ESHRE guideline: number of embryos to transfer during IVF/ICSI†.

STUDY QUESTION: Which clinical and embryological factors should be considered to apply double embryo transfer (DET) instead of elective single embryo transfer (eSET)? SUMMARY ANSWER: No clinical or embryological factor per se justifies a recommendation of DET instead of eSET in IVF/ICSI. WHAT IS KNOWN ALREADY: DET is correlated with a higher rate of multiple pregnancy, leading to a subsequent increase in complications for both mother and babies. These complications include preterm birth, low birthweight, and other perinatal adverse outcomes. To mitigate the risks associated with multiple pregnancy, eSET is recommended by international and national professional organizations as the preferred approach in ART. STUDY DESIGN, SIZE, DURATION: The guideline was developed according to the structured methodology for development and update of ESHRE guidelines. Literature searches were performed in PUBMED/MEDLINE and Cochrane databases, and relevant papers published up to May 2023, written in English, were included. Live birth rate, cumulative live birth rate, and multiple pregnancy rate were considered as critical outcomes. PARTICIPANTS/MATERIALS, SETTING, METHODS: Based on the collected evidence, recommendations were discussed until a consensus was reached within the Guideline Development Group (GDG). A stakeholder review was organized after the guideline draft was finalized. The final version was approved by the GDG and the ESHRE Executive Committee. MAIN RESULTS AND THE ROLE OF CHANCE: The guideline provides 35 recommendations on the medical and non-medical risks associated with multiple pregnancies and on the clinical and embryological factors to be considered when deciding on the number of embryos to transfer. These recommendations include 25 evidence-based recommendations, of which 24 were formulated as strong recommendations and one as conditional, and 10 good practice points. Of the evidence-based recommendations, seven (28%) were supported by moderate-quality evidence. The remaining recommendations were supported by low (three recommendations; 12%), or very low-quality evidence (15 recommendations; 60%). Owing to the lack of evidence-based research, the guideline also clearly mentions recommendations for future studies. LIMITATIONS, REASONS FOR CAUTION: The guideline assessed different factors one by one based on existing evidence. However, in real life, clinicians' decisions are based on several prognostic factors related to each patient's case. Furthermore, the evidence from randomized controlled trials is too scarce to formulate high-quality evidence-based recommendations. WIDER IMPLICATIONS OF THE FINDINGS: The guideline provides health professionals with clear advice on best practice in the decision-making process during IVF/ICSI, based on the best evidence currently available, and recommendations on relevant information that should be communicated to patients. In addition, a list of research recommendations is provided to stimulate further studies in the field. STUDY FUNDING/COMPETING INTEREST(S): The guideline was developed and funded by ESHRE, covering expenses associated with the guideline meetings, the literature searches, and the dissemination of the guideline. The guideline group members did not receive payment. DPB declared receiving honoraria for lectures from Merck, Ferring, and Gedeon Richter. She is a member of ESHRE EXCO, and the Mediterranean Society for reproductive medicine and the president of the Croatian Society for Gynaecological Endocrinology and Reproductive Medicine. CDG is the past Chair of the ESHRE EIM Consortium and a paid deputy member of the Editorial board of Human Reproduction. IR declared receiving reimbursement from ESHRE and EDCD for attending meetings. She holds an unpaid leadership role in OBBCSSR, ECDC Sohonet, and AER. KAR-W declared receiving grants for clinical researchers and funding provision to the institution from the Swedish Cancer Society (200170F), the Senior Clinical Investigator Award, Radiumhemmets Forskningsfonder (Dnr: 201313), Stockholm County Council FoU (FoUI-953912) and Karolinska Institutet (Dnr 2020-01963), NovoNordisk, Merck and Ferring Pharmaceuticals. She received consulting fees from the Swedish Ministry of Health and Welfare. She received honoraria from Roche, Pfizer, and Organon for chairmanship and lectures. She received support from Organon for attending meetings. She participated in advisory boards for Merck, Nordic countries, and Ferring. She declared receiving time-lapse equipment and grants with payment to institution for pre-clinical research from Merck pharmaceuticals and from Ferring. SS-R received research funding from Roche Diagnostics, Organon/MSD, Theramex, and Gedeo-Richter. He received consulting fees from Organon/MSD, Ferring Pharmaceuticals, and Merck Serono. He declared receiving honoraria for lectures from Ferring Pharmaceuticals, Besins, Organon/MSD, Theramex, and Gedeon Richter. He received support for attending Gedeon Richter meetings and participated in the Data Safety Monitoring Board of the T-TRANSPORT trial. He is the Deputy of ESHRE SQART special interest group. He holds stock options in IVI Lisboa and received equipment and other services from Roche Diagnostics and Ferring Pharmaceuticals. KT declared receiving payment for honoraria for giving lectures from Merck Serono and Organon. She is member of the safety advisory board of EDQM. She holds a leadership role in the ICCBBA board of directors. ZV received reimbursement from ESHRE for attending meetings. She also received research grants from ESHRE and Juhani Aaltonen Foundation. She is the coordinator of EHSRE SQART special interest group. The other authors have no conflicts of interest to declare. DISCLAIMER: This guideline represents the views of ESHRE, which were achieved after careful consideration of the scientific evidence available at the time of preparation. In the absence of scientific evidence on certain aspects, a consensus between the relevant ESHRE stakeholders has been obtained. Adherence to these clinical practice guidelines does not guarantee a successful or specific outcome, nor does it establish a standard of care. Clinical practice guidelines do not replace the need for application of clinical judgement to each individual presentation, nor variations based on locality and facility type. ESHRE makes no warranty, express or implied, regarding the clinical practice guidelines and specifically excludes any warranties of merchantability and fitness for a particular use or purpose (full disclaimer available at https://www.eshre.eu/Guidelines-and-Legal).

Sperm Selection Using Zona Pellucida-Binding Enhanced Embryo Morphokinetic and Clinical Outcomes in ICSI: A Sibling Oocytes Study.

The objective was to investigate the embryo morphokinitics using a time-lapse monitoring (TLM) system and assessment of clinical outcomes following intracytoplasmic sperm injection (ICSI) with zona pellucida (ZP)-bound sperm selection and conventional methods. A total of 371 metaphase II (MII) oocytes from 50 ICSI cycles were studied. Sibling oocytes were randomly divided into control (n = 199) and ZP-bound group (n = 172). All resulting zygotes were cultured and monitored in the TLM system up to Day 3 after ICSI. Fertilization rate, early embryo development, and clinical outcomes were evaluated. No significant differences were found in fertilization rate, time-lapse qualitative and quantitative measures, pronuclear fading time (PNF) t2, t3, t4, t5, t6, and t7 (times of cleavage to 2, 3, 4, 5, 6, and 7 cells), respectively. However, the t8 (time of cleavage to eight cells) and cc3 (duration of third cell cycle) revealed a significant difference between control and ZP-bound groups (p < .05). A significant difference between the two groups (p < .05) in the rates of Grade A embryos (according to Basile algorithm), chemical pregnancy, clinical pregnancy, and implantation was observed. Sperm selection using biological materials, such as ZP, improved both embryo quality and pregnancy outcomes, despite not affecting the early embryo development and morphokinetic parameters up to t8. This prospective randomized sibling oocyte trial was registered in October 2020 to January 2022 (IRCT20200705048021N1).

The effect of an educational animation on knowledge of testicular health and fertility of adolescents.

STUDY QUESTION: Does the provision of an educational animation, developed with young people, about testicular health and fertility impact the knowledge of these topics among adolescents? SUMMARY ANSWER: The development and provision of education on testicular health and fertility were welcomed by adolescents and associated with a significant increase in knowledge. WHAT IS KNOWN ALREADY: Young people may know less than they should about testicular health and male fertility topics. Lack of knowledge can have implications for health including late medical help-seeking for signs and symptoms of scrotal disorders, such as torsion, for which late presentation frequently results in testicular damage. STUDY DESIGN, SIZE, DURATION: A mixed methods experimental pre- and post-design was used with embedded qualitative data collection. High school students completed a pre-animation questionnaire, watched four animations on testicular health and fertility, and completed a post-animation questionnaire. Data were collected during Personal Social and Health Education lessons across a 2-week period. PARTICIPANTS/MATERIALS, SETTING, METHODS: Four animations on testicular health and fertility, informed by andrologists, academics, designers, boys, and young men, were developed. Eligible participants were boys and girls in the UK school years 8 and 9 (age 13-14 years). Participants completed a Time 1 (T1) survey (fertility knowledge, demographics) prior to watching the animations and a Time 2 (T2) survey (fertility knowledge, perceptions of the animations) immediately after the animations. Perceptions were rated on 10-point response scales (higher scores better). Participants additionally expressed in their own words positive and negative aspects of the animations. ANOVA was used to examine the effects of the animations using a 2 (time: T1, T2)×2 (gender: male, female) design on topic knowledge, perceived importance, usefulness, and style of the animations according to gender. Regression analysis examined the associations between gender, disability, class year, and knowledge at T2 while controlling for knowledge at T1. Qualitative data on perceptions of the animations were analyzed using inductive thematic analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Results showed that the animations significantly increased testicular health and fertility-related knowledge from T1 (x̄=41.84 ± 24.72) to T2 (x̄=79.15, ±15.04). Boys had significantly higher levels of knowledge compared to girls at T1 (x̄=44.74, SD = 25.16 versus x̄=37.79 ± 23.49, respectively) and T2 (x̄=80.07, SD = 15.68 versus x̄=77.89 ± 14.30, respectively) but knowledge gain from T1 to T2 was not significantly different according to gender (P = 0.11) as shown by non-significant gender×time interaction. There were no significant gender differences in the perceived usefulness and importance of the animations or liking of the style of the animations, with both genders considering the animations as useful, important, and likable. Regression analysis showed only knowledge at T1 to be significantly associated with knowledge at T2. Qualitative data showed three main themes: accessibility of important and useful information; information engagement and help-seeking behaviour; and inclusivity of information. LIMITATIONS, REASONS FOR CAUTION: This was a pre- and post-study with a sample of young people from a selected educational institution without a control group. Only short-term effects of the animations were recorded. WIDER IMPLICATIONS OF THE FINDINGS: Adolescents are interested in and learn from the provision of engaging fertility-related information. Boys and men should be considered as being a relevant target population for fertility education, not just girls and women. STUDY FUNDING/COMPETING INTEREST(S): This research was carried out in partnership with the British Fertility Society, was financially supported by an Economic and Social Research Council Impact Acceleration Award (520792) and commercial sponsorship from iMediCare Ltd, Bayer AG, Merck Group, Cryos International given to the British Fertility Society, and a financial contribution from Orchid Cancer Appeal. The authors are fully responsible for the content of the animations and this manuscript, and the views and opinions described in the publication reflect solely those of the authors. J.B. reports a grant from Merck Serono Ltd outside the submitted work. C.H., G.G., A.D., E.B., U.G., M.L, B.W., and M.H. declare no conflict of interest. K.M. reports honoraria from Bayer and Merck. A.P. reports paid consultancy for Cryos International, Cytoswim Ltd, Exceed Health, and Merck Serono in the last 2 years, but all monies have been paid to the University of Sheffield. TRIAL REGISTRATION NUMBER: N/A.

Dimensions of beliefs without strong supporting evidence and reasons for holding them.

Beliefs without strong supporting evidence (BWSSE) are commonplace, such as religious beliefs and conspiracy theories. The goals of the current study were to identify dimensions of BWSSE in the general public and study how reasons for holding each dimension depend on the strength of the belief. Participants completed a BWSSE questionnaire online, and principal component analysis suggested that the questionnaire captured 6 dimensions of beliefs that range in strength: New Age Spiritual, Traditional Spiritual, Nonconformist, Science, Mythical, and Conspiracy Theory. Mixed-model analyses of variance showed that while high-strength believers in both New Age and Traditional Spiritual shifted their reasons-for-belief away from 'just believe' and towards personal experience, only Traditional Spiritual shifted away from 'just believe' to culture. In contrast, for Conspiracy Theory and Mythical, the dominant reason for belief was media, but for Conspiracy Theory only, there was a shift from media to education/personal research for high-strength believers. This demonstrates that although spiritual beliefs are strengthened by personal experience, conspiracy theory beliefs are strengthened by information gathering. Understanding the source of an existing belief is important for debiasing attempts to move people towards beliefs with strong supporting evidence, including greater acceptance of evidence provided by experts, a likely requirement for negotiating global humanitarian emergencies in the not-so-distant future. Statement of Relevance. Beliefs without strong supporting evidence (BWSSE) have been gaining attention in mainstream society; particularly, the sources of information that may contribute to their formation and resistance to correction. Understanding the source of an existing belief is important for debiasing attempts to move people towards beliefs with strong supporting evidence, including greater acceptance of evidence provided by experts, a likely requirement for negotiating global humanitarian emergencies in the not-so-distant future.

Ethical issues surrounding the use of assisted reproductive technologies in Ghana: An analysis of the experiences of clients and service providers.

Since the advent of Assisted Reproductive Technologies (ART) in Ghana about three decades ago, IVF and ICSI treatments have enabled infertile Ghanaian couples to fulfill their aspirations and dreams of having children. In this extremely pronatalist society, ART has provided relief to many childless couples by reducing, if not eliminating, the shame of childlessness that they would otherwise have to suffer. However, as the provision and utilization of ART continue to increase, so do worries regarding the ethical difficulties surrounding this field of medicine, which challenge cultural ideals and personal desires. The study explores client and service provider experiences with ART in urban Ghana. Observation and in-depth interviews were employed to collect data, and the ethical dimensions of people's experiences relative to Ghanaian cultural and ethical values were analyzed. The results show that the provision of ART services for heterosexual couples in marital unions, the availability of PGT for sickle cell clients, the preference for multiple births emerging from embryo transfers, the lower preference for cryopreservation, the high cost of ART treatment, and the need for regulation of the provision of ART services in Ghana were some of the ethical concerns expressed by both clients and service providers.

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.

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.

Free centrifuge sorting method for high-count sperm preparation improves biological characteristics of human spermatozoa and clinical outcome: A sibling oocytes study.

Sperm processing for assisted reproductive technologies (ART) aims to separate immotile and debris from the motile spermatozoa in the semen. The purpose of this study was to assess the effect of free centrifuge sorting (FCS) approach based on a combination of rheotaxis and swim-up on sperm biological characteristics and ICSI clinical outcomes. Each semen sample was splitted into two equal parts for 67 ICSI cycles with donation oocytes. Parts were processed with the Direct Swim Up (DSU) (control) and with the FCS method (experimental). Sperm quality was assessed in terms of motility, fine morphology, mitochondrial membrane potential, lipid peroxidation and sperm DNA fragmentation. Also Following ICSI, the clinical outcomes were compared between the groups. Sperm progressive motility (93.5 ± 4.1% vs. 78.6 ± 8.2%; p < 0.001), the fraction of Class I (good) morphology (30.2 ± 9.4% vs. 23.7 ± 8.5%; p < 0.0001) and the rate of mitochondrial membrane potential (77.4 ± 7.2% vs. 66.9 ± 5.7%; p < 0.0001) were significantly higher in the FCS compared to DSU groups. The level of lipid peroxidation (0.5 ± 0.05% vs. 0.6 ± 0.06%; p < 0.0001) and concentration of DNA fragmentation (DF) (7.4 ± 1.6% vs. 15.4 ± 2.6%; p < 0.0001) were lower in sperm from the FCS group compared to DSU group. There were higher rates of high-quality embryo formation (p < 0.001), implantation and clinical pregnancy rates (p = 0.03) in the FCS group compared to the control group. The processing of seminal samples using FCS collected spermatozoa with better biological quality and resulted in higher reproductive outcomes in ICSI cycles.

Morphokinetic evaluation of embryos generated from vitrified oocytes maintaining the meiotic spindle.

Vitrification is a technique for preservation of human oocytes. There is still a lack of basic research about the possible effects of vitrification on subsequent embryos following oocyte vitrification. The purpose of this study was to evaluate the embryo morphokinetic parameters formed after fertilization of vitrified-warmed oocytes, where an intact meiotic spindle (MS) was observed pre- and post-cryopreservation. Matured oocytes after in vitro maturation were collected and MS evaluation was performed. The oocytes with MS were divided into two groups: fresh and post vitrification. After intra-cytoplasmic sperm injection, the oocytes were cultured in time lapse monitoring (TLM) and time of second polar body extrusion (SPBE), pronuclei appearance (tPNA), pronuclei fading (tPNF), formation of two to eight cells (t2 to t8), and irregular cleavage events [direct cleavage (DC), reverse cleavage (RC)] and vacuolation were assessed. The fertilization rate was not significantly different between the groups, although the rate of abnormal fertilization was higher in vitrification group compared with fresh group (23.5% VS 7.7%). Analysis of the TLM showed a significant delay in time points, including SPBE, tPNA, tPNF, t 2-cells cleavage in vitrification group (p = 0.02, p = 0.00, p = 0.002, P = 0.00, P = 0.01, respectively). In addition, t3 and t4 time points tended to be delayed in vitrification group (p = 0.05). Moreover, the higher level of DC, RC and vacuolation were noticed in the vitrification group (P˂0.05). In conclusion, despite MS maintenance after warming, TLM evaluation showed both a delay and abnormal cleavage patterns in generated embryos.

IUI needs fairer appraisal to improve patient and stakeholder choices.

Information supporting IVF at the expense of intrauterine insemination (IUI) has become commonplace, but it lacks critical analyses. Data from poorly practiced IUI, without an equivalent comparison to IVF, has been generalised to recommend a total abandonment of IUI in favour of IVF treatment. Our intention with this paper is to reappraise and balance arguments so that patients and stakeholders can have an unbiased informed choice. We provide information that reveals IUI to predominate over IVF in terms of integrated success, risks and cost to deliver one live birth whilst obviating the maternal and neonatal costs. Exceptional cost savings are demonstrated for IUI over IVF for fee-paying agencies and patients with lowered risks of maternal and neonatal care along with other risks including OHSS, fetal reduction and termination of pregnancies. This analysis supports the view that patients and stakeholders can choose IUI instead of IVF in most instances, except with bilateral tubal blockage and severe male factor infertility. It is apparent that fertility clinics need to re-evaluate and reconsider this field, and IUI can be of benefit to both subfertile patients and the stakeholders.

ESHRE guideline: medically assisted reproduction in patients with a viral infection/disease.

STUDY QUESTION: What is the recommended management for medically assisted reproduction (MAR) in patients with a viral infection or disease, based on the best available evidence in the literature? SUMMARY ANSWER: The ESHRE guideline on MAR in patients with a viral infection/disease makes 78 recommendations on prevention of horizontal and vertical transmission before, during and after MAR, and the impact on its outcomes, and these also include recommendations regarding laboratory safety on the processing and storage of gametes and embryos testing positive for viral infections. WHAT IS KNOWN ALREADY: The development of new and improved anti-viral medications has resulted in improved life expectancy and quality of life for patients with viral infections/diseases. Patients of reproductive age are increasingly exploring their options for family creation. STUDY DESIGN SIZE DURATION: The guideline was developed according to the structured methodology for the development of ESHRE guidelines. After the formulation of nine key questions for six viruses (hepatitis B virus, hepatitis C virus, human immunodeficiency virus, human papilloma virus, human T-lymphotropic virus I/II and Zika virus) by a group of experts, literature searches and assessments were performed. Papers published up to 2 November 2020 and written in English were included in the review. Evidence was analyzed by female, male or couple testing positive for the virus. PARTICIPANTS/MATERIALS SETTING METHODS: Based on the collected evidence, recommendations were formulated and discussed until consensus was reached within the guideline group. There were 61 key questions to be answered by the guideline development group (GDG), of which 12 were answered as narrative questions and 49 as PICO (Patient, Intervention, Comparison, Outcome) questions. A stakeholder review was organized after the finalization of the draft. The final version was approved by the GDG and the ESHRE Executive Committee. MAIN RESULTS AND THE ROLE OF CHANCE: This guideline aims to help providers meet a growing demand for guidance on the management of patients with a viral infection/disease presenting in the fertility clinic.The guideline makes 78 recommendations on prevention of viral transmission before and during MAR, and interventions to reduce/avoid vertical transmission to the newborn. Preferred MAR treatments and interventions are described together with the effect of viral infections on outcomes. The GDG formulated 44 evidence-based recommendations-of which 37 were formulated as strong recommendations and 7 as weak-33 good practice points (GPP) and one research only recommendation. Of the evidence-based recommendations, none were supported by high-quality evidence, two by moderate-quality evidence, 15 by low-quality evidence and 27 by very low-quality evidence. To support future research in the field of MAR in patients with a viral infection/disease, a list of research recommendations is provided. LIMITATIONS REASONS FOR CAUTION: Most interventions included are not well-studied in patients with a viral infection/disease. For a large proportion of interventions, evidence was very limited and of very low quality. More evidence is required for these interventions, especially in the field of human papilloma virus (HPV). Such future studies may require the current recommendations to be revised. WIDER IMPLICATIONS OF THE FINDINGS: The guideline provides clinicians with clear advice on best practice in MAR for patients with a viral infection/disease, based on the best evidence currently available. In addition, a list of research recommendations is provided to stimulate further studies in the field. STUDY FUNDING/COMPETING INTERESTS: The guideline was developed and funded by ESHRE, covering expenses associated with the guideline meetings, with the literature searches and with the dissemination of the guideline. The guideline group members did not receive any financial incentives, all work was provided voluntarily. A.D. reports research fees from Ferring and Merck, consulting fees from Ferring, outside the submitted work. C.P. reports speakers fees from Merck and MSD outside the submitted work. K.T. reports speakers fees from Cooper Surgical and Ferring and consultancy fees as member of the advisory board BioTeam of Ferring, outside the submitted work. The other authors have no conflicts of interest to declare. DISCLAIMER: This guideline represents the views of ESHRE, which were achieved after careful consideration of the scientific evidence available at the time of preparation. In the absence of scientific evidence on certain aspects, a consensus between the relevant ESHRE stakeholders has been obtained. Adherence to these clinical practice guidelines does not guarantee a successful or specific outcome, nor does it establish a standard of care. Clinical practice guidelines do not replace the need for application of clinical judgment to each individual presentation, nor variations based on locality and facility type. ESHRE makes no warranty, express or implied, regarding the clinical practice guidelines and specifically excludes any warranties of merchantability and fitness for a particular use or purpose. (Full disclaimer available at  www.eshre.eu/guidelines.).

Does in vitro application of pentoxifylline have beneficial effects in assisted male reproduction?

Application of nonspecific phosphodiesterases inhibitors, such as pentoxifylline (PTX), is a strategy utilised to aid sperm selection from immotile sperm samples prior to ICSI. No extensive studies have yet been performed to verify the safety of the clinical outcomes of ICSI after PTX administration. In this article, we summarise the data reported in the literature that assess the implication of in vitro usage of PTX on sperm parameters, as well as clinical outcomes during assisted male reproduction programme.

ESHRE Clinical Embryologist certification: the first 10 years.

STUDY QUESTION: What has the ESHRE programme 'ESHRE Certification for Clinical Embryologists' achieved after 10 years? SUMMARY ANSWER: The post-exam analysis showed a pass rate of 60% for Clinical and 50% for Senior Clinical Embryologists and a high level of internal consistency of all exams, leading to a total of 773 certified Clinical and 493 Senior Clinical Embryologists over the decade. WHAT IS KNOWN ALREADY: In an ESHRE survey on the educational and professional status of Clinical Embryology in Europe, it was found that education of laboratory personnel working in the field of assisted reproduction is highly variable between countries. In 2008, ESHRE introduced a programme, curriculum and certification in the field of Clinical Embryology. Knowledge gained by postgraduate study of recommended literature, following a clear curriculum, is verified by a written two-level exam for obtaining a certificate for Clinical (basic) or Senior Clinical (advanced) Embryologists. With a total of 1266 certificates awarded over a period of 10 years and recognition by the Union Européenne des Médecins Spécialistes and their Council for European Specialists Medical Assessment, the ESHRE Clinical Embryology exams have become an internationally recognized educational standard in the field of Clinical Embryology. STUDY DESIGN SIZE DURATION: A retrospective analysis of all applications for ESHRE Clinical (2009-2018) and Senior Clinical Embryologist Certification (2008-2018) and exam results of the first decade was carried out by the Steering Committee for Clinical Embryologist Certification. PARTICIPANTS/MATERIALS SETTING METHODS: A total of 2894 applications for ESHRE Certification for Clinical Embryologists and the results of 10 exams for the Clinical (1478 candidates) and 11 exams for Senior Clinical (987 candidates) levels were analysed. A detailed post-exam retrospective analysis was performed regarding difficulty, discrimination and reliability levels of 1600 multiple-choice questions (MCQs) with a single best answer among four options, from eight different curriculum topics (Basic cell biology, Genetics, Developmental biology, Female reproduction, Male reproduction, IVF laboratory, Cryopreservation and Laboratory management), representing the core theoretical knowledge of Clinical Embryology. Difficulty levels of the MCQs were subsequently compared regarding each topic and each yearly exam. The participation and success rates in the ESHRE Clinical Embryology exams were also assessed in terms of the educational and geographic backgrounds of candidates. MAIN RESULTS AND THE ROLE OF CHANCE: Over the 10 years studied, the mean pass rate for the Clinical Embryologist exam was 60% (range 41-86%), and for the Senior Clinical Embryologist exam was 50% (range 34-81%). On average, 63% European candidates and 35% non-European candidates passed the Clinical Embryologist exam, while 52% European candidates and 31% non-European candidates passed the Senior Clinical Embryologist exam. The candidates' educational level impacted on the success of the Clinical Embryologist exam but not of the Senior Clinical Embryologist exam. The mean difficulty indices by study topic showed that in the period of 10 years, there were no statistically significant differences between topics, for either the Clinical or Senior Clinical Embryologist exams. However, the overall exam difficulty varied between years. Reassuringly, the exam MCQ discrimination and reliability indices always showed a high level of internal consistency in all exams. LIMITATIONS REASONS FOR CAUTION: Some data from the initial ESHRE certification programme were not obtained electronically, in particular data for education, implying tables and figures reflect the specified valid data periods. Several countries exhibit different study profiles for those working in ART laboratories, such that laboratory technicians/technologists predominate in some countries, while in others only biologists and medical doctors are allowed to work with human embryos. Such differences could consequently affect the exam performance of candidates from specific countries. WIDER IMPLICATIONS OF THE FINDINGS: The ESHRE exams on Clinical Embryology are the most widely, internationally accepted tests of knowledge in the rapidly growing area of human reproduction. Clinical Embryology is increasingly recognized as a specific discipline for scientific staff who are collaborating closely with clinicians in managing human infertility through medically assisted reproduction. The analysis of the first 10 years of application of a two-level exam for Clinical Embryology shows a consistent high quality and reliability of the exam and MCQs used. These results represent an important follow-up of the quality of the ESHRE Certification programme for Clinical Embryologists, and convincingly position Clinical Embryology in the wider group of health disciplines that are harmonized through professional bodies such as ESHRE and European Board & College of Obstetrics and Gynaecology. The exams provide a clear step towards the increasing professional recognition and establishment of Clinical Embryology within health systems at both European and international level. STUDY FUNDING/COMPETING INTERESTS: No competing interest. All costs of the Steering Committee meetings were covered by ESHRE.

Association between early embryo morphokinetics plus transcript levels of sperm apoptotic genes and clinical outcomes in IMSI and ICSI cycles of male factor patients.

PURPOSE: The aim was to assess the correlation of sperm apoptotic transcript levels with cleavage stage embryokinetic and pregnancy outcomes of intracytoplasmic morphologically selected sperm injection (IMSI) and ICSI methods in patients with male factor infertility. MATERIAL AND METHODS: Eighty male factor cases were divided into ICSI and IMSI groups. ICSI was done routinely, and for IMSI, sperm was selected at high magnification and injected. On day 3, time-lapse parameters were evaluated, and the best embryos were transferred and followed to delivery. In addition, sperm DNA fragmentation and apoptotic transcript levels were quantified using reverse transcription Q-PCR between the groups. RESULTS: IMSI selected spermatozoa had lower DNA fragmentation and apoptotic transcript levels compared with ICSI (p < 0.0001). Moreover, all cytokinetic variables and cleavage abnormalities were noticeably different between groups (p < 0.0001); the rates of clinical outcomes were higher in the IMSI group. The transcript levels of Caspase 3 showed a moderate negative correlation with s2 and s3 (rs = - 0.57, P = 0.008 and rs = - 0.51, p = 0.021, respectively) in the IMSI group. However, there was no relationship between sperm apoptotic transcript levels and clinical outcomes in two groups. CONCLUSIONS: Sperms selected at high magnification showed lower DNA fragmentation and apoptosis genes transcript. Also, better embryo kinetics and clinical outcomes were confirmed in IMSI than ICSI groups. Some time-lapse parameters may be associated with transcript levels of apoptosis genes. Therefore, these noninvasive techniques may be unique in assisting couples with male factor infertility. TRIAL REGISTRATION: This trial retrospectively registered on 4 July 2020 (IRCT20180130038561N1).

GM-CSF (granulocyte macrophage colony-stimulating factor) supplementation in culture media for women undergoing assisted reproduction.

BACKGROUND: GM-CSF (granulocyte macrophage colony-stimulating factor) is a growth factor that is used to supplement culture media in an effort to improve clinical outcomes for those undergoing assisted reproduction. It is worth noting that the use of GM-CSF-supplemented culture media often adds a further cost to the price of an in vitro fertilisation (IVF) cycle. The purpose of this review was to assess the available evidence from randomised controlled trials (RCTs) on the effectiveness and safety of GM-CSF-supplemented culture media. OBJECTIVES: To assess the effectiveness and safety of GM-CSF-supplemented human embryo culture media versus culture media not supplemented with GM-CSF, in women or couples undergoing assisted reproduction. SEARCH METHODS: We used standard methodology recommended by Cochrane. We searched the Cochrane Gynaecology and Fertility Group Trials Register, CENTRAL, MEDLINE, Embase, CINAHL, LILACS, DARE, OpenGrey, PubMed, Google Scholar, and two trials registers on 15 October 2019, checked references of relevant papers and communicated with experts in the field. SELECTION CRITERIA: We included RCTs comparing GM-CSF (including G-CSF (granulocyte colony-stimulating factor))-supplemented embryo culture media versus any other non-GM-CSF-supplemented embryo culture media (control) in women undergoing assisted reproduction. DATA COLLECTION AND ANALYSIS: We used standard methodological procedures recommended by Cochrane. The primary review outcomes were live birth and miscarriage rate. The secondary outcomes were clinical pregnancy, multiple gestation, preterm birth, birth defects, aneuploidy, and stillbirth rates. We assessed the quality of the evidence using GRADE methodology. We undertook one comparison, GM-CSF-supplemented culture media versus culture media not supplemented with GM-CSF, for those undergoing assisted reproduction. MAIN RESULTS: We included five studies, the data for three of which (1532 participants) were meta-analysed. We are uncertain whether GM-CSF-supplemented culture media makes any difference to the live-birth rate when compared to using conventional culture media not supplemented with GM-CSF (odds ratio (OR) 1.19, 95% confidence interval (CI) 0.93 to 1.52, 2 RCTs, N = 1432, I2 = 69%, low-quality evidence). The evidence suggests that if the rate of live birth associated with conventional culture media not supplemented with GM-CSF was 22%, the rate with the use of GM-CSF-supplemented culture media would be between 21% and 30%. We are uncertain whether GM-CSF-supplemented culture media makes any difference to the miscarriage rate when compared to using conventional culture media not supplemented with GM-CSF (OR 0.75, 95% CI 0.41 to 1.36, 2 RCTs, N = 1432, I2 = 0%, low-quality evidence). This evidence suggests that if the miscarriage rate associated with conventional culture media not supplemented with GM-CSF was 4%, the rate with the use of GM-CSF-supplemented culture media would be between 2% and 5%. Furthermore, we are uncertain whether GM-CSF-supplemented culture media makes any difference to the following outcomes: clinical pregnancy (OR 1.16, 95% CI 0.93 to 1.45, 3 RCTs, N = 1532 women, I2 = 67%, low-quality evidence); multiple gestation (OR 1.24, 95% CI 0.73 to 2.10, 2 RCTs, N = 1432, I2 = 35%, very low-quality evidence); preterm birth (OR 1.20, 95% CI 0.70 to 2.04, 2 RCTs, N = 1432, I2 = 76%, very low-quality evidence); birth defects (OR 1.33, 95% CI 0.59 to 3.01, I2 = 0%, 2 RCTs, N = 1432, low-quality evidence); and aneuploidy (OR 0.34, 95% CI 0.03 to 3.26, I2 = 0%, 2 RCTs, N = 1432, low-quality evidence). We were unable to undertake analysis of stillbirth, as there were no events in either arm of the two studies that assessed this outcome. AUTHORS' CONCLUSIONS: Due to the very low to low quality of the evidence, we cannot be certain whether GM-CSF is any more or less effective than culture media not supplemented with GM-CSF for clinical outcomes that reflect effectiveness and safety. It is important that independent information on the available evidence is made accessible to those considering using GM-CSF-supplemented culture media. The claims from marketing information that GM-CSF has a positive effect on pregnancy rates are not supported by the available evidence presented here; further well-designed, properly powered RCTs are needed to lend certainty to the evidence.

Time-lapse imaging of inner cell mass splitting with monochorionic triamniotic triplets after elective single embryo transfer: a case report.

RESEARCH QUESTION: Can detailed scrutiny of time-lapse imaging (TLI) of inner cell mass (ICM) splitting help to reduce the frequency of multiple pregnancies following elective single embryo transfer (eSET)? DESIGN: Retrospective analysis of time-lapse images of an embryo in vitro, which resulted in a monochorionic triamniotic pregnancy following eSET on Day 5 of development. RESULTS: A 37-year-old female patient underwent a frozen embryo transfer cycle whereby a single vitrified/warmed embryo was transferred at the hatching blastocyst stage. The subsequent pregnancy scan revealed a monochorionic triamniotic pregnancy. Because the blastocyst was cultured in an incubator incorporating TLI, retrospective scrutiny of the digital recordings demonstrated two distinct ICM structures splitting apart, which formed during the '8-shaped hatching'. CONCLUSIONS: Assisted reproductive techniques and in-vitro culture have been associated with an increased frequency of embryo splitting. This has been postulated to be linked to the in-vitro hatching method observed at the blastocyst stage of development. This case report highlights the need to objectively assess any splitting of the ICM, beyond standard grading of the quality of the ICM and the trophectoderm. Such assessments of ICM splitting should be routine practice in clinical embryology when selecting embryos for transfer.

Cryopreservation of Low Number of Human Spermatozoa; Which is Better: Vapor Phase or Direct Submerging in Liquid Nitrogen?

Cryopreservation and subsequent survival of semen samples with low numbers of human spermatozoa in assisted reproductive technology (ART) facilities can be challenging. The aim of this study was to compare the quality of warmed human spermatozoa following vitrification using direct submerging (DS) in liquid nitrogen (LN2) or with LN2 vapour (V). Normozoospermic ejaculates were prepared by the swim-up technique and the motile sperm fraction was divided into three groups: (i) fresh (control); (ii) DS methods in LN2 (DS Group); or (iii) cryopreserved in V (Group V). Cryopreservation was performed in a small volume using a Cryotech device. After warming, sperm parameters (motility, viability, acrosome, DNA fragmentation and chromatin integrity) were assessed using cytochemical assays. Progressive motility, viability, chromatin integrity and DNA fragmentation differed significantly after warming when compared with the control. Sperm progressive motility and total motility rates were significantly higher in the DS Group compared to Group V. However, normal morphology, acrosome integrity and DNA damage were not significantly different between two groups. Also, sperm chromatin condensation using chromomycin-A3 (CMA3) and Aniline Blue (AB) staining showed fewer alterations in the DS Group compared to Group V. The rates of spermatozoa with an intact acrosome decreased significantly after thawing from 81.30 ± 6.76% to 68.84 ± 14.70% in the DS Group and to 60.92 ± 8.06% in Group V. Cryopreservation of a small number of spermatozoa with the DS method showed superior outcomes with regard to motility, viability and chromatin configuration.

GDF9-ß promotes folliculogenesis in sheep ovarian transplantation onto the chick embryo chorioallantoic membrane (CAM) in cryopreservation programs.

PURPOSE: Ovarian tissue (OT) cryopreservation is a treatment option for fertility preservation among young cancer patients. However, the procedure may involve a reduction in the GDF9-ß expression and a delay in follicular growth after thawing and transplantation. The aim of this study was to evaluate whether supplementation of GDF9-ß can compensate the reduction of this factor during the cryopresevation process and promote folliculogenesis after transplantation of thawed sheep ovarian tissue. METHODS: Sheep OT was cryopreserved using two methods of vitrification and slow freezing. Fresh and thawed OTs were then transplanted onto chick embryo chorioallantoic membrane (CAM) and then divided into two groups based on the addition of GDF9-ß to the grafted tissue. After 5 days of culture, both histological and immunohistological (Ki-67) assessments were performed to evaluate follicular structure, development, and proliferation. The fibrotic and necrotic areas were measured using MICROVISIBLE software. RESULTS: Folliculogenesis took place in all culture groups, but was significantly improved only in the +GDF9-ß cultured group. Also, better follicular structure was preserved in the aforementioned group (p < 0.05). When GDF9-ß was supplemented to the culture medium, more neovascularization (p < 0.05) and better transplantation (p > 0.05) was observed. Furthermore, the areas of fibrosis and necrosis were lower in this group rather than the controls. Follicular proliferative activity was significantly higher only in the slow freezing +GDF9-ß cultured group. CONCLUSIONS: GDF9-ß, as a stimulatory factor, not only promoted the folliculogenesis in the fresh ovarian transplant, but also compensated for its reduction during the cryopreservation process.