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.

Matrix Metalloproteinases in Human Decidualized Endometrial Stromal Cells.

Cyclic changes, such as growth, decidualization, shedding, and regeneration, in the human endometrium are regulated by the reciprocal action of female hormones, such as estradiol (E2), and progesterone (P4). Matrix metalloproteases (MMPs) and tissue inhibitors of MMPs (TIMPs) control the invasion of extravillous trophoblast cells after implantation. Several MMPs and TIMPs function in the decidua and endometrial stromal cells (ESCs). Here, we aimed to systematically investigate the changes in MMPs and TIMPs associated with ESC decidualization. We evaluated the expression of 23 MMPs, four TIMPs, and four anti-sense non-coding RNAs from MMP loci. Primary ESC cultures treated with E2 + medroxyprogesterone acetate (MPA), a potent P4 receptor agonist, showed significant down-regulation of MMP3, MMP10, MMP11, MMP12, MMP20, and MMP27 in decidualized ESCs, as assessed by quantitative reverse transcription PCR. Further, MMP15 and MMP19 were significantly upregulated in decidualized ESCs. siRNA-mediated silencing of Heart and Neural Crest Derivatives Expressed 2 (HAND2), a master transcriptional regulator in ESC decidualization, significantly increased MMP15 expression in untreated human ESCs. These results collectively indicate the importance of MMP15 and MMP19 in ESC decidualization and highlight the role of HAND2 in repressing MMP15 transcription, thereby regulating decidualization.

Transcriptional regulation of LGALS9 by HAND2 and FOXO1 in human endometrial stromal cells in women with regular cycles.

Uterine natural killer cells are regulated via surface inhibitory receptors for IL15 and galectin-9 (LGALS9) secreted by endometrial stromal cells (ESCs). However, the mechanism that regulates LGALS9 mRNA levels in ESCs is unclear. The aim of this study is to clarify the transcriptional regulation of LGALS9 in ESCs. Here, LGALS9 mRNA expression levels significantly decreased in the endometrial tissue in the early- to mid-secretory phase, and recovered in the mid- to late-secretory phase, compared to that in the proliferative phase. In ESCs, LGALS9 mRNA expression significantly decreased following estradiol + medroxyprogesterone acetate treatment for 1 day and increased after 12 days compared to that in the control. The transcriptional activity of the LGALS9 upstream region was upregulated by heart and neural crest derivatives expressed 2 (HAND2) and downregulated by forkhead box O1 (FOXO1). In ESCs, HAND2 expression significantly increased throughout the 12 days treatment with steroid hormones, whereas FOXO1 expression significantly increased on Day 1, reached a plateau, and significantly increased again after 6 days of treatment. Levels of FOXO1 phosphorylation (pFOXO1) remained unchanged after a 3-day treatment of ESCs with steroid hormones, but significantly increased following a 12-day treatment. pFOXO1 could not bind to the DNA and was thus unable to directly suppress LGALS9 transcription. Therefore, expression level of HAND2 and phosphorylation status of FOXO1 may determine LGALS9 mRNA expression. This study provides a novel molecular mechanism underlying the transcriptional regulation of LGALS9 mRNA in ESCs, which could be valuable in the treatment of diseases associated with decidualization failure.

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.

The transcription factor HAND2 up-regulates transcription of the IL15 gene in human endometrial stromal cells.

Cyclic changes of the human endometrium, such as proliferation, secretion, and decidualization, occur during regular menstrual cycles. Heart- and neural crest derivatives-expressed transcript 2 (HAND2) is a key transcription factor in progestin-induced decidualization of human endometrial stromal cells (ESCs). It has been suggested that HAND2 regulates interleukin 15 (IL15), a key immune factor required for the activation and survival of uterine natural killer (uNK) cells. Activated uNK cells can promote spiral artery remodeling and secrete cytokines to induce immunotolerance. To date, no studies have evaluated the transcription factors that regulate IL15 expression in human ESCs. In the present study, we examined whether HAND2 controls IL15 transcriptional regulation in human ESCs. Quantitative RT-PCR and histological analyses revealed that HAND2 and IL15 levels increase considerably in the secretory phase of human endometrium tissues. Results from ChIP-quantitative PCR suggested that HAND2 binds to a putative HAND2 motif, which we identified in the upstream region of the human IL15 gene through in silico analysis. Using a luciferase reporter assay, we found that the upstream region of the human IL15 gene up-regulates reporter gene activities in response to estradiol and a progestin representative (medroxyprogesterone) in ESCs. The upstream region of the human IL15 gene also exhibited increasing responsiveness to transfection with a HAND2 expression vector. Of note, deletion and substitution variants of the putative HAND2 motif in the upstream region of IL15 did not respond to HAND2 transfection. These findings confirm that HAND2 directly up-regulates human IL15 transcription in ESCs.

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.

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.

Ovarian hyperstimulation syndrome following surgical removal of a complete hydatidiform mole: a case report.

BACKGROUND: Generally, ovarian hyperstimulation syndrome develops after superovulation caused by ovulation-inducing drugs in infertile patients. However, ovarian hyperstimulation syndrome associated with natural pregnancy is rare, and most cases of ovarian hyperstimulation syndrome have been associated with a hydatidiform mole. CASE PRESENTATION: We describe a case of a 16-year-old Japanese girl with a complete hydatidiform mole. The patient was referred for intensive examination and treatment of the hydatidiform mole and underwent surgical removal of the hydatidiform mole at 9 weeks, 5 days of gestation. Histopathological examination revealed a complete hydatidiform mole. The patient's blood human chorionic gonadotropin level decreased from 980,823 IU/L to 44,815 IU/L on postoperative day 4, and it was below the cutoff level on postoperative day 64. Transvaginal ultrasonography on postoperative day 7 revealed a multilocular cyst measuring 82 × 43 mm in the right ovary and a multilocular cyst measuring 66 × 50 mm in the left ovary. Both ovarian cysts enlarged further. Magnetic resonance imaging on postoperative day 24 revealed that the right multilocular ovarian cyst had enlarged to 10 × 12 cm and that the left multilocular ovarian cyst had enlarged to 25 × 11 cm. Blood examination showed an elevated estradiol level as high as 3482 pg/ml. We diagnosed the patient with bilateral giant multilocular cysts accompanied by ovarian hyperstimulation syndrome because of the rapid increase in the size of the cysts. The patient complained of mild abdominal bloating; however, symptoms such as nausea, vomiting, dyspnea, and abdominal pain were not observed. Therefore, we chose spontaneous observation in the outpatient clinic. The cysts gradually decreased and disappeared on postoperative day 242. CONCLUSION: Physicians should be aware that ovarian cysts can occur and can increase rapidly after abortion of a hydatidiform mole. However, the ovarian cyst can return to its original size spontaneously even if it becomes huge.