A Prospective Cohort Study of Metformin as an Adjuvant Therapy for Infertile Women With Endometrial Complex Hyperplasia/Complex Atypical Hyperplasia and Their Subsequent Assisted Reproductive Technology Outcomes.

Objective: To investigate the adjuvant efficacy of metformin treatment to achieve pathological complete response (CR) in patients with endometrial complex hyperplasia (CH) and complex atypical hyperplasia (CAH), and secondarily, to evaluate their pregnancy outcomes after following assisted reproductive technology (ART). Study Design: This prospective cohort study analyzed 219 patients diagnosed with infertility and CH/CAH from January 2016 to December 2020. Among these patients, 138 were assigned to the control group (progesterone alone) and 81 were assigned to the study group (progesterone+metformin). After 8/12 weeks of therapy, the treatment responses were assessed by histological examination of curettage specimens obtained by hysteroscopy. Once the pathological results indicated CR, the patients were able to receive ART. The ART treatment and follow-up data of these patients were collected and analyzed. Results: 116 patients in the control group achieved CR, compared with 76 patients in the study group. The CR rate in the control group was significantly lower than that in the study group (P=0.034). We then divided the patients into subgroups to compare the treatment responses. In the subgroup analyses, patients with body mass index (BMI) ≥25 kg/m2 and patients with polycystic ovarian syndrome (PCOS) had higher CR rates in the metformin group compared with the control group (P=0.015, P=0.028 respectively). Subsequently, 68 patients in the control group and 47 patients in the study group received an ART cycle. We examined the pregnancy indications and found no significant differences in the clinical pregnancy rate and live birth rate between the two groups (P>0.05). Conclusion: Regression of CH/CAH may be improved by progesterone+metformin compared with progesterone alone. The effect was particularly pronounced in patients with BMI ≥25 kg/m2 and patients with PCOS. Metformin had no obvious effect on subsequent ART outcomes. The trial is registered on the publicly accessible website. Clinical Trial Registration: http://www.chictr.org.cn/showproj.aspx?proj=15372, identifier ChiCTR-ONR-16009078.

Dusp6 inhibits epithelial-mesenchymal transition in endometrial adenocarcinoma via ERK signaling pathway.

Background Endometrial adenocarcinoma (EAC) is one of the most commonly diagnosed gynaecological malignancies among female population of the developed countries. DUSP6 is a negative regulator of ERK signaling, which is a molecular switch involved in MAPK signaling during the progress of malignancies. DUSP6 was previously found to inhibit tumorigenesis and EMT-associated properties in several cancers, however, its exact role in EAC remains unclear Methods The level of DUSP6, (E-cad) and (N-cad) in EAC cancerous tissues and respective adjacent non-cancerous tissues were examined by western-blot or immunohistochemistry. The cell growth, invasion and migration abilities were measured in Ishikawa 3H12 endometrial cancer cell lines with overexpressed or knock down DUSP6. Protein levels of EMT-associated markers E-cadherin, N-cadherin and Vimentin were also determined. The impacts of DUSP6 on ERK signaling was assessed by detection of ERK and p-ERK. Results Down-regulation of DUSP6 was observed in EAC compared with the normal controls. The overexpression of DUSP6 significantly attenuated tumor cell growth, invasion, migration abilities and inhibited EMT-associated markers, while knock down of DUSP6 showed opposite trends. Overexpression of DUSP6 also down-regulated p-ERK and the knock down of DUSP6 inversely up-regulated p-ERK level. Conclusions DUSP6 inhibited cell growth, invasion and migration abilities in Ishikawa 3H12 cells as well as attenuating EMT-associated properties. This tumor suppressive effect of DUSP6 in EAC is achieved by inhibiting ERK signaling pathway.

A research on the protein expression of p53, p16, and MDM2 in endometriosis.

This study aims to examine the expression of p53, p16, and murine double minute 2 (MDM2) protein in normal endometrium and endometriosis, in order to discuss the role of p53, p16, and MDM2 protein and apoptosis in the pathogenesis and development of endometriosis, and provide a theoretical basis for clinical diagnosis and treatment.The immunohistochemical streptavidin-biotin peroxidase method was used to detect the expression of p53, p16, and MDM2 in tissue samples obtained from 30 women with pathologically confirmed ovarian endometriosis and 29 women with pathologically confirmed normal endometrium. The relationship between p53, p16, and MDM2 expression and apoptosis was analyzed.In normal endometrium, the positive rate of p53 in the secretory phase was higher than that in the proliferative phase (P < .05). Furthermore, the positive rate of p53 in normal endometrium was higher than that in ovarian endometriosis (P < .05). There was a significant difference between normal endometrium and ovarian endometriosis.The positive rate of p16 in normal endometrium was higher than that in ovarian endometriosis (P < .05). Furthermore, there was a significant difference between normal endometrium and ovarian endometriosis. The positive rate of MDM2 in normal endometrium was lower than that in ovarian endometriosis (P < .05).In ovarian endometriosis, the expression of p53 and p16 was positively correlated with each other (r = 0.611, P < .01). However, the expression of p53 and MDM2 was negatively correlated with each other (r = -0.541, P < .01). Furthermore, the expression of p16 and MDM2 might not be relevant in the endometriosis (r = 0.404, P > .05).As important apoptosis regulatory genes, p53, p16, and MDM2 might be involved in the pathogenesis and development of endometriosis.

Identifying risk factors for cesarean scar pregnancy: a retrospective study of 79 cases.

OBJECTIVES: To explore the possible risk factors for cesarean scar pregnancy (CSP), the incidence of which is increasing rapidly in China. MATERIAL AND METHODS: 79 patients with CSP and 69 non-CSP expectant mothers with at least 1 previous cesarean section were employed in the study. The obstetric histories of the participants were collected and analyzed using Chi square test. RESULTS: We found that 77.2% CSP patients had ≥ 3 pregnancies and only 36.2% women had ≥ 3 pregnacies in non-CSP group. During the previous cesarean delivery, 21.5% of CSP patients had entered the first stage of labor, which was 43.5% in non-CSP group (P < 0.05). Cephalopelvic disproportion occurred in 51.9% of CSP patients, which was significantly higher than that (23.2%) in non-CSP group (P < 0.01). 11.4% of CSP patients had undergone cesarean section due to breech and shoulder presentation in the past, which was only 1.4% in non-CSP group. However, no significance was noted (P > 0.05). We did not find significant differences between the CSP and non-CSP patients in maternal age, multiple cesarean sections, gestational age, emergency or elective caesarean section. CONCLUSIONS: Multiple pregnancies, absence of the first stage of labor, and cephalopelvic disproportion might be the risk factors for the occurrence of CSP.

Inhibitory effect of 2'-o-methoxyethyl-modified antisense oligonucleotides targeting vascular endothelial growth factor A on SKOV3 human ovarian cancer cells.

BACKGROUND: Ovarian cancers are often at an advanced stage at diagnosis because early detection is difficult. The poor prognosis of ovarian cancers highlights the crucial need to develop better therapeutic agents and strategies. The objective of this study was to investigate the inhibitory effects of a new modified antisense oligonucleotides targeting vascular endothelial growth factor A (VEGF-A) in SKOV3 ovarian cancer cells. METHODS: Antisense oligonucleotides targeting VEGF-A was designed, synthesized and transfected into SKOV3 ovarian cancer cells. Western blotting and real-time RT-PCR were used to analyze the inhibitory effects of antisense oligonucleotides on VEGF-A protein and mRNA expression. Transwell matrix assay was used to detect cell migration inhibition. RESULTS: The antisense oligonucleotides targeting VEGF-A significantly decreased VEGF-A protein and mRNA expression and inhibited cell migration in SKOV3 ovarian cancer cells. CONCLUSIONS: This new modified antisense oligonucleotides targeting VEGF-A can decrease VEGF-A expression and inhibit cell migration in SKOV3 ovarian cancer cells. This new oligonucleotides may be a promising therapeutic agent for ovarian cancers.

[Sequencing analysis of the control region and intron1 region and characterization of two novel microsatellites in obese gene of pig].

For the first time, a 16 kb fragment of the porcine Ob gene, namely intron1, exon1, 5' region of Ob gene, was restrictively analyzed and sequenced by the primers designed in the portion of the swine Ob sequence. The first small 38 bp untranslated exon1 is located 11.1 kb upstream of the initiator ATG codon, and two novel microsatellites SW200 and SW160 are found in intron1. Promoter analysis of several putative binding sites revealed that this 300 bp promoter located at -1 to -300, including C/EBP and two Sp1, may be as effective as the longer promoter in directing leptin transcription. To examine microsatellites association with important economic traits, we statistically analyzed genotypes and alleles of the two microsatellites. Statistical analysis carried out by SAS 8.2 revealed significant positive correlation between the two microsatellites genotypes and the litter size in first parity of Erhualian.

[Influence of mifepristone and lilopristone on proliferation and expression of nuclear factor-kappa B of ectopic stromal cells in vitro].

OBJECTIVE: To evaluate the effects of antiprogestins mifepristone and lilopristone on proliferation and expressions of nuclear factor-kappa B (NF-kappaB) of ectopic stromal cells in vitro. METHODS: The ectopic stromal cells of ovary were cultured in vitro. Methyl thiazolyl tetrazolium method was used to evaluate proliferative activity of ectopic stromal cells. Cells were divided into five groups according to drug concentration: control group, group I and group II of mifepristone and of lilopristone. The expressions of NF-kappaB P65 and NF-kappaB P65 mRNA of ectopic stromal cells were determined by immunocytochemistry and in situ hybridization of cell slice. RESULTS: Antiprogestins mifepristone and lilopristone were able to significantly suppress the proliferation of ectopic stromal cells in a time- and dose-dependent manner in vitro. The expressions of NF-kappaB P65 and NF-kappaB P65 mRNA of ectopic stromal cells in the control group were higher than that of group I and group II. Their expressions in mifepristone groups were higher than that of lilopristone groups. CONCLUSIONS: Antiprogestin mifepristone and lilopristone could significantly suppress the proliferation of ectopic stromal cells in a time- and dose-dependent manner in vitro. The action mechanisms may be associated with the suppression of expression of NF-kappaB P65 mRNA and NF-kappaB P65.

[Identification and analysis of a novel microsatellite marker flanking porcine myostatin gene (MSTN)].

In animal breeding, microsatellite marker plays an important role in constructing genetic maps, QTL mapping and function analysis of structural genes. Myostatin, also known as GDF8, is a negative regulator of skeletal muscle mass and, in swine, it is evidenced to be related to birth weight and average daily gain from 60 kg to 100 kg of body weight. In present study, by subcloning and sequencing,we identified a novel microsatellite marker which is useful for fine QTL mapping for meat traits. A BAC clone containing porcine MSTN was extracted and digested with EcoR I to recover the fragment of > 4 kb for subcloning in pGEM-3zf (+). Sequencing and alignment results showed that this subcloned fragment was not from porcine MSTN, but included a tandem repeat of (TG) 13, which is a novel microsatellite marker (GenBank accession number: AF454400) flanking MSTN. To exclude its vector origin we designed specific primers flanking this marker and successfully amplified this fragment from porcine genome. Through a pedigree analysis of a double-muscled Yorshire strain, we found that it is inherited in a co-dominant manner. We also checked the gene frequencies of this locus in 381 unrelated individuals of 7 pig breeds, namely Laiwu,Landrace, Yorkshire,Duroc, Peterian, Min and Erhualian. Only two alleles were detected, the repeating number of which are 13 (allele A) and 19 (allele B) respectively, which indicated that it is a low poly morphic microsatellite marker. In addition, the frequencies of the two alleles are different between the two types of pig breeds, while allele A is dominant in Chinese local breeds, allele B is dominant in imported breeds. Alignment with AY208121 indicate that this locus is located 42 kb downstream of porcine MSTN. We speculate that this microsatellite DNA is an important marker both in fine QTL mapping for meat traits and in the expression study of porcine MSTN.

[Construction and identification on enriched microsatellite library from yak genome].

We constructed the first microsatellite-enriched library of yak according to the strong affinity between biotin and streptavidin. The method included ligation of 300 approximately 1 000 bp enzyme-digested fragments and adaptors, affinity capture of microsatellite repeat using biotinylated oligoprobe ((CA)12, (CCG) 8, (CAG)8, (TTTC) 8) attached to streptavidin-coated magnetic beads, PCR amplification using the 21-mer adaptor oligonucleotide as primer to obtain double-stranded targeted fragments, religated into pMD18-T vector and transformed to DH5alpha. The results of sequencing showed that 37 of 48 readable sequences contained microsatellites indicating a high degree of microsatellite enrichment. The new polymorphic microsatellite markers we have identified and characterized will contribute to the yak genetic linkage mapping, molecular evolution and phylogenetic studies, marker assistant selection and QTLs location of yak main economic traits.

[Studies of the gene expression and structure alteration of vimentin during differentiation of rat preadipocytes].

Observations on structure and distribution of vimentin as determined by indirect immunofluorescence were showed that the arrangement of vimentin intermediate filments (IFs) changed from an extended fibrillar state to a complex cage formation tightly associated with the forming lipid globules during adipose conversion of rat preadipocytes. The effects of differentiation of rat preadipocytes into adipocytes on vimentin mRNA levels as determined by in situ hybridization (ISH) using oligonucleotides probes to vimentin mRNA labeled by digoxigenin (DIG), and on vimentim protein expression as determined by Western blotting were researched. The results suggested that the expressions of vimentin with molecular weight of about 57 kD at the levels of both mRNA and protein was existed throughout the predipocyte differentiation and downregulation of vimentin is required for adipose conversion. The specificity of the association of lipid globules with vimentin IFs during adipose conversion was taken as an example of a possible IF function in relation to a preadipocytes differentiation process, especially discussed as a functional relationship in supporting adiposgenesis.

[Influence of infusion of Duroc blood on characteristics of chromosomes in Gansu black pig].

The variation of chromosomes of Gansu Black pig was caused by infusion of Duroc blood. The chromosome length ratios of No.1,6,7,13 chromosomes in the line I of Gansu Black pig were higher than those in the line II. It showed that the homogeneity in line I was higher than in line II. The number of Ag-staining nucleolus organizers (Ag-NOR) was between 1-4 in the line I and II, the average No. of Ag-NOR was different obviously between the line I and the line II. This indicated that the activity of translation in rRNA in the line I was different in the line II.

[Study of effect of mifepristone on apoptosis of human ovarian cancer cell line 3AO].

OBJECTIVE: To investigate the effect of mifepristone on the activity of proliferation and the apoptosis, the expression of estrogen receptor (ER), progesterone receptor (PR) protein and morphology changes of human ovarian carcinoma cell line 3AO and SKOV3 in vitro. METHODS: The proliferative activity of 3AO and SKOV3, which were cultured in vitro, was measured by tetrazolium-based colorimetric assay (MTT assay). Flow cytormetry (FCM) was used to measure the expressive rate of ER, PR, p53 protein, bcl-2 protein, cell apoptotic rate and cell proliferative cycle of 3AO cells, which were cultured with different concentration and duration of mifepristone. The morphologic and ultrastructure changes of apoptotic 3AO cells was observed by the light and electron microscopy. RESULTS: Mifepristone inhibited significantly the proliferation of 3AO cells in dose-time dependent manner in vitro. The inhibitory rate of 3AO cells growth, which were cultured with different concentration of mifepristone (5, 10, 20, 40, 80 micro mol/L) and duration (24, 48, 72 h) was from 1.7% to 75.0% (P < 0.01), but the proliferation activity of SKOV3 cells in vitro had not significant changes (P > 0.05). 3AO cells apoptosis activity appeared the positive correlation with the dose of mifepristone and cultured duration (P < 0.01). Mifepristone blocked 3AO cells proliferative cycle at the G(0)-G(1) stage, decreased the cell radio of S stage. Mifepristone induced the apoptosis of 3AO cells in vitro. The light and electron microscopy demonstrated that 3AO cells cultured with mifepristone appeared typical morphological characteristics of apoptosis including the compaction and margination of the chromosomes, nuclear fragments and formation of apoptotic bodies. Mifepristone up-regulated significantly the expression of p53 protein, but down-regulated the expression of bcl-2 protein (P < 0.01). The expressive rates of p53 and bcl-2 proteins were (54.8 +/- 4.0)% and (10.1 +/- 1.2)%, respectively, when 3AO cells was cultured with mifepristone of 10 micro mol/L for 24 h. Compared with the expression rates of control group (27.1 +/- 3.3)% and (17.6 +/- 1.0)%, the difference was significant (P < 0.01). The expressive rate of PR protein of 3AO cells cultured with mifepristone of 10 micro mol/L for 48 h was (12.7 +/- 1.4)%, which was decreased compared with the expressive rate of control group (44.9 +/- 1.4)% (P < 0.01). CONCLUSIONS: Mifepristone inhibited significantly the proliferation and induced the cell apoptosis of cell line 3AO in dose-time dependent manner in vitro. The anti-tumor effect was related to down-regulation the expression of PR protein and bcl-2 protein, and to up-regulation the expression of p53 protein of 3AO cells.

[Single nucleotide polymorphism analysis in chicken insulin-like growth factor-II gene and its associations with growth and carcass traits].

In this experiment, F2 chicken derived from Broilers crossing with Silky was used to study the effect of insulin-like growth factor-II gene on growth and carcass traits. The partial gene was amplified by two pairs of primers, and single nucleotide polymorphism (SNPs) was detected by the technique of restriction fragment length polymorphism (RFLP), and then confirmed by DNA sequencing. The mutation was found in the exon-2 of the gene, and can be clarified by cutting of restriction enzyme Aci-I. The result of least square analysis showed the gene was significantly related with growth and carcass traits. It implied that the insulin-like growth factor-II gene could be a genetic locus or linked to a major gene affecting greatly the growth and carcass traits in chicken.

[Analysis of succeed percentage on sequencing ESTs and construction of porcine muscle cDNA library].

Using Longissimus Dorsi muscle as material and Lambda ZAP II as Vector, Xiang Pig Longissimus Dorsi muscle cDNA library has been constructed in our study. The results showed that the titration of the library was 3.4 x 10(7) pfu/ml, the recombinant percentage was 94%, and the fragment length of inserted average cDNA were 1.5 kb. The study pointed out that the more than 30 T insertion is the major factor for low percentage if sequencing the 3'-end.