The role of regulatory T-cells in the development of endometriosis.

Endometriosis is a benign disease of the female reproductive tract, characterized by the process of chronic inflammation and alterations in immune response. It is estimated to affect 2-19% of women in the general population and is commonly associated with symptoms of chronic pelvic pain and infertility. Regulatory T cells (Treg) are a subpopulation of T lymphocytes that are potent suppressors of inflammatory immune response, essential in preventing destructive immunity in all tissues. In endometriosis, several studies have investigated the possible role of Treg cells in the development of the disease. Most studies to date are heterogeneous in methodology and are based on a small number of cases, which means that it is impossible to define their exact role at present. Based on current knowledge, it seems that disturbed Treg homeostasis, leading to increased systemic and local inflammation within ectopic and eutopic endometrium, is present in women who eventually develop endometriosis. It is also evident that different subsets of human Treg cells have different roles in suppressing the immune response. Recent studies in patients with endometriosis have investigated naive/resting FOXP3lowCD45RA+ Treg cells, which upon T cell receptor stimulation, differentiate into activated/effector FOXP3highCD45RA- Treg cells, characterized by a strong immunosuppressive activity. In addition, critical factors controlling expression of Treg/effector genes, including reactive oxygen species and heme-responsive master transcription factor BACH2, were found to be upregulated in endometriotic lesions. As shown recently for cancer microenvironments, microbial inflammation may also contribute to the local composition of FOXP3+ subpopulations in endometriotic lesions. Furthermore, cytokines, such as IL-7, which control the homeostasis of Treg subsets through the tyrosine phosphorylation STAT5 signalling pathway, have also been shown to be dysregulated. To better understand the role of Treg in the development of endometriosis, future studies should use clear definitions of Tregs along with specific characterization of the non-Treg (FOXP3lowCD45RA-) fraction, which itself is a mixture of follicular Tregs and cells producing inflammatory cytokines.

Transcriptomics of receptive endometrium in women with sonographic features of adenomyosis.

BACKGROUND: Women with uterine adenomyosis seeking assisted reproduction have been associated with compromised endometrial receptivity to embryo implantation. To understand the mechanisms involved in this process, we aimed to compare endometrial transcriptome profiles during the window of implantation (WOI) between women with and without adenomyosis. METHODS: We obtained endometrial biopsies LH-timed to the WOI from women with sonographic features of adenomyosis (n=10) and controls (n=10). Isolated RNA samples were subjected to RNA sequencing (RNA-seq) by the Illumina NovaSeq 6000 platform and endometrial receptivity classification with a molecular tool for menstrual cycle phase dating (beREADY®, CCHT). The program language R and Bioconductor packages were applied to analyse RNA-seq data in the setting of the result of accurate endometrial dating. To suggest robust candidate pathways, the identified differentially expressed genes (DEGs) associated with the adenomyosis group in the receptive phase were further integrated with 151, 173 and 42 extracted genes from published studies that were related to endometrial receptivity in healthy uterus, endometriosis and adenomyosis, respectively. Enrichment analyses were performed using Cytoscape ClueGO and CluePedia apps. RESULTS: Out of 20 endometrial samples, 2 were dated to the early receptive phase, 13 to the receptive phase and 5 to the late receptive phase. Comparison of the transcriptomics data from all 20 samples provided 909 DEGs (p<0.05; nonsignificant after adjusted p value) in the adenomyosis group but only 4 enriched pathways (Bonferroni p value < 0.05). The analysis of 13 samples only dated to the receptive phase provided suggestive 382 DEGs (p<0.05; nonsignificant after adjusted p value) in the adenomyosis group, leading to 33 enriched pathways (Bonferroni p value < 0.05). These included pathways were already associated with endometrial biology, such as "Expression of interferon (IFN)-induced genes" and "Response to IFN-alpha". Data integration revealed pathways indicating a unique effect of adenomyosis on endometrial molecular organization (e.g., "Expression of IFN-induced genes") and its interference with endometrial receptivity establishment (e.g., "Extracellular matrix organization" and "Tumour necrosis factor production"). CONCLUSIONS: Accurate endometrial dating and RNA-seq analysis resulted in the identification of altered response to IFN signalling as the most promising candidate of impaired uterine receptivity in adenomyosis.

To collapse or not to collapse blastocysts before vitrification? A matched case-control study on single vitrified-warmed blastocyst transfers.

RESEARCH QUESTION: Does laser-induced artificial blastocoel collapse result in better blastocyst cryopreservation survival and a higher live birth rate (LBR) in comparison with intact counterparts? DESIGN: Half of the supernumerary blastocysts from IVF cycles were randomly selected before vitrification for laser-induced artificial collapsing or vitrification in intact form. A matched case-control study of first transfers of single blastocysts artificially collapsed (case) or intact (control) before vitrification was conducted. Controls were matched to cases on a 1:1 ratio by female age, parity, fresh and vitrified cycle protocol, blastocyst age and quality, resulting in 309 case-control pairs. RESULTS: The two groups were comparable in terms of their characteristics. Survival rates in the case and control groups (97.8% and 95.7%; P = 0.133) were comparable, but the optimal survival rate was higher in the case group (78.2% and 69.3%; P = 0.03). Clinical pregnancy rates (38.2% and 35.3%; P = 0.518), miscarriage rates (15.2% and 22%; P = 0.190), LBR per transfer (32.4% and 27.5%; P = 0.221) and LBR per warmed blastocyst (31.6% and 26.3%; P = 0.137) were not statistically different between the case and control groups. No significant difference in preterm births (11.1% versus 15.7%), birthweights (3333 ± 723 g versus 3304 ± 609 g) or sex ratio (49.3% versus 50.7% boys) was observed between the two groups. No major malformations were detected in the study population. CONCLUSIONS: Compared with vitrification of intact blastocysts, collapsed blastocysts resulted in a significantly higher optimal survival rate, and although they resulted in a 5% higher LBR, this was not significant for the chosen sample size. Neonatal outcomes were comparable in the two groups.

Patient Outcomes Following Extra-Osseous Talo-Tarsal Stabilization for Foot Hyperpronation.

The aim is to present a single-center case series of patients with symptomatic hyperpronated feet treated with arthroereisis by using a second generation extra-osseous talo-tarsal stabilization device. This case series enrolled 123 feet in 87 patients (20 [6-75] years) treated with arthroereisis, either isolated (76 cases) or combined procedure (47 cases). At their final follow-up, a patient reported questionnaire (overall satisfaction, foot stability and shape, activities of daily living, pain level, and analgesics usage) was distributed. The average postoperative follow-up period was 30 (13-55) months. Nineteen (15%) cases required at least one revision surgery: the implant was manipulated in 5 (4%), while 14 cases (11%) required definitive implant removal. The predominant reason for implant removal was pain (50%), followed by implant migration (27%). The pediatric population with isolated procedure showed lowest revision rate (5%), while adults with combined ankle/hindfoot procedures demonstrated revision rate of 50%. The overall patient satisfaction after arthroereisis was 84%. The patients' perceived improvement in foot stability was 75%, foot shape 85%, and activities of daily living 64%. Eighty-two percent of cases reported no analgesics usage in the last month and mean visual analogue scale (0-10) pain level decreased from 5.5 to 2.2 (p < .001). The subgroup analyses of patient-reported questionnaires revealed the best outcome in the pediatric-isolated cases, while adults with combined procedures reported the lowermost outcome. Extra-osseous talo-tarsal stabilization demonstrated a low rate of revisions surgery and a high satisfaction rate as an isolated procedure. Patients with conjoined procedures experienced more revisions and considerably lower satisfaction rates.

Molecular signature of eutopic endometrium in endometriosis based on the multi-omics integrative synthesis.

PURPOSE: To synthesise data from genome-wide studies reporting molecular signature of eutopic endometrium through the phases of the menstrual cycle in endometriosis. METHODS: Extraction of data from publications reporting genetic signatures characterising endometrium associated with endometriosis. The nomenclature of extracted differentially expressed transcripts and proteins was adopted according to the HUGO Gene Nomenclature Committee (HGNC). Loci were further sorted according to the different phases of the menstrual cycle, i.e. menstrual (M), proliferative (P), secretory (S), early-secretory (ES), mid-secretory (MS), late-secretory (LS), and not specified (N/S) if the endometrial dating was not available. Enrichment analysis was performed using the DAVID bioinformatics tool. RESULTS: Altered molecular changes were reported by 21 studies, including 13 performed at the transcriptomic, 6 at proteomic, and 2 at epigenomic level. Extracted data resulted in a catalogue of total 670 genetic causes with available 591 official gene symbols, i.e. M = 3, P = 188, S = 81, ES = 82, MS = 173, LS = 36, and N/S = 28. Enriched pathways included oestrogen signalling pathway, extracellular matrix organization, and endothelial cell chemotaxis. Our study revealed that knowledge of endometrium biology in endometriosis is fragmented due to heterogeneity of published data. However, 15 genes reported as dysregulated by at least two studies within the same phase and 33 significantly enriched GO-BP terms/KEGG pathways associated with different phases of the menstrual cycle were identified. CONCLUSIONS: A multi-omics insight into molecular patterns underlying endometriosis could contribute towards identification of endometrial pathological mechanisms that impact fertility capacities of women with endometriosis.

Could polymorphisms of some hormonal receptor genes, involved in folliculogenesis help in predicting patient response to controlled ovarian stimulation?

PURPOSE: The aim of this study was to investigate whether single nucleotide polymorphisms (SNPs) in selected genes, responsible for hormonal regulation of folliculogenesis, are associated with response to controlled ovarian hyperstimulation (COH) and clinical characteristics of women enrolled in in vitro fertilization (IVF) programs. METHODS: In a cross-sectional study, 60 (IVF) patients underwent COH by using gonadotropin-releasing hormone (GnRH) antagonist and recombinant follicle-stimulating hormone (rFSH) protocol. Patients were classified into three groups: poor-responders (according to Bologna criteria), normo-responders (≤ 15 oocytes), and hyper-responders (> 15 oocytes). Genotyping of SNPs AMH rs10407022, AMHR rs3741664, FSHR rs1394205 and rs6166, and ESR1 rs2234693 was performed using high-resolution melting analysis (HRMA). Basal FSH (bFSH), estradiol (E2), and anti-Müllerian hormone (AMH) were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Patients with GG genotype of FSHR rs1394205 had significantly lower AMH level (P = 0.016) and required higher rFSH dose per oocyte compared to women with AA or AG genotype (P = 0.036). We also found higher frequency of GG genotype of FSHR rs1394205 in poor- (76.5%) than in hyper-responders (37.5%, P = 0.002). Patients with AA genotype of FSHR rs6166 had higher level of measured bFSH compared to those with AG or GG genotypes (P = 0.043). Women with GG genotype of AMHR rs3741664 required higher rFSH dose in comparison with patients carrying genotypes AA or AG (P = 0.028). CONCLUSIONS: The GG genotype at position rs1394205 is associated with poor ovarian response to COH. Patients with this genotype may require higher doses of rFSH for ovulation induction.

Artificial blastocoel collapse of human blastocysts before vitrification and its effect on re-expansion after warming - a prospective observational study using time-lapse microscopy.

Vitrified human blastocysts show varied re-expansion capacity after warming. This prospective observational study compared behaviour of artificially collapsed blastocysts (study group patients, n = 69) to that of blastocysts that were vitrified without artificial collapse (control group patients, n = 72). Warmed blastocysts were monitored by time-lapse microscopy and blastocoel re-expansion speed and growth patterns compared between study and control groups. These parameters were also retrospectively compared between blastocysts that resulted in live birth and those that failed. Artificially collapsed blastocysts re-expanded on average 15.01 µm2/min faster than control blastocysts (P = 0.0013). Warmed blastocysts expressed four different patterns of blastocoel growth. The pattern showing contractions at the end of culture was observed to have a lower prevalence in control blastocysts, which coincided with the lower incidence of hatching in this group. Re-expansion speed and prevalence of growth patterns were comparable between blastocysts that did and did not result in a live birth. This was seen in the study and control groups. Despite faster re-expansion and different growth patterns of artificially collapsed blastocysts, live birth rate did not differ between groups. However, this result should be interpreted with caution due to the small sample size and high risk of bias.

Cumulative live birth rate after GnRH agonist trigger and elective cryopreservation of all embryos in high responders.

Elective embryo cryopreservation after using gonadotrophin-releasing hormone (GnRH) antagonist protocols and GnRH agonist triggering is becoming an increasingly important part of medically assisted reproduction. We designed a single-centre retrospective study to assess the cumulative probability of achieving a live birth through consecutive transfers of vitrified-warmed blastocysts after elective embryo cryopreservation in high-responding patients. Hence, 123 women identified to be at high risk for developing ovarian hyperstimulation syndrome were included. They were stimulated using GnRH antagonist protocol, and GnRH agonist was used to trigger final oocyte maturation. All embryos were vitrified at the blastocyst stage and transferred in the subsequent menstrual cycles. Using the Kaplan-Meier survival analysis, a total of 65.9% (95% CI 57.5 to 74.3) women achieved a live birth after a maximum of six embryo transfer cycles using the 'conservative' approach. Applying the 'optimistic' approach, presuming that women who still had cryopreserved embryos and did not return for embryo transfer had the same chance of achieving a live birth as those returning for transfer, the cumulative live birth rate estimated in six embryo transfer cycles was 76.6% (95% CI 69.1 to 84.1). No cases of severe ovarian hyperstimulation syndrome were recorded.

Endometrial injury, the quality of embryos, and blastocyst transfer are the most important prognostic factors for in vitro fertilization success after previous repeated unsuccessful attempts.

PURPOSE: The purpose of this study was to find out the most important prognostic factors for achieving a pregnancy after in vitro fertilization (IVF) in women with history of repeated unsuccessful IVF attempts. METHODS: We analyzed factors affecting pregnancy rate in a retrospective study including 429 IVF/ICSI cycles performed in women younger than 40 years with at least three previous consecutive failed IVF/ICSI attempts. RESULTS: Clinical pregnancy was observed in 140/429 (32.6%) cycles. Clinical pregnancy rate (CPR) was significantly higher in cycles with LEI compared to cycles without LEI before embryo transfer (44.4 vs 26.54%, p = 0.007). The CPR was also higher in cycles with day 5 blastocyst- compared with day 3 cleavage-stage embryo transfers (45.51 vs 26.54%, p < 0.001). In multivariate logistic regression model, only transfer of at least one good quality embryo (OR = 4.32, 95% CI 2.41-7.73), local endometrial injury (OR = 1.73, 95% CI 1.02-2.92), and transfer on day 5 (OR = 3.02, 95% CI 1.53-5.94) remained important independent prognostic factors for clinical pregnancy. CONCLUSIONS: These results suggest that hysteroscopy with local injury to the endometrium prior to ovarian stimulation for IVF/ICSI can improve implantation and pregnancy rates in women experiencing recurrent IVF failure. However, large studies are needed to confirm these findings.

What is the value of anti-Müllerian hormone in predicting the response to ovarian stimulation with GnRH agonist and antagonist protocols?

BACKGROUND: Anti-Müllerian hormone (AMH) is a marker of the ovarian reserve with promising prognostic potential in reproductive medicine. We aimed to evaluate the prognostic ability of AMH for predicting excessive or poor responses to ovarian stimulation using gonadotrophin-releasing hormone (GnRH) agonist and GnRH antagonist protocols in patients undergoing medically assisted reproduction (MAR) procedures. METHODS: This retrospective analysis included 623 women who underwent ovarian stimulation for medically assisted reproduction. AMH level measurements were acquired from all couples within six months of the initiation of ovarian stimulation. RESULTS: AMH was significantly correlated with the number of retrieved oocytes, and age was not relevant in a multivariate regression analysis (unstandardized regression coefficient of 1.130, 95 % confidence interval 0.977-1.283). AMH was a better predictor of both excessive (>19 oocytes) and poor (<4 oocytes) ovarian response than age (areas under the curve (AUCs) of 0.882 and 0.816, respectively). When stratified according to the stimulation protocol (a long GnRH agonist versus a GnRH antagonist protocol), AMH retained its high predictive value for excessive and poor responses in both groups. Serum AMH levels exhibited a strong correlation with the level of the response to ovarian stimulation. CONCLUSIONS: AMH is an independent and an accurate predictor of excessive and poor responses to GnRH agonist and GnRH antagonist protocols for ovarian stimulation.

Embryos from Prepubertal Hyperglycemic Female Mice Respond Differentially to Oxygen Tension In Vitro.

Reduced oxygen during embryo culture in human ART prevents embryo oxidative stress. Oxidative stress is also the major mechanism by which maternal diabetes impairs embryonic development. This study employed induced hyperglycemia prepubertal mice to mimic childhood diabetes to understand the effects of varying oxygen tension during in vitro embryonic development. The oocytes were fertilized and cultured at low (≈5%) oxygen (LOT) or atmospheric (≈20%) oxygen tension (HOT) for up to 96 h. Embryo development, apoptosis in blastocysts, inner cell mass (ICM) outgrowth proliferation, and Hif1α expression were assessed. Though the oocyte quality and meiotic spindle were not affected, the fertilization rate (94.86 ± 1.18 vs. 85.17 ± 2.81), blastocyst rate (80.92 ± 2.92 vs. 69.32 ± 2.54), and ICM proliferation ability (51.04 ± 9.22 vs. 17.08 ± 3.05) of the hyperglycemic embryos were significantly higher in the LOT compared to the HOT group. On the other hand, blastocysts from the hyperglycemic group, cultured at HOT, had a 1.5-fold increase in apoptotic cells compared to the control and lower Hif1α transcripts in ICM outgrowths compared to the LOT. Increased susceptibility of embryos from hyperglycemic mice to higher oxygen tension warrants the need to individualize the conditions for embryo culture systems in ART clinics, particularly when an endogenous maternal pathology affects the ovarian environment.

Secretomes from Conventional Insemination and Intra-Cytoplasmic Sperm Injection Derived Embryos Differentially Modulate Endometrial Cells In Vitro.

Conventional Insemination (CI) and Intra-Cytoplasmic Sperm Injection (ICSI) are routinely used insemination methods in clinical Assisted Reproductive Technologies (ART) settings. However, the existing data on the developmental competence and implantation potential of CI and ICSI derived embryos are not unequivocal. This prospective study on 23 patients undergoing ART treatment explored whether the secretomes of CI- and ICSI-derived embryo differentially alter the expression of integrins (αv and ß3 integrin) and MUCIN-1 (MUC-1) in a human endometrial epithelial cell line (Ishikawa). Immunocytochemical data demonstrated that the secretome of CI-derived top quality (GI) embryos induced higher (p < 0.05) expression of ɑv ß3 compared to sibling ICSI derived G1 embryos in Ishikawa cells. Though, relative levels of the transcript for MUC-1, anti-adhesion molecule did not show a significant difference between the study groups, immunocytochemical analysis demonstrated significantly (p < 0.0001) higher expression of MUC-1 in cells treated with ICSI-derived embryo secretome, compared to that treated with CI -derived embryo secretome. These results suggest that secretomes from CI and ICSI embryos differentially modulate the endometrial cells in vitro. This hints at differences in the ability of CI- and ICSI- derived embryos to alter endometrial profile.

Comparison of embryo transfer strategies and assisted reproduction outcome in Slovenian and cross-border patients.

A reduction in the number of embryos transferred is the most important step in decreasing multiple gestation rates after medically assisted reproduction. Slovenia has implemented insurance company regulations that regulate single-embryo transfer in selected good-prognosis couples. The aim of the present study was to evaluate its effects on the Slovenian population compared with cross-border patients, who are not affected by the insurance company policy. Ultimately, 2403 couples undergoing IVF or intracytoplasmic sperm injection were included in the retrospective analysis. Patients were classified according to their origin. The decision about the number of embryos transferred and the treatment success were evaluated. The implementation of the policy favouring single-embryo transfer resulted in a significant decrease in the twin birth rate in Slovenian patients (24.4% before policy versus 6.7% after policy implementation, P<0.001). Although in cross-border patients twin birth rates have declined through the study period, they remained significantly higher compared with Slovenian patients (23.1% versus 6.7%, P<0.001). The data demonstrate that insurance company policies favouring single-embryo transfer are an effective tool in decreasing multiple gestation rates. Similar mechanisms should be implemented in the cross-border patient population.

Training and competency assessment of Clinical Embryologists and licensing of the profession in European countries.

STUDY QUESTION: How is the acquisition and testing of theoretical and practical knowledge in Clinical Embryology and the licensing of ART laboratory personnel carried out in European countries? SUMMARY ANSWER: Twelve out of 31 European countries have established some kind of verification of laboratory competency and skills in ART: in 7 countries, this was related to licensing, but where organized education for Clinical Embryologists existed, there were vast differences in the way these processes were undertaken. WHAT IS KNOWN ALREADY: In 2015, a report by the ESHRE Embryology Certification Committee concluded that regardless of the large number of people working in IVF laboratories, Clinical Embryology was only recognized as an official profession in 3 out of 27 European national health systems. In most countries, Clinical Embryologists needed to be officially registered under an alternative profession and there were limited opportunities for organized education in this specialist field. Five years after this report, the ESHRE Working Group on Embryologist Training Analysis conducted a survey to collect detailed information about how Clinical Embryologists from different European countries are acquiring their theoretical knowledge and practical skills in ART, and how their level of education and competence in Clinical Embryology is verified. STUDY DESIGN SIZE DURATION: Two questionnaires about the possibilities for acquiring the education and training needed to work in ART and verification of this knowledge were prepared by the ESHRE Working Group on Embryologist Training Analysis. The first was sent in 2020 to a panel of invited lead European Embryologists who attended an Expert Meeting held in Rome, Italy. In order to have a more comprehensive and updated picture, in 2021 the same survey was also sent to the ESHRE Committee of National Representatives (CNRs). At the end of 2021, the second survey with specific questions, more focused on Clinical Embryologists' training and licencing, was sent to the CNRs who reported on verification of education in Clinical Embryology. PARTICIPANTS/MATERIALS SETTING METHODS: The first survey consisted of 17 questions. It was initially submitted to 14 lead Embryologists and then resubmitted to the 34 ESHRE CNRs. Representatives from 31 countries responded. A second survey with 23 questions was sent to the 12 ESHRE CNRs who reported an established national system of verification of education in Clinical Embryology, with specific questions focused on the training of Clinical Embryologists. All 12 CNRs responded. MAIN RESULTS AND THE ROLE OF CHANCE: Analysis showed that European national education programmes in Clinical Embryology could be split into 4 categories: non-existent (13 countries), recommended (5 countries), simple compulsory (9 countries), and complex compulsory (4 countries). A national document stating the minimum education requirements for staff to work in an IVF laboratory was reported by 19 respondents. The requirement to follow a prescribed theoretical and laboratory training programme in ART was compulsory in 9 and 10 countries, respectively. Some form of verification of laboratory skills, theoretical knowledge in ART, and continuing professional development was required in 12, 10, and 9 countries, respectively. A national trainee's logbook format was reported by seven respondents and a national tutorial system was available in six countries. Only seven countries had official licensing of ART laboratory staff. The title of Clinical Embryologist was not recognized in 13 countries and in 6 countries, it was used only by professional bodies, while in 12 countries the profession was at least cited in governmental regulations. The ESHRE Clinical Embryologist Certificate was officially recognized in eight countries. LIMITATIONS REASONS FOR CAUTION: The survey took place in two steps and the results were then combined to provide a representative picture for most of the European countries sampled. The vast majority, but not all, of the CNRs answered the request to participate in the survey. WIDER IMPLICATIONS OF THE FINDINGS: The professional recognition of Clinical Embryology within Europe is steadily evolving. However, it remains a concern that many countries continue to not recognize Clinical Embryology as a profession, with a vast difference in the reported organization of educational and training programmes and verification of skills. It is recommended that a training programme for Clinical Embryology and ART in Europe should be standardized and relevant issues should be addressed by competent authorities and European Union institutions. ESHRE is best placed to take a leading role in this educational process. STUDY FUNDING/COMPETING INTERESTS: The Working Group members who are the authors of this article did not receive payments for the completion of this study. The authors have no conflicts of interest to declare.

An annotated human blastocyst dataset to benchmark deep learning architectures for in vitro fertilization.

Medical Assisted Reproduction proved its efficacy to treat the vast majority forms of infertility. One of the key procedures in this treatment is the selection and transfer of the embryo with the highest developmental potential. To assess this potential, clinical embryologists routinely work with static images (morphological assessment) or short video sequences (time-lapse annotation). Recently, Artificial Intelligence models were utilized to support the embryo selection procedure. Even though they have proven their great potential in different in vitro fertilization settings, there is still considerable room for improvement. To support the advancement of algorithms in this research field, we built a dataset consisting of static blastocyst images and additional annotations. As such, Gardner criteria annotations, depicting a morphological blastocyst rating scheme, and collected clinical parameters are provided. The presented dataset is intended to be used to train deep learning models on static morphological images to predict Gardner's criteria and clinical outcomes such as live birth. A benchmark of human expert's performance in annotating Gardner criteria is provided.

ESHRE certification of ART centres for good laboratory and clinical practice.

STUDY QUESTION: Three years after the start of the ESHRE ART Centre Certification (ARTCC) programme, what is the current state of the system, in terms of the interest expressed in it and experiences during the assessment of ART services? SUMMARY ANSWER: As of 1 December 2021, 25 European ART centres have been involved in the various stages of certification and the most common recommendations from inspectors were the need for documented training, verification of competencies for all staff members, verification of laboratory and clinical performance indicators, implementation of a quality management system and avoidance of overusing ICSI and add-ons. WHAT IS KNOWN ALREADY: European Union (EU) legislation has included ART activities in the EU Tissue and Cells Directives (EUTCDs). Following inspections by national EUTCD authorities, many details regarding documentation, laboratory environment, handling of reproductive cells and tissues, traceability, coding and patient testing have become standardized. However, the EUTCDs do not cover all ART-specific aspects. For this reason, the ARTCC was established to focus on peculiar areas, including relevant staff qualifications, training, continuing professional development, workload, equipment suitability, (non)-evidence-based laboratory and clinical methods used, treatment approaches according to ESHRE guidelines, recommendations and laboratory and clinical key performance indicators. STUDY DESIGN SIZE DURATION: The article reviews the state-of-the-art of the ESHRE certification of ART centres for good clinical and laboratory practice over an initial 3-year period of operation, including the number of ART centres involved in the different stages of certification and the most common recommendations by inspectors. PARTICIPANTS/MATERIALS SETTING METHODS: In 2016, the ARTCC working group began to establish a new ESHRE ARTCC programme. Since then, the working group has organized 4 preparatory courses and appointed 37 inspectors (19 clinicians, 17 embryologists and one paramedical). A tool to verify compliance with ESHRE recommendations for good laboratory and clinical practice was developed. The ARTCC has been open for applications since September 2018. In Step 1, the applicant enters basic information about the ART centre, staff and ART activities into the application platform. After review and approval, the applicant is given the opportunity to enter Step 2 and provide detailed online checklists on general, laboratory, clinical services and clinical outcomes. Two inspectors (one clinician and one embryologist) independently evaluate the submitted checklists. The condition to proceed to evaluation is a positive mean score (at least 66%) from each of the four checklists. In Step 3, a live site visit (or virtual owing to the coronavirus disease 2019 (COVID-19) pandemic) is organized and the inspectors prepare a final report with appropriate recommendations. The application may be rejected at any time if the criteria required to advance to the next stage are not met. The ARTCC programme is currently available for European countries listed in ESHRE internal rules, available on the ESHRE website. The certificate is valid for 3 years, after which an application for renewal can be submitted. MAIN RESULTS AND THE ROLE OF CHANCE: Over a 3-year period (until 1 December 2021), 63 ART centres from 25 countries started applying through an online platform. So far, 38 applications did not progress owing to lack of completion of the initial application within a 1-year period or because applications came from non-European countries. Of the remaining 25 applications, 8 centres have been inspected and 7 centres have been certified. The most common recommendations given by inspectors to assessed centres were the need for documented training, verification of competencies, skills and continuing professional development for all staff members, verification of laboratory and clinical performance indicators and implementation of a quality management system. The inspectors identified some recurring areas of medically assisted reproduction that deviate from good practice: the overuse of ICSI, preimplantation genetic testing for aneuploidies, freeze-all and other add-ons. They often reported that the clinical outcomes could not be objectively assessed because of non-inclusion of the started cycles or the frequent use of freeze-all cycles. LIMITATIONS REASONS FOR CAUTION: No major modifications have been made to the application platform and checklists since the early stages of the certification programme. However, in this short time, quite a few changes in clinical practice have occurred, especially concerning the more frequent use of the 'freeze-all' strategy. As a result, problems arose in the evaluation of clinical outcomes. In addition, because of the COVID-19 pandemic, site visits were substituted by the implementation of virtual visits. While this enabled the certification programme to continue, it is possible that certain critical details that would have been noticed during a traditional site visit may have been overlooked. WIDER IMPLICATIONS OF THE FINDINGS: Regular monitoring of the observations of ARTCC inspectors and analysis of their reports is certainly useful to harmonize inspectors' criteria in the assessment process and to identify chronic deficiencies in clinical and laboratory practice. Non-conformities can be addressed by ESHRE through guidelines and recommendations, as well as through discussion with EU institutions and competent authorities. STUDY FUNDING/COMPETING INTERESTS: The ARTCC programme was developed and funded by ESHRE, covering expenses associated with the meetings. The Steering Committee members who are the authors of this article did not receive payments for the completion of this study. The inspectors were remunerated for their work with an honorarium. The authors have no conflicts of interest to declare.

Experimentally Induced Hyperglycemia in Prepubertal Phase Impairs Oocyte Quality and Functionality in Adult Mice.

Reproductive abnormalities in women with a history of childhood diabetes are believed to be partially attributed to hyperglycemia. Prolonged hyperglycemia can negatively affect ovarian function and fertility during reproductive life. To address this in an experimental setting, the present study used streptozotocin-induced hyperglycemic prepubertal mouse model. The impact of prolonged hyperglycemic exposure during prepubertal life on ovarian function, oocyte quality, and functional competence was assessed in adult mice. The ovarian reserve was not significantly altered; however, the in vitro maturation potential (P < 0.001), mitochondrial integrity (P < 0.01), and meiotic spindle assembly (P < 0.05-0.001) in oocytes were significantly affected in hyperglycemic animals in comparison to control groups. The results from the study suggest that prepubertal hyperglycemia can have adverse effects on the oocyte functional competence and spindle integrity during the reproductive phase of life. Because these changes can have a significant impact on the genetic integrity and developmental potential of the embryos and fetus, the observation warrants further research both in experimental and clinical settings.

ICSI in non-male factor infertility patients does not alter metabolomic signature in sibling embryos as evidenced by sensitivity enhanced nuclear magnetic resonance (NMR) spectroscopy.

Intracytoplasmic sperm injection (ICSI) was developed to overcome male factor infertility, however, there recently has been an increasing trend in ICSI usage irrespective of the etiology, demonstrating an overuse of this insemination technique. There is a limited knowledge on the behaviour of ICSI derived embryos in non-male factor infertility patients. Metabolomic assessment of preimplantation embryos in conjunction with morphological evaluation can provide better understanding of embryonic behaviour. Hence, this study was undertaken to explore if there are any metabolomic differences between IVF and ICSI derived sibling day-5 blastocysts from non-male factor infertility patients. This prospective study included nineteen couples with non-male factor infertility undergoing Assisted Reproductive Technology. The sibling oocytes retrieved from each patient were randomly assigned to two groups and inseminated either by IVF or ICSI. Spent culture media (SCM) in which embryos were cultured up to day 5 were collected and investigated using sensitivity enhanced NMR based metabolite profiling utilizing high resolution (800 MHz) NMR equipped with cryogenically cooled micro-coil (1.7 mm) probe. The metabolomic signature between IVF and ICSI derived sibling blastocysts was assessed. A significant reduction in the concentrations of pyruvate, citrate, glucose and lysine were observed in both IVF and ICSI sibling embryos compared to medium control (P< 0.05-0.001). Further, histidine and valine level was found lower in ICSI embryos compared to medium control (P<0.05) during 96 hours of in vitro culture. Notably, between IVF and ICSI SCM, no significant difference in the concentration of the metabolites was found. Our results suggest that ICSI in non-male factor does not alter the SCM metabolomic signature during 96 hours of embryonic development.