Do assisted reproductive technologies and in vitro embryo culture influence the epigenetic control of imprinted genes and transposable elements in children?

STUDY QUESTION: Do assisted reproductive technologies (ART) and in vitro embryo culture influence the epigenetic control of imprinted genes (IGs) and transposable elements (TEs) in children? SUMMARY ANSWER: Significant differences in the DNA methylation of IGs or transposon families were reported between ART and naturally conceived children, but there was no difference between culture media. WHAT IS KNOWN ALREADY: There is concern that ART may play a role in increasing the incidence of adverse health outcomes in children, probably through epigenetic mechanisms. It is crucial to assess epigenetic control, especially following non-optimal in vitro culture conditions and to compare epigenetic analyses from ART-conceived and naturally conceived children. STUDY DESIGN, SIZE, DURATION: This follow-up study was based on an earlier randomized study comparing in vitro fertilization outcomes following the use of two distinct culture media. We compared the epigenetic profiles of children from the initial randomized study according to the mode of conception [i.e. ART singletons compared with those of a cohort of naturally conceived singleton children (CTL)], the type of embryo culture medium used [global medium (LifeGlobal) and single step medium (Irvine Scientific)] and the mode of in vitro fertilization (i.e. IVF versus ICSI). PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 57 buccal smears were collected from 7- to 8-year-old children. The DNA methylation profiles of four differentially methylated regions (DMRs) of IGs (H19/IGF2: IG-DMR, KCNQ1OT1: TSS-DMR, SNURF: TSS-DMR, and PEG3: TSS-DMR) and two TEs (AluYa5 and LINE-1) were first assessed by pyrosequencing. We further explored IGs and TEs' methylation changes through methylation array (Human MethylationEPIC BeadChip referred as EPIC array, Illumina). MAIN RESULTS AND THE ROLE OF CHANCE: Changes in the IGs' DNA methylation levels were found in ART children compared to controls. DNA methylation levels of H19/IGF2 DMR were significantly lower in ART children than in CTL children [52% versus 58%, P = 0.003, false discovery rate (FDR) P = 0.018] while a significantly higher methylation rate was observed for the PEG3 DMR (51% versus 48%, P = 0.007, FDR P = 0.021). However, no differences were found between the culture media. After observing these targeted modifications, analyses were performed at wider scale. Again, no differences were detected according to the culture media, but imprinted-related DMRs overlapping promoter region near the genes major for the development (MEG3, BLCAP, and DLX5) were detected between the ART and CTL children. LIMITATIONS, REASONS FOR CAUTION: The sample size could seem relatively small, but the high consistency of our results was ensured by the homogeneity of the cohort from the initial randomized study, the standardized laboratory techniques and the robust statistical analyses accounting for multiple testing. WIDER IMPLICATIONS OF THE FINDINGS: Although this study did not report DNA methylation differences depending on the culture medium, it sheds light on epigenetic changes that could be observed in some children conceived by ART as compared to CTL children. The clinical relevance of such differences remains largely unknown, and it is still unclear whether such changes are due to some specific ART procedures and/or to parental infertility. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by funding from the Agence Nationale pour la Recherche ('CARE'-ANR JCJC 2017). The authors have no conflicts of interest. TRIAL REGISTRATION NUMBER: Not concerned.

Can novel early non-invasive biomarkers of embryo quality be identified with time-lapse imaging to predict live birth?

STUDY QUESTION: Can time-lapse imaging systems make it possible to identify novel early non-invasive biomarkers to predict live birth? SUMMARY ANSWER: 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 (MNB2cell), were potentially associated with live birth. WHAT IS KNOWN ALREADY: 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. STUDY DESIGN, SIZE, DURATION: 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. PARTICIPANTS/MATERIALS, SETTING, METHODS: 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. MAIN RESULTS AND THE ROLE OF CHANCE: 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 MNB2cell 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. LIMITATIONS, REASONS FOR CAUTION: 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. WIDER IMPLICATIONS OF THE FINDINGS: 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). STUDY FUNDING/COMPETING INTEREST(S): 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.

The epigenetic control of transposable elements and imprinted genes in newborns is affected by the mode of conception: ART versus spontaneous conception without underlying infertility.

STUDY QUESTION: Do assisted reproductive technologies alter DNA methylation and/or transcription of transposable elements and imprinted genes in cord blood and placenta? SUMMARY ANSWER: After ART, DNA methylation and/or transcription changes of some transposable elements and imprinted genes were found in placenta samples while transcription modifications for some transposable elements were also discovered in cord blood. WHAT IS KNOWN ALREADY: Recent studies have confirmed the increased risk of placenta-related adverse pregnancy outcomes and the excess of imprinted disorders with abnormal methylation patterns after ART, which raises the issue of a potential ART-induced epigenetic risk. STUDY DESIGN, SIZE, DURATION: A total of 51 IVF/ICSI (15 conventional and 36 ICSI) singleton pregnancies were prospectively included from January 2013 to April 2015 and compared to 48 spontaneously conceived singleton pregnancies. PARTICIPANTS/MATERIALS, SETTING, METHODS: The DNA methylation and transcription of three imprinted loci (H19/IGF2, KCNQ1OT1 and SNURF DMRs) and four transposon families (LINE-1, ERVFRD, AluYa5 and ERVW) in cord blood and placenta obtained at birth were assessed by pyrosequencing and quantitative RT-PCR, respectively. All data were adjusted for gestational age at delivery, sex of the newborn, parity and maternal age. MAIN RESULTS AND THE ROLE OF CHANCE: DNA methylation levels of H19/IGF2, KCNQ1OT1, LINE-1Hs and ERVFRD-1 were significantly lower in IVF/ICSI placentas than in control placentas, while there was no difference for cord blood. Moreover, the expression of ERVFRD-1 and LINE-1 ORF2 in cord blood and ERVFRD-1 in placenta was lower in the IVF/ICSI group than in controls. The expression of ERVFRD-1 in placenta correlated positively with birth weight and placenta weight, but only in the control group, thus pointing to the potential deregulation of syncytin function after ART. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The control group of fertile couples having conceived within 1 year prevented us from deciphering the distinct roles of ART and infertility. WIDER IMPLICATIONS OF THE FINDINGS: These novel findings of ERVFRD (syncytin-2) expression correlating with birth weight and placenta weight suggest that more research on syncytins and pregnancy-associated diseases could lead to them being used as biomarkers or even as therapeutic targets. The epigenetic modifications in placenta for sequences involved in foetal development raise the question of their potential effects on pregnancy and future life. These results should encourage us to analyse the exact causes and consequences of epigenetic changes and strive to minimize these variations in the interests of epigenetic safety after ART. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by a grant from Besançon and Dijon University Hospitals. The authors have no conflicts of interest to declare.

[Immunosuppressive therapy and fertility preservation: Indications and methods]. / Traitements immunosuppresseurs et préservation de la fertilité : indications et modalités pratiques.

Fertility preservation is routinely performed in cancerology but less systematically used in the field of immune diseases, even though the use of gonadotoxic treatments in young patients may be required and even though the disease itself can alter fertility. This review aimed to clarify the indications and methods of fertility preservation in this context. Cyclophosphamide is the only immunosuppressive drug requiring fertility preservation in women. In men, fertility preservation should be proposed before treatment with cyclophosphamide, methotrexate, mycophenolate mofetil or mTOR inhibitors. Other factors inherent to the disease or the patient may alter fertility. Thus, screening for infertility and fertility preservation have to be implemented as much as possible to increase the chances of successful procreation in patients with immune disease. For women, the choice between the different preservation methods depends on the patient's age, disease activity, the time available before the start of treatment, the possibility of future pregnancy and the woman's and even couple's wishes. Before puberty, the only accepted method is cryopreservation of ovarian tissue. After puberty, the first-line method is the cryopreservation of mature oocytes. If the treatment has to be started in an emergency, if ovarian hyperstimulation/oocyte retrieval is contraindicated or if the patient refuses this option, cryopreservation of ovarian tissue or GnRH agonists could be proposed. For men, the accepted method is sperm cryopreservation. For prepubertal boys, the cryopreservation of spermatogonia after testicular biopsy is still experimental.

Diagnostic genetic screening for assisted reproductive technologies patients with macrozoospermia.

Macrozoospermia is characterized by a high proportion of abnormal spermatozoa with enlarged heads. So far, it has been associated with mutations only in the Aurora Kinase C gene (AURKC) in some cases. Although many publications have reported failure to conceive in couples with macrozoospermia, a few others have described successful pregnancies, thus raising questions as to whether ICSI and AURKC genetic screening should be recommended in all patients with macrozoospermia. First, we report on two monozygotic twins presenting macrozoospermia for whom the genetic status was explored (Aurora Kinase C sequencing) and whole semen and gradient-selected spermatozoa were analyzed, using Fluorescent In Situ Hybridization (FISH), Electron Microscopy and flow cytometry. Additionally, FISH analysis was performed on individually selected uniflagellate spermatozoa with normal sized heads. Second, we also provide an updated review of patients with macrozoospermia gathering the percentage of enlarged head spermatozoa, the genetic status and pregnancy outcomes. Both twins carried a homozygous mutation of AURKC. Spermocytograms showed means of 86% and 83.5% of enlarged head forms. FISH analyses showed that normal head size, uniflagellate spermatozoa had an aneuploid or polyploid nucleus despite a high level of selection. SEM analysis also showed special intranuclear inclusions in enlarged head spermatozoa. Our data together with cases reported in the literature allowed us to recommend that the AURKC gene should be sequenced when the sperm contains 30% or more of enlarged head spermatozoa, and when a mutation is found, ART should not be performed. Our analyses provide information that could greatly help practitioners in their decision-making with regard to optimal care of patients with macrozoospermia.