PRDM14 extinction enables the initiation of trophoblast stem cell formation.

Trophoblast stem cells (TSCs) can be chemically converted from embryonic stem cells (ESCs) in vitro. Although several transcription factors (TFs) have been recognized as essential for TSC formation, it remains unclear how differentiation cues link elimination of stemness with the establishment of TSC identity. Here, we show that PRDM14, a critical pluripotent circuitry component, is reduced during the formation of TSCs. The reduction is further shown to be due to the activation of Wnt/ß-catenin signaling. The extinction of PRDM14 results in the erasure of H3K27me3 marks and chromatin opening in the gene loci of TSC TFs, including GATA3 and TFAP2C, which enables their expression and thus the initiation of the TSC formation process. Accordingly, PRDM14 reduction is proposed here as a critical event that couples elimination of stemness with the initiation of TSC formation. The present study provides novel insights into how induction signals initiate TSC formation.

Comprehensive preimplantation genetic testing for balanced insertional translocation carriers.

BACKGROUND: Balanced insertional translocations (BITs) can increase the risk of infertility, recurrent miscarriages or neonatal birth defects due to chromosomal imbalances in gametes. However, studies on preimplantation genetic testing (PGT) for patients carrying BITs are inadequate. METHODS: A preimplantation genetic genotyping and haplotype analysis approach was developed and implemented in this study. Genome-wide SNP genotyping was performed, followed by core family-based haplotype analysis. The balanced insertion segments in euploid embryos were inferred from the haplotypes inherited from the carrier parent. RESULTS: A total of 10 BIT carrier couples were enrolled in our study. 15 in vitro fertilisation cycles were conducted, resulting in 73 blastocysts biopsied and subjected to PGT analysis. Among these, 20 blastocysts displayed rearrangement-related imbalances, 13 exhibited de novo aneuploidies, 15 presented a complex anomaly involving both imbalances and additional aneuploidies, while 25 were euploid. Within the euploid embryos, 12 were balanced carrier embryos and 13 were non-carrier embryos. To date, eight non-carrier and one carrier embryos have been transferred, resulting in seven clinical pregnancies. All pregnancies were recommended to perform prenatal diagnosis, our date revealed complete concordance between fetal genetic testing results and PGT results. Presently, five infants have been born from these pregnancies, and two pregnancies are still ongoing. CONCLUSION: The proposed method facilitates comprehensive chromosome screening and the concurrent identification of balanced insertions or normal karyotypes in embryos. This study offers an effective and universally applicable strategy for BIT carriers to achieve a healthy pregnancy and prevent the transmission of BITs to their offspring.

Sodium oligomannate's amelioration of reproductive and metabolic phenotypes in a letrozole-induced PCOS-like mouse model depends on the gut microbiome†.

It has been well established that there is a connection between polycystic ovary syndrome pathology and gut microbiome dysbiosis. A marine-derived oligosaccharide, GV-971, has been reported to alter gut microbiota and alleviate Aß amyloidosis. In this study, the effects of GV-971 on polycystic ovary syndrome-like mice were explored. Mice were randomly assigned into four groups: control, letrozole, letrozole + GV-971, and control + GV-971. Glucose metabolism in polycystic ovary syndrome-like mice was ameliorated by GV-971, while the reproductive endocrine disorder of polycystic ovary syndrome-like mice was partially reversed. The messenger ribonucleic acid levels of steroidogenic enzymes in ovaries of polycystic ovary syndrome-like mice were improved. GV-971 restored the fertility of polycystic ovary syndrome-like mice and significantly increase the number of litters. Furthermore, GV-971 treatment effectively mitigated abnormal bile acid metabolism. Notably, after GV-971 intervention, gut microbiota alpha-diversity was considerably raised and the relative abundance of Firmicutes was reduced. In conclusion, the hyperinsulinemia and hyperandrogenemia of polycystic ovary syndrome-like mice were alleviated by GV-971 intervention, which was associated with mitigating bile acid metabolism and modulating gut microbiota.

Detection of cryptic balanced chromosomal rearrangements using high-resolution optical genome mapping.

BACKGROUND: Chromosomal rearrangements have profound consequences in diverse human genetic diseases. Currently, the detection of balanced chromosomal rearrangements (BCRs) mainly relies on routine cytogenetic G-banded karyotyping. However, cryptic BCRs are hard to detect by karyotyping, and the risk of miscarriage or delivering abnormal offspring with congenital malformations in carrier couples is significantly increased. In the present study, we aimed to investigate the potential of single-molecule optical genome mapping (OGM) in unravelling cryptic chromosomal rearrangements. METHODS: Eleven couples with normal karyotypes that had abortions/affected offspring with unbalanced rearrangements were enrolled. Ultra-high-molecular-weight DNA was isolated from peripheral blood cells and processed via OGM. The genome assembly was performed followed by variant calling and annotation. Meanwhile, multiple detection strategies, including FISH, long-range-PCR amplicon-based next-generation sequencing and Sanger sequencing were implemented to confirm the results obtained from OGM. RESULTS: High-resolution OGM successfully detected cryptic reciprocal translocation in all recruited couples, which was consistent with the results of FISH and sequencing. All high-confidence cryptic chromosomal translocations detected by OGM were confirmed by sequencing analysis of rearrangement breakpoints. Moreover, OGM revealed additional complex rearrangement events such as inverted aberrations, further refining potential genetic interpretation. CONCLUSION: To the best of our knowledge, this is the first study wherein OGM facilitate the rapid and robust detection of cryptic chromosomal reciprocal translocations in clinical practice. With the excellent performance, our findings suggest that OGM is well qualified as an accurate, comprehensive and first-line method for detecting cryptic BCRs in routine clinical testing.

Targeted Imaging of Endometriosis and Image-Guided Resection of Lesions Using Gonadotropin-Releasing Hormone Analogue-Modified Indocyanine Green.

Objective: In this study, we utilized gonadotropin-releasing hormone analogue-modified indocyanine green (GnRHa-ICG) to improve the accuracy of intraoperative recognition and resection of endometriotic lesions. Methods: Gonadotropin-releasing hormone receptor (GnRHR) expression was detected in endometriosis tissues and cell lines via immunohistochemistry and western blotting. The in vitro binding capacities of GnRHa, GnRHa-ICG, and ICG were determined using fluorescence microscopy and flow cytometry. In vivo imaging was performed in mouse models of endometriosis using a near-infrared fluorescence (NIRF) imaging system and fluorescence navigation system. The ex vivo binding capacity was determined using confocal fluorescence microscopy. Results: GnRHa-ICG exhibited a significantly stronger binding capacity to endometriotic cells and tissues than ICG. In mice with endometriosis, GnRHa-ICG specifically imaged endometriotic tissues (EMTs) after intraperitoneal administration, whereas ICG exhibited signals in the intestine. GnRHa-ICG showed the highest fluorescence signals in the EMTs at 2 h and a good signal-to-noise ratio at 48 h postadministration. Compared with traditional surgery under white light, targeted NIRF imaging-guided surgery completely resected endometriotic lesions with a sensitivity of 97.3% and specificity of 77.8%. No obvious toxicity was observed in routine blood tests, serum biochemicals, or histopathology in mice. Conclusions: GnRHa-ICG specifically recognized and localized endometriotic lesions and guided complete resection of lesions with high accuracy.

Sox6 impairs the adipogenic commitment of mesenchymal stem cells by targeting lysyl oxidase and preadipocyte factor 1.

The commitment of mesenchymal stem cells (MSCs) to preadipocytes and the termination of differentiation to adipocytes are critical for maintaining systemic energy homeostasis. However, our knowledge of the molecular mechanisms governing the commitment of MSCs to preadipocytes and the subsequent termination of their differentiation into adipocytes remain limited. Additionally, the role of Sox6 sex-determining region Y (SRY)-box6 (Sox6), a transcription factor that regulates gene transcription, is reportedly involved in various cellular processes, including adipogenesis; however, its function in regulating preadipocyte development and the factors involved in the termination of adipogenic differentiation remain unexplored. Therefore, we investigated the role of Sox6 in regulating the differentiation of adipocytes by monitoring the effects of its overexpression in C3H10T1/2 cells (in vitro) and C57BL/6J mouse (in vivo) models of adipogenesis. We observed lower Sox6 expression in the adipose tissue of obese mice than that in control mice. Sox6 overexpression inhibited the differentiation of MSC by directly binding to the lysyl oxidase (Lox) and preadipocyte factor 1 (Pref1) promoters, which was potentiated by histone deacetylase-1(HDAC1). Our findings suggest that Sox6 is a key regulator of MSC commitment to adipocytes; therefore, targeting the Sox6-mediated regulation of this process could offer potential therapeutic avenues for addressing obesity and related metabolic disorders.

Dominant-negative transforming growth factor-ß receptor-armoured mesothelin-targeted chimeric antigen receptor T cells slow tumour growth in a mouse model of ovarian cancer.

Ovarian cancer is a major cause of death among all gynaecological cancers. Although surgery, chemotherapy and targeted therapy have yielded successful outcomes, the 5-year survival rate remains < 30%. Adoptive immunotherapy, particularly chimeric antigen receptor (CAR) T-cell therapy, has demonstrated improved survival in acute lymphoblastic leukaemia with manageable toxicity. We explored CAR T-cell therapy in a preclinical mouse model of ovarian cancer. Second-generation CAR T cells were developed targeting mesothelin (MSLN), which is abundantly expressed in ovarian cancer. Cytotoxicity experiments were performed to verify the lethality of CAR T cells on target cells via flow cytometry. The in vivo antitumour activity of MSLN CAR T cells was also verified using a patient-derived xenograft (PDX) mouse model with human tumour-derived cells. We also evaluated the potency of CAR T cells directed to MSLN following co-expression of a dominant-negative transforming growth factor-ß receptor type II (dnTGFßRII). Our data demonstrate that anti-MSLN CAR T cells specifically eliminate MSLN-expressing target cells in an MSLN density-dependent manner. This preclinical research promises an effective treatment strategy to improve outcomes for ovarian cancer, with the potential for prolonging survival while minimizing risk of on-target off-tumour toxicity.

Genetic variability analysis of human papillomavirus 58: Novel sublineage identification and persistent infection association.

This study aims to characterize the genetic variability of HPV58, identify novel lineages and sublineages, and explore the association between persistent/multiple HPV58 infections and genetic variation. In this study, samples from 124 women with HPV58 infection in Eastern China were collected and 81 isolates of E6 and L1 full-length genes were successfully amplified from 55 samples. We evaluated the diversity of genetic variants and performed correlation analyses between genetic variability and pathology, vaccination, multiple infections, and persistent infections. Among the E6 and L1 gene sequences collected, the dominant prevailing sublineages were A1 (46.2%) and A2 (23.1%). In addition, we found two potential novel sublineages denoted as the A4 and A5 sublineage. A total of 50 nucleotide substitutions, including 28 synonymous substitutions and 22 nonsynonymous substitutions, were observed in the E6 and L1 genes. Among them, variants with A388C/K93N substitutions in the E6 gene correlated with persistent infection (≥1 and ≥2 years) (p < 0.005), and C307T/C66C was associated with persistent infection (≥2 years) (p < 0.005). Notably, two mutations above were detected in the isolate from the patient with breakthrough vaccine infection. Our study found two novel sublineages and sites of genetic variability in multiple and persistent infection variants. In addition, we identified two mutational sites associated with persistent infection. This study provides new insight into the clinical characteristics of HPV 58 genetic variations and offers new ideas for research on next-generation vaccines in Eastern China.

The molecular regulatory mechanisms of meiotic arrest and resumption in Oocyte development and maturation.

In human female primordial germ cells, the transition from mitosis to meiosis begins from the fetal stage. In germ cells, meiosis is arrested at the diplotene stage of prophase in meiosis I (MI) after synapsis and recombination of homologous chromosomes, which cannot be segregated. Within the follicle, the maintenance of oocyte meiotic arrest is primarily attributed to high cytoplasmic concentrations of cyclic adenosine monophosphate (cAMP). Depending on the specific species, oocytes can remain arrested for extended periods of time, ranging from months to even years. During estrus phase in animals or the menstrual cycle in humans, the resumption of meiosis occurs in certain oocytes due to a surge of luteinizing hormone (LH) levels. Any factor interfering with this process may lead to impaired oocyte maturation, which in turn affects female reproductive function. Nevertheless, the precise molecular mechanisms underlying this phenomenon has not been systematically summarized yet. To provide a comprehensive understanding of the recently uncovered regulatory network involved in oocyte development and maturation, the progress of the cellular and molecular mechanisms of oocyte nuclear maturation including meiosis arrest and meiosis resumption is summarized. Additionally, the advancements in understanding the molecular cytoplasmic events occurring in oocytes, such as maternal mRNA degradation, posttranslational regulation, and organelle distribution associated with the quality of oocyte maturation, are reviewed. Therefore, understanding the pathways regulating oocyte meiotic arrest and resumption will provide detailed insight into female reproductive system and provide a theoretical basis for further research and potential approaches for novel disease treatments.

Prognostic significance of positive peritoneal cytology in endometrial carcinoma based on ESGO/ESTRO/ESP risk classification: A multicenter retrospective study.

OBJECTIVE: This study aimed to determine the prognostic significance of positive peritoneal cytology (PC) on endometrial carcinoma (EC) patients under the ESGO/ESTRO/ESP risk classification. METHODS: This study retrospectively analyzed EC patients from 27 medical centers in China from 2000 to 2019. Patients were divided into three ESGO risk groups: low-risk, intermediate-risk and high-intermediate risk, and high-risk groups. The covariates were balanced by using the propensity score-based inverse probability of treatment weighting (PS-IPTW). The prognostic significance of PC was assessed by Kaplan-Meier curves and multivariate Cox regression analysis. RESULTS: A total of 6313 EC patients with PC results were included and positive PC was reported in 384 women (6.1%). The multivariate Cox analysis in all patients showed the positive PC was significantly associated with decreased PFS (hazard ratio [HR] 2.20, 95% confidence interval [CI] 1.55-3.13, P < 0.001) and OS (HR 2.25, 95% CI 1.49-3.40, P < 0.001),and the Kaplan-Meier curves also showed a poor survival in the intermediate and high-intermediate risk group (5-year PFS: 75.5% vs. 93.0%, P < 0.001; 5-year OS: 78.3% vs. 96.4%, P < 0.001); While in the low-risk group, there were no significant differences in PFS and OS between different PC status (5-year PFS: 93.1% vs. 97.3%, P = 0.124; 5-year OS: 98.6% vs. 98.2%, P = 0.823); in the high-risk group, significant difference was only found in PFS (5-year PFS: 62.5% vs. 77.9%, P = 0.033). CONCLUSION: Positive PC was an adverse prognostic factor for EC, especially in the intermediate and high-intermediate risk patients. Gynecologic oncologists should reconsider the effect of positive PC on different ESGO risk groups.

Bowel wall thickness measured by MRI is useful for early diagnosis of bowel endometriosis.

OBJECTIVE: To evaluate MRI features of bowel endometriosis (BE) and verify its clinical significance compared with pathological diagnosis. MATERIALS AND METHODS: Since 2018, patients clinically diagnosed with deep endometriosis (DE) and planned to undergo surgery were enrolled prospectively. MRI parameters including traction, thickening sign of the rectum, obliteration of the Douglas Pouch, sign of adenomyosis, and pelvic adhesion were extracted. Uni- and multi-variate analyses were performed to explore their association with pathological diagnosis of BE. ROC curve was utilized to ascertain the appropriate cutoff value for predicting the presence and assessing the severity of BE. RESULTS: A total of 226 patients with DE were recruited, and 154 BE cases were pathologically confirmed. Logistic regression analysis revealed that thickness of the rectal wall, traction sign of the rectum, and obliteration of the Douglas Pouch were independent factors to predict the presence of BE with the OR 1.59 (95% CI: 1.29-1.96), 0.24 (95% CI: 0.09-0.67), and 0.17 (95% CI: 0.07-0.40), respectively (p all < 0.01). A cutoff value of 6.0 mm for the thickness of rectal wall resulted in the highest predictive value of BE (specificity: 90.3%; sensitivity: 78.6%). For patients with measured thickness of the rectal wall over 6.0 mm, 72.1% (93/129) was confirmed BE with lesions infiltrated more than muscular layer. CONCLUSION: This prospective study indicates that based on precise definition of visualized features on MRI images, BE could be recognized pre-operatively. DE patients with thickness of rectal wall exceeding 6.0 mm have a greater probability of BE. CLINICAL RELEVANCE STATEMENT: Based on precise definition of visualized features and accurate measurement on MRI images, bowel infiltrating among deep endometriosis patients could be recognized pre-operatively. KEY POINTS: • Precise definition of measurable MRI parameters made it possible for early detection of bowel endometriosis. • Thickening sign, traction sign of the rectum, and obliteration of the Douglas Pouch were typical radiological indicators for bowel endometriosis. • Bowel involvement is more sensitive to be detected among pelvic deep endometriosis patients with the thickness of the rectal wall over 6.0 mm.

RAD51D Secondary Mutation-Mediated Resistance to PARP-Inhibitor-Based Therapy in HGSOC.

Ovarian cancer is the leading cause of gynecologic cancer-related death, and PARP inhibitors (PARPis) are becoming a promising treatment option, as demonstrated by recent clinical trials. After PARPi exposure, somatic reversion mutations in the homologous recombination genes may be a mechanism of PARPi resistance in ovarian carcinoma. We present an ovarian cancer case of a 61-year-old woman, who underwent routine tumor reduction surgery followed by platinum and PARPis. She demonstrated a good response to PARPis for 15 months before recurrence and secondary tumor reduction surgery. However, post-surgery platinum and PARPi treatment only kept the disease stable for 5 months. A potential molecular mechanism for PARPi resistance was investigated using next-generation sequencing, immunohistochemical (IHC) staining, and other functional assays. A germline RAD51D loss-of-function mutation was found in the reported case (LRG_516t1:c.270_271dup p1:p.(Lys91fs*13)). Subsequently, a secondary mutation (LRG_516t1:c.271_282 del) was identified in the same locus of the germline duplication in the post-progression biopsies and ctDNA. The IHC staining supported low expression of RAD51D in the initial tumor tissue, but the expression was restored after the correction of the open reading frame by the secondary mutation. The in vitro results supported that the loss-of-function mutation of RAD51D was the basis for the initial response to the platinum and PARPi therapy, while the newly acquired reversion mutation could be attributed to the observed PARPi resistance. An acquired mutation can reverse a loss-of-function change in RAD51D and can result in PARPi resistance in a hereditary ovarian cancer patient. Liquid biopsy could be considered for longitudinal monitoring in ovarian patients under PARPi-based therapy, which can identify acquired resistant mutations earlier and facilitate precision management.

Chronic Stress Blocks the Endometriosis Immune Response by Metabolic Reprogramming.

Studies have shown that the occurrence and development of endometriosis are closely linked to long-term psychological stress. The specific contribution of chronic stress to the metabolic adaptations in patients with endometriosis is still unknown. Lesions were removed from ten endometriosis patients during an operation, and the participants were divided into two groups using a psychological questionnaire. An mRNA Human Gene Expression Microarray analysis was applied to compare the mRNA expression profiles between the chronic stress group and the control group. In addition, the reliability of the mRNA Human Gene Expression Microarray analysis was verified by using research on metabolites based on both the liquid chromatography (LC-MS/MS) technique and quantitative reverse transcription polymerase chain reaction (RT-PCR). A microarray analysis of significantly up-regulated, differentially expressed genes between the chronic stress and the control groups showed genes that were principally related to metabolism-related processes and immune-related processes, such as the immune response process, negative regulation of T cell proliferation, the leucine metabolic process, and the L-cysteine metabolic process (p < 0.05). LC-MS showed that the differential metabolites were primarily concerned with arginine and proline metabolism, D-glutamine and D-glutamate metabolism, aspartate metabolism, glycine, serine metabolism, and tyrosine metabolism (p < 0.05). The possibility of chronic stress blocks the endometriosis immune response through metabolic reprogramming. Chronic stress reduces the supply of energy substrates such as arginine and serine, down-regulates T immune cell activation, and affects the anti-tumor immune response, thereby promoting the migration and invasion of endometriosis lesions in patients with chronic stress.

Out of step societal and Darwinian adaptation during evolution is the cause of multiple women's health issues.

During human evolution, major changes in our societal conditions and environment took place without sufficient time for concomitant genetic alterations, leading to out of step adaptation and diseases in women. We first discuss recent societal adaptation mismatch (menstrual bleeding; increases in cancers of reproductive organs, endometriosis; mother's nursing; polycystic ovarian syndrome; transgenerational epigenetic modifications), followed by Darwinian out of step adaptation (labor difficulties; sex chromosomes, human diseases and sex disparity in genomic DNA). We discuss the evolutionary basis of menstrual bleeding, followed by recent increases in cancers of reproductive organs and endometriosis. The importance of breastfeeding by mothers is also emphasized. Earlier onset of menarche, decreased rates of childbirths and breastfeeding resulted in increased number of menstrual cycles in a lifetime, coupled with excess estrogen exposure and incessant ovulation, conditions that increased the susceptibility to mammary and uterine cancers as well as ovarian epithelial cancer and endometriosis. Shorter lactation duration in mothers also contributed to more menstrual cycles. We further discuss the evolutionary basis of the prevalent polycystic ovary syndrome. During the long-term Darwinian evolution, difficulties in childbirth evolved due to a narrowed pelvis, our upright walking and enlarged fetal brain sizes. Because there are 1.5% genomic DNA differences between woman and man, it is of significance to investigate sex-specific human physiology and diseases. In conclusion, understanding out of step adaptation during evolution could allow the prevention and better management of female reproductive dysfunction and diseases.

Tackling cellular senescence by targeting miRNAs.

Cellular senescence, which is characterized by permanent proliferation arrest, has become an important target for the amelioration of various human diseases. The activity of senescent cells is mainly related to the senescence-associated secretory phenotype (SASP). The SASP can cause chronic inflammation in local tissues and organs through autocrine and paracrine mechanisms, and a series of factors secreted by senescent cells can deteriorate the cellular microenvironment, promoting tumor formation and exacerbating aging-related diseases. Therefore, avoiding the promotion of cancer is an urgent problem. In recent years, increased attention has been given to the mechanistic study of microRNAs in senescence. As important posttranscriptional regulators, microRNAs possess unique tissue-specific expression in senescence. MicroRNAs can regulate the SASP by regulating proteins in the senescence signaling pathway, the reverse transcriptase activity of telomerase, the generation of reactive oxygen species and oxidative damage to mitochondria. Numerous studies have confirmed that removing senescent cells does not cause significant side effects, which also opens the door to the development of treatment modalities against senescent cells. Herein, this review discusses the double-edged sword of cellular senescence in tumors and aging-related diseases and emphasizes the roles of microRNAs in regulating the SASP, especially the potential of microRNAs to be used as therapeutic targets to inhibit senescence, giving rise to novel therapeutic approaches for the treatment of aging-associated diseases.

Expression profiling of lncRNAs and mRNAs in placental site trophoblastic tumor (PSTT) by microarray.

As a rare type of gestational trophoblastic disease, placental site trophoblastic tumor (PSTT) is originated from intermediate trophoblast cells. Long noncoding RNAs (lncRNAs) regulate numerous biological process. However, the role of lncRNAs in PSTT remains poorly understood. In the present study, expression levels of lncRNAs and mRNAs in four human PSTT tissues and four normal placental villi were investigated. The results of microarray were validated by the reverse transcription and quantitative real-time polymerase reaction (RT-qPCR) and immunohistochemistry analyses. Furthermore, GO and KEGG pathway analyses were performed to identify the underlying biological processes and signaling pathways of aberrantly expressed lncRNAs and mRNAs. We also conducted the coding-non-coding gene co-expression (CNC) network to explore the interaction of altered lncRNAs and mRNAs. In total, we identified 1247 up-regulated lncRNAs and 1013 down-regulated lncRNAs as well as 828 up-regulated mRNAs and 1393 down-regulated mRNAs in PSTT tissues compared to normal villi (fold change ≥ 2.0, p < 0.05). GO analysis showed that mitochondrion was the most significantly down-regulated GO term, and immune response was the most significantly up-regulated term. A CNC network profile based on six confirmed lncRNAs (NONHSAT114519, NR_103711, NONHSAT003875, NONHSAT136587, NONHSAT134431, NONHSAT102500) as well as 354 mRNAs was composed of 497 edges. GO and KEGG analyses indicated that interacted mRNAs were enriched in the signal-recognition particle (SRP)-dependent cotranslational protein targeting to membrane and Ribosome pathway. It contributes to expand the understanding of the aberrant lncRNAs and mRNAs profiles of PSTT, which may be helpful for the exploration of new diagnosis and treatment of PSTT.

PLODs are overexpressed in ovarian cancer and are associated with gap junctions via connexin 43.

Procollagen-lysine, 2-oxoglutarate 5-dioxygenases (PLODs) play important roles in cancer progression, but their role in ovarian cancer remains elusive. In silico analysis of expression of PLODs in ovarian cancer was performed with reproduction of The Cancer Genome Atlas dataset. PLOD-enriched pathways and related gene(s) were validated by immunohistochemistry (IHC) in 80 ovarian cancer tissue blocks and in vivo xenograft murine models. PLODs (PLOD-1, -2, and -3) were overexpressed in ovarian cancer tissue. Overexpression of individual PLODs showed mutual exclusivity. Each of the three PLODs was differentially expressed between normal and cancer tissue of the ovary. PLOD1 was not prognostic, whereas lower PLOD2 and higher PLOD3 expression were associated with worsened prognosis, respectively. Cases with PLOD overexpression showed enrichment in gap junctions. GJA1 (connexin 43) was significantly overexpressed in cases with PLOD overexpression. IHC in tissue showed the strongest positive correlation between PLOD3 and connexin 43 expression, followed by PLOD2. As per Harmonizome, we selected SKOV3 and CAOV3 cell lines based on constitutive high PLOD1 and PLOD2/PLOD3 expression, respectively for in vitro and in vivo modeling. Only knockdown of PLOD3 was significantly associated with decreased GJA1 expression level in both cell lines. IHC in murine xenograft tumors also showed significantly lower connexin 43 in PLOD3-KD SKOV3 tumors. We conclude that PLODs are generally overexpressed in ovarian cancer and each PLOD may be functionally non-redundant. Association between PLOD3 and gap junctions warrants further investigation.

Impaired decidualization of human endometrial stromal cells from women with adenomyosis†.

Differentiation of endometrial stromal cells (ESCs) into secretory decidualized cells (dESCs) is essential for embryo implantation. Adenomyosis is a common benign gynecological disease that causes infertility. However, whether adenomyosis affects decidualization of human ESCs is elusive. Primary eutopic ESCs were obtained from patients with adenomyosis (n = 9) and women with nonendometrial diseases (n = 12). We determined the capacity of decidualization of human ESCs by qRT-PCR, Edu proliferation assay, cytokine array, and ELISA assay. We found that the expression of decidualization markers (IGFBP1 and PRL) in ESCs of adenomyosis was reduced, concomitant with increased cell proliferation. Differential secretion of cytokines in dESCs, including CXCL1/2/3, IL-6, IL-8, MCP-1, VEGF-A, MIP-3α, OPN, SDF-1α, HGF, and MMP-9, was observed between adenomyosis and nonadenomyosis. Moreover, the expression of decidualization regulators (HOXA10 at both mRNA and protein levels, FOXO1, KLF5, CEBPB, and HAND2 at mRNA levels) in the eutopic endometrium of adenomyosis was lower than that of nonadenomyosis. We propose that ESCs from adenomyosis have defected ability to full decidualization, which may lead to a nonreceptive endometrium.

Scribble downregulation in adenomyosis compromises endometrial stromal decidualization by decreasing FOXO1 expression.

STUDY QUESTION: Does Scribble (SCRIB) contribute to aberrant decidualization of endometrial stromal cells (ESC) in adenomyosis? SUMMARY ANSWER: SCRIB knockdown impairs decidualization of ESC by decreasing Fork-head box O1A (FOXO1) expression through the protein kinase B (AKT) and atypical protein kinase C (aPKC) activated pathways. WHAT IS KNOWN ALREADY: Stromal SCRIB is required for primary decidual zone formation and pregnancy success in mice. In our previous studies, decidualization was dampened in ESC isolated from adenomyosis patients, yet the underlying molecular mechanisms remain elusive. STUDY DESIGN, SIZE, DURATION: Eutopic endometrium tissue samples from diffuse adenomyosis and non-adenomyosis patients in proliferative, early-secretory and mid-secretory phase (n = 10 per phase for each group) were explored. In parallel, in vitro decidualization studies were carried out in ESC isolated from non-adenomyosis women (n = 8). PARTICIPANTS/MATERIALS, SETTING, METHODS: The endometrial SCRIB expression was analyzed using immunohistochemistry staining and western blot. Quantitative RT-PCR (qRT-PCR), western blot and immunofluorescence staining were used to explore the expression of SCRIB in ESC during in vitro decidualization. siRNA-mediated SCRIB knockdown followed by decidual markers expression analysis, flow cytometry for cell cycle analysis and phalloidin staining for morphological analysis were performed to examine the function of SCRIB in ESC decidualization. RNA-sequencing was performed to examine the SCRIB-mediated transcriptional changes in decidualized ESC (DSC). Rescue experiments using an AKT inhibitor MK2206 and aPKC inhibitor NSC37044 were used to investigate the signaling pathways through which could mediate SCRIB-regulated FOXO1 protein expression and ESC decidualization. MAIN RESULTS AND THE ROLE OF CHANCE: We found that the expression of SCRIB in the mid-secretory phase eutopic endometrial stroma of adenomyosis patients was significantly lower than that of non-adenomyosis. SCRIB knockdown reduced the expression of decidual markers, abrogated the epithelioid-like morphological changes, inhibited the mesenchymal-to-epithelial transitions process and promoted the cell cycle progression of ESC during in vitro decidualization. SCRIB knockdown-induced decidualization defects were attributed to a decrease in expression of transcription factor FOXO1, known to regulate decidualization. Furthermore, we found that SCRIB knockdown induced the aberrant activation of AKT and aPKC, which led to FOXO1 phosphorylation and degradation. Rescue assay confirmed that restoring the expression of FOXO1 effectively reversed the decidualization defects and cell cycle progression caused by SCRIB knockdown. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: In this study, it was demonstrated that SCRIB knockdown mediated the activation of AKT and aPKC, contributing to FOXO1 degradation and aberrant decidualization, however, the molecular link between AKT and aPKC signaling was not determined, and still requires further exploration. WIDER IMPLICATIONS OF THE FINDINGS: Our findings support the hypothesis that adenomyosis interferes with embryo implantation due to insufficient endometrial receptivity. Abnormal decidualization of the endometrial stroma may clarify the possible association between adenomyosis and infertility. Our findings may be clinically useful for counseling and treatment of infertile adenomyosis patients. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Natural Science Foundation of China (82001523 and 82171639). The authors have no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER: N/A.

lnc003875/miR-363/EGR1 regulatory network in the carcinoma -associated fibroblasts controls the angiogenesis of human placental site trophoblastic tumor (PSTT).

The rare gestational trophoblastic neoplasia placental site trophoblastic tumor (PSTT) frequently demonstrates a high degree of vascularization, which may facilitate the tumor metastasis. However, the underlying mechanisms remain largely unknown. In the present study, we found that early growth response 1 (EGR1) was highly expressed in the carcinoma-associated fibroblasts (CAFs) of PSTT tissues. Further data showed that miR-363 down-regulated EGR1 expression whereas long non-coding RNA NONHSAT003875 (lnc003875) up-regulated EGR1 expression in PSTT derived CAFs. lnc003875 exerted no effect on miR-363 expression, but it recovered the decrease of EGR1 caused by miR-363 mimic. The conditioned media from PSTT CAFs treated with miR-363 mimic abrogated the tube formation capacity of human umbilical vein endothelial cells (HUVECs), which can be partially restored by lnc003875 over-expression. Moreover, over-expression of EGR1 promoted the secretion of Angiopoietin-1 (Ang-1) in PSTT derived CAFs and improved the tube formation of HUVECs, which could be effectively abrogated by Ang-1 siRNAs. In vivo vasculogenesis assay demonstrated that lnc003875/EGR1 in PSTT derived CAFs promoted the vasculogenesis of HUVECs in C57BL/6 mice. Collectively, these findings indicated that lnc003875/miR-363/EGR1/Ang-1 in CAFs may be crucial for the angiogenesis of PSTT.