Blastocysts from partial compaction morulae are not defined by their early mistakes.

RESEARCH QUESTION: Is partial compaction during morula formation associated with an embryo's developmental ability and implantation potential? DESIGN: Retrospective analysis of data from 196 preimplantation genetic testing for aneuploidy (PGT-A) cycles. Embryos starting compaction were grouped according to the inclusion or not of all the blastomeres in the forming morula (full compaction or partial compaction). The possible effect of maternal age and ovarian response on compaction was analysed. Morphokinetic characteristics, blastocyst formation rate, morphology and cytogenetic constitution of the obtained blastocysts were compared. Comparisons of reproductive outcomes after the transfer of euploid blastocysts from both groups were established. Finally, in a subset of embryos, the chromosomal constitution concordance of the abandoned cells and the corresponding blastocyst through trophectoderm biopsies was assessed. RESULTS: A total of 430 embryos failed to include at least one cell during compaction (partial compaction group [49.3%]), whereas the 442 remaining embryos formed a fully compacted morula (full compaction group [50.7%]). Neither female age nor the number of oocytes collected affected the prevalence of partial compaction morulae. Morphokinetic parameters were altered in embryos from partial compaction morulae compared with full compaction. Although an impairment in blastocyst formation rate was observed in partial compaction morulae (57.2% versus 70.8%, P < 0.001), both chromosomal constitution (euploidy rate: partial compaction [38.4%] versus full compaction [34.2%]) and reproductive outcomes (live birth rate: partial compaction [51.9%] versus full compaction [46.2%]) of the obtained blastocysts were equivalent between groups. A high ploidy correlation of excluded cells-trophectoderm duos was observed. CONCLUSIONS: Partial compaction morulae show a reduced developmental ability compared with full compaction morulae. Resulting blastocysts from both groups, however, have similar euploidy rates and reproductive outcomes. Cell exclusion might be a consequence of a compromised embryo development regardless of the chromosomal constitution of the excluded cells.

Do reproductive history and information given through genetic counselling influence patients' decisions on mosaic embryo transfer?

OBJECTIVE: To assess patients' and embryonic characteristics that may have an influence on the decision to transfer a mosaic embryo. METHOD: Single centre retrospective cohort study including 1247 PGT-A cycles. Demographic and clinical factors associated with a decision to transfer a mosaic embryo were studied. Female age, number of previous cycles, previous availability of euploid embryos, history of miscarriages and parity as well as percentage of mosaicism, type of anomaly and chromosome risk were studied in relation to decision-making. Outcomes after mosaic embryo transfer were assessed. RESULTS: To date, in 7.9% of cycles (99/1247), patients have had to make a decision on the fate of their mosaic embryos. In 23.2% of cycles (23/99), patients decided to transfer. In most cases (79.8%; 79/99), patients underwent genetic counselling before the decision. None of the variables analysed were associated with the patients' decision, although parity and the high-degree mosaicism (>50%) seemed to be negatively associated with the decision to transfer (18.2% vs. 29.8%, p = 0.294; 10% vs. 32.2%, p = 0.052). CONCLUSIONS: Neither reproductive history nor information on mosaic embryo characteristics through counselling seems to be determinative for patients when deciding to transfer a mosaic embryo. Promising and increasing data on clinical outcomes after mosaic embryo transfer will be of utmost importance to soften risk perception regarding mosaic embryos and give a better, simplified and more evidence-based counselling.

The effect of trophectoderm biopsy technique and sample handling on artefactual mosaicism.

PURPOSE: To determine whether embryo mosaicism prevalence in preimplantation genetic testing for aneuploidy (PGT-A) cycles is associated with the trophectoderm biopsy technique used (a. number of laser pulses or b. the use of flicking or pulling) or the time to tubing. METHODS: Prospective observational study performed in a single IVF-PGT-A setting from May 2019 to May 2021. Trophectoderm biopsies were analysed by next-generation sequencing. Mosaicism was analysed in relation to the biopsy methodology (number of laser pulses and pulling vs flicking), time elapsed from biopsy to tubing (min), and time of sample cryostorage from tubing to amplification (days). As a secondary objective, the number of laser pulses and biopsy methodology were studied in relation to clinical outcomes of transferred euploid blastocysts. RESULTS: None of the analysed variables were associated to mosaicism prevalence. Multivariable regression analysis demonstrated that mosaicism prevalence was comparable either when  > 3 laser pulses were used as compared to ≤ 3 (13.9% vs 13.8%, aOR = 0.8726 [0.60-1.28]) and pulling compared to flicking (13.1% vs 14.0%, aOR = 0.86 [0.60-1.23]). Moreover, neither the number of laser pulses during biopsy (> 3 vs ≤ 3) nor the technique used (pulling vs flicking) were associated with clinical pregnancy after the transfer of frozen-thawed euploid blastocysts (54.9% vs 55.2%, aOR = 1.05 [0.53-2.09]; 61.1% vs 52.9%, aOR = 1.11 [0.55-2.25], respectively). CONCLUSION: Our results suggest that, as long as the biopsy and tubing procedures are performed following standardized high quality procedures, no specific approach would increase the generation of artefactual mosaicism as a result of trophectoderm biopsy. Trophectoderm biopsies should be performed regardless of the methodology but always aiming on minimising blastocyst manipulation.

Prevalence, types and possible factors influencing mosaicism in IVF blastocysts: results from a single setting.

RESEARCH QUESTION: Are intrinsic or extrinsic factors associated with embryo mosaicism prevalence in IVF cycles? DESIGN: Retrospective cohort study of preimplantation genetic testing for aneuploidy (PGT-A) cycles carried out at a university-affiliated IVF clinic between October 2017 and October 2019. Trophectoderm biopsies were analysed by next generation sequencing. Mosaicism prevalence, type of anomaly and the chromosomes involved were analysed. Intrinsic and extrinsic factors potentially inducing mosaicism were studied: maternal and paternal age, antral follicle count, cumulus-oocyte complexes retrieved, female body mass index, PGT-A indication, sperm concentration, total dosage of gonadotrophins, embryo quality and day of blastocyst formation, single-step commercial media used and biopsy operator. RESULTS: Overall prevalence of mosaicism in our PGT-A setting was 13.9%. In segmental mosaicism, larger chromosomes tended to be more affected, which was not observed in whole-chromosome mosaicism. Additionally, segmental mosaicism was mostly observed in monosomy (69.6%; P < 0.01) compared with whole-chromosome mosaicism (49.7% monosomies versus 50.3% trisomies; P = 0.83). Although a high inter-patient variability was observed, only paternal age showed a positive association with mosaicism (adjusted OR 1.26, 95% CI 1.02 to 1.54) among the analysed variables. CONCLUSIONS: Our results suggest remarkable differences in the mechanisms generating segmental and whole-chromosome mosaicism, indicating that they may deserve different consideration when studying them and when prioritizing them for transfer. Male factor seems to be associated with mosaicism and may be worthy of specific assessment in future studies.

Morphokinetics and in vitro developmental potential of monopronucleated ICSI zygotes until the blastocyst stage.

The aim of this study was to provide a more comprehensive understanding of 1PN intracytoplasmic sperm injection (ICSI) zygotes. To achieve this objective, we assessed whether all 1PN-derived embryos showed a similar morphokinetic pattern, and if the morphokinetic behaviour of 1PN-derived embryos was comparable with that of 2PN-derived embryos. In total, 149 1PN ICSI zygotes (study group) and 195 2PN ICSI zygotes (control group) were included in the study. Embryo development potential was evaluated in terms of blastocyst rate. Morphokinetic parameters, including the pronucleus diameter and kinetics of in vitro development, were also analyzed. Embryos derived from 1PN ICSI zygotes showed impaired development compared with 2PN-derived embryos, with blastocyst rates of 28.9% and 67.2%, respectively. The diameter of the pronucleus of 1PN zygotes was larger than that of 2PN zygotes. When compared with 2PN-derived embryos, those derived from 1PN zygotes had a visible pronucleus for a shorter time, in addition to a longer syngamy time and slower kinetic behaviour from two to nine cells. When 1PN-derived blastocysts and 2PN-derived blastocysts were compared, the developmental kinetics were similar in both groups, except for a delayed and longer duration of the compaction phase in 1PN-derived embryos. In conclusion, monopronucleated ICSI zygotes present differences in developmental capacity and morphokinetic behaviour compared with 2PN ICSI zygotes, showing particular morphokinetic parameters related to pronucleus formation. Only the 1PN ICSI-derived embryos that reached the blastocyst stage have similar morphokinetic development to blastocysts from 2PN zygotes.

Inconclusive results in preimplantation genetic testing: go for a second biopsy?

The transition in biopsy timing from blastomere to trophectoderm biopsy has led to a remarkable decrease in the percentage of undiagnosed blastocysts. However, patients with few or no euploid blastocysts can be affected by this residual percentage of diagnosis failure. The aim of this study is to assess whether blastocyst rebiopsy and revitrification is an efficient and safe procedure to be applied in cases of no results after analysis. Fifty-three patients agreed to the warming of 61 blastocysts to perform a second biopsy and PGT-A by aCGH. Only 75.4% of the blastocysts survived, reexpanded, and could be rebiopsied. After the second biopsy and analysis, 95.6% of the blastocysts were successfully diagnosed with an euploidy rate of 65.9%. Eighteen euploid blastocysts were warmed and transferred to 18 patients with a 100% survival and reexpansion rate. Seven clinical pregnancies have been achieved with 4 live births, 1 ongoing pregnancy, and 2 miscarriages. Thus, although few transfers of rebiopsied and revitrified blastocysts have been performed till date, our preliminary results show that this approach is efficient and safe to be applied for undiagnosed blastocysts, as it ultimately allows the transfer of euploid blastocysts and good clinical outcomes.

Transition from blastomere to trophectoderm biopsy: comparing two preimplantation genetic testing for aneuploidies strategies.

SummaryShortly after the implementation of comprehensive chromosome screening (CCS) techniques for preimplantation genetic testing for aneuploidies (PGT-A), the discussion about the transition from day 3 to blastocyst stage biopsy was initiated. Trophectoderm biopsy with CCS is meant to overcome the limitations of cleavage-stage biopsy and single-cell analysis. The aim of this study was to assess the results obtained in our PGT-A programme after the implementation of this new strategy. Comparisons between the results obtained in 179 PGT-A cycles with day 3 biopsy (D+3) and fresh embryo transfer, and 204 cycles with trophectoderm biopsy and deferred (frozen-thawed) embryo transfer were established. Fewer embryos were biopsied and a higher euploidy rate was observed in the trophectoderm biopsy group. No differences in implantation (50.3% vs. 61.4%) and clinical pregnancy rate per transfer (56.1% vs. 65.3%) were found. Although the mean number of euploid embryos per cycle did not differ between groups (1.5 ± 1.7 vs. 1.7 ± 1.8), the final number of euploid blastocysts available for transfer per cycle was significantly higher in the trophectoderm biopsy group (1.1 ± 1.3 vs. 1.7 ± 1.8). This factor led to an increased cumulative live birth rate in this last group (34.1% vs. 44.6%). Although both strategies can offer good results, trophectoderm biopsy offers a more robust diagnosis and the intervention is less harmful for the embryos so more euploid blastocysts are finally available for transfer and/or vitrification.

Chromosome positioning and male infertility: it comes with the territory.

The production of functional spermatozoa through spermatogenesis requires a spatially and temporally highly regulated gene expression pattern, which in case of alterations, leads to male infertility. Changes of gene expression by chromosome anomalies, gene variants, and epigenetic alterations have been described as the main genetic causes of male infertility. Recent molecular and cytogenetic approaches have revealed that higher order chromosome positioning is essential for basic genome functions, including gene expression. This review addresses this issue by exposing well-founded evidences which support that alterations on the chromosome topology in spermatogenetic cells leads to defective sperm function and could be considered as an additional genetic cause of male infertility.

Normalization matters: tracking the best strategy for sperm miRNA quantification.

STUDY QUESTION: What is the most reliable normalization strategy for sperm microRNA (miRNA) quantitative Reverse Transcription Polymerase Chain Reactions (qRT-PCR) using singleplex assays? SUMMARY ANSWER: The use of the average expression of hsa-miR-100-5p and hsa-miR-30a-5p as sperm miRNA qRT-PCR data normalizer is suggested as an optimal strategy. WHAT IS KNOWN ALREADY: Mean-centering methods are the most reliable normalization strategies for miRNA high-throughput expression analyses. Nevertheless, specific trustworthy reference controls must be established in singleplex sperm miRNA qRT-PCRs. STUDY DESIGN, SIZE DURATION: Cycle threshold (Ct) values from previously published sperm miRNA expression profiles were normalized using four approaches: (i) Mean-Centering Restricted (MCR) method (taken as the reference strategy); (ii) expression of the small nuclear RNA RNU6B; (iii) expression of four miRNAs selected by the Concordance Correlation Restricted (CCR) algorithm: hsa-miR-100-5p, hsa-miR-146b-5p, hsa-miR-92a-3p and hsa-miR-30a-5p; (iv) the combination of two of these miRNAs that achieved the highest proximity to MCR. PARTICIPANTS/MATERIALS, SETTING, METHODS: Expression profile data from 736 sperm miRNAs were taken from previously published studies performed in fertile donors (n = 10) and infertile patients (n = 38). For each tested normalizer molecule, expression ubiquity and uniformity across the different samples and populations were assessed as indispensable requirements for being considered as valid candidates. The reliability of the different normalizing strategies was compared to MCR based on the set of differentially expressed miRNAs (DE-miRNAs) detected between populations, the corresponding predicted targets and the associated enriched biological processes. MAIN RESULTS AND THE ROLE OF CHANCE: All tested normalizers were found to be ubiquitous and non-differentially expressed between populations. RNU6B was the least uniformly expressed candidate across samples. Data normalization through RNU6B led to dramatically misguided results when compared to MCR outputs, with a null prediction of target genes and enriched biological processes. Hsa-miR-146b-5p and hsa-miR-92a-3p were more uniformly expressed than RNU6B, but their results still showed scant proximity to the reference method. The highest resemblance to MCR was achieved by hsa-miR-100-5p and hsa-miR-30a-5p. Normalization against the combination of both miRNAs reached the best proximity rank regarding the detected DE-miRNAs (Area Under the Curve = 0.8). This combination also exhibited the best performance in terms of the target genes predicted (72.3% of True Positives) and their corresponding enriched biological processes (70.4% of True Positives). LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: This study is focused on sperm miRNA qRT-PCR analysis. The use of the selected normalizers in other cell types or tissues would still require confirmation. WIDER IMPLICATIONS OF THE FINDINGS: The search for new fertility biomarkers based on sperm miRNA expression using high-throughput assays is one of the upcoming challenges in the field of reproductive genetics. In this context, validation of the results using singleplex assays would be mandatory. The normalizer strategy suggested in this study would provide a universal option in this area, allowing for normalization of the validated data without causing meaningful variations of the results. Instead, qRT-PCR data normalization by RNU6B should be discarded in sperm-miRNA expression studies. STUDY FUNDING/COMPETING INTERESTS: This work was supported by the 2014/SGR00524 project (Agència de Gestió d'Ajuts Universitaris i de Recerca, Generalitat de Catalunya, Spain) and UAB CF-180034 grant (Universitat Autònoma de Barcelona). Celia Corral-Vazquez is a recipient of a Personal Investigador en Formació grant UAB/PIF2015 (Universitat Autònoma de Barcelona). The authors report no conflict of interest.

Altered bivalent positioning in metaphase I human spermatocytes from Robertsonian translocation carriers.

PURPOSE: The study aims to determine whether there is an altered bivalent positioning in metaphase I human spermatocytes from Robertsonian translocation carriers. METHODS: Metaphase I human spermatocytes from three 45,XY,der(13;14)(q10;q10) individuals and a 45,XY,der(14;15)(q10;q10) individual were analyzed. Proximity relationships of bivalents were established by analyzing meiotic preparations combining Leishman staining and multiplex-FISH procedures. Poisson regression model was used to determine proximity frequencies between bivalents and to assess associations with chromosome size, gene density, acrocentric morphology, and chromosomes with heterochromatic blocks. The hierarchical cluster Ward method was used to characterize the groups of bivalents with preferred proximities in a cluster analysis. Bivalent groups obtained were individually compared with those obtained in normal karyotype individuals evaluated in a previous study. RESULTS: A total of 1288 bivalents were examined, giving a total of 2289 proximity data. Only four positive significant proximities were detected for each type of Robertsonian translocation. Significant bivalent associations were only observed by small-size chromosomes for MI,22,XY,III(13q14q). These results were clearly divergent from 46,XY individuals. Moreover, cluster analysis revealed that about 30 % of the bivalents showed changes in their proximity relationships in metaphase I. CONCLUSIONS: The territorial organization of bivalents in metaphase I human spermatocytes changes in the presence of a Robertsonian translocation.

Altered segregation pattern and numerical chromosome abnormalities interrelate in spermatozoa from Robertsonian translocation carriers.

The aim of this study was to assess whether there is a relationship between numerical chromosome abnormalities and certain segregation modes in spermatozoa from Robertsonian translocation carriers. A sequential fluorescence in-situ hybridization protocol based on two successive hybridization rounds was performed on sperm samples from one t(13;22) and ten t(13;14) carriers. Patient inclusion criteria included the presence of a positive interchromosomal effect (ICE). In the first round, numerical abnormalities for chromosomes 15/22, 18, 21, X and Y were analysed. In the second round, the segregation outcome of the rearranged chromosomes was evaluated in the numerically abnormal spermatozoa detected in the first round, as well as in randomly assessed spermatozoa. Aneuploid spermatozoa showed statistical differences in all segregation modes when compared with randomly assessed spermatozoa: alternate (50.7% versus 84.3%), adjacent (36.6% versus 14.6%) and 3:0 (10.2% versus 1%). Diploid/multiple disomic spermatozoa showed differences in alternate (3.7% versus 84.3%) and 3:0 (67.6% versus 1%). We concluded that in Robertsonian translocation carriers that exhibit ICE, numerically abnormal spermatozoa preferentially contain unbalanced segregation products. This might be explained by heterosynapsis acting as a rescue mechanism that would lead to aberrant recombination, which is a predisposing factor for non-disjunction events.

Sequential FISH allows the determination of the segregation outcome and the presence of numerical anomalies in spermatozoa from a t(1;8;2)(q42;p21;p15) carrier.

PURPOSE: To find out the meiotic segregation behaviour of the t(1;8;2)(q42;p21;p15), to evaluate the occurrence of interchromosomal effects, and to determine whether there is an accumulation of unbalanced products in aneuploid/diploid gametes. METHODS: A sequential FISH protocol based on two successive hybridization rounds over the same spermatozoa was performed to determine the segregation outcome of the rearranged chromosomes. The presence of numerical abnormalities for 13, 18, 21, X and Y was also evaluated by sperm FISH. Those aneuploid/diploid gametes were subsequently relocalized and analyzed for their segregation content through additional hybridization rounds. RESULTS: The segregation pattern observed reported a very low production of normal/balanced gametes (11.7 %). Significant increased frequencies of diploidies and disomies for chromosomes X/Y and 18 were detected (p < 0.001). Aneuploid and diploid spermatozoa displayed significant increases of 5:1, 6:0 and other unexpected disjunction modes (p < 0.001). CONCLUSIONS: The strategy developed in this study is a reliable new approach to establish the full segregation pattern of complex chromosome rearrangements (CCR). Results corroborate the low number of normal/balanced spermatozoa produced by CCR carriers and support previous findings regarding an altered segregation pattern in gametes with numerical abnormalities. Altogether this confirms the importance of PGD as a tool to prevent the transmission of chromosomal abnormalities to the offspring in CCR patients.

A transcriptomic insight into the human sperm microbiome through next-generation sequencing.

The purpose of this study is to provide novel information through Next Generation Sequencing (NGS) for the characterization of viral and bacterial RNA cargo of human sperm cells from healthy fertile donors. For this, RNA-seq raw data of poly(A) RNA from 12 sperm samples from fertile donors were aligned to microbiome databases using the GAIA software. Species of viruses and bacteria were quantified in Operational Taxonomic Units (OTU) and filtered by minimal expression level (>1% OTU in at least one sample). Mean expression values (and their standard deviation) of each species were estimated. A Hierarchical Cluster Analysis (HCA) and a Principal Component Analysis (PCA) were performed to detect common microbiome patterns among samples. Sixteen microbiome species, families, domains, and orders surpassed the established expression threshold. Of the 16 categories, nine corresponded to viruses (23.07% OTU) and seven to bacteria (2.77% OTU), among which the Herperviriales order and Escherichia coli were the most abundant, respectively. HCA and PCA displayed four clusters of samples with a differentiated microbiome fingerprint. This work represents a pilot study into the viruses and bacteria that make up the human sperm microbiome. Despite the high variability observed, some patterns of similarity among individuals were identified. Further NGS studies under standardized methodological procedures are necessary to achieve a deep knowledge of the semen microbiome and its implications in male fertility.

Sperm rates of 7q11.23, 15q11q13 and 22q11.2 deletions and duplications: a FISH approach.

Genomic disorders are human diseases caused by meiotic chromosomal rearrangements of unstable regions flanked by Low Copy Repeats (LCRs). LCRs act as substrates for Non-Allelic Homologous Recombination (NAHR) leading to deletions and duplications. The aim of this study was to assess the basal frequency of deletions and duplications of the 7q11.23, 15q11-q13 and 22q11.2 regions in spermatozoa from control donors to check differences in the susceptibility to generate anomalies and to assess the contribution of intra- and inter-chromatid NAHR events. Semen samples from ten control donors were processed by FISH. A customized combination of probes was used to discriminate among normal, deleted and duplicated sperm genotypes. A minimum of 10,000 sperm were assessed per sample and region. There were no differences in the mean frequency of deletions and duplications (del + dup) among the 7q11.23, 15q11-q13 and 22q11.2 regions (frequency ± SEM, 0.37 ± 0.02; 0.46 ± 0.07 and 0.27 ± 0.07%, respectively) (P = 0.122). Nevertheless, hierarchical cluster analysis reveals interindividual differences suggesting that particular haplotypes could be the main source of variability in NAHR rates. The mean frequency of deletions was not different from the mean frequency of duplications in the 7q11.23 (P = 0.202) and 15q11-q13 (P = 0.609) regions, indicating a predominant inter-chromatid NAHR. By contrast, in the 22q11.2 region the frequency of deletions slightly exceed duplications (P = 0.032), although at the individual level any donor showed differences. Altogether, our results support the inter-chromatid NAHR as the predominant mechanism involved in the generation of sperm deletions and duplications.

Impact of sperm DNA fragmentation on the outcome of IVF with own or donated oocytes.

A prospective study was performed to assess the impact of sperm DNA fragmentation on the outcome of IVF with own or donated oocytes. The study population included 178 couples (62 cycles of IVF, 116 of intracytoplasmic sperm injection (ICSI)) with own (n=77) and donor (n=101) oocytes. DNA fragmentation was evaluated by TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling assay. Correlation between DNA damage to oocyte fertilization, embryo quality and clinical pregnancy, implantation and miscarriage rates was evaluated. DNA fragmentation was not related to fertilization rates in either IVF (r=0.08) or ICSI (r=-0.04) cycles. DNA fragmentation was similar in patients with <50% embryo utilization rate compared with ≥50%, in cancelled and in embryo transfer cycles and in miscarriages and in successful deliveries. Moreover, DNA fragmentation was similar in pregnant and non-pregnant women as well as in IVF with own or donor oocytes. In the multivariable analysis, the odds ratio of DNA after controlling by age was 1.0. Using a 36% sperm fragmentation threshold, results did not vary. It is concluded that DNA damage was not related to outcomes of IVF or ICSI with own or donor oocytes.

Meiotic behavior of three D;G Robertsonian translocations: segregation and interchromosomal effect.

Robertsonian translocations are one of the most frequent reorganizations in humans. Their segregational behavior and their implication in the occurrence of interchromosomal effects (ICEs) has been an important topic of research for the past 10 years. Most of the cases analyzed correspond to rearrangements with chromosomes from the D-group (chromosomes 13, 14 and 15), whereas some rare Robertsonian translocations are scarcely found in the literature, mainly those with both chromosomes from the G-group (chromosomes 21 and 22) and those involving chromosomes from both groups (D;G translocations). Results supporting/rejecting the existence of the ICE phenomenon have been reported, showing the need for more studies to characterize its distribution. In this study, sperm fluorescent in situ hybridization studies have been performed in three D;G Robertsonian translocation carriers: two men with the translocation t(14;21) and a third individual with the rare t(13;22) reorganization. Segregation and ICE results have been considered in relation to their cytogenetic features and all previously published data. The compiled information discards a particular segregation behavior related to the chromosomes involved. In contrast with this segregational homogeneity, heterogeneous ICE results were observed, indicating a significant but random distribution of such phenomenon.

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.

Sperm microRNA pairs: new perspectives in the search for male fertility biomarkers.

OBJECTIVE: To identify candidates of fertility biomarkers among pairs of human sperm microRNAs. DESIGN: Expression data of 736 sperm microRNAs from fertile and infertile individuals characterized in previous published studies by means of TaqMan quantitative polymerase chain reaction (PCR) were reexamined. A set of microRNA pairs with the best biomarker potential were selected and validated by means of quantitative real-time (qRT) PCR in an independent cohort. SETTING: University laboratory. PATIENT(S): Semen samples were obtained from fertile (n = 10) and infertile (asthenozoospermia, n = 10; teratozoospermia, n = 10; oligozoospermia, n = 10; unexplained male infertility [UMI], n = 8) individuals. The validation cohort included 9 fertile donors and 14 infertile patients with different seminal alterations. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Spearman test was used to select microRNA pairs with a correlated expression in fertile individuals and a noncorrelated expression in each infertile group. The biomarker potential of these pairs was determined with the use of receiver operating characteristic curves. The differential relative expression of each pair in fertile and infertile populations was verified (Mann-Whitney test). Those pairs with best results were validated by qRT-PCR. RESULT(S): Forty-eight pairs showed significant correlations in the fertile group. The pairs that were uncorrelated in the infertile populations and displayed the best biomarker potential were hsa-miR-942-5p/hsa-miR-1208 (asthenozoospermia), hsa-miR-296-5p/hsa-miR-328-3p (teratozoospermia), hsa-miR-139-5p/hsa-miR-1260a (oligozoospermia), and hsa-miR-34b-3p/hsa-miR-93-3p (UMI). The hsa-miR-942-5p/hsa-miR-1208 pair showed the greatest potential for detecting seminal alterations in the validation cohort (85.71% true positives). CONCLUSION(S): The pairs hsa-miR-942-5p/hsa-miR-1208 and hsa-miR-34b-3p/hsa-miR-93-3p have the potential to become new molecular biomarkers that could help to diagnose male infertility, especially in cases of UMI or when seminal parameters are close to the threshold values.

Reciprocal translocations: tracing their meiotic behavior.

PURPOSE: Segregation and interchromosomal effect studies have been performed in reciprocal translocation carriers by sperm-fluorescent in situ hybridization reporting a great heterogeneity. The divergences have been attributed to the particular cytogenetic characteristics of each rearrangement. Nevertheless, there is no consensus in the factors that are responsible for such variability. The purpose of this study was to determine which cytogenetic features influence in the segregation and interchromosomal effect outcome. METHODS: Segregation and interchromosomal effects analyses were performed in 14 reciprocal translocation carriers, selected because they presented very different cytogenetic features regarding the tetravalent pairing geometry. In each segregation study, a customized combination of probes was used to identify all the segregation products. In the interchromosomal effect study, we used a triple-color fluorescent in situ hybridization for chromosomes X, Y, and 18. RESULTS: A preferential segregation pattern with a gradually decreasing production of Alternate, Adjacent I, Adjacent II, and 3:1 segregation was observed in the segregation analysis. Some specific features have been observed to influence this distribution: size of the translocated and centric segments and the presence of centromeres from acrocentric chromosomes in the center of the cross. Aneuploidy/diploidy screening revealed increased frequencies of numerical anomalies in seven carriers. CONCLUSIONS: Our data suggest that reciprocal translocations display a more homogeneous behavior than described in the literature. The interchromosomal effects represent an additional source of imbalances in these carriers.

Chromosomal analysis of blastocyst derived from monopronucleated ICSI zygotes: approach by double trophectoderm biopsy.

OBJECTIVE: This study aims to increase the knowledge about monopronucleated ICSI-derived blastocysts, analyzing trophectoderm biopsies by aCGH and FISH to evaluate their chromosome constitution. METHODS: Fifteen monopronucleated ICSI-derived blastocysts were studied. Double trophectoderm biopsy was performed and analyzed by FISH and aCGH. The blastocysts were classified according to chromosome constitution. Disagreements between the two techniques were assessed. RESULTS: Results obtained after FISH and aCGH analyses showed the following: 20% (3/15) and 60% (9/15) diploid females, respectively; 26.7% (4/15) and 26.7% (4/15) diploid males, respectively; and 53.3% (8/15) and 13.3% (2/15) mosaics, respectively. No mosaic male embryos were found using FISH or aCGH. There were disagreements in 40% (6/15) of the cases due to the higher detection of mosaicism by FISH compared to aCGH. CONCLUSIONS: The combination of FISH and aCGH has been shown to be a suitable approach to increase the knowledge about monopronucleated ICSI-derived embryos. FISH analysis of blastocysts derived from monopronucleated ICSI zygotes enabled us to conclude that aCGH underestimates haploidy. Some diploid embryos diagnosed by aCGH are in fact mosaic. In cases where these embryos would be used for reproductive purposes, extra analysis of parental genome origin is recommended.