Ruiwei Jiang

miR-181a increases FoxO1 acetylation and promotes granulosa cell apoptosis via SIRT1 downregulation

Oxidative stress impairs follicular development by inducing granulosa cell (GC) apoptosis, which involves enhancement of the transcriptional activity of the pro-apoptotic factor Forkhead box O1 (FoxO1). However, the mechanism by which oxidative stress promotes FoxO1 activity is still unclear. Here, we found that miR-181a was upregulated in hydrogen peroxide (H2O2)-treated GCs and a 3-nitropropionic acid (NP)-induced in vivo model of ovarian oxidative stress. miR-181a overexpression promoted GC apoptosis, whereas knockdown of endogenous miR-181a blocked H2O2-induced cell apoptosis. Moreover, we identified that Sirtuin 1 (SIRT1), a deacetylase that suppresses FoxO1 acetylation in GCs, was downregulated by miR-181a and reversed the promoting effects of H2O2 and miR-181a on FoxO1 acetylation and GC apoptosis. Importantly, decreased miR-181a expression in the in vivo ovarian oxidative stress model inhibited apoptosis by upregulating SIRT1 expression and FoxO1 deacetylation. Together, our results suggest that miR-181a mediates oxidative stress-induced FoxO1 acetylation and GC apoptosis by targeting SIRT1 both in vitro and in vivo.

Activating transcription factor 3 promotes embryo attachment via up-regulation of leukemia inhibitory factor in vitro

BackgroundA receptive endometrium is essential for maternal-embryonic molecular communication during implantation. However, the specific molecular regulatory mechanisms of the endometrial capacity remain poorly understood. Here, we examined activating transcription factor 3 (ATF3) expression in human endometria and the functional effect of ATF3 on embryo attachment in vitro.MethodsImmunohistochemistry (IHC) was used to assess the ATF3 expression patterns in human endometria. Quantitative real-time PCR (qRT-PCR), western blotting and immunofluorescence (IF) studies were applied to explore ATF3 expression in Ishikawa cells upon estrogen (E2) and medroxyprogesterone acetate (MPA) treatment. qRT-PCR and western blotting were performed to inspect LIF (leukemia inhibitory factor) expression after enhancement or inhibition of ATF3, and a luciferase reporter assay and ChIP-PCR were used to confirm the regulatory mechanism of ATF3 to LIF. Endometrial epithelial capacity was assessed by an in vitro model of attachment of BeWo spheroids to Ishikawa cells. Western blotting was performed to compare the expression of ATF3 in endometrial samples of recurrent implantation failure (RIF) patients with that in samples from fertile women (FER) who had undergone no less than one successful embryo transplantation.ResultsATF3 was located in human endometrial epithelial cells and stromal cells and was significantly induced by E2 and MPA in Ishikawa cells. Adenovirus-mediated overexpression of ATF3 in Ishikawa cells activated LIF promoter activity and enhanced its expression. Accordingly, the stimulation of BeWo spheroid adhesion promoted by ATF3 was inhibited by pretreatment with a specific antibody against LIF via the antibody-blocking assay. Moreover, ATF3 was aberrantly decreased in the endometria of RIF patients.ConclusionsOur findings suggest that ATF3 plays a significant role in regulating human endometrial receptivity and embryo attachment in vitro via up-regulation of leukemia inhibitory factor.Trial registrationConstruction and management of the Nanjing multi-center biobank. No. 2013-081-01. Registered 10 Dec. 2013.

Different Angiogenic Potentials of Mesenchymal Stem Cells Derived from Umbilical Artery, Umbilical Vein, and Wharton’s Jelly

Human mesenchymal stem cells derived from the umbilical cord (UC) are a favorable source for allogeneic cell therapy. Here, we successfully isolated the stem cells derived from three different compartments of the human UC, including perivascular stem cells derived from umbilical arteries (UCA-PSCs), perivascular stem cells derived from umbilical vein (UCV-PSCs), and mesenchymal stem cells derived from Whartons jelly (WJ-MSCs). These cells had the similar phenotype and differentiation potential toward adipocytes, osteoblasts, and neuron-like cells. However, UCA-PSCs and UCV-PSCs had more CD146+ cells than WJ-MSCs (P < 0.05). Tube formation assay in vitro showed the largest number of tube-like structures and branch points in UCA-PSCs among the three stem cells. Additionally, the total tube length in UCA-PSCs and UCV-PSCs was significantly longer than in WJ-MSCs (P < 0.01). Microarray, qRT-PCR, and Western blot analysis showed that UCA-PSCs had the highest expression of the Notch ligand Jagged1 (JAG1), which is crucial for blood vessel maturation. Knockdown of Jagged1 significantly impaired the angiogenesis in UCA-PSCs. In summary, UCA-PSCs are promising cell populations for clinical use in ischemic diseases.

Enhanced HOXA10 sumoylation inhibits embryo implantation in women with recurrent implantation failure

HOXA10 has emerged as an important molecular marker of endometrial receptivity. Recurrent implantation failure (RIF) after in vitro fertilization-embryo transplantation (IVF-ET) treatment is associated with impaired endometrial receptivity, but the exact underlying mechanism of this phenomenon remains elusive. Here we found that HOXA10 was modified by small ubiquitin like-modifier 1 (SUMO1) at the evolutionarily conserved lysine 164 residue. Sumoylation inhibited HOXA10 protein stability and transcriptional activity without affecting its subcellular localization. SUMO1-modified HOXA10 expression was decreased in estradiol- and progesterone-treated Ishikawa cells. Sumoylation inhibited the accelerant role of HOXA10 in BeWo spheroid and mouse embryo attachment to Ishikawa cells. Importantly, aberrantly high SUMO1-HOXA10 expression was detected in mid-secretory endometria of women with RIF compared with that of the control fertile women. Together, our results suggest that HOXA10 sumoylation impairs the process of embryo implantation in vitro and takes part in the development of RIF.

Transplantation of UC-MSCs on collagen scaffold activates follicles in dormant ovaries of POF patients with long history of infertility

Premature ovarian failure (POF) is a refractory disease for clinical treatment with the goal of restoring fertility. In this study, umbilical cord mesenchymal stem cells on a collagen scaffold (collagen/UC-MSCs) can activate primordial follicles in vitro via phosphorylation of FOXO3a and FOXO1. Transplantation of collagen/UC-MSCs to the ovaries of POF patients rescued overall ovarian function, evidenced by elevated estradiol concentrations, improved follicular development, and increased number of antral follicles. Successful clinical pregnancy was achieved in women with POF after transplantation of collagen/UC-MSCs or UC-MSCs. In summary, collagen/UC-MSC transplantation may provide an effective treatment for POF.

Increased Krüppel-like factor 12 impairs embryo attachment via downregulation of leukemia inhibitory factor in women with recurrent implantation failure

Recurrent implantation failure (RIF) caused by various etiological factors remains a challenge for fertility clinicians using assisted reproductive technology (ART) worldwide. Dysregulation of leukemia inhibitory factor (LIF) in the endometria of women with RIF is involved in impaired endometrial receptivity and embryo adhesion. However, the mechanism through which LIF expression is regulated in women with RIF is still poorly understood. Our previous study noted that the abnormally increased endometrial Krüppel-like factor 12 (KLF12) in RIF women led to impaired decidualization and embryo implantation. Here, we further found that KLF12 inhibited embryo adhesion in vivo and in vitro by repressing LIF expression. Mechanistically, KLF12 bound to conserved sites (CAGTGGG, −6771 to −6765 and −7115 to −7109) within the LIF promoter region and repressed LIF transcription directly. Exogenous LIF significantly reversed the KLF12-mediated repression of BeWo spheroid adhesion. KLF12 expression was reduced significantly in Ishikawa cells treated with progestogen, which was due to the activation of Akt signaling. These findings may provide novel potential therapeutic regimens for patients with RIF and disrupted endometrial receptivity.

Calpain7 impairs embryo implantation by downregulating β3-integrin expression via degradation of HOXA10

Endometriosis (ENDO) is a common gynecological disease that causes infertility in many women. Previous studies noted that the dysregulation of Homeo box A10 (HOXA10) in the endometrium of women with ENDO was involved in the failure of embryo implantation. However, the mechanism by which HOXA10 expression is reduced in women with ENDO is still poorly understood. Here we found that a member of the calcium (Ca2+)-dependent cysteine protease family calpain7 (CAPN7), negatively correlated with HOXA10, was highly expressed in the endometrium of infertile women with ENDO and was significantly downregulated during the window of embryo implantation in mice. Overexpression of CAPN7 in Ishikawa cells or in the uterus of mice inhibited embryo implantation in vitro and in vivo. In the current study, we identified a sequence rich in proline, glutamic acid, serine, and threonine (PEST sequence) that enhanced the Ca2+-dependent degradation of HOXA10 by CAPN7. Furthermore, the interaction between HOXA10 and CAPN7 repressed the transcriptional activity and protein stability of HOXA10. In contrast, the administration of the calpain inhibitor ALLN reversed the CAPN7-induced HOXA10 degradation. Moreover, truncation of the PEST motif in HOXA10 abolished its CAPN7-dependent proteolysis. These studies reveal a novel pattern of HOXA10 regulation via PEST sequence-mediated calpain proteolysis that was demonstrated to be reversed by a calpain inhibitor. Thus, the inhibition of CAPN7-induced HOXA10 degradation may represent a novel potential therapeutic method to improve impaired embryo implantation in women with ENDO.

Increased Krüppel-like factor 12 in recurrent implantation failure impairs endometrial decidualization by repressing Nur77 expression

BackgroundDecidualization is a prerequisite for successful implantation and the establishment of pregnancy. A critical role of impaired decidualization in subfertility has been established. In human endometrial stromal cells (hESCs), Krüppel-like factor 12 (KLF12) and Nur77 are novel regulators of decidualization. We investigated whether KLF12 impaired the decidualization of hESCs in recurrent implantation failure (RIF) patients.MethodsEndometrial tissues and hESCs were collected from RIF patients (n = 34) and fertile controls (n = 30) for in vitro analysis. Primary hESCs isolated from RIF endometrial tissues were used to evaluate the biological functions of KLF12 and Nur77. In addition, their molecular mechanisms were investigated by adenovirus-mediated overexpression. Gene expression regulation was examined by real-time-quantitative PCR (qRT-PCR), immunostaining and luciferase reporter assay. Further, blastocyst-like spheroid (BLS) and blastocyst implantation models were performed to examine the roles of KLF12 and Nur77 during embryo expansion on hESCs.ResultshESCs from the RIF patients showed a poor decidual response, mainly characterized by decreased decidual prolactin (dPRL) secretion, impaired transformation and limited BLS expansion. In addition, KLF12 expression was increased in endometrial tissues from the RIF patients compared with those from the fertile controls, especially in stromal compartments. The opposite results were observed for Nur77 expression in these tissues. KLF12 repressed hESC decidualization by decreasing Nur77 expression. Mechanistically, KLF12 bound to a conserved site in the Nur77 promoter region. Nur77 overexpression significantly reversed the KLF12-mediated repression of dPRL expression, decidual transformation and BLS/blastocyst expansion.ConclusionsKLF12 impairs endometrial decidualization by transcriptionally repressing Nur77, and Nur77 overexpression reverses the poor decidual response of hESCs in RIF patients.

Abnormal ratio of CD57+ cells to CD56+ cells in women with recurrent implantation failure

To define a more precise parameter for a better understanding of natural killer (NK) cells and its relation with regulatory T cells (Tregs) in women with recurrent implantation failure (RIF).