Josephine G. Lemmen

The rat, mouse and human estrogen receptor (ER) exists as two subtypes, ER alpha and ER beta, which differ in the C-terminal ligand-binding domain and in the N-terminal transactivation domain. In this study, we investigated the estrogenic activity of environmental chemicals and phytoestrogens in competition binding assays with ER alpha or ER beta protein, and in a transient gene expression assay using cells in which an acute estrogenic response is created by cotransfecting cultures with recombinant human ER alpha or ER beta complementary DNA (cDNA) in the presence of an estrogen-dependent reporter plasmid. Saturation ligand-binding analysis of human ER alpha and ER beta protein revealed a single binding component for [3H]-17beta-estradiol (E2) with high affinity [dissociation constant (Kd) = 0.05 - 0.1 nM]. All environmental estrogenic chemicals [polychlorinated hydroxybiphenyls, dichlorodiphenyltrichloroethane (DDT) and derivatives, alkylphenols, bisphenol A, methoxychlor and chlordecone] compete with E2 for binding to both ER subtypes with a similar preference and degree. In most instances the relative binding affinities (RBA) are at least 1000-fold lower than that of E2. Some phytoestrogens such as coumestrol, genistein, apigenin, naringenin, and kaempferol compete stronger with E2 for binding to ER beta than to ER alpha. Estrogenic chemicals, as for instance nonylphenol, bisphenol A, o, p'-DDT and 2',4',6'-trichloro-4-biphenylol stimulate the transcriptional activity of ER alpha and ER beta at concentrations of 100-1000 nM. Phytoestrogens, including genistein, coumestrol and zearalenone stimulate the transcriptional activity of both ER subtypes at concentrations of 1-10 nM. The ranking of the estrogenic potency of phytoestrogens for both ER subtypes in the transactivation assay is different; that is, E2 >> zearalenone = coumestrol > genistein > daidzein > apigenin = phloretin > biochanin A = kaempferol = naringenin > formononetin = ipriflavone = quercetin = chrysin for ER alpha and E2 >> genistein = coumestrol > zearalenone > daidzein > biochanin A = apigenin = kaempferol = naringenin > phloretin = quercetin = ipriflavone = formononetin = chrysin for ER beta. Antiestrogenic activity of the phytoestrogens could not be detected, except for zearalenone which is a full agonist for ER alpha and a mixed agonist-antagonist for ER beta. In summary, while the estrogenic potency of industrial-derived estrogenic chemicals is very limited, the estrogenic potency of phytoestrogens is significant, especially for ER beta, and they may trigger many of the biological responses that are evoked by the physiological estrogens.

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.

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.

Abstract 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 INTEREST(S) The study has no external funding and all costs were covered by ESHRE. There are no competing interests.

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 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.