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.

Binding of the androgen receptor to the nuclear matrix of human foreskin.

The nuclear matrix (NM) is a salt and nuclease-resistant nuclear substructure. It is associated with active DNA transcription and has been shown to contain acceptor sites for steroid receptors in a number of specific target tissues. We have investigated the presence of acceptor sites for the androgen receptor (AR) in the NM of human newborn foreskin. The NM was prepared from the 800 g pellet by successive treatments with detergent, DNase and high salt extraction. It contained 13 +/- 7% of total proteins and 10 +/- 6% of total DNA. After extensive washing, the NM spheres were incubated in the presence of cytosol and [3H]methyltrienolone +/- 200-fold excess of unlabeled steroid. Maximal binding of the AR to NM was reached in 30 min and decreased slightly thereafter to reach an equilibrium which was maintained for 18 h. Binding was saturable. In the absence of AR, the steroid did not bind to NM. When Scatchard analysis was performed on cytosol previously incubated with NM, cytosolic binding capacity significantly decreased relative to preincubation values (3.6 +/- 1.9 to 1.3 +/- 1.2 fmol/mg protein, P less than 0.05, n = 6). In contrast, apparent binding affinity was not changed. 0.8 mg of NM protein could bind AR from 2.4 mg of cytosol protein. In conclusion, NM from human foreskin binds the AR with high affinity. This binding is rapid and is maintained for at least 18 h. This is consistent with a potential role of NM in the mechanism of action of androgens in their target tissues.

Decreased urinary 5 alpha-androstane-3 alpha,17 beta-diol glucuronide excretion in patients with benign prostatic hyperplasia.

Urinary testosterone and 3 alpha-androstanediol (3 alpha diol G) glucuronides together with plasma testosterone, 5 alpha-dihydrotestosterone (DHT), and delta 4-androstenedione (delta 4) were measured in 43 normal young men (18-36 yr old), 23 elderly men without clinically evident prostatic pathology (54-89 yr old), 68 elderly men with benign prostatic hyperplasia (BPH group; 54-91 yr old), and 26 elderly men with well differentiated cancer of the prostate (K group; 63-97 yr old). Plasma testosterone decreased slightly with age in all 3 elderly groups (from 591 to 438, 479, and 444 ng/100 ml, respectively). Plasma DHT, on the contrary, was significantly (P less than 0.01) higher in the BPH group than in the other three groups (68 vs. 30, 37, and 32 ng/100 ml, respectively). Plasma delta 4 was significantly lower (P less than 0.01) in the elderly K group than in all other groups (59 vs. 109, 83, and 78 ng/100 ml, respectively). Urinary testosterone glucuronide decreased with age in all 3 elderly groups (from 109 to 55, 38, and 44 micrograms/24 h, respectively) as a result of decreased androgen production rates with age. All 3 elderly groups also had decreased urinary 3 alpha diol G, from 194 to 123, 55, and 118 micrograms/24 h, respectively. The group of elderly patients with BPH had the lowest mean urinary 3 alpha diol G excretion together with the highest mean plasma DHT. This low urinary 3 alpha diol G excretion, which reflects a decrease in both androgen production and DHT metabolism, suggests a decrease in 3 alpha-hydroxysteroid dehydrogenase activity, which, in turn, could explain the increased DHT availability and tissue retention in most target organs. Moreover, the extent of these modifications in androgen metabolism specific to the BPH condition raises the question of an overall alteration of androgen metabolism in patients with BPH which could be the cause of the disease.

Androgen binding capacity and 5 alpha-reductase activity in pubic skin fibroblasts from hirsute patients.

We have measured the total (cytosolic plus nuclear) androgen binding capacity of pubic skin fibroblasts from nine patients with hirsutism of various origin. Confluent intact cell monolayers were incubated with increasing concentrations (0.05-2 nM) of [3H]dihydrotestosterone ([3H]DHT) with or without a 200-fold excess of unlabeled DHT. The androgen binding capacities (mean +/- SD) were similar in normal men (411 +/- 171 fmol/mg DNA), women (310 +/- 103 fmol/mg DNA), and hirsute patients (313 +/- 141 fmol/mg DNA) regardless of the plasma androgen levels. In contrast, the 5 alpha-reductase level in pubic skin fibroblasts (mean +/- SD) was, as previously described, higher in hirsute women (3.3 +/- 2.6 fmol/micrograms DNA . h) than in normal women (1.1 +/- 0.6 fmol/microgram DNA . h; P less than 0.05). We conclude from these data that: 1) increased androgen binding capacity cannot be held responsible for hypersensitivity to androgens in hirsutism; 2) the androgen receptor is not regulated by androgens in human skin, as similar levels are observed in men, women, and hirsute patients; 3) this contrasts with 5 alpha-reductase activity and emphasizes the importance of this enzyme as an amplifier of androgen action in areas where it is stimulated by androgens, such as pubic skin.