Significance of placental CD200 expression in patients with preeclampsia: Comparison between early­ and late­onset patients.

Preeclampsia, characterized by high blood pressure and proteinuria during pregnancy, causes serious complications in both the mother and the fetus. Although there have been several studies on the causes of preeclampsia, the detailed mechanism of this disease remains unclear. Moreover, a few reports have focused on the causes of preeclampsia in number of weeks at onset. The present study aimed to elucidate the differences between early­ and late­onset preeclampsia. This study enrolled patients with preeclampsia from January 2014 to December 2020. They were classified into early­ (<34 weeks) and late­onset (≥34 weeks) preeclampsia groups. The expression profiles of 770 immune­related genes were studied in the placental tissue from five patients each in the early­ and late­onset groups. The expression of CD200 in the trophoblasts of the placenta of 26 and 27 patients in early­ and late­onset groups, respectively, was also analyzed using immunostaining. Analysis of extracted RNA indicated that CD200 was significantly upregulated in the early­onset group compared with late­onset group and normal control. Immunostaining for CD200 demonstrated a significantly increased expression in the early­onset group compared with the late­onset group. The present study demonstrated that upregulation of CD200, which belongs to the immunoglobulin superfamily and is recognized as a molecule that acts in immune tolerance via inhibition of classical macrophage activation, may be associated with early­onset preeclampsia, although it remains unknown whether upregulation of CD200 expression is a cause or effect of the development of early­onset preeclampsia. Early­onset preeclampsia might have a different mechanism from that of late­onset; thus, further studies are needed to clarify the mechanism of these conditions for adequate treatment.

NDRG1 is expressed in human granulosa cells: An implicative role of NDRG1 in the ovary.

Purpose: N-myc downstream-regulated gene 1 (NDRG1) is expressed in various human tissues and plays a role in regulating cellular proliferation, angiogenesis, and hypoxia sensing. However, the role of NDRG1 in the ovary remains poorly understood. Therefore, we investigated NDRG1 expression and the role of NDRG1 in the human ovary. Methods: Follicular fluid (FF) and luteinized granulosa cells were collected from follicles during oocyte retrieval. KGN cells were cultured with cobalt chloride (CoCl2, a hypoxia-mimicking agent) and/or echinomycin. mRNA, protein levels and secretion, and localization were assessed by real-time PCR, Western blotting, ELISA, and immunohistochemical analysis, respectively. KGN cells were also transfected with NDRG1 siRNA for 72 h. Results: NDRG1 protein was expressed in luteinized granulosa cells. NDRG1 concentration was positively correlated with vascular endothelial growth factor (VEGF) and progesterone concentrations in FF. CoCl2-induced hypoxic stress significantly increased NDRG1 and VEGF mRNA and protein and hypoxia-inducible factor-1α expression compared with those in the controls. The CoCl2-induced overexpression of NDRG1 and VEGF was suppressed by echinomycin. Transfection with NDRG1 siRNA significantly suppressed the release of progesterone in the culture medium. Conclusions: These results indicate that ovarian NDRG1 may play important roles in follicular development, especially in the early luteinization of pre-ovulatory follicles.

Hypoxia: Role of SIRT1 and the protective effect of resveratrol in ovarian function.

BACKGROUND: Ovarian function is closely related to the degree of vascular network development surrounding the ovary. Maternal aging-related construction defects in this vascular network can cause ovarian hypoxia, which impedes oocyte nutrient supply, leading to physiological changes in the ovaries and oocytes. The anti-aging gene Sirtuin 1 (SIRT1) senses and adapts to ambient stress and is associated with hypoxic environments and mitochondrial biogenesis. METHODS: The present study is a literature review focusing on investigations involving the changes in SIRT1 and mitochondrial expression during hypoxia and the cytoprotective effects of the SIRT1 activator, resveratrol. MAIN FINDINGS: Hypoxia suppresses SIRT1 and mitochondrial expression. Resveratrol can reverse the hypoxia-induced decrease in mitochondrial and SIRT1 activity. Resveratrol suppresses the production of hypoxia-inducible factor-1α and vascular endothelial growth factor proteins. CONCLUSION: Resveratrol exhibits protective activity against hypoxic stress and may prevent hypoxia- or aging-related mitochondrial dysfunction. Resveratrol treatment may be a potential option for infertility therapy.

Exposure to cigarette smoke affects endometrial maturation including angiogenesis and decidualization.

PURPOSE: To elucidate the effects of cigarette smoking on human endometrial maturation for reproductive function, the authors examined the in vitro effects of cigarette smoke extract (CSE) on angiogenesis and decidualization in primary human endometrial stromal cells (ESCs). METHODS: Endometrial stromal cells were cultured with CSE and/or estradiol-17ß (E2) and medroxyprogesterone acetate (MPA). The mRNA, protein levels, and protein secretion of the angiogenic factors and decidual specific factors were assessed using real-time polymerase chain reaction, Western blot analysis, and enzyme-linked immunosorbent assay, respectively. Decidualization was also monitored by the changes in cellular morphology. RESULTS: Endometrial stromal cell proliferation substantially decreased after dose-dependent treatments with CSE at concentrations above 1%, whereas cell death was induced at treatment concentrations above 1% CSE. Treatments above 0.025% CSE led to increased vascular endothelial growth factor mRNA through hypoxia-inducible factor-1α accumulation. CSE concentrations at 0.01% and 0.025% increased the prolactin expression levels after treatment with E2 and MPA, whereas 0.1% and 0.25% CSE concentrations suppressed prolactin. Similar tendencies were observed in cellular morphology and other decidual specific factors. CONCLUSION: These results suggest that exposure to cigarette smoke affects endometrial appropriate maturation including the processes of angiogenesis and decidualization in the reproductive system.

Glucose transporter 1 is important for the glycolytic metabolism of human endometrial stromal cells in hypoxic environment.

AIM: The study aimed to elucidate the glycolytic metabolism of human endometrial stromal cells (hESCs) in hypoxic environment. MAIN METHODS: The hESCs were cultured in hypoxic environment, and their metabolic pathways were analyzed using metabolomics. We assessed glucose uptake using 2-deoxyglucose (2-DG) assay. The expression of glucose transporters (GLUTs) required for glucose uptake was determined using real-time quantitative polymerase chain reaction (qPCR) and western blotting. Furthermore, we knocked down GLUT1 and examined the uptake of 2-DG. KEY FINDINGS: Under hypoxia, glucose-6-phosphate, fructose-6-phosphate, and fructose-1,6-diphosphate were significantly elevated in hESCs (P < 0.05). This finding indicated enhancement in glycolysis. The volume of glucose uptake increased significantly under hypoxia (P < 0.05). Hypoxia simultaneously induced the expression of GLUT1 and GLUT3 mRNA (P < 0.05) and attenuated the expression of GLUT8 (P < 0.05). Glucose uptake was significantly inhibited upon knockdown of GLUT1 (P < 0.0001). SIGNIFICANCE: These results demonstrated a very important role of glucose transport under hypoxia. Also, hESCs utilize glycolysis to adapt to hypoxic conditions that could occur in menstrual and implantation period. These findings pave the way to study implantation failure and tumors originating from the endometrium.

Resveratrol protects mitochondrial quantity by activating SIRT1/PGC-1α expression during ovarian hypoxia.

PURPOSE: Resveratrol is a well-known potent activator of sirtuin-1 (SIRT1). We investigated the direct effects of hypoxia and resveratrol on SIRT1/ peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α) pathways, vascular endothelial growth factor (VEGF), hypoxia-inducible factor (HIF)-1α, and mitochondrial quantity in a steroidogenic human ovarian granulosa-like tumor cell line (KGN) cells. METHODS: KGN cells were cultured with cobalt chloride (CoCl2; a hypoxia-mimicking agent) and/or resveratrol. The mRNA and protein levels, protein secretion, and intracellular localization were assessed by real-time PCR, Western blot analysis, ELISA, and immunofluorescence staining, respectively. Mitochondrial quantity was measured based on the mitochondrial DNA (mtDNA) copy number. RESULTS: CoCl2 simultaneously attenuated the levels of SIRT1 and mtDNA expression, and induced the levels of VEGF protein production. In contrast, resveratrol significantly increased the levels of SIRT1 and mtDNA copy number, but reduced VEGF production in normoxia. Resveratrol could recover CoCl2-suppressed SIRT1 and mtDNA expression and antagonize CoCl2-induced VEGF production. CoCl2 treatment resulted in a downregulation of PGC-1α expression, and this effect was recovered by resveratrol. Resveratrol significantly suppressed the production of the CoCl2-induced HIF-1α and VEGF proteins. CONCLUSION: These results suggest that resveratrol improves mitochondrial quantity by activating the SIRT1/PGC-1α pathway and inhibits VEGF induction through HIF-1α under hypoxic conditions.

Thyroid Hormone Facilitates in vitro Decidualization of Human Endometrial Stromal Cells via Thyroid Hormone Receptors.

Endometrial stromal cells differentiate into decidual cells through the process of decidualization. This differentiation is critical for embryo implantation and the successful establishment of pregnancy. Recent epidemiological studies have suggested that thyroid hormone is important in the endometrium during implantation, and it is commonly believed that thyroid hormone is essential for proper development, differentiation, growth, and metabolism. This study aimed to investigate the impact of thyroid hormone on decidualization in human endometrial stromal cells (hESCs) and define its physiological roles in vitro by gene targeting. To identify the expression patterns of thyroid hormone, we performed gene expression profiling of hESCs during decidualization after treating them with the thyroid hormone levothyroxine (LT4). A major increase in decidual response was observed after combined treatment with ovarian steroid hormones and thyroid hormone. Moreover, LT4 treatment also affected the regulation of many transcription factors important for decidualization. We found that type 3 deiodinase, which is particularly important in fetal and placental tissues, was upregulated during decidualization in the presence of thyroid hormone. Further, it was observed that progesterone receptor, an ovarian steroid hormone receptor, was involved in thyroid hormone-induced decidualization. In the absence of thyroid hormone receptor (TR), due to the simultaneous silencing of TRα and TRß, thyroid hormone expression was unchanged during decidualization. In summary, we demonstrated that thyroid hormone is essential for decidualization in the endometrium. This is the first in vitro study to find impaired decidualization as a possible cause of infertility in subclinical hypothyroidism (SCH) patients.

Concentration of stromal cell-derived factor-1 (SDF-1/CXCL12) in the follicular fluid is associated with blastocyst development.

PURPOSE: To study the association between stromal cell-derived factor-1 (SDF-1/CXCL12) and vascular endothelial growth factor (VEGF) concentrations in individual human ovarian follicles and IVF outcomes. METHODS: Concentrations of SDF-1 and VEGF in 261 follicular fluid samples were measured with enzyme-linked immunosorbent assay. IVF outcome parameters were included in fertilization rate, cleavage rate, embryo morphology on day 3, and blastocyst morphology on day 5. RESULTS: The follicular concentration of SDF-1 and VEGF was not significantly associated with fertilization and cleavage outcome, and embryo morphology. The rates of full blastocysts and good-quality blastocysts were significantly higher in follicles with an SDF-1 concentration of 275-350 pg/mL than in the follicles with SDF-1 concentrations of <200 and ≥350 pg/mL (P < 0.05). The follicular concentration of VEGF was not associated with the blastocyst morphology. CONCLUSION: Our findings showed that follicular concentration of SDF-1, and not VEGF, may be a valuable biochemical marker of blastocyst development.

Effects of the hypoxia-inducible factor-1 inhibitor echinomycin on vascular endothelial growth factor production and apoptosis in human ectopic endometriotic stromal cells.

Recent evidence points to a possible role for hypoxia-inducible factor (HIF)-1 in the pathogenesis and development of endometriosis. The objectives of this study were to investigate the critical role of HIF-1 in endometriosis and the effect of the HIF-1 inhibitor echinomycin on human ectopic endometriotic stromal cells (eESCs). Ectopic endometriotic tissues were obtained from 20 patients, who received an operation for ovarian endometriomas. We examined vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1) production, HIF-1 expression, cell proliferation and apoptosis of eESCs. Cobalt chloride (CoCl2) significantly induced expression of HIF-1α protein and VEGF production in a time-dependent manner in eESCs, but reduced SDF-1 production. VEGF production was significantly suppressed by treatment of 100 nM echinomycin without causing cell toxicity, but 0.1-10 nM echinomycin or 100 nM progestin had no significant effect. SDF-1 production was not affected by echinomycin treatment at any dose. Echinomycin inhibited cell proliferation and induced apoptotic cell death of the eESCs, and significantly inhibited expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL. Echinomycin inhibits VEGF production and induces apoptosis of eESCs by suppression of Bcl-2 and Bcl-xL. These findings suggest the unique therapeutic potential for echinomycin as an inhibitor of HIF-1 activation for endometriosis treatment.

Lipid-soluble fraction of Shakuyaku-kanzo-to inhibits myometrial contraction in pregnant women.

AIM: Shakuyaku-kanzo-to, a Kampo medicine composed equally of shakuyaku and kanzo, is an antispasmodic drug that can inhibit contraction of uterine smooth muscles in pregnant women and rats. We aimed to test the inhibitory effects of water- and lipid-soluble extracts of shakuyaku-kanzo-to, shakuyaku, and kanzo in order to identify the fraction responsible for inhibiting uterine smooth muscle contraction in pregnancy. MATERIAL AND METHODS: Myometrial tissues were obtained from pregnant women and rats. The water- and lipid-soluble fractions of shakuyaku-kanzo-to, shakuyaku, and kanzo were obtained using the method of Bligh and Dyer. Lipid-soluble fractions were also partially purified using thin-layer chromatography (TLC) with a chloroform : methanol : water (65:25:4 by volume) solvent system to yield four TLC fractions. The effect of each fraction on oxytocin-induced myometrial contraction was examined in vitro. RESULTS: Lipid-soluble fractions obtained from shakuyaku-kanzo-to and kanzo inhibited myometrial contraction; water-soluble fractions had no effect. Of the four TLC fractions, the inhibitory effect was greatest with TLC fraction 1 (0.75 < Rf value ≤ 1.0). Neither the water-soluble nor the lipid-soluble fraction from shakuyaku inhibited myometrial contraction. CONCLUSIONS: These results suggest that lipid-soluble substances with low polarity derived from kanzo are responsible for the inhibitory effect of shakuyaku-kanzo-to on myometrial contraction.

Regulation of decidualization and angiogenesis in the human endometrium: mini review.

AIM: The human endometrium is a dynamic tissue that undergoes regular cycles of menstruation, menstrual repair, proliferation and secretory differentiation in response to hypoxia and the female sex hormones. METHODS: We identified new target genes that are regulated by progesterone during the decidualization of human endometrial stromal cells (ESC), including interleukin-15 (IL-15), fibulin-1 (FBLN-1), and heart and neural crest derivatives expressed transcript 2 (HAND2). RESULTS: IL-15 is deeply involved in the hormonal control of the human endometrium by progesterone and may be important in embryo implantation. FBLN-1 has been shown to be an important extracellular matrix that mediates progesterone action in human ESC differentiation toward implantation. Moreover, progestin-induced HAND2 is a transcription factor that contributes to the increased levels of FBLN-1 in human ESC. Several mediators, including vascular endothelial growth factor (VEGF), angiopoietin (ANGPT) and stromal cell-derived factor 1 (SDF-1), regulate human endometrial angiogenesis. Hypoxia increased the expression of VEGF and decreased the expression of SDF-1 in ESCs. Furthermore, hypoxia reduced ANGPT1 levels in ESC; however, ANGPT2 levels were unaffected. Estradiol simultaneously induced the expressions of VEGF and SDF-1, suppressing ANGPT1 production. Therefore, hypoxia and estradiol caused an increase in the ANGPT2/ANGPT1 ratio. CONCLUSION: Hypoxia and female sex hormones are involved in the regulation of angiogenic factors in an independent manner in human ESC. Analysis of the process of decidualization and angiogenesis in the human endometrium would provide useful information for the fields of reproductive biology, regenerative medicine and tissue engineering.

Two-step inhibitory effect of kanzo on oxytocin-induced and prostaglandin F2α-induced uterine myometrial contractions.

We previously reported that shakuyaku-kanzo-to, a kampo medicine consisting of shakuyaku and kanzo, has an inhibitory effect on myometrial contractions in pregnant women. In this study, we evaluated the effects of kanzo, glycyrrhizin (a major component of kanzo), glycyrrhetinic acid (GA; a major metabolite of glycyrrhizin), shakuyaku, and paeoniflorin (a major component of shakuyaku) on agonist-induced contractions of the uterus of pregnant humans and rats. We prepared myometrial strips from the uterus of pregnant humans and rats and induced contractions with oxytocin (50 µU/mL) or prostaglandin F2α (PGF2α) (10(-7) or 10(-6) M). Kanzo (250 µg/mL) and GA (5 × 10(-6) M) inhibited the oxytocin-induced and PGF2α-induced contractions in pregnant human and rat myometrium, but shakuyaku (250 µg/mL), paeoniflorin (10(-5) M), and glycyrrhizin (10(-5) M) did not inhibit contractions in either. Interestingly, kanzo and GA showed an inhibitory effect after temporarily enhancing the PGF2α-induced contractions in the rat myometrium, but not in the human myometrium. These results suggest that kanzo has at least a two-step inhibitory effect on the myometrial contractions that originate from the kanzo itself and a metabolite of glycyrrhizin in kanzo. Furthermore, kanzo was found to be safe for inhibiting PGF2α-induced contractions in humans because it did not temporarily enhance PGF2α-induced contractions.

Requirement of heart and neural crest derivatives-expressed transcript 2 during decidualization of human endometrial stromal cells in vitro.

OBJECTIVE: To investigate the role of heart and neural crest derivatives-expressed transcript 2 (HAND2) during decidualization of human endometrial stromal cells (ESCs). DESIGN: In vitro experiment. SETTING: Research laboratory. PATIENT(S): Twenty-six patients undergoing hysterectomy for benign reasons. INTERVENTION(S): ESCs were cultured for 12 days with HAND2 small interfering RNA (siRNA) or nonsilencing RNA during decidualization by medroxyprogesterone acetate (MPA) and E2. MAIN OUTCOME MEASURE(S): Decidualization was monitored by changes in cellular morphology and the expression of several decidual-specific genes. RESULT(S): HAND2 siRNA effectively suppressed HAND2 levels in ESCs after 12 days of E2 + MPA treatment. ESCs cultured with HAND2 siRNA retained a long fibroblast-like shape, whereas the cells cultured with control siRNA transformed into enlarged polygonal cells. Silencing of HAND2 expression significantly reduced connexin-43 involved in the morphologic changes. HAND2 silencing significantly reduced the mRNA levels of fibulin-1, prolactin, tissue inhibitor of metalloproteinase 3, interleukin-15, and forkhead box O1A (FOXO1A), but had no effect on the mRNA levels of dickkopf-1, serum glucocorticoid kinase 1, and insulin-like growth factor-binding protein 5. HAND2 siRNA effectively suppressed the levels of nuclear FOXO1A protein as a regulator of decidualization. CONCLUSION(S): These results suggest that HAND2 plays a key role in the regulation of progestin-induced decidualization of human ESCs.

Effects of ovarian hormone treatment on the gene expression of muscarinic acetylcholine receptors in the ovariectomized rat myometrium.

We investigate the effects of ovarian hormone on the gene expression of muscarinic acetylcholine receptors (M1-M5) in the myometrium using real-time PCR and evaluate the relationships between their expression and that of ovarian hormone receptors (ERα, ERß, and PgR). Wistar rats were sham operated (SO) or ovariectomized (OVX) and treated with vehicle, estradiol (E2), progesterone (P4), or both E2 and P4 for 2 days beginning on postoperative day 33. M1 and M4 mRNA expressions were not detected in the myometrium. M2 mRNA expression did not change significantly in the OVX and OVX+P4 groups compared to the SO group, but increased significantly in the OVX+E2 group and was normalized in the OVX+E2P4 group. M3 mRNA expression increased significantly in the OVX and OVX+P4 groups compared to the SO group, but was normalized in the OVX+E2 and OVX+E2P4 groups. M5 mRNA expression did not change significantly in all experimental groups. ERα mRNA expression increased significantly in the OVX, OVX+E2, and OVX+P4 groups compared to the SO group, but was normalized in the OVX+E2P4 group. The changes in ERß mRNA expression were similar to those of M3 mRNA expression in all experimental groups. In contrast, the changes in PgR mRNA expression did not correspond with that of M2, M3, or M5 mRNA expression in any of the experimental groups. Additionally, we evaluated the relationship between the expression of muscarinic acetylcholine receptors and ovarian hormone receptors in estrus cycle. M2 mRNA expression increased significantly in diestus and metaestrus compared in proestrus and estrus. M3 mRNA expression increased significantly in only diestrus compared in the other stages. In contrast, M5 mRNA expression did not change in estrus cycle. The changes in ERα mRNA expression appeared to be similar to those of M2 in estrus cycle, but no significant difference was found. The changes in ERß mRNA expression were similar to those of M3 mRNA expression. The change in PgR mRNA expression increased significantly in diestrus compared in metaestrus, but did not correspond with that of M2, M3, or M5 mRNA expression in estrus cycle. When acetylcholine sensitivity in the myometrium was compared between diestrus and estrus, the sensitivity is significantly lower in estrus than in diestrus. These results suggest that ovarian hormones influence the expression of M2 and M3 in the myometrium by regulating the expression of hormone receptors. E2 may upregulate M2 via ERα, but P4 may downregulate M2 by inhibiting ERα via PgR. E2 may downregulate M3 by inhibiting ERß, but P4 may not regulate the expression of M3 and ERß. M5 may be a constitutive muscarinic receptor in the myometrium because neither E2 nor P4 influence the expression of M5. The combination of E2 and P4 may contribute the reproduction by quieting down the acetylcholine-induced myometrial contraction.

Progestin-induced heart and neural crest derivatives expressed transcript 2 is associated with fibulin-1 expression in human endometrial stromal cells.

OBJECTIVE: To investigate whether heart and neural crest derivatives expressed transcript 2 (HAND2) regulates fibulin-1 (FBLN1) expression during decidualization of human endometrial stromal cells (ESCs). DESIGN: In vitro experiment. SETTING: Research laboratory. PATIENT(S): Twenty-four patients undergoing hysterectomy for benign reasons. INTERVENTION(S): ESCs were cultured with various progestins (medroxyprogesterone acetate [MPA], norethisterone, levonorgestrel, dienogest, and P), E(2), dexamethasone, and/or 8-bromoadenosine 3', 5'-cyclic monophosphate (8-Br-cAMP). HAND2 and FBLN1 were silenced by small interfering RNA technology. MAIN OUTCOME MEASURE(S): HAND2 and FBLN1 expression levels were assessed by real-time polymerase chain reaction and Western blot analysis. RESULT(S): MPA or E(2) + MPA increased HAND2 mRNA levels in ESCs in a time- and dose-dependent manner, and this stimulatory effect was blocked by RU-486 (P receptor antagonist). HAND2 was increased by E(2) + MPA earlier than FBLN1. Simultaneous MPA and 8-Br-cAMP treatment synergistically enhanced HAND2 mRNA levels. P and all the progestins significantly increased HAND2 mRNA levels, whereas E(2), 8-Br-cAMP, or dexamethasone alone had no effect. Silencing of HAND2 expression significantly reduced FBLN1 expression, whereas FBLN1 silencing had no effect on HAND2 expression. CONCLUSION(S): These results suggest that progestin-induced HAND2 contributes to FBLN1 expression in human ESCs.

The concentration of human follicular fluid stromal cell-derived factor-1 is correlated with luteinization in follicles.

Stromal cell-derived factor-1 (SDF-1/CXCL12) and vascular endothelial growth factor (VEGF) are angiogenic factors that have possible roles in ovarian function. The objectives of this study were to investigate the association between the individual concentrations of SDF-1 and VEGF and sex steroid hormones in human preovulatory follicles and to verify the SDF-1 expression in ovarian follicles. Follicular fluid (FF) and luteinizing granulosa cells (LGCs) were collected from follicles at the time of oocyte retrieval. The concentrations of SDF-1, VEGF, estradiol (E2) and progesterone (P4) were determined by biochemical assay. The expression levels of SDF-1 mRNA and protein were analyzed by RT-PCR and immunohistochemical analysis, respectively. A total of 177 follicles were analyzed. The FF concentrations of SDF-1 and VEGF positively correlated with P4 concentrations (r = 0.457 and p < 0.01, r = 0.698 and p < 0.01, respectively), but did not correlate with E2 concentrations in FF. Furthermore, we confirmed that SDF-1 mRNA was expressed in LGCs and SDF-1 protein is present in the granulosa cells of the human ovary. Our findings suggest that SDF-1, as well as VEGF, may play important modulatory roles in early luteinization of human preovulatory follicles.

Hypoxic stress simultaneously stimulates vascular endothelial growth factor via hypoxia-inducible factor-1α and inhibits stromal cell-derived factor-1 in human endometrial stromal cells.

BACKGROUND: Hypoxia of the human endometrium is a physiologic event occurring during the perimenstrual period and the local stimulus for angiogenesis. The aim of this study was to investigate the effects of hypoxic stress on the regulation of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1/CXCL12), and the potential role of hypoxia-inducible factor-1α (HIF-1α) in the endometrium. METHODS: Human endometrial stromal cells (ESCs, n= 22 samples) were studied in vitro. ESCs were cultured under hypoxic and normoxic conditions and treated with cobalt chloride (CoCl2; a hypoxia-mimicking agent) and/or echinomycin, a small-molecule inhibitor of HIF-1α activity. The mRNA levels and production of VEGF and SDF-1 were assessed by real-time PCR and ELISA, respectively. The HIF-1α protein levels were measured using western blot analysis. RESULTS: Hypoxia simultaneously induced the expression of mRNA and production of VEGF and attenuated the expression and production of SDF-1 from ESCs in a time-dependent manner. Similar changes were observed in the ESCs after stimulation with CoCl2 in a dose-dependent manner. CoCl2 significantly induced the expression of HIF-1α protein, and its highest expression was observed at 6 h. Echinomycin inhibited hypoxia-induced VEGF production without affecting the HIF-1α protein level and cell toxicity and had no effect on SDF-1 secretion (P < 0.01). CONCLUSIONS: Hypoxia simultaneously acts to increase VEGF via HIF-1α and to decrease SDF-1 in a HIF-1α-independent manner in ESCs. These results indicate a potential mechanism for the action of hypoxic conditions that could influence angiogenesis in the human endometrium.

Angiopoietin 1 and angiopoietin 2 in follicular fluid of women undergoing a long protocol.

OBJECTIVE: To determine the concentrations of angiopoietin 1 (ANGPT1) and ANGPT2 in individual human preovulatory follicles in relation to their diameter or volume to clarify the role of these molecules in folliculogenesis. DESIGN: Prospective study. SETTING: Research laboratory at Kansai Medical University, Osaka, Japan. PATIENT(S): Twenty-three women undergoing IVF. INTERVENTION(S): On the day of oocyte retrieval, serum samples and follicular fluid (FF) from individual follicles were collected. We analyzed 348 follicles. MAIN OUTCOME MEASURE(S): ANGPT1 and ANGPT2 concentrations in FF and serum and oocyte recovery rates. RESULT(S): On average, ANGPT1 concentrations in FF were 150 times lower than those in serum, whereas ANGPT2 concentrations in FF were 8 times higher than those in serum. The concentrations of ANGPT1 in follicles with a diameter ≤17 mm were significantly higher than those in follicles with a diameter ≥18 mm. On the other hand, the concentrations of ANGPT2 in follicles with a diameter ≤17 mm were significantly lower than those in follicles with a diameter ≥18 mm. The ANGPT2/ANGPT1 ratio increased with enlargement of follicular diameter. ANGPT1 concentrations in FF decreased with follicular volume. ANGPT2 concentrations and the ANGPT2/ANGPT1 ratio in FF rose with follicular volume. The ANGPT2/ANGPT1 ratio in FF from the oocyte recovery group was significantly higher than that from the nonrecovery group. CONCLUSION(S): Our data suggested that the change in ANGPT1 and ANGPT2 levels may be associated with follicular growth and angiogenesis during the preovulatory period.

Concentrations of stromal cell-derived factor-1 and vascular endothelial growth factor in relation to the diameter of human follicles.

OBJECTIVE: To determine the concentrations of stromal cell-derived factor-1 (SDF-1/CXCL12) and vascular endothelial growth factor (VEGF) in individual human preovulatory follicles in relation to their diameter or volume for clarifying the role of these molecules in folliculogenesis. DESIGN: Prospective study. SETTING: Research laboratory at Kansai Medical University. PATIENT(S): Twenty-seven women undergoing IVF. INTERVENTION(S): Follicular fluid (FF) was collected from individual follicles. A total of 373 follicles were analyzed. MAIN OUTCOME MEASURE(S): The concentrations of SDF-1 and VEGF in FF and oocyte recovery rates. RESULT(S): The concentrations of SDF-1 and VEGF in follicles with a diameter ≤ 14 mm were significantly lower than those in follicles with a diameter ≥ 15 mm. The concentrations of SDF-1 and VEGF in FF increased with follicular diameters or volume, with concentrations peaking in follicles with a diameter of 18-20 mm or a volume of 3.6-5.0 mL. Furthermore, we found that there exists a positive correlation between the concentrations of SDF-1 and VEGF in FF from follicles ≤ 20 mm in diameter. The oocyte recovery rates increased with concentrations of SDF-1 and VEGF in FF. CONCLUSION(S): Our data suggest that SDF-1, as well as VEGF, may play an important role in follicular growth and development.