Can time-lapse imaging systems make it possible to identify novel early non-invasive biomarkers to predict live birth? From mostly high-grade embryos, out of 35 morphometric, morphologic and morphokinetic variables, only pronuclei (PN) position at time of PN juxtaposition and the absence of multinucleated blastomeres at the 2-cell stage (MNB^2cell), were potentially associated with live birth. Previous studies indicate that some kinetic markers may be predictive of blastocyst development and embryonic implantation. Certain teams have suggested including some of them in decisional algorithms for embryo transfers. Using a time-lapse incubator (EmbryoScope, Unisense FertiliTech), we retrospectively explored the associations between the morphometric, morphologic and morphokinetic parameters of oocytes, zygotes and embryos, and their associations with live birth. This study assessed 232 embryos from single embryo transfers after ICSI cycles performed between January 2014 and December 2017. The morphometric, morphologic and morphokinetic parameters (18, 4 and 13, respectively) of oocytes, zygotes and early embryos were studied retrospectively. The associations between these parameters were examined using a Spearman’s correlation, Mann–Whitney or chi-squared test as appropriate. We examined whether these parameters were associated with outcomes in univariate and multivariate logistic regression analyses. Central PN juxtaposition was associated with a 2-fold increase in the odds of live birth (OR = 2.20; 95% CI, [1.26–3.89]; P = 0.006), while the presence of MNB^2cell was associated with half the odds of live birth (OR = 0.51; 95% CI, [0.27–0.95]; P = 0.035). These two parameters were independent of embryo kinetics. The 33 remaining parameters had no significant association with the capacity of transferred embryos to develop to term. Even though the population size was relatively small, our analyses were based on homogeneous cycles, i.e. young women whose transferred embryos were found to be high-grade according to conventional morphology evaluation. In addition, our conclusions were established from a specific, highly selected population, so other study populations, such as women in an older age bracket, may yield different results. Finally, because we assessed day 2/3 transfers, our findings cannot be generalized to embryos cultured up to the blastocyst stage. It would be interesting to explore, prospectively, whether PN localisation is a relevant measure to predict embryo development when added into further algorithms and whether this parameter could be suitable for use in other IVF clinics. Further studies are needed, notably to explore the added value of timing evaluation in cohorts of embryos with low or intermediate morphology grade, as well as in other maternal populations (i.e. older women). No external funding was used for this study. P. Sagot received funding from the following commercial companies: Merck Serono, Finox Biotech, Ferring, MSD France SAS, Teva Sante ́ SAS, Allergan France, Gedeon Richter France, Effik S.A., Karl Storz Endoscopie France, GE Medical Systems SCS, Laboratoires Genevrier, H.A.C. Pharma and Ipsen. All the authors confirm that none of this funding was used to support the research in this study. There are no patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the journal policies on sharing data and materials.

Children conceived by assisted reproductive technologies (ART) have a moderate risk for a number of adverse events and conditions. The question whether this additional risk is associated with specific procedures used in ART or whether it is related to the intrinsic biological factors associated with infertility remains unresolved. One of the main hypotheses is that laboratory procedures could have an effect on the epigenome of gametes and embryos. This suspicion is linked to the fact that ART procedures occur precisely during the period when there are major changes in the organization of the epigenome. Oocyte freezing protocols are generally considered safe; however, some evidence suggests that vitrification may be associated with modifications of the epigenetic marks. In this manuscript, after describing the main changes that occur during epigenetic reprogramming, we will provide current information regarding the impact of oocyte vitrification on epigenetic regulation and the consequences on gene expression, both in animals and humans. Overall, the literature suggests that epigenetic and transcriptomic profiles are sensitive to the stress induced by oocyte vitrification, and it also underlines the need to improve our knowledge in this field.

The many manipulations and processes used in ART coincide with the timing of epigenetic reprogramming and imprinting during female gametogenesis and pre-implantation embryo development, leading to concerns that the actual ART could negatively affect epigenetic reprogramming and imprinting in gametes and early embryos. A growing body of literature suggests that ART may affect epigenetic marks, such as DNA methylation, in the fetus and placenta. Potentially, this may be responsible later in life for the increased risk of adverse outcomes associated with ART. Unfortunately, the conclusions are inconsistent and, despite the increasing usage of ART, its safety at the epigenetic level is still not established. To examine whether ART is associated with DNA methylation modifications and if these modifications persist throughout life, we provide an update on the current understanding of epigenetic reprogramming in human gametes and embryos, and then focus on the assessment of fetal and postnatal DNA methylation modifications that may remain until adulthood following the use of ART in humans. We reviewed studies using targeted or epigenome-wide techniques to assess the DNA methylation patterns of the conceptus after ART compared with natural conceptions. A search for relevant studies was performed in the PubMed and EMBASE databases on 15 July 2021 with an extensive search equation. Studies on animals, gametes and embryos were subsequently excluded. After an in-depth review of full-text articles, studies on specific populations with imprinting disorders were removed and not further discussed. Before comprehensive analysis, the risk of bias of each included study was assessed with the Newcastle–Ottawa scale and quality of evidence was graded using the Grading of Recommendations, Assessment, Development and Evaluations criteria. In total, 928 records were initially identified, and 51 were finally included in the systematic review. Given the variability in the genomic scale at which DNA methylation was measured in the different studies, they were separated into two categories: targeted DNA methylation or genome-wide DNA methylation study. The present systematic review has made it possible to assess a substantial number of children since more than 4000 DNA methylation profiles of ART concepti were compared to more than 7000 controls. There is evidence that ART conception is associated with aberrant DNA methylation in imprinted loci and other genes in various tissues. One isolated modification notably occur in the paternally expressed gene 1/mesoderm-specific transcript homologue (PEG1/MEST) region, and we cannot rule out other studied sequences owing to the heterogeneity of the evidence base. Differences in DNA methylation after ART conceptions are modest, and the functional relevance in adult tissues is unknown. Functional effects in terms of gene expression as well as the roles of other epigenetic marks need to be further explored. Moreover, there is little overlap of findings obtained in targeted and genome-scale analyses owing to the lack of comparability of CpGs analyzed between both techniques. This issue also stems from small sample sizes and marked differences in methodology and cohort characteristics. Standardization of methodologies and large collaborative efforts are required to reduce the inconsistency of results and increase the robustness of findings. Finally, further studies are required to determine the contribution of parental infertility per se from the ART treatment.

Testicular germ cell tumor such as seminoma is strongly associated with male reproductive problems commonly associated with the alteration of sperm parameters as described in testicular dysgenesis syndrome. Interestingly, numerous studies have reported that the precursor of germ cell cancer, germ cell neoplasia in situ (GCNIS), present similarities to fetal gonocytes, specifically characterized by global DNA hypomethylation particularly on imprinting sequences. These disorders may have a common origin derived from perturbations of embryonal programming during fetal development. Presently, there is no available information concerning the sperm DNA methylation patterns of testicular cancer patients. For the first time, we evaluated the sperm imprinting of seminoma patients. A total of 92 cryopreserved sperm samples were included, 31 before seminoma treatment (S): 23 normozoospermic (SN) and 8 oligozoospermic (SO) and 61 sperm controls samples: 31 normozoospermic (N) and 30 oligozoospermic (O). DNA methylation levels of seven differentially methylated regions (DMRs) of imprinted genes [H19/IGF2: IG-DMR (CTCF3 and CTCF6 of H19 gene); IGF2-DMRs (DMR0 and DMR2); MEG3/DLK1:IG-DMR; SNURF:TSS-DMR; KCNQ1OT1:TSS-DMR] were assessed by pyrosequencing. All comparative analyses were adjusted for age. Comparisons of sperm DNA methylation levels between seminoma (S) and normozoospermic (N) samples showed a significant difference for the SNURF sequence (p = 0.017), but after taking into account the sperm parameters, no difference was observed. However, we confirmed a significant association between oligozoospermia (O) and imprinting defects for H19/IGF2-CTCF6 (p = 0.001), MEG3/DLK1 (p = 0.017), IGF2-DMR2 (p = 0.022), and SNURF (p = 0.032) in comparison with control groups (N). This study highlights the high risk of sperm imprinting defects in cases of oligozoospermia and shows for the first time that seminoma patients with normal spermatogenesis present sperm imprinting integrity. These data suggest a low probability of the involvement of a common imprinting defect in fetal cells leading to both TGCT and subfertility.

Early life periconceptional exposures during assisted reproductive technology (ART) procedures could alter the DNA methylation profiles of ART children, notably in imprinted genes and repetitive elements. At the genome scale, DNA methylation differences have been reported in ART conceptions at birth, but it is still unclear if those differences remain at childhood. Here, we performed an epigenome-wide DNA methylation association study using Illumina InfiniumEPIC BeadChip to assess the effects of the mode of conception on the methylome of buccal cells from 7- to 8-year-old children (48 children conceived after ART or naturally (control, CTL)) and according to the embryo culture medium in which they were conceived. We identified 127 differentially methylated positions (DMPs) and 16 differentially methylated regions (DMRs) (FDR < 0.05) with low delta beta differences between the two groups (ART vs. CTL). DMPs were preferentially located inside promoter proximal regions and CpG islands and were mostly hypermethylated with ART. We highlighted that the use of distinct embryo culture medium was not associated with DNA methylation differences in childhood. Overall, we bring additional evidence that children conceived via ART display limited genome-wide DNA methylation variation compared with those conceived naturally.

Surgical treatment or varicocele embolization (VE) with sclerosing or mechanical embolic agents have been shown to improve the semen parameters of infertile men. The aim of this study was to evaluate the impact of VE using N-butyl cyanoacrylate (NBCA) glue on semen parameters in infertile men. From January 2014 to June 2018, infertile adult patients with stage 3 varicocele and an initial semen analysis showing at least one abnormal semen parameter, and who were successfully embolized with NBCA Glubran^®2 glue, were retrospectively recruited. The availability of a second semen analysis after VE was mandatory for patient inclusion. The primary endpoint was the change in total sperm number (TSN) after VE. The other parameters of interest were progressive and total sperm motilities (Smot) at 1 h (H1), sperm vitality (SV) and morphology (SMor). One hundred and two patients were included. Eight patients presented null TSN before and after VE. Among the remaining 94 patients, a significant improvement in the median TSN after VE was shown (31.79 × 10⁶/ejaculate [IQR: 11.10–127.40 × 10⁶/ejaculate] versus 62.24 × 10⁶/ejaculate [IQR: 17.90–201.60 × 10⁶/ejaculate], p = 0.0295). Significant improvement in TSN was found for the 60 oligo- or azoospermic patients (p = 0.0007), whereas no significant change was found for the 42 patients with normal initial TSN (p = 0.49). Other parameters, such as progressive and total SMot, SV and SMor, also significantly improved after VE (p = 0.0003, 0.0013, 0.0356 and 0.007, respectively). The use of NBCA glue as an embolic agent for VE in infertile men with stage 3 varicocele significantly improves the semen parameters.