Isolation and characterization of rabbit limbal niche cells.

Limbal niche cells (LNCs) are one of the most important supporting cells for corneal epithelial stem cells (CES), however, research on LNCs has been mostly limited to humans and rats previously. To expand the research work into the rabbit animal model, one of the most often used animals in stem cell study, this study was carried out for the in vitro isolation and identification of rabbit LNCs. Rabbit LNCs were isolated by collagenase A digestion method and single cells were obtained, the cells were then seeded on 5% Matrigel-coated plastic surface and cultured in modified embryonic stem cell medium (MESCM). Three biological replicates of the isolating and characterization were recorded from New Zealand White rabbits aged from 2.5 months to 5 months. LNC markers (VIM/CD90/CD105/SCF/PDGFRß) were analyzed using tyramide signal amplification (TSA) staining, immunohistochemical staining (IHC), western blotting (WB), and real-time reverse transcription polymerase chain reaction (qPCR). TSA staining suggested that VIM was highly expressed in rabbit limbus stroma, which was confirmed by WB, and P63α was expressed in the basal limbus epithelium. Pan-CK and CK12 were highly expressed in the central corneal epithelium but lightly expressed in the limbal epithelium. The WB result indicated that PDGFRß and VIM expressions in rabbit-LNCs P4 were higher than in P1 and P7. In addition, rabbit corneal epithelium highly expressed Paired Box 6 (PAX6) and Epidermal growth factor-like domain 6(EGFL6). For the three repeat experiments, the cell expansion activity of rabbit-LNC was highest at P4. Rabbit-LNCs were passaged from P0 to P7, and the number of cell doublings (NCD) of P4 for the three repeat experiments was 2.816, 2.737, and 2.849. qPCR showed that high mRNA expression levels of VIM, CD90, CD105, SCF, and PDGFRß in rabbit-LNCs P4. In conclusion, rabbit-LNCs could be successfully isolated by the collagenase A digestion method as used in human tissue. There were similar characteristics between rabbit and human LNCs (VIM+/CD90+/CD105+/SCF+/PAX6+/PDGFRß+).

Isolation and Identification of Limbal Niche Cells.

Here we report a standard procedure for the isolation and identification of limbal niche cells (LNCs). Limbus tissue obtained from an eye bank was used for LNCs isolation. The tissue was divided into 12 pieces under aseptic conditions and digested for 18 h at 37 °C in the cell culture incubator using collagenase A to obtain cell clusters with LNCs and limbal epithelial progenitor cells. The cell clusters were further digested for 15 min at 37 °C using 0.25% trypsin-EDTA to obtain single cells and then cultured in modified embryonic stem cell medium (MESCM) on a plastic surface coated with 5% Matrigel. Cells were passaged upon 70% confluence, and LNCs were identified using immunofluorescence, real-time quantitative PCR (qPCR), and flow cytometry. Primary LNCs were isolated and passaged more than 12 times. The proliferation activity of LNCs from P4 to P6 was the highest. LNCs expressed higher stem cell markers than BMMSCs (SCF, Nestin, Rex1, SSEA4, CD73, CD90, MSX1, P75NTR, and PDGFRß). Furthermore, results showed that P4 LNCs uniformly expressed VIM, CD90, CD105, and PDGFRß, but not Pan-CK, which could be used as a marker for the identification of LNCs. Flow cytometric analysis showed that approximately 95%, 97%, 92%, and 11% of LNCs expressed CD73, CD90, CD105, and SCF respectively, while they were 68%, 99%, 20%, and 3% in BMMSCs. The standard process for LNC isolation and identification could provide a reliable laboratory basis for the widespread use of LNCs.

Role of estrogen in the regulation of central and peripheral energy homeostasis: from a menopausal perspective.

Estrogen plays a prominent role in regulating and coordinating energy homeostasis throughout the growth, development, reproduction, and aging of women. Estrogen receptors (ERs) are widely expressed in the brain and nearly all tissues of the body. Within the brain, central estrogen via ER regulates appetite and energy expenditure and maintains cell glucose metabolism, including glucose transport, aerobic glycolysis, and mitochondrial function. In the whole body, estrogen has shown beneficial effects on weight control, fat distribution, glucose and insulin resistance, and adipokine secretion. As demonstrated by multiple in vitro and in vivo studies, menopause-related decline of circulating estrogen may induce the disturbance of metabolic signals and a significant decrease in bioenergetics, which could trigger an increased incidence of late-onset Alzheimer's disease, type 2 diabetes mellitus, hypertension, and cardiovascular diseases in postmenopausal women. In this article, we have systematically reviewed the role of estrogen and ERs in body composition and lipid/glucose profile variation occurring with menopause, which may provide a better insight into the efficacy of hormone therapy in maintaining energy metabolic homeostasis and hold a clue for development of novel therapeutic approaches for target tissue diseases.

Elevated adipose differentiation-related protein level in ovariectomized mice correlates with tissue-specific regulation of estrogen.

OBJECTIVE: Redistribution of adipose tissue in the abdomen during the menopausal transition is attributable mostly to estrogen drop with aging. Adipose differentiation-related protein (ADRP), a major component of lipid droplets, is closely related to the onset of lipid accumulation. We hypothesized that estrogen exerted its tissue-specific effect in reducing abdominal fat accumulation by regulation of ADRP. METHODS: Twenty-four female C57/BL6 mice aged 8 weeks were randomly divided into 3 groups: sham operation (Sham), bilateral ovariectomy (OVX), and OVX plus 17ß-estradiol (OVX + E2). After being fed 8 weeks of a high-fat diet, plasma lipid profiles including total cholesterol (TC), total triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) levels, body weight gain, visceral, and subcutaneous adipose tissue, adipocyte size, and ADRP expression were measured. RESULTS: In comparison to sham-operated mice, OVX mice presented a weight gain with higher plasma TC, TG, LDL-C levels, and lower HDL-C levels. E2 supplement ameliorated the increase in weight and lipid profiles. Elevated ADRP expression was observed in visceral adipose tissue of OVX mice, whereas treatment of estrogen suppressed the ADPR expression and reversed the fat accumulation in the abdomen. However, no significant difference of ADRP expression in subcutaneous adipose tissue was detected between sham, OVX, and OVX + E2 mice. CONCLUSIONS: Our findings suggested that enhanced ADRP expression in ovariectomized mice correlates with the tissue-specific regulation of estrogen, which may provide useful clues for further exploring the regulatory mechanism and corresponding anti-abdominal obesity treatment in postmenopausal women.

Single-cell transcriptome analysis uncovers the molecular and cellular characteristics of thin endometrium.

Infertility is a social and medical problem around the world and the incidence continues to rise. Thin endometrium (TE) is a great challenge of infertility treatment, even by in vitro fertilization and embryo transfer. It is widely believed that TE impairs endometrium receptivity. However, only a few studies have explained the molecular mechanism. Herein, in order to reveal the possible mechanism, we sampled endometrium from a TE patient and a control volunteer and got a transcriptomic atlas of 18 775 individual cells which was constructed using single-cell RNA sequencing, and seven cell types have been identified. The cells were acquired during proliferative and secretory phases, respectively. The proportion of epithelial cells and stromal cells showed a significant difference between the TE group and the control group. In addition, differential expressed genes (DEGs) in diverse cell types were revealed, the enriched pathways of DEGs were found closely related to the protein synthesis in TE of both proliferative and secretory phases. Some DEGs can influence cell-type ratio and impaired endometrial receptivity in TE. Furthermore, divergent expression of estrogen receptors 1 and progesterone receptors in stromal and epithelial cells were compared in the TE sample from the control. The cellular and molecular heterogeneity found in this study provided valuable information for disclosing the mechanisms of impaired receptivity in TE.

The Performance of Transvaginal Two-Dimensional Fundamental Sonosalpingography Combined with Saline Infusion Pelvic Sonosalpingography for Assessing Fimbrial Part's Morphology and Function of the Fallopian Tubes.

OBJECTIVES: The purpose of this study was to evaluate the diagnostic efficacy of transvaginal two-dimensional fundamental sonosalpingography (2D-FS) combined with saline infusion pelvic sonosalpingography (SIPS) for assessing fimbrial part's morphology and function of the fallopian tubes. METHODS: One hundred and sixty-nine cases underwent 2D-FS combined with SIPS. Among them, 18 cases received laparoscopy and dye test (LDT) within 3 months after the examination and the results were regarded as reference standard. RESULTS: Excluding proximal or middle segment obstructed tubes, the remaining fimbrial parts' display rate by using 2D-FS combined with SIPS was 75.1%. According to the ultrasonic appearance, the fimbrial parts were classified into 4 types: normal, abnormal, suspected abnormal, and unclassifiable. Normal fimbrial parts accounted for 73.8% when the tubes were patent; abnormal fimbrial parts accounted for 74.1% when the tubes were partial obstructed; all became abnormal when the tubes were distal complete obstructed. The fimbrial parts which had been classified by 2D-FS combined with SIPS were compared with LDT further. This combination's accuracy (ACC), sensitivity (SEN), specificity (SPE), positive predictive value (PPV), negative predictive value (NPV), and Youden's index (YI) were 86.4, 87.5, 85.7, 77.8, 92.3, and 0.73%, respectively. The result of consistency analysis showed the combination was essentially consistent with LDT result (Kappa = 0.713). CONCLUSION: 2D-FS combined with SIPS can be a preferred method for assessment of the fimbrial part's morphology and function, with its advantages of non-invasive, intuition, and accuracy. This combination could provide an objective imaging basis for choosing clinical treatment strategies and predicting prognosis.

Influence of vitamin D supplementation on lipid levels in polycystic ovary syndrome patients: a meta-analysis of randomized controlled trials.

OBJECTIVE: Observational studies have shown that circulating vitamin D (VitD) deficiency is associated with atherogenic lipid patterns among polycystic ovary syndrome (PCOS) patients. However, interventional studies have shown inconsistent results. The aim of this meta-analysis was to investigate how VitD supplementation influences lipid indices in PCOS patients. METHODS: The authors searched four electronic databases through August 2019 to identify randomized controlled trials (RCTs) that assessed the effect of VitD intervention on serum lipids among PCOS patients. Mean differences were generated for statistical evaluation. RESULTS: We included eight studies and performed nine comparisons across 467 participants. VitD supplementation reduced serum triglyceride levels (-11.88 mg/dL; 95% confidence interval [CI]: -17.03 to -6.73), total cholesterol (-9.09 mg/dL; 95% CI: -14.90 to -3.29), low-density lipoprotein cholesterol (-5.22 mg/dL; 95% CI: -10.32 to -0.13), and very-low-density lipoprotein cholesterol (-2.43 mg/dL; 95% CI: -3.69 to -1.17) compared with no VitD supplementation. However, high-density lipoprotein cholesterol levels showed no differences with or without VitD supplementation (-0.39 mg/dL; 95% CI: -1.39 to 0.61). CONCLUSIONS: VitD supplementation improved serum lipid levels among PCOS patients, but serum high-density lipoprotein cholesterol levels were not changed. VitD intervention might benefit PCOS patients who are at high risk of an atherogenic lipid profile.

Laparoscopic tumorectomy for a primary ovarian leiomyoma during pregnancy: A case report.

Few reports have explored laparoscopic adnexal tumorectomy as a treatment for large and symptomatic ovarian leiomyomas during pregnancy. The current study presents the case of a patient with a large and symptomatic ovarian leiomyoma at 14 weeks of pregnancy. A laparoscopic adnexal tumorectomy was performed without complications. The laparoscopic management of large primary ovarian leiomyoma during pregnancy has not been reported in literature. Therefore, laparoscopy may be considered as a minimally invasive and feasible alternative to laparotomy for the treatment of large ovarian solid tumors during pregnancy, resulting in reduced postoperative pain, a smaller scar and shorter recovery time. By contrast, with respect to the ovarian solid tumor, surgery prior to gestation is advised, even for tumors of <3 cm in diameter, due to the probability of rapid growth of the tumor during pregnancy.