Diagnostic Benefit of the Detection of Mitotic Figures in Endometriotic Lesions

Geburtsh Frauenheilk 2022; 82: 85 –92 DOI 10.1055/a-1580-0601 ISSN 0016‑5751 © 2022. The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial-License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/) Georg Thieme Verlag KG, Rüdigerstraße 14, 70469 Stuttgart, Germany Correspondence Maren Goeckenjan M. D. Department of Obstetrics and Gynecology, Technische Universität Dresden Fetscherstraße 74, 01307 Dresden, Germany [email protected]

Objectives Endometriosis is a chronic disease which is diag- nosed by surgical intervention combined with a histological work-up. Current international and national recommenda- tions do not require the histological determination of the pro- liferation rate. The diagnostic and clinical importance of the mitotic rate in endometriotic lesions still remains to be eluci- dated.

In this retrospective study, the mitotic rates and clinical data of 542 patients with histologically diagnosed endometriosis were analyzed. The mean patient age was 33.5 ± 8.0 (17 –72) years, and the mean reproductive lifespan was 21.2 ± 7.8 (4 –41) years. Patients were divided into two groups and patients ʼ reproductive history and clinical endo- metriosis characteristics were compared between groups. The study group consisted of women with confirmed mitotic figures (n = 140, 25.83 %) and the control group comprised women without proliferative activity according to their mi- totic rates (n = 402, 74.27 %).

Women with endometriotic lesions and histologically confirmed mitotic figures were significantly more likely to have a higher endometriosis stage (p = 0.001), deep infiltrat- ing endometriosis (p 0 was seen significantly less often in cases with incidental findings of endometriosis (p = 0.031). The presence of symptoms and basic characteristics such as age, age at onset of menarche, reproductive lifespan and parity did not differ between the group with and the group without mitotic figures.

This study shows that a simple histological as- sessment of the mitotic rate offers additional diagnostic value for the detection of advanced stages of endometriosis. The possible role as a predictive marker for the recurrence of en- dometriosis or the development of endometriosis-associated cancer will require future study. GebFra Science | Original Article 85Wetzk M et al. Diagnostic Benefit of … Geburtsh Frauenheilk 2022; 82: 85 –92 | © 2022. The author(s). Article published online: 2022-01-10

Endometriosis is a common gynecological disease which is typi- cally diagnosed by a histological examination after laparoscopy. Around 10 % of women aged 15 –50 years suffer from dysmenor- rhea, dyspareunia, abdominal and pelvic pain as well as infertility and are diagnosed with endometriosis [1]. The presentation of symptoms in women with endometriosis is suggestive for the lo- cation of the endometriotic lesions and the clinical stage [2], but up to now, the definitive diagnosis of endometriosis needs sur- gery. Serum markers for endometriosis are being investigated but have not yet been clinically established [3, 4]. International and national guidelines recommend a histologi- cal assessment after surgical procedures to make a diagnosis of endometriosis [5, 6]. An initiative to evaluate the quality of care in endometriosis centers in German-speaking countries in Europe was recently published [7]. It reported that all certified endome- triosis centers took biopsies to confirm endometriosis histologi- cally. Nevertheless, the current guidelines give no clear statements about the specific procedures for the histological assessment [5, 6]. Standardized quality indicators for diagnosis and therapy have not been implemented to date [7]. Mitotic figures as a marker of cell proliferation in endometriotic lesions were initially described by Nisolle and Donnez [8, 9]. The detection of mitotic figures and determination of the mitotic rate per 10 high-power fields after staining with hematoxylin and eo- sin is an established marker of cellular proliferation in oncology [6]. The disadvantages of counting mitotic figures are the high in- ter- and intra-observer variation rates and limited reproducibility [10, 11]. Another marker of cell proliferation proposed for the di- agnosis of endometriosis is Ki-67 [12]. Using the mitotic rate as an additional histopathological as- sessment of cellular proliferation is an established, cost-efficient procedure and part of the routine histological examination. It is routinely used not only in oncology but also for the histological diagnosis of endometriosis. Atypical ovarian endometriosis is known to be especially asso- ciated with an increased risk of malignancy [13]. Currently, molec- ular mechanisms and biomarkers are being carefully investigated in women with endometriosis to find a diagnostic algorithm for the prediction and possibly prevention or treatment of women with an increased risk of endometriosis-associated malignancies [14]. Histological parameters of cell proliferation may have an ad- ditional diagnostic value when assessing the severity of endome- triosis but could also serve as predictive markers for the develop- ment of malignant epithelial tumors. ZUSAMMENFASSUNG Ziele Endometriose ist eine chronische Erkrankung, die durch einen chirurgischen Eingriff und einen histologischen Nach- weis diagnostiziert wird. Laut den aktuellen internationalen und nationalen Empfehlungen ist der histologische Nachweis der Proliferationsrate nicht nötig. Die diagnostische und kli- nische Bedeutung der Mitoserate in endometriotischen Läsio- nen ist immer noch ungeklärt. Methoden In dieser retrospektiven Studie wurden die Mito- seraten und die klinischen Daten von 542 Patientinnen mit histologisch nachgewiesener Endometriose analysiert. Das durchschnittliche Alter der Patientinnen betrug 33,5 ± 8,0 (17–72) Jahre, und ihre durchschnittliche Reproduktions- spanne lag bei 21,2 ± 7,8 (4 –41) Jahren. Die Reproduktions- geschichte und klinischen Endometriosemerkmale wurden verglichen. Die Studiengruppe bestand aus Frauen, bei denen Mitosefiguren nachgewiesen wurden (n = 140, 25,83 %), und die Kontrollgruppe bestand aus Frauen ohne Proliferations- aktivität (n = 402, 74,27 %). Ergebnisse Frauen mit endometriotischen Läsionen und einem histologischen Nachweis von Mitosefiguren hatten sig- nifikant häufiger ein klinisch höheres Endometriosestadium (p = 0,001), tief infiltrierende Endometriose (p 0 (p = 0,031). Es gab keine Unterschiede in den Sympto- men und Charakteristika wie Alter, Menarche, Reproduktions- spanne und Parität zwischen der Gruppe mit und der Gruppe ohne Mitosefiguren. Schlussfolgerung Diese Studie zeigt, dass eine einfache his- tologische Evaluierung der Mitoserate für die Erkennung einer Endometriose im fortgeschrittenen Stadium einen diagnosti- schen Mehrwert bietet. Um die Bedeutung der Mitoserate als prädiktiven Marker für das Wiederauftreten einer Endo- metriose bzw. für die Entwicklung von endometrioseassoziier- tem Krebs zu evaluieren, werden weitere Untersuchungen be- nötigt. n = 812 Patients with a diagnosis of endometriosis (ICD N80.0–N80.9) at a certified university endometriosis center (study period: 01/01/2013–31/12/2016) n=5 1 Repeat surgical treatmentn=1 0 Missing surgical data n = 180 Missing clinical datan=1 6 Missing mitotic index n=1 3 Endometriosis not histologically confirmed n = 542 Patients with histologically confirmed endometriosis and mitotic index (66.75%) ▶ Fig. 1 Flowchart showing the inclusion into the retrospective study of patients treated for endometriosis at a university center. 86 Wetzk M et al. Diagnostic Benefit of … Geburtsh Frauenheilk 2022; 82: 85 –92 | © 2022. The author(s). GebFra Science | Original Article This study aimed to assess the diagnostic impact of detected mitotic figures as a marker of proliferation in endometriotic le- sions in a retrospective study carried out at a tertiary university endometriosis center.

A total of 812 patients with a diagnosis of endometriosis were treated at the Department of Gynecology and Obstetrics in a ter- tiary university center during the study period from 2013 to 2016. The patients ʼ clinical data were analyzed after ethical approval of the study protocol (by the local Ethics Committee of TU Dresden, EK 189062018) and the consent of study participants treated in the University Endometriosis Center was obtained. Patient characteristics After patients without biopsies showing endometriosis and a his- tological assessment which included a mitotic figure count in standardized HPF areas after H & E staining were excluded, a total of 542 patients remained and were included in this study. No ad- ditional staining to evaluate cellular proliferation, e.g., using Ki‑67, was performed. Each patient was only included once in the study even if they underwent repeat surgical treatment in the same hospital. The inclusion chart of the study is shown in ▶ Fig. 1. Measurements The following parameters were assessed: diagnosis based on the international classification of diseases, symptoms, medical history including medication, previous pregnancies, abortions and births, organ manifestations, endometriosis stage based on the rASRM score (revised score of the American Society of Reproductive Medicine) [15], histology, surgical intervention and recom- mended endocrine treatment. The ENZIAN classification was used for deep infiltrating endometriosis [16]. As not all lesions were documented photographically, further classification of each en- dometriotic lesion into typical or atypical as proposed by Nisolle et al. [17] could not be performed systematically in the setting of a retrospective study. The mitotic figure count was determined after H & E staining and performed as a standardized procedure used for histological assessment in oncology. The method was described by Barry et al. in 2001 [11]. Statistical analysis Statistical analysis was performed using SPSS V. 25.0 (IBM, Ar- monk, NY, USA). χ 2 and Fisher ʼse x a c tt e s t sw e r eu s e df o rt h ea n a l - ysis of categorical variables. Quantitative variables were com- pared using Mann-Whitney U test. A p-value < 0.05 was consid- ered statistically significant.

A total of 542 patients with histologically confirmed endometrio- sis were included in the study. The mean age of these patients was 33.5 ± 8.0 years. The majority of the women were nulligravida (n = 313, 58.29 %); 350 (66.04 %) had not previously given birth. ▶ Table 1 Baseline characteristics of women with histologically con- firmed endometriosis (n = 542). Data are presented as mean ± standard deviation (median, range) or number (%). Clinical characteristics Patients Age (years) 33.5 ± 8.0 (17 –72) Obstetric history ▪ No prior pregnancies (n = 537) 313 (58.29 %) ▪ No prior births (n = 530) 350 (66.04 %) Age at onset of menarche (years) 13.0 ± 1.50 (9 –17) Reproductive lifespan (years) 21.2 ± 7.8 (4 –41) Mean duration of menstrual cycle (n = 286) 29.5 ± 7.6 (13 –90) Mean duration of menstrual bleeding (n = 314)  5.7 ± 1.8 (2–15) Previous history of endometriosis ▪ No, first diagnosis 404 (74.54 %) ▪ Yes, recurrent disease 128 (23.61 %) Main reason for surgery ▪ Acute or chronic pain 192 (35.42 %) ▪ Infertility 130 (23.99 %) ▪ Incidental finding 100 (18.45 %) Symptoms prior to surgery ▪ Cyclic pelvic pain 238 (43.91 %) ▪ Dysmenorrhea  56 (10.33 %) ▪ Dysuria  42 (7.75 %) ▪ Dyspareunia 103 (19.00 %) ▪ Primary and secondary infertility 210 (38,74 %) ▪ Abnormal uterine bleeding  80 (14.8 %) Concomitant gynecological diagnoses ▪ Uterine malformations  32 (5.90 %) Surgery ▪ Outpatient treatment 152 (28.04 %) ▪ Hospitalization after surgery 390 (71.96 %) ▪ Laparoscopy 496 (91.51 %) ▪ Laparotomy  36 (6.60 %) Occurrence of endometriosis ▪ Adenomyosis  58 (10.70 %) ▪ Ovarian endometriosis 180 (33.21 %) ▪ Deep infiltrating endometriosis 103 (19.00 %) ▪ Peritoneal adhesions 185 (34.13 %) rASRM classification (n = 523) 1 ▪ Stage I 214 (40.92 %) ▪ Stage II  95 (18.16 %) ▪ Stage III 111 (21.22 %) ▪ Stage IV 103 (19.69 %) Hormonal treatment at the time of surgery 102 (18.82 %) Mean number of probes for histological assessment  3.2 ± 1.8 (1–13) Mean mitotic rate  0.4 ± 0.8 (0–7) rASRM: revised classification of the American Society of Reproductive Medicine [9]. 1 In 19 cases no rARSM classification was given; 9 of those women had endometriosis of the abdominal wall without laparoscopy and 10 women had adenomyosis. The ENZIAN score was used for those clinical entities. 87Wetzk M et al. Diagnostic Benefit of … Geburtsh Frauenheilk 2022; 82: 85 –92 | © 2022. The author(s). Analysis and mitotic figure count After mitotic figures were counted, 402 women (74.27 %) were found to have no mitotic figures in 10 microscopic high-power fields (HPF). These women were assigned to the control group of women without proliferative activity based on mi totic figures. 140 women (25.83 %) had at least one mitotic figure in 10 HPFs and were diagnosed as having endometriosis with proliferative ac- tivity based on the presence of mitotic figures. The mean number of mitotic figures was 0.4 ± 0.8 (0 –7) for all patients included in the study. Six women had three mitotic figures, four and five mi- toses were counted in three women respectively, and seven mi- totic figures were counted in one woman. As the number of wom- en with a higher mitotic index was small, no additional statistical analysis was performed to assess the association between mitotic index and disease severity. The baseline characteristics of the patients are shown in ▶ Table 1 . Presence of mitotic figures and clinical characteristics The clinical data of the two subgroups were compared based on the presence of mitotic figures in up to 10 HPF. The results of this comparison are shown in ▶ Table 2 . Surgery was planned signifi- cantly more often because of acute or chronic pain (p = 0.018) in the subgroups with mitotic figures. The predominant reason for surgery was infertility, and there were no significant differences between the two subgroups with regard to infertility, whereas a diagnosis of endometriosis during surgery for other reasons was more frequent in the group without mi totic figures (p = 0. 031). We found no statistical difference between the two groups with regard to self-reported symptoms prior to surgery. Only in- fertility was more common in women with mitotic figures (45.0 %) than in the subgroup without detected mi totic figures (36,4 %). The mean reproductive lifespan from the onset of men- arche to surgery for endometriosis was significantly shorter in women with detected mitotic figures in endometriotic lesions (19.4 vs. 21.9 years, cf. ▶ Table 2 ). Significantly more lesions showed mitotic figures in the group of women with deep infiltrating endometriosis (p < 0.001), ovar- ian endometrioma (p = 0.012) and infertility (p = 0.049). Mi totic figures and countable mitotic rates were more common in wom- en with multiple endometriotic lesions (p < 0.001). Surgery was performed in the majority of cases via laparoscopy (91.51 %). Our results showed that there was no significant difference with regard to the presence of mitotic figures in the biopsies of women undergoing concurrent hormonal treatment at the time of surgery compared to women without endocrine treatment (p = 0.210). Forty-nine women were treated with combined oral contraceptives, 12 (24.5 %) of whom had mitotic activity. Twenty-six patients took progestin only pills, mainly dienogest (n = 19), and three of them had mitotic activity (15.8 %). Only one woman was treated with long-acting agonists of gonadotro- pin-releasing hormone (GnRH); no mitosis was detected in this woman. In the group receiving endocrine treatment, no differ- ence was detected with regard to different medications (p = 0.793). The percentages for the different stages based on the rASRM and ENZIAN scores for the groups with inactive and active prolif- eration are shown in ▶ Fig. 2 a and b. rASRM stages differed signif- icantly between the two subgroups (p = 0.001). In spite of the higher occurrence of deep infiltrating endometriosis, the distribu- tion of the lesions classified using the ENZIAN score does not show significant differences ( ▶ Fig. 2 b).

Since the first description of mitotic figures in endometriotic le- sions [8, 9] only a few studies have been published on the topic of active proliferation in endometriotic lesions. The detection of mitotic figures as a marker of active proliferation in cancer cells after H & E staining is a standardized procedure [18]. Assessment is easy to perform without further staining or additional costs. Usually, 10 HPF are controlled and the number of mitotic figures is counted and documented. It appears that proliferative cellular activity does not change during the menstrual cycle in the ectopic endometrial tissue of women with endometriosis [17]. Nisolle et al. identified the mitotic rate as a histological marker for the intra- operative appearance of endometriotic lesions during surgery and as a morphological criterion for typical and atypical lesions [17]. The mitotic rate was a histological correlate for the macroscopic appearance of proliferation. In our study, we defined one mitotic figure and more in 10 HPFs as a sign of active cellular proliferation. In this retrospective analysis of clinical data from one single cen- ter, we examined the correlation between baseline characteristics and the presence of mitotic figures in the tissue of endometriotic lesions from more than 500 women. The presence of mitotic figures had a significant additional di- agnostic value with regard to the severity of endometriosis. After comparing the characteristics of women in our two subgroups, we found a statistically significant higher risk of severe endome- triosis, with higher rASRM stages and deep infiltrating endome- triosis, in the group with confirmed mitotic figures. No statistically significant differences were seen between the two groups with regard to medical history, mean age at surgery, age at onset of menarche, menstrual cycle, or typical endometrio- sis-associated symptoms. The study shows the representative clinical baseline data of women with endometriosis, which were comparable to former studies on the clinical characteristics of en- dometriosis [18 – 20]. Interestingly, we found a statistically signifi- cant difference with regard to the interval between onset of men- arche and surgery as a surrogate marker for reproductive lifespan and mitotic rate, with shorter reproductive lifespan in women with higher rates. It could be hypothesized that a longer repro- ductive lifespan with a longer cumulative exposure to estrogen may be correlated with active cellular proliferation. But our results suggest that endometriosis is diagnosed significantly earlier in women with active proliferative disease. According to current guidelines and clinical algorithms, the in- dication for surgery is usually based on three indicators: pain, in- fertility, and clinical signs of endometriosis [21]. In our study, pain was the main reason for surgery significantly more often in the subgroup of women with detected mitotic figures, which corre- sponded to more advanced stages of endometriosis. Although detection of mitotic figures correlated with clinical stage and especially with deep infiltrating endometriosis, no significant dif- 88 Wetzk M et al. Diagnostic Benefit of … Geburtsh Frauenheilk 2022; 82: 85 –92 | © 2022. The author(s). GebFra Science | Original Article ▶ Table 2 Clinical characteristics of the subgroups without and with mitotic figures as a marker of active proliferation in 542 women with histologically confirmed endometriosis. Clinical characteristics Endometriosis without mitotic figures (n = 402) Endometriosis with a mitotic rate ≥ 1( n=1 4 0 ) p-value Age (years)  33.9 ± 8.2 (17–72)  32.5 ± 7.2 (19–55)  0.134 Obstetric history ▪ No prior pregnancies (n = 537) 233 (58.25%)  80 (58.39 %)  0.772 ▪ No prior births (n = 530) 263 (65.91%)  87 (63.50 %)  0.925 Age at onset of menarche (years)  13.1 ± 1.5 (9–17)  12.9 ± 1.4 (9–16)  0.667 Reproductive lifespan (years)  21.9 ± 8.1 (4–41)  19.4 ± 6.5 (5–35)  0.023* Mean duration of menstrual cycle (n = 286)  29.3 ± 7.3 (13–90)  29.9 ± 8.2 (21–90)  0.643 Mean duration of menstrual bleeding (n = 314)   5.6 ± 1.8 (2–14)   5.9 ± 2.0 (3–15)  0.138 Previous history of endometriosis (n = 397) ▪ No, first diagnosis 305 (76.83%)  99 (73.31 %)  0.239 ▪ Yes, recurrent disease  92 (23.17%)  36 (26.69 %) Predominant reason for surgery ▪ Acute or chronic pain 150 (37.78%)  42 (31.11 %)  0.018* ▪ Infertility  90 (22.67%)  40 (29.62 %)  0.415 ▪ Diagnosis of endometriosis during surgery for other indications 1  83 (20.64%)  17 (12.14 %)  0.031* Symptoms prior to surgery ▪ Deep pelvic pain 255 (63.43%)  86 (61.42 %)  0.685 ▪ Dysmenorrhea 178 (44.28%)  60 (42.9 %)  0.843 ▪ Dysuria  35 (8.71%)   7( 5 . 0 0 % )  0.199 ▪ Dyspareunia  82 (20.40%)  21 (15.00 %)  0.171 ▪ Dyschezia  44 (10.95%)  12 (8.57 %)  0.268 ▪ Primary and secondary infertility 147 (36.37%)  63 (45.00 %)  0.049* ▪ Abnormal uterine bleeding  63 (15.67%)  17 (12.14 %)  0.336 Surgery ▪ Laparoscopy 373 (93.02%) 123 (87.86 %)  0.036* ▪ Laparotomy  23 (5.74%) 123 (9.29 %) rASRM classification (n = 523) ▪ Stage I 179 (45.90 %)  35 (26.32 %) ▪ Stage II  67 (17.18%)  28 (21.05 %)  0.001* ▪ Stage III  74 (18.97%)  37 (27.82 %) ▪ Stage IV  70 (17.95%)  33 (24.81 %) Hormonal treatment  81 (20.15%)  21 (15.00 %)  0.210 ▪ Combined oral contraceptives  55 (13.7%)   18 (12.9 %)  0.886 Only one biopsy for histological assessment  71 (17.67%)  12 (8.56 %) < 0.001** Occurrence of endometriosis (n = 542) ▪ Adenomyosis  42 (10.42%)  16 (11.43 %)  0.752 ▪ Ovarian endometrioma 121 (30.31%)  59 (42.14 %)  0.012* ▪ Deep infiltrating endometriosis according to the ENZIAN score  37 (9.20%)  32 (22.86 %) < 0.001* Peritoneal adhesions 140 (34.83%)  45 (32.14 %)  0.606 Other ovarian cysts  49 (12.19%)  15 (10.71 %)  0.761 Uterine malformations (n = 451)  27 (6.72%)   5( 3 . 5 7 % )  0.214 Significant differences: * p < 0.05, ** p < 0.001. 1 Other indications for surgery were: ovarian cysts not suspicious for endometriosis (n = 45), surgery for fibroids (n = 29), hysterectomy (n = 14), ectopic pregnancy (n = 6), suspected uterine malformation (n = 3), cryopreservation of ovarian tissue (n = 2), tubal sterilization (n = 1). 89Wetzk M et al. Diagnostic Benefit of … Geburtsh Frauenheilk 2022; 82: 85 –92 | © 2022. The author(s). ference was seen with regard to overall clinical symptoms (▶ Table 2). This may be explained by the high variability of symp- toms. The results of studies investigating endometriosis-related pain and clinical staging or localization are conflicting. In an Italian study with 1000 patients, Vercellini et al. reported a higher sever- ity of pain symptoms in patients with higher rASRM stages [22]. In contrast, a study by the US-American group of Sinai et al. with a similar sample size found that women with lower rASRM stages (I–II) suffered more frequently from dyspareunia. There were no differences with regard to other symptoms between the group with low rASRM stages and the group with a high rASRM stage [19]. The heterogenous clinical symptoms of endometriosis may rASRM I ENZIAN A ENZIAN B a b 45.9 17.2 19 17.9 26.3 22.2 52.8 36.1 30.6 25 36.1 2.8 50 29 45.2 19.3 19.3 6.4 45.2 6.4 p = 0.592 p = 0.809 p = 1.000 p = 0.052 p = 0.401 p = 0.176 p = 0.627 p = 0.581 48.4 21.1 27.8 24.8 rASRM classification (n = 423) ENZIAN classification (n = 67) rASRM II ENZIAN C rASRM III FA – adenomyosis FB – bladder FI – intestine FU – ureter FO – other locations rASRM IV Percentage of stages (%) Percentage of stages (%) 60 55 50 45 40 35 30 25 20 15 10 5 0 60 55 50 45 40 35 30 25 20 15 10 5 0 No proliferative activity (n = 390) No proliferative activity (n = 36) Proliferative activity (n = 133) Proliferative activity (n = 31) ▶ Fig. 2 Comparison of stages using the rASRM ( a) and the ENZIAN score ( b) in patients without and with detected mitotic figures as a marker of proliferative activity. 90 Wetzk M et al. Diagnostic Benefit of … Geburtsh Frauenheilk 2022; 82: 85 –92 | © 2022. The author(s). GebFra Science | Original Article explain why our study did not find any statistical differences in the mitotic rate with regard to most symptoms. A prospective multi- center study evaluating longitudinal clinical data in combination with repeated standardized pain questionnaires might find statis- tical differences. Women with symptoms of infertility have higher stages of endometriosis after an invasive diagnosis [20, 24]. There was no significant difference in the number of women with adenomyosis and typical clinical symptoms of dysmenorrhea and abnormal uterine bleeding in the groups with and without ac- tive proliferation (p = 0.752). Adeno myosis is a highly proliferative disease of the uterus, and higher Ki-67 cell indices were found in uterine myometrial tissue [25]. Nevertheless, we calculated the cellular proliferative rate for adenomyosis and endometriotic tis- sue. The strengths of this study are the large sample size and the detailed information on symptoms, reproductive health and med- ical history. Although the data was collected retrospecti vely, infor- mation about the extent of disease was obtained from surgical re- cords, and staging of the disease was standardized. The study was performed in a single certified endometriosis center. While this is a strength, it may also lead to a selection bias with a tendency to higher stages. This can be seen in the percentage of women with deep infiltrating endometriosis in our study (19.0 %). A recent publication reported a lower percentage (14.4 %) of deep infiltrat- ing endometriosis in patients with endometriosis [26]. A detailed analysis of ENZIAN stages and the distribution of endometriotic lesions in deep infiltrating endometriosis did not show significant differences. The long-term follow-up is of special interest in view of the data on recurrence and the possible development of cancerous le- sions. The lack of follow-up is one of the limitations of this study. A prospective follow-up study is currently being planned. The presence of mitotic figures as a marker of severe disease could be an additional factor when recommending that women receive a longer follow-up and treatment to prevent recurrence [27]. We propose that women in whom mitotic figures are de- tected should have shorter postoperative follow-up intervals and that the time to pregnancy in cases of infertility should be short- ened. Assisted reproductive techniques could be proposed earlier for women with confirmed mitotic figures. Although this retrospective study did not find an association between the mitotic rate and recurrence, further prospective val- idating studies could clarify the prognostic value of mitotic figures and their impact on recurrence. Endometriosis can lead to epithelial cancer, although the underlying mechanisms are still not understood [28]. The mitotic rate could serve as an early prognostic marker for women who have a higher risk of developing endometriosis-associated cancer and who therefore require intensified long-term follow-up.

The mitotic rate can be used as a simple additional histological as- sessment for endometriosis. Histology is already routinely used to investigate endometriosis, and this approach would not need ad- ditional training of the pathologist or additional staining of the specimen. We can confirm that the mitotic rate can be an effec- tive diagnostic tool in women with more severe endometriosis based on their surgical findings. Our data strongly suggest that further studies should be carried out to investigate the association between mitotic rate and the rate of recurrence after the initial diagnosis of endometriosis and pregnancy outcomes in women with infertility. Funding No funding was used for the study or preparation of the manu- script. Conflict of Interest The authors declare that they have no conflict of interest.

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