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Study question Does the presence of mild adenomyosis, a common acquired uterine anomaly, affect the endometrial gene expression levels during window of receptivity? Summary answer Mild adenomyosis has no significant influence on gene expression signature in the window of implantation (WOI). What is known already The improvements in imaging techniques have led to frequent detection of adenomyosis in women undergoing investigations for infertility. Although the data are conflicting, some clinical studies have shown that the presence of adenomyosis may interfere with embryo implantation and lead to poor pregnancy outcomes. The knowledge of molecular background that would lead to the phenomenon of altered endometrial receptivity in women with adenomyosis is limited and mainly demonstrated by selected candidate genes. Next-generation sequencing platforms enable genome-wide transcriptomic profiling of desired tissue samples and present a powerful tool to identify differentially expressed genes (DEGs) between women with adenomyosis and controls. Study design, size, duration We designed a prospective case-control study comparing women with sonographic evidence of mild adenomyosis (n = 10) and women with normal uteri seeking assisted reproduction due to male factor infertility as the control group (n = 10). All eligible women underwent infertility treatment at the Department of Reproductive Medicine and Gynaecological Endocrinology, University Medical Centre Maribor, Slovenia between years 2018 and 2020. For the present study, they were scheduled for cycle monitoring by urinary luteinizing hormone (LH) tests. Participants/materials, setting, methods Each endometrial biopsy was obtained in the presumed window of implantation (WOI) on days LH + 7 to LH + 9 after LH surge (LH + 0). Isolated total RNA was applied for mRNA + lncRNA sequencing (RNA-seq) by Illumina Novaseq 6000. An aliquot of RNA samples was used to verify the WOI by the endometrial receptivity test “beREADY” (CCHT, Estonia). Gene Ontology and Reactome pathway enrichment analyses were conducted in ClueGO bioinformatics tool to study biological role behind obtained DEGs. Main results and the role of chance The R program language and Bioconductor packages were used to align generated RNA-seq reads on the human reference genome assembly (hg19) and to calculate gene expression differences between study groups using normalized counts per million (CPM)>10 in at least 10 samples. A total 233 DEGs (p Limitations, reasons for caution Only mild adenomyosis was considered in this study, which is most commonly detected in women. The results could differ in women in severe cases of adenomyosis. Multicellular whole-tissue endometrial samples that were used for RNA isolation could mask gene expression differences of specific cell types between study groups. Wider implications of the findings: According to our results of transcriptome analysis, the presence of mild adenomyosis has no significant influence on the gene expression signature during endometrial receptivity in natural menstrual cycle. Women being investigated for infertility can be reassured that this common acquired anomaly does not significantly influence the chances of successful conception. Trial registration number 0120–259/2018/16

Study question Does laser-induced artificial blastocoel collapse result in better blastocyst cryopreservation survival and higher live birth rate (LBR) in comparison with intact counterparts? Summary answer Compared to vitrification of intact blastocysts, collapsed blastocysts resulted in higher survival and for 5% higher LBR. Neonatal outcomes were comparable in both groups. What is known already Blastocysts have long been considered a stage that is suboptimal for freezing-thawing procedures due to their high fluid content and different cell types. The development of a modified vitrification technique has enabled blastocysts to better survive cryopreservation compared to a slow freezing procedure. Many studies on the optimization of cryopreservation of blastocysts have mentioned the need for artificial collapsing of the blastocoel prior to cryopreservation, thereby reducing the risk of intracellular ice-crystals formation. However, the effectiveness of artificial collapsing on blastocyst survival rate, single vitrified-warmed blastocyst transfer (SVBT) outcome and on safety of such intervention remains to be confirmed. Study design, size, duration A retrospective matched case-control study of transfers of single blastocysts being artificially collapsed (case) or intact (control) before vitrification. A sample size of 306 cycles in both arms was needed to achieve 80% power to detect a difference between the groups of 10% with P Participants/materials, setting, methods Artificial collapsing was introduced into clinical practice gradually. In fresh IVF cycles (performed in university clinic from 2012 until 2014) with supernumerary blastocysts, half of the blastocysts were randomly selected before vitrification for laser-induced artificial collapsing. The other half was vitrified in intact form. Only the first transfers of a single vitrified-warmed blastocyst (n = 818) were included in the study. By matching, 309 pairs of collapsed (study) and intact (control) SVBTs were identified. Main results and the role of chance Both groups were comparable by their characteristics in indications, female age, type and length of ovarian hyperstimulation, insemination method in fresh cycle, protocol for warmed blastocyst transfer, blastocyst quality and day of blastocyst vitrification. Survival rates in case and control group ((309/316) 97.8% and (309/323) 95.7%; P = 0.13) were comparable, but optimal survival rates (100% survival and re-expansion after warming) was significantly higher in artificial collapse group ((247/316) 78.2% and (225/323) 69.7%; P = 0.01). Clinical pregnancy rates ((120/309) 38.8% and (110/309) 35.6%; P = 0.4), miscarriage rates ((15/120) 12.5% and (24/110) 21.8%; P = 0.06) and LBR per transfer ((100/309) 32.4% and (85/309) 27.5%; P = 0.19) or LBR per warmed blastocyst ((100/316) 31.6% and (85/323) 26.3%; P = 0.14) were not statistically different between case and control groups. Since the study was powered to detect a 10% difference, the possibility of type 2 error cannot be excluded. Perinatal outcomes were available for 175 live births. There were 10.5% (10/95) preterm births in the study group vs. 16.3% (13/80) in control group (P > 0.05). Birth weights (3,308 g (SD 592 g) vs 3,308 g (SD 738 g) and sex ratio (50.7% vs 49.2% boys) were also comparable between both groups (P > 0.05). There were no major malformations detected in the study population. Limitations, reasons for caution The research is retrospective, but the cycles from both groups were performed in the same time period. The groups were balanced according to all possible confounders. Blastocysts for vitrification were first categorized by quality groups and embryos from each category were randomized for collapsing or for remaining intact. Wider implications of the findings: No significant difference was found in live births by this sample size. Nevertheless, increasing the success by 5% with the introduction of artificial collapsing can be an important step towards optimizing of blastocyst cryopreservation. To confirm a 5% improvement in results, a sample size of > 2500 cases would be needed. Trial registration number The study has been approved by the National Ethics Committee of the Republic of Slovenia (0120–204/2016–2).

Study question What is the recognition of clinical embryology and the current status of clinical embryologists in European countries, regarding educational levels, responsibilities and workload, and need for a formal education in assisted reproductive technology (ART)? Summary answer It is striking that the profession of clinical embryology, almost 40 years after the introduction of IVF, is still not officially recognized in most European countries. What is known already Reproductive medicine has developed into a sophisticated multidisciplinary medical branch since the birth of Louise Brown 37 years ago. The European Board & College of Obstetrics and Gynaecology (EBCOG) has recognized reproductive medicine as a subspeciality and has developed a subspeciality training for gynaecologists in collaboration with the European Society for Human Reproduction and Embryology (ESHRE). However, nothing similar exists for the field of clinical embryology or for clinical embryologists. Study design, size, duration A questionnaire about the situation in clinical embryology in the period of 2012-2013 in the respective European country was sent to ESHRE National representatives (basic scientists only) in December 2013. At this time, 28 European countries had at least one basic scientist in the ESHRE Committee of National Representatives. Participants/materials, setting, methods The survey consisted of 46 numeric, dichotomous (yes/no) or descriptive questions. Answers were obtained from 27 out of 28 countries and the data were tabulated. Data about the numbers of 'ESHRE Certified Embryologists' were taken from the ESHRE Steering Committee for Embryologist Certification. Main results and the role of chance In 2012, more than 7000 laboratory staff from 1349 IVF clinics in 27 European countries performed over 700 000 fresh and frozen ART cycles. Despite this, clinical embryology is only recognized as an official profession in 3 out of 27 national health systems. In most countries clinical embryologists need to be registered under another profession, and have limited possibilities for organized education in clinical embryology. Mostly they are trained for practical work by senior colleagues. ESHRE embryologist certification so far constitutes the only internationally recognized qualification; however this cannot be considered a subspecialization. Limitations, reasons for caution Data were obtained through different methods, by involving national embryologist societies and cycle registers, collecting information from centre to centre, and in some cases by individual assessment of the situation. For these reasons, the results should be interpreted with caution. Wider implications of the findings This paper presents the current status of clinical embryology and clinical embryologists in Europe and is an important step towards implementation of clinical embryology as an officially recognized profession. Study funding/competing interests None. Trial registration number No.

BACKGROUND: The aim of the study was to test the influence of 2- and 5-day cultivation of a single oocyte on the pregnancy rate in a non-stimulated cycle. METHODS: A retrospective chart review of 391 consecutive patients undergoing IVF and intracytoplasmic sperm injection in unstimulated cycles was performed. The embryos were kept in MediCult universal IVF medium for day 2 transfers and in BlastAssist System for day 5 transfers. RESULTS: The oocyte recovery rate in the group for 2-day cultivation and in the group for 5-day cultivation was similar, being 79.4 (162/204) and 83.6% (154/187) respectively. The same is true of the fertilization rate (73.8 versus 77.7%). The blastulation rate was 52.8%. The embryo transfer rate per cycle was higher when day 2 embryos were transferred: 64.8% (105/162) compared with 35.7% (55/154) if blastocyst-stage embryos were transferred. The pregnancy rate per transferred embryo was higher when a blastocyst was transferred (40.0%) instead of a day 2 embryo (23.8%). CONCLUSION: The expected pregnancy rate calculated per embryo available on day 2 of cultivation was similar in both groups (23.8 versus 22.2%) and it was not affected by oocyte culture to the blastocyst stage.