Anne-Sophie Boudy
email: annesophie.boudy@aphp.fr

Elie Vesale

Alexandra Arfi

Clementine Owen

Aude Jayot

Sonia Zilberman

Sofiane Bendifallah

Emile Darai

Jayot Aude

Prevesical peritoneum interposition to prevent risk of rectovaginal fistula after en bloc colorectal resection with hysterectomy for endometriosis: Results of a pilot study

Keywords: Bowel endometriosis, Colorectal resection, Deep infiltrating endometriosis (DE), Hysterectomy, Rectovaginal fistula

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

3

Introduction.

Endometriosis is a benign gynecologic disorder defined by the presence of endometrial glands and stroma outside the uterus [START_REF] Koninckx | Suggestive evidence that pelvic endometriosis is a progressive disease, whereas deeply infiltrating endometriosis is associated with pelvic pain[END_REF]. It affects 3% of the general female population and about 10% of women of reproductive age [START_REF] Koninckx | Suggestive evidence that pelvic endometriosis is a progressive disease, whereas deeply infiltrating endometriosis is associated with pelvic pain[END_REF][START_REF] Koninckx | Deep endometriosis: definition, diagnosis, and treatment[END_REF]. Bowel endometriosis is estimated to occur in 5.3% to 12% of women with endometriosis overall [START_REF] Macafee | Intestinal endometriosis. A report of 29 cases and a survey of the literature[END_REF][START_REF] Weed | Endometriosis of the bowel[END_REF] but in as many as 35% of women with deep endometriosis (DE) managed in specialized centers [START_REF] Bazot | Deep pelvic endometriosis: MR imaging for diagnosis and prediction of extension of disease[END_REF].

The classic treatment of colorectal endometriosis is based on hormonal therapy [START_REF] Geoffron | Endometriosis medical treatment: Hormonal treatment for the management of pain and endometriotic lesions recurrence. CNGOF-HAS Endometriosis Guidelines[END_REF].

However, this approach is frequently associated with side effects and incomplete symptom relief and surgery is often required. In this specific setting, various surgical techniques have been advocated such as rectal shaving and discoid or segmental colorectal resection depending on the location, the size of the lesion and the multifocality [START_REF] Andres | Transvaginal Ultrasound for the Diagnosis of Adenomyosis: Systematic Review and Meta-Analysis[END_REF][START_REF] Kho | Surgical treatment of different types of endometriosis: Comparison of major society guidelines and preferred clinical algorithms[END_REF][START_REF] Johnson | World Endometriosis Society consensus on the classification of endometriosis[END_REF].

For patients with no desire to preserve fertility and with associated uterine disorders including myomas and/or adenomyosis, radical surgery including en bloc hysterectomy and colorectal resection (H-CR) is an option. This approach improves symptoms and quality of life [START_REF] Daraï | Laparoscopic versus laparotomic radical en bloc hysterectomy and colorectal resection for endometriosis[END_REF][START_REF] Touboul | Long-term symptoms, quality of life, and fertility after colorectal resection for endometriosis: extended analysis of a randomized controlled trial comparing laparoscopically assisted to open surgery[END_REF] and limits the risk of recurrence. Around 12% of patients with endometriosis require a hysterectomy [START_REF] Rizk | Recurrence of endometriosis after hysterectomy[END_REF].

In addition to the risk of voiding dysfunction, H-CR particularly involving the rectum and the recto-sigmoid junction exposes patients to the need for defunctioning stoma to limit the risk of rectovaginal fistula even if the effectiveness of this surgical procedure remains a matter of debate [START_REF] Belghiti | Role of protective defunctioning stoma in colorectal resection for endometriosis[END_REF][START_REF] Agnifili | The value of omentoplasty in protecting colorectal anastomosis from leakage. A prospective randomized study in 126 patients[END_REF][START_REF] Merad | Omentoplasty in the prevention of anastomotic leakage after colonic or rectal resection: a prospective randomized study in 712 patients. French Associations for Surgical Research[END_REF]. Other surgical techniques have been suggested to reduce the risk of rectovaginal fistula such as omental flap interposition (epiploplasty). However, this procedure is not always feasible due to the anatomical characteristics of the omentum.

Moreover, the efficacy of epiploplasty in the context of concomitant opening of the vagina and colorectal resection of endometriosis is questionable.

Therefore, the objective of this pilot study was to evaluate the potential role of prevesical peritoneum interposition between the vagina and the colorectum after H-CR for DE to limit the risk of rectovaginal fistula while avoiding systematic defunctioning stoma.

Material and Methods.

The present retrospective pilot study was based on the analysis of a prospective database of patients who underwent colorectal surgery for DE from June 2016 to June 2018 at Tenon University Hospital, Expert Center in Endometriosis (Paris), Sorbonne University.

We identified all patients who had undergone H-CR with prevesical peritoneum interposition.

All the patients had given their informed consent for H-CR surgery after being informed about the risk of protective defunctioning stoma. The anatomical locations of endometriosis and extent of colorectal endometriosis were also recorded especially evaluating external adenomyosis and the presence of uterine myomas. All the patients underwent pelvis ultrasonography, MRI, and rectal endoscopic sonography before surgery. DE was diagnosed according to the morphology of the lesions observed by imaging techniques and in accordance with previously described criteria [START_REF] Balleyguier | Comparison of magnetic resonance imaging and transvaginal ultrasonography in diagnosing bladder endometriosis[END_REF][START_REF] Bazot | Ultrasonography compared with magnetic resonance imaging for the diagnosis of adenomyosis: correlation with histopathology[END_REF][START_REF] Bazot | Transvaginal sonography and rectal endoscopic sonography for the assessment of pelvic endometriosis: a preliminary comparison[END_REF][START_REF] Wood | Laparoscopic surgical techniques for endometriosis and adenomyosis[END_REF][START_REF] Roseau | Rectosigmoid endometriosis: endoscopic ultrasound features and clinical implications[END_REF]. Colorectal endometriosis was defined as DE with infiltration of at least the rectal muscularis [START_REF] Agnifili | The value of omentoplasty in protecting colorectal anastomosis from leakage. A prospective randomized study in 126 patients[END_REF]. Associated adenomyosis was defined as previously published by Bazot et al. [START_REF] Bazot | Ultrasonography compared with magnetic resonance imaging for the diagnosis of adenomyosis: correlation with histopathology[END_REF]. The ENZIAN classification was used to described MRI distribution of lesions [START_REF] Tuttlies | ENZIAN-score, a classification of deep infiltrating endometriosis[END_REF].

Preoperative Assessment of DE

Surgical Procedure

All the patients received GnRH analogues for 3 months before surgery and were operated on under general anesthesia in the dorsolithotomy position. The abdominal and pelvic cavities were explored to identify all locations of endometriosis to evaluate the ENZIAN Score. All the colorectal resections were laparoscopically assisted and performed with the objective of complete resection, as previously described associating both hysterectomy and colorectal resection [START_REF] Belghiti | Role of protective defunctioning stoma in colorectal resection for endometriosis[END_REF][START_REF] Fedele | Long-term follow-up after conservative surgery for rectovaginal endometriosis[END_REF]. Procedures included adnexal surgery (ovarian cystectomy or salpingo-oophorectomy); uterosacral ligament, parametrium, or vaginal resection; ureterolysis; and ureteral reimplantation when required. No omental flap interposition was used between the vaginal suture and rectal staple line.

To prevent the risk of rectovaginal fistula and to avoid protective defunctioning stoma, a prevesical peritoneum interposition was performed between the vaginal and digestive scars. For this procedure, the prevesical peritoneum was mobilized then sutured to the posterior vaginal wall by absorbable stitches over the vaginal suture.

For the colorectal resection, lesions under 3 centimeters in diameter underwent a discoid resection after removal of the exophytic portion keeping the infiltrating portion on the rectum. Discoid resection was then performed using an automatic transanal stapler (CDH33A, Endo-Surgery, Ethicon, France). The vagina was sutured with absorbable sutures.

For the segmental colorectal resection consisted of sectioning with endo GIA 60 by laparoscopy (Auto Suture; Tyco S.A., Elancourt, France) after mobilizing the rectum and left colon. The uterus was extracted by the vaginal route and the vagina then closed with absorbable sutures. The median suprapubic trocar was withdrawn and the incision enlarged to 3 cm to allow resection of the colorectum and placement of the anvil of the automatic stapler (CCEA forceps, Auto Suture; Tyco S.A., France).

Study Variables

Data including epidemiologic characteristics age, body mass index (BMI), previous surgery, details of radiologic and surgical findings were recorded from the patients' medical records. The distribution of DE lesions was scored according to the ENZIAN classification.

The size of the histological specimen was also recorded. All intra-and postoperative complications were recorded. In accordance with the Clavien-Dindo classification, complications were classified as minor when of grade I-II (deviation from the normal postoperative course without the need for surgical, endoscopic or radiological interventions) and major when of grade IIIa (requiring surgical, endoscopic or radiological intervention without general anesthesia), IIIb (requiring surgical, endoscopic or radiological intervention under general anesthesia), IV (life-threatening complication, including central nervous system complications or requiring intermediate or intensive care unit management) and V (death). In addition, de-novo voiding dysfunction requiring self-catheterization lasting more than 1 month was considered a major complication. The Ethical Review Committee (CEROG) approved this study (CEROG 2018-GYN-0201). Written informed consent was obtained from all patients.

Statistical Analysis

Quantitative variables were expressed in median and interquartile range (IQR) and nominal variables in proportion. Univariate analysis was performed using the Wilcoxon test for continuous variables and the Fisher exact test for categorical variables. All reported p values were 2-sided. The significance threshold was set at .05. All statistical analysis was performed using commercially available software JMP v.13.0.0 software © (SAS Institute Inc., Cary, NC, United-States).

Results.

Epidemiological and surgical characteristics of the population

During the study period, 160 patients with DE underwent surgery for colorectal endometriosis. Among them, 27 patients (16.8%) underwent an H-CR with prevesical peritoneum interposition.

The characteristics of the study population are summarized in Table 1. The median age was 45 years (IQR 41-47.5), and median BMI was 25.6 kg/m2 (IQR 22.7-28.9). Thirtyseven percent of the patients (n=10) had a history of surgery for endometriosis: 19% (n=5) for DE and 26% (n=7) for ovarian endometriosis.

The main symptoms of the population were dysmenorrhea (96%), dyspareunia (63%), transit disorder (44%), chronic pelvic pain (44%), pain on defecation (26%) and dysuria (11%). Thirty percent (n=8) of the patients were nulliparous and 30% (n=8) had a prior history of IVF. The median gravidity was 1 (IQR 0-2) and the median parity was 1 (IQR 0-2).

According to the ENZIAN classification based on MRI description (Table 2), absence of vaginal involvement (A0) was found in 56% of the patients (n=15), involvement of the lateral compartment between 1 and 3 cm (B2) in 59% (n=16), and rectal involvement between 1 and 3 cm (C2) in 33% (n=9). Bladder endometriosis was described in one patient (4%), and ureter endometriosis in one patient (4%). Associated adenomyosis was found in 52% of the patients (n=14) and other intestine involvement (sigmoid, caecum, appendix and ileum) in 15%.

All procedures were performed by the laparoscopic route. Laparoscopic and MRI evaluation of the ENZIAN score were recorded (Table 2).

Segmental resection and discoid excision were performed in 15 (56%) and 12 patients (44%), respectively (Table 3). Ninety-three percent (n=25) of the patients underwent a torus or uterosacral ligament resection. Bilateral ureterolysis was performed for all the patients. Parametrial resection was performed in 14 cases (52%) (unilateral in six cases and bilateral in eight). Associated procedures were ovarian cystectomy in 12 cases (44%), appendectomy in two (7%), ileocecal resection in five (19%), and a partial bladder resection in one (4%) (Table 3). No patients had primary protective defunctioning stoma.

Endometriosis was confirmed by surgery and histology in all the patients. The median size of the rectal nodules on the histological specimen was 15 mm (IQR 10-25).

Surgical complications and follow-up

The median length of hospital stay was 7 days (IQR 6.5-10.5). Median follow-up was 4). No grade IV-V complications occurred.

The patient with a suspicion of rectovaginal fistula underwent both rectal shaving and colorectal resection associated with bilateral salpingectomy, bilateral ureterolysis and left parametrectomy. She experienced chest pain in the early postoperative period and was investigated by an angio CT scan. No emboli were detected but pneumothorax with mediastinal emphysema was found. As the patient did not have a diaphragmatic location of endometriosis or a high intraabdominal pressure, the presumed diagnosis was esophageal injury linked to the nasogastric tube. Thoracic drainage was performed with a favorable outcome. Scanner guided drainage of a pelvic collection was also performed on postoperative day 8. The patient was re-hospitalized 3 weeks after this procedure for vaginal discharge with complete vaginal dehiscence. CT scan exhibited multiple pelvis abscesses with suspicion of a rectovaginal fistula. A second surgery was required by laparoscopy with conversion to laparotomy with extensive adhesiolysis, drainage of multiple pelvic abscesses and resection of 25 cm of the small bowel. No signs of rectovaginal fistula were found on rectal examination and intraoperative rectal blue test. Pathological analysis of the small bowel revealed an inflammatory reaction. A protective defunctioning stoma was performed.

Three months later, the vagina had healed and the stoma could be closed after a CT scan confirmed absence of rectovaginal fistula. Seven (26%) patients experienced postoperative voiding dysfunction and required self-bladder catheterization: five patients for less than 1 month, one for 45 days and one for 90 days).

Discussion.

This pilot study supports the practice of prevesical peritoneum interposition between the vagina and the colorectum after H-CR for DE as a simple and reproducible procedure which may avoid systematic digestive defunctioning stoma.

In a sub-analysis of a randomized study, H-CR was associated with a significant improvement in dysmenorrhea, dyspareunia and asthenia [START_REF] Daraï | Laparoscopic versus laparotomic radical en bloc hysterectomy and colorectal resection for endometriosis[END_REF]. A trend for improvement was found for diarrhea and back pain, while no improvement in constipation, bowel movement pain or cramping, and dyschesia was observed [START_REF] Daraï | Laparoscopic versus laparotomic radical en bloc hysterectomy and colorectal resection for endometriosis[END_REF]. No difference in urinary function was observed pre-and postoperatively [START_REF] Daraï | Laparoscopic versus laparotomic radical en bloc hysterectomy and colorectal resection for endometriosis[END_REF]. All the SF-36 quality-of-life items apart from physical functioning were significantly improved by surgery [START_REF] Daraï | Laparoscopic versus laparotomic radical en bloc hysterectomy and colorectal resection for endometriosis[END_REF].

However, an important question that deserves to be addressed is whether our results reflect a real and reproducible benefit of prevesical peritoneum interposition in this setting or rather a coincidence due to the relatively low prevalence of the complication. We hypothesize the interest of this simple technique since we found no case of rectovaginal fistula after segmental or discoid colorectal resection in a subgroup of so-called high-risk patients. In these patients the rate of rectovaginal fistula can be as high as 10%, justifying the use of systematic protective defunctioning stoma for several authors [START_REF] Darai | Feasibility and clinical outcome of laparoscopic colorectal resection for endometriosis[END_REF]. In the present study, the only case of suspected rectovaginal fistulae was observed after rectal shaving associated with a segmental colorectal resection complicated by a pelvic abscess with subsequent vaginal dehiscence. No fistula was seen intraoperatively.

In a review published in 2008, Vercellini et al reported that the incidence of rectovaginal fistula after rectovaginal septum surgery (with and without colorectal resection) was between 2 and 10% depending on the series and the surgical procedure [START_REF] Vercellini | The effect of surgery for symptomatic endometriosis: the other side of the story[END_REF]. In a metaanalysis of 49 series focusing on colorectal resection, Meuleman et al observed that the incidence of rectovaginal fistula was 2.7% after segmental resection and 0.7% for the mixed group composed of patients undergoing rectal shaving or discoid resection [START_REF] Meuleman | Surgical treatment of deeply infiltrating endometriosis with colorectal involvement[END_REF]. Finally, in a recent systematic review of the literature involving 3079 colorectal resections, Balla et al reported similar results with 2.4% of rectovaginal fistula overall (2.8% for the open approach, 2.2% for the laparosopic approach and 7.4% for the robotic approach) [START_REF] Balla | Outcomes after rectosigmoid resection for endometriosis: a systematic literature review[END_REF]. However, despite the interest of these reviews, it is difficult to evaluate the true incidence of rectovaginal fistula in routine practice as most publications are from experienced teams and this could constitute a selection bias. In a French multicenter study involving 56 departments of gynecology with 1135 colorectal resections performed in 2015, Roman H. on behalf of the FRIENDS group reported a similar rate of rectovaginal fistula [START_REF] Roman | French coloRectal Infiltrating ENDometriosis Study group). A national snapshot of the surgical management of deep infiltrating endometriosis of the rectum and colon in France in 2015: A multicenter series of 1135 cases[END_REF]. However, a sub-analysis of data from the FRIENDS group by Bendifallah et al showed that this rate varied according to the hospital case volume: 4.95% of rectovaginal fistula for centers managing fewer than 10 procedures per year and 2.77% for centers with more than 40 procedures per year, with a significant cut-off at more than 20 procedures per year [START_REF] Bendifallah | Impact of hospital and surgeon case volume on morbidity in colorectal endometriosis management: a plea to define criteria for expert centers[END_REF].

Several surgical procedures have been advocated to decrease the risk of rectovaginal fistulae after colorectal resection. Belghiti et al reported that protective defunctioning stoma

was associated with a decrease in the number of rectovaginal fistulas in women undergoing partial colpectomy and low colorectal resection for endometriosis from 27% to 15% but without reaching significance [START_REF] Belghiti | Role of protective defunctioning stoma in colorectal resection for endometriosis[END_REF]. However, in this specific setting, the new French guidelines for the management of colorectal endometriosis stated that, due to the lack of sufficient high-level evidence, no recommendation for a systematic protective defunctioning stoma cannot be formulated (30). In a prospective randomized study including 126 patients, Agnifili et al suggested that omentoplasty seemed to be effective in lowering the rate and the severity of anastomotic leakage after colorectal surgery. However, they reported a high incidence of anastomotic leakage of 14.3% in the whole population (6.4% in the omentoplasty group vs 21.9% in the non-omentoplasty group) [START_REF] Agnifili | The value of omentoplasty in protecting colorectal anastomosis from leakage. A prospective randomized study in 126 patients[END_REF]. Conversely, in a prospective randomized study of 712 patients undergoing colonic or rectal resection, Merad et al demonstrated that omentoplasty decreased neither the rate nor the severity of anastomotic failure [START_REF] Merad | Omentoplasty in the prevention of anastomotic leakage after colonic or rectal resection: a prospective randomized study in 712 patients. French Associations for Surgical Research[END_REF]. Finally, a meta-analysis showed that no statistically significant difference was found between the omentoplasty group and the no omentoplasty group in radiological anastomotic leakage (RR 0.76, 95% CI 0.41 to 1.40), death (RR 1.01, 95% CI 0.55 to 1.86), and repeat operation (RR 0.60, 95% CI 0.35 to 1.05) except for clinical anastomotic leakage (RR 0.36, 95% CI 0.16 to 0.78) [START_REF] Hao | Omentoplasty in the prevention of anastomotic leakage after colorectal resection: a meta-analysis[END_REF]. The authors stated that there was not enough evidence to say whether or not omentoplasty should be used to reduce anastomotic leakage after colorectal resection [START_REF] Hao | Omentoplasty in the prevention of anastomotic leakage after colorectal resection: a meta-analysis[END_REF].

Despite the high feasibility rate of prevesical peritoneum interposition between the vagina and the colorectum, the technique is not feasible in patients undergoing partial colpectomy with uterine conservation and is difficult in those with a concomitant bladder resection or with vesico-uterine fold endometriosis imposing extensive peritoneal resection.

In this specific setting, several surgical techniques to minimize the risk of rectovaginal fistula have been advocated [START_REF] Belghiti | Role of protective defunctioning stoma in colorectal resection for endometriosis[END_REF][START_REF] Agnifili | The value of omentoplasty in protecting colorectal anastomosis from leakage. A prospective randomized study in 126 patients[END_REF][START_REF] Merad | Omentoplasty in the prevention of anastomotic leakage after colonic or rectal resection: a prospective randomized study in 712 patients. French Associations for Surgical Research[END_REF][START_REF] Hao | Omentoplasty in the prevention of anastomotic leakage after colorectal resection: a meta-analysis[END_REF]. For instance, some authors recommend a subtotal hysterectomy although this surgical option is associated with a risk of endometriosis recurrence requiring a second surgery [START_REF] Schuster | Endometriosis after laparoscopic supracervical hysterectomy with uterine morcellation: a case control study[END_REF][START_REF] Lieng | Long-term outcomes following laparoscopic supracervical hysterectomy[END_REF]. Another option is the use of a protective stoma. However, this technique is often poorly accepted by patients and requires a second surgery. Akladios et al suggested that, in the absence of partial colpectomy, ileostomy may be omitted in patients with low colorectal anastomosis which were more than 5 cm from the anal verge, and with no adverse intraoperative events [START_REF] Akladios | Is ileostomy always necessary following rectal resection for deep infiltrating endometriosis?[END_REF]. After multivariate regression analysis and adjusting for major clinical, demographic, and surgical characteristics, Milone et al underlined that the only factor associated with complicated cases was the distance of the endometriotic localization from the lower rectum [START_REF] Milone | Colorectal resection in deep pelvic endometriosis: Surgical technique and post-operative complications[END_REF]. In a large series of patients undergoing colorectal resection for endometriosis, Belghiti et al confirmed that all rectovaginal fistulas occurred in patients with a low colorectal anastomosis (p<.001) and 88% in patients with a partial colpectomy (p<.001) [START_REF] Belghiti | Role of protective defunctioning stoma in colorectal resection for endometriosis[END_REF]. Protective defunctioning stoma was associated with a decrease, albeit non-significant, in the number of rectovaginal fistulas in women undergoing partial colpectomy and low colorectal resection (from 27% to 15%) [START_REF] Belghiti | Role of protective defunctioning stoma in colorectal resection for endometriosis[END_REF]. Data from randomized trials evaluating the contribution of epiploplasty in limiting the risk of digestive complications after colorectal resection in indications other than endometriosis, are controversial [START_REF] Agnifili | The value of omentoplasty in protecting colorectal anastomosis from leakage. A prospective randomized study in 126 patients[END_REF][START_REF] Merad | Omentoplasty in the prevention of anastomotic leakage after colonic or rectal resection: a prospective randomized study in 712 patients. French Associations for Surgical Research[END_REF]. However, in a meta-analysis, Hao et al emphasized that there is not enough evidence to claim whether or not epiploplasty should be used to reduce complication rates after colorectal resection [START_REF] Hao | Omentoplasty in the prevention of anastomotic leakage after colorectal resection: a meta-analysis[END_REF].

From another point of view, it has been stated that this major complication can be avoided by leaving the uterus in situ [START_REF] Darai | Feasibility and clinical outcome of laparoscopic colorectal resection for endometriosis[END_REF], with the risk of postoperative recurrence due to incomplete removal of endometriosis [START_REF] Fedele | Long-term follow-up after conservative surgery for rectovaginal endometriosis[END_REF]. The most recent French guidelines suggest that hysterectomy is an option after failure of conservative medical and surgical therapies or in cases of associated uterine disorders such as myomas and external and internal adenomyosis [START_REF] Collinet | Management of endometriosis CNGOF/HAS clinical practice guidelines short version[END_REF][START_REF] Bazot | Role of transvaginal sonography and magnetic resonance imaging in the diagnosis of uterine adenomyosis[END_REF]. This may explain why more than half of our population operated on for colorectal endometriosis underwent an H-CR. Although highly selected, 19 of the 27 women (70%) in our series had had a previous pregnancy with a median gravidity of 1 (IQR 0-2) underlining our center's policy concerning radical hysterectomy. Some limits of this pilot study deserve to be mentioned. First, the low sample size could imply a selection bias as our subgroup of patients were at high risk of complications due to associated disorders such as both internal and external adenomyosis [START_REF] Bazot | Role of transvaginal sonography and magnetic resonance imaging in the diagnosis of uterine adenomyosis[END_REF][START_REF] Bazot | Diagnosis of deep endometriosis: clinical examination, ultrasonography, magnetic resonance imaging, and other techniques[END_REF].

Second, comparison with the literature is difficult as few data have focused on this specific population. In a previous study, we found a high risk of complications in this subpopulation of patients with colorectal endometriosis [START_REF] Touboul | Long-term symptoms, quality of life, and fertility after colorectal resection for endometriosis: extended analysis of a randomized controlled trial comparing laparoscopically assisted to open surgery[END_REF]. Third, our surgical procedure is not applicable in certain cases, for example: in patients with concomitant large bladder involvement or 13 extensive vesico-uterine fold involvement requiring removal of the prevesical peritoneum; and in patients requiring a colpectomy without associated hysterectomy which represents the vast majority of patients with vaginal endometriosis.

Despite the small sample size, the present pilot study supports the practice of prevesical peritoneum interposition to limit the risk of rectovaginal fistula in patients who undergo H-CR for deep endometriosis. Further studies are necessary to confirm these results and to identify good candidates for this simple technique.

Disclosure statement: The authors declare that they have no conflicts of interest and nothing 306 to disclose.

Table 1. Epidemiological characteristics of the population.

Characteristics

  DE was diagnosed clinically by two experienced surgeons (E.D. and S.B.) on the basis of the following criteria: visible dark blue nodules on the posterior vaginal fornix at speculum examination or infiltration associated with palpable induration at vaginal and digital rectal examination. Patients were then referred to the Department of Radiology for confirmation of the diagnosis. All the patients underwent transvaginal ultrasonography followed by magnetic resonance imaging (MRI) to assess the presence of colorectal lesions, the uni-or multifocality of bowel endometriosis, and location of associated DE lesions (2,13,16).

14. 6

 6 months (IQR 10.5-20.2). No patient was lost to follow-up. Nine (33.3%) patients experienced intra-or postoperative complications. One patient (3.7%) experienced a Clavien-Dindo grade I complication (parietal abscess), four (15%) a grade II complication (two urinary infections treated by antibiotics, one pelvic abscess requiring a treatment by antibiotics and one vesicovaginal fistula requiring a prolonged bladder drainage by a Foley catheter for 21 days), two (7.4%) a grade IIIA complication (pelvic abscess requiring radiological drainage, ureteral injury requiring a double-J stent), and two (7.4%) a grade IIIB complication requiring a second surgery (due to a hemorrhage linked to ureteral vessel injury in one case, and for multiple pelvic abscesses with suspicion of rectovaginal fistula in the other) (Table

  value, mean, ng/mL ** 0.56

2017;108(6):886-94.417

 26)

(0)

Parametrial resection, n (%) -unilateral -bilateral

(30)

Ovarian ablation, n (%) (44)

Appendicectomy, n (%)

Partial bladder resection, n (%)


D Canel

B J Skalli

B Paniel

C Haddad

Touboul

Y Dabi

Canel V

Paniel Bj

M.D. Ph.D Cyril Touboul

Postoperative evaluation of chronic pain in patients with Mayer -Rokitansky -Küster -Hauser (MRKH) syndrome and uterine horn remnant: Experience

Keywords: Chronic pain, Mayer -Rokitansky -Küster -Hauser (MRKH) syndrome, uterine horn remnant, surgical management, uterine malformation

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Mayer -Rokitansky -Küster -Hauser (MRKH) syndrome is the second most common cause of primary amenorrhea and occurs in around 1 of 4500 female lives birth [START_REF] Rall | Uterine rudiments in patients with Mayer-Rokitansky-Küster-Hauser syndrome consist of typical uterine tissue types with predominantly basalis-like endometrium[END_REF]. It results from interrupted embryonic development of the paramesonephronic (müllerian) ducts and associates both uterus and the upper two-thirds of the vagina aplasia. Two distinct forms of this syndrome have been described: 1) Type 1 (or simple) with isolated utero -vaginal aplasia 2) Type 2 (or complex), more frequent, with other malformations associated, mostly of the urinary tract.

The diagnostic is usually performed in patients with primary amenorrhea or following first sexual intercourse failure [2][START_REF] Morcel | Programme de Recherches sur les Aplasies Müllériennes, Guerrier D. Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome[END_REF][START_REF] Morcel | The Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome: clinical description and genetics[END_REF]. Patients with MRKH syndrome often have a reduced quality of life due to chronic pelvic pain and current understanding of the origin of this pain remains poor [START_REF] Rall | Uterine rudiments in patients with Mayer-Rokitansky-Küster-Hauser syndrome consist of typical uterine tissue types with predominantly basalis-like endometrium[END_REF]. This pain could result from a secretory endometrium in the uterine remnant leading to painful hematometra [START_REF] Elliott | Presurgical management of dysmenorrhea and endometriosis in a patient with Mayer-Rokitansky-Kuster-Hauser syndrome[END_REF][START_REF] Marsh | Uterine remnants and pelvic pain in females with Mayer-Rokitansky-Küster-Hauser syndrome[END_REF] but this hypothesis has never been proved completely. As MRKH is a rare disease, scientific literature is mainly composed of either case reports or limited -sized cohort always limiting the extent of the conclusions reached.

The aim of our study was to further investigate the different components of this pelvic pain in patients with MRKH undergoing pelvic surgery. Our second objective was to assess the evolution of this pain in patients undergoing surgical removal of their uterine horn remnant.

Material and Methods

We conducted a retrospective study within a tertiary referring gynaecologic department in the Centre Hospitalier Intercommunal de Creteil, in France. Between 1991 and 2013, all patients treated with an MRKH syndrome and with at least one uterine horn remnant were included. Patients with total utero -vaginal aplasia (no uterine remnant) were excluded. Formal written consent of the patients was not required nor asked considering our study design. The research protocol was approved by the Institutional Review Board (IRB) of the French College of Obstetrics and Gynaecology (CEROG 2019-GYN-0301).

All the patients included had their diagnostic confirmed by a clinical examination and an imaging exam, pelvic ultrasonography and / or magnetic resonance imaging (MRI).

All relevant data were extracted from their charts, which included general demographic characteristics, disease history and treatments received, pain description and analgesic consumption. As some patients were referred from other centres, some have had genital surgery before and this information was recorded as well.

Decision to proceed to surgery was taken in multidisciplinary committee. The first step of any surgery performed within our centre consisted in pelvic and abdominal exploration and confirmation of the diagnostic if needed. All additional findings during this exploratory step were recorded, which included other pelvic lesions, peritoneal endometriosis or any other specificity. The gestures performed were based on both presurgical and per-operative data, at surgeon discretion. Surgical horn removal decision was taken in cases its aspect was dilated on pre operative workup and the symptoms (and especially the pain) were compatible with the pain. All specimen removed were addressed for pathological analysis. The presence on the uterus remnant of a secretary endometrium was specified.

All patients had a postoperative visit 4 weeks following surgery, which included a clinical examination. Postoperative pain was evaluated at this time in all patients and was assessed using simple words ("better" / "worse") and / or analgesic consumption evaluation.

The database was managed using Excel (Microsoft Corporation, Redmond, WA, USA).

Considering the size of the cohort, no comparative statistics were performed as they would be meaningless. Descriptive statistics were performed using R software ((3.3.1 version, available online).

Results

Between 1991 and 2013, 21 patients fitted our inclusion criteria and were included in our tertiary referring department. The main characteristics of the patients are presented in table 1. All patients had female phenotype with normal secondary sexual characteristics. Three patients (14%) had a family history of genital malformation (bicornuate or septate uterus, but no MRKH syndrome). Four patients (19%) also had urinary tract malformation and one of them had a polymalformative syndrome.

Before surgery, 20 patients (95%) declared having chronic pelvic pain and in 16 among them (76%), this pain occurred cyclically every month and lasted 2 or 3 days. Thirteen patients (62%) had regular analgesic consumption for this pain and one of them even required opioids on a regular basis. Nine patients (42%) had hormonal therapy for this indication and pain relief was obtained in 6 of them.

Data regarding the surgical findings is presented in table 2. Twenty patients (95%) had bilateral uterine horn remnants. In 13 patients (62%), per-operative additional findings were reported, mostly peritoneal endometriosis (8 patients). Fourteen patients (67%) had surgical removal of uterine horn remnant and 6 patients (29%) had other surgical gestures. One patient had surgical removal of a subserous myoma developed from the uterine horn remnant. Another one had a strong desire of pregnancy and have undergone unification of her two uterine horn remnant as well as an uterine -vaginal anastomosis. She successfully got pregnant after multiple attempts of in vitro fertilisation but had uterine rupture at 18 weeks of amenorrhea.

Mean length of hospitalization was 2.3 ± 1.8 days (Table 3). One patient (5%) had postoperative complication: a short -term recurrence of a recto -vaginal fistula after surgical treatment. Pathological analysis retrieved secretary endometrium in 11 patients and confirmed peritoneal endometriosis in 5 patients. Only 3 patients (21%) had persistent residual pain following surgery (Table 3).

Discussion

Twenty-one patients with MRKH syndrome and uterine horn remnant underwent surgery in our tertiary referring centre and almost all of them (95%) described chronic pelvic pain, on a cyclic basis for 2 to 3 days. Only 3 patients (21%) that underwent surgical removal of uterine horns remnant had residual pain after surgery. This cyclic pain alters those patients quality of life and so far, despite some hypotheses from small cohorts, no consensus has been reached to explain their origin.

The hypothesis of a secretary endometrium mimicking regular periods is plausible and if valid, continuous hormonal therapy should improve the pain by preventing endometrial decidualization. Eliott et al. reported a case of a MRKH patient successfully improved with low dose monophasic oral contraceptive [START_REF] Elliott | Presurgical management of dysmenorrhea and endometriosis in a patient with Mayer-Rokitansky-Kuster-Hauser syndrome[END_REF]. However, in our experience, only 66% of patients felt improved by such treatment. There is two ways to interpret failure of hormonal therapy in some patients: either a secretary endometrium is not responsible (alone) for the pain or there is a residual bleeding in atrophic endometrium. Furthermore, if secretary endometrium was the only factor responsible for cyclic pelvic pain, patients undergoing surgical removal of uterine horns remnant should fully recover. This was not the experience we had in our centre with only half patients with no residual pain following surgery. Those arguments supports the idea secretary endometrium is not the sole responsible factor for chronic pelvic pain in those patients but might be part of it.

Another factor probably implicated is the presence of peritoneal endometriosis [START_REF] Elliott | Presurgical management of dysmenorrhea and endometriosis in a patient with Mayer-Rokitansky-Kuster-Hauser syndrome[END_REF]. Peritoneal endometriosis could provoke cyclic chronic pain by itself and it has been described in patients with müllerian abnormalities more than 30 years ago [START_REF] Olive | Endometriosis and mullerian anomalies[END_REF]. In our cohort, peritoneal endometriosis was discovered during surgery in 8 patients (38%).

Continuous hormonal therapy should improve those patients too. A more radical treatment is the surgical removal of the uterine horns remnant, easily feasible by laparoscopy with a low rate of complications [START_REF] Will | Surgical pearls: laparoscopic removal of uterine remnants in patients with Mayer-Rokitansky-Küster-Hauser[END_REF]. In our cohort, 14 patients had such surgery and none experienced postoperative complications. Besides, patients with MRKH are often operated multiple times and postoperative adhesions could partly be responsible for their pain.

Finally, peritoneal irritation following ovulation or organic lesions such as ovarian cyst and uterine myoma could induce pelvic pain in those patients [START_REF] Olive | Endometriosis and mullerian anomalies[END_REF]. Those findings were not anecdotic in our cohort (29%). In our experience, pelvic MRI had a higher sensitivity than ultrasonography for the diagnostic of the MRKH syndrome. Some of those lesions could be diagnosed prior to surgery and this highlights the importance of a meticulous preoperative workup.

It is far more likely all the parameters aforementioned are somehow combined in pelvic pain. The aim of our study was to further investigate the origins of those patients' pain and we recognize some limits to our analysis. Firstly, when studying a rare disease, prospective design is not feasible and some biases are unavoidable. While we report one of the largest cohorts ever, the number of patients included remains low; all the more we restricted inclusion to patients with MRKH syndrome and at least one uterine horn remnant. Our results are strictly descriptive. Since the literature is mainly composed of case -reports and limited cohorts, a meta -analysis would be of great interest to take fully advantage of the work of different teams worldwide. Secondly, we were not able to report long -term evaluation of those patients pain following surgery and our analysis is limited to postoperative evaluation. This is all the more unfortunate that it would be of great interest to evaluate pain reduction following surgery over more than one cycle, and this could bias our results. As a tertiary referring centre for this disease, we often manage patients from many different centres across France and a long term follow up is delicate. Eventually, pain evaluation was not systematically assessed using a reproductive validated scale, neither was recorded the analgesic consumption at the time of the postoperative visit, which limits the precision of the analysis of the surgical impact.

Conclusion

We report a cohort of 21 patients with MRKH syndrome and at least one uterine horn remnant. Only 3 of them (21%) had residual disease following surgical removal of their uterine horn remnant. Management of those patients in relative high -volume centres is of paramount importance to keep improving our understanding of their symptoms and decide the proper management.

Table 1 : Initial characteristics of the patients included N = 21

 1 syndrome. J Pediatr Adolesc Gynecol. 2013;26(4):224 7. 224 225

	Age at diagnosis (years)	18.9 ± 4.8
	Height (cm)	160.8 ± 4.8
	Weight (kg)	55.0 ± 8.1
	IMC (kg/M2) (mean (±SD))	21.1 ± 3.4
	Family history of genital malformation	3 (14)
	Cyclic pelvic pain	20 (95)
	Cyclic pain	16 (76)
	Regular analgesic use	13 (62)
	Hormonal therapy	9 (42)
	Sexual intercourses
	Spontaneous	9 (42)
	After surgical procedure	6 (29)
	Initial assessment of the syndrome
	Pelvic ultrasonography	19 (90)
	Pelvic MRI	13 (62)
	Urinary malformation associated	4 (19)
	Multiple congenital malformation	1 (5)
	Data are given as mean ± standard deviation or n (%)


Xavier Mesmin

Anne-Marie Cortesero

Loïc Daniel

Manuel Plantegenest

Vincent Faloya

Anne Le Ralec
email: anne.leralec@agrocampus-ouest.fr.

Anne Le

Influence of soil tillage on natural regulation of the cabbage root fly Delia radicum in brassicaceous crops

Keywords: ground dwelling predators, pest regulation service, cabbage root fly

Ground dwelling predators provide regulation services of several insect pests.

Enhancing these services may be a step toward integrated crop protection. Many studies have shown that soil tillage is deleterious to ground dwelling predators but pest regulation processes and services have rarely been measured. We performed an experiment to study whether simplifying soil tillage before the establishment of spring broccoli enhanced ground dwelling predator populations and the control they provide on Delia radicum. The direct effect of tillage on arthropods was assessed by comparing their emergence rates in plots differing in soil tillage management. The natural regulation service was assessed by comparing a control and an exclusion treatment in which predators were removed. The effect of soil tillage on carabids, spiders and staphylinids did not match the gradient of disturbance induced by tillage treatments. Tillage did not appear to affect the predators that likely contribute to D. radicum regulation. Consistently, the number of pests suppressed and the root injuries were unaffected by tillage treatments. The main deleterious effect of soil tillage was on the emergence of those carabid species that overwinter partly as larvae, suggesting that spring tillage could affect pest control in the following crops.

Introduction

Farmers have long used soil tillage, mostly for weed management and refinement of the soil structure to favor crop germination [START_REF] El Titi | Soil tillage in agroecosystems[END_REF]. However, many studies have shown that intensive tillage practices generally lead to a reduction in the abundance of soilinhabiting macroorganisms including potential predators of weed seeds or arthropod pests, i.e. mainly spiders, carabid beetles and staphylinid beetles [START_REF] Holland | The environmental consequences of adopting conservation tillage in Europe: reviewing the evidence[END_REF][START_REF] Kendall | Soil tillage and epigeal predatory arthropods[END_REF]. This reduction results from direct effects (e.g. direct mortality from the tilling, desiccation; [START_REF] Kendall | Soil tillage and epigeal predatory arthropods[END_REF] and/or from indirect effects, i.e. from different arthropods colonizing the field after tillage (e.g. in response to modified plant cover, to alternative prey availability; [START_REF] Petersen | Effects of non-inverting deep tillage vs. conventional ploughing on collembolan populations in an organic wheat field[END_REF][START_REF] Thorbek | Reduced numbers of generalist arthropod predators after crop management[END_REF]). To date, most studies did not distinguish between these direct and indirect effects, mainly because it is difficult to design and carry out an experiment allowing these two effects to be disentangled [START_REF] Thorbek | Reduced numbers of generalist arthropod predators after crop management[END_REF].

The effects of soil tillage on ground dwelling predators depend on several traits of the studied organisms including their development stage at tillage [START_REF] Purvis | Emergence of Carabidae (Coleoptera) from pupation: A technique for studying the "productivity" of carabid habitats[END_REF], burrowing depth [START_REF] Lorenz | Mechanische Unkrautbekämpfungsverfahren in Zuckerrübenkulturen und ihre Nebenwirkungen auf Laufkäfer (Coleoptera, Carabidae) und andere epigäische Arthropoden[END_REF] or body size [START_REF] Hatten | Effects of Tillage on the Activity Density and Biological Diversity of Carabid Beetles in Spring and Winter Crops[END_REF]. Therefore, carabids, staphylinids and spiders respond differently to soil tillage: tillage generally does not have much of an impact on staphylinids whereas carabids and spiders are more affected [START_REF] Wardle | Impacts of Disturbance on Detritus Food Webs in Agro-Ecosystems of Contrasting Tillage and Weed Management Practices[END_REF]. Consequently, soil tillage is likely to modify pest natural regulation processes because ground dwelling predators abundance [START_REF] Symondson | Can Generalist Predators Be Effective Biocontrol Agents?[END_REF] and traits [START_REF] Wood | Functional traits in agriculture: agrobiodiversity and ecosystem services[END_REF] are involved in their predation potential.

Many articles reported the effect of soil tillage on pest predation using sentinel preys or predation cards (e.g. [START_REF] Petit | Interactions between conservation agricultural practice and landscape composition promote weed seed predation by invertebrates[END_REF][START_REF] Tamburini | Conservation tillage mitigates the negative effect of landscape simplification on biological control[END_REF], generally showing that reduced tillage had a positive impact on predation potential. However, such methods only give indications on the potential natural regulation obtained and have considerable limitations for several pests [START_REF] Zou | Video monitoring of brown planthopper predation in rice shows flaws of sentinel methods[END_REF]. Direct measurements, e.g. using exclusion protocols [START_REF] Luck | Experimental Methods for Evaluating Arthropod Natural Enemies[END_REF], are still needed to obtain a more realistic estimate of the effect of soil tillage on the natural regulation provided by pest predators. There is also a need to go further and to test whether tillage simplification leads to fewer injuries and/or damage to the crop in order to assess the potential of soil tillage simplification as a conservation biological control practice [START_REF] Rusch | Conservation Biological Control in Agricultural Landscapes[END_REF].

The main pest of brassicaceous vegetables in northwestern Europe is the cabbage root fly Delia radicum L. (Diptera: Anthomyiidae), that may inflict 40-60% plant mortality [START_REF] Estorgues | Maladies et ravageurs des légumes de plein champ en Bretagne. Chambres d'agriculture de Bretagne[END_REF] on spring vegetables without insecticide protection. The flies lay their eggs at the base of plant stems, on or slightly below ground [START_REF] Hughes | Natural mortality of Erioischia brassicae (Bouché) (Diptera, Anthomyiidae) during the immature stages of the first generation[END_REF]. Then the larvae develop below ground, feeding on, and thus inflicting injuries to plant roots.

Metamorphosis occurs inside a pupa, also buried in the ground, near plant roots [START_REF] Hughes | Natural mortality of Erioischia brassicae (Bouché) (Diptera, Anthomyiidae) during the immature stages of the first generation[END_REF]. The cabbage root fly suffers high mortality rates between egg and pupa, mostly at the egg stage (about 80-90%; [START_REF] Hughes | Natural mortality of Erioischia brassicae (Bouché) (Diptera, Anthomyiidae) during the immature stages of the first generation[END_REF]. Part of this mortality is due to predation by ground dwelling arthropods. The most significant predators are probably carabids [START_REF] Andersen | Carabidae and Staphylinidae (Col.) as predators of eggs of the turnip root fly Delia floralis Fallén (Diptera, Anthomyiidae) in cage experiments[END_REF][START_REF] Coaker | The Importance of Some Carabidae and Staphylinidae as Predators of the Cabbage Root Fly, Erioischia Brassicae (Bouché)[END_REF] with a strong positive relationship between their body size and their regulation potential [START_REF] Finch | Effect of beetle size on predation of cabbage root fly eggs by ground beetles[END_REF]. Fewer staphylinid species are involved in D. radicum natural regulation but Aleochara (Gravenhorst) species are particularly efficient egg predators [START_REF] Andersen | Carabidae and Staphylinidae (Col.) as predators of eggs of the turnip root fly Delia floralis Fallén (Diptera, Anthomyiidae) in cage experiments[END_REF]. Moreover, they contribute to D. radicum natural regulation as parasitoids of the pupae [START_REF] Hughes | Natural mortality of Erioischia brassicae (Bouché) (Diptera, Anthomyiidae) during the immature stages of the first generation[END_REF]. Finally the role of spiders has never been reported but their significance as predators of arthropod eggs could be greater than currently expected [START_REF] Nyffeler | Spiders as predators of arthropod eggs[END_REF].

The aim of the present study was to compare the levels of natural regulation of D. radicum by ground dwelling predators under common tillage practices. We tested the following hypotheses: 1) intensive soil tillage significantly decreases the abundance of ground dwelling predators emerging inside the field; 2) The natural regulation provided by these ground dwelling predators decreases with soil tillage intensity and root injuries consequently increase. For this we monitored natural populations of D. radicum on broccoli Brassica oleracea var. italica Plenck (Brassicales: Brassicaceae) in an exclusion experiment performed under various tillage regimes.

Materials and methods

Study site and experimental design

The experiment was carried out in northwestern France during spring 2016, at the INRA experimental station ("Domaine expérimental de la Motte", UE 0787) of Le Rheu (48°07'N, 1°47'W), inside a 6.3 ha field sown with Triticale the previous year. The field was sown with an intercrop (mix Phacelia -buckwheat) in September 2015 and the intercrop was mown and the residues removed in February 2016. In the five years preceding the experiment, the field was under non-inversion tillage practices (i.e. no plowing). An area of 0.6 ha of the study field was split into four blocks separated by 10m bare ground passageways. Each block was split into three plots, each measuring 30m x 15m. Three tillage treatments were allocated to the plots in a randomized complete block design along a gradient of tillage intensity: plowed and harrowed, only harrowed or not tilled at all. Plowing was performed on March 21 st using a four-bottom rollover moldboard plow equipped with skim coulters (Grégoire Besson SAS, Sèvremoine, France; depth ~ 25 cm) and harrowing was performed on March 22 nd using a rotary harrow (RABE Agri GmbH, Bad Essen, Germany; rotors at 420 rpm, depth ~ 10 cm). On the 20th and 21st of April, the field was planted with broccoli (cv. 'Marathon') plants at the stage of 2-3 true leaves, every 0.50 m in rows 0.75 apart. The field was kept free of weeds at least until early June using herbicides (0.6 L/ha of metazachlore on April 29 th followed by 1kg/ha of pyridate on May 20 th ) and by manual weeding in the experimental subplots on May 24 th .

To minimize the homogenizing effect of ground dwelling arthropods migrating from surrounding habitats after soil tillage [START_REF] Thorbek | Reduced numbers of generalist arthropod predators after crop management[END_REF], we excluded them in all treatments. Two "predator treatments" were implemented per plot. The control "exclusion" treatment, consisted of 25 m² square subplots surrounded by partially buried barriers (40cm below ground and 20cm above ground, Greenborder, Nortene ®). The "removal" treatment consisted of 9 m² square subplots in which 13 pitfall traps were added to remove the ground dwelling arthropods. This resulted in 1.4 traps per m² in "removal" subplots, a density exceeding most of the studies reporting efficient exclusion (e.g. 0.3 traps / m² on average in [START_REF] Chiverton | Effects of exclusion barriers and inclusion trenches on polyphagous and aphid specific predators in spring barley[END_REF][START_REF] Holland | The effectiveness of exclusion barriers for polyphagous predatory arthropods in wheat[END_REF], to ensure that we obtained an efficient removal of ground dwelling predators. Although subplot surfaces differed, plant density and plant growing conditions were identical as space within and between rows were the same. The traps were half-filled with water to which a few drops of odorless detergent were added and renewed weekly. Exclusion barriers and pitfall traps were set up immediately after soil tillage, on March 22 nd and 23 rd , to avoid arthropod immigration into experimental subplots.

Data collection

Soil tillage and ground dwelling arthropods emergence

Emerging arthropods abundance was monitored using emergence tents (60cm x 60cm

x 60cm, MegaView Science Co.). Two tents were set up in each plot, immediately after tillage (between the 22 nd and the 29 th of March), so that there was a total of eight tents per tillage treatment. The lateral flaps of the emergence traps were buried vertically to a depth of about 10-15cm, as in [START_REF] Hanson | Agricultural Land Use Determines the Trait Composition of Ground Beetle Communities[END_REF], to avoid immigration or emigration of ground dwelling arthropods. Each tent included one pitfall and one aerial collector to collect ground dwelling and climbing or flying arthropods, respectively, emerging inside the tent. Both collectors were half-filled with water containing a few drops of odorless detergent. The trapping period began on March 30 th and the traps were collected weekly until June 27 th . Carabids, staphylinids and spiders were sorted and counted. The cumulative numbers of carabids, staphylinids and spiders trapped per tent from March 30 th to June 27 th were used as response variables in data analysis. Carabids were identified at the species level based on [START_REF] Roger | Clé de détermination des Carabidae -Paysages agricoles du Nord Ouest de la France[END_REF] because: i/ their sensibility to soil tillage has been shown to depend on their overwintering stage [START_REF] Purvis | Emergence of Carabidae (Coleoptera) from pupation: A technique for studying the "productivity" of carabid habitats[END_REF] and ii/ they are thought to contribute much to D. radicum natural regulation [START_REF] Coaker | The Importance of Some Carabidae and Staphylinidae as Predators of the Cabbage Root Fly, Erioischia Brassicae (Bouché)[END_REF]. Their overwintering stage (only as adults vs. partly or only as larvae) was assigned following [START_REF] Ribera | A comparative study of the morphology and life traits of Scottish ground beetles (Coleoptera, Carabidae)[END_REF] and the public database carabids.org [START_REF] Homburg | Carabids.org -a dynamic online database of ground beetle species traits (Coleoptera, Carabidae)[END_REF]; and their mean body size was taken from [START_REF] Roger | Clé de détermination des Carabidae -Paysages agricoles du Nord Ouest de la France[END_REF] (Table S1 in Supplementary materials). For each tent, mean carabid body length was calculated using community weighted means (CWM).

Soil tillage and Delia radicum natural regulation

Delia radicum egg laying was monitored using felt traps strapped at the base of broccoli stems [START_REF] Freuler | Le piège à oeufs, nouveau moyen de prévision d'attaque pour la mouche du chou, Delia radicum (brassicae)[END_REF]). Four traps were set up in each removal subplot at planting, aligned along a diagonal to have two traps close to the barriers and two in the center of the subplots. They were recorded weekly until June 20 th . Eggs laid on the traps were counted, removed and the trap was then replaced around the same plant. Plants used for egg monitoring were not used for any other measurement. The cumulated number of eggs laid per plant throughout the experiment (i.e. from April 21 st to June 20 th ) was used as a response in data analysis.

When eggs had theoretically reached the pupal stage (about 300 degree days between egg laying and pupation; [START_REF] Collier | Thermal requirements for cabbage root fly, Delia radicum, development[END_REF] i.e. about 27 days after the end of egg laying in our experiment), soil samples (12cm in diameter, 13.5 ± 0.4cm in depth) were taken from around plant roots (including the root system after cutting the broccolis at the base of plant stem) and the number of pupae was counted after washing the samples through a 1mm x 1mm square mesh sieve. Ten samples were taken in every subplot on June 30 th , on randomly selected plants, making a total of 240 samples, i.e. 40 per combination of tillage treatmentpredator treatment. The same plants were used to assess root injuries caused by D. radicum larvae feeding following the qualitative ordinal notation proposed by [START_REF] Dosdall | Susceptibilities of Species and Cultivars and Canola and Mustard to Infestation by Root Maggots (Delia spp.) (Diptera, Anthomyiidae)[END_REF]: 0 = no root damage; 1 = small feeding channels on the root comprising less than 10% of the root surface area; 2 = 11-25%; 3 = 26-50%; 4 = 51-75%; and 5 = 76-100% of the tap root surface area injured. In figures, this qualitative ordinal scale was transformed into a continuous variable representing the fraction of the tap root surface injured (using the median of each class) to facilitate the interpretations.

Data analysis

We tested the effect of soil tillage on ground dwelling predators abundance and on carabid traits and we tested the effects of soil tillage, predator treatment and of their interaction on the number of D. radicum eggs laid, on the number of pupae and on root injuries. All statistical analyses were performed using R software (R core team, 2017). We used generalized linear mixed models with a distribution appropriate to the type of the response variable (functions 'lmer', 'glmer' or 'glmer.nb' of the package 'lme4'; [START_REF] Bates | Fitting Linear Mixed-Effects Models using lme4[END_REF]. Considered distributions were: negative binomial (response variables: cumulative density of carabids, staphylinids and spiders; number of D. radicum eggs per plant; number of D. radicum pupae per plant), binomial (response variable: proportion of carabids overwintering partly as larvae) and Gaussian (response variable: carabid body length). In every model, the experimental block was added as a random factor. Finally, the qualitative ordinal variable of root injuries was analyzed with a cumulative link mixed model (function 'clmm', package 'ordinal'; [START_REF] Christensen | ordinal -Regression models for ordinal data[END_REF]. The significance of the fixed effects was tested using type II Wald chi-square tests, except for root injuries where a likelihood-ratio chisquare test was used (function 'Anova', package 'car'; Fox and Weisberg, 2011 and package 'RVAideMemoire';[START_REF] Hervé | RVAideMemoire: Diverse basic statistical and graphical functions[END_REF]. When the effect of tillage treatment was significant, pairwise comparisons of the estimated marginal means of each treatment were performed using the Tukey method (function 'emmeans' from the 'emmeans' package; [START_REF] Lenth | emmeans: Estimated Marginal Means, aka Least-Squares Means[END_REF].

Results

Soil tillage and ground dwelling arthropods emergence

Between March 30 th and June 27 th , we trapped 380 spiders, 5171 staphylinids and 1069 carabids in the emergence tents (see Table S2 and Figure S1 in Supplementary materials for details on species composition and emergence dynamic). The cumulative number of carabid beetles and the cumulative number of spiders trapped per tent did not differ between tillage treatments (Table 1) but tillage treatment had a significant effect on the number of staphylinids, which was significantly lower in the "no-tillage" treatment than in the other two (Table 1). There was no effect of tillage treatment on the CWM carabid body length but the proportion of carabids overwintering at least partly as larvae was significantly lower in the tents set up in the "only harrowed" treatment than in the two others (Table 1). Trechus gr. quadristriatus (85.9% ± 1.4%) was highly dominant in this group of carabid species that overwintered at least partly as larvae.

Soil tillage and Delia radicum natural regulation

The number of eggs laid per plant did not differ significantly between tillage treatments (χ² = 4.52, df = 2, P = 0.104). We collected significantly more pupae in the "removal" than in the "exclusion" treatment (χ² = 6.26, df = 1, P = 0.012; Figure 1A). There was no significant effect of tillage treatment on the number of collected pupae (χ² = 1.97, df = 2, P = 0.372) and the interaction between the removal and tillage treatments was not significant (χ² = 0.81, df = 2, P = 0.667). The number of pupae was reduced on average by 22.7% in the presence of ground-dwelling predators.

D. radicum larvae inflicted significantly more root injuries in the "removal" than the "exclusion" treatment (χ² = 11.43, df = 1, P < 0.001; Figure 1B) but there was again no significant difference among soil tillage treatments (χ² = 0.73, df = 2, P = 0.693) and no interaction between these two factors (χ² = 3.51, df = 2, P = 0.173).

Discussion

Arthropod emergence in the three tillage treatments tested did not fit the expected gradient of disturbance. Our results are therefore not in agreement with the general consensus that ground dwelling arthropod abundance decreases when soil tillage intensity increases [START_REF] Rusch | Conservation Biological Control in Agricultural Landscapes[END_REF][START_REF] Wardle | Impacts of Disturbance on Detritus Food Webs in Agro-Ecosystems of Contrasting Tillage and Weed Management Practices[END_REF]. However, most studies published to date did not distinguish between the direct and indirect effects of soil tillage. The discrepancy between our results and previous reports may then come from the removal of indirect effects in our experimental setup. Using similar methods on the same taxa, [START_REF] Thorbek | Reduced numbers of generalist arthropod predators after crop management[END_REF] also found that the direct effects of soil tillage were minimal. Nevertheless, among carabids we showed that the proportion of species overwintering at least partly as larvae was lowest when plots had been only harrowed. This suggests that overwintering larvae are more sensitive than adults to the rotary harrow, but not if the field is previously plowed. The higher sensibility of larvae had already been pointed out in carabids [START_REF] Purvis | Emergence of Carabidae (Coleoptera) from pupation: A technique for studying the "productivity" of carabid habitats[END_REF] but our results further indicate that the direct effects of soil tillage probably depend on the sequence of applied practices: the negative effect of harrowing was suppressed when applied after plowing. The deepest tillage operation (i.e. often plowing) is thus not necessarily the most injuring and could even have a protective effect on overwintering larvae against more damaging practices. This is in line with the fact that some overwintering larvae live at a shallow depth, as shown for Pseudoophonus rufipes for instance (10-15cm in [START_REF] Hartke | Seed Feeding, Seed Caching, and Burrowing Behaviors of Harpalus rufipes De Geer Larvae (Coleoptera: Carabidae) in the Maine Potato Agroecosystem[END_REF], and thus could have been buried by plowing [START_REF] Roger-Estrade | Modelling vertical and lateral weed seed movements during mouldboard ploughing with a skim-coulter[END_REF] and protected from the harrow. Finally, it should be noted that some effects of tillage such as modified soil structure [START_REF] Bronick | Soil structure and management: a review[END_REF] and modified soil organic matter distribution in the tilled horizon [START_REF] Balesdent | Relationship of soil organic matter dynamics to physical protection and tillage[END_REF] are not immediate. They may have consequences on microorganisms [START_REF] Bronick | Soil structure and management: a review[END_REF] and on higher-order trophic levels (such as the predators monitored here) in the long term that we could not assess due to the short duration of tillage treatments differentiation. To date the response, over several years, of ground dwelling predators following tillage changes remains largely unexplored (but see [START_REF] Wardle | Effects of agricultural intensification on soil-associated arthropod population dynamics, community structure, diversity and temporal variability over a seven-year period[END_REF].

Regardless of tillage practice, we found that the number of pupae per plant and the intensity of root injuries were reduced in the presence of locally emerging ground dwelling predators. On the other hand, the number of pests suppressed or the level of root injuries did not differ between tillage treatments. The ground dwelling predators that overwintered inside the field therefore provided the same level of regulation in every tillage condition. This is consistent with the limited effect of soil tillage on the emergence of the ground dwelling arthropods. Especially, soil tillage had no effect on two characteristics of carabid communities which potentially determine their predation potential: i/ body size, which is a key factor in prey -predator relationships in general [START_REF] Brose | Consumer-Resource Body-Size Relationships in Natural Food Webs[END_REF] and in this biological system in particular [START_REF] Finch | Effect of beetle size on predation of cabbage root fly eggs by ground beetles[END_REF]; and ii/ their abundance, especially at the beginning of egg laying, which is probably the appropriate time for D. radicum regulation [START_REF] Mesmin | Carabid predation on Delia radicum: the early bird catches the worm[END_REF].

Conversely, the two features of ground dwelling arthropods emergence that were impacted by soil tillage were unlikely to lead to different levels of predation. First, the observed effect on overwintering traits of carabids was related to the massive emergence of T. gr. quadristriatus, a species that emerged too late to contribute to D. radicum egg predation. Secondly, although emerging staphylinids were not determined to species, there were very probably few Aleochara among them as only 63 D. radicum pupae out of 1713 were parasitized by Aleochara spp. (results not shown), a weak abundance that is usual in the region studied (e.g. [START_REF] Lamy | Artificially applied plant volatile organic compounds modify the behavior of a pest with no adverse effect on its natural enemies in the field[END_REF]. Other potential D. radicum predators may also have rapidly dispersed by air after emergence as most staphylinids have good flying abilities [START_REF] Levesque | Abundance, diversity and dispersal power of rove beetles (Coleoptera: Staphylinidae) in a raspberry plantation and adjacent sites in Eastern Canada[END_REF].

To conclude, soil tillage before crop establishment did not have a significant impact on the natural regulation of D. radicum in spring broccoli. This finding is consistent with the fact that the ground dwelling predators that are likely to provide the service in spring did not suffer from soil tillage. However, we showed that shallow tillage affected the species that overwinter partly as larvae, suggesting that the natural regulation services these species provide later in summer and autumn crops could be weakened. Furthermore, the harmlessness of plowing vs. the deleterious effect of harrowing on these taxa raises the question of the relative impact, on ground dwelling predators, of one deep soil disturbance vs. the multiple shallow disruptions that can be necessary to control weeds in no-plow systems.

Table 1 :

 1 Values and significance of Type-II Wald chi square tests performed on the cumulative number of carabids, spiders and staphylinids trapped per tent from March 30 th to June 27 th and on two functional traits of carabids trapped during this period: the proportion of each overwintering stage and the community weighted mean body length. The backtransformed estimated marginal means (± SE) are given for each tillage treatment (PH: plowed and harrowed, OH: only harrowed, Ø: not tilled) and the results of pairwise comparisons are shown when chi square tests were significant: tillage treatments denoted with different letters are significantly different (p < 0.05).

Figure 1 :

 1 Figure 1: Mean number (± SE) of pupae collected per plant at harvest (A) and mean

Acknowledgements

We thank Marie Vincent and Paul Adam for their helpful contribution to the field work. We thank Franck Duval for sorting and identifying the arthropods. We thank the "Unité Expérimentale de la Motte" (INRA, Le Rheu) for the setup of the experimental field. We thank Dr. Leigh Gebbie who revised the English of this manuscript.

Funding This work was supported by the Région Bretagne (grant n° 0461/COH14000/00001042) and the Chaire AEI (grant n° 2016 0190).

Declaration of interest: none