Publications

Unbiased In Silico Analysis of Gene Expression Pinpoints Circulating miRNAs Targeting KIAA1324, a New Gene Drastically Downregulated in Ovarian Endometriosis.

Objective: To identify circulating miRNAs associated with ovarian endometriosis (OMA), and to analyze candidate genes targeted by these miRNAs. Methods: Putative regulating miRNAs were identified through an original bioinformatics approach. We first queried the miRWalk 2.0 database to collect putative miRNA targets. Then, we matched it to a transcriptomic dataset of OMA. Moving from gene expression in the tissue to possible alterations in the patient plasma, a selection of these miRNAs was quantified by qRT-PCR in plasma samples from 93 patients with isolated OMA and 95 patients surgically checked as free from endometriosis. Then, we characterized the genes regulated by more than one miRNA and validated them by immunohistochemistry and transfection experiments on endometrial cell primary cultures obtained from endometrial biopsies of 10 women with and without endometriosis with miRNA mimics. Stromal and epithelial cells were isolated and cultured separately and gene expression levels were measured by RT-qPCR. Results: Eight miRNAs were identified by bioinformatics analysis. Two of them were overexpressed in plasma from OMA patients: let-7b-5p and miR-92a-3p (p < 0.005). Three miRNAs, let-7b and miR-92a-3p, and miR-93-5p potentially targeted KIAA1324, an estrogen-responsive gene and one of the most downregulated genes in OMA. Transfection experiments with mimics of these two miRNAs showed a strong decrease in KIAA1324 expression, up to 40%. Immunohistochemistry revealed a moderate-to-intense staining for KIAA1324 in the eutopic endometrium and a faint-to-moderate staining in the ectopic endometrium for half of the samples, which is concordant with the transcriptomic data. Discussion and Conclusion: Our results suggested that KIAA1324 might be involved in endometriosis through the downregulating action of two circulating miRNAs. As these miRNAs were found to be overexpressed, their quantification in plasma could provide a tool for an early diagnosis of endometriosis.

ENDOCELL-Seud: a Delphi protocol to harmonise methods in endometrial cell culturing.

IN VITRO: culturing of endometrial cells obtained from the uterine mucosa or ectopic sites is used to study molecular and cellular signalling relevant to physiologic and pathologic reproductive conditions. However, the lack of consensus on standard operating procedures for deriving, characterising and maintaining primary cells in two- or three-dimensional cultures from eutopic or ectopic endometrium may be hindering progress in this area of research. Guidance for unbiased in vitro research methodologies in the field of reproductive science remains essential to increase confidence in the reliability of in vitro models. We present herein the protocol for a Delphi process to develop a consensus on in vitro methodologies using endometrial cells (ENDOCELL-Seud Project). A steering committee composed of leading scientists will select critical methodologies, topics and items that need to be harmonised and that will be included in a survey. An enlarged panel of experts (ENDOCELL-Seud Working Group) will be invited to participate in the survey and provide their ratings to the items to be harmonised. According to Delphi, an iterative investigation method will be adopted. Recommended measures will be finalised by the steering committee. The study received full ethical approval from the Ethical Committee of the Maastricht University (ref. FHML-REC/2021/103). The study findings will be available in both peer-reviewed articles and will also be disseminated to appropriate audiences at relevant conferences. LAY SUMMARY: Patient-derived cells cultured in the lab are simple and cost-effective methods used to study biological and dysfunctional or disease processes. These tools are frequently used in the field of reproductive medicine. However, the lack of clear recommendations and standardised methodology to guide the laboratory work of researchers can produce results that are not always reproducible and sometimes are incorrect. To remedy this situation, we define here a method to ascertain if researchers who routinely culture cells in the lab agree or disagree on the optimal laboratory techniques. This method will be used to make recommendations for future researchers working in the field of reproductive biology to reproducibly culture endometrial cells in the laboratory.

Potential competing risk of death in older high-risk endometrial carcinoma patients: Results from a multicentric retrospective cohort.

INTRODUCTION: Adjuvant therapeutic decisions in older endometrial carcinoma (EC) patients are challenged by a balance between more frequent aggressive EC and comorbidities. We assessed whether EC and comorbidities are competing or cumulative risks in older EC patients. METHODS: All consecutive patients treated for FIGO stage I-IV EC in two University Hospitals in Paris between 2010 and 2017 were retrospectively included. Patients were categorized as: <70 years (y), >70y without comorbidity (fit), and > 70y with a Charlson comorbidity index>3 (comorbid). Association between high-risk EC (2021-ESGO-ETRO-ESP) or comorbidity, and disease-specific-survival (DSS), was evaluated using Cox model (estimation of cause-specific hazard ratio (CSHR), and Fine-Gray model (subdistribution HR) to account for competing events (death unrelated with EC). RESULTS: Overall, 253 patients were included (median age = 67y, IQR[59-77], median follow-up = 61.5 months, [44.4-76.8]). Among them, 109 (43%) were categorized at high-risk (proportion independent of age), including 67 (26%) who had TP53-mutated tumors. Comorbidity and high-risk group were both associated with all-cause mortality (HR = 4.09, 95%CI[2.29; 7.32] and HR = 3.21, 95%CI [1.69; 6.09], respectively). By multivariate analysis, patients with high-risk EC exhibited poorer DSS, regardless of age/comorbidity (Adjusted-CSHR = 6.62, 95%CI[2.53;17.3]; adjusted-SHR = 6.62 95%CI[2.50;17.5]). Patients>70y-comorbid with high-risk EC had 5-years cumulative incidences of EC-related and EC-unrelated death of 29% and 19%, respectively. In patients <70y, 5-years cumulative incidence of EC-related and EC-unrelated death were 25% and < 1% (one event), respectively. CONCLUSION: High-risk EC patients are exposed to poorer DSS regardless of age/comorbidities, comorbidities and cancer being two cumulative rather than competing risks. Our results suggest that age/comorbidity alone should not lead to underestimate EC-specific survival.

Presence of adenomyosis at MRI reduces live birth rates in ART cycles for endometriosis.

STUDY QUESTION: What is the impact of adenomyosis on the live birth rate (LBR) in women affected by endometriosis women undergoing ART? SUMMARY ANSWER: For women undergoing ART, the presence of adenomyosis at MRI, especially T2 high-signal intensity spots within the myometrium, has a negative impact on the LBR. WHAT IS KNOWN ALREADY: Adenomyosis is a common gynecological disease. The development of imaging techniques for the diagnosis has led to several adenomyosis phenotypes being described, and fertility issues appear to vary according to the characteristics of the lesions. What makes assessment of the impact of adenomyosis on fertility issues even more difficult is its frequent association with endometriosis, which is another known risk factor of infertility. Although data suggest that adenomyosis may worsen the ART prognosis, there is no clear consensus regarding the impact of adenomyosis on ART outcomes in women affected by endometriosis. STUDY DESIGN, SIZE, DURATION: This was an observational study that included phenotyped patients with endometriosis, aged between 18 and 42 years, who underwent IVF/ICSI treatment in a tertiary care center between June 2015 and July 2018. Only women who had undergone a pelvic MRI during the pre-therapeutic ART workup were retained for this study. The MRI data were interpreted by radiologists who had expertise in gynecological MRI. PARTICIPANTS/MATERIALS, SETTING, METHODS: A continuous series of 202 women affected by endometriosis was included. The women were monitored until four ART cycles had been completed, until delivery, or until discontinuation of treatment before the completion of four cycles. The primary outcome was the delivery of at least one live infant after up to four IVF/ICSI cycles. The patient and the MRI characteristics were compared between the women who achieved a live birth versus those who did not. MAIN RESULTS AND THE ROLE OF CHANCE: The patients' mean age was 32.5 ± 3.7 years. Deep infiltrating endometriosis was present in 90.1% (182/202) of the included population. Adenomyosis (lesions of the internal and/or the external myometrium) was found in 71.8% (145/202) of the included women. The cumulative LBR was 57.4% (116/202). The women who gave birth were significantly younger (32.0 ± 3.3 versus 33.3 ± 4.1, P = 0.026) and had significantly better ovarian reserve parameters (anti-Müllerian hormone levels, antral follicle count) than those who did not. The presence of adenomyosis, irrespective of the phenotype (76/116 (65.5%) versus 69/86 (80.2%), respectively, P = 0.022) and the presence of T2 high-signal intensity myometrial spots (27/116 (23.3%) and 37/86 (43.0%), respectively, P = 0.003) was significantly less frequent in the group of women who gave birth versus those who did not. After multivariate analysis, the presence of adenomyosis (odds ratio (OR): 0.48, 95% CI (0.29-0.99), P = 0.048) and the presence of T2 high-signal intensity myometrial spots (OR: 0.43, 95% CI (0.22-0.86), P = 0.018) were independently found to be associated with a decrease in the cumulative chance of live birth. LIMITATIONS, REASONS FOR CAUTION: The inclusion of patients from a referral center specialized in the management of women affected by endometriosis could constitute a selection bias, as these women may have had particularly severe forms of adenomyosis and/or endometriosis. A sensitive issue is that there is no consensual classification of adenomyosis and several lesions of adenomyosis can co-exist. Therefore, a comparison of fertility outcomes between women with and without adenomyosis is difficult to perform in practice. WIDER IMPLICATIONS OF THE FINDINGS: In women exhibiting endometriosis, the practitioner should perform an appropriate imaging workup to search for adenomyosis, identify prognostic factors, and personalize the patient management strategy in the setting of ART. STUDY FUNDING/COMPETING INTEREST(S): No funding was obtained and there were no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Highly Specific Droplet-Digital PCR Detection of Universally Methylated Circulating Tumor DNA in Endometrial Carcinoma.

BACKGROUND: No circulating biomarker is available for endometrial carcinoma (EC). We aimed to identify DNA positions universally hypermethylated in EC, and to develop a digital droplet PCR (ddPCR) assay for detection of hypermethylated circulating tumor DNA (meth-ctDNA) in plasma from patients with EC. METHODS: DNA positions hypermethylated in EC, and without unspecific hypermethylation in tissue/cell types releasing circulating cell-free DNA in plasma, were identified in silico from TCGA/Gene Expression Omnibus (GEO) data. A methylation-specific ddPCR (meth-ddPCR) assay following bisulfite conversion of DNA extracted from plasma was optimized for detection of meth-ctDNA according to dMIQE guidelines. Performances were validated on a retrospective cohort (n = 78 tumors, n = 30 tumor-adjacent tissues), a prospective pilot cohort (n = 33 stage I-IV patients), and 55 patients/donors without cancer. RESULTS: Hypermethylation of zinc finger and SCAN domain containing 12 (ZSCAN12) and/or oxytocin (OXT) classified EC samples from multiple noncancer samples with high diagnostic specificity/sensitivity [>97%; area under the curve (AUC) = 0.99; TCGA/GEO tissues/blood samples]. These results were confirmed in the independent retrospective cohort (AUC = 0.99). Meth-ddPCR showed a high analytical specificity (limit of blank = 2) and sensitivity (absolute lower threshold of detection = 50 pgmethDNA/mLplasma). In the pilot cohort, meth-ctDNA was detected in pretreatment plasma samples from 9/11 and 5/20 patients with advanced and non-advanced EC, respectively. 2 of 9 patients had ctDNA detected after macroscopic complete surgery and experienced progression within 6 months. No healthy donors had any copy of hypermethylated DNA detected in plasma. CONCLUSIONS: Meth-ddPCR of ZSCAN12/OXT allows a highly specific and sensitive detection of ctDNA in plasma from patients with EC and appears promising for personalized approaches for these patients.

Pregnancy, fertility concerns and fertility preservation procedures in a national study of French breast cancer survivors.

RESEARCH QUESTION: What are the real-life oncofertility practices in young women diagnosed with breast cancer? DESIGN: The FEERIC (FErtility, prEgnancy, contRaceptIon after breast Cancer in France) study is a web-based cohort study launched with the French collaborative research platform Seintinelles. The current work is based on the enrolment self-administered questionnaire of 517 patients with prior breast cancer diagnosis, free from relapse and aged 18 to 43 years at inclusion (from 12 March 2018 to 27 June 2019). RESULTS: Median age at breast cancer diagnosis was 33.6 years and 424 patients (82.0%) received chemotherapy. Overall, 236 (45.6%) patients were offered specialized oncofertility counselling, 181 patients underwent at least one fertility preservation procedure (FPP); 125 (24.2%) underwent one or more FPP with material preservation (oocytes n = 108, 20.9%; embryos n = 31, 6.0%; ovarian cryopreservation n = 6, 1.2%) and 78 patients received gonadotrophin-releasing hormone agonists (15.1%). With a median follow-up of 26.9 months after the end of treatments, 133 pregnancies had occurred in 85 patients (16.4%), including 20 unplanned pregnancies (15.0%). Most of the pregnancies were natural conceptions (n = 113, 87.6%), while 16 (12.4%) required medical interventions. For the planned pregnancies, median time to the occurrence of an ongoing pregnancy was 3 months. Patients who had an unplanned pregnancy reported lower rates of information on the consequences of the treatments on fertility (P = 0.036) at diagnosis. CONCLUSIONS: Most of the patients were not offered proper specialized oncofertility counselling at the time of breast cancer diagnosis. Naturally conceived pregnancies after breast cancer were much more frequent than pregnancies resulting from the use of cryopreserved gametes. Adequate contraceptive counselling seems as important as information about fertility and might prevent unplanned pregnancies.

[How I do… an ultrasound guided drainage of a complicated pelvic inflammatory disease?].

Development and validation of a RNAseq signature for prognostic stratification in endometrial cancer.

BACKGROUND: Despite recent advances in endometrial carcinoma (EC) molecular characterization, its prognostication remains challenging. We aimed to assess whether RNAseq could stratify EC patient prognosis beyond current classification systems. METHODS: A prognostic signature was identified using a LASSO-penalized Cox model trained on TCGA (N = 543 patients). A clinically applicable polyA-RNAseq-based work-flow was developed for validation of the signature in a cohort of stage I-IV patients treated in two Hospitals [2010-2017]. Model performances were evaluated using time-dependent ROC curves (prediction of disease-specific-survival (DSS)). The additional value of the RNAseq signature was evaluated by multivariable Cox model, adjusted on high-risk prognostic group (2021 ESGO-ESTRO-ESP guidelines: non-endometrioid histology or stage III-IVA orTP53-mutated molecular subgroup). RESULTS: Among 209 patients included in the external validation cohort, 61 (30%), 10 (5%), 52 (25%), and 82 (40%), had mismatch repair-deficient, POLE-mutated, TP53-mutated tumors, and tumors with no specific molecular profile, respectively. The 38-genes signature accurately predicted DSS (AUC = 0.80). Most disease-related deaths occurred in high-risk patients (5-years DSS = 78% (95% CI = [68%-89%]) versus 99% [97%-100%] in patients without high-risk). A composite classifier accounting for the TP53-mutated subgroup and the RNAseq signature identified three classes independently associated with DSS: RNAseq-good prognosis (reference, 5-years DSS = 99%), non-TP53 tumors but with RNAseq-poor prognosis (adjusted-hazard ratio (aHR) = 5.75, 95% CI[1.14-29.0]), and TP53-mutated subgroup (aHR = 5.64 [1.12-28.3]). The model accounting for the high-risk group and the composite classifier predicted DSS with AUC = 0.84, versus AUC = 0.76 without (p = 0.01). CONCLUSION: RNA-seq profiling can provide an additional prognostic information to established classification systems, and warrants validation for potential RNAseq-based therapeutic strategies in EC.

Low serum progesterone affects live birth rate in cryopreserved blastocyst transfer cycles using hormone replacement therapy.

RESEARCH QUESTION: Does serum progesterone concentration on the day of vitrified-warmed embryo transfer affect live birth rate (LBR) with hormonal replacement therapy (HRT) cycles? DESIGN: Observational cohort study of patients (n = 915) undergoing single autologous vitrified-warmed blastocyst transfer under HRT using vaginal micronized progesterone. Women were included once, between January 2019 and March 2020. Serum progesterone concentration was measured by a single laboratory on the morning of embryo transfer. The primary end point was LBR. Univariate and multivariate logistic regression models were used for statistical analyses. RESULTS: Median (25th-75th percentile) serum progesterone concentration on the day of embryo transfer was 12.5 ng/ml (9.8-15.3). The LBR was 31.5% (288/915) in the overall population. No significant differences were found in implantation rates (40.7% versus 44.9%); LBR was significantly lower in women with a progesterone concentration ≤25th percentile (≤9.8 ng/ml) (26.1% versus 33.2%, P = 0.045) versus women with a progesterone concentration >25th percentile. This correlated with a significantly higher early miscarriage rate (35.9% versus 21.6%, P = 0.005). After adjusting for potential confounding factors in multivariate analysis, low serum progesterone levels (≤9.8 ng/ml) remained significantly associated with lower LBR (OR 0.68 95% CI 0.48 to 0.97). CONCLUSION: A minimum serum progesterone concentration is needed to optimize reproductive outcomes in HRT cycles with single autologous vitrified-warmed blastocyst transfer. Whether modifications of progesterone administration routes, dosage, or both, can improve pregnancy rates needs further study so that treatment of patients undergoing HRT cycles can be further individualized.

Association Between Endometriosis Phenotype and Preterm Birth in France.

IMPORTANCE: Endometriosis is an inflammatory disease with a heterogeneous presentation that affects women of childbearing age. Given the limitations of previous retrospective studies, it is still unclear whether endometriosis has adverse implications for pregnancy outcomes. OBJECTIVE: To evaluate the association between the presence of endometriosis and preterm birth and whether the risk varied according to the disease phenotype. DESIGN, SETTING, AND PARTICIPANTS: This cohort study with exposed and unexposed groups was conducted in 7 maternity units in France from February 4, 2016, to June 28, 2018. Participants included women with singleton pregnancies who were followed up before 22 weeks' gestation along with their newborns delivered at or after 22 weeks' gestation. The final follow-up occurred in July 2019. Data were analyzed from October 7, 2020, to February 7, 2021. EXPOSURES: Women in the endometriosis group had a documented history of endometriosis and were classified according to 3 endometriosis phenotypes: isolated superficial peritoneal endometriosis (SUP), ovarian endometrioma (OMA; potentially associated with SUP), and deep endometriosis (DE; potentially associated with SUP and OMA). Women in the control group did not have a history of clinical symptoms of endometriosis before their current pregnancy. MAIN OUTCOMES AND MEASURES: The primary outcome was preterm birth between 22 weeks and 36 weeks 6 days of gestation. Association between endometriosis and the primary outcome was assessed through univariate and multivariate logistic regression analyses and was adjusted for the following risk factors associated with preterm birth: maternal age, body mass index (calculated as weight in kilograms divided by height in meters squared) before pregnancy, country of birth, parity, previous cesarean delivery, history of myomectomy and hysteroscopy, and preterm birth. The same analysis was performed according to the 3 endometriosis phenotypes (SUP, OMA, and DE). RESULTS: Of the 1351 study participants (mean [SD] age, 32.9 [5.0] years) who had a singleton delivery after 22 weeks of gestation, 470 were assigned to the endometriosis group (48 had SUP [10.2%], 83 had OMA [17.7%], and 339 had DE [72.1%]) and 881 were assigned to the control group. No difference was observed in the rate of preterm deliveries before 37 weeks 0 days of gestation between the endometriosis and control groups (34 of 470 [7.2%] vs 53 of 881 [6.0%]; P = .38). After adjusting for confounding factors, endometriosis was not associated with preterm birth before 37 weeks' gestation (adjusted odds ratio, 1.07; 95% CI, 0.64-1.77). The results were comparable for the different disease phenotypes (SUP: 6.2% [3 of 48]; OMA: 7.2% [6 of 83]; and DE: 7.4% [25 of 339]; P = .84). CONCLUSIONS AND RELEVANCE: This cohort study found no association between endometriosis and preterm birth, and the disease phenotype did not appear to alter the result. Monitoring the pregnancy beyond the normal protocols or changing management strategies for women with endometriosis may not be warranted to prevent preterm birth.