Predicting the Invasiveness of Pulmonary Adenocarcinomas in Pure Ground-Glass Nodules Using the Nodule Diameter: A Systematic Review, Meta-Analysis, and Validation in an Independent Cohort.

The nodule diameter was commonly used to predict the invasiveness of pulmonary adenocarcinomas in pure ground-glass nodules (pGGNs). However, the diagnostic performance and optimal cut-off values were inconsistent. We conducted a meta-analysis to evaluate the diagnostic performance of the nodule diameter for predicting the invasiveness of pulmonary adenocarcinomas in pGGNs and validated the cut-off value of the diameter in an independent cohort. Relevant studies were searched through PubMed, MEDLINE, Embase, and the Cochrane Library, from inception until December 2022. The inclusion criteria comprised studies that evaluated the diagnostic accuracy of the nodule diameter to differentiate invasive adenocarcinomas (IAs) from non-invasive adenocarcinomas (non-IAs) in pGGNs. A bivariate mixed-effects regression model was used to obtain the diagnostic performance. Meta-regression analysis was performed to explore the heterogeneity. An independent sample of 220 pGGNs (82 IAs and 128 non-IAs) was enrolled as the validation cohort to evaluate the performance of the cut-off values. This meta-analysis finally included 16 studies and 2564 pGGNs (761 IAs and 1803 non-IAs). The pooled area under the curve, the sensitivity, and the specificity were 0.85 (95% confidence interval (CI), 0.82-0.88), 0.82 (95% CI, 0.78-0.86), and 0.73 (95% CI, 0.67-0.78). The diagnostic performance was affected by the measure of the diameter, the reconstruction matrix, and patient selection bias. Using the prespecified cut-off value of 10.4 mm for the mean diameter and 13.2 mm for the maximal diameter, the mean diameter showed higher sensitivity than the maximal diameter in the validation cohort (0.85 vs. 0.72, p < 0.01), while there was no significant difference in specificity (0.83 vs. 0.86, p = 0.13). The nodule diameter had adequate diagnostic performance in differentiating IAs from non-IAs in pGGNs and could be replicated in a validation cohort. The mean diameter with a cut-off value of 10.4 mm was recommended.

The solid component within part-solid nodules: 3-dimensional quantification, correlation with the malignant grade of nonmucinous pulmonary adenocarcinomas, and comparisons with 2-dimentional measures and semantic features in low-dose computed tomography.

BACKGROUND: There is no consensus on 3-dimensional (3D) quantification method for solid component within part-solid nodules (PSNs). This study aimed to find the optimal attenuation threshold for the 3D solid component proportion in low-dose computed tomography (LDCT), namely the consolidation/tumor ratio of volume (CTRV), basing on its correlation with the malignant grade of nonmucinous pulmonary adenocarcinomas (PAs) according to the 5th edition of World Health Organization classification. Then we tested the ability of CTRV to predict high-risk nonmucinous PAs in PSNs, and compare its performance with 2-dimensional (2D) measures and semantic features. METHODS: A total of 313 consecutive patients with 326 PSNs, who underwent LDCT within one month before surgery and were pathologically diagnosed with nonmucinous PAs, were retrospectively enrolled and were divided into training and testing cohorts according to scanners. The CTRV were automatically generated by setting a series of attenuation thresholds from - 400 to 50 HU with an interval of 50 HU. The Spearman's correlation was used to evaluate the correlation between the malignant grade of nonmucinous PAs and semantic, 2D, and 3D features in the training cohort. The semantic, 2D, and 3D models to predict high-risk nonmucinous PAs were constructed using multivariable logistic regression and validated in the testing cohort. The diagnostic performance of these models was evaluated by the area under curve (AUC) of receiver operating characteristic curve. RESULTS: The CTRV at attenuation threshold of -250 HU (CTRV- 250HU) showed the highest correlation coefficient among all attenuation thresholds (r = 0.655, P < 0.001), which was significantly higher than semantic, 2D, and other 3D features (all P < 0.001). The AUCs of CTRV- 250HU to predict high-risk nonmucinous PAs were 0.890 (0.843-0.927) in the training cohort and 0.832 (0.737-0.904) in the testing cohort, which outperformed 2D and semantic models (all P < 0.05). CONCLUSIONS: The optimal attenuation threshold was - 250 HU for solid component volumetry in LDCT, and the derived CTRV- 250HU might be valuable for the risk stratification and management of PSNs in lung cancer screening.

FHL2 deficiency impairs follicular development and fertility by attenuating EGF/EGFR/YAP signaling in ovarian granulosa cells.

Female subfertility is an increasing reproductive issue worldwide, which is partially related to abnormal ovarian follicular development. Granulosa cells (GCs), by providing the necessary physical support and microenvironment for follicular development, play critical roles in maintaining female fertility. We previously showed that ectopic expression of four and a half LIM domains 2 (FHL2) promoted ovarian granulosa cell tumor progression. However, its function in follicular development and fertility remains unknown. Here, we confirmed that FHL2 is highly expressed in human and mouse ovaries. FHL2 immunosignals were predominantly expressed in ovarian GCs. A Fhl2 knockout (KO) mouse model was generated to examine its roles in follicular development and fertility. Compared with wildtype, knockout of Fhl2 significantly decreased female litter size and offspring number. Furthermore, Fhl2 deficiency reduced ovarian size and impaired follicular development. RNA-sequencing analysis of GCs isolated from either KO or WT mice revealed that, Fhl2 deletion impaired multiple biological functions and signaling pathways, such as Ovarian Putative Early Atresia Granulosa Cell, ErbB, Hippo/YAP, etc. In vitro studies confirmed that FHL2 silencing suppressed GCs growth and EGF-induced GCs proliferation, while its overexpression promoted GC proliferation and decreased apoptosis. Mechanistic studies indicated that FHL2, via forming complexes with transcriptional factors AP-1 or NF-κB, regulated Egf and Egfr expression, respectively. Besides, FHL2 depletion decreased YAP1 expression, especially the active form of YAP1 (nuclear YAP1) in GCs of growing follicles. EGF, serving as an autocrine/paracrine factor, not only induced FHL2 expression and nuclear accumulation, but also stimulated YAP1 expression and activation. Collectively, our study suggests that FHL2 interacts with EGFR and Hippo/YAP signaling to regulate follicular development and maintain fertility. This study illuminates a novel mechanism for follicular development and a potential therapeutic target to address subfertility.

An ordinal radiomic model to predict the differentiation grade of invasive non-mucinous pulmonary adenocarcinoma based on low-dose computed tomography in lung cancer screening.

OBJECTIVES: To construct a radiomic model of low-dose CT (LDCT) to predict the differentiation grade of invasive non-mucinous pulmonary adenocarcinoma (IPA) and compare its diagnostic performance with quantitative-semantic model and radiologists. METHODS: A total of 682 pulmonary nodules were divided into the primary cohort (181 grade 1; 254 grade 2; 64 grade 3) and validation cohort (69 grade 1; 99 grade 2; 15 grade 3) according to scanners. The radiomic and quantitative-semantic models were built using ordinal logistic regression. The diagnostic performance of the models and radiologists was assessed by the area under the curve (AUC) of the receiver operating characteristic curve and accuracy. RESULTS: The radiomic model demonstrated excellent diagnostic performance in the validation cohort (AUC, 0.900 (95%CI: 0.847-0.939) for Grade 1 vs. Grade 2/Grade 3; AUC, 0.929 (95%CI: 0.882-0.962) for Grade 1/Grade 2 vs. Grade 3; accuracy, 0.803 (95%CI: 0.737-0.857)). No significant difference in diagnostic performance was found between the radiomic model and radiological expert (AUC, 0.840 (95%CI: 0.779-0.890) for Grade 1 vs. Grade 2/Grade 3, p = 0.130; AUC, 0.852 (95%CI: 0.793-0.900) for Grade 1/Grade 2 vs. Grade 3, p = 0.170; accuracy, 0.743 (95%CI: 0.673-0.804), p = 0.079), but the radiomic model outperformed the quantitative-semantic model and inexperienced radiologists (all p < 0.05). CONCLUSIONS: The radiomic model of LDCT can be used to predict the differentiation grade of IPA in lung cancer screening, and its diagnostic performance is comparable to that of radiological expert. KEY POINTS: • Early identifying the novel differentiation grade of invasive non-mucinous pulmonary adenocarcinoma may provide guidance for further surveillance, surgical strategy, or more adjuvant treatment. • The diagnostic performance of the radiomic model is comparable to that of a radiological expert and superior to that of the quantitative-semantic model and inexperienced radiologists. • The radiomic model of low-dose CT can be used to predict the differentiation grade of invasive non-mucinous pulmonary adenocarcinoma in lung cancer screening.

Radiomic and quantitative-semantic models of low-dose computed tomography for predicting the poorly differentiated invasive non-mucinous pulmonary adenocarcinoma.

PURPOSE: Poorly differentiated invasive non-mucinous pulmonary adenocarcinoma (IPA), based on the novel grading system, was related to poor prognosis, with a high risk of lymph node metastasis and local recurrence. This study aimed to build the radiomic and quantitative-semantic models of low-dose computed tomography (LDCT) to preoperatively predict the poorly differentiated IPA in nodules with solid component, and compare their diagnostic performance with radiologists. MATERIALS AND METHODS: A total of 396 nodules from 388 eligible patients, who underwent LDCT scan within 2 weeks before surgery and were pathologically diagnosed with IPA, were retrospectively enrolled between July 2018 and December 2021. Nodules were divided into two independent cohorts according to scanners: primary cohort (195 well/moderate differentiated and 64 poorly differentiated) and validation cohort (104 well/moderate differentiated and 33 poorly differentiated). The radiomic and quantitative-semantic models were built using multivariable logistic regression. The diagnostic performance of the models and radiologists was assessed by area under curve (AUC) of receiver operating characteristic (ROC) curve, accuracy, sensitivity, and specificity. RESULTS: No significant differences of AUCs were found between the radiomic and quantitative-semantic model in primary and validation cohorts (0.921 vs. 0.923, P = 0.846 and 0.938 vs. 0.911, P = 0.161). Both the models outperformed three radiologists in the validation cohort (all P < 0.05). CONCLUSIONS: The radiomic and quantitative-semantic models of LDCT, which could identify the poorly differentiated IPA with excellent diagnostic performance, might provide guidance for therapeutic decision making, such as choosing appropriate surgical method or adjuvant chemotherapy.

Quantitative parameters of enhanced dual-energy computed tomography for differentiating lung cancers from benign lesions in solid pulmonary nodules.

Objectives: This study aimed to investigate the ability of quantitative parameters of dual-energy computed tomography (DECT) and nodule size for differentiation between lung cancers and benign lesions in solid pulmonary nodules. Materials and Methods: A total of 151 pathologically confirmed solid pulmonary nodules including 78 lung cancers and 73 benign lesions from 147 patients were consecutively and retrospectively enrolled who underwent dual-phase contrast-enhanced DECT. The following features were analyzed: diameter, volume, Lung CT Screening Reporting and Data System (Lung-RADS) categorization, and DECT-derived quantitative parameters including effective atomic number (Zeff), iodine concentration (IC), and normalized iodine concentration (NIC) in arterial and venous phases. Multivariable logistic regression analysis was used to build a combined model. The diagnostic performance was assessed by area under curve (AUC) of receiver operating characteristic curve, sensitivity, and specificity. Results: The independent factors for differentiating lung cancers from benign solid pulmonary nodules included diameter, Lung-RADS categorization of diameter, volume, Zeff in arterial phase (Zeff_A), IC in arterial phase (IC_A), NIC in arterial phase (NIC_A), Zeff in venous phase (Zeff_V), IC in venous phase (IC_V), and NIC in venous phase (NIC_V) (all P < 0.05). The IC_V, NIC_V, and combined model consisting of diameter and NIC_V showed good diagnostic performance with AUCs of 0.891, 0.888, and 0.893, which were superior to the diameter, Lung-RADS categorization of diameter, volume, Zeff_A, and Zeff_V (all P < 0.001). The sensitivities of IC_V, NIC_V, and combined model were higher than those of IC_A and NIC_A (all P < 0.001). The combined model did not increase the AUCs compared with IC_V (P = 0.869) or NIC_V (P = 0.633). Conclusion: The DECT-derived IC_V and NIC_V may be useful in differentiating lung cancers from benign lesions in solid pulmonary nodules.

Anti-Müllerian Hormone Inhibits FSH-Induced Cumulus Oocyte Complex In Vitro Maturation and Cumulus Expansion in Mice.

Anti-Müllerian hormone (AMH) is secreted by the ovaries of female animals and exerts its biological effects through the type II receptor (AMHR2). AMH regulates follicular growth by inhibiting the recruitment of primordial follicles and reducing the sensitivity of antral follicles to FSH. Despite the considerable research on the actions of AMH in granulosa cells, the effect of AMH on the in vitro maturation of oocytes remains largely unknown. In the current study, we showed that AMH is only expressed in cumulus cells, while AMHR2 is produced in both cumulus cells and oocytes. AMH had no significant effect on COCs nuclear maturation, whereas it inhibited the stimulatory effects of FSH on COCs maturation and cumulus expansion. Moreover, AMH treatment effectively inhibited the positive effect of FSH on the mRNA expressions of Hyaluronan synthase 2 (Has2), Pentraxin 3 (Ptx3), and TNF-alpha-induced protein 6 (Tnfaip 6) genes in COCs. In addition, AMH significantly decreased the FSH-stimulated progesterone production, but did not change estradiol levels. Taken together, our results suggest that AMH may inhibit the effects of FSH-induced COCs in vitro maturation and cumulus expansion. These findings increase our knowledge of the functional role of AMH in regulating folliculogenesis.

Development and validation of qualitative and quantitative models to predict invasiveness of lung adenocarcinomas manifesting as pure ground-glass nodules based on low-dose computed tomography during lung cancer screening.

Background: Due to different management strategy and prognosis of different subtypes of lung adenocarcinomas appearing as pure ground-glass nodules (pGGNs), it is important to differentiate invasive adenocarcinoma (IA) from adenocarcinoma in situ/minimally invasive adenocarcinoma (AIS/MIA) during lung cancer screening. The aim of this study was to develop and validate the qualitative and quantitative models to predict the invasiveness of lung adenocarcinoma appearing as pGGNs based on low-dose computed tomography (LDCT) and compare their diagnostic performance with that of intraoperative frozen section (FS). Methods: A total of 223 consecutive pathologically confirmed pGGNs from March 2018 to December 2020 were divided into a primary cohort (96 IAs and 64 AIS/MIAs) and validation cohort (39 IAs and 24 AIS/MIAs) according to scans (Brilliance iCT and Somatom Definition Flash) performed at Sichuan Cancer Hospital and Institute. The following LDCT features of pGGNs were analyzed: the qualitative features included nodule location, shape, margin, nodule-lung interface, lobulation, spiculation, pleural indentation, air bronchogram, vacuole, and vessel type, and the quantitative features included the diameter, volume, and mean attenuation. Multivariate logistic regression analysis was used to build a qualitative model, quantitative model, and combined qualitative and quantitative model. The diagnostic performance was assessed according to the following factors: the area under curve (AUC) of the receiver operating characteristic (ROC) curve, sensitivity, specificity, and accuracy. Results: The AUCs of the qualitative model, quantitative model, combined qualitative and quantitative model, and the FS diagnosis were 0.854, 0.803, 0.873, and 0.870, respectively, in the primary cohort and 0.884, 0.855, 0.875, and 0.946, respectively, in the validation cohort. No significant difference of the AUCs was found among the radiological models and the FS diagnosis in the primary or validation cohort (all corrected P>0.05). Among the radiological models, the combined qualitative and quantitative model consisting of vessel type and volume showed the highest accuracy in both the primary and validation cohorts (0.831 and 0.889, respectively). Conclusions: The diagnostic performances of the qualitative and quantitative models based on LDCT to differentiate IA from AIS/MIA in pGGNs are equivalent to that of intraoperative FS diagnosis. The vessel type and volume can be preoperative and non-invasive biomarkers to assess the invasive risk of pGGNs in lung cancer screening.

Improvement of image quality of laryngeal squamous cell carcinoma using noise-optimized virtual monoenergetic image and nonlinear blending image algorithms in dual-energy computed tomography.

BACKGROUND: Dual-energy computed tomography (DECT) has been used to improve image quality of head and neck squamous cell carcinoma (SCC). This study aimed to assess image quality of laryngeal SCC using linear blending image (LBI), nonlinear blending image (NBI), and noise-optimized virtual monoenergetic image (VMI+) algorithms. METHODS: Thirty-four patients with laryngeal SCC were retrospectively enrolled between June 2019 and December 2020. DECT images were reconstructed using LBI (80 kV and M_0.6), NBI, and VMI+ (40 and 55 keV) algorithms. Contrast-to-noise ratio (CNR), tumor delineation, and overall image quality were assessed and compared. RESULTS: VMI+ (40 keV) had the highest CNR and provided better tumor delineation than VMI+ (55 keV), LBI, and NBI, while NBI provided better overall image quality than VMI+ and LBI (all corrected p < 0.05). CONCLUSIONS: VMI+ (40 keV) and NBI improve image quality of laryngeal SCC and may be preferable in DECT examination.

Melatonin Administration Accelerates Puberty Onset in Mice by Promoting FSH Synthesis.

Although melatonin has been extensively studied in animal reproduction, the mechanism of melatonin in puberty remains elusive. This study was designed to explore the effect of intraperitoneal administration of melatonin on puberty onset in female mice. The injection of melatonin into postnatal days 10 mice at a dose of 15 mg/kg accelerated the puberty onset in mice. Mechanistically, there was no difference in physical growth and serum Leptin levels after melatonin administration. Meanwhile, the serum levels of reproductive hormones involved in hypothalamic-pituitary-ovarian axis, such as FSH and estrogen level in serum were increased. The mRNA levels of GnRH and GnRHr were not affected by melatonin, while the expressions of FSHß in pituitary and Cyp19a1 in ovary were significantly up-regulated. In addition, melatonin still promoted FSH synthesis after ovariectomy. Furthermore, the enhanced activity of ERK1/2 signaling verified that the expression of FSHß increased in pituitary. We confirmed that melatonin promoted the FSH synthesis in pituitary, thereby increased serum estrogen levels and ultimately accelerated puberty onset. However, these effects of melatonin may be pharmacological due to the high dose. This study would help us to understand the functions of melatonin in pubertal regulation comprehensively.

Comparison of Radiomic Models Based on Low-Dose and Standard-Dose CT for Prediction of Adenocarcinomas and Benign Lesions in Solid Pulmonary Nodules.

OBJECTIVES: This study aimed to develop radiomic models based on low-dose CT (LDCT) and standard-dose CT to distinguish adenocarcinomas from benign lesions in patients with solid solitary pulmonary nodules and compare the performance among these radiomic models and Lung CT Screening Reporting and Data System (Lung-RADS). The reproducibility of radiomic features between LDCT and standard-dose CT were also evaluated. METHODS: A total of 141 consecutive pathologically confirmed solid solitary pulmonary nodules were enrolled including 50 adenocarcinomas and 48 benign nodules in primary cohort and 22 adenocarcinomas and 21 benign nodules in validation cohort. LDCT and standard-dose CT scans were conducted using same acquisition parameters and reconstruction method except for radiation dose. All nodules were automatically segmented and 104 original radiomic features were extracted. The concordance correlation coefficient was used to quantify reproducibility of radiomic features between LDCT and standard-dose CT. Radiomic features were selected to build radiomic signature, and clinical characteristics and radiomic signature were combined to develop radiomic nomogram for LDCT and standard-dose CT, respectively. The performance of radiomic models and Lung-RADS was assessed by area under curve (AUC) of receiver operating characteristic curve, sensitivity, and specificity. RESULTS: Shape and first order features, and neighboring gray tone difference matrix features were highly reproducible between LDCT and standard-dose CT. No significant differences of AUCs were found among radiomic signature and nomogram of LDCT and standard-dose CT in both primary and validation cohort (0.915 vs. 0.919 vs. 0.898 vs. 0.909 and 0.976 vs. 0.976 vs. 0.985 vs. 0.987, respectively). These radiomic models had higher specificity than Lung-RADS (all correct P < 0.05), while there were no significant differences of sensitivity between Lung-RADS and radiomic models. CONCLUSIONS: The diagnostic performance of LDCT-based radiomic models to differentiate adenocarcinomas from benign lesions in solid pulmonary nodules were equivalent to that of standard-dose CT. The LDCT-based radiomic model with higher specificity and lower false-positive rate than Lung-RADS might help reduce overdiagnosis and overtreatment of solid pulmonary nodules in lung cancer screening.

Accuracy of Pulmonary Nodule Volumetry at Different Exposure Parameters in Low-Dose Computed Tomography: A Phantom Study.

OBJECTIVE: To explore the exposure parameters with minimized radiation dose for accurate pulmonary nodule volumetry using low-dose computed tomography (LDCT). METHODS: An anthropomorphic chest phantom with 11 pulmonary nodules (6 solid nodules and 5 ground-glass opacities) was scanned using 256-slice multidetector computed tomography scanner at various tube voltage and current (combinations of 80, 100 and 120 kV with 10 to 30 mAs). Raw data sets were reconstructed using the hybrid iterative reconstruction method and nodule volume was calculated by a semiautomatic software. The absolute percentage error (APE) of nodule volume relating to the reference acquisition and contrast-to-noise ratio was measured. RESULTS: Nodule characteristic and tube voltage (P < 0.0001) as well as the interaction between nodule characteristic and tube voltage (P = 0.0026) contributed significantly to the mean difference of APE, while tube current did not (P = 0.21). Post hoc analysis revealed no significant difference was found between the APE at 100 kV and 120 kV in both solid nodules (2.3 ± 0.4% vs 1.8 ± 0.6%, P = 0.14) and ground-glass opacities (6.0 ± 0.5% vs 4.9 ± 0.6%, P = 0.11). Exploratory analyses further showed that the APE at 100 kV with 10 mAs did not differ from that at 120 kV with 30 mAs in both solid nodules (2.5 ± 0.5% vs 1.7 ± 0.3%, P = 0.025, corrected P = 0.20) and ground-glass opacities (6.4 ± 0.4% vs 4.8 ± 1.0%, P = 0.0084, corrected P = 0.068). CONCLUSIONS: In our study, the exposure parameters with minimized radiation dose for accurate pulmonary nodule volumetry were found at 100 kV with 10 mAs, and the estimated effect radiation dose was as low as 0.2 mSv, suggesting the feasibility of further reducing radiation dose by decreasing tube voltage and current in LDCT lung screening.

Screening baseline characteristics of early lung cancer on low-dose computed tomography with computer-aided detection in a Chinese population.

OBJECTIVES: This study investigated appropriate baseline characteristics for screening a Chinese population at high risk of early lung cancer, assisted by low-dose computed tomography (LDCT) with computer-aided detection (CAD). Included is a discussion of the viability of using LDCT in the screening guideline and optimizing the guideline. METHODS: In 2014, 1016 individuals from Sichuan Province were enrolled who satisfied the criteria for high risk according to the 2013 National Comprehensive Cancer Network (NCCN) Guidelines for Non-Small Cell Lung Cancer. From 2014 to 2018, each subject was followed using LDCT with CAD, and pathologically confirmed lung cancers and baseline nodule characteristics (size and density) were recorded. Positive risk was considered a non-calcified solid or part-solid nodule on LDCT with diameter ≥5 mm and ground-glass nodule ≥8 mm, as newly recommended by the China National Lung Cancer Screening Guideline. RESULTS: From 2014-2018, 13 cases of lung cancer were detected; 5 of these were early stage (38.5%). According to the NCCN criteria, 54 women were included and one of these (1.8%) developed lung cancer. The prevalence of lung cancer was 0.7% at baseline. For the entire population (excluding subjects with a tumor mass at baseline, n = 4), the rate of positivity was 20.4% at baseline; applying the Chinese criteria, the false positive rate was 19.5% (197/1012). CONCLUSIONS: Further studies are warranted to establish appropriate eligible criteria and management strategies for Chinese populations.

Alpha-ketoglutarate affects murine embryo development through metabolic and epigenetic modulations.

α-Ketoglutarate (α-KG) is an intermediary metabolite in the tricarboxylic acid (TCA) cycle and functions to inhibit ATPase and maintain the pluripotency of embryonic stem cells (ESCs); however, little is known regarding the effects of α-KG on the development of preimplantation embryos. Herein, we report that α-KG (150 µM) treatment significantly promoted the blastocyst rate, the number of inner cell mass (ICM) cells and foetal growth after embryo transfer. Mechanistic studies revealed two important pathways involved in the α-KG effects on embryo development. First, α-KG modulates mitochondria function by inducing relatively low ATP production without modification of mitochondrial copy number. The relatively low energy metabolism preserves the pluripotency and competence of the ICM. Second, α-KG modifies epigenetics in embryos cultured in vitro by affecting the activity of the DNA demethylation enzyme TET and the DNA methylation gene Dnmt3a to increase the ratio of 5hmC/5mC ratio. Elevation of the 5hmC/5mC ratio not only promotes the pluripotency of the ICM but also leads to a methylation level in an in vitro embryo close to that in an in vivo embryo. All these functions of α-KG collectively contribute to an increase in the number of ICM cells, leading to greater adaptation of cultured embryos to in vitro conditions and promoting foetal growth after embryo transfer. Our findings provide basic knowledge regarding the mechanisms by which α-KG affects embryo development and cell differentiation.

Aanat Knockdown and Melatonin Supplementation in Embryo Development: Involvement of Mitochondrial Function and DNA Methylation.

Aims: In addition to pineal gland, many cells, tissues, and organs also synthesize melatonin (N-acetyl-5-methoxytryptamine). Embryos are a group of special cells and whether they can synthesize melatonin is still an open question. However, melatonin application promoted embryo development in many species in in vitro condition. The purpose of this study was to investigate whether embryos can synthesize melatonin; if it is so, what are the impacts of the endogenously produced melatonin on embryo development and the associated molecular mechanisms. These have never been reported previously. Results: Melatonin synthesis was observed at different stages of embryonic development. Aanat (aralkylamine N-acetyltransferase), a rate-limiting enzyme for melatonin production, was found to mostly localize in the mitochondria. Aanat knockdown significantly impeded embryonic development, and melatonin supplementation rescued it. The potential mechanisms might be that melatonin preserved mitochondrial intact and its function, thus providing sufficient adenosine 5'-triphosphate for the embryo development. In addition, melatonin scavenged intracellular reactive oxygen species (ROS) and reduced the DNA mutation induced by oxidative stress. In the molecular level, Aanat knockdown reduced tet methylcytosine dioxygenase 2 (Tet2) expression and DNA demethylation in blastocyst and melatonin supplementation rescued these processes. Innovation: This is the first report to show that embryos synthesize melatonin, and its synthetic enzyme Aanat was located in the mitochondria of embryos. An effect of melatonin is to maintain Tet2 expression and normal methylation status, and thereby promote embryonic development. Conclusion: Embryos can produce melatonin that reduces ROS production, preserves mitochondrial function, and maintains Tet2 expression and the normal DNA methylation.

Melatonin Improves the Quality of Inferior Bovine Oocytes and Promoted Their Subsequent IVF Embryo Development: Mechanisms and Results.

The inferior oocytes (IOs), which are not suitable for embryo development, occupy roughly one-third or more of the collected immature bovine oocytes. The IOs are usually discarded from the in vitro bovine embryo production process. Improving the quality of the inferior oocytes (IOs) and make them available in in vitro embryo production would have important biological, as well as commercial, value. This study was designed to investigate whether melatonin could improve the quality of IOs and make them usable in the in vitro maturation (IVM) and subsequent (in vitro fertilization) IVF embryo development. The results indicated that: the maturation rate of IOs and their subsequent IVF embryo developments were impaired compared to cumulus-oocyte complexes and melatonin treatment significantly improved the quality of IOs, as well as their IVF and embryo developments. The potential mechanisms are that: (1) melatonin reduced reactive oxygen species (ROS) and enhanced glutathione (GSH) levels in the IOs, thereby protecting them from oxidative stress; (2) melatonin improved mitochondrial normal distribution and function to increase ATP level in IOs; and (3) melatonin upregulated the expression of ATPase 6, BMP-15, GDF-9, SOD-1, Gpx-4, and Bcl-2, which are critical genes for oocyte maturation and embryo development and downregulated apoptotic gene expression of caspase-3.

Knockdown of melatonin receptor 1 and induction of follicle-stimulating hormone on the regulation of mouse granulosa cell function.

Melatonin receptor 1 (MT1) performs a critical role in the regulation of the animal reproductive system, particularly in follicular growth, and has a considerable effect on reproductive performance. However, the role that MT1 plays in regulating hormones associated with reproduction remains unclear. This study was designed to examine the physiological role of constitutive MT1 silencing and follicle stimulating hormone (FSH) treatment in reproduction, making use of mouse granulosa cells (mGCs) as a model. To understand the constitutive role of MT1 in ovarian physiology, the RNAi-Ready pSIREN-RETROQ-ZsGreen Vector mediated recombinant pshRNA was used to silence MT1 gene expression. Furthermore, we observed that the expression of MT1 was successfully inhibited both at the protein and mRNA levels (P<0.001). We demonstrated that RNAi-B-mediated MT1 down-regulation significantly promoted apoptosis (P<0.001), inhibited proliferation, and regulated the cell cycle at the S-phase; conversely, FSH treatment partially aided the apoptotic effect and improved proliferation but showed a significant effect at the S-phase of the cell cycle. Transitory knockdown of MT1 proved essential in the function of mGCs, as it significantly decreased cyclic adenosine monophospahte (cAMP) level and increased cell apoptosis. Following knockdown of MT1, the expression of Bax was significantly up-regulated (P<0.001), but Bcl-2 was slightly down-regulated, both at the transcriptional and at translational levels. Moreover, the silencing of MT1 and its constitutive effect on FSH significantly promoted an increase in estradiol (P<0.001) and slightly decreased the concentration of progesterone. Together, our data indicates that MT1 suppression leads to interference in the normal physiological function of the ovary by enhancing follicular apoptosis, inhibiting proliferation, and influencing hormonal signaling, whereas constitutive FSH treatment counteracted the negative down-regulatory effects of MT1 on mGCs.

The Regulatory Mechanism of MLT/MT1 Signaling on the Growth of Antler Mesenchymal Cells.

Melatonin (MLT) plays an important role in regulating the physiological cycle of seasonal breeding animals. Melatonin receptor I (MT1) is effectively expressed in the cambium layer of deer antler. However, the function and metabolic mechanism of MLT/MT1 signaling in the mesenchymal cells of sika deer remain to be further elucidated. In this work, we detected the effects of MLT/MT1 signaling on mesenchymal cells proliferation and the interaction between MLT/MT1 and IGF1/IGF1-R signaling. The results show that (1) deer antler mesenchymal cells actually express MT1; (2) exogenous melatonin significantly promotes mesenchymal cells proliferation, while MT1 knock-down significantly impairs the positive effects of melatonin; and (3) melatonin significantly enhanced IGF1/IGF1-R signaling, as both the expression of IGF1 and IGF-1R increased, while MT1 knock-down significantly decreased IGF1-R expression and IGF1 synthesis. In summary, these data verified that MLT/MT1 signaling plays a crucial role in antler mesenchymal proliferation, which may be mediated by IGF1/IGF1-R.

Beneficial Effects of Melatonin on the In Vitro Maturation of Sheep Oocytes and Its Relation to Melatonin Receptors.

(1) Background: The binding sites of melatonin, as a multifunctional molecule, have been identified in human, porcine, and bovine samples. However, the binding sites and mechanisms of melatonin have not been reported in sheep; (2) Methods: Cumulus-oocyte complexes (COCs) were cultured in TCM-199 supplemented with melatonin at concentrations of 0, 10-3, 10-5, 10-7, 10-9, and 10-11 M. Melatonin receptors (MT1 and MT2) were evaluated via immunofluorescence and Western blot. The effects of melatonin on cumulus cell expansion, nuclear maturation, embryo development, and related gene (GDF9, DNMT1, PTX3, HAS2, and EGFR) expression were investigated. The level of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were evaluated in oocytes and cumulus, respectively; (3) Results: Both MT1 and MT2 were expressed in oocytes, cumulus cells, and granulosa cells. Melatonin with a concentration of 10-7 M significantly enhanced the rates of nuclear maturation, cumulus cells expansion, cleavage, and blastocyst. Melatonin enhanced the expression of BMP15 in oocytes and of PTX3, HAS2, and EGFR in cumulus cells. Melatonin decreased the cAMP level of oocytes but enhanced the cGMP level in oocytes and cumulus cells; (4) Conclusion: The higher presence of MT1 in GV cumulus cells and the beneficial effects of melatonin indicated that its roles in regulating sheep oocyte maturation may be mediated mainly by the MT1 receptor.

Resveratrol compares with melatonin in improving in vitro porcine oocyte maturation under heat stress.

BACKGROUND: Resveratrol, an important phyto-antioxidant commonly found in grapes, mulberry, and other plants, has a variety of functions including anti-aging, anti-cancer and anti-inflammatory activities. In the current study, we investigated the beneficial effects of resveratrol on in vitro porcine oocyte maturation under heat stress (HS). The effect of resveratrol, melatonin and their combination on alleviating HS was compared according to the maturation rate of oocytes and the development competence of embryos after parthenogenetic activation (PA). RESULTS: Supplementation with resveratrol (2.0 µmol/L) not only improved the nuclear maturation but also raised the blastocyst rate of porcine embryos' PA from oocytes that underwent HS by increasing their glutathione (GSH) level, reducing reactive oxygen species (ROS) and up-regulating the expression of Sirtuin 1 (SIRT1). It was also found that melatonin (10(-7) mol/L) and the combination of resveratrol (2.0 µmol/L) plus melatonin (10(-7) mol/L) exhibited more potent effects than resveratrol alone regarding their protective activities on oocyte maturation under HS. CONCLUSIONS: This study compared the efficiencies of resveratrol, melatonin and their combination for protecting porcine oocytes from heat stress. The mechanisms are attributed to the fact that each treatment may have different ability to regulate the synthesis of steroid hormones and the expression of mature related genes.