The prevalence of endometriosis in unexplained infertility: a systematic review.

In 15-30% of couples with infertility, no abnormalities are found after the initial diagnostic work-up. The aim of this study was to investigate the prevalence of endometriosis in patients with unexplained infertility undergoing diagnostic laparoscopy in the current era of improved imaging and assisted reproductive technology. A systematic search of PubMed, Embase and Cochrane Central was conducted to identify all studies reporting on pelvic pathologies found by laparoscopy in couples diagnosed with unexplained infertility. Normal ovulatory cycles, normal semen analysis and an infertility period of ≥12 months were the minimum requirements for a study population to be included. The prevalence of endometriosis was 44%, and most lesions were classified as minimal or mild (74%). The prevalence rates of tubal factors and adhesions were 20% and 16%, respectively. The detection rate for pelvic abnormalities was higher in women with prior fertility treatment (75%) compared with women without prior fertility treatment (53%). Despite the significant improvements in imaging for the diagnosis of endometriosis and tubal factors over the last decades, the prevalence rates of endometriosis and tubal abnormalities remain high in patients with unexplained infertility. The high prevalence of endometriosis in this population is important for decision-making in patients who also suffer from pain symptoms suggestive of endometriosis.

Circulating microRNAs as Non-Invasive Biomarkers in Endometriosis Diagnosis-A Systematic Review.

The aim of this systematic review is to assess the power of circulating miRNAs as biomarkers as a diagnostic tool in endometriosis. In endometriosis-suspected women with uncertain imaging, the only way to confirm or exclude endometriosis with certainty is currently laparoscopy. This creates a need for non-invasive diagnostics. We searched the literature through the PubMed database using the Mesh terms 'endometriosis' and 'miRNAs'. Some, but limited, overlap was found between the 32 articles included, with a total of 20 miRNAs reported as dysregulated in endometriosis in two or more studies. MiR-17-5p was reported as dysregulated in six studies, followed by miR-451a and let-7b-5p in four studies and miR-20a-5p, miR-143-3p, miR-199a-5p and miR-3613-5p in three studies. Furthermore, a possible impact of the menstrual phase on miRNA expression was noted in five studies, while no influence of hormonal intake was observed in any included study. The modest reproducibility between studies may be attributable to biological variability as well as to the lack of universal protocols, resulting in pre- and analytical variability. Despite the identification of several suitable candidate biomarkers among the miRNAs, the need for high-quality studies with larger and well-defined population cohorts and the use of standardized protocols lingers.

Correlation Between Surgical Phenotype and Pain Improvement After Endometriosis Surgery.

STUDY OBJECTIVE: To examine pain improvement after endometriosis surgery and whether it can be predicted by the observed surgical phenotype. DESIGN: Prospective longitudinal survey study. SETTING: A University hospital. PATIENTS: A total of 125 patients completed a preoperative questionnaire (response rate: n = 227 of 277, 81.9%), had surgically confirmed endometriosis (n = 202 of 227), and returned a second postoperative questionnaire (response rate: n = 125 of 202, 61.9%). INTERVENTIONS: All patients underwent complete laparoscopic removal of the endometriotic lesions. Surgical phenotype was classified according to the rASRM and #Enzian classification. MEASUREMENTS AND MAIN RESULTS: The intensity of 5 specific pain symptoms was questioned by postal paper-pencil questionnaires with a numerical rating scale (0-10) both preoperatively (3.01 ± 2.72 months before surgery) and one year after surgery (12.62 ± 1.59 months). A postoperative pain improvement score was computed (postoperative pain-preoperative pain) for each specific pain symptom (0-10) and for the overall/global pain sum score (0-50). The mean intensity of all pain scores was lower postoperatively as compared with preoperatively (p <.0001). A statistically significant weak correlation was observed between the surgical phenotype "rectovaginal endometriosis" and postoperative improvement of dyspareunia (r = .265; p = .003). The other 11 hypothesized correlations between surgical phenotype and postoperative improvement of pain intensity failed to reach statistical significance. CONCLUSION: Clinicians can inform patients that surgery is effective in reducing endometriosis-related pain symptoms and the overall/global pain scores at 12 months postoperatively. From our data, we can conclude that patients with rectovaginal endometriosis might benefit the most from the reduction of their sexual pain. On the basis of these results, we could suggest that deep dyspareunia (even if present as an isolated symptom) might be a valid indication for surgery. Further research could explore the association between a certain surgical phenotype and more detailed assessments of sexual functioning, as well as explore whether feedback from the surgeon on expected pain improvement affects patient-reported outcomes.

Similar long-term recurrence rates with cystectomy and CO2 laser vaporization for endometrioma: a retrospective study.

RESEARCH QUESTION: Is there a difference in recurrence rate of endometrioma(s) after cystectomy versus CO2 laser vaporization of the cyst wall? DESIGN: This single-centre retrospective study included 270 patients undergoing laparoscopic surgery for endometriomas between January 2010 and December 2014, stratified according to the surgical technique used. All 270 included patients underwent complete laparoscopic surgery for endometrioma(s): 155 underwent cystectomy, 63 complete CO2 laser vaporization of the cyst wall and 52 a mixed technique. The primary outcome studied was the difference in recurrence rate between the cystectomy group and the CO2 laser vaporization group. RESULTS: The mean duration of follow-up was 58 (±34) months. Imaging-based recurrence (any cyst size) was reported in 9.9% of patients (n = 12/121) treated with cystectomy and in 13.3% of patients (n = 6/45) who underwent a vaporization (P = 0.577). The need for reintervention for endometrioma(s) was also similar in both groups, with a rate of 3.2% (n = 5/155) after cystectomy and 4.8% (n = 3/63) after vaporization (P = 0.476). Of 160 women who wanted to conceive immediately after surgery, 73.8% became pregnant (72.6% [77/106] in the cystectomy group and 75.9% [41/54] in the vaporization group [P = 0.310]). Conception occurred mostly by assisted reproductive technology (57.1% [44/77] in the cystectomy group and 70.7% [29/41] in the vaporization group [P = 0.074]). CONCLUSIONS: Similar rates of recurrence for endometrioma(s) were observed after cystectomy versus CO2 laser vaporization. As other studies have suggested that CO2 laser vaporization may be less harmful to the normal ovarian tissue, it can be considered as a safe alternative for cystectomy in women wishing to preserve their reproductive potential.

Endometriotic lung cyst causing catamenial hemoptysis; a case report and review of literature.

We report a rare case of an endometriotic lung cyst in a 47-year woman with recurrent catamenial hemoptysis. Chest computed tomography (CT) obtained outside the menstruation in October 2019 revealed a cystic lesion (2.5 cm) located in the right inferior lobe near the distal esophagus and the inferior pulmonary vein. Compared to CT abdomen in May 2019, this lesion had increased with a larger volume and a thicker wall. An endometrial lung cyst was suspected as episodes of hemoptysis no longer occurred after initiating hormonal treatment with nomegestrol acetate. Exploratory video-assisted thoracoscopic surgery with wedge resection of the cyst was performed. Histopathologic examination confirmed the diagnosis of an endometriotic cystic lesion. Postoperative course was uneventful with no further symptoms since then.

Prophylactic ureteral catheterization in the intraoperative diagnosis of iatrogenic ureteral injury.

BACKGROUND: Iatrogenic ureteral injury (IUI) is a rare but feared complication in pelvic surgery. Prophylactic ureteral catheterization (PUC) is inconsistently used to reduce this risk, however no strong evidence exists for this practice. The objective is to investigate whether prophylactic ureteral catheterization can enhance intraoperative detection of IUI and reduce associated patient morbidity. METHODS: The database of our tertiary referral hospital was retrospectively queried for ureter repairs due to iatrogenic injuries. The search yielded 845 unique patient files. After application of exclusion criteria and manual review of files, 155 individual cases remained. Statistical analysis was performed on the following parameters: timing of ureteral injury discovery, duration until catheter removal and postoperative complications. RESULTS: Prophylactic ureteral catheterization was able to significantly enhance intraoperative diagnosis of IUI (OR = 5.09; 95%CI = 2.26-11.48). The number needed to treat is 2.6 patients. Furthermore, when the IUI was diagnosed during surgery, a significant reduction in postoperative complications was observed (RR = 0.64; 95%CI = 0.42-0.98). CONCLUSION: Although the beneficial effects of PUC on IUI incidence remain controversial, when a ureteral injury occurs during pelvic surgery, the presence of prophylactic ureteral catheters can significantly reduce both diagnostic delay and postoperative morbidity. By promoting an immediate repair, ureteral catheterization reduces need for further diagnostics and secondary interventions.

Outcome after surgery for deep endometriosis infiltrating the rectum.

OBJECTIVE: To evaluate the rate of postoperative complications between conservative surgery and segmental resection in patients with rectal endometriosis. DESIGN: Single-center retrospective study. SETTING: University hospital. PATIENT(S): A total of 232 women undergoing surgery for deep endometriosis infiltrating the rectum up to 15 cm from the anus with at least involvement of the muscularis layer, stratified into two arms according to surgical technique. Subgroup analysis was performed in patients without previous therapeutic laparoscopy for endometriosis (n = 108). A propensity-score approach was used to correct for group differences. INTERVENTION(S): All patients underwent CO2-laser laparoscopic surgery: 61 underwent conservative surgery, and 171 had a segmental resection. MAIN OUTCOME MEASURE(S): Postoperative complication rate (Clavien-Dindo classification). RESULT(S): Clavien-Dindo type 1 and 2 complications did not differ between both groups. Clavien-Dindo type 3 complications were more frequent in the segmental resection group (1/61 [1.6%] conservative vs. 18/171 [10.5%] segmental), after propensity analysis only a trend was retained. In the subgroup analysis, no difference or trend was found (1/27 [3.7%] conservative vs. 5/81 [6.2%] segmental). A low rate of temporary diverting stoma was recorded: 24/232 (10.3%). CONCLUSION(S): A higher major complication (Clavien-Dindo ≥3) rate for segmental resections compared with conservative surgical treatment was shown in the overall population, although after correction for group differences this was attenuated to a trend only. However, in patients without previous therapeutic laparoscopy no significant difference or trend was found regardless of the surgical technique used. This not only suggests that redo/repeated surgery has a potentially increased morbidity, but also emphasizes the importance of a well executed primary surgery.

Patient-derived organoids from endometrial disease capture clinical heterogeneity and are amenable to drug screening.

Endometrial disorders represent a major gynaecological burden. Current research models fail to recapitulate the nature and heterogeneity of these diseases, thereby hampering scientific and clinical progress. Here we developed long-term expandable organoids from a broad spectrum of endometrial pathologies. Organoids from endometriosis show disease-associated traits and cancer-linked mutations. Endometrial cancer-derived organoids accurately capture cancer subtypes, replicate the mutational landscape of the tumours and display patient-specific drug responses. Organoids were also established from precancerous pathologies encompassing endometrial hyperplasia and Lynch syndrome, and inherited gene mutations were maintained. Endometrial disease organoids reproduced the original lesion when transplanted in vivo. In summary, we developed multiple organoid models that capture endometrial disease diversity and will provide powerful research models and drug screening and discovery tools.

Functional expression of the mechanosensitive PIEZO1 channel in primary endometrial epithelial cells and endometrial organoids.

Successful pregnancy requires the establishment of a complex dialogue between the implanting embryo and the endometrium. Knowledge regarding molecular candidates involved in this early communication process is inadequate due to limited access to primary human endometrial epithelial cells (EEC). Since pseudo-pregnancy in rodents can be induced by mechanical scratching of an appropriately primed uterus, this study aimed to investigate the expression of mechanosensitive ion channels in EEC. Poking of EEC provoked a robust calcium influx and induced an increase in current densities, which could be blocked by an inhibitor of mechanosensitive ion channels. Interestingly, RNA expression studies showed high expression of PIEZO1 in EEC of mouse and human. Additional analysis provided further evidence for the functional expression of PIEZO1 since stimulation with Yoda1, a chemical agonist of PIEZO1, induced increases in intracellular calcium concentrations and current densities in EEC. Moreover, the ion channel profile of human endometrial organoids (EMO) was validated as a representative model for endometrial epithelial cells. Mechanical and chemical stimulation of EMO induced strong calcium responses supporting the hypothesis of mechanosensitive ion channel expression in endometrial epithelial cells. In conclusion, EEC and EMO functionally express the mechanosensitive PIEZO1 channel that could act as a potential target for the development of novel treatments to further improve successful implantation processes.

Evaluation of Haptic Feedback on Bimanually Teleoperated Laparoscopy for Endometriosis Surgery.

Robotic minimal invasive surgery is gaining acceptance in surgical care. In contrast with the appreciated three-dimensional vision and enhanced dexterity, haptic feedback is not offered. For this reason, robotics is not considered beneficial for delicate interventions such as the endometriosis. Overall, haptic feedback remains debatable and yet unproven except for some simple scenarios such as fundamentals of laparoscopic surgery exercises. OBJECTIVE: This work investigates the benefits of haptic feedback on more complex surgical gestures, manipulating delicate tissue through coordination between multiple instruments. METHODS: A new training exercise, "endometriosis surgery exercise" (ESE) has been devised approximating the setting for monocular robotic endometriosis treatment. A bimanual bilateral teleoperation setup was designed for laparoscopic laser surgery. Haptic guidance and haptic feedback are, respectively, offered to the operator. User experiments have been conducted to assess the validity of ESE and examine possible advantages of haptic technology during execution of bimanual surgery. RESULTS: Content and face validity of ESE were established by participating surgeons. Surgeons suggested ESE also as a mean to train lasering skills, and interaction forces on endometriotic tissue were found to be significantly lower when a bilateral controller is used. Collisions between instruments and the environment were less frequent and so were situations marked as potentially dangerous. CONCLUSION: This study provides some promising results suggesting that haptics may offer a distinct advantage in complex robotic interventions were fragile tissue is manipulated. SIGNIFICANCE: Patients need to know whether it should be incorporated. Improved understanding of the value of haptics is important as current commercial surgical robots are widely used but do not offer haptics.

Genome-wide haplotyping embryos developing from 0PN and 1PN zygotes increases transferrable embryos in PGT-M.

STUDY QUESTION: Can genome-wide haplotyping increase success following preimplantation genetic testing for a monogenic disorder (PGT-M) by including zygotes with absence of pronuclei (0PN) or the presence of only one pronucleus (1PN)? SUMMARY ANSWER: Genome-wide haplotyping 0PNs and 1PNs increases the number of PGT-M cycles reaching embryo transfer (ET) by 81% and the live-birth rate by 75%. WHAT IS KNOWN ALREADY: Although a significant subset of 0PN and 1PN zygotes can develop into balanced, diploid and developmentally competent embryos, they are usually discarded because parental diploidy detection is not part of the routine work-up of PGT-M. STUDY DESIGN, SIZE, DURATION: This prospective cohort study evaluated the pronuclear number in 2229 zygotes from 2337 injected metaphase II (MII) oocytes in 268 cycles. PGT-M for 0PN and 1PN embryos developing into Day 5/6 blastocysts with adequate quality for vitrification was performed in 42 of the 268 cycles (15.7%). In these 42 cycles, we genome-wide haplotyped 216 good quality embryos corresponding to 49 0PNs, 15 1PNs and 152 2PNs. The reported outcomes include parental contribution to embryonic ploidy, embryonic aneuploidy, genetic diagnosis for the monogenic disorder, cycles reaching ETs, pregnancy and live birth rates (LBR) for unaffected offspring. PARTICIPANTS/MATERIALS, SETTING, METHODS: Blastomere DNA was whole-genome amplified and hybridized on the Illumina Human CytoSNP12V2.1.1 BeadChip arrays. Subsequently, genome-wide haplotyping and copy-number profiling was applied to investigate the embryonic genome architecture. Bi-parental, unaffected embryos were transferred regardless of their initial zygotic PN score. MAIN RESULTS AND THE ROLE OF CHANCE: A staggering 75.51% of 0PN and 42.86% of 1PN blastocysts are diploid bi-parental allowing accurate genetic diagnosis for the monogenic disorder. In total, 31% (13/42) of the PGT-M cycles reached ET or could repeat ET with an unaffected 0PN or 1PN embryo. The LBR per initiated cycle increased from 9.52 to 16.67%. LIMITATIONS, REASONS FOR CAUTION: The clinical efficacy of the routine inclusion of 0PN and 1PN zygotes in PGT-M cycles should be confirmed in larger cohorts from multicenter studies. WIDER IMPLICATIONS OF THE FINDINGS: Genome-wide haplotyping allows the inclusion of 0PN and 1PN embryos and subsequently increases the cycles reaching ET following PGT-M and potentially PGT for aneuploidy (PGT-A) and chromosomal structural rearrangements (PGT-SR). Establishing measures of clinical efficacy could lead to an update of the ESHRE guidelines which advise against the use of these zygotes. STUDY FUNDING/COMPETING INTEREST(S): SymBioSys (PFV/10/016 and C1/018 to J.R.V. and T.V.), the Horizon 2020 WIDENLIFE: 692065 to J.R.V., T.V., E.D., A.D. and M.Z.E. M.Z.E., T.V. and J.R.V. co-invented haplarithmisis ('Haplotyping and copy-number typing using polymorphic variant allelic frequencies'), which has been licensed to Agilent Technologies. H.M. is fully supported by the (FWO) (ZKD1543-ASP/16). The authors have no competing interests to declare.

Functional Expression of TRP Ion Channels in Endometrial Stromal Cells of Endometriosis Patients.

Endometriosis is a common gynecological disease that is characterized by the presence of functional endometrial-like lesions in the abdominal cavity. Aside from epithelial cells, these lesions consist of stromal cells that have the capacity to migrate, adhere, proliferate, and induce neuro- and lymphangiogenesis, which allows them to survive at ectopic locations. However, the exact underlying mechanisms that regulate these changes are yet to be elucidated. The common ground of these processes, however, is the second messenger, calcium. In this regard, members of the superfamily of transient receptor potential (TRP) ion channels, which are known to be calcium-permeable and expressed in the endometrium, have emerged as key regulators. Here, we assessed the molecular and functional expression of TRP channels in stromal cells isolated from the eutopic endometrium of endometriosis patients and controls. Using RT-qPCR, high mRNA levels of TRPV2, TRPV4, TRPM4, TRPM7, TRPC1, TRPC3, TRPC4, and TRPC6 were observed in the whole endometrium throughout the menstrual cycle. Additionally, and in line with previous reports of control patients, TRPV2, TRPV4, TRPC1/4, and TRPC6 were present in human endometrial stromal cells (hESC) from endometriosis patients both at the molecular and functional level. Moreover, proliferation and migration assays illustrated that these parameters were not affected in stromal cells from endometriosis patients. Furthermore, comparison between eutopic and ectopic endometrial samples revealed that the RNA expression pattern of TRP channels did not differ significantly. Collectively, although a functional expression of specific ion channels in hESCs was found, their expression did not correlate with endometriosis.

Cost-effectiveness of ovarian stimulation with gonadotrophin and clomiphene citrate in an intrauterine insemination programme for subfertile couples.

Ovarian stimulation with low-dose human menopausal gonadotrophin (HMG) is superior to clomiphene citrate in intrauterine insemination (IUI) cycles with respect to clinical pregnancy rate, but it is unclear whether HMG is also the more cost-effective option. The aim of this study was to compare the cost-effectiveness of ovarian stimulation with low-dose subcutaneously administred HMG (37.5-75 IU per day) to orally administred clomiphene citrate (50 mg/day from day 3-7) in an IUI programme for subfertile couples. A cost-effectiveness analysis was conducted using the results of a randomized trial, including 620 IUI cycles. The primary outcome was the incremental cost-effectiveness ratio (ICER) of using HMG versus clomiphene citrate. Results are presented from the healthcare payer perspective. The total cost per patient associated with one IUI treatment with HMG is €764, whereas it is €558 if clomiphene citrate is used, resulting in an incremental cost of €206 for HMG per treatment. The incremental clinical pregnancy rate of using HMG instead of clomiphene citrate, however, is also 5.7 percentage points higher, resulting in an ICER of HMG versus clomiphene citrate of €3615 per additional clinical pregnancy achieved. On average, HMG was found to be more cost-effective than clomiphene citrate.

Evaluation of Total, Active, and Specific Myeloperoxidase Levels in Women with and without Endometriosis.

Myeloperoxidase (MPO) is a proinflammatory enzyme and a marker for neutrophil activation and oxidative stress. Since oxidative stress and inflammation are linked to the pathogenesis of endometriosis, we hypothesized that the total, active, and specific (active/total) MPO levels were significantly different in plasma of women with and without endometriosis. Samples were selected from our biobank from women with endometriosis (n = 212) and controls without endometriosis (n = 121) across the menstrual cycle. Total MPO plasma levels were measured by immunoassay and MPO activity by enzymatic assay. Total and active MPO levels did not differ significantly among endometriosis cases and controls, whereas the specific MPO activity was significantly lower in women with endometriosis than that in controls (p = 0.0159). After the subdivision of control patients into women with a normal pelvis and women with other benign gynecological disorders, a significant difference was observed only between women with endometriosis and women with other benign gynecological disorders (p = 0.0266). In conclusion, systemic MPO levels may not be suited as a single biomarker for endometriosis. Our data support the involvement of MPO in other gynecological disorders but do not provide any evidence for an association with endometriosis.

Development of organoids from mouse and human endometrium showing endometrial epithelium physiology and long-term expandability.

The endometrium, which is of crucial importance for reproduction, undergoes dynamic cyclic tissue remodeling. Knowledge of its molecular and cellular regulation is poor, primarily owing to a lack of study models. Here, we have established a novel and promising organoid model from both mouse and human endometrium. Dissociated endometrial tissue, embedded in Matrigel under WNT-activating conditions, swiftly formed organoid structures that showed long-term expansion capacity, and reproduced the molecular and histological phenotype of the tissue's epithelium. The supplemented WNT level determined the type of mouse endometrial organoids obtained: high WNT yielded cystic organoids displaying a more differentiated phenotype than the dense organoids obtained in low WNT. The organoids phenocopied physiological responses of endometrial epithelium to hormones, including increased cell proliferation under estrogen and maturation upon progesterone. Moreover, the human endometrial organoids replicated the menstrual cycle under hormonal treatment at both the morpho-histological and molecular levels. Together, we established an organoid culture system for endometrium, reproducing tissue epithelium physiology and allowing long-term expansion. This novel model provides a powerful tool for studying mechanisms underlying the biology as well as the pathology of this key reproductive organ.

Principles guiding embryo selection following genome-wide haplotyping of preimplantation embryos.

STUDY QUESTION: How to select and prioritize embryos during PGD following genome-wide haplotyping? SUMMARY ANSWER: In addition to genetic disease-specific information, the embryo selected for transfer is based on ranking criteria including the existence of mitotic and/or meiotic aneuploidies, but not carriership of mutations causing recessive disorders. WHAT IS KNOWN ALREADY: Embryo selection for monogenic diseases has been mainly performed using targeted disease-specific assays. Recently, these targeted approaches are being complemented by generic genome-wide genetic analysis methods such as karyomapping or haplarithmisis, which are based on genomic haplotype reconstruction of cell(s) biopsied from embryos. This provides not only information about the inheritance of Mendelian disease alleles but also about numerical and structural chromosome anomalies and haplotypes genome-wide. Reflections on how to use this information in the diagnostic laboratory are lacking. STUDY DESIGN, SIZE, DURATION: We present the results of the first 101 PGD cycles (373 embryos) using haplarithmisis, performed in the Centre for Human Genetics, UZ Leuven. The questions raised were addressed by a multidisciplinary team of clinical geneticist, fertility specialists and ethicists. PARTICIPANTS/MATERIALS, SETTING, METHODS: Sixty-three couples enrolled in the genome-wide haplotyping-based PGD program. Families presented with either inherited genetic variants causing known disorders and/or chromosomal rearrangements that could lead to unbalanced translocations in the offspring. MAIN RESULTS AND THE ROLE OF CHANCE: Embryos were selected based on the absence or presence of the disease allele, a trisomy or other chromosomal abnormality leading to known developmental disorders. In addition, morphologically normal Day 5 embryos were prioritized for transfer based on the presence of other chromosomal imbalances and/or carrier information. LIMITATIONS, REASONS FOR CAUTION: Some of the choices made and principles put forward are specific for cleavage-stage-based genetic testing. The proposed guidelines are subject to continuous update based on the accumulating knowledge from the implementation of genome-wide methods for PGD in many different centers world-wide as well as the results of ongoing scientific research. WIDER IMPLICATIONS OF THE FINDINGS: Our embryo selection principles have a profound impact on the organization of PGD operations and on the information that is transferred among the genetic unit, the fertility clinic and the patients. These principles are also important for the organization of pre- and post-counseling and influence the interpretation and reporting of preimplantation genotyping results. As novel genome-wide approaches for embryo selection are revolutionizing the field of reproductive genetics, national and international discussions to set general guidelines are warranted. STUDY FUNDING/COMPETING INTEREST(S): The European Union's Research and Innovation funding programs FP7-PEOPLE-2012-IAPP SARM: 324509 and Horizon 2020 WIDENLIFE: 692065 to J.R.V., T.V., E.D. and M.Z.E. J.R.V., T.V. and M.Z.E. have patents ZL910050-PCT/EP2011/060211-WO/2011/157846 ('Methods for haplotyping single cells') with royalties paid and ZL913096-PCT/EP2014/068315-WO/2015/028576 ('Haplotyping and copy-number typing using polymorphic variant allelic frequencies') with royalties paid, licensed to Cartagenia (Agilent technologies). J.R.V. also has a patent ZL91 2076-PCT/EP20 one 3/070858 ('High throughout genotyping by sequencing') with royalties paid. TRIAL REGISTRATION NUMBER: N/A.

The Presence of Endometrial Cells in Peritoneal Fluid of Women With and Without Endometriosis.

To reinforce Sampson's theory of retrograde menstruation in the pathogenesis of endometriosis, proof should be provided that during menstruation endometrial cells are present in peritoneal fluid (PF). We hypothesize that the prevalence of PF samples containing endometrial cells is higher in patients with endometriosis than in controls without endometriosis during menstruation. We selected from our biobank PF samples of 17 reproductive-age women with (n = 9) or without (n = 8) endometriosis who had received a diagnostic laparoscopy for investigation of pain/infertility. Peritoneal fluid had been collected during laparoscopy in the menstrual phase of the cycle, centrifuged, and the resulting pellet was stored at -80°C. About 5-µm sections of frozen PF pellets were stained using the Dako Envision Flex system with primary antibodies against epithelial cell adhesion molecule (Ep-CAM; endometrial epithelial cells), CD10 (endometrial stromal cells), prekeratin (epithelial/mesothelial cells), vimentin (endometrial/mesothelial/immune cells), calretinin (mesothelial cells), and CD68 (macrophages). The PF cells positive for Ep-CAM were detected in 5 of 9 patients with endometriosis and 6 of 8 controls ( P = .62). CD10 stained positively in 6 of the 9 patients with endometriosis and 3 of the 8 controls ( P = .35). Calretinin and prekeratin staining showed the presence of mesothelial cells in all pellets. Vimentin stained approximately 100% of the PF cells. CD68+ macrophages represented >50% of cells in all pellets. The prevalence of PF samples containing endometrial epithelial and stromal cells was not higher in patients with endometriosis than in controls without endometriosis during menstruation. Our findings question the relevance of endometrial cells in PF for the pathogenesis of endometriosis and support the importance of other mechanisms such as immune dysfunction and/or endometrial stem cells.

Vascular endothelial growth factor pathway in endometriosis: genetic variants and plasma biomarkers.

OBJECTIVE: To study single nucleotide polymorphisms (SNPs) involved in angiogenesis (VEGF, PLGF, VEGFR1, VEGFR2, HIF-1α) and plasma levels of the corresponding proteins (VEGF, PLGF, sVEGFR1, sVEGFR2) in women with and without endometriosis. DESIGN: Allele frequencies of vascular endothelial growth factor (VEGF) pathway SNPs and plasma levels of the corresponding proteins were investigated in patients with endometriosis and in controls. SETTING: University hospital. PATIENT(S): Samples of DNA from 1,931 Caucasian patients were included (1,109 patients with endometriosis and 822 controls). An additional study group included 973 DNA samples from volunteers, self-reported to be healthy without laparoscopic evaluation. INTERVENTION(S): Women who underwent a laparoscopy for subfertility and/or pain and healthy volunteers without laparoscopic evaluation. MAIN OUTCOME MEASURE(S): Functional SNPs of the VEGF, VEGFR1, VEGFR2, HIF-1α genes and Hap Map tagging SNPs of the PLGF gene were genotyped by using iPLEX technology on a Sequenom MassArray and TaqMan SNP Genotyping Assay. The VEGF levels were determined in ethylenediaminetetraacetic acid plasma samples by using Bio-Plex Protein Array System. PLGF, sVEGFR1, and sVEGFR2 levels were measured in ethylenediaminetetraacetic acid plasma samples by using ELISA Quantikine kits. RESULT(S): A significant association was found between the rs2268613 polymorphism in the PLGF gene and PLGF plasma levels. In all study subjects, women with the AA variant of the rs2268613 PLGF gene had significantly lower PLGF plasma levels (median [interquartile range] 9.36 [8.19-10.43] pg/mL) than those with the AG variant (12.1 [11.81-20.84] pg/mL; P(a)=.0085, P(b)=.04), both before and after multiple testing. Plasma levels of VEGF were elevated in endometriosis patients (especially in minimal-mild endometriosis during the menstrual cycle phase) compared with laparoscopic controls but had a moderate diagnostic performance (area under the curve, 0.73) in this discovery dataset. At a cut-off plasma level of VEGF >3.88 pg/mL, minimal-mild stages of endometriosis were diagnosed with a sensitivity of 74% and a specificity of 80% during the menstrual phase of cycle. The associations between the presence of endometriosis and SNPs in PLGF (rs2268614), HIF-1α (rs11549465), and VEGFR1 (rs9582036) genes lost statistical significance after multiple testing. CONCLUSION(S): Genetic variants in the PLGF rs2268613 gene may influence plasma levels of the corresponding protein. Plasma levels of VEGF were elevated in endometriosis patients compared with controls. The associations between the presence of endometriosis and SNPs in PLGF (rs2268614), HIF-1α (rs11549465), and VEGFR1 (rs9582036) genes lost statistical significance after multiple testing.

Challenges in the development of novel therapeutic strategies for treatment of endometriosis.

INTRODUCTION: Endometriosis is an estrogen-dependent disease that results in pelvic pain and infertility. Its treatment is often frustrating due to limited medical treatment options, complex surgical treatment and high recurrence rates. Despite the advances in our understanding of the pathogenesis over the last decades and the consequent novel therapeutic strategies, no new drugs have been introduced in daily clinical practice. AREAS COVERED: In the first part we present an overview of the pathogenesis of endometriosis. In the second part we discuss how new insights have led to the development of novel nonhormonal strategies for the treatment of endometriosis, focusing on anti-inflammatory and anti-angiogenic agents. In the third part we describe the problems encountered in the translation from experimental drugs to routine medicine for the treatment of endometriosis. EXPERT OPINION: Despite the multitude of agents that have been tested in preclinical trials, only few drugs have passed to the stage of clinical testing and none have been introduced into clinical practice. It is our opinion that the major challenges in the translation from novel agents for endometriosis is due to the use of inadequate rodent models and a lack of standardization in the design and reporting of preclinical endometriosis models.

Characteristics of the menstrual cycle in 13-year-old Flemish girls and the impact of menstrual symptoms on social life.

UNLABELLED: This study aimed to investigate the characteristics of symptoms related to the menstrual cycle and their impact on social activities in young teenage girls. Between March and June 2009, all girls born in 1996 who were residents of eight regions in Flanders (Belgium) received a semi-structured questionnaire, including questions about the age of menarche, characteristics of the menstrual cycle, and its impact on social activities. Participants were 792 13-year-old girls (15.7 % of the target population). Out of 363 (47.2 % of participants) postmenarcheal girls, 41.6 % (95 % confidence interval (CI) 36.4-47.0 %) reported painful menstruations. The proportion of girls with painful menstrual periods decreased approximately 16 % with each year the age at menarche increased (relative risk (RR) = 0.84; 0.73-0.98; p < 0.05) and was positively correlated with the amount of blood loss (RR = 0.33; 0.16-0.67; p < 0.05 when little and 1.85; 1.49-2.31; p < 0.001 when abundant, compared to average). One in four (25.4 %) postmenarcheal girls indicated a negative impact of menstruation on social activities, but this proportion was significantly higher in girls who experienced menstruation as painful (41.3 %) compared to those who did not (14.2 %). CONCLUSION: Early menstrual complaints are common in young adolescent girls and the likelihood of pain increased significantly with lower menarcheal age. What is Known? • Menstrual cycle-related symptoms may negatively interfere with school absence and social activities. • Early menarche and severe dysmenorrhea are correlated with endometriosis. What is New? • In this large population-based study on the characteristics of the menstrual cycle in young teenage girls at or shortly after menarche, painful menstruation was highly prevalent (41.7 %), but related school absenteeism was low (3.2 %). The likelihood of pain increased significantly with lower menarcheal age. • The findings support the need for a systematic evaluation of the characteristics of the menstrual cycle shortly after menarche.