Xiao-Ling Hu

High Maternal Serum Estradiol Environment in the First Trimester Is Associated With the Increased Risk of Small-for-Gestational-Age Birth

CONTEXT There are increasing concerns that a disrupted endocrine environment may disturb the growth of the fetus. Assisted reproductive technology (ART) situates gamete/embryo in a supraphysiological estradiol (E2) environment and, thus, provides an ideal model to investigate this problem. OBJECTIVE Our objective was to investigate whether the maternal high-E2 environment in the first trimester increases the risks of low birth weight (LBW) and small-for-gestational-age (SGA) birth. METHODS In total, 8869 singletons born after fresh embryo transfer (ET) (n = 2610), frozen ET (n = 1039), and natural conception (NC) (n = 5220) and their mothers were included. Birth weight, LBW, SGA, and maternal serum E2 levels were investigated. RESULTS The mean serum E2 levels of women undergoing fresh ET at 4 and 8 weeks of gestation were significantly higher than those of the women undergoing frozen ET and the women with NC (P < .01). Serum E2 levels of women undergoing fresh ET at 4 and 8 weeks of gestation were positively correlated to those on the day of human chorionic gonadotropin (hCG) administration (r = 0.5 and r = 0.4, respectively; P < 0.01). The birth weight after fresh ET was significantly lower than that after frozen ET and NC (P < 0.01), with increased incidence of LBW and SGA (P < .05). Furthermore, in the fresh ET group, singletons of mothers with high E2 levels (≥10460 pmol/L on the day of hCG administration) had higher risks of LBW (P < .01) and SGA (P < .01) than those with low E2 levels, and maternal serum E2 level on the day of hCG administration negatively correlated with the birth weight (P < .01). CONCLUSIONS The maternal high-E2 environment in the first trimester is correlated with increased risks of LBW and SGA. Evaluation of serum E2 before ET should be adopted to reduce the possibility of high E2 exposure to gamete/embryo.

Aquaporin-1 Plays a Crucial Role in Estrogen-Induced Tubulogenesis of Vascular Endothelial Cells

CONTEXT Aquaporin-1 (AQP1) has been proposed as a mediator of estrogen-induced angiogenesis in human breast cancer and endometrial cancer. Elucidation of the molecular mechanisms governing AQP1-mediated, estrogen-induced angiogenesis may contribute to an improved understanding of tumor development. OBJECTIVE Our objective was to identify the estrogen-response element (ERE) in the promoter of the Aqp1 gene and investigate the effects and mechanisms of AQP1 on estrogen-induced tubulogenesis of vascular endothelial cells. SETTING The study was conducted in a university hospital in eastern China. MAIN OUTCOME MEASURES Immunohistological, real-time PCR and Western blot analyses were used to determine the expression AQP1 mRNA and protein in vascular endothelial cells. Chromatin immunoprecipitation analyses and luciferase reporter assays identified ERE-like motif in the promoter of the Aqp1 gene. RESULTS Expression of AQP1 in blood vessels of human breast and endometrial carcinoma tissues were significantly higher than controls. Estradiol (E2) dose-dependently increased the expression levels of AQP1 mRNA and protein in human umbilical vein endothelial cells (HUVECs). A functional ERE-like motif was identified in the promoter of the Aqp1 gene. AQP1 colocalized with ezrin, a component of the ezrin/radixin/moesin protein complex, and, ezrin colocalized with filamentous actin in HUVECs. Knockdown of AQP1 or ezrin with specific small interfering RNA significantly attenuated the formation of transcytoplasmic filamentous actin stress fibers induced by E2 and inhibited E2-enhanced cell proliferation, migration, invasion, and tubule formation of HUVECs. CONCLUSIONS Estrogen induces AQP1 expression by activating ERE in the promoter of the Aqp1 gene, resulting in tubulogenesis of vascular endothelial cells. These results provide new insights into the molecular mechanisms underpinning the angiogenic effects of estrogen.

Expression of aquaporins in human embryos and potential role of AQP3 and AQP7 in preimplantation mouse embryo development.

Background/Aims: Water channels, also named aquaporins (AQPs), play crucial roles in cellular water homeostasis. Methods: RT-PCR indicated the mRNA expression of AQPs 1-5, 7, 9, and 11-12, but not AQPs 0, 6, 8, and 10 in the 2∼8-cell stage human embryos. AQP3 and AQP7 were further analyzed for their mRNA expression and protein expression in the oocyte, zygote, 2-cell embryo, 4-cell embryo, 8-cell embryo, morula, and blastocyst from both human and mouse using RT-PCR and immunofluorescence, respectively. Results: AQP3 and AQP7 were detected in all these stages. Knockdown of either AQP3 or AQP7 by targeted siRNA injection into 2-cell mouse embryos significantly inhibited preimplantation embryo development. However, knockdown of AQP3 in JAr spheroid did not affect its attachment to Ishikawa cells. Conclusion: These data demonstrate that multiple aquaporins are expressed in the early stage human embryos and that AQP3 and AQP7 may play a role in preimplantation mouse embryo development.

High Maternal Serum Estradiol Levels Induce Dyslipidemia in Human Newborns via a Hepatic HMGCR Estrogen Response Element

While the intrauterine environment is essential for the health of offspring, the impact of high maternal serum estradiol (E2) on lipid metabolism in offspring and the mechanisms are unknown. We found that ovarian stimulation (OS) could result in high E2 levels in women throughout pregnancy. Strikingly, their newborns showed elevated total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) levels that were positively related with E2 in newborns. In vitro, E2 dose-dependently stimulated TC and LDL-C secretion, and increased expression of the cholesterol synthesis rate-limiting enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) in HepG2 cells and mouse fetal hepatocytes. In vivo, high maternal E2 was detected and fetal livers also showed significantly higher HMGCR expression in an OS mouse model. Notably, an estrogen response element (ERE) was identified in the HMGCR promoter, indicating that high maternal serum E2 could up-regulate HMGCR expression in fetal hepatocytes via an ERE that in turn induces elevated levels of TC and LDL-C in offspring. Conclusion: OS can induce a high maternal E2 environment, which up-regulates HMGCR expression in fetal hepatocytes via an ERE in the promoter, and induces elevated levels of TC and LDL-C in newborns that may be related to increased risk of metabolic disease in adulthood.

Binding of FGF2 to FGFR2 in an autocrine mode in trophectoderm cells is indispensable for mouse blastocyst formation through PKC-p38 pathway

Fibroblast growth factors (FGF1, FGF2 and FGF4) and fibroblast growth factor receptors (FGFR1, FGFR2, FGFR3 and FGFR4) have been reported to be expressed in preimplantation embryos and be required for their development. However, the functions of these molecules in trophectoderm cells (TEs) that lead to the formation of the blastocyst as well as the underlying mechanism have not been elucidated. The present study has demonstrated for the first time that endogenous FGF2 secreted by TEs can regulate protein expression and distribution in TEs via the FGFR2-mediated activation of PKC and p38, which are important for the development of expanded blastocysts. This finding provides the first explanation for the long-observed phenomenon that only high concentrations of exogenous FGFs have effects on embryonic development, but in vivo the amount of endogenous FGFs are trace. Besides, the present results suggest that FGF2/FGFR2 may act in an autocrine fashion and activate the downstream PKC/p38 pathway in TEs during expanded blastocyst formation.

Assisted Reproductive Technology and Gamete/Embryo-Fetal Origins of Diseases

Suboptimal intrauterine conditions may produce small for gestational age (SGA), and low birth weight (LBW) babies, that are predisposed to develop cardiovascular and metabolic disease in later life [1]. During assisted reproductive technology (ART) treatment, gametes and zygotes are exposed to a series of non-physiological processes and culture media with increasing evidence that offspring of ART have increasing chances of being low birth weight. An increasing incidence of early-onset hypertension has also been reported in the population of ART offspring. It is therefore important to understand whether ART plays any specific role in disadvantaging a fetus, and, whether any such disadvantage carries long-term consequences.

Impact of Galectin-1 on Trophoblast Stem Cell Differentiation and Invasion in In Vitro Implantation Model

Trophoblast stem cells (TSCs) differentiate in an orderly manner, which plays an important role in the process of embryo implantation, placentation, and early pregnancy maintenance. At the maternal–fetal interface, the dialogue is crucial between trophoblast cells and endometrial epithelial cells. Previous studies suggested that galectin-1 (Gal-1) may play an important role in placental development. In this study, we used Ishikawa (IK) cells–TSC coculture model to simulate the maternal–fetal interface and induce the differentiation of TSCs by differentiation media. The messenger RNA level of each cell type markers, fusion markers, and Gal-1 was detected by quantitative reverse transcription polymerase chain reaction during the differentiation of TSCs. Wound healing and transwell invasion assays were used to detect the migration and invasion ability in each group. We found that coculture with IK cells or conditioned media from IK cells could promote the differentiation and invasion of TSCs and increase Gal-1 expression in TSCs. Furthermore, recombinant Gal-1 could also promote the differentiation and invasion of TSCs, suggesting that some of IK cells secretion increase the expression of Gal-1 in TSCs during implantation, which then induced trophoblast differentiation and invasion in vitro. These findings provide significant insights into the biology of embryo–maternal interactions with the importance of Gal-1 in TSCs for the successful establishment and maintenance of pregnancy.

MiR-320d suppresses the progression of breast cancer via lncRNA HNF1A-AS1 regulation and SOX4 inhibition

MicroRNA-320d (miR-320d) is a novel cancer-related miRNA and functions as a tumor suppressor in human cancers. However, the expression pattern and function of miR-320d in breast cancer remain largely unknown. In the present study, we found that the expression level of miR-320d in breast cancer tissues and cells was significantly lower than in non-tumor tissues and MCF-10A cells. Decreased miR-320d was associated with poor overall survival in patients with breast cancer. Overexpression of miR-320d inhibited proliferation, migration, and invasion and promoted apoptosis of breast cancer cells. In addition, the long non-coding RNA, HNF1A antisense RNA 1 (HNF1A-AS1) was up-regulated in both breast cancer tissues and cell lines. HNF1A-AS1 suppressed the expression and function of miR-320d. Moreover, SRY-related HMG-box 4 (SOX4) was speculated and confirmed as a target of miR-320d. We also demonstrated that HNF1A-AS1 may function as a sponge competitive endogenous RNA for miR-320d, and thus regulate the expression of SOX4. Taken together, our study has identified a novel signaling pathway through which miR-320d exerts its anti-carcinogenic roles and suggested that the HNF1A-AS1/miR-320d/SOX4 may be a potential target for the therapy of breast cancer.

The Presence of Hepatitis B and C Virus in Human Gametes and Embryos

Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are major causes of chronic liver disease in both children and adults worldwide. Although the use of the HBV vaccine and hepatitis B immunoglobulin in the perinatal stage has effectively prevented most perinatal transmissions of HBV, 5–10 % of infections still cannot be prevented using these methods. More and more studies have shown that the viruses are not restricted to any particular organ, but occur in many extrahepatic tissues, including host somatic cells, oocytes, spermatozoa and embryos. As there is no ethical reason to reject in vitro fertilisation treatment in HBV/HCV-infected patients, many experts suggest that these patients should be given the opportunity to have children. However, the main question is that infertility treatment in couples where one or both parents are infected with hepatitis raises many concerns about transmission of the infection to the baby, laboratory technicians, and medical staff, and contamination of other gametes/embryos that are from virus-free parents in the same laboratory. In this chapter, we discuss the evidence related to HBV/HCV-infected gametes and embryos, and assisted reproductive technology for HBV/HCV-infected infertility couples.

Impact of Axis of GHRH and GHRH Receptor on Cell Viability and Apoptosis of the Placental Choriocarcinoma Cell Line.

Although GHRH and GHRH-R are recognized as key factors in placental development, little is known about the mechanism(s) of the regulation in trophoblastic cells during placental development. The objective of this study is to determine the potential relationship between the expression levels of GHRH-R and the placental and JEG-3 cell function. Furthermore, we aim to investigate the downstream pathways of GHRH/GHRH-R axis in the control of the JEG-3 cell viability and apoptosis. In this study, we detected the expression pattern of GHRH-R in human chorionic villous tissues and JEG-3 cell. Then, we evaluated the effects of GHRH/GHRH-R and the downstream pathways by using GHRH antagonist (JMR-132) on JEG-3 cell. Our present study found the expressions of GHRH-R in placental villous tissues and JEG-3 cell, and the expression levels of GHRH-R was significantly lower in villous tissues of early pregnancy loss when compared to normal controls. JMR-132 inhibited cellular viability and induced apoptosis in JEG-3 cell in a time and dosedependent manners through activation of caspase-3, p38, and p53, as well as inhibition of phosphorylation of Akt. Interestingly, ER stress markers such as GRP78, ubiquitinated proteins and phospho-eIF2α were significantly increased in JEG-3 cell after being treated with JMR-132. Conversely, pretreated with salubrinal (a selective inhibition of protein phosphatase 1-mediated eIF2α dephosphorylation), JEG-3 cells were rescued from JMR-132-mediated cell growth inhibition, and abolished JMR-132-induced cleaved caspase-3, CHOP, phospho-p53, and ubiquitinated proteins accumulation. Knockdown of endogenous GHRH-R significantly abolished the JMR-132-induced cleaved caspase-3 and activation of p38. In conclusion, our results, for the first time, demonstrated the expression levels of GHRH-R were closely related to the placental function. Inhibition of GHRH-R by using GHRH antagonist in JEG-3 cell may reduce cell viability and induce apoptosis through inactivation of Akt and ER stress via phosphorylation of eIF2α. These observations have enriched our understanding on the function of GHRH/GHRH-R axis and the downstream pathways in the control of the placental development. The Most Important Aspect of the Paper: Our present study for the first time provided evidences that GHRH and GHRH-R loops involve in JEG-3 cell viability and apoptosis through Akt and eIF2α pathways.