized into three time points, i.e. before surgery (timepoint 0), 3-6month after surgery (time point 1), and 9-12month after surgery (time point 2). If women had multiple assay during these periods, lower value were selected for analysis. In some women conceived during the follow-up periods, serum AMH levels were determined until they got conceived.
Measurement of cytokines and chemokines in peritoneal fluids.
Proinflammatory cytokines and chemokines, which may relate to the pathogenesis of endometriosis [14,15], such as interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor (TNF)-α, monocyte chemotactic protein (MCP)-1 in peritoneal fluids were measured by electrochemiluminescence immunoassays (MESO QuickPlex SQ 120, MSD, Tokyo, Japan) at out-sourcing laboratory (KPSL, Fukuoka, Japan). One mL aliquot of centrifugated peritoneal fluids were used for the assay. We calculated the percent changes of measured cytokines and chemokines levels by the formula described below.
(value of SLL – value of FLL) / value of SLL*100
The decline was defined if value at second look laparoscopy (after 3 months of oral dienogest) was decreased more than 10% comparing to the value at FLL.
Statistical analysis
Continuous variables are compared with paired or unpaired student-t test and Wilcoxon signed-rank test. Categorical variables are compared with kai square test and Fisher’s exact test. All statistical analysis was performed with computer software (JMP Pro 14.0.0, SAS institute Japan, Tokyo). P value under 0.05 were considered as statistical significance.