Liya Wang

Evaluation of DNA methylation status at differentially methylated regions in IVF-conceived newborn twins

OBJECTIVE To examine the effect of assisted reproductive technology on the stability of DNA methylation at differentially methylated regions (DMRs) in twins conceived by IVF. DESIGN Prospective clinical observational study. SETTING IVF center, university-affiliated teaching hospital. PATIENT(S) Fifty-nine pairs of twins were recruited, including 29 pairs conceived through IVF and 30 pairs of naturally conceived twins. INTERVENTION(S) Collection of umbilical cord blood samples. MAIN OUTCOME MEASURE(S) DNA was extracted from umbilical cord blood. Two maternally methylated regions (KvDMR1 and PEG1) and one paternally methylated region (H19/IGF2 DMR) were analyzed using bisulfite-based technologies. RESULT(S) Although H19/IGF2 DMR and KvDMR1 showed slightly more variable levels of methylation in IVF cases than in spontaneous cases, methylation indices did not reveal significant differences at three DMRs between IVF-conceived and naturally conceived twins. CONCLUSION(S) Our results suggest no significant increase in imprint variability at these DMRs, but the greater variance in the IVF twins has a biologically meaningful consequence and may be a topic for future investigation. Large cohorts are needed to systematically assess the potential epigenetic risk in twins conceived with IVF.

Assisted reproductive technologies impair the expression and methylation of insulin-induced gene 1 and sterol regulatory element-binding factor 1 in the fetus and placenta.

OBJECTIVE To evaluate the cholesterol metabolism linked to assisted reproductive technology (ART) by analyzing the expression levels and DNA methylation patterns of the insulin-induced gene (INSIG), sterol regulatory element-binding protein (SREBP), and SREBP cleavage-activating protein in the fetus and placenta. DESIGN Experimental research study. SETTING An IVF center, university-affiliated teaching hospital. PATIENT(S) Four patients groups were recruited: pregnancies after IVF/intracytoplasmic sperm injection (ICSI) (n = 55), natural pregnancies (n = 40), multifetal reduction after IVF/ICSI (n = 56), and multifetal reduction after controlled ovarian hyperstimulation (COH) (n = 42). INTERVENTION(S) Expression and DNA methylation of INSIG-SREBP- SREBP cleavage-activating protein in the fetus and placenta samples were determined. MAIN OUTCOME MEASURE(S) The expression and DNA methylation patterns were tested by real-time quantitative polymerase chain reaction (PCR) and pyrosequencing. RESULT(S) In the ICSI treatment group, significantly higher levels of triglycerides and apolipoprotein-B were observed in cord blood compared with controls. Meanwhile, in ICSI-conceived fetuses, the expression of INSIG1 was significantly higher, and methylation rates were lower, than in the IVF and control groups. Furthermore, in the placenta, the INSIG1 and SREBF1 transcripts were also significantly higher with lower methylation rates in the ICSI group than in the IVF and control groups. CONCLUSION(S) Our results indicated that the dysregulation of INSIG1 and SREBF1 caused by ART were observed not only in the fetus but also in the placenta, primarily in the ICSI group. However, the long-term sequelae of this dysregulation should be closely followed.

Genome-wide DNA methylation patterns in IVF-conceived mice and their progeny: A putative model for ART-conceived humans ☆

The aim of this study was to use a mouse model to gain an understanding of the safety of reproduction between humans conceived through assisted reproductive technology (ART). Mice derived from in vitro fertilization and embryo transfer (IVF-ET) were crossed. Their behavior, morphology, histology and genome-wide DNA methylation status in the brain were examined by the Morris water maze, H&E staining and methylated DNA immunoprecipitation coupled with DNA methylation microarrays. Although no significant differences in behavior or morphology were observed, we did find small clusters of CpG islands and promoters that were aberrantly methylated. Hypermethylation was more common than hypomethylation in each of the two generations. Some of the aberrant methylated promoters were validated by bisulfite sequencing. Our results show that IVF may slightly modify the somatic methylation pattern and that some of this aberrant methylation might be inherited by the following generation.

Alteration of fatty acid metabolism in the liver, adipose tissue, and testis of male mice conceived through assisted reproductive technologies: fatty acid metabolism in ART mice

BackgroundLipid metabolism plays important roles in the whole process of pregnancy. Previous studies have demonstrated abnormalities of lipid metabolism in the placentas of pregnancies obtained by assisted reproductive technology (ART). Therefore, we hypothesized that ART micromanipulation may affect lipid metabolism in offspring, and focused on the fatty acid metabolism in ART male offspring in this study.MethodsThe fatty acid metabolism in the liver, adipose tissue and testis was detected. The comparison between naturally conceived (NC), controlled ovarian hyperstimulation (COH), in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) mice was made to analyze the effect of ART on offspring. The mice models in this study included two age groups: adult group and old group. The fatty acid composition and the expression of lipid metabolism-related genes were analyzed by GC-MS and qRT-PCR.ResultsThe fatty acid composition in the liver and adipose tissue were significantly altered in ART mice, but no significant difference was found in the testis. In adipose tissue, ART mice showed decreased monounsaturated fatty acids (MUFAs) and increased polyunsaturated fatty acids (PUFAs) in both adult and old mice, while the alteration of saturated fatty acids (SFAs) in the adult disappeared in the old. In liver, the changes were much complex in adult mice, while increased MUFAs and decreased PUFAs were found in ART old mice. The activities of fatty acid metabolism-related enzymes and the expression of lipogenic and lipolytic proteins changed in ART groups, with the adult mice and old mice showing inconsistent alterations. Further analysis indicated that SFAs was closely associated with the alterations of fatty acid metabolism-related enzyme activities and the expression of lipogenic and lipolytic proteins. Furthermore, we also found that the effect of separated ART treatments on fatty acid metabolism varied with different ages and tissues.ConclusionsART treatments had effect on the fatty acid composition in adipose tissue and liver of male mice. The alteration of SFAs content was crucial for the regulation of fatty acid composition. These changes might have potential effects on the health of ART male offspring which need further investigation.

Human Sperm Devoid of Germinal Angiotensin-Converting Enzyme Is Responsible for Total Fertilization Failure and Lower Fertilization Rates by Conventional In Vitro Fertilization

ABSTRACT In conventional in vitro fertilization (IVF), complete failure of fertilization occurs in 5% to 15% of treatments. Although the causes may be unclear, sperm defects appear to be the major contributor. However, a convincing test is not yet available that can predict the risk of fertilization failure. In this study, we found that germinal angiotensin-converting enzyme (gACE) (also called testicular ACE) was undetectable in sperm from patients who had total fertilization failure (TFF) and lower fertilization rates (LFRs) by IVF based on Western blot and indirect immunofluorescence analyses. Additionally, almost all of the patients without gACE on sperm (23 of 25) manifested a TT genotype of the rs4316 single-nucleotide polymorphism of ACE. Overall, our results indicate that the absence of gACE expression is responsible for TFF and LFRs by IVF. The rs4316 polymorphism of ACE might be associated with infertility in those patients. We conclude that sperm lacking gACE may be recognized before commencing IVF and that the patients may be directed instead to consider intracytoplasmic sperm injection.

Altered expression of Armet and Mrlp51 in the oocyte, preimplantation embryo and brain of mice following oocyte in vitro maturation but postnatal brain development and cognitive function are normal

Despite the efforts to recapitulate the follicle environment, oocytes from in vitro maturation (IVM) have poorer developmental potential than those matured in vivo and the effects on the resultant offspring are of concern. The aim of this study was to determine altered gene expression in oocytes following IVM and to evaluate the expression of the arginine rich, mutated in early stage of tumors gene (Armet) and mitochondrial ribosomal protein L51 (Mrpl51) in embryos and brains of fetal/postnatal mice and the brain development of IVM offspring. An IVM mouse model was established while oocytes matured in vivo were used as the controls. Suppressive subtractive hybridization (SSH) and RT-PCR/western blot were used to analyze the differential expression of genes/proteins between IVM and the control group. HE staining and water maze were used to assess the histological changes in brain tissue and cognition of the offspring. The rates of fertilization, cleavage, and live birth were significantly decreased in IVM group. Thirteen genes were upregulated in IVM oocytes compared with the control, including Armet and Mrpl51. The higher level of Armet in IVM oocytes was retained in brain of newborn mice, which could be related to the upregulation of activating transcription factor 6 (Atf6) and X-box binding protein 1 (Xbp1), while Mrpl51 was expressed normally in brain of postnatal mice. No significant differences were detected in brain weight, neuronal counts, and the cognition in the offspring between the two groups. The present results suggested that IVM could affect the pregnancy outcome and the Armet and Mrpl51 gene/protein expression. The change in Armet expression lasted while the change of Mrpl51 disappeared after birth. However, the brain development of the offspring seemed to be unaffected by IVM.

Persistence and intergenerational transmission of differentially expressed genes in the testes of intracytoplasmic sperm injection conceived mice.

Intracytoplasmic sperm injection (ICSI) is commonly used to solve male infertility problems. Previous studies showed that early environmental exposure of an embryo may influence postnatal development. To detect whether ICSI operations affect the reproductive health of a male or his offspring, we established assisted reproductive technologies (ART) conceived mouse models, and analyzed gene expression profiles in the testes of both ICSI and naturally conceived (NC) newborn F1 mice using micro-array analysis. Among the differentially expressed genes, we focused on the expression of eight male reproduction-related genes. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) was used to analyze the expression of these genes in the testes of both adult and old F1 generation mice and adult F2 generation mice. Our results showed that down-regulated and somatic cell-expressed genes in newborn mice retained their differential expression patterns in adult and old F1 generation individuals, implying the persistence and fetal origin of the alteration in the expression of these genes. The intergenerational transmission of differential gene expression was observed, but most changes tended to be reduced in adult F2 generations. Controlled ovarian hyperstimulation (COH) and in vitro fertilization (IVF) mice models were added to explore the precise factors contributing to the differences in ICSI offspring. The data demonstrated that superovulation, in vitro culture, and mechanical stimulation involved in ICSI had a cumulative effect on the differential expression of these male reproductive genes.

Altered expressions and DNA methylation of imprinted genes in chromosome 7 in brain of mouse offspring conceived from in vitro maturation.

OBJECTIVES The aim of this research was to investigate the effects of in vitro maturation (IVM) on the expression of imprinted genes in offspring, and to identify possible regulatory mechanisms for these genes. RESULTS By using an IVM mouse model, mRNA expression of H19 was found down-regulated and Kcnq1ot1 was up-regulated in brains of IVM offspring. Of the differentially methylated regions (DMRs) of H19, CpG island 3 was found to have higher DNA methylation levels in IVM group compared with the control group; in contrast, Kcnq1ot1 showed a significant reduction in DNA methylation in IVM mice by using the bisulfite sequence PCR and pyrosequencing. However, Igf2 and Snrpn showed no significant differences in expression of mRNA or DNA methylation levels between the IVM and control groups. In conclusion, IVM could affect the expression of imprinted genes in the brains of IVM offspring, which could be induced by DNA methylation alterations.

Serum and Glucocorticoid Regulated Kinase 1 in Sodium Homeostasis

The ubiquitously expressed serum and glucocorticoid regulated kinase 1 (SGK1) is tightly regulated by osmotic and hormonal signals, including glucocorticoids and mineralocorticoids. Recently, SGK1 has been implicated as a signal hub for the regulation of sodium transport. SGK1 modulates the activities of multiple ion channels and carriers, such as epithelial sodium channel (ENaC), voltage-gated sodium channel (Nav1.5), sodium hydrogen exchangers 1 and 3 (NHE1 and NHE3), sodium-chloride symporter (NCC), and sodium-potassium-chloride cotransporter 2 (NKCC2); as well as the sodium-potassium adenosine triphosphatase (Na+/K+-ATPase) and type A natriuretic peptide receptor (NPR-A). Accordingly, SGK1 is implicated in the physiology and pathophysiology of Na+ homeostasis. Here, we focus particularly on recent findings of SGK1’s involvement in Na+ transport in renal sodium reabsorption, hormone-stimulated salt appetite and fluid balance and discuss the abnormal SGK1-mediated Na+ reabsorption in hypertension, heart disease, edema with diabetes, and embryo implantation failure.

Normal epigenetic inheritance in mice conceived by in vitro fertilization and embryo transfer

An association between assisted reproductive technology (ART) and neurobehavioral imprinting disorders has been reported in many studies, and it seems that ART may interfere with imprint reprogramming. However, it has never been explored whether epigenetic errors or imprinting disease susceptibility induced by ART can be inherited transgenerationally. Hence, the aim of this study was to determine the effect of in vitro fertilization and embryo transfer (IVF-ET) on transgenerational inheritance in an inbred mouse model. Mice derived from IVF-ET were outcrossed to wild-type C57BL/6J to obtain their female and male line F2 and F3 generations. Their behavior, morphology, histology, and DNA methylation status at several important differentially methylated regions (DMRs) were analyzed by Morris water maze, hematoxylin and eosin (H&E) staining, and bisulfite genomic sequencing. No significant differences in spatial learning or phenotypic abnormality were found in adults derived from IVF (F1) and female and male line F2 and F3 generations. A borderline trend of hypomethylation was found in H19 DMR CpG island 3 in the female line-derived F3 generation (0.40±0.118, P=0.086). Methylation status in H19/Igf2 DMR island 1, Igf2 DMR, KvDMR, and Snrpn DMR displayed normal patterns. Methylation percentage did not differ significantly from that of adults conceived naturally, and the expression of the genes they regulated was not disturbed. Transgenerational integrity, such as behavior, morphology, histology, and DNA methylation status, was maintained in these generations, which indicates that exposure of female germ cells to hormonal stimulation and gamete manipulation might not affect the individuals and their descendents.