Landscapes of gut bacterial and fecal metabolic signatures and their relationship in severe preeclampsia.

BACKGROUND: Preeclampsia is a pregnancy-specific disease leading to maternal and perinatal morbidity. Hypertension and inflammation are the main characteristics of preeclampsia. Many factors can lead to hypertension and inflammation, including gut microbiota which plays an important role in hypertension and inflammation in humans. However, alterations to the gut microbiome and fecal metabolome, and their relationships in severe preeclampsia are not well known. This study aims to identify biomarkers significantly associated with severe preeclampsia and provide a knowledge base for treatments regulating the gut microbiome. METHODS: In this study, fecal samples were collected from individuals with severe preeclampsia and healthy controls for shotgun metagenomic sequencing to evaluate changes in gut microbiota composition. Quantitative polymerase chain reaction analysis was used to validate the reliability of our shotgun metagenomic sequencing results. Additionally, untargeted metabolomics analysis was performed to measure fecal metabolome concentrations. RESULTS: We identified several Lactobacillaceae that were significantly enriched in the gut of healthy controls, including Limosilactobacillus fermentum, the key biomarker distinguishing severe preeclampsia from healthy controls. Limosilactobacillus fermentum was significantly associated with shifts in KEGG Orthology (KO) genes and KEGG pathways of the gut microbiome in severe preeclampsia, such as flagellar assembly. Untargeted fecal metabolome analysis found that severe preeclampsia had higher concentrations of Phenylpropanoate and Agmatine. Increased concentrations of Phenylpropanoate and Agmatine were associated with the abundance of Limosilactobacillus fermentum. Furthermore, all metabolites with higher abundances in healthy controls were enriched in the arginine and proline metabolism pathway. CONCLUSION: Our research indicates that changes in metabolites, possibly due to the gut microbe Limosilactobacillus fermentum, can contribute to the development of severe preeclampsia. This study provides insights into the interaction between gut microbiome and fecal metabolites and offers a basis for improving severe preeclampsia by modulating the gut microbiome.

Iron overload increases the sensitivity of endometriosis stromal cells to ferroptosis via a PRC2-independent function of EZH2.

Given the high concentration of iron in the micro-environment of ovarian endometriosis, it is plausible to hypothesize that ectopic endometrial cells may be more susceptible to undergoing ferroptosis. Manipulation of ferroptosis has been explored as a potential therapeutic strategy to treat related diseases. In this study, we examined the impact on ectopic endometrial stromal cells (EESCs) of iron overload and an inducer of ferroptosis. We found that the iron concentration in the ovarian endometriosis was much higher than control samples. Treatment of cultured EESCs with ferric ammonium citrate (FAC) increase the sensitivity to undergo ferroptosis. By analyzing the RNA-seq results, it was discovered that zeste 2 polycomb repressive complex 2 subunit (EZH2) was significantly downregulated in ferroptosis induced EESCs. Moreover, overexpression of EZH2 effectively prevented the induction of ferroptosis. In addition, the activity or expression of EZH2 is directly and specifically inhibited by the methyltransferase inhibitor GSK343, which raises the sensitivity of stromal cells to ferroptosis. Taken together, our findings revealed that EZH2 act as a suppressor in the induced cell ferroptosis through a PRC2-independent methyltransferase mechanism. Therefore, blocking EZH2 expression and inducing ferroptosis may be effective treatment approaches for ovarian endometriosis.

Mutational analysis of minichromosome maintenance complex component (MCM) family genes in Chinese Han women with polycystic ovarian syndrome.

PURPOSE: To investigate whether mutations in the minichromosome maintenance complex component (MCM) family genes were present in patients with polycystic ovary syndrome (PCOS) of Chinese descent. METHODS: A total of 365 Chinese patients with PCOS and 860 women without PCOS as control who underwent with assisted reproductive technology were enrolled. Genomic DNA was extracted from the peripheral blood of these patients for PCR and Sanger sequencing. The potential damage of these mutations/rare variants was analyzed through evolutionary conservation analysis and bioinformatic programs. RESULTS: Twenty-nine missense or nonsense mutations/rare variants in the MCM genes were identified in 365 patients with PCOS (7.9%, 29/365), all these mutations/rare variants were predicted to be 'disease causing' by SIFT and PolyPhen2 programs. Among those, four mutations were reported here for the first time, p.S7C (c.20C > G) in MCM2 (NM_004526.3), p.K350R (c.1049A > G) in MCM5 (NM_006739.3), p.K283N (c.849G > T) in MCM10 (NM_182751.2), and p.S1708F (c.5123C > T) in MCM3AP (NM_003906.4). All of these novel mutations were not found in our 860 control women, or also absent in public databases. In addition, the evolutionary conservation analysis results suggested that these novel mutations caused highly conserved amino acid substitutions among 10 vertebrate species. CONCLUSION: This study identified a high frequency of potential pathogenic rare variants/mutations in MCM family genes in Chinese women with PCOS, which further expands the genotype spectrum in PCOS.

Identification of a novel ESR1 mutation in a Chinese PCOS woman with estrogen insensitivity in IVF treatment.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a complex reproductive disorder, that affects approximately 5-10% of women of reproductive age. The disease is complex because its evolution may be impacted by genetic, lifestyle and environmental factors. Previous studies have emphasized the important roles of estrogen receptors in the pathogenesis of PCOS. OBJECTIVE: To use whole exome sequencing (WES) to assess possible pathogenic factors in a PCOS patient who exhibited estrogen insensitivity during hormone replacement therapy (HRT) treatment. METHODS: Genome sequencing and variant filtering via WES were performed in a patient with PCOS. DNA extraction from 364 unrelated female controls without PCOS was followed by PCR amplification, Sanger sequencing and sequence alignment. Evolutionary conservation analysis, protein structural modelling and in silico prediction were applied to analyse the potential pathogenicity of the novel ESR1 mutation. RESULT(S): During the controlled ovarian hyperstimulation (COH) period of an IVF cycle, the patient experienced markedly prolonged ovarian stimulation due to a poor response to gonadotropins (Gn) and elevated serum FSH. A novel heterozygous ESR1 mutation, c.619G > A/p.A207T, leading to the replacement of a highly conserved alanine with a threonine, was identified in this patient, via WES analysis. This novel variant was not identified in 364 unrelated female controls without PCOS, or in the Exome Aggregation Consortium (ExAC) or 1000 Genome Project. CONCLUSION(S): We identified a novel heterozygous ESR1 mutation in a Han Chinese PCOS woman exhibiting clinical signs of estrogen insensitivity. This study may provide new strategies for IVF therapy, especially for patients who exhibit estrogen insensitivity during IVF cycle.

miR-218-5p in endometrial microenvironment prevents the migration of ectopic endometrial stromal cells by inhibiting LASP1.

BACKGROUND: Our previous two-dimensional electrophoresis experiment showed that the expression of LASP1 in patients with endometriosis was significantly higher than that of control endometrium. However, the molecular mechanism by which LASP1 is regulated in endometriosis/adenomyosis is unknown. METHODS: Herein, qPCR was performed to analyze the expression levels of LASP1 and miR-218-5p between endometriosis (Ems) cells and control cells. Fluorescence in situ hybridization was carried out to measure the expression level of miR-218-5p in ectopic endometrium versus normal endometrium. After miR-218-5p mimic or inhibitor were transfected, the transwell experiment was carried out to see the effect of miR-218-5p on the migration of endometrial stromal cells (ESCs). EdU was used to measure cell proliferation rate. Dual-luciferase reporter assay was used to verify the binding of hsa-miR-218-5p to the 3'UTR of LASP1. Western blot and immunofluorescence analysis were carried out to identify the protein expression pattern of LASP1 and EMT markers in endometrial tissue. RESULTS: The miR-218-5p is mainly secreted from blood vessels and expressed in the muscle layer around the endometrium, which inhibits the expression level of LASP1 by binding the 3'UTR region of LASP1 in normal ESCs. Overexpression of miR-218-5p impedes the epithelial-to-mesenchymal transition (EMT) and prevents the migration of ESCs and the expression of Vimentin in Ems. CONCLUSIONS: Our findings revealed that miR-218-5p in endometrial microenvironment prevents the migration of ectopic endometrial stromal cells by inhibiting LASP1.

Whole-exome sequencing and functional validation reveal a rare missense variant in MMP7 that confers ovarian endometriosis risk.

Prior studies have shown that genetic factors play important roles in ovarian endometriosis. Herein, we first analyzed the whole-exome sequencing data from 158 patients with ovarian endometriosis and 385 local control women without endometriosis. Among which, a rare missense variant in the MMP7 (p.I79T, rs150338402) gene exhibited a significant frequency difference. This rare variant was screened in an additional 1176 patients and 600 control women via direct DNA sequencing. Meanwhile, a total of 38 available clinical characteristics were collected. Our results showed 45 out of 1334 (3.37%) patients, while 15 out of 985 control women (1.52%) (P = 0.0076) harbored this rare variant, respectively. This rare variant was associated with clinical features such as follicle-stimulating hormone (Padj = 0.0342), luteinizing hormone (Padj = 0.0038), progesterone (Padj = 1.4e-7), testosterone (Padj = 0.0923), total bilirubin (Padj = 0.0699), carcinoembryonic antigen (Padj = 0.0665) and squamous cell carcinoma antigen (Padj = 0.0817), respectively. Functional assays showed that this rare variant could promote cell migration, invasion, epithelial-mesenchymal transition (EMT) and increase the proteolytic protein activity of MMP7, implicating that the increased capacities of cell invasion, migration and EMT might be mediated by enhanced proteolytic activity of MMP7 mutant. These results showed that the MMP7 rare missense variant (p.I79T) played important roles in the pathogenesis of ovarian endometriosis. In conclusion, we identified, for the first time, a significantly enriched MMP7 rare variant in ovarian endometriosis; this rare variant was closely associated with certain clinical features in ovarian endometriosis; thus, it could be a promising early diagnostic biomarker for this disease.

Frequent DYSF rare variants/mutations in 152 Han Chinese samples with ovarian endometriosis.

PURPOSE: Endometriosis is a common chronic gynecological disease greatly affecting women health. Prior studies have implicated that dysferlin (DYSF) aberration might be involved in the pathogenesis of ovarian endometriosis. In the present study, we explore the potential presence of DYSF mutations in a total of 152 Han Chinese samples with ovarian endometriosis. METHODS: We analyze the potential presence of DYSF mutations by direct DNA sequencing. RESULTS: A total of seven rare variants/mutations in the DYSF gene in 10 out of 152 samples (6.6%) were identified, including 5 rare variants and 2 novel mutations. For the 5 rare variants, p.R334W and p.G941S existed in 2 samples, p.R865W, p.R1173H and p.G1531S existed in single sample, respectively; for the two novel mutations, p.W352* and p.I1642F, they were identified in three patients. These rare variants/mutations were absent or existed at extremely low frequency either in our 1006 local control women without endometriosis, or in the China Metabolic Analytics Project (ChinaMAP) and Genome Aggregation Database (gnomAD) databases. Evolutionary conservation analysis results suggested that all of these rare variants/mutations were evolutionarily conserved among 11 vertebrate species from Human to Fox. Furthermore, in silico analysis results suggested these rare variants/mutations were disease-causing. Nevertheless, we find no significant association between DYSF rare variants/mutations and the clinical features in our patients. To our knowledge, this is the first report revealing frequent DYSF mutations in ovarian endometriosis. CONCLUSION: We identified a high frequency of DYSF rare variants/mutations in ovarian endometriosis for the first time. This study suggests a new correlation between DYSF rare variants/mutations and ovarian endometriosis, implicating DYSF rare variants/mutations might be positively involved in the pathogenesis of ovarian endometriosis.

Endometrial stromal cell proteomic analysis reveals LIM and SH3 protein 1 (LASP1) plays important roles in the progression of adenomyosis.

Adenomyosis is one of the most common gynecological disorders that the molecular events underlying its pathogenesis remain not fully understood. Prior studies have shown that endometrial stromal cells (ESCs) played crucial roles in the pathogenesis of adenomyosis. In this study, we utilized two-dimensional gel electrophoresis combined with protein identification by mass spectrometry (2D/MS) proteomics analysis to compare the differential protein expression profile between the paired eutopic and ectopic ESCs (EuESCs and EcESCs) in adenomyosis, and a total of 32 significantly altered protein spots were identified. Among which, the expression of LIM and SH3 protein 1 (LASP1) was increased significantly in EcESCs compared to EuESCs. Immunohistochemical assay showed that LASP1 was overexpressed in the stromal cells of ectopic endometriums compared to eutopic endometriums; further functional analyses revealed that LASP1 overexpression could enhance cell proliferation, migration and invasion of EcESCs. Furthermore, we also showed that the dysregulated expression of LASP1 in EcESCs was associated with DNA hypermethylation in the promoter region of the LASP1 gene. However, the detailed molecular mechanisms of enhancing cell proliferation, invasion and migration caused by upregulated LASP1 in adenomyosis needs further study. For the first time, our data suggested that LASP1 plays important roles in the pathogenesis of adenomyosis, and could serve as a prognostic biomarker of adenomyosis.

ITRAQ-based proteomics analysis of tanshinone IIA on human ectopic endometrial stromal cells of adenomyosis.

PURPOSE: Adenomyosis is a diffuse or localized disease. Our previous study has indicated that tanshinone IIA (TSIIA) inhibits the proliferation, migration, and induces apoptosis of ectopic endometrial stromal cells (EESCs) of adenomyosis. However, the complex molecular mechanism of TSIIA in adenomyosis remains unclear. The objective of this study was to explore the complex molecular mechanism of TSIIA on EESCs. METHODS: In our present study, we used the proteomics approach iTRAQ (isobaric tags for relative and absolute quantitation) combined with LC-MS/MS (liquid chromatography-mass spectrometry) to investigate changes in the protein profile of EESCs treated with TSIIA. Differential proteins were analyzed by employing bioinformatics tools and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. In TSIIA treated EESCs, the protein expression levels of TNFRSF10D, PLEKHM1, FECH, and TPM1A were detected by western blotting. RESULTS: Quantitative results revealed 267 significantly differential proteins in TSIIA pretreated EESCs. Gene Ontology (GO) analysis presented an overview of dysregulated proteins in the biological process (BP), cell component (CC), and molecular function (MF) categories. Interestingly, we observed that differential proteins in the extracellular matrix (ECM)-receptor interaction pathway and estrogen signaling pathway were all involved in the focal adhesion pathway, which plays essential roles in the TSIIA-mediated inhibition of EESC proliferation and migration. Furthermore, some significantly differential proteins, which may be potential targets for the treatment of adenomyosis in the future, were validated by western blotting. CONCLUSIONS: Our study provides a useful method to detect the detailed mechanism underlying the efficacy of TSIIA on EESCs.

[MGRN1 affects the mitophagy of spermatogonial stem cells in mice].

Objective: To study the effect of mahogunin ring finger-1 (MGRN1) on the mitophagy of the spermatogonial stem cells (SSC) in mice. METHODS: SSCs cultured in vitro were divided into three groups: empty vector control, MGRN1 (MGRN1 in SSCs knocked down by RNAi), and MGRN1 + FCCP (inducing mitophagy with carbonyl cyanide p-trifluoromethoxyphenylhydrazone ï¼»FCCPï¼½ in the SSCs with down-regulated MGRN1). The expressions of mitochondrial function-related proteins (Cytochromo c and COX IV) and mitophagy-related proteins (LC3, P62, FUNDC1 and CK2) and the phosphorylation of FUNDC1 were detected by Western blot. Mitochondria and mitochondrial autophagosomes in the SSCs were observed under the electron microscope. RESULTS: Compared with the empty vector control group, the MGRN1 and MGRN1 + FCCP groups showed significantly down-regulated expressions of Cytochromo c, Cox IV, LC3 and P62, increased phosphorylation level of FUNDC1, and up-regulated expression of CK2 in the SSCs (P < 0.05). No statistically significant differences were found in the expressions of Cytochromo c, Cox IV, LC3, P62 and CK2 or in the phosphorylation level of FUNDC1 between the MGRN1 and MGRN1 + FCCP groups (P > 0.05). Electron microscopy manifested increased mitochondrial damage and reduced mitochondrial autophagosomes in the SSCs in the MGRN1 and MGRN1 + FCCP groups compared with those in the control group. CONCLUSIONS: MGRN1 affects mitophagy in the SSCs of mice, which may be associated with the effect of CK2 on the phosphorylation of FUNDC1, and its molecular mechanism needs to be further studied.

[Influence of MGRN1 on the autophagy of Sertoli cells in mice].

OBJECTIVE: To study the effect of mahogunin ring finger-1 (MGRN1) on the autophagy of Sertoli cells in mice. METHODS: Using RNA interference, we down-regulated the expression of MGRN1 in the mouse TM4 Sertoli cells cultured in vitro, determined the expressions of the autophagy-related proteins LC3-II/I, ATG-5 and ATG-7 by Western blot, and detected the autophagosomes in the TM4 cells by immunofluorescence and electron microscopy. RESULTS: Western blot showed increased expressions of LC3-II/I, ATG-5 and ATG-7 in the mouse TM4 Sertoli cells after knockdown of MGRN1. Fluorescence microscopy revealed significantly more autophagosomes in the TM4 cells than in the control group (P < 0.05). CONCLUSIONS: MGRN1 affects the autophagy of mouse Sertoli cells, and its specific molecular mechanism needs to be further studied.

FoxM1 promotes the migration of ovarian cancer cell through KRT5 and KRT7.

Forkhead box M1(FoxM1) played an important role in the pathogenesis of ovarian cancer, but its downstream molecular network is mysterious. Here, we combined ChIP-seq with RNA-seq analysis and identified 687 FoxM1-binding regions and 182 genes regulated by FoxM1. The above data pointed out that KRT5 and KRT7 were downstream target genes of FoxM1. Next, we used qPCR and Western blot to verify that FoxM1 knockdown inhibited the expression levels of KRT5 and KRT7. We also demonstrated that FoxM1 regulated KRT5 and KRT7 genes expression through binding a consensus AP-2 cis element, and showed that KRT5 and KRT7 deficiency could prevent the migration but not proliferation of SK-OV-3 cells. Finally, tissue microarray results indicated that KRT5 and KRT7 were highly expressed in ovarian cancer and positively correlated with FoxM1 expression. TCGA database showed that high expression of KRT5 and KRT7 could significantly reduce the survival rate of patients with ovarian cancer. The above results clarify the specific downstream molecular network of FoxM1 to promote the pathogenesis of ovarian cancer, and provide a basis experiment for the judgment of ovarian cancer prognosis and the design of drug targets.

Suppression of PRDX4 inhibits cell proliferation and invasion of ectopic endometrial stromal cells in endometriosis.

Oxidative stress (OS) has been proposed to play a role in the development of EMs. Peroxiredoxins are a family of antioxidant proteins that exhibit peroxidase activity in a thioredoxin-dependent manner, protecting cells against OS. The Western blotting results showed that the relative expression of PRDX4 was significantly increased in ectopic endometria compared with the normal endometria of EMs-free (p < .05). The H2O2 concentration was also significantly higher in the ectopic endometrium. PRDX4 siRNA was transfected into primary ectopic endometrial stromal cells (EESCs). The viability of the transfected EESCs was measured by CCK-8 assay, and the results showed significantly decreased cell viability. Furthermore, the apoptosis rate and ROS generation in flow cytometry assays were significantly increased after the knockdown of PRDX4 expression (p < .05). Scratch assays and transwell assays revealed that decreased expression of PRDX4 mediated by siRNA inhibited EESC migration and invasion. In conclusion, these findings indicate the potential role of PRDX4 in the development of EMs and PRDX4 as a possible therapeutic target for EMs treatment.

Novel MYH8 mutations in 152 Han Chinese samples with ovarian endometriosis.

Endometriosis is a common gynecological disease affecting up to 10% of women at reproductive age. Prior combined studies implied that MYH8 mutations might exist in endometriosis. Here, 152 Han Chinese samples with ovarian endometriosis were analyzed for the presence of MYH8 mutations. Two heterozygous missense mutations in the MYH8 gene, c.1441A > C (p.I481L) and c.4057G > A (p.E1353K), were identified in our samples. These mutations were neither found in public databases nor detected in our 485 Han Chinese control women without endometriosis. The p.I481L-mutated sample belonged to 34-year-old, who had slightly elevated serum CA 125 (42.09 U/mL); while the sample with p.E1353K mutation belonged to 25 years old, who had a markedly increased serum CA125 (89.86 U/mL). The evolutionary conservation analysis results suggested that these MYH8 mutations caused highly conserved amino acid substitutions among vertebrate species. Both the mutations were predicted to be 'disease causing' by MutationTaster and SIFT programs. In addition, no association was observed between MYH8 mutations and the available clinical data. In summary, the present study identified two novel potential pathogenic mutations in the MYH8 gene in samples with ovarian endometriosis for the first time, implying that MYH8 mutations might play a positive role in the pathogenesis of endometriosis.

The Expression of MBD6 Is Associated with Tumor Size in Uterine Leiomyomas.

Background: Uterine leiomyoma (UL) is the most common benign smooth muscle tumor of the uterus in reproductive women. Prior studies indicated that methyl-CpG-binding domain proteins (MBDs) may be involved in the pathogenesis of UL. Materials and Methods: In this study, UL tissues and paired adjacent myometrium were collected from a total of 51 patients. The expression of MBD mRNAs and their cognate proteins were analyzed via quantitative polymerase chain reaction assays and western blotting, respectively. The relationships between the MBD expression levels and the patients' clinicopathologic variables were assessed using Student's t test, nonparametric tests, or Pearson χ2 methods. Results: Our results show that both the mRNA and protein levels of MBD2 were significantly decreased in ULs compared to the adjacent myometrium. In addition, MBD6 protein expression was also decreased significantly in UL samples when compared to the adjacent myometrium. There was, however, no significant difference on the mRNA expression of MBD6 between these two groups. Neither the mRNA nor the protein levels of the other MBD members (MBD1, MBD3, MBD4, MBD5, and MeCP2) showed any significant differences between ULs and the adjacent myometria. The decreased expression of the MBD6 protein was correlated with the tumor size of ULs. Conclusions: These results suggest that the dysregulated expression of MBD2 and MBD6 in ULs may play a role in their development; however, a larger sample size together with cellular functional assays should be carried out to further elucidate the precise role of MBD6 in ULs.

FBXW7 Mutations Promote Cell Proliferation, Migration, and Invasion in Cervical Cancer.

Aim: Cervical cancer is the most common gynecological cancer. Recent studies have revealed that the F-box and WD repeat domain containing 7 (FBXW7) gene, which encodes a subunit of Skp1-Cul1-F-box protein (SCF) ubiquitin ligase, is frequently mutated in cervical squamous cell carcinomas. In this study, we investigated whether Chinese cervical cancer cells also harbor these mutations. Methods: Using PCR and sequencing assays, a total of 190 specimens from Han Chinese patients with cervical cancer were analyzed for FBXW7 mutations. Results: Two FBXW7 mutations (p.R479P and p.L443H), were identified from a study of 145 (1.4%) cervical squamous cell carcinomas. The p.L443H somatic mutation has not been previously reported. Functional assays showed that both of these FBXW7 mutations could promote cell proliferation, migration, and invasion. Conclusion: A low frequency (1.4%) of cervical squamous cell carcinomas were identified with FBXW7 mutations. We did, however, identify a novel FBXW7 mutation. Our results also demonstrated that the identified FBXW7 mutations could promote cell proliferation, migration, and invasion in cervical cancer cells.

Inhibition of formin like 2 promotes the transition of ectopic endometrial stromal cells to epithelial cells in adenomyosis through a MET-like process.

EMT (Epithelial-Mesenchymal Transition) is one of the factors in the pathogenesis of adenomyosis. FMNL2 induced invasion of cancer cell through promoting EMT, but it is unclear the role of FMNL2 in the adenomyosis. By IHC staining, we found the expression level of FMNL2 was significantly higher in the ectopic endometrial stromal cells from women with adenomyosis when compared with normal endometrial stromal cells. Knockdown of FMNL2 inhibited the invasion and migration of ectopic endometrial stromal cells and promoted the protein levels of E-cadherin and Vimentin. Meanwhile, inhibition of FMNL2 could induce the cell membrane localization of E-cadherin. Our findings reveal that the aberrant activation of FMNL2 promotes the pathogenesis of adenomyosis through inducing the EMT process. On the contrary, inhibition of FMNL2 promotes the transition of ectopic endometrial stromal cells to epithelial cells in adenomyosis through a MET-like process.

The mitochondrial DNA 4977-bp deletion and copy number alteration in Han Chinese samples with uterine fibroids.

Uterine fibroids (UFs) are the most common benign neoplasms, but their pathogenesis is not completely understood. Thus far, alterations in the mitochondrial DNA (mtDNA) content and the mtDNA 4977-bp deletion level in UFs, as well as the corresponding nontumorous tissue, have remained elusive. To test whether large mtDNA deletions and mtDNA content are involved in the pathogenesis of UFs, a total of 309 UF tissues and 28 paired adjacent myometrium from 270 UF patients were enrolled for the analysis of large mtDNA deletions and mtDNA content through the use of nested PCR and qPCR techniques, respectively. In our samples, a 4977-bp deletion was identified: 36 out of 309 UF tissues (11.56%) and 15 out of 28 (53.57%) paired adjacent myometrium were detected to harbor the 4977-bp deletion. In addition, a novel 4838-bp mtDNA deletion was identified in three UF tissues, and other different sizes of deleted fragments (4910, 4926, 5135-bp) were also found in UFs for the first time. Furthermore, older age was significantly associated with an mtDNA large deletion in the paired adjacent myometrium. We also found that increased mtDNA content and higher expression of ND1 occurred in solitary fibroids compared to adjacent myometrium. In conclusion, we identified a lower frequency of mtDNA large deletions and some novel large deletion in UFs for the first time. Furthermore, there was a general increase of mtDNA copy number during solitary UF development. Although the definite mechanism by which mtDNA was altered is supposed to be further confirmed, it will be helpful for further studies on the pathological mechanism of UFs.

Mutation analysis of ZP1, ZP2, ZP3 and ZP4 genes in 152 Han Chinese samples with ovarian endometriosis.

Endometriosis is characterized by the ectopic implant of endometrial tissue outside the uterine cavity and found in ˜35-50% of subfertile women. Previous studies have found that endometriosis had frequent defects in zona pellucida (ZP), and mutations in ZP genes could lead to ZP defects, raising the possibility that mutations in ZP genes might exist in endometriosis. We analyzed a total of 152 Han Chinese samples with ovarian endometriosis for the presence of mutations in the ZP1, ZP2, ZP3 and ZP4 genes. Two novel nonsynonymous ZP4 mutations were identified in three out of 152 (2.0%) samples: a p.M1?/(c.3 G > C) mutation in a 27- and 35-year-old sample, respectively, and a p.A433 V (c.1298C > T) mutation in a 31-year-old patient. No mutations were detected in ZP1, ZP2 or ZP3 genes; furthermore, no mutations in ZP genes were identified in 85 female control samples without endometriosis. The p.M1?/(c.3 G > C) mutation could lead to the usage of a downstream translation initiation site, while the evolutionary conservation and protein structural modeling analyses suggested that the p.A433 V mutation might be functionally important. However, there were strikingly different fertility outcomes among the three samples with ZP4 mutations: the p.A433V-mutated sample had no problem in fertility; while the p.M1?-mutated samples presented with paradoxical effects on fertility: the 35-year-old patient had a child while the 27-year-old patient was infertile, who underwent two spontaneous abortions and an implantation failure after IVF treatment. These results suggested that the potential role of ZP4 mutations on human fertility might be more complex than we thought, and other genetic and environment factors might play a role. In conclusion, we identified two novel mutations in the ZP4 gene in 2.0% of Han Chinese patients with ovarian endometriosis for the first time, our results suggested that mutations in ZP4, but not ZP1, ZP2 and ZP3, might play active roles in the pathogenesis of ovarian endometriosis, despite the mutation-carriers present with complex fertility outcomes.

Analysis of CARD10 and CARD11 somatic mutations in patients with ovarian endometriosis.

Endometriosis is a complex and heterogeneous pre-malignant inflammatory disease harboring multiple gene mutations. Previous studies have suggested that caspase recruitment domain family member (CARD)10 and CARD11 mutations may exist in endometriosis. In the present study, a collection of endometriotic lesions and paired peripheral blood from 101 patients with ovarian endometriosis were obtained, and the entire coding sequences of the CARD10 and CARD11 genes were sequenced. Evolutionary conservation analysis and online prediction programs were applied to analyze the disease-causing potential of the identified mutations. A total of 4 novel somatic mutations were identified in 4 out of the 101 (4.0%) samples: 2 in-frame deletions in CARD10 (c.785_790delAGGAGA, p.K272_E273delKE; c.785_802delAGGAGAAGGAGAAGGAGA, p.K272_V277delKEPDNV) and 2 heterozygous missense mutations in CARD11 (c.49G>T, p.D17Y; c.160G>C, p.E54Q). The sample with CARD10 p.K272_E273delKE deletion was obtained from a 47-year-old patient who was also diagnosed with uterine leiomyoma, while the CARD10 p.K272_V277delKEPDNV-mutated sample was from a 43-year-old patient exhibiting a decreased blood eosinophil granulocyte ratio (0.3%) and an elevated serum creatine kinase level (314 U/l). The patient with the CARD11 p.D17Y mutation was 38 years old and exhibited an increased level of cancer antigen 125 (45.4 U/ml), while the patient with the CARD11 p.E54Q mutation was 46 years old and exhibited no other gynecological conditions. Evolutionary conservation analysis and online prediction programs suggested that these mutations may be disease-causing. In summary, 4 novel somatic mutations in the CARD10 and CARD11 genes were identified from amongst 101 cases of ovarian endometriosis for the first time, these mutations may serve active roles in the development of ovarian endometriosis.