Increased hepatic INSIG-SCAP-SREBP expression is associated with cholesterol metabolism disorder in assisted reproductive technology-conceived aged mice.

Although most children conceived by assisted reproductive technology (ART) are healthy, there are concerns regarding the potential long-term health implications of ART. It has been reported that alterations in insulin-induced gene (INSIG), sterol regulatory element binding protein (SREBP), and SREBP cleavage-activating protein (SCAP) are involved in cardiometabolic changes. Thus, ART mouse models were established via in vitro fertilization (IVF), intracytoplasmic injection (ICSI), and in vitro oocyte maturation (IVM). A significantly higher systolic blood pressure was identified in the IVM aged female mice. In addition, abnormalities in the blood lipids and liver function were identified in the IVM- or ICSI-conceived elderly mice. Furthermore, ICSI or IVM significantly affected the hepatic expression and methylation of INSIG-SCAP-SREBP from a young to old age. Our animal data indicated that ICSI or IVM result in a higher risk of cholesterol metabolism dysfunction in older mice, which may be associated with long-term alterations of INSIG-SCAP-SREBP.

Superovulation Induced Changes of Lipid Metabolism in Ovaries and Embryos and Its Probable Mechanism.

This research was intended to investigate the fetal origins of changed birth weight of the offspring born through assisted reproductive technology (ART). The association between hormone and lipid metabolism or body weight has been generally accepted, and as the basic and specific treatment in ART procedure, gonadotropin stimulation might have potential effects on intrauterine lipid metabolism. In our studies, the mice were superovulated with two doses of gonadotropin. The cholesterol metabolism in ovaries and the triglyceride metabolism in embryos were analyzed. The results showed gonadotropin probably accelerated luteinization and induced a longer time follicle development and ovulation, which resulted in histological and morphological alteration of ovary, and increased the cholesterol content and the expressions of steroidogenesis-related genes. In embryos, gonadotropin increased lipid accumulation and decreased fatty acid synthesis in a dose-dependent manner. Moreover, the changes of fatty acid composition were also shown in superovulation groups. Our studies firstly provided the evidence that the superovulation might affect the maternal and fetal lipid metabolism. These variations of lipid metabolism in our results may be associated with birth weight of ART infants.

Decreased expression of SAM68 in human testes with spermatogenic defects.

OBJECTIVE: To assess the expression patterns of SAM68 in the testes of azoospermic patients with normal and abnormal spermatogenesis. DESIGN: Retrospective study and in vitro study. SETTING: University hospital. PATIENT(S): Testicular biopsies of azoospermic men with normal spermatogenesis (OAZ; n=20), with maturation arrest at the spermatocyte stage (MA; n=20), and with Sertoli cell-only syndrome (SCOS; n=10). INTERVENTION(S): No interventions with patients. Knockdown of Sam68 was performed in the GC-2spd(ts) cell line. MAIN OUTCOME MEASURE(S): SAM68 expression was analyzed using quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot, and immunohistochemistry analysis in tissues. Moreover, Sam68 was knocked down in GC-2spd(ts) cells. Cell viability was measured using the MTT assay, and the apoptosis rate was detected using flow cytometry with the Annexin V-FITC kit. RESULT(S): Using qRT-PCR, the expression level of testicular SAM68 mRNA in MA and SCOS patients was statistically reduced compared with in OAZ patients. In addition, using qRT-PCR, Western blot, and immunohistochemistry analyses, mRNA and protein expressions of SAM68 were absent or barely detectable in testicular tissues in 45% (9 of 20) of patients with MA and in all patients with SCOS. Furthermore, decreased expression of Sam68 suppressed germ cell proliferation and induced apoptosis in transfected GC-2spd(ts) cells. CONCLUSION(S): Deficient SAM68 expression was observed in the human testis with MA at the spermatocyte stage and SCOS. These results may offer new perspectives on the molecular basis of abnormal spermatogenesis.

Human sperm devoid of germinal angiotensin-converting enzyme is responsible for total fertilization failure and lower fertilization rates by conventional in vitro fertilization.

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.

Epigenetic inheritance of paternally expressed imprinted genes in the testes of ICSI mice.

Worse reproductive health in the men born through intracytoplasmic sperm injection (ICSI) or other assisted reproductive techniques (ART) has been reported in many studies. However, owing to the interference of genetic and environmental factors, it is difficult to identify whether ICSI method would affect male reproductive health. Therefore, ART mouse models were established in this study. Besides semen quality, serum testosterone and histological analysis of testes, 6 paternally expressed imprinted genes were chosen to detect their expressions and methylation levels in testes of adult F1 and F2 mice. Although the phenotypic abnormalities weren't found, Kcnq1ot1, Mest, Peg3, Plagl1 and Snrpn in ICSI group showed lower expressions than those in naturally conceived (NC) group. The expressions of Kcnq1ot1, Peg3 and Snrpn in in vitro fertilization (IVF) conceived mice was lower than those in controlled ovarian hyperstimulation (COH) conceived mice, but higher than those in ICSI mice. Most differences between NC and ICSI group and between IVF and ICSI group were also represented in F2 generations. During the methylation analysis, no matter there was significant difference between compared groups, the changing trends of methylation level were almost opposite to their corresponding gene expressions. These results indicated that the differential expressions of paternally expressed genes occurred in testes of ICSI mice, which may be mediated by methylation modification. Both ICSI procedure and mechanical stimulation can induce intergenerational transmission of the epigenetic changes. In vitro culture and mechanical stimulation were the main factors inducing the down regulation of paternally expressed imprinted genes in testes.

An overview of studies on psychological well-being in children born following assisted reproductive technologies.

Over the course of the past 35 years, assisted reproductive technologies (ARTs) have been increasingly used worldwide, while debates on their safety have been generated. Birth defects and imprinting disorders were reported in previous research. Thus, the psychological development of children born following ARTs has become a major concern nowadays. This review gives a systematic view of psychological well-being of children conceived by different types of ART, including in vitro fertilization, intracytoplasmic sperm injection (ICSI), preimplantation genetic diagnosis/screening, and in vitro maturation. The previous studies are analyzed in three sections: (1) cognitive, motor, and language developments, (2) behavior problems and socio-emotional development, and (3) parent-child relationship. We conclude that although the majority of the studies on cognitive, motor, and language developments reported comparable achievements in the ART group vs. the naturally conceived group, lower intelligence quotient (IQ) scores, worse visual-motor ability or locomotor development, and delayed receptive language competence were found in the ART group. The results on the socio-emotional development were reassuring. As for the behavior problems, a higher prevalence of behavior problems existed in ART children; moreover, ICSI children were found to be at a higher risk of autism than the general population. Meanwhile, ART parents tended to have positive parental attitudes and be more protective of their children. Some suggestions for further research are also given in this review.

Alterations in the frequency of trinucleotide repeat dynamic mutations in offspring conceived through assisted reproductive technology.

STUDY QUESTION: How does the frequency of trinucleotide repeat dynamic mutations in offspring conceived through assisted reproductive technology (ART) compare with the frequency of these mutations in control offspring conceived from spontaneous pregnancies? SUMMARY ANSWER: There is a slight increase in dynamic mutation instability in offspring conceived through ART compared with the naturally conceived offspring. WHAT IS KNOWN ALREADY: There is evidence to suggest that ART can increase the risk of birth defects and karyotypic abnormalities. However, the accumulating evidence of an association between ART and de novo genetic aberrations is controversial. STUDY DESIGN, SIZE, DURATION: A prospective clinical observational study was performed on 246 families recruited from an in vitro fertilisation (IVF) centre at a tertiary-care, university-affiliated teaching hospital from 2008 to 2012. The study included 147 ART families [75 IVF and 72 intracytoplasmic sperm injection (ICSI)] in the study group and 99 natural-conception families in the control group. PARTICIPANTS, SETTING, METHODS: Parental, umbilical cord and infant peripheral blood samples were collected, and the trinucleotide repeats of the ATN1, AR, ATXN1, ATXN3, Huntington, DMPK and FMR-1 genes were investigated between the generations; these genes were chosen due to their ability to undergo dynamic mutation. The frequencies and sizes of the mutational repeats, as well as the intergenerational instability, were measured. MAIN RESULTS AND THE ROLE OF CHANCE: In 2466 transmissions identified in the ART offspring, 2.11% (n = 52/2466) of the alleles were unstable upon transmission, while in the control group offspring, the frequency of dynamic mutation was 0.77% (n = 10/1300); this difference was statistically significant (P < 0.01). The unstable transmission alleles were detected in 32 (2.48%) of the 1288 alleles from the IVF offspring and in 20 (1.70%) of the 1178 alleles from the ICSI offspring; both of these frequencies were significantly different from that of naturally conceived offspring (0.77%) (P < 0.01 and P < 0.05, respectively). However, there were no significant differences in the sizes of the mutational repeats or in the rates of expansion or contraction among the three groups (P > 0.05). The repeat copy numbers of the examined genes were found to be within the normal ranges in all parents and infants. LIMITATIONS, REASONS FOR CAUTION: One strength of our study is the relatively large sample size; we were able to detect mutations in seven common dynamic genes, and this large sample size allowed us to detect unstable alleles. Although we observed a clear alteration in the frequency of dynamic mutation in the ART offspring compared with controls, further studies are urgently needed to confirm this observation and determine the cause of this phenomenon. WIDER IMPLICATIONS OF THE FINDINGS: DNA microsatellite analysis provides an important tool to assess genomic instability. In this study, we report an association between ART and the frequency of dynamic mutation. The instability could be a reflection of the core infertility problem, the controlled ovarian hyperstimulation and/or the in vitro culture conditions.

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.

In vitro fertilization alters growth and expression of Igf2/H19 and their epigenetic mechanisms in the liver and skeletal muscle of newborn and elder mice.

Epidemiological studies have reported a higher incidence of growth disorders among newborns conceived by in vitro fertilization (IVF), suggesting that IVF may be disruptive to the process of embryonic and fetal growth. However, the long-term effects of IVF on the growth and molecular mechanisms remain unclear. Therefore, we evaluated the body weight of IVF mice from birth to the age of 1.5 yr. In addition, we analyzed gene expression of insulin-like growth factor 2 (Igf2), H19, Igf2 receptor (Igf2r), and miR-483 and their DNA methylation status using real-time quantitative PCR, Western blot, and pyrosequencing. The results showed that when compared with the in vivo group, the body weight of IVF mice was significantly higher at birth, but lower at 3 wk; in addition, gene expression of Igf2 was significantly up-regulated, with down-regulated expression of H19 and miR-483 in both liver and skeletal muscle. At the same time, there were significant differences in the DNA methylation rates of Igf2/H19 differentially methylated regions (DMRs) and the IGF2 protein expression between the two groups. In the IVF treatment group, the differences in growth and expression disappeared at 10 wk. However, at 1.5 yr of age, aberrant expressions of Igf2/H19, Igf2r, and miR-483 and changes in DNA methylation rates in the liver or skeletal muscle were again observed in IVF mice. Our results indicate that IVF causes alterations in mouse growth during the postnatal periods that may be associated with alterations in Igf2/H19 expression and likely involve the regulation of miR-483 and the methylation status of Igf2/H19 DMRs.

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.

BACKGROUND: Lipid 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. METHODS: The 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. RESULTS: The 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. CONCLUSIONS: ART 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.

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.

Poor ovarian response to gonadotropin stimulation is associated with low expression of follicle-stimulating hormone receptor in granulosa cells.

OBJECTIVE: To explore the importance of follicle-stimulating hormone receptor (FSHR) in granulosa cells in the ovarian response to gonadotropin stimulation. DESIGN: Prospective study. SETTING: A women's hospital in China. PATIENT(S): One hundred infertile women undergoing ovarian stimulation with recombinant follicle-stimulating hormone (rFSH). INTERVENTION(S): These women were divided into three groups: poor, moderate, and high responders, according to the number of follicles with diameter >/=14 mm. The FSHR expression at both mRNA and protein levels was determined by either reverse transcription-polymerase chain reaction or Western blot in granulosa cells. E(2) concentrations in serum and FSH levels in serum/follicular fluid (FF) were measured by electrochemiluminescence immunoassay. MAIN OUTCOME MEASURE(S): Relative expression of mRNA and protein of FSHR in granulosa cells, serum E(2), FSH level in serum and FF, and the number of mature follicles. RESULT(S): The expression of FSHR, at both the mRNA and protein levels, was significantly different among the three groups, with the lowest expression in the poor responders. The level of FSHR protein was positively correlated with the peak level of serum E(2) and the number of mature oocytes. FSH levels in FF and the dosage of rFSH used were significantly different among the three groups, with the highest values in the poor responders. CONCLUSION(S): Different levels of FSHR expression in granulosa cells result in different ovarian response, and lower expression of FSHR may account for poor ovarian response to gonadotropin stimulation, which suggests the critical role of FSHR in the ovarian response to gonadotropin stimulation.

[Follicle-stimulating hormone receptor levels in relation to ovarian cycling response from gonadotropin hormone stimulation]

OBJECTIVE: To investigate the relationship between the ovarian response, induced by gonadotropin hormone, and levels of follicle-stimulating hormone receptor (FSHR). METHODS: The level of ovarian response was divided into three groups based on the number of follicles obtained during oocyte retrieval: poor responders (<4 follicles), normal responders (4 approximate, equals 13) and high responders(>13 follicles). FSHR mRNA was measured using reverse transcription polymerase chain reaction (RT-PCR). RESULTS: The expression of FSHR mRNA in the poor responders was 0.54+/-0.07. This was much lower than that of the normal responders who were at 0.90+/-0.17,and the high responders who were at 1.20+/-0.45(P<0.05). CONCLUSION: Ovarian response induced by gonadotropin hormone stimulation is correlated with the level of FSHR mRNA in the granulosa cells.