RETRACTED: Dye diffusion during laparoscopic tubal patency tests may suggest a lymphatic contribution to dissemination in endometriosis: A prospective, observational study

Endometriosis is an estrogen-dependent disease characterized by endometrial tissue abnormally growing outside the uterus. It contributes significantly to pelvic pain and infertility and affects up to 10% of women of reproductive age and 30 to 50% of infertile women [ 1 , 2 ]. Endometriosis and secondary adhesions in advanced stages can affect all pelvic structures leading to profound changes in pelvic anatomy that contribute heavily to symptoms (pain and infertility) and make imaging diagnosis [ 3 – 5 ] and surgery [ 2 ] harder. Although peritoneal superficial lesions and ovarian endometriomas represent the majority of endometriotic implants within the pelvis, deep infiltrating endometriosis and extra pelvic endometriosis are the most challenging conditions to face off. Despite medical therapy is a valid approach to reduce symptoms [ 6 ], often a surgical treatment with complete eradication [ 7 ] using a nerve- and vascular-sparing approach [ 8 – 10 ] is needed to improve clinical symptoms (pain unresponsive to medical treatment) and to restore the normal pelvic anatomy and its functions (when non-sexual organs are compromised or to improve fertility) [ 11 ]. The pathophysiology of endometriosis remains unclear as different theories have been proposed although peritoneal implants of endometrial tissues derived from retrograde menstruation in women with a genetic predisposition and immune dysfunction seems to be the most widely accepted hypothesis [ 2 , 12 ]. Certainly, deep infiltrating endometriosis [ 13 ] without peritoneal involvement and distant localizations [ 14 ] cannot be easily explained by this model but are probably due to lymphatic/hematogenic dissemination or metaplastic transformation [ 15 ]. Lymphatic spread was hypothesized based on histological findings as endometrial cells were found in lymph nodes in women with endometriosis [ 16 ]. Angiogenesis and lymphangiogenesis in endometriosis and adenomyosis were shown to play a role in the histological and clinical presentation of the disease [ 17 ]. Adenomyosis is a specific form of endometriosis characterized by endometrial tissue within the myometrium and contributes to dysmenorrhea, pain during sexual intercourse, chronic pelvic pain, and subfertility [ 18 ]. It is characterized by endometrial glands and stroma that break through the myometrium and induce hypertrophy and hyperplasia of the surrounding myometrium associated with an abnormal distribution of uterine vessels [ 18 , 19 ]. The correlation between adenomyosis and endometriosis is still debated as cases with isolated adenomyosis are reported [ 18 , 19 ]. Nevertheless, it is very often found in cases of extrapelvic endometriosis [ 12 ]. Ueki [ 20 ] demonstrated ectatic lymphatic vessels in the myometrium of adenomyotic uteri after hysterectomy and proposed that the lymphatic system could carry endometrial tissue away from the uterus promoting endometriosis of ovaries and pelvic organs. This study was to assess the diffusion of the dye during a laparoscopic tubal patency test through the uterine wall and to extrauterine structures especially where endometriosis foci lay.

Between September 2016 and January 2018, 191 patients underwent laparoscopy for endometriosis ( Table 1 ). All patients were adults (range 18 to 43 years of age). In 104 cases (55%), a tubal permeability test (dye test) was carried out as the patients were infertile. It is not unusual to observe during a laparoscopic tubal dye test a color change, at least in some areas, of the adenomyotic uterus from pale to deep blue. In fact, a change of color of the uterus was noted in 27 cases (26%) that in most cases (93%) extended to extrauterine organs. A clear perfusion through the lymphatic vessels of the methylene blue solution from the uterine cavity to extrauterine structures was seen and this was not related to tubal patency as at least unilateral unobstructed tube was confirmed in 24 out of the 27 cases. Other 7 women with suspect of adenomyosis at preoperatory assessment (ultrasound or MRI) and underwent tubal dye test did not show a macroscopic color chance of the uterus wall. Fig 2 and S1 Video show diffused blue color of the fundus and focal changes of the posterior wall of the uterus ( Fig 2A ) with a clear staining of the proximal part of the right uterosacral ligament ( Fig 2B ). Lymphatic vessels surrounding the ovarian artery and vein in the infundibulopelvic ligament were stained ( Fig 2C ), with a single spot close to the internal iliac vein (a lymph node) ( Fig 2D ). In some cases, the dye permeated the uterine fundus and flowed slowly into small subperimetrial vessels ( Fig 3A and 3B ) before reaching lymphatics surrounding the utero-ovarian vessels ( Fig 3C ) and the external layer of the right ovary ( Fig 3D ). In some cases, endometriosis lesions of subperitoneal lymphatic vessels and distal endometriosis (diaphragmatic endometriosis) can be found ( Fig 2E and 2F ). In other cases, a blue staining of the subperitoneal structures could be seen during dissection ( Fig 4A and 4B ). Endometriosis lesions (endometriotic tissue and/or endometriosis-related reactive fibrosis) were all confirmed at histology and by the assessment of the uterine biopsy ( Fig 4C ) revealed ectatic lymphatic vessels surrounding endometrial glands within the myometrium ( Fig 4D ) as described by other authors as a typical feature of adenomyosis. Adenomyotic tissue reached the outer layer of the myometrium without perimetrium infiltration. It is interesting to note that in two cases of symptomatic women (infertility, dysmenorrhea, and prolonged menstrual bleeding) with unremarkable pelvic ultrasound and office hysteroscopy, the whole uterus shaded into a “bizarre” blue color during a blue tubal test that toned down in a few minutes ( Fig 5 ), notwithstanding unobstructed tubes and without any pelvic endometriosis lesion. Adenomyosis was confirmed at biopsy and a diffuse adenomyosis was confirmed by subsequent MRI. The uterus showed a transient scattered blue color and unilateral right tubal permeability was demonstrated (A). A deep blue color of the endometriosis nodule of the right uterosacral ligament appeared after a few seconds (B) along with a progressive staining of lymphatic vessels of the infundibulopelvis ligament (C). A blue spot was seen where lymph nodes of the internal iliac vein lie (D). The photo sequence (A to D) shows the methylene blue solution that seeped through the tubes and that permeated the uterine wall (A); after a short while, only sub-perimetrial vessels remained dyed (B) and a few seconds later, a clear staining of the lymphatics surrounding di utero-ovarian artery and vein was evident (C). The dye took some minutes to reach the ovary that showed a faint blue color of the cortex beneath the tunica albuginea (D). Photo E shows an endometriosis lesion within a lymphatic vessel of the lateral abdominal wall (the inset at the lower right corner shows a higher magnification thereof) as demonstrated at immunohistochemistry. Two endometriosis foci deeply invaded the peritoneum that lined the right diaphragm (F). Clustered areas of the uterus were permeated by the methylene blue during a dye test that showed bilateral permeability of the tubes (A). After opening the peritoneum of the anterior broad ligament, a blue staining of the connective tissue beneath the round ligament (*) was found (B). In this case, a deep biopsy of the uterine wall where the color changed was taken and adenomyosis with ectatic lymphatic vessels was demonstrated. Deparaffinized 4-μm sections were immunostained with antibodies against CD34 and podoplanin (C and D, respectively) to confirm that the ectatic vessels were lymphatics. An apparently normal uterus (A) shaded into a peculiar blue color during a methylene blue test in a case of diffuse adenomyosis (C). Bilateral tubal permeability was demonstrated (B).

The dissemination of endometriosis is likely to be multifactorial and the lymphatic network contributes to the reflux theory. During menstruation, exfoliated menstrual cells can pour into lymphatic vessels and spread to close and distant structures and organs. These findings throw a different light on the pathogenesis of this common disease and may prompt new researches in this direction, perhaps focusing on translational models [ 41 ] to test new drugs and early molecular targets for the treatment of endometriosis [ 42 ].

This study took the cue from an observation in September 2016: During a laparoscopic procedure in an infertile woman with presurgical evidence of endometriosis, the dye test to verify tubal permeability showed a peculiar effect on the uterine wall. Some areas of the external layer of the myometrium showed a pale blue color and, notwithstanding both tubes were unobstructed, the drainage of the dye from the uterine cavity was noted along the infundibulopelvic ligament through lymphatic vessels. Subsequently, all women undergoing laparoscopy for endometriosis were enrolled and the diffusion sites of the dye during a tubal patency test were recorded. All laparoscopies were performed at the Sacro Cuore Don Calabria Hospital, Negrar, Italy, by an expert gynecologic surgeon (MS) with more than 10 years of experience in laparoscopic gynecological surgery according to our previously described surgical approach [ 21 ]. All patients undergoing laparoscopy for endometriosis were considered for this study and those with infertility problems were enrolled. According to our internal protocol [ 7 , 21 ], all the patients were not on hormonal therapy at the time of surgery as we ask to suspend estroprogestin/progestin treatment one month before surgery. The evaluation of tubal patency for routine assessment of infertility during laparoscopy can be easily obtained injecting a diluted methylene blue solution (0.1%) into the uterine cavity [ 22 ]. An hysterometer was used to sound the uterus length and an appropriate manipulator tip was used (1 cm shorter than the uterus length to avoid uterine perforation/trauma). A uterine manipulator with die injection system was inserted into the cervix under direct vision at beginning of the surgical procedure and used to drive the methylene blue solution into the uterine cavity to verify tubal permeability. Laparoscopic uterine biopsy was performed where the dye reached the uterine external layer ( Fig 1 ) to confirm the presence of adenomyosis [ 23 ] in cases where the preoperative evaluation (ultrasound and MRI) did not suggested adenomyosis. A scattered blue area (arrow) after a dye test (A) that was biopsied (B). All specimens were evaluated in a blind manner by two different pathologists following the same protocol. Surgical specimens were fixed in 10% buffered formalin for 12 hours, paraffin embedded blocks were sectioned and processed with a standard technique followed by hematoxylin/eosin staining. For immunohistochemistry, deparaffinized 4-μm sections were incubated with the following monoclonal antibodies using standard reagents and techniques on Ventana Bench Marks ULTRA: CD34 (QBend 10 RTU) and podoplanin (D2-40 RTU). CD34 is a commonly used marker of hematopoietic progenitor cells and endothelial cells; podoplanin identifies lymphatic endothelium and is not expressed in vascular blood vessel endothelial cells. The clinical trial was registered on UMIN Clinical Trials Registry ( https://www.umin.ac.jp/ctr/ ; registration number is UMIN000034585). Our internal review board considered not necessary a formal approval as the tubal dye test is routinely performed during laparoscopy in adult women suffering from endometriosis and infertility. According to our surgical approach to endometriosis, when a suspect of adenomyosis is made, a biopsy may be taken to complete the diagnosis. Furthermore, a specific preoperative written consent for the removal of all endometriosis lesions (radical approach) and the tubal dye test was obtained by all patients as well as a written clearance for imaging use for research/teaching.

A deliberately full-length video (with an accidentally activated video setting box) was reported to demonstrate that the blue color permeated through tissues and lymphatics and the bluish areas were not due to superficial deposition of the dye from tubal extravasation. Staining of the lymphatic vessels of the uterosacral ligaments, the right infundibulopelvic ligament and the round ligaments can be seen as they appear and progress. (MP4)