Horizons in Endometriosis: Proceedings of the Montreux Reproductive Summit, 14-15 July 2023.

Endometriosis is a complex and chronic gynaecological disorder that affects millions of women worldwide, leading to significant morbidity and impacting reproductive health. This condition affects up to 10% of women of reproductive age and is characterised by the presence of endometrial-like tissue outside the uterus, potentially leading to symptoms such as chronic pelvic pain, dysmenorrhoea, dyspareunia, and infertility. The Montreux summit brought a number of experts in this field together to provide a platform for discussion and exchange of ideas. These proceedings summarise the six main topics that were discussed at this summit to shed light on future directions of endometriosis classification, diagnosis, and therapeutical management. The first question addressed the possibility of preventing endometriosis in the future by identifying risk factors, genetic predispositions, and further understanding of the pathophysiology of the condition to develop targeted interventions. The clinical presentation of endometriosis is varied, and the correlation between symptoms severity and disease extent is unclear. While there is currently no universally accepted optimal classification system for endometriosis, several attempts striving towards its optimisation - each with its own advantages and limitations - were discussed. The ideal classification should be able to reconcile disease status based on the various diagnostic tools, and prognosis to guide proper patient tailored management. Regarding diagnosis, we focused on future tools and critically discussed emerging approaches aimed at reducing diagnostic delay. Preserving fertility in endometriosis patients was another debatable aspect of management that was reviewed. Moreover, besides current treatment modalities, potential novel medical therapies that can target underlying mechanisms, provide effective symptom relief, and minimise side effects in endometriotic patients were considered, including hormonal therapies, immunomodulation, and regenerative medicine. Finally, the question of hormonal substitution therapy after radical treatment for endometriosis was debated, weighing the benefits of hormone replacement.

ART in Europe, 2018: results generated from European registries by ESHRE.

STUDY QUESTION: What are the data and trends on ART and IUI cycle numbers and their outcomes, and on fertility preservation (FP) interventions, reported in 2018 as compared to previous years? SUMMARY ANSWER: The 22nd ESHRE report shows a continued increase in reported numbers of ART treatment cycles and children born in Europe, a decrease in transfers with more than one embryo with a further reduction of twin delivery rates (DRs) as compared to 2017, higher DRs per transfer after fresh IVF or ICSI cycles (without considering freeze-all cycles) than after frozen embryo transfer (FET) with higher pregnancy rates (PRs) after FET and the number of reported IUI cycles decreased while their PR and DR remained stable. WHAT IS KNOWN ALREADY: ART aggregated data generated by national registries, clinics or professional societies have been gathered and analysed by the European IVF-monitoring Consortium (EIM) since 1997 and reported in 21 manuscripts published in Human Reproduction and Human Reproduction Open. STUDY DESIGN SIZE DURATION: Data on medically assisted reproduction (MAR) from European countries are collected by EIM for ESHRE on a yearly basis. The data on treatment cycles performed between 1 January and 31 December 2018 were provided by either national registries or registries based on initiatives of medical associations and scientific organizations or committed persons of 39 countries. PARTICIPANTS/MATERIALS SETTING METHODS: Overall, 1422 clinics offering ART services in 39 countries reported a total of more than 1 million (1 007 598) treatment cycles for the first time, including 162 837 with IVF, 400 375 with ICSI, 309 475 with FET, 48 294 with preimplantation genetic testing, 80 641 with egg donation (ED), 532 with IVM of oocytes and 5444 cycles with frozen oocyte replacement (FOR). A total of 1271 institutions reported data on IUI cycles using either husband/partner's semen (IUI-H; n = 148 143) or donor semen (IUI-D; n = 50 609) in 31 countries and 25 countries, respectively. Sixteen countries reported 20 994 interventions in pre- and post-pubertal patients for FP including oocyte, ovarian tissue, semen and testicular tissue banking. MAIN RESULTS AND THE ROLE OF CHANCE: In 21 countries (21 in 2017) in which all ART clinics reported to the registry, 410 190 treatment cycles were registered for a total population of ∼ 300 million inhabitants, allowing a best estimate of a mean of 1433 cycles performed per million inhabitants (range: 641-3549). Among the 39 reporting countries, for IVF, the clinical PR per aspiration slightly decreased while the PR per transfer remained similar compared to 2017 (25.5% and 34.1% in 2018 versus 26.8% and 34.3% in 2017). In ICSI, the corresponding rates showed similar evolutions in 2018 compared to 2017 (22.5% and 32.1% in 2018 versus 24.0% and 33.5% in 2017). When freeze-all cycles were not considered for the calculations, the clinical PRs per aspiration were 28.8% (29.4% in 2017) and 27.3% (27.3% in 2017) for IVF and ICSI, respectively. After FET with embryos originating from own eggs, the PR per thawing was 33.4% (versus 30.2% in 2017), and with embryos originating from donated eggs 41.8% (41.1% in 2017). After ED, the PR per fresh embryo transfer was 49.6% (49.2% in 2017) and per FOR 44.9% (43.3% in 2017). In IVF and ICSI together, the trend towards the transfer of fewer embryos continues with the transfer of 1, 2, 3 and ≥4 embryos in 50.7%, 45.1%, 3.9% and 0.3% of all treatments, respectively (corresponding to 46.0%, 49.2%. 4.5% and 0.3% in 2017). This resulted in a reduced proportion of twin DRs of 12.4% (14.2% in 2017) and similar triplet DR of 0.2%. Treatments with FET in 2018 resulted in twin and triplet DRs of 9.4% and 0.1%, respectively (versus 11.2% and 0.2%, respectively in 2017). After IUI, the DRs remained similar at 8.8% after IUI-H (8.7% in 2017) and at 12.6% after IUI-D (12.4% in 2017). Twin and triplet DRs after IUI-H were 8.4% and 0.3%, respectively (in 2017: 8.1% and 0.3%), and 6.4% and 0.2% after IUI-D (in 2017: 6.9% and 0.2%). Among 20 994 FP interventions in 16 countries (18 888 in 13 countries in 2017), cryopreservation of ejaculated sperm (n = 10 503, versus 11 112 in 2017) and of oocytes (n = 9123 versus 6588 in 2017) were the most frequently reported. LIMITATIONS REASONS FOR CAUTION: The results should be interpreted with caution as data collection systems and completeness of reporting vary among European countries. Some countries were unable to deliver data about the number of initiated cycles and/or deliveries. WIDER IMPLICATIONS OF THE FINDINGS: The 22nd ESHRE data collection on ART, IUI and FP interventions shows a continuous increase of reported treatment numbers and MAR-derived livebirths in Europe. Although it is the largest data collection on MAR in Europe, further efforts towards optimization of both the collection and reporting, with the aim of improving surveillance and vigilance in the field of reproductive medicine, are awaited. STUDY FUNDING/COMPETING INTERESTS: The study has received no external funding and all costs are covered by ESHRE. There are no competing interests.

ART in Europe, 2017: results generated from European registries by ESHRE.

STUDY QUESTION: What are the data on ART and IUI cycles, and fertility preservation (FP) interventions reported in 2017 as compared to previous years, as well as the main trends over the years? SUMMARY ANSWER: The 21st ESHRE report on ART and IUI shows the continual increase in reported treatment cycle numbers in Europe, with a decrease in the proportion of transfers with more than one embryo causing an additional slight reduction of multiple delivery rates (DR) as well as higher pregnancy rates (PR) and DR after frozen embryo replacement (FER) compared to fresh IVF and ICSI cycles, while the number of IUI cycles increased and their outcomes remained stable. WHAT IS KNOWN ALREADY: Since 1997, ART aggregated data generated by national registries, clinics or professional societies have been gathered and analyzed by the European IVF-monitoring Consortium (EIM) and communicated in a total of 20 manuscripts published in Human Reproduction and Human Reproduction Open. STUDY DESIGN SIZE DURATION: Data on European medically assisted reproduction (MAR) are collected by EIM for ESHRE on a yearly basis. The data on treatments performed between 1 January and 31 December 2017 in 39 European countries were provided by either National Registries or registries based on personal initiatives of medical associations and scientific organizations. PARTICIPANTS/MATERIALS SETTING METHODS: Overall, 1382 clinics offering ART services in 39 countries reported a total of 940 503 treatment cycles, including 165 379 with IVF, 391 379 with ICSI, 271 476 with FER, 37 303 with preimplantation genetic testing (PGT), 69 378 with egg donation (ED), 378 with IVM of oocytes, and 5210 cycles with frozen oocyte replacement (FOR). A total of 1273 institutions reported data on 207 196 IUI cycles using either husband/partner's semen (IUI-H; n = 155 794) or donor semen (IUI-D; n = 51 402) in 30 countries and 25 countries, respectively. Thirteen countries reported 18 888 interventions for FP, including oocyte, ovarian tissue, semen and testicular tissue banking in pre- and postpubertal patients. MAIN RESULTS AND THE ROLE OF CHANCE: In 21 countries (20 in 2016) in which all ART clinics reported to the registry, 473 733 treatment cycles were registered for a total population of approximately 330 million inhabitants, allowing a best-estimate of a mean of 1435 cycles performed per million inhabitants (range: 723-3286).Amongst the 39 reporting countries, the clinical PR per aspiration and per transfer in 2017 were similar to those observed in 2016 (26.8% and 34.6% vs 28.0% and 34.8%, respectively). After ICSI the corresponding rates were also similar to those achieved in 2016 (24% and 33.5% vs 25% and 33.2% in 2016). When freeze all cycles were removed, the clinical PRs per aspiration were 30.8% and 27.5% for IVF and ICSI, respectively.After FER with embryos originating from own eggs the PR per thawing was 30.2%, which is comparable to 30.9% in 2016, and with embryos originating from donated eggs it was 41.1% (41% in 2016). After ED the PR per fresh embryo transfer was 49.2% (49.4% in 2016) and per FOR 43.3% (43.6% in 2016).In IVF and ICSI together, the trend towards the transfer of fewer embryos continues with the transfer of 1, 2, 3 and ≥4 embryos in 46.0%, 49.2%, 4.5% and in 0.3% of all treatments, respectively (corresponding to 41.5%, 51.9%. 6.2% and 0.4% in 2016). This resulted in a reduced proportion of twin DRs of 14.2% (14.9% in 2016) and stable triplet DR of 0.3%. Treatments with FER in 2017 resulted in a twin and triplet DR of 11.2% and 0.2%, respectively (vs 11.9% and 0.2% in 2016).After IUI, the DRs remained similar at 8.7% after IUI-H (8.9% in 2016) and at 12.4% after IUI-D (12.4.0% in 2016). Twin and triplet DRs after IUI-H were 8.1% and 0.3%, respectively (in 2016: 8.8% and 0.3%) and 6.9% and 0.2% after IUI-D (in 2016: 7.7% and 0.4%). Amongst 18 888 FP interventions in 13 countries, cryopreservation of ejaculated sperm (n = 11 112 vs 7877 from 11 countries in 2016) and of oocytes (n = 6588 vs 4907 from eight countries in 2016) were the most frequently reported. LIMITATIONS REASONS FOR CAUTION: As the methods of data collection and levels of reporting vary amongst European countries, interpretation of results should remain cautious. Some countries were unable to deliver data about the number of initiated cycles and deliveries. WIDER IMPLICATIONS OF THE FINDINGS: The 21st ESHRE report on ART, IUI and FP interventions shows a continuous increase of reported treatment numbers and MAR-derived livebirths in Europe. Being already the largest data collection on MAR in Europe, efforts should continue to optimize data collection and reporting with the perspective of improved quality control, transparency and vigilance in the field of reproductive medicine. STUDY FUNDING/COMPETING INTERESTS: The study has received no external funding and all costs are covered by ESHRE. There are no competing interests.

Anti-adhesion Gel versus No gel following Operative Hysteroscopy prior to Subsequent fertility Treatment or timed InterCourse (AGNOHSTIC), a randomised controlled trial: protocol.

STUDY QUESTIONS: Does the application of anti-adhesion gel, compared to no gel, following operative hysteroscopy to treat intrauterine pathology in women wishing to conceive increase the chance of conception leading to live birth? WHAT IS KNOWN ALREADY: Intrauterine adhesions (IUAs) following operative hysteroscopy may impair reproductive success in women of reproductive age. Anti-adhesion barrier gels may decrease the occurrence of IUAs, but the evidence on their effectiveness to improve reproductive outcomes is sparse and of low quality. STUDY DESIGN SIZE DURATION: This multicentre, parallel group, superiority, blinded and pragmatic randomised controlled trial is being carried out in seven participating centres in Belgium. Recruitment started in April 2019. Women will be randomly allocated to treatment with anti-adhesion gel (intervention group) or no gel (control group). Sterile ultrasound gel will be applied into the vagina as a mock-procedure in both treatment arms. The patient, fertility physician and gynaecologist performing the second-look hysteroscopy are unaware of the allocated treatment. Power analysis, based on a target improvement of 15% in conception leading to live birth using anti-adhesion gel, a power of 85%, a significance level of 5%, and a drop-out rate of 10%, yielded a number of 444 patients to be randomised. The baseline rate of conception leading to live birth in the control group is expected to be 45%. PARTICIPANTS/MATERIALS SETTING METHODS: Women of reproductive age (18-47 years), wishing to conceive (spontaneously or by fertility treatment) and scheduled for operative hysteroscopy to treat intrauterine pathology (endometrial polyps, myomas with uterine cavity deformation, uterine septa, IUAs or retained products of conception) are eligible for recruitment. Women may try to conceive from 3 to 6 weeks after receiving allocated treatment with follow-up ending at 30 weeks after treatment. If the woman fails to conceive within this timeframe, a second-look hysteroscopy will be scheduled within 2-6 weeks to check for IUAs. The primary endpoint is conception leading to live birth, measured at 30 weeks after randomisation. The secondary endpoints are time to conception, clinical pregnancy, miscarriage and ectopic pregnancy rates, measured at 30 weeks after receiving allocated treatment. The long-term follow-up starts when the patient is pregnant and she will be contacted every trimester. STUDY FUNDING/COMPETING INTERESTS: This work is funded by the Belgian Healthcare Knowledge Centre (KCE). The anti-adhesion gel is supplied at no cost by Nordic Pharma and without conditions. Dr. Tomassetti reports grants and non-financial support from Merck SA, non-financial support from Ferring SA, personal fees and non-financial support from Gedeon-Richter, outside the submitted work. None of the other authors have a conflict of interest.

Long-term follow-up of boys who have undergone a testicular biopsy for fertility preservation.

STUDY QUESTION: What is the long-term reproductive health outcome of patients who have undergone testicular sampling for fertility preservation (FP) before and during the pubertal transition period? SUMMARY ANSWER: In long-term follow-up after testicular sampling for FP, hormonal data showed that 33% of patients had primary seminiferous tubule insufficiency (high FSH) while semen analyses showed 52% of patients having a severe reduction in total sperm counts or complete absence of ejaculated sperm. WHAT IS KNOWN ALREADY: During childhood and adolescence, both treatments for cancer and benign haematological diseases that require a bone marrow transplantation, can be detrimental to spermatogenesis by depleting the spermatogonial stem cell population. A testicular biopsy prior to chemotherapy or radiotherapy, even though still an experimental procedure, is now recommended for FP by European and USA oncofertility societies if performed within an institutional research setting. While short-term follow-up studies showed little to no post-operative complications and a normal testicular development after 1 year, data regarding the long-term follow-up of boys who have undergone this procedure are still lacking. STUDY DESIGN, SIZE, DURATION: This is a longitudinal retrospective cohort study that reports on the long-term follow-up of pre- and peri-pubertal boys who have undergone a testicular biopsy for FP between May 2005 and May 2020. All the patients included in this study were referred to our programme by haematologists-oncologists who are part of a regional multi-centric collaborative care pathway. PARTICIPANTS/MATERIALS, SETTING, METHODS: Of the 151 boys referred to our FP programme, 139 parents/legal guardians accepted that their child undergo a testicular biopsy. Patient characteristics (i.e. age at biopsy, urogenital history, pubertal status at diagnosis), indications (disease type and dosage of gonadotoxic treatments), operative and post-operative data (biopsy volume, surgical complications), anatomopathological analyses (presence/absence of spermatogonia, Johnsen score) and reproductive data (semen analyses, FSH, LH, testosterone levels) were collected from the institutions' FP database and medical records or from the 'Brussels Health Network'. Cumulative alkylating agent treatment was quantified using the cyclophosphamide equivalent dose (CED). Patients who were 14 years or older at the time of the follow-up and in whom the testicular tissue was shown to contain spermatogonia were included in the reproductive outcome analysis. Comparison of the sperm count findings (absence/presence of spermatozoa) and FSH levels (high (≥10 IU/l)/normal) between patients who were either pre- (Tanner 1) or peri-pubertal (Tanner >1) at the time of the biopsy was done using the Mann-Whitney U or Fisher's tests. A multiple logistic regression was used to study the relationship between the hormone reproductive outcome (high versus normal FSH), as a proxy marker for fertility, and both the pubertal status (Tanner 1 versus Tanner >1) and Johnsen score at the time of the biopsy, while adjusting for CED. MAIN RESULTS AND THE ROLE OF CHANCE: A testicular biopsy was performed in 139 patients either before (129/139) or after (10/139) the start of a gonadotoxic treatment. Post-operative complications occurred in 2.1% (3/139). At the time of the procedure, 88% (122/139) of patients were pre-pubertal and 12% (17/139) were peri-pubertal. The presence of spermatogonia was documented in 92% (128/139) of cases. Follow-up data were available for 114 patients after excluding 23 deceased and two patients lost to follow-up. A paediatric endocrinologist's follow-up including clinical examination and data on reproductive hormones was available for 57 patients (age ≥14) and 19 (33%) of these were found to have high FSH levels (20 ± 8.8 IU/l). There were 37 patients who had returned to the reproductive specialist's consultation for post-treatment fertility counselling and results on semen analysis were available in 27 of these cases; 14/27 (52%) had severely impaired semen parameters including 8 who were azoospermic. Among patients who received an alkylating agent-based treatment (n = 42), a peri-pubertal status (Tanner >1) at the time of diagnosis/biopsy was found to be associated with a higher risk of having primary testicular failure (defined by an FSH ≥ 10 IU/l) after treatment completion with an OR of 6.4 (95% CI 1.22-33.9; P = 0.03). Of all the patients, 2.6% had already fulfilled their wish to build a family or were actively seeking parenthood. LIMITATIONS, REASONS FOR CAUTION: Although this is the largest cohort with follow-up data providing proxy markers of the reproductive potential of boys in whom a testicular biopsy for FP was performed before puberty or during the pubertal transition period, the amount of data provided is limited, and originating from a single programme. Further data collection to confirm the observations in other settings is therefore awaited. WIDER IMPLICATIONS OF THE FINDINGS: Testicular sampling for FP should be offered to boys at risk of losing their fertility (and is recommended for those at high risk) as part of ethically approved research programmes. Long-term follow-up data on increasing numbers of boys who have undergone an FP procedure will help improve patient care in the future as patient-specific factors (e.g. urogenital history, age at gonadotoxic therapy) appear to influence their reproductive potential after gonadotoxic therapies. STUDY FUNDING/COMPETING INTEREST(S): FNRS-Télévie, the Salus Sanguinis Foundation and the Belgian Foundation against Cancer supported the studies required to launch the FP programme. The authors declare that they have no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Impact of ARTs on oncological outcomes in young breast cancer survivors.

STUDY QUESTION: What is the risk of recurrence in young breast cancer survivors who undergo ARTs following completion of anticancer treatment? SUMMARY ANSWER: ART in breast cancer survivors does not appear to have a negative impact on disease-free survival. WHAT IS KNOWN ALREADY: In healthy women, fertility treatment does not increase the risk of developing breast cancer. At the time of breast cancer diagnosis and before starting anticancer treatments, several studies have shown the safety of performing ART. However, the safety of ART in breast cancer survivors following completion of anticancer treatment remains under-investigated. In general, breast cancer survivors are counselled to avoid any hormonal treatment but there are limited data available on the effect of short exposure to high oestradiol levels during ART. The largest study in this regard included 25 breast cancer survivors exposed to ART and did not show a detrimental effect of ART on patient survival. Hence, taking into account that pregnancy after breast cancer does not affect cancer prognosis, defining the safety of ART in breast cancer survivors remains a priority. STUDY DESIGN, SIZE, DURATION: We conducted a retrospective multicentric matched cohort study including a cohort of breast cancer survivors who underwent ART (exposed patients) between January 2006 and December 2016. Exposed patients who were eligible for the study were matched according to known breast cancer prognostic factors. Matched breast cancer survivors did not undergo ART (non-exposed patients) and were disease-free for a minimum time that was not less than the time elapsed between breast cancer diagnosis and first ART for the matched ART-exposed patients. PARTICIPANTS/MATERIALS, SETTING, METHODS: Data were retrieved from all survivors who had been diagnosed with breast cancer in eight participating centres at an age of ≤40 years, without metastasis, ongoing pregnancy, pre-existing neoplasia or ovarian failure. ART included ovarian stimulation for IVF/ICSI, clomiphene citrate treatment and hormone replacement therapy for embryo transfer. Data were collected from an oncological database for the selection of breast cancer patients in the non-exposed group. Exposed patients were matched (1:2) for germline BRCA status, tumour stage, anticancer treatment and age, whenever feasible. Matched groups were compared at baseline according to characteristics using conditional logistic regression. Kaplan-Meier curves were constructed to compare time to recurrence between groups, with the time of ART as starting point that has been adjusted in the non-exposed group. The analyses were performed using Stata IC/15.1. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 39 breast cancer patients in the ART group were eligible for the analysis and were matched with 73 controls. There was no statistical difference between the two groups for the presence of BRCA mutation, tumour characteristics, use of (neo)adjuvant chemotherapy and of adjuvant endocrine therapy. Exposed patients were younger than non-exposed patients (mean age 31.8 vs 34.3 years, respectively; P < 0.001). In the ART group, 89.7% were nulliparous at diagnosis compared to 46.6% of controls (P < 0.001). ART was performed at a mean age of 37.1 years old, after a median time of 4.1 years following breast cancer diagnosis (range: 1.5-12.5). Median anti-Müllerian hormone at the time of ART was 0.28 ng/ml (range: 0-4.4) and median serum oestradiol peak level was 696.5 pg/ml (range: 139.7-4130). Median follow-up time from first attempt of ART was 4.6 years (range: 2.4-12.5) in the ART group. Adjusted follow-up time for the non-exposed group was 6.9 years (range: 1.1-16.5 years) (P = 0.004). In the ART group, 59% of patients had a pregnancy after breast cancer compared to 26% in the non-exposed patients (P = 0.001). Breast cancer relapsed in 7.7% versus 20.5% women in the ART and non-exposed groups, respectively (hazard ratio 0.46, 95% CI 0.13-1.62, P = 0.23). Median time to relapse was 1.3 (range: 0.3-2.7) years versus 4.5 (range: 0.4-11.1) years after ART and adjusted time in the ART and non-exposed groups, respectively (P = 0.14). LIMITATIONS, REASONS FOR CAUTION: Although this is the first and largest multicentric study addressing the impact of ART on breast cancer recurrence to provide data on oestrogen exposure, only a small number of patients could be included. This reflects the reluctance of breast cancer survivors and/or oncologists to perform ART, and highlights the need for a prospective data registry to confirm the safety of this approach. This would offer the possibility for these patients, who are at a high risk of infertility, to fully benefit from ART. WIDER IMPLICATIONS OF THE FINDINGS: Although recent studies have proven that pregnancy after breast cancer has no detrimental impact on prognosis, counselling patients about the safety of ART remains challenging. Our study provides reassuring data on the use of ART in breast cancer survivors with favourable prognostic factors, for when natural conception fails. STUDY FUNDING/COMPETING INTEREST(S): M.C. and I.D. are funded by FNRS, Télévie-FNRS and Fonds Erasme. M.D.V. is a CooperSurgical scientific advisory board member and receives lecture fees for MSD, Gedeon-Richter and Ferring, outside the submitted work. M.L. has acted as a consultant for Roche and Novartis and has received honoraria from Theramex, Roche, Lilly, Pfizer, Novartis and Takeda, outside the submitted work. I.D. has acted as a consultant for ROCHE and has received speaker's fees from Novartis, outside the submitted work. E.d.A. has received honoraria and is a Roche/GNE, Novartis, SeaGen and Zodiac scientific advisory board member, has received travel grants from Roche/GNE and GSK/Novartis, and has received research grants from Roche/GNE, Astra-Zeneca, GSK/Novartis and Servier, outside the submitted work. A.D. is a recipient of a research grant from Ferring Pharmaceuticals and receives lecture and/or consultancy fees from Merck, Gedeon-Richter and Ferring Pharmaceuticals, outside the submitted work. The remaining authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

20 years of the European IVF-monitoring Consortium registry: what have we learned? A comparison with registries from two other regions.

STUDY QUESTION: How has the performance of the European regional register of the European IVF-monitoring Consortium (EIM)/European Society of Human Reproduction and Embryology (ESHRE) evolved from 1997 to 2016, as compared to the register of the Centres for Disease Control and Prevention (CDC) of the USA and the Australia and New Zealand Assisted Reproduction Database (ANZARD)? SUMMARY ANSWER: It was found that coherent and analogous changes are recorded in the three regional registers over time, with a different intensity and pace, that new technologies are taken up with considerable delay and that incidental complications and adverse events are only recorded sporadically. WHAT IS KNOWN ALREADY: European data on ART have been collected since 1997 by EIM. Data collection on ART in Europe is particularly difficult due to its fragmented political and legal landscape. In 1997, approximately 78.1% of all known institutions offering ART services in 23 European countries submitted data and in 2016 this number rose to 91.8% in 40 countries. STUDY DESIGN, SIZE, DURATION: We compared the changes in European ART data as published in the EIM reports (2001-2020) with those of the USA, as published by CDC, and with those of Australia and New Zealand, as published by ANZARD. PARTICIPANTS/MATERIALS, SETTING, METHODS: We performed a retrospective analysis of the published EIM data sets spanning the 20 years observance period from 1997 to 2016, together with the published data sets of the USA as well as of Australia and New Zealand. By comparing the data sets in these three large registers, we analysed differences in the completeness of the recordings together with differences in the time intervals on the occurrence of important trends in each of them. Effects of suspected over- and under-reporting were also compared between the three registers. X2 log-rank analysis was used to assess differences in the data sets. MAIN RESULTS AND THE ROLE OF CHANCE: During the period 1997-2016, the numbers of recorded ART treatments increased considerably (5.3-fold in Europe, 4.6-fold in the USA, 3.0-fold in Australia and New Zealand), while the number of registered treatment modalities rose from 3 to 7 in Europe, from 4 to 10 in the USA and from 5 to 8 in Australia and New Zealand, as published by EIM, CDC and ANZARD, respectively. The uptake of new treatment modalities over time has been very different in the three registers. There is a considerable degree of underreporting of the number of initiated treatment cycles in Europe. The relationship between IVF and ICSI and between fresh and thawing cycles evolved similarly in the three geographical areas. The freeze-all strategy is increasingly being adopted by all areas, but in Europe with much delay. Fewer cycles with the transfer of two or more embryos were reported in all three geographical areas. The delivery rate per embryo transfer in thawing cycles bypassed that in fresh cycles in the USA in 2012, in Australia and New Zealand in 2013, but not yet in Europe. As a result of these changing approaches, fewer multiple deliveries have been reported. Since 2012, the most documented adverse event of ART in all three registers has been premature birth (<37 weeks). Some adverse events, such as maternal death, ovarian hyperstimulation syndrome, haemorrhage and infections, were only recorded by EIM and ANZARD. LIMITATIONS, REASONS FOR CAUTION: The methods of data collection and reporting were very different among European countries, but also among the three registers. The better the legal background on ART surveillance, the more complete are the data sets. Until the legal obligation to report is installed in all European countries together with an appropriate quality control of the submitted data the reported numbers and incidences should be interpreted with caution. WIDER IMPLICATIONS OF THE FINDINGS: The growing number of reported treatments in ART, the higher variability in treatment modalities and the rising contribution to the birth rates over the last 20 years point towards the increasing impact of ART. High levels of completeness in data reporting have been reached, but inconsistencies and inaccuracies still remain and need to be identified and quantified. The current trend towards a higher diversity in treatment modalities and the rising impact of cryostorage, resulting in improved safety during and after ART treatment, require changes in the organization of surveillance in ART. The present comparison must stimulate all stakeholders in ART to optimize surveillance and data quality assurance in ART. STUDY FUNDING/COMPETING INTEREST(S): This study has no external funding and all costs are covered by ESHRE. There are no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Corrigendum. ART in Europe, 2015: results generated from European registries by ESHRE.

[This corrects the article DOI: 10.1093/hropen/hoz038.].

ART in Europe, 2016: results generated from European registries by ESHRE.

STUDY QUESTION: What are the reported data on cycles in ART, IUI and fertility preservation (FP) interventions in 2016 as compared to previous years, as well as the main trends over the years? SUMMARY ANSWER: The 20th ESHRE report on ART and IUI shows a progressive increase in reported treatment cycle numbers in Europe, with a decrease in the number of transfers with more than one embryo causing a reduction of multiple delivery rates (DR), as well as higher pregnancy rates and DR after frozen embryo replacement (FER) compared to fresh IVF and ICSI cycles, while the outcomes for IUI cycles remained stable. WHAT IS KNOWN ALREADY: Since 1997, ART aggregated data generated by national registries, clinics or professional societies have been collected, analysed by the European IVF-monitoring Consortium (EIM) and reported in 19 manuscripts published in Human Reproduction and Human Reproduction Open. STUDY DESIGN SIZE DURATION: Yearly collection of European medically assisted reproduction (MAR) data by EIM for ESHRE. The data on treatments performed between 1 January and 31 December 2016 in 40 European countries were provided by either National Registries or registries based on personal initiatives of medical associations and scientific organizations. PARTICIPANTS/MATERIALS SETTING METHODS: In all, 1347 clinics offering ART services in 40 countries reported a total of 918 159 treatment cycles, involving 156 002 with IVF, 407 222 with ICSI, 248 407 with FER, 27 069 with preimplantation genetic testing, 73 927 with egg donation (ED), 654 with IVM of oocytes and 4878 cycles with frozen oocyte replacement (FOR). European data on IUI using husband/partner's semen (IUI-H) and donor semen (IUI-D) were reported from 1197 institutions offering IUI in 29 and 24 countries, respectively. A total of 162 948 treatments with IUI-H and 50 467 treatments with IUI-D were included. A total of 13 689 FP interventions from 11 countries including oocyte, ovarian tissue, semen and testicular tissue banking in pre-and postpubertal patients were reported. MAIN RESULTS AND THE ROLE OF CHANCE: In 20 countries (18 in 2015) with a total population of approximately 325 million inhabitants, in which all ART clinics reported to the registry, a total of 461 401 treatment cycles were performed, corresponding to a mean of 1410 cycles per million inhabitants (range 82-3088 per million inhabitants). In the 40 reporting countries, after IVF the clinical pregnancy rates (PR) per aspiration and per transfer in 2016 were similar to those observed in 2015 (28.0% and 34.8% vs 28.5% and 34.6%, respectively). After ICSI, the corresponding rates were also similar to those achieved in 2015 (25% and 33.2% vs 26.2% and 33.2%). After FER with own embryos, the PR per thawing is still on the rise, from 29.2% in 2015 to 30.9% in 2016. After ED, the PR per fresh embryo transfer was 49.4% (49.6% in 2015) and per FOR 43.6% (43.4% in 2015). In IVF and ICSI together, the trend towards the transfer of fewer embryos continues with the transfer of 1, 2, 3 and ≥4 embryos in 41.5%, 51.9%, 6.2% and 0.4% of all treatments, respectively (corresponding to 37.7%, 53.9%, 7.9% and 0.5% in 2015). This resulted in a proportion of singleton, twin and triplet DRs of 84.8%, 14.9% and 0.3%, respectively (compared to 83.1%, 16.5% and 0.4%, respectively in 2015). Treatments with FER in 2016 resulted in twin and triplet DR of 11.9% and 0.2%, respectively (vs 12.3% and 0.3% in 2015). After IUI, the DRs remained similar at 8.9% after IUI-H (7.8% in 2015) and at 12.4% after IUI-D (12.0% in 2015). Twin and triplet DRs after IUI-H were 8.8% and 0.3%, respectively (in 2015: 8.9% and 0.5%) and 7.7% and 0.4% after IUI-D (in 2015: 7.3% and 0.6%). The majority of FP interventions included the cryopreservation of ejaculated sperm (n = 7877 from 11 countries) and of oocytes (n = 4907 from eight countries). LIMITATIONS REASONS FOR CAUTION: As the methods of data collection and levels of completeness of reported data vary among European countries, the results should be interpreted with caution. A number of countries failed to provide adequate data about the number of initiated cycles and deliveries. WIDER IMPLICATIONS OF THE FINDINGS: The 20th ESHRE report on ART and IUI shows a continuous increase of reported treatment numbers and MAR-derived livebirths in Europe. Being already the largest data collection on MAR in Europe, continuous efforts to stimulate data collection and reporting strive for future quality control of the data, transparency and vigilance in the field of reproductive medicine. STUDY FUNDING/COMPETING INTERESTS: The study has no external funding and all costs were covered by ESHRE. There are no competing interests.

Fertility preservation in boys: recent developments and new insights †.

BACKGROUND: Infertility is an important side effect of treatments used for cancer and other non-malignant conditions in males. This may be due to the loss of spermatogonial stem cells (SSCs) and/or altered functionality of testicular somatic cells (e.g. Sertoli cells, Leydig cells). Whereas sperm cryopreservation is the first-line procedure to preserve fertility in post-pubertal males, this option does not exist for prepubertal boys. For patients unable to produce sperm and at high risk of losing their fertility, testicular tissue freezing is now proposed as an alternative experimental option to safeguard their fertility. OBJECTIVE AND RATIONALE: With this review, we aim to provide an update on clinical practices and experimental methods, as well as to describe patient management inclusion strategies used to preserve and restore the fertility of prepubertal boys at high risk of fertility loss. SEARCH METHODS: Based on the expertise of the participating centres and a literature search of the progress in clinical practices, patient management strategies and experimental methods used to preserve and restore the fertility of prepubertal boys at high risk of fertility loss were identified. In addition, a survey was conducted amongst European and North American centres/networks that have published papers on their testicular tissue banking activity. OUTCOMES: Since the first publication on murine SSC transplantation in 1994, remarkable progress has been made towards clinical application: cryopreservation protocols for testicular tissue have been developed in animal models and are now offered to patients in clinics as a still experimental procedure. Transplantation methods have been adapted for human testis, and the efficiency and safety of the technique are being evaluated in mouse and primate models. However, important practical, medical and ethical issues must be resolved before fertility restoration can be applied in the clinic.Since the previous survey conducted in 2012, the implementation of testicular tissue cryopreservation as a means to preserve the fertility of prepubertal boys has increased. Data have been collected from 24 co-ordinating centres worldwide, which are actively offering testis tissue cryobanking to safeguard the future fertility of boys. More than 1033 young patients (age range 3 months to 18 years) have already undergone testicular tissue retrieval and storage for fertility preservation. LIMITATIONS REASONS FOR CAUTION: The review does not include the data of all reproductive centres worldwide. Other centres might be offering testicular tissue cryopreservation. Therefore, the numbers might be not representative for the entire field in reproductive medicine and biology worldwide. The key ethical issue regarding fertility preservation in prepubertal boys remains the experimental nature of the intervention. WIDER IMPLICATIONS: The revised procedures can be implemented by the multi-disciplinary teams offering and/or developing treatment strategies to preserve the fertility of prepubertal boys who have a high risk of fertility loss. STUDY FUNDING/COMPETING INTERESTS: The work was funded by ESHRE. None of the authors has a conflict of interest.

ART in Europe, 2015: results generated from European registries by ESHRE.

STUDY QUESTION: What are the European trends and developments in ART and IUI in 2015 as compared to previous years? SUMMARY ANSWER: The 19th ESHRE report on ART shows a continuing expansion of treatment numbers in Europe, and this increase, the variability in treatment modalities and the rising contribution to the birth rates in most participating countries all point towards the increasing impact of ART on European society. WHAT IS KNOWN ALREADY: Since 1997, the ART data generated by national registries have been collected, analysed and reported in 18 manuscripts published in Human Reproduction. STUDY DESIGN SIZE DURATION: Collection of European data by the European IVF-Monitoring Consortium (EIM) for ESHRE. The data for treatments performed between 1 January and 31 December 2015 in 38 European countries were provided by national registries or on a voluntary basis by clinics or professional societies. PARTICIPANTS/MATERIALS SETTINGS METHODS: From 1343 institutions in 38 countries offering ART services a total of 849 811 treatment cycles, involving 155 960 with IVF, 385676 with ICSI, 218098 with frozen embryo replacement (FER), 21 041 with preimplantation genetic testing (PGT), 64 477 with egg donation (ED), 265 with IVM and 4294 with FOR were recorded. European data on IUI using husband/partner's semen (IUI-H) and donor semen (IUI-D) were reported from 1352 institutions offering IUI in 25 countries and 21 countries, respectively. A total of 139 050 treatments with IUI-H and 49 001 treatments with IUI-D were included. MAIN RESULTS AND THE ROLE OF CHANCE: In 18 countries (14 in 2014) with a population of approximately 286 million inhabitants, in which all institutions contributed to their respective national registers, a total of 409 771 treatment cycles were performed, corresponding to 1432 cycles per million inhabitants (range: 727-3068 per million). After IVF the clinical pregnancy rates (PRs) per aspiration and per transfer were slightly lower in 2015 as compared to 2014, at 28.5 and 34.6% versus 29.9 and 35.8%, respectively. After ICSI, the corresponding PR achieved per aspiration and per transfer in 2015 were also slightly lower than those achieved in 2014 (26.2 and 33.2% versus 28.4 and 35.0%, respectively). On the other hand, after FER with own embryos the PR per thawing continued to rise from 27.6% in 2014 to 29.2% in 2015. After ED a slightly lower PR per embryo transfer was achieved: 49.6% per fresh transfer (50.3% in 2014) and 43.4% for FOR (48.7% in 2014). The delivery rates (DRs) after IUI remained stable at 7.8% after IUI-H (8.5% in 2014) and at 12.0% after IUI-D (11.6% in 2014). In IVF and ICSI together, 1, 2, 3 and ≥4 embryos were transferred in 37.7, 53.9, 7.9 and in 0.5% of all treatments, respectively (corresponding to 34.9, 54.5, 9.9 and in 0.7% in 2014). This evolution towards the transfer of fewer embryos in both IVF and ICSI resulted in a proportion of singleton, twin and triplet DR of 83.1, 16.5 and 0.4%, respectively (compared to 82.5, 17.0 and 0.5%, respectively, in 2014). Treatments with FER in 2015 resulted in twin and triplet DR of 12.3 and 0.3%, respectively (versus 12.4 and 0.3% in 2014). Twin and triplet delivery rates after IUI-H were 8.9 and 0.5%, respectively (in 2014: 9.5 and 0.3%), and 7.3 and 0.6% after IUI-D (in 2014: 7.7 and 0.3%). LIMITATIONS REASONS FOR CAUTION: The methods of data collection and reporting vary among European countries. The EIM receives aggregated data from various countries with variable levels of completeness. Registries from a number of countries have failed to provide adequate data about the number of initiated cycles and deliveries. As long as incomplete data are provided, the results should be interpreted with caution. WIDER IMPLICATIONS OF THE FINDINGS: The 19th EIM report on ART shows a continuing expansion of treatment numbers in Europe. The number of treatments reported, the variability in treatment modalities and the rising contribution to the birth rates in most participating countries point towards the increasing impact of ART on reproduction in Europe. Being the largest data collection on ART worldwide, detailed information about ongoing developments in the field is provided. STUDY FUNDING/COMPETING INTERESTS: The study has no external funding and all costs are covered by ESHRE. There are no competing interests.

Survey on ART and IUI: legislation, regulation, funding and registries in European countries: The European IVF-monitoring Consortium (EIM) for the European Society of Human Reproduction and Embryology (ESHRE).

STUDY QUESTION: How are ART and IUI regulated, funded and registered in European countries? SUMMARY ANSWER: Of the 43 countries performing ART and IUI in Europe, and participating in the survey, specific legislation exists in only 39 countries, public funding (also available in the 39 countries) varies across and sometimes within countries and national registries are in place in 31 countries. WHAT IS KNOWN ALREADY: Some information devoted to particular aspects of accessibility to ART and IUI is available, but most is fragmentary or out-dated. Annual reports from the European IVF-Monitoring (EIM) Consortium for ESHRE clearly mirror different approaches in European countries regarding accessibility to and efficacy of those techniques. STUDY DESIGN SIZE DURATION: A survey was designed using the online SurveyMonkey tool consisting of 55 questions concerning three domains-legal, funding and registry. Answers refer to the countries' situation on 31 December 2018. PARTICIPANTS/MATERIALS SETTINGS METHODS: All members of EIM plus representatives of countries not yet members of the Consortium were invited to participate. Answers received were checked, and initial responders were asked to address unclear answers and to provide any additional information they considered important. Tables of individual countries resulting from the consolidated data were then sent to members of the Committee of National Representatives of ESHRE, asking for a second check. Conflicting information was clarified by direct contact. MAIN RESULTS AND THE ROLE OF CHANCE: Information was received from 43 out of the 44 European countries where ART and IUI are performed. Thirty-nine countries reported specific legislation on ART, and artificial insemination was considered an ART technique in 35 of them. Accessibility is limited to infertile couples in 11 of the 43 countries. A total of 30 countries offer treatments to single women and 18 to female couples. In five countries ART and IUI are permitted for treatment of all patient groups, being infertile couples, single women and same sex couples, male and female. Use of donated sperm is allowed in 41 countries, egg donation in 38, the simultaneous donation of sperm and egg in 32 and embryo donation in 29. Preimplantation genetic testing (PGT) for monogenic disorders or structural rearrangements is not allowed in two countries, and PGT for aneuploidy is not allowed in 11; surrogacy is accepted in 16 countries. With the exception of marital/sexual situation, female age is the most frequently reported limiting criteria for legal access to ART-minimal age is usually set at.18 years and maximum ranging from 45 to 51 years with some countries not using numeric definition. Male maximum age is set in very few countries. Where permitted, age is frequently a limiting criterion for third-party donors (male maximum age 35 to 55 years; female maximum age 34 to 38 years). Other legal constraints in third-party donation are the number of children born from the same donor (in some countries, number of families with children from the same donor) and, in 10 countries, a maximum number of egg donations. How countries deal with the anonymity is diverse-strict anonymity, anonymity just for the recipients (not for children when reaching legal adulthood age), mixed system (anonymous and non-anonymous donations) and strict non-anonymity.Public funding systems are extremely variable. Four countries provide no financial assistance to patients. Limits to the provision of funding are defined in all the others i.e. age (female maximum age is the most used), existence of previous children, maximum number of treatments publicly supported and techniques not entitled for funding. In a few countries, reimbursement is linked to a clinical policy. The definition of the type of expenses covered within an IVF/ICSI cycle, up to what limit and the proportion of out-of-pocket costs for patients is also extremely dissimilar.National registries of ART and IUI are in place in 31 out of the 43 countries contributing to the survey, and a registry of donors exists in 18 of them. LIMITATIONS REASONS FOR CAUTION: The responses were provided by well-informed and committed individuals and submitted to double checking. Since no formal validation was in place, possible inaccuracies cannot be excluded. Also, results are a cross section in time and ART and IUI legislations within European countries undergo continuous evolution. Finally, several domains of ART activity were deliberately left out of the scope of this ESHRE survey. WIDER IMPLICATIONS OF THE FINDINGS: Results of this survey offer a detailed view of the ART and IUI situation in European countries. It provides updated and extensive answers to many relevant questions related to ART usage at national level and could be used by institutions and policymakers in planning services at both national and European levels. STUDY FUNDING/COMPETING INTERESTS: The study has no external funding, and all costs were covered by ESHRE. There were no competing interests.ESHRE Pages are not externally peer reviewed. This article has been approved by the Executive Committee of ESHRE.

ART in Europe, 2014: results generated from European registries by ESHRE: The European IVF-monitoring Consortium (EIM) for the European Society of Human Reproduction and Embryology (ESHRE).

STUDY QUESTION: What are the European trends and developments in ART and IUI in 2014 as compared to previous years? SUMMARY ANSWER: The 18th ESHRE report on ART shows a continuing expansion of both treatment numbers in Europe and more variability in treatment modalities resulting in a rising contribution to the birth rates in most participating countries. WHAT IS KNOWN ALREADY: Since 1997, ART data generated by national registries have been collected, analysed by the European IVF-monitoring (EIM) Consortium and reported in 17 manuscripts published in Human Reproduction. STUDY DESIGN, SIZE, DURATION: Continuous collection of European data by the EIM for ESHRE. The data for treatments performed in 2014 between 1 January and 31 December in 39 European countries were provided by national registries or on a voluntary basis by clinics or professional societies. PARTICIPANTS/MATERIALS, SETTING, METHODS: From 39 countries and 1279 institutions offering ART services, a total of 776 556 treatment cycles, involving 146 148 with IVF, 362 285 with ICSI, 192 027 with frozen embryo replacement (FER), 15 894 with PGT, 56 516 with egg donation (ED), 292 with IVM and 3404 with frozen oocyte replacement (FOR) were reported. European data on IUI using husband/partner's semen (IUI-H) and donor semen (IUI-D) were reported from 1364 institutions offering IUI in 26 countries and 21 countries, respectively. A total of 120 789 treatments with IUI-H and 49 163 treatments with IUI-D were included. MAIN RESULTS AND THE ROLE OF CHANCE: In 14 countries (17 in 2013), where all institutions contributed to their respective national registers, a total of 291 235 treatment cycles were performed in a population of ~208 million inhabitants, corresponding to 1925 cycles per million inhabitants (range: 423-2978 per million inhabitants). After treatment with IVF the clinical pregnancy rates (PR) per aspiration and per transfer were marginally higher in 2014 than in 2013, at 29.9 and 35.8% versus 29.6 and 34.5%, respectively. After treatment with ICSI the PR per aspiration and per transfer were also higher than those achieved in 2013 (28.4 and 35.0% versus 27.8 and 32.9%, respectively). After FER with own embryos the PR continued to rise, from 27.0% in 2013 to 27.6% in 2014. After ED a similar trend was observed with PR reaching 50.3% per fresh transfer (49.8% in 2013) and 48.7% for FOR (46.4% in 2013). The delivery rates (DR) after IUI remained stable at 8.5% after IUI-H (8.6% in 2013) and at 11.6% after IUI-D (11.1% in 2013). In IVF and ICSI together, 1, 2, 3 and ≥4 embryos were transferred in 34.9, 54.5, 9.9 and in 0.7% of all treatments, respectively (corresponding to 31.4%, 56.3, 11.5% and 1% in 2013). This evolution in embryo transfer strategy in both IVF and ICSI resulted in a singleton, twin and triplet DR of 82.5, 17.0 and 0.5%, respectively (compared to 82.0, 17.5 and 0.5%, respectively, in 2013). Treatments with FER in 2014 resulted in a twin and triplet DR of 12.4 and 0.3%, respectively (versus 12.5 and 0.3% in 2013). Twin and triplet DR after IUI were 9.5 and 0.3%, respectively, after IUI-H (in 2013:9.5 and 0.6%) and 7.7 and 0.3% after IUI-D (in 2013: 7.5 and 0.3%). LIMITATION, REASONS FOR CAUTION: The method of data collection and reporting varies among European countries. The EIM receives aggregated data from various countries with variable levels of completeness. Registries from a number of countries have failed to provide adequate data about the number of initiated cycles and deliveries. As long as incomplete data are provided, the results should be interpreted with caution. WIDER IMPLICATIONS OF THE FINDINGS: The 18th ESHRE report on ART shows a continuing expansion of treatment numbers in Europe. The number of treatments reported, the variability in treatment modalities and the rising contribution to the birth rates in most participating countries point towards the increasing impact of ART on reproduction in Europe. Being the largest data collection on ART, the report gives detailed information about ongoing developments in the field. STUDY FUNDING/COMPETING INTEREST(S): The study has no external funding and all costs are covered by ESHRE. There are no competing interests.

In vitro formation of the blood-testis barrier during long-term organotypic culture of human prepubertal tissue: comparison with a large cohort of pre/peripubertal boys.

STUDY QUESTION: How does the formation of the blood-testis barrier (BTB), as reflected by the expression of connexin 43 and claudin 11 proteins during the pubertal transition period, take place in vitro compared to samples from a large cohort of pre/peripubertal boys? SUMMARY ANSWER: The BTB connexin 43 and claudin 11 expression patterns appeared to be partially achieved in organotypic culture when compared to that in samples from 71 pre/peripubertal patients. WHAT IS KNOWN ALREADY: Although alterations in the protein expression patterns of the BTB, whose main components are connexin 43 and claudin 11, are known to be associated with impaired spermatogenesis in mice and adult men, there is a lack of knowledge on its formation in pre-peripubertal human tissue both in vitro and in vivo. Moreover, despite Sertoli cell (SC) maturation during long-term organotypic culture of immature testicular tissue (ITT), initiation of spermatogenesis has not yet been achieved. STUDY DESIGN, SIZE, DURATION: Histological sections from 71 pre-peripubertal patients were evaluated for the formation of the BTB acting as in vivo controls according to age, SC maturation, clinical signs of puberty and germ cell differentiation. Testicular tissue fragments retrieved from three prepubertal boys were cultured in a long-term organotypic system to analyze the BTB formation and expression pattern in correlation with SC maturation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Testicular histological sections from 71 patients aged 0-16 years who underwent a biopsy between 2005 and 2014 to preserve their fertility before gonadotoxic treatment were examined. Immunohistochemistry (IHC) results for connexin 43 and claudin 11 as BTB markers, using a semi-quantitative score for their expression, and for Anti-Mullerian hormone (AMH), as SC maturation marker, were analyzed. Germ cell differentiation was evaluated on Hematoxylin-Eosin sections. Tanner stages at the time of biopsy were recorded from medical files. A longitudinal analysis of connexin 43, claudin 11 and AMH expressions on immunohistological sections of organotypic cultured testicular tissue from three prepubertal boys who underwent a biopsy for fertility preservation was performed. Immunostaining was evaluated at culture Days 0, 1, 3, 10, 16, 27, 32, 53, 64 and 139 for two different types of culture media. MAIN RESULTS AND THE ROLE OF CHANCE: Immunohistochemical control sections showed progressive maturation of SCs, as shown by the decrease in AMH expression, with increasing age (P ≤ 0.01) and the AMH expression was negatively correlated with the expression of connexin 43 and claudin 11 (P ≤ 0.01 for both proteins). Androgen receptor (AR) expression increased with age (P ≤ 0.01) and was significantly correlated with the expression of connexin 43 (P = 0.002) and claudin 11 (P = 0.03). A statistical correlation was also found between the reduction of AMH expression and both the advancement of Tanner stages (P ≤ 0.01) and the differentiation of germ cells (P ≤ 0.01). Furthermore, positive correlations between BTB formation (using connexin 43 and claudin 11 expression) and age (P ≤ 0.01 for both the proteins), higher Tanner stages (P ≤ 0.001 and P ≤ 0.01 for connexin 43 and claudin 11, respectively), and presence of more advanced germ cells (P ≤ 0.001 for both proteins) were observed. In the subanalysis on organotypic cultured ITT, where a significant decrease in AMH expression as a marker of SC maturation was already reported, we showed the onset of expression of connexin 43 at Day 16 (P ≤ 0.001) and a constant expression of claudin 11 from Days 0 to 139, for all three patients, without differences between the two types of culture media. LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: Accessibility of prepubertal human testicular tissue is a major limiting factor to the analysis of cultured tissue samples from a wide number of patients, as would be needed to assess the in vitro development of the BTB according to the age. The impossibility of performing longitudinal studies on in vivo BTB formation in the same patient prevents a comparison of the time needed to achieve effective BTB formation and protein expression patterns in vivo and in vitro. WIDER IMPLICATIONS OF THE FINDINGS: To the best of our knowledge, this is the first report describing the expression of two BTB proteins in samples from a cohort of prepubertal and peripubertal boys, for the in vivo pattern, and in cultured ITT from a few prepubertal boys, for the in vitro evaluation. Since the formation of this barrier is essential for spermatogenesis and because little is known about its protein expression patterns and development in humans, a deeper understanding of the testicular microenvironment is essential to improve ITT in vitro culture conditions. The final aim is to restore fertility by acheiving in vitro differentiation of spermatogonial stem cells, using cryopreserved ITT collected before gonadotoxic therapies. STUDY FUNDING AND COMPETING INTEREST(S): Funding was received from Fonds National de la Recherche Scientifique de Belgique (Grant Télevie Nos. 7.4554.14F and 7.6511.16) and Fondation Salus Sanguinis. No conflict of interest has to be disclosed.

Assisted reproductive technology in Europe, 2013: results generated from European registers by ESHRE.

STUDY QUESTION: Are there any changes in the treatments involving ART and IUI initiated in Europe during 2013 compared with previous years? SUMMARY ANSWER: An increase in the overall number of ART cycles resulting from a higher number of countries reporting data was evident, the pregnancy rates (PRs) in 2013 remained stable compared with those reported in 2012, the number of transfers with multiple embryos (3+) was lower than ever before yet the multiple delivery rates (DRs) remained unchanged, and IUI activity and success rates were similar to those of last years. WHAT IS KNOWN ALREADY: Since 1997, ART data in Europe have been collected and reported in 16 manuscripts, published in Human Reproduction. STUDY DESIGN, SIZE, DURATION: Retrospective data collection of European ART data by the European IVF-monitoring Consortium for ESHRE. Data for cycles between 1 January and 31 December 2013 were collected from National Registers, when existing, or on a voluntary basis by personal information. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: From 38 countries (+4 compared with 2012), 1169 clinics reported 686 271 treatment cycles including 144 299 of IVF, 330 367 of ICSI, 154 712 of frozen embryo replacement (FER), 40 244 of egg donation (ED), 247 of IVM, 9791 of PGD/PGS and 6611 of frozen oocyte replacements. European data on intrauterine insemination using husband/partner's semen (IUI-H) and donor semen (IUI-D) were reported from 1095 IUI labs in 22 countries. A total of 175 467 IUI-H and 43 785 IUI-D cycles were included. MAIN RESULTS AND THE ROLE OF CHANCE: In 17 countries where all clinics reported to their ART register, a total of 374 177 ART cycles were performed in a population of around 310 million inhabitants, corresponding to 1175 cycles per million inhabitants (range, 235-2703 cycles per million inhabitants). For all IVF cycles, the clinical PRs per aspiration and per transfer were stable with 29.6% (29.4% in 2012) and 34.5% (33.8% in 2012), respectively. For ICSI, the corresponding rates also were stable with 27.8% (27.8% in 2012) and 32.9% (32.3% in 2012). In FER-cycles, the PR per thawing/warming increased to 27.0% (23.1% in 2012). In ED cycles, the PR per fresh transfer increased to 49.8% (48.4% in 2012), to 38.5% (35.9% in 2012) per thawed transfer, and to 46.4% for transfers after FOR (45.1% in 2012). The DRs after IUI remained stable at 8.6% (8.5% in 2012) after IUI-H and was slightly lower after IUI-D (11.1% versus 12.0% in 2012). In IVF and ICSI cycles, 1, 2, 3 and 4+ embryos were transferred in 31.4, 56.3, 11.5, and 1.0% of the cycles, respectively (corresponding numbers were 30.2, 55.4, 13.3 and 1.1% in 2012). The proportions of singleton, twin and triplet deliveries after IVF and ICSI (added together) were 82., 17.5 and 0.5%, respectively, resulting in a total multiple DR of 18.0% compared to 17.9% in 2012. In FER-cycles, the multiple DR was 12.8% (12.5% twins and 0.3% triplets), nearly the same as in 2012 (12.5, 12.2 and 0.3% respectively). Twin and triplet DRs associated with IUI cycles were 9.5%/0.6% and 7.5%/0.3%, following treatment with husband/donor semen, respectively. LIMITATIONS, REASONS FOR CAUTION: The method of reporting varies among countries, and registers from a number of countries have been unable to provide some of the relevant data such as initiated cycles and deliveries. As long as data are incomplete and generated through different methods of collection, the results should be interpreted with caution. WIDER IMPLICATIONS OF THE FINDINGS: The 17th ESHRE report on ART shows a continuing expansion of the number of treatment cycles in Europe, with more than 685 000 cycles reported in 2013 and an increasing contribution to birth rate in many countries. However, the need to improve and standardize the national registries, and to establish validation methodologies, remains manifest. STUDY FUNDING/COMPETING INTEREST(S): The study has no external funding; all costs are covered by ESHRE. There are no competing interests.

Efficacy of ovarian tissue cryopreservation for fertility preservation: lessons learned from 545 cases.

STUDY QUESTION: How effective is ovarian tissue cryopreservation (OTC)? SUMMARY ANSWER: In our cohort of patients who underwent OTC, premature ovarian failure (POF) rates, return rates and pregnancy rates after autotransplantation were 31.5, 4.4 and 33%, respectively. WHAT IS KNOWN ALREADY: OTC for fertility purposes has been performed for >20 years now. With over 86 live births reported worldwide and success rates of ~30% after autotransplantation of frozen-thawed ovarian cortex, the procedure should no longer be considered experimental. However, very few publications report the efficacy of this procedure. STUDY DESIGN, SIZE, DURATION: Cases of ovarian tissue cryobanking for fertility preservation performed between 1997 and 2013 in a single institution were reviewed by analysis of the cryobank database and a prospective questionnaire sent out in March 2015. PARTICIPANTS/MATERIALS, SETTING, METHODS: There were 545 patients who underwent OTC during this period. The analysis included indications for OTC, survival rates, ovarian function and spontaneous pregnancies after OTC, come-back rates for ovarian tissue transplantation, pregnancy rates after transplantation, and complication and satisfaction rates. MAIN RESULTS AND THE ROLE OF CHANCE: OTC was performed in this cohort at a mean age of 22.3 ± 8.8 years for oncological indications (79%), benign gynecological pathologies (17.5%) and genetic risks of POF (3.5%). Of the 545 patients, 29% were under 18 years of age at the time of OTC and 15% were prepubertal. While 10% of patients died from their disease, 21 patients (3.9%) underwent autotransplantation, 7 of whom delivered a healthy baby, yielding a post-transplantation live birth rate of 33%. Of 451 patients who were sent the questionnaire, 143 agreed to respond (32%). Nevertheless, ovarian function could not be evaluated in 36% of those who answered. Of 92 evaluable patients, 31.5% were menopausal and 68.5% showed persistent ovarian function. Of 52 women who attempted to conceive naturally, 37 were successful (71%). Among 140 patients who answered the questionnaire, 96% were satisfied with the procedure and only 1 major complication (intra-abdominal hemorrhage) was encountered. Among all the patients, 12% have donated their ovarian cortex for research purposes or have had it destroyed. LIMITATIONS, REASONS FOR CAUTION: The questionnaire participation rate (32%), limited follow-up (mean 7.6 ± 3.5 years) and use of only clinical criteria for evaluation of ovarian function made it difficult to accurately assess the risk of POF and efficiency of OTC. WIDER IMPLICATIONS OF THE FINDINGS: Our findings confirm a 30% pregnancy rate after ovarian cortex autotransplantation but also stress the difficulties of evaluating the real efficacy of OTC. STUDY FUNDING/COMPETING INTEREST(S): No funding was sought for this study and none of the authors have any conflict of interest. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov Registration ID: CRYOFONOV01.

Trends over 15 years in ART in Europe: an analysis of 6 million cycles.

STUDY QUESTION: Was the European IVF Monitoring (EIM) Consortium, established in 1999 by ESHRE, able to monitor the trend over time of ART in Europe? SUMMARY ANSWER: The initial aims of the EIM programme (to collect and publish regional European data on census and trends on ART utilization, effectiveness, safety and quality) have been achieved. WHAT IS ALREADY KNOWN: ART data in Europe have been collected and reported annually in Human Reproduction. STUDY DESIGN SIZE DURATION: A retrospective data analysis and summary of the first 15 years of ART activity in Europe (1997-2011) was carried out, using the key figures from the annual ESHRE reports and focusing on how the practice of ART has evolved over the years. PARTICIPANTS/MATERIALS SETTING METHOD: A total of 5 919 320 ART cycles are reported, including IVF, ICSI, frozen embryo relacment and egg donation, resulting in the birth of more than 1 million infants. A total of 1 548 967 IUIs are also reported, including husband/partner's semen and donor semen cycles. The most relevant and complete data are analysed and discussed. MAIN RESULTS AND THE ROLE OF CHANCE: With some fluctuations, the number of countries and clinics reporting to EIM increases significantly from 1997 to 2011. A constant increase was also registered in the number of annual cycles reported. Since 2005, the estimation of the EIM coverage on the total European activity was >80%. In countries with 100% of coverage, the mean availability of ART increased from 765 cycles per million inhabitants in 1997 to 1269 cycles per million inhabitants in 2011, and the proportion of ART infants of the total number of infants born in the country increased from 1.3% to 2.4%. The proportion of women aged > 39 years undergoing IVF and ICSI cycles gradually increased. For 12 consecutive years, the proportion of ICSI versus IVF cycles showed a marked increase before reaching a plateau from 2008. The proportion of transfers with three or more embryos decreased constantly and the proportion of SETs increased over the time period. The triplets deliveries were reduced from 3.7% in 1997 to less than 1% since 2005 (0.6% in 2011). The effectiveness (evaluated as clinical pregnancy rate per aspiration and per embryo transfer) increased until 2007, then the figure remained stable. The cumulative percentage of documented pregnancy losses was 17%. No differences have been noted in terms of outcomes in the IUI cycles. LIMITATIONS REASONS FOR CAUTION: The data presented are accumulated from countries with different collection systems, regulations, insurance coverage and different practices. Each year a number of countries have been unable to provide some of the data. WIDER IMPLICATIONS OF THE FINDINGS: The first summary of 15 years of the EIM reports offers interesting data on census and trends on ART utilization, safety and quality in Europe. The primary aim of the ESHRE effort in supporting European data collection has been reached. Owing to its importance inside and outside the professional community, European data collection and publication on ART have to be supported and implemented. STUDY FUNDING/COMPETING INTERESTS: None.

Preserved seminiferous tubule integrity with spermatogonial survival and induction of Sertoli and Leydig cell maturation after long-term organotypic culture of prepubertal human testicular tissue.

STUDY QUESTION: Is an organotypic culture system able to provide the appropriate testicular microenvironment for in-vitro maturation of human immature testicular tissue (ITT)? SUMMARY ANSWER: Our organotypic culture system provided a microenvironment capable of preserving seminiferous tubule (ST) integrity and Leydig cell (LC) functionality and inducing Sertoli cell (SC) maturation. WHAT IS KNOWN ALREADY: Cryopreservation of human ITT is a well-established strategy to preserve fertility in prepubertal boys affected by cancer, with a view for obtaining sperm. While spermatogenesis in mice has been replicated in organotypic culture, yielding reproductively efficient spermatozoa, this process has not yet been achieved in humans. STUDY DESIGN, SIZE, DURATION: The aim of this study was to in vitro mature frozen-thawed ITT. To this end, 1 mm3 tissue fragments from three prepubertal patients aged 2 (P1), 11 (P2) and 12 (P3) years were placed in organotypic culture for 139 days. Culture media, supplemented with either testosterone or hCG, were compared. PARTICIPANTS/MATERIALS, SETTING, METHODS: ST integrity and tissue viability were assessed by histological score and lactate dehydrogenase (LDH) levels in supernatants. Spermatogonia (SG), proliferating cells and proliferating SG were identified by the use of MAGE-A4 and Ki67 immunohistochemical markers. Glial cell line-derived neurotrophic factor (GDNF) was used as a marker of SC functionality, while SC maturation was evaluated by androgen receptor (AR), anti-Müllerian hormone (AMH) immunohistochemistry (IHC) and AMH immunoenzymatic assay. LC functionality was determined by testosterone levels in supernatants and by 3ß-hydroxysteroid dehydrogenase (3ß-HSD) IHC. Apoptosis was studied by IHC with active caspases 3 and 8 and by TUNEL (terminal deoxynubocleotidyl transferase-mediated dUTP nick end labeling) analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Tissue viability was preserved, as demonstrated by the decrease in and stabilization of LDH release, and evolution of ST scoring, with the percentage of well-preserved STs showing no statistical differences during culture in either medium. GDNF was expressed until Day 139, demonstrating SC functionality. Moreover, a significant reduction in AMH expression and release indicated SC maturation. Testosterone concentrations in supernatants increased in both culture media, demonstrating LC functionality with paracrine interactions. SG were present up to Day 139, although the ratio between MAGE-A4-positive cells and well-preserved tubules was significantly reduced over the course of culture (P ≤ 0.001). SCs exhibited a decreased proliferation rate over time (P ≤ 0.05). The proliferation rate of SG remained stable until Day 64, but over the total culture period (139 days), it was found to have decreased (P ≤ 0.05). The number of apoptotic cells did not vary during culture, nor was any statistical difference observed between the two culture media for any of the studied parameters. LARGE SCALE DATA: N/A LIMITATIONS, REASONS FOR CAUTION: Loss of SG constitutes a limitation for evaluating full functionality of spermatogonial stem cells and warrants further investigation. The scarcity of human immature material is the reason for the limited amount of tissue available for experiments, precluding more comprehensive analysis. WIDER IMPLICATIONS OF THE FINDINGS: Our culture system, mimicking the peripubertal testicular microenvironment with SC maturation, LC functionality and preserved paracrine interactions, and the first to use human ITT, opens the door to a deeper understanding of niche and culture conditions to obtain sperm from cryostored ITT, with the ultimate goal of restoring fertility after gonadotoxic treatments. STUDY FUNDING/COMPETING INTERESTS: This project was supported by a grant from the Fond National de la Recherche Scientifique de Belgique (grant Télevie N° 7.4554.14F and N° 7.4512.15F) and the Fondation Salus Sanguinis. No conflict of interest is declared.

Data collection systems in ART must follow the pace of change in clinical practice.

In assisted reproductive technology (ART), quality control necessitates the collection of outcome data and occurring complications. Traditional quality assurance is based on data derived from single ART centres and more recently from national registries, both recording outcome parameters during well-defined observation periods. Nowadays, ART is moving towards much more diverse approaches, with sequential activities including short- or long-term freezing of gametes, gonadal tissues and embryos, and cross-border reproductive care. Hence, long-term cumulative treatment rates and an international approach are becoming a necessity. We suggest the initiation of an easy access European Reproductive Coding System, through which each ART recipient is allocated a unique reproductive care code. This code would identify individuals (and reproductive material) during case to case data reporting to national ART data collecting institutions and to a central European ART monitoring agency. For confidentiality reasons, the identity of the individuals should remain with the local ART provider. This way, cumulative and fully reliable reproductive outcome data can be constructed with follow-up over prolonged time periods.