The need for longitudinal data on the value of fertility treatment for infertile patients' quality of life: A review and pilot study.

Infertility threatens the life goal of parenthood and, hence, quality of life (QoL) of (wo)men, but the fertility clinic trajectory might be burdensome. This review of longitudinal studies and pilot longitudinal study examines the impact of the pre-in vitro fertilization (IVF) fertility clinic trajectory on patient-reported outcome measures (PROMs) for emotional well-being, including QoL. A publication found that the diagnostic workup decreases men's infertility-specific distress while publications disagree whether it decreases (wo)men's anxious and depressive reactions. Intrauterine insemination (IUI) was found to increase (wo)men's depressive reactions. Publications on infertility-specific, health-related, and overall QoL were missing. The pilot indicated that (wo)men's overall QoL is not affected by the diagnostic workup but is decreased by the time of the third IUI. Longitudinal studies on the impact of starting the fertility clinic trajectory on PROMs are needed as they are essential for patient-centered clinical decision-making and patient-centered policy-level decision-making.

Ovarian stimulation strategies for intrauterine insemination in couples with unexplained infertility: a systematic review and individual participant data meta-analysis.

BACKGROUND: Intrauterine insemination with ovarian stimulation (IUI-OS) is a first-line treatment for unexplained infertility. Gonadotrophins, letrozole and clomiphene citrate (CC) are commonly used agents during IUI-OS and have been compared in multiple aggregate data meta-analyses, with substantial heterogeneity and no analysis on time-to-event outcomes. Individual participant data meta-analysis (IPD-MA) is considered the gold standard for evidence synthesis as it can offset inadequate reporting of individual studies by obtaining the IPD, and allows analyses on treatment-covariate interactions to identify couples who benefit most from a particular treatment. OBJECTIVE AND RATIONALE: We performed this IPD-MA to compare the effectiveness and safety of ovarian stimulation with gonadotrophins, letrozole and CC and to explore treatment-covariate interactions for important baseline characteristics in couples undergoing IUI. SEARCH METHODS: We searched electronic databases including MEDLINE, EMBASE, CENTRAL, CINAHL, and PsycINFO from their inception to 28 June 2021. We included randomized controlled trials (RCTs) comparing IUI-OS with gonadotrophins, letrozole and CC among couples with unexplained infertility. We contacted the authors of eligible RCTs to share the IPD and established the IUI IPD-MA Collaboration. The primary effectiveness outcome was live birth and the primary safety outcome was multiple pregnancy. Secondary outcomes were other reproductive outcomes, including time to conception leading to live birth. We performed a one-stage random effects IPD-MA. OUTCOMES: Seven of 22 (31.8%) eligible RCTs provided IPD of 2495 couples (62.4% of the 3997 couples participating in 22 RCTs), of which 2411 had unexplained infertility and were included in this IPD-MA. Six RCTs (n = 1511) compared gonadotrophins with CC, and one (n = 900) compared gonadotrophins, letrozole and CC. Moderate-certainty evidence showed that gonadotrophins increased the live birth rate compared to CC (6 RCTs, 2058 women, RR 1.30, 95% CI 1.12-1.51, I2 = 26%). Low-certainty evidence showed that gonadotrophins may also increase the multiple pregnancy rate compared to CC (6 RCTs, 2058 women, RR 2.17, 95% CI 1.33-3.54, I2 = 69%). Heterogeneity on multiple pregnancy could be explained by differences in gonadotrophin starting dose and choice of cancellation criteria. Post-hoc sensitivity analysis on RCTs with a low starting dose of gonadotrophins (≤75 IU) confirmed increased live birth rates compared to CC (5 RCTs, 1457 women, RR 1.26, 95% CI 1.05-1.51), but analysis on only RCTs with stricter cancellation criteria showed inconclusive evidence on live birth (4 RCTs, 1238 women, RR 1.15, 95% CI 0.94-1.41). For multiple pregnancy, both sensitivity analyses showed inconclusive findings between gonadotrophins and CC (RR 0.94, 95% CI 0.45-1.96; RR 0.81, 95% CI 0.32-2.03, respectively). Moderate certainty evidence showed that gonadotrophins reduced the time to conception leading to a live birth when compared to CC (6 RCTs, 2058 women, HR 1.37, 95% CI 1.15-1.63, I2 = 22%). No strong evidence on the treatment-covariate (female age, BMI or primary versus secondary infertility) interactions was found. WIDER IMPLICATIONS: In couples with unexplained infertility undergoing IUI-OS, gonadotrophins increased the chance of a live birth and reduced the time to conception compared to CC, at the cost of a higher multiple pregnancy rate, when not differentiating strategies on cancellation criteria or the starting dose. The treatment effects did not seem to differ in women of different age, BMI or primary versus secondary infertility. In a modern practice where a lower starting dose and stricter cancellation criteria are in place, effectiveness and safety of different agents seem both acceptable, and therefore intervention availability, cost and patients' preferences should factor in the clinical decision-making. As the evidence for comparisons to letrozole is based on one RCT providing IPD, further RCTs comparing letrozole and other interventions for unexplained infertility are needed.

Great expectations of IVF patients: the role of gender, dispositional optimism and shared IVF prognoses.

STUDY QUESTION: Which success rates do female and male IVF patients expect, what determines their expectations and do patients reconsider their expectations after receiving a personal IVF prognosis at the expense of anxious reactions? SUMMARY ANSWER: Female and male IVF patients have unrealistic high expectations which are positively associated with their dispositional optimism, and which are only reconsidered by patients receiving a less than average IVF prognosis, which leads to more anxious reactions in females. WHAT IS KNOWN ALREADY: Female patients undergoing IVF are known to have unrealistic expectations of the success of their own IVF cycle. The available evidence suggests women expect above average performance of their fertility clinic and (family) reproductive systems. The association of gender and personality trait dispositional optimism, with expectations of IVF success and the impact of providing couples with their IVF prognosis have not been studied previously. STUDY DESIGN, SIZE, DURATION: A total of 148 partnered individuals participated in this prospective survey at two separate points in treatment: following oocyte aspiration (T1) and embryo transfer (T2) (2019-2020, participation rate = 85%). At the time of embryo transfer, gynaecologists provided couples with their IVF prognosis, calculated with the Adapted van Loendersloot model. Women and their male partners completed questionnaires independently and immediately following oocyte aspiration and embryo transfer. PARTICIPANTS/MATERIALS, SETTING, METHODS: Dispositional optimism ('LOT-R' questionnaire) and expectations of IVF success (numerical rating scale) were assessed in eligible couples commencing a 2nd-6th IVF cycle on T1. Expectations of IVF success and anxiety ('Spielberger State-Anxiety Inventory') were (re)assessed on T2. The inter-partner correlation of expectations of IVF success was examined. Linear mixed models examined hypothesized determinants of expectations of IVF success (T1) and explored (determinants of) whether participants reconsidered their expectations after receiving their IVF prognosis (T1-T2) and whether couple's IVF prognosis was associated with anxious reactions (T2). MAIN RESULTS AND THE ROLE OF CHANCE: The mean of the IVF success rates expected by patients immediately after oocyte aspiration was 59.1% (±20.0), irrespective of gender (P = 0.077). Partners expectations of IVF success were moderately correlated (r = 0.483; P < 0.001). Expectations of IVF success were positively associated with the participant's dispositional optimism (P < 0.001) but were not associated with their partner's dispositional optimism, women's age and their previous (un)successful IVF experiences. Gynaecologists gave couples their calculated IVF prognosis ranging from 4.8% to 69.2% (mean = 30.9%) at the time of embryo transfer. Gender did not influence whether participants reconsidered their expectations after receiving their prognosis. In contrast to the subgroup (n = 78), who received at least an average IVF prognosis and that did not reconsider their expectations of IVF success, the subgroup (n = 70) receiving a below average IVF prognosis lowered their expectations of IVF success (interaction effect: P < 0.001) from 55% to 46%. A below average IVF prognosis was associated with anxious reactions in women but not in men (interaction effect: P = 0.011). LIMITATIONS, REASONS FOR CAUTION: The study design and sample size were more optimal for examining hypothesized determinants of patient's expectations of IVF success than for studying the impact of sharing prognoses with patients. Whether (reconsidering) expectations influences IVF discontinuation rates and achieved live birth rates has yet to be followed-up. WIDER IMPLICATIONS OF THE FINDINGS: Clinics are advised to offer patients the opportunity of receiving their IVF prognosis. Providing prognoses is in line with patient preferences and tempers the unrealistic high expectations of both partners in couples with a less than average prognosis. A sensitive communication style is indicated, as lower prognoses are associated with mild anxious reactions in women. STUDY FUNDING/COMPETING INTEREST(S): E.A.F.D. holds a postdoctoral fellowship of the Research Foundation-Flanders (12H9819N) and this study was funded by the Research Council of the KU Leuven (C14/18/106; project of J.V., K.P. and E.A.F.D.) and as an investigator sponsored study of K.P. and E.A.F.D. by Merck nv/sa Belgium, an affiliate of Merck KGaA, Darmstadt, Germany. The authors declare no conflict of interest related to this study. TRIAL REGISTRATION NUMBER: N/A.

The stepwise development of an interactive web-based sex education programme for subfertile couples: the Pleasure & Pregnancy programme.

STUDY QUESTION: Can we develop a web-based sex education programme (programme running in a web browser) that addresses the needs of subfertile couples who are advised expectant management for at least 6 months? SUMMARY ANSWER: The 'Pleasure & Pregnancy' programme addresses couples' needs, is likely to improve couples' sexual functioning, and is subsequently hypothesised to improve the chance of natural pregnancy. WHAT IS KNOWN ALREADY: According to professional guidelines (e.g. the Netherlands and UK) couples with unexplained subfertility and a good chance of natural pregnancy, should be advised at least 6 months of expectant management. Adherence to expectant management is challenging as couples and gynaecologist prefer a more active approach. Targeting sexuality may be useful as subfertility is a risk factor for decreased sexual functioning. STUDY DESIGN, SIZE, DURATION: A novel programme was developed according to the three steps of the Medical Research Councils' (MRC) framework. First, relevant literature was explored. Second, an interdisciplinary expert panel developed a theory (based on a systematic literature review and patient interviews) on how the chance of natural conception can be improved. Third, the expected process and outcomes were modelled. PARTICIPANTS/MATERIALS, SETTING, METHODS: Two licenced clinical sexologists, two gynaecologists, a clinical embryologist and two midwife-researchers, all from Belgium and the Netherlands, proposed components for the sex education programme. PubMed was searched systematically for randomised controlled trials (RCTs) evaluating the proposed components in different patient populations. The needs of 12 heterosexual Dutch or Belgian couples who were advised expectant management were explored with in-depth interviews. The content and delivery characteristics of the novel programme were described in detail with the aid of 'Intervention Taxonomy'. To model the outcomes, a protocol for an RCT was designed, registered and submitted for publication. MAIN RESULTS AND THE ROLE OF CHANCE: To help maintain or improve sexual functioning, mainly pleasure, and hence increase pregnancy rates, the web-based Pleasure & Pregnancy programme contains a combination of psychosexual education and couple communication, mindfulness and sensate focus exercises. Information on the biology of conception and interaction with fertility clinic staff and peers were added based on couples' needs to increase potential acceptability. LIMITATIONS AND REASON FOR CAUTION: This paper outlines the development phase of a sex education programme according to the MRC-framework. Whether the Pleasure & Pregnancy programme actually is acceptable, improves sexual functioning, increases pregnancy rates and is cost-effective remains to be determined. WIDER IMPLICATIONS OF THE FINDINGS: No previous interactive web-based sex education programme has aimed to increase the natural pregnancy rate of subfertile couples by targeting their sexual pleasure. The Pleasure & Pregnancy programme addresses couples' needs and its effect on sexual functioning and pregnancy rate is plausible but remains to be demonstrated by an RCT which is currently ongoing. STUDY FUNDING/COMPETING INTEREST(S): Funding was provided by The Netherlands Organisation for Health Research and Development (ZonMw), Flanders Research Foundation and the University of Amsterdam. C.B.L. is editor-in-chief of Human Reproductionbut was blinded to all parts of the peer review process. The remaining authors have no conflict of interest to report. TRIAL REGISTRATION NUMBER: Not applicable.

Patient-centeredness and endometriosis: Definition, measurement, and current status.

The patient-centeredness of care is important for health care quality, especially because it was recently associated with health-related quality of life, which is increasingly being recognized as the ultimate outcome parameter of health care. Therefore, insight into the definition, measurement, and current status of the patient-centeredness of clinics is important for all health care professionals caring for patients with endometriosis. The definition of patient-centered endometriosis care is based on research into the most common preferences, needs, and values of patients with endometriosis and can be summarized in ten dimensions. One valid and reliable questionnaire exists, the ENDOCARE questionnaire, which allows measuring the patient-centeredness status of an endometriosis clinic. This questionnaire has been used to benchmark centers in different countries and has allowed the identification of ten targets for improving the patient-centeredness of endometriosis care. The next step would be to use the ENDOCARE questionnaire before and after an improvement project.

Vitrification of cleavage stage day 3 embryos results in higher live birth rates than conventional slow freezing: a RCT.

STUDY QUESTION: Is the live birth rate (LBR) per embryo thawed/warmed higher when Day 3 cleavage stage embryos are cryopreserved by vitrification compared with slow freezing? SUMMARY ANSWER: The LBR per embryo thawed/warmed was higher after vitrification than after slow freezing on Day 3, based on better embryo survival, quality and availability of embryos in the vitrification group. WHAT IS KNOWN ALREADY: Post-thawing survival rate of cleavage-stage embryos has been reported to be higher after vitrification than after slow freezing. STUDY DESIGN, SIZE, DURATION: This RCT was performed in an academic tertiary center between September 2011 and March 2013. If supernumerary embryos were available on Day 3, patients were randomized at the time of cryopreservation using a computerized system to determine a simple allocation to the vitrification group or the slow freezing group and all embryos were frozen with the same technique. The primary outcome of this study was the LBR per embryo thawed/warmed. Power calculation revealed that 184 thawed embryos were needed in each group (ß = 0.8, α < 0.05) to test the hypothesis that the LBR per embryo thawed/warmed was significantly higher (16%) after vitrification than after slow freezing (6%). PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients <40 years old undergoing their first oocyte retrieval (OR), with embryo transfer and with supernumerary embryos on Day 3, were randomized. Day 3 embryos with ≥6 cells, <25% fragmentation and morphologically equal blastomeres were cryopreserved by slow freezing (using 1,2-propanediol and 0.1 M sucrose as cryoprotectant) or by closed vitrification using commercially available freezing/vitrification media. Survival was defined as ≥50% cells were intact after thawing. Thawed embryos were further cultured overnight. In total, 307 patients were randomized to slow freezing (155 patients, 480 embryos) or vitrification (152 patients, 495 embryos). MAIN RESULTS AND THE ROLE OF CHANCE: By March 2013, 200 embryos were thawed after slow freezing in 95 cycles for 79 patients and 217 embryos were warmed after vitrification in 121 cycles in 90 patients. The LBR per embryo thawed/warmed was significantly higher after vitrification (16.1% (35/217)) than after slow freezing (5.0% (10/200); P < 0.0022; relative risk (RR) 3.23; 95% confidence interval (CI) 1.64-6.35). Similarly, the implantation rate per embryo thawed/warmed was higher after vitrification (20.7% (45/217) than after slow freezing (7.5% (15/200); P = 0.0012; RR 2.76; CI 1.59-4.81). The survival rate was significantly higher after vitrification (84.3% (183/217) than after slow freezing (52.5% (105/200); P < 0.0001). Significantly more embryos were fully intact after vitrification (75.4% (138/183) than after slow freezing (28.6% (30/105); P < 0.0001). The number of transfers was significantly higher after vitrification (90.1% (109/121)) than after slow freezing (73.7% (70/95); P = 0.0024). LIMITATIONS, REASONS FOR CAUTION: Survival rates in the slow freezing group were low in this study. Additional RCTs are needed to compare reproductive outcome after vitrification and after slow freezing with 1,2-propanediol and 0.2 M sucrose, since this method has been reported to have better survival than the method used in our study. Our findings are only applicable to the specific slow freezing cryopreservation medium used in our study, and not to any other commercially available media. WIDER IMPLICATIONS OF THE FINDINGS: When compared with slow freezing using 1,2-propanediol and 0.1 M sucrose as cryoprotectant, vitrification of Day 3 cleavage stage embryos resulted in a higher LBR per embryo warmed, and may therefore result into a higher cumulative delivery rate after one oocyte retrieval. STUDY FUNDING/COMPETING INTERESTS: None. TRIAL REGISTRATION NUMBER: NCT02013024.

Quality of life outcomes in women with endometriosis are highly influenced by recruitment strategies.

STUDY QUESTION: To what extent are outcome measures in endometriosis-related quality of life studies influenced by the setting in which patient recruitment is performed? SUMMARY ANSWER: Quality of life outcomes in women with endometriosis are highly influenced by recruitment strategies. WHAT IS KNOWN ALREADY: Most studies on quality of life in women with endometriosis are conducted in tertiary care centres or patient associations. It is conceivable that the setting in which patient recruitment is performed influences the quality of life results. This has not been investigated before. STUDY DESIGN, SIZE, DURATION: Retrospective questionnaire based cohort study (part of the World Endometriosis Research Foundation (WERF) EndoCost study). The investigated women were recruited in three settings: a tertiary care centre for endometriosis (n = 135); five secondary care centres (n = 63); an endometriosis patient association (n = 291). PARTICIPANTS/MATERIALS, SETTING, METHODS: The secondary and tertiary care population included women with a laparoscopic and/or histological diagnosis of endometriosis. The patient association population consisted of women with a self-reported diagnosis of surgically confirmed endometriosis. MAIN RESULTS AND THE ROLE OF CHANCE: The populations did not differ in terms of age, co-morbidities and education level. Delay of diagnosis was the longest in the patient association (median 7 years) (tertiary care 2 years; secondary care 1.5 years) (P < 0.001). The tertiary care population reported more laparotomies (64%) than the other populations (secondary care 43%; patient association 47%) (P = 0.002). Affected job was least prevalent in the secondary care setting (35%) (patient association 64%; tertiary care 56%) (P < 0.001). Affected relationships were most prevalent in the patient association setting (52%) (tertiary care 38%; secondary care 22%) (P < 0.001). Chronic pain was least prevalent in patients in secondary care (44%) (tertiary care 65%; patient association 61%) (P = 0.009). Substantial differences in quality of life were detected between secondary care (median physical component 50.4, mental component 49.6); tertiary care (physical component 46.2, mental component 46.2) and the patient association (physical component 45.0, mental component 44.6) (P < 0.001, P = 0.018). LIMITATIONS, REASONS FOR CAUTION: The response rate was relatively low (35%). Analysis of the hospital populations revealed that non-responders and responders did not differ with respect to age or revised American Fertility Society classification, indicating that the non-responder bias is limited. However, other factors, such as social and marital status or symptomatology, might be different for non-responders. Missing values were analysed as if the symptom was not present. Missing values never exceeded 10%, except for one value. Therefore, it can be expected that the effect of missing data on the outcome is negligible. Twenty-five patients belonged to more than one category. A sensitivity analysis showed that the influence of assigning patients to another category was limited. WIDER IMPLICATIONS OF THE FINDINGS: Outcomes regarding quality of life are highly influenced by recruitment strategy. None of the groups appeared to be a representative selection of the total population of women with endometriosis. An alternative strategy for creating a representative population for cost and quality of life studies is probably to recruit women who live in a specific geographic area rather than women that visit a specific hospital or are a member of a patient association. STUDY FUNDING/COMPETING INTERESTS: The WERF EndoCost study was funded by the World Endometriosis Research Foundation. The sponsors did not have a role in the design and conduct of this study: collection, management, analysis, interpretation of the data; preparation, review, approval of the manuscript. L.H. is the chief executive and T.M.D. was a board member of WERF at the time of funding. T.M.D holds the Merck-Serono Chair and the Ferring Chair in Reproductive Medicine in Leuven, Belgium and has served as consultant for Merck-Serono, Schering-Plough, Astellas, and Arresto. TRIAL REGISTRATION NUMBER: Not applicable.

Low-dose human menopausal gonadotrophin versus clomiphene citrate in subfertile couples treated with intrauterine insemination: a randomized controlled trial.

STUDY QUESTION: Can controlled ovarian stimulation with low-dose human menopausal gonadotrophin (hMG) improve the clinical pregnancy rate when compared with ovarian stimulation with clomiphene citrate (CC) in an intrauterine insemination (IUI) programme for subfertile couples? SUMMARY ANSWER: Ovarian stimulation with low-dose hMG is superior to CC in IUI cycles with respect to clinical pregnancy rate. WHAT IS KNOWN ALREADY: IUI after ovarian stimulation is an effective treatment for mild male subfertility, unexplained subfertility and minimal-mild endometriosis, but it is unclear which medication for ovarian stimulation is more effective. STUDY DESIGN, SIZE, DURATION: A total of 330 women scheduled for IUI during 657 cycles (September 2004-December 2011) were enrolled in an open-label randomized clinical trial to ovarian stimulation with low-dose hMG subcutaneous (n = 334, 37.5-75 IU per day) or CC per oral (n = 323, 50 mg/day from Day 3-7). Assuming a difference of 10% in 'clinical pregnancy with positive fetal heart beat', we needed 219 cycles per group (alpha-error 0.05, power 0.80). PARTICIPANTS/MATERIALS, SETTING, METHODS: We studied subfertile couples with mild male subfertility, unexplained subfertility or minimal-mild endometriosis. Further inclusion criteria were failure to conceive for ≥12 months, female age ≤42 years, at least one patent Fallopian tube and a total motility count (TMC) ≥5.0 million spermatozoa after capacitation. The primary end-point was clinical pregnancy. Analysis was by intention to treat and controlled for the presence of multiple measures, as one couple could have more randomizations in multiple cycles. Linear mixed models were used for continuous measures. For binary outcomes we estimated the relative risk using a Poisson model with log link and using generalized estimating equations. MAIN RESULTS AND THE ROLE OF CHANCE: When compared with ovarian stimulation with CC, hMG stimulation was characterized by a higher clinical pregnancy rate (hMG 48/334 (14.4%) versus CC 29/323 (9.0%), relative risk (RR) 1.6 (95% confidence interval (CI) 1.1-2.4)), higher live birth rate (hMG 46/334 (13.8%) versus CC 28/323 (8.7%), RR 1.6 (95% CI 1.0-2.4)), low and comparable multiple live birth rate (hMG 3/46 (6.5%) versus CC 1/28 (3.6%), P > 0.99), lower number of preovulatory follicles (hMG 1.2 versus CC 1.5, P < 0.001), increased endometrial thickness (hMG 8.5 mm versus CC 7.5 mm, P < 0.001), and a lower cancellation rate per started cycle (hMG 15/322 (4.7%) versus CC 46/298 (15.4%), P < 0.001). LIMITATIONS, REASONS FOR CAUTION: We randomized patients at a cycle level, and not at a strategy over multiple cycles. WIDER IMPLICATIONS OF THE FINDINGS: This study showed better reproductive outcome after ovarian stimulation with low-dose gonadotrophins. A health economic analysis of our data is planned to test the hypothesis that ovarian stimulation with low-dose hMG combined with IUI is associated with increased cost-effectiveness when compared with ovarian stimulation with CC. STUDY FUNDING/ COMPETING INTERESTS: T.M.D. and K.P. were supported by the Clinical Research Foundation of UZ Leuven, Belgium. This study was also supported by the Ferring company (Copenhagen, Denmark) which provide free medication (Menopur) required for the group of patients who were randomized in the hMG COS group. The Ferring company was not involved in the study design, data analysis, writing and submission of the paper. TRIAL REGISTRATION NUMBER: NCT01569945 (ClinicalTrials.gov).

The impact of legally restricted embryo transfer and reimbursement policy on cumulative delivery rate after treatment with assisted reproduction technology.

STUDY QUESTION: What is the impact of the Belgian legislation (1 July 2003), coupling reimbursement of six assisted reproduction technology (ART) cycles per patient to restricted embryo transfer policy, on cumulative delivery rate (CDR) per patient? SUMMARY ANSWER: The introduction of Belgian legislation in ART had no negative impact on the CDR per patient based on realistic estimates within six cycles or 36 months. WHAT IS KNOWN ALREADY: The introduction of Belgian legislation limiting the number of embryos for transfer resulted in a reduction of the multiple pregnancy rate (MPR) per cycle by 50%. STUDY DESIGN, SIZE, DURATION: A retrospective cohort study with a study group after implementation of the new ART legislation (July 2003 to June 2006) and the control group, before legislation (July 1999 to June 2002). PARTICIPANTS/MATERIALS, SETTING, METHODS: CDR was compared in an academic tertiary setting between a study group after legislation (n = 795 patients, 1927 fresh and 383 frozen-thawed embryo transfer (FET) cycles) and a control group before legislation (n = 463 patients, 876 fresh and 185 FET cycles) within six cycles or 36 months, delivery or discontinuation of treatment. The CDR was estimated using life table analysis considering pessimistic, optimistic and realistic scenarios and compared after adjustment for confounding variables. In the realistic scenario we included information on embryo quality to define the prognosis of each patient discontinuing treatment. MAIN RESULTS AND THE ROLE OF CHANCE: In the realistic scenario, CDR within 36 months was comparable (all ages, P = 0.221) in study group (60.8%) and control group (65.6%), as well as in different age groups (<36 years, P = 0.242; 36-39 years, P = 0.851; 40-42 years, P = 0.840). In the realistic scenario applied to six cycles, we found lower CDRs in the study group than in the control group within the two first cycles (all ages, P = 0.009; <36 years, P = 0.007) but no difference in CDRs between the two groups within the four subsequent cycles (all ages P = 0.232; <36 years, P = 0.198). The CDR within six cycles was 60 and 65.3% for study group and control group, respectively, for all ages, and 65.8 and 70.4%, respectively, in the subgroup younger than 36 years. In women ≥36 years, CDR within six cycles was comparable in both groups (36-39 years, 43% in study versus 44.4% in control group, P = 0.730; 40-42 years, 21% in study versus 23% in control group, P = 0.786). LIMITATIONS, REASONS FOR CAUTION: A retrospective cohort study design was the only way to study the impact of legislation on CDR. Owing to the retrospective nature of this analysis over a long period of time, our data are potentially influenced by improvements in techniques and therefore improved success rates in ART over time. WIDER IMPLICATIONS OF THE FINDINGS: This 'Belgian model' can now be considered for application worldwide in countries with the aim to reduce the main ART side effect (high MPR) and its associated costs without a negative effect on the main intended effect (high CDR). STUDY FUNDING/COMPETING INTEREST(S): The authors have no conflict of interest to declare. No funding was obtained for this study.

The significant effect of endometriosis on physical, mental and social wellbeing: results from an international cross-sectional survey.

STUDY QUESTION: To what extent do the management of endometriosis and the symptoms that remain after treatment affect the quality of life in women with the disease? SUMMARY ANSWER: Many women with endometriosis had impaired quality of life and continued to suffer from endometriosis-associated symptoms even though their endometriosis has been managed in tertiary care centres. WHAT IS KNOWN ALREADY: The existing literature indicates that quality of life and work productivity is reduced in women with endometriosis. However, most studies have small sample sizes, are treatment related or examine newly diagnosed patients only. STUDY DESIGN, SIZE, DURATION: A cross-sectional questionnaire-based survey among 931 women with endometriosis treated in 12 tertiary care centres in 10 countries. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women diagnosed with endometriosis who had at least one contact related to endometriosis-associated symptoms during 2008 with a participating centre were enrolled into the study. The study investigated the effect of endometriosis on education, work and social wellbeing, endometriosis-associated symptoms and health-related quality of life, by using questions obtained from the World Endometriosis Research Foundation (WERF) GSWH instrument (designed and validated for the WERF Global Study on Women's Health) and the Short Form 36 version 2 (SF-36v2). MAIN RESULTS AND THE ROLE OF CHANCE: Of 3216 women invited to participate in the study, 1450 (45%) provided informed consent and out of these, 931 (931/3216 = 29%) returned the questionnaires. Endometriosis had affected work in 51% of the women and affected relationships in 50% of the women at some time during their life. Dysmenorrhoea was reported by 59%, dyspareunia by 56% and chronic pelvic pain by 60% of women. Quality of life was decreased in all eight dimensions of the SF-36v2 compared with norm-based scores from a general US population (all P < 0.01). Multivariate regression analysis showed that number of co-morbidities, chronic pain and dyspareunia had an independent negative effect on both the physical and mental component of the SF-36v2. LIMITATIONS, REASONS FOR CAUTION: The fact that women were enrolled in tertiary care centres could lead to a possible over-representation of women with moderate-to-severe endometriosis, because the participating centres typically treat more complex and referred cases of endometriosis. The response rate was relatively low. Since there was no Institute Review Board approval to do a non-responder investigation on basic characteristics, some uncertainty remains regarding the representativeness of the investigated population. WIDER IMPLICATIONS OF THE FINDINGS: This international multicentre survey represents a large group of women with endometriosis, in all phases of the disease, which increases the generalizability of the data. Women still suffer from frequent symptoms, despite tertiary care management, in particular chronic pain and dyspareunia. As a result their quality of life is significantly decreased. A patient-centred approach with extensive collaboration across disciplines, such as pain specialists, psychologists, sexologists and social workers, may be a valuable strategy to improve the long-term care of women with endometriosis. STUDY FUNDING/COMPETING INTEREST(S): The WERF EndoCost study is funded by the World Endometriosis Research Foundation (WERF) through grants received from Bayer Schering Pharma AG, Takeda Italia Farmaceutici SpA, Pfizer Ltd and the European Society of Human Reproduction and Embryology. The sponsors did not have a role in the design and conduct of the study; collection, management, analysis and interpretation of the data; and preparation, review or approval of the manuscript. L.H. is the chief executive and T.D. was a board member of WERF at the time of funding. T.D. holds the Merck-Serono Chair in Reproductive Medicine and Surgery, and the Ferring Chair in Reproductive Medicine at the Katholieke Universiteit Leuven in Belgium and has served as consultant/research collaborator for Merck-Serono, Schering-Plough, Astellas and Arresto.

Prevalence of endometriosis diagnosed by laparoscopy in adolescents with dysmenorrhea or chronic pelvic pain: a systematic review.

BACKGROUND Endometriosis associated with pain symptoms in adolescents has been extensively reported, but the exact prevalence is unclear because pain symptoms may be atypical and endometriosis can only be diagnosed by laparoscopy. The aim of this paper is to provide a systematic review of the prevalence of endometriosis diagnosed by laparoscopy in adolescents. METHODS A systematic literature search was carried out for relevant articles published between 1980 and 2011 in the databases PUBMED and EMBASE, based on the keywords 'endometriosis', 'laparoscopy', 'adolescents' and 'chronic pelvic pain (CPP)'. In addition, the reference lists of the selected articles were examined. RESULTS Based on 15 selected studies, the overall prevalence of visually confirmed endometriosis was 62% (543/880; range 25-100%) in all adolescent girls undergoing laparoscopic investigation, 75% (237/314) in girls with CPP resistant to treatment, 70% (102/146) in girls with dysmenorrhea and 49% (204/420) in girls with CPP that is not necessarily resistant to treatment. Among the adolescent girls with endometriosis, the overall prevalence of American Society of Reproductive Medicine classified moderate-severe endometriosis was 32% (82/259) in all girls, 16% (17/108) in girls with CPP resistant to treatment, 29% (21/74) in girls with dysmenorrhea and 57% (44/77) in girls with CPP that is not necessarily resistant to treatment. Due to the quality of the included papers an overestimation of the prevalence and/or severity of endometriosis is possible. CONCLUSIONS About two-thirds of adolescent girls with CPP or dysmenorrhea have laparoscopic evidence of endometriosis. About one-third of these adolescents with endometriosis have moderate-severe disease. The value of early detection of endometriosis in symptomatic adolescents and the indications for laparoscopic investigation in adolescents require more research.

Quality indicators for all dimensions of infertility care quality: consensus between professionals and patients.

STUDY QUESTION: What is the relative importance of the six dimensions of quality of care according to different stakeholders and can a quality indicator set address all six quality dimensions and incorporate the views from professionals working in different disciplines and from patients? SUMMARY ANSWER: Safety, effectiveness and patient centeredness were the most important quality dimensions. All six quality dimensions can be assessed with a set of 24 quality indicators, which is face valid and acceptable according to both professionals from different disciplines and patients. WHAT IS KNOWN ALREADY: To our knowledge, no study has weighted the relative importance of all quality dimensions to infertility care. Additionally, there are very few infertility care-specific quality indicators and no quality indicator set covers all six quality dimensions and incorporated the views of professionals and patients. STUDY DESIGN, SIZE AND DURATION: A three-round iterative Delphi survey including patients and professionals from four different fields, conducted in two European countries over the course of 2011 and 2012. PARTICIPANTS/MATERIALS, SETTINGS AND METHODS: Dutch and Belgian gynaecologists, embryologists, counsellors, nurses/midwifes and patients took part (n = 43 in round 1 and finally 30 in round 3). Respondents ranked the six quality dimensions twice for importance and their agreement was evaluated. Furthermore, in round 1, respondents gave suggestions, which were subsequently uniformly formulated as quality indicators. In rounds 2 and 3, respondents rated the quality indicators for preparedness to measure and for importance (relation to quality and prioritization for benchmarking). Providing feedback allowed selecting indicators based on consensus between stakeholder groups. Measurable indicators, important to all stakeholder groups, were selected for each quality dimension. MAIN RESULTS: All stakeholder groups and most individuals agreed that safety, effectiveness and patient centeredness were the most important quality dimensions. A total of 498 suggestions led to the development of 298 indicators. Professionals were sufficiently prepared to measure 204 of these indicators. Based on importance, 52 (7-15 per dimension; round 2) and finally 24 (4 per dimension; round 3) quality indicators were selected. LIMITATIONS, REASONS FOR CAUTION: The final quality indicator set does not cover the entire care process, but rather takes a 'sample' of each quality dimension. Although the quality indicators are face valid and acceptable, their psychometric characteristics need to be tested by further research. WIDER IMPLICATIONS OF THE FINDINGS: Quality management should focus on safety, effectiveness and patient centeredness of care. Clinics can use the quality indicator set to assess all quality dimensions of their care.

Semi-automated morphometric analysis of human embryos can reveal correlations between total embryo volume and clinical pregnancy.

STUDY QUESTION: Is there a link between morphometric characteristics measured by a computer-assisted scoring system and clinical pregnancy outcome? SUMMARY ANSWER: The results confirm that computer-assisted assessment of the total embryo volume is associated with clinical pregnancy outcome and can be used to complement current procedures of embryo selection. WHAT IS KNOWN ALREADY: Morphometric analysis of a large group of embryos has revealed the potential to optimize algorithms for image-analysis systems for the grading of embryos and predicting pregnancy outcomes. STUDY DESIGN, SIZE, DURATION: Oocytes and embryos were obtained from 458 patients who underwent single embryo transfer on Day 3 after IVF/ICSI, between September 2006 and December 2010 at the Leuven University Fertility Center, Belgium. In total, the data set contained 2796 embryos including 458 embryos that were transferred on Day 3. Ongoing pregnancy was defined as the presence of at least one intrauterine gestational sac at 20 weeks. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients included in this study were younger than 36 years, entering their first (n = 375) or second (n = 83) IVF/ICSI cycle and were only included once. Patients were excluded if the cycle included biopsy for PGD or if donor sperm/donor oocytes were used. Based on the 26 sequential images of the same embryo taken at one time point in different planes, the software calculates the total cytoplasmic volume for each time point, from which any reduction or change in the volume with time can be assessed (which helps interpret the degree of fragmentation) and the size of blastomeres. The diameter of the smallest and largest blastomere and the total volume of each embryo were extracted from the computer-assisted scoring system database and the coefficient of diversity was calculated for Days 1, 2 and 3. A logistic regression analysis was performed to determine the range of embryo volume associated with an increased chance of pregnancy. MAIN RESULTS AND THE ROLE OF CHANCE: On Day 3, blastomeres of 8-cell stage embryos were less divergent in size than those of 6-, 7-, 9-cell stage embryos. Although, the coefficients of diversity (ratio of the largest:smallest blastomeres) of implanted embryos tended to be lower than for non-implanted embryos, the difference was only significant for 6-cell stage embryos (P = 0.02). After logistic regression, an association between total embryo volume and pregnancy was observed which had a quadratic nature: both lower and higher volumes were associated with a lower probability of successful pregnancy. A significant association was identified between total embryo volume and pregnancy rate on both Days 2 (P = 0.003) and 3 (P = 0.0003). Diagnostic measures (sensitivity, specificity, positive predictive value, accuracy and c-statistics) of the defined volume range were relatively poor. However, results showed a good negative predictive value [76.86% (95% confidence interval 71.03-82.02) on Day 3]. LIMITATIONS, REASONS FOR CAUTION: A general disadvantage of studies evaluating the impact of a characteristic on the implantation potential of an embryo is the fact that the best embryo is chosen for transfer. No comparisons can therefore be made with the other embryos. Moreover, the decision process is currently based on a non-automated, standard scoring system, which means that a 'bias' in the selection process is always present. WIDER IMPLICATIONS OF THE FINDINGS: Our results are an important step towards the development of an automated computer-assisted scoring system for the morphological characteristics of human embryos to improve embryo selection for optimizing implantation potential. Total embryo volume appears to be one of the objective characteristics that should be included. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: Not applicable.

Replication of endometriosis-associated single-nucleotide polymorphisms from genome-wide association studies in a Caucasian population.

STUDY QUESTION: Is it possible to replicate the previously identified genetic association of four single-nucleotide polymorphisms (SNPs), rs12700667, rs7798431, rs1250248 and rs7521902, with endometriosis in a Caucasian population? SUMMARY ANSWER: A borderline association was observed for rs1250248 and endometriosis (P = 0.049). However, we could not replicate the other previously identified endometriosis-associated SNPs (rs12700667, rs7798431 and rs7521902) in the same population. WHAT IS KNOWN ALREADY: Endometriosis is considered a complex disease, influenced by several genetic and environmental factors, as well as interactions between them. Previous studies have found genetic associations with endometriosis for SNPs at the 7p15 and 2q35 loci in a Caucasian population. STUDY DESIGN, SIZE, DURATION: Allele frequencies of SNPs were investigated in patients with endometriosis and controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: Blood samples and peritoneal biopsies were taken from a Caucasian female population consisting of 1129 patients with endometriosis and 831 controls. DNA was extracted for genotyping. The study was performed at a University hospital and research laboratories. MAIN RESULTS AND THE ROLE OF CHANCE: A weak association with endometriosis (all stages) was observed for rs1250248 (P = 0.049). No significant associations were observed for the SNPs rs12700667, rs7798431 and rs7521902. A non-significant trend towards the association of rs1250248 with moderate/severe endometriosis was observed (odds ratio 1.18, 95% confidence interval 0.97-1.44). LIMITATIONS, REASONS FOR CAUTION: The inability to confirm all previous findings may result from differences between populations and type II errors. WIDER IMPLICATIONS OF THE FINDINGS: Our result demonstrates the difficulty of identifying common genetic variants in complex diseases. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Karolinska Institutet and Stockholm City County/Karolinska Institutet (ALF), Stockholm, Sweden, Swedish Medical Research Council (K2007-54X-14212-06-3, K2010-54X-14212-09-3), Stockholm, Sweden, Leuven University Research Council (Onderzoeksraad KU Leuven), the Leuven University Hospitals Clinical Research Foundation (Klinisch onderzoeksfonds) and by the National Scientific Foundation (Fonds voor Wetenschappelijk Onderzoek, FWO). The authors have no conflict of interest.

Dutch translation of the ICMART-WHO revised glossary on ART terminology.

BACKGROUND: A standardized set of definitions was needed in the field of medically assisted reproduction (MAR) to standardize and harmonize international data acquisition and to monitor the availability, efficacy, and safety of assisted reproductive technology (ART) worldwide. In order to provide accurate national data, the use of a terminology list which was composed and negotiated by the International Committee for Monitoring Assisted Reproductive Technology (ICMART) is essential, and a translation into Dutch was crucial for its implementation in Belgium and the Netherlands. METHOD: The authors of the Dutch article translated the English publication that appeared simultaneously in Human Reproduction and Fertility and Sterility in 2009. A consensus text was obtained after evaluation by experts in the field of MAR both in the Netherlands and in Belgium and then by the board of the respective organizations of obstetrics and gynecology. It was then sent to the World Health Organization (WHO) for approval of publication. RESULT: A translation into Dutch of the ICMART terminology of 2009 was obtained after consensus was reached on clinical and laboratory procedures, outcome variables, and birth. CONCLUSION: The availability and use of standardized terminology and its translation into Dutch will add to a more standardized communication between professionals responsible for the practice of ART and for those responsible for national, regional, and international registries.

The ENDOCARE questionnaire guides European endometriosis clinics to improve the patient-centeredness of their care.

STUDY QUESTION: How patient-centered are two included specialized endometriosis clinics relative to each other and how can they improve the patient-centeredness of their care? SUMMARY ANSWER: The validated ENDOCARE questionnaire (ECQ) reliably concluded that the adjusted overall patient-centeredness did not differ between the clinics, that each clinic was significantly more patient-centered for 2 out of 10 dimensions of patient-centered endometriosis care and that clinics 1 and 2 had to improve 8 and 13 specific care aspects, respectively. WHAT IS KNOWN ALREADY: Patient-centered endometriosis care is essential to high-quality care and is defined by 10 dimensions. The ECQ was developed, validated and proved to be reliable in a European setting of self-reported endometriosis patients but had not yet been used at a clinic level for quality management. STUDY DESIGN, SIZE, DURATION: A cross-sectional survey was disseminated in 2011 to all 514 women diagnosed with endometriosis during a laparoscopy indicated for pain and/or infertility during a retrospective 2-year period (2009-2010) in two university clinics from two different European countries. PARTICIPANTS/MATERIALS, SETTING, METHODS: In total 337 patients completed the ECQ (216 and 121 per clinic). Respondents had a mean age of 34.3 years. Three in four reported a surgical diagnosis of moderate or severe endometriosis and the majority reported surgical treatment by a multidisciplinary team. The ECQ assessed the 10 dimensions of patient-centeredness, more specifically whether the health-care performance, as perceived by patients, measured up to what is important to patients in general. MAIN RESULTS: The ECQ was completed by 337 respondents (response rate = 65.6%). Reliability and validity of the ECQ for use on clinic level were confirmed. Clinics did not differ in overall mean importance scores; importance rankings of the ECQ dimensions were almost identical. The overall patient-centeredness scores (PCS), adjusted for education level, did not discriminate between the clinics. However, the adjusted PCS for the dimensions 'clinic staff' and 'technical skills' were significantly better in clinic 1, whereas the dimensions 'physical comfort' and 'access to care' were significantly better in clinic 2. There were 8 (clinic 1) and 13 (clinic 2) targets identified for joint and cross-clinic improvement. LIMITATIONS, REASONS FOR CAUTION: Response rates were relatively high. Recall bias was the most important limitation and research in more clinics is needed to define the statistical discriminative value of the ECQ. WIDER IMPLICATIONS OF THE FINDINGS: European endometriosis clinics can use the validated ECQ for reliable assessment of their 'patient-centeredness', for comparison with others and for setting specific targets to improve the patient-centeredness of their endometriosis care, to plan interventions, and to evaluate their effectiveness. STUDY FUNDING/COMPETING INTEREST: This work was funded by KU Leuven and European Network of Endometriosis (ENE), supported by the European Commission (Public Health Executive Agency). No competing interests are declared.

Evaluation of a panel of 28 biomarkers for the non-invasive diagnosis of endometriosis.

BACKGROUND: At present, the only way to conclusively diagnose endometriosis is laparoscopic inspection, preferably with histological confirmation. This contributes to the delay in the diagnosis of endometriosis which is 6-11 years. So far non-invasive diagnostic approaches such as ultrasound (US), MRI or blood tests do not have sufficient diagnostic power. Our aim was to develop and validate a non-invasive diagnostic test with a high sensitivity (80% or more) for symptomatic endometriosis patients, without US evidence of endometriosis, since this is the group most in need of a non-invasive test. METHODS: A total of 28 inflammatory and non-inflammatory plasma biomarkers were measured in 353 EDTA plasma samples collected at surgery from 121 controls without endometriosis at laparoscopy and from 232 women with endometriosis (minimal-mild n = 148; moderate-severe n = 84), including 175 women without preoperative US evidence of endometriosis. Surgery was done during menstrual (n = 83), follicular (n = 135) and luteal (n = 135) phases of the menstrual cycle. For analysis, the data were randomly divided into an independent training (n = 235) and a test (n = 118) data set. Statistical analysis was done using univariate and multivariate (logistic regression and least squares support vector machines (LS-SVM) approaches in training- and test data set separately to validate our findings. RESULTS: In the training set, two models of four biomarkers (Model 1: annexin V, VEGF, CA-125 and glycodelin; Model 2: annexin V, VEGF, CA-125 and sICAM-1) analysed in plasma, obtained during the menstrual phase, could predict US-negative endometriosis with a high sensitivity (81-90%) and an acceptable specificity (68-81%). The same two models predicted US-negative endometriosis in the independent validation test set with a high sensitivity (82%) and an acceptable specificity (63-75%). CONCLUSIONS: In plasma samples obtained during menstruation, multivariate analysis of four biomarkers (annexin V, VEGF, CA-125 and sICAM-1/or glycodelin) enabled the diagnosis of endometriosis undetectable by US with a sensitivity of 81-90% and a specificity of 63-81% in independent training- and test data set. The next step is to apply these models for preoperative prediction of endometriosis in an independent set of patients with infertility and/or pain without US evidence of endometriosis, scheduled for laparoscopy.

Combined mRNA microarray and proteomic analysis of eutopic endometrium of women with and without endometriosis.

BACKGROUND: An early semi-invasive diagnosis of endometriosis has the potential to allow early treatment and minimize disease progression but no such test is available at present. Our aim was to perform a combined mRNA microarray and proteomic analysis on the same eutopic endometrium sample obtained from patients with and without endometriosis. METHODS: mRNA and protein fractions were extracted from 49 endometrial biopsies obtained from women with laparoscopically proven presence (n= 31) or absence (n= 18) of endometriosis during the early luteal (n= 27) or menstrual phase (n= 22) and analyzed using microarray and proteomic surface enhanced laser desorption ionization-time of flight mass spectrometry, respectively. Proteomic data were analyzed using a least squares-support vector machines (LS-SVM) model built on 70% (training set) and 30% of the samples (test set). RESULTS: mRNA analysis of eutopic endometrium did not show any differentially expressed genes in women with endometriosis when compared with controls, regardless of endometriosis stage or cycle phase. mRNA was differentially expressed (P< 0.05) in women with (925 genes) and without endometriosis (1087 genes) during the menstrual phase when compared with the early luteal phase. Proteomic analysis based on five peptide peaks [2072 mass/charge (m/z); 2973 m/z; 3623 m/z; 3680 m/z and 21133 m/z] using an LS-SVM model applied on the luteal phase endometrium training set allowed the diagnosis of endometriosis (sensitivity, 91; 95% confidence interval (CI): 74-98; specificity, 80; 95% CI: 66-97 and positive predictive value, 87.9%; negative predictive value, 84.8%) in the test set. CONCLUSION: mRNA expression of eutopic endometrium was comparable in women with and without endometriosis but different in menstrual endometrium when compared with luteal endometrium in women with endometriosis. Proteomic analysis of luteal phase endometrium allowed the diagnosis of endometriosis with high sensitivity and specificity in training and test sets. A potential limitation of our study is the fact that our control group included women with a normal pelvis as well as women with concurrent pelvic disease (e.g. fibroids, benign ovarian cysts, hydrosalpinges), which may have contributed to the comparable mRNA expression profile in the eutopic endometrium of women with endometriosis and controls.

Paired-box gene 2 is down-regulated in endometriosis and correlates with low epidermal growth factor receptor expression.

BACKGROUND: Paired-box 2 (Pax2) is involved in the development of the female genital tract and has been associated with endometrial pathologies. The expression of Pax2 is induced by epidermal growth factor (EGF) and estrogens. In the present study, Pax2 expression and regulation were investigated in endometriosis. METHODS AND RESULTS: Pax2 protein expression was assessed by immunohistochemistry in the eutopic (i.e. inside the uterus) and ectopic tissue (endometriosis) from 11 patients. Immunoreactivity was high in the endometrium, with strong epithelial and weaker stromal staining. Similar expression patterns of Pax2 were observed in the endometrium of women without endometriosis (n = 12). The mRNA level of Pax2 was assessed by real-time PCR in the eutopic and ectopic endometria of 14 patients and in the endometrium from women without endometriosis (n = 20). Pax2 expression was lower in endometriotic lesions than that in the eutopic endometrium of patients (P< 0.001) and controls (P= 0.007). Three possible mechanisms determining low Pax2 expression were investigated: EGF signalling, CpG DNA methylation of the Pax2 promoter and steroid response. The mRNA level of the EGF receptor (EGFR1) was assessed in the samples used for Pax2 mRNA assessment. A significant correlation between EGFR1 and Pax2 in both eutopic and ectopic tissues was observed (R = 0.58; slope regression line, 0.81; 95% CI: 0.09-1.52 and R = 0.54; slope regression line, 2.51; 95% CI: 0.02-4.99, respectively). CpG DNA methylation was analyzed by methyl-specific PCR in two regions of the Pax2 promoter but they were unmethylated in all samples. Steroid responsiveness was assessed using endometrial explant cultures and Pax2 was not regulated by either 17ß-estradiol or progesterone. CONCLUSIONS: In endometriosis patients, Pax2 is down-regulated in the lesions compared with the eutopic tissue, possibly due to low EGF signalling.