Does underlying infertility in natural conception modify the epigenetic control of imprinted genes and transposable elements in newborns?

RESEARCH QUESTION: Does the epigenetic control of imprinted genes and transposable elements at birth differ according to time to conception in natural conception and after intrauterine insemination (IUI)? DESIGN: A total of 144 singletons were included in four groups: 50 natural pregnancies obtained within 6 months after stopping contraception (group 1); 34 natural pregnancies with infertility period between 6 and 12 months (group 2); 36 pregnancies with an infertility period of more than 12 months (group 3) and 24 pregnancies obtained after IUI (group 4). RESULTS: The placental DNA methylation levels of H19/IGF2 and KCNQ1OT1 were lower in groups 2, 3 and 4 than in group 1 (P = 0.025 in the overall comparison). The DNA methylation rate for LINE-1 was higher in placentas from group 2 than in group 1 (P = 0.022). In cord blood, DNA methylation levels were not significantly different between groups except for H19/IGF2 for which the DNA methylation levels were higher in group 2 than in group 1 (H19/IGF2-seq1 and seq2: P = 0.023 and P = 0.002, respectively). In placenta tissue, compared with group 1, relative expression for SNRPN and for LINE-1 was significantly higher in group 2 (P = 0.002 and P < 0.001, respectively). The relative expression of KCNQ1 in placenta was lower in group 4 than in group 1 (P = 0.013). In cord blood, compared with group 1, the relative expression for H19 was significantly higher in group 3 (P = 0.026), and the relative expression of LINE-1 was higher in groups 2 and 3 and in group 4 (P < 0.001). CONCLUSIONS: Infertility itself, and not only ART techniques, could contribute to potential epigenetic risks for children.

Effect of paternal smoking on pre-implantation embryonic development: a prospective cohort study.

CONTEXT: Recent studies have failed to demonstrate the negative impact of male tobacco smoking on embryo development, raising the question of its actual implication on natural fecundity and assisted reproductive techniques outcomes. AIMS: To assess the impact of paternal smoking on embryo development. METHODS: In this prospective cohort study, 252 men from couples undergoing in vitro fertilisation (IVF) were included. Each patient was interviewed and took a carbon monoxide breath-test, creating three groups: non-smokers (n =113), former smokers (n =81) and active smokers (n =58). The Top-grade embryo ratio (primary endpoint), embryo morphokinetic parameters and clinical outcomes were assessed. KEY RESULTS: In a multivariate analyses based on 1521 embryos, no significant difference was found in the top-grade embryo ratio between the groups. Tobacco smoking had no impact on clinical outcomes. Compared to non-smokers the time to the pronuclei fading (tPNf, P =0.006) and the time to the first embryonic cleavage (t2, P =0.002) were shorter in smokers, and the t2 was also slightly shorter in former smokers (P =0.045). No other differences were found in the morphokinetic parameters. CONCLUSION: Even if a few differences were observed in the first timing of embryonic events, this study did not highlight a major embryonic and clinical impact of the paternal smoking status. IMPLICATION: The results obtained here are reassuring towards IVF outcomes. As maternal smoking is highly controlled in the IVF patients in this study, we speculate that the sperm selection process may limit the adverse effects of tobacco consumption on embryo development.

Vps13b is required for acrosome biogenesis through functions in Golgi dynamic and membrane trafficking.

The sperm acrosome is a lysosome-related organelle that develops using membrane trafficking from the Golgi apparatus as well as the endolysosomal compartment. How vesicular trafficking is regulated in spermatids to form the acrosome remains to be elucidated. VPS13B, a RAB6-interactor, was recently shown involved in endomembrane trafficking. Here, we report the generation of the first Vps13b-knockout mouse model and show that male mutant mice are infertile due to oligoasthenoteratozoospermia. This phenotype was explained by a failure of Vps13b deficient spermatids to form an acrosome. In wild-type spermatids, immunostaining of Vps13b and Rab6 revealed that they transiently locate to the acrosomal inner membrane. Spermatids lacking Vps13b did not present with the Golgi structure that characterizes wild-type spermatids and showed abnormal targeting of PNA- and Rab6-positive Golgi-derived vesicles to Eea1- and Lamp2-positive structures. Altogether, our results uncover a function of Vps13b in the regulation of the vesicular transport between Golgi apparatus, acrosome, and endolysosome.

Effects of assisted reproductive technologies on transposon regulation in the mouse pre-implanted embryo.

STUDY QUESTION: Do assisted reproductive technologies (ARTs) impact on the expression of transposable elements (TEs) in preimplantation embryos? SUMMARY ANSWER: The expression of all TE families is globally increased with mouse embryo culture with differences according to culture medium composition. WHAT IS KNOWN ALREADY: Mammalian genomes are subject to global epigenetic reprogramming during early embryogenesis. Whether ARTs could have consequences on this period of acute epigenetic sensitivity is the matter of intense research. So far, most studies have examined the impact of ARTs on the regulation of imprinted genes. However, very little attention has been given to the control of TEs, which exceed by far the number of genes and account for half of the mammalian genomic mass. This is of particular interest as TEs have the ability to modulate gene structure and expression, and show unique regulatory dynamics during the preimplantation period. STUDY DESIGN, SIZE, DURATION: Here, we evaluated for the first time the impact of ART procedures (superovulation, in-vitro fertilisation and embryo culture) on the control of different TE types throughout preimplantation development of mouse embryos. We also made use of a mouse model carrying a LINE-1 retrotransposition-reporter transgene to follow parental patterns of transmission and mobilisation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Hybrid B6CBA/F1 mice were used for the expression analyses. Relative TE expression was evaluated by using the nCounter quantification methodology (Nanostring®). This quantitative method allowed us to simultaneously follow 15 TE targets. Another technique of quantification (RTqPCR) was also used.A mouse model carrying a LINE-1 retrotransposition-reporter transgene (LINE-1 GF21) was used to follow parental patterns of transmission and mobilisation. MAIN RESULTS AND THE ROLE OF CHANCE: We found that the superovulation step did not modify the dynamics nor the level of TE transcription across the preimplantation period. However, upon in-vitro culture, TE expression was globally increased at the blastocyst stage in comparison with in-vivo development. Finally, by monitoring the transmission and mobilisation of a transgenic LINE-1 transposon, we found that in-vitro fertilisation may alter the mendelian rate of paternal inheritance. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Even though the Nanostring results concerning the dynamics of transcription throughout preimplantation development were based on pools of embryos originating from several females, only two pools were analysed per developmental stage. However, at the blastocyst stage, consistent expressional results were found between the Nanostring technology and the other technique of quantification used, RTqPCR. WIDER IMPLICATIONS OF THE FINDINGS: Our findings highlight the sensitivity of TEs to the ART environment and their great potential as biomarkers of culture medium-based effects. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by funding from the 'Agence de la Biomedecine', 'Conseil Régional de Bourgogne' and 'RCT grant from INSERM-DGOS'. The authors have no conflicts of interest to declare.

Assessing the influence of distinct culture media on human pre-implantation development using single-embryo transcriptomics.

The use of assisted reproductive technologies is consistently rising across the world. However, making an informed choice on which embryo culture medium should be preferred to ensure satisfactory pregnancy rates and the health of future children critically lacks scientific background. In particular, embryos within their first days of development are highly sensitive to their micro-environment, and it is unknown how their transcriptome adapts to different embryo culture compositions. Here, we determined the impact of culture media composition on gene expression in human pre-implantation embryos. By employing single-embryo RNA-sequencing after 2 or 5 days of the post-fertilization culture in different commercially available media (Ferticult, Global, and SSM), we revealed medium-specific differences in gene expression changes. Embryos cultured pre-compaction until day 2 in Ferticult or Global media notably displayed 266 differentially expressed genes, which were related to essential developmental pathways. Herein, 19 of them could have a key role in early development, based on their previously described dynamic expression changes across development. When embryos were cultured after day 2 in the same media considered more suitable because of its amino acid enrichment, 18 differentially expressed genes thought to be involved in the transition from early to later embryonic stages were identified. Overall, the differences were reduced at the blastocyst stage, highlighting the ability of embryos conceived in a suboptimal in vitro culture medium to mitigate the transcriptomic profile acquired under different pre-compaction environments.

DNA methylation profiles after ART during human lifespan: a systematic review and meta-analysis.

BACKGROUND: 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. OBJECTIVE AND RATIONALE: 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. SEARCH METHODS: 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. OUTCOMES: 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. WIDER IMPLICATIONS: 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.

Comparison of oocyte vitrification using a semi-automated or a manual closed system in human siblings: survival and transcriptomic analyses.

BACKGROUND: Indications of oocyte vitrification increased substantially over the last decades for clinical and ethical reasons. A semi-automated vitrification system was recently developed making each act of vitrification reproducible. In this study, we evaluated the efficiency of the semi-automated technique of oocyte vitrification by survival rate, morphometric assessment and resistance to empty micro-injection gesture as compared with a manual method. Additionally, we intended to evaluate transcriptomic consequences of both techniques using single-cell RNA-seq technology. RESULTS: Post-warming survival rate, oocyte surfaces and resistance to empty micro-injection were comparable between semi-automated and manual vitrification groups. Both oocyte vitrification techniques showed limited differences in the resulting transcriptomic profile of sibling oocytes since only 5 differentially expressed genes were identified. Additionally, there was no difference in median transcript integrity number or percentage of mitochondrial DNA between the two groups. However, a total of 108 genes were differentially expressed between fresh and vitrified oocytes (FDR < 0.05) and showed over-represented of genes related to important cellular process. CONCLUSIONS: Our results provide reassurance about the influence of semi-automation as compared with the manual vitrification method. Concerning oocyte vitrification itself, no tight common transcriptomic signature associated has been observed across studies. TRIAL REGISTRATION: NCT03570073.

Do frozen embryo transfers modify the epigenetic control of imprinted genes and transposable elements in newborns compared with fresh embryo transfers and natural conceptions?

OBJECTIVE: To determine whether the epigenetic control of imprinted genes (IGs) and transposable elements (TEs) differs at birth between fresh or frozen embryo transfers and natural conceptions. DESIGN: Prospective study. SETTING: University hospital. PATIENT(S): A total of 202 singleton births were divided into three groups: 84 natural pregnancies (controls), 66 in vitro fertilization/intracytoplasmic sperm injection with fresh embryo transfers, and 52 vitro fertilization/intracytoplasmic sperm injection with frozen embryo transfers. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Pyrosequencing was used to assess the DNA methylation profiles of three IGs (H19/IGF2:IG-DMR [two sequences], KCNQ1OT1:TSS-DMR, and SNURF:TSS-DMR) and two TEs (LINE-1 and HERV-FRD) in cord blood and placenta. The quantitative reverse transcriptase polymerase chain reaction was used to study the transcription of three IGs (H19, KCNQ1, and SNRPN) and two TEs (LINE-1 and ORF2). RESULT(S): After adjustment, the placental DNA methylation levels of H19/IGF2 were lower in the fresh embryo transfer group than in the control (H19/IGF2-seq1) and frozen embryo transfer (H19/IGF2-seq2) groups. The DNA methylation rate for LINE-1 was lower in placentas from the fresh embryo transfer group than in placentas from the control and frozen embryo transfer groups and for HERV-FRD compared with controls. In cord blood, DNA methylation levels were not significantly associated with the mode of conception. The relative expression of LINE-1 and ORF2 was decreased in both cord blood and placental tissues from fresh embryo transfer conceptions compared with natural conceptions and frozen embryo transfer conceptions. CONCLUSION(S): Compared with natural conceptions and frozen embryo transfers, fresh embryo transfers were associated with methylation and/or transcription changes in some TEs and IGs, mostly in placental samples, which could indicate altered placental epigenetic regulation resulting from ovarian stimulation protocols.

Genome-Wide Analysis of DNA Methylation in Buccal Cells of Children Conceived through IVF and ICSI.

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.

What impact does oocyte vitrification have on epigenetics and gene expression?

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.

Sperm imprinting integrity in seminoma patients?

BACKGROUND: 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. RESULTS: 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). CONCLUSIONS: 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.