Clinical Efficacy of Assisted Reproductive Technology Combined with Progesterone Capsules in the Treatment of Infertility Caused by Diminished Ovarian Reserve and Its Influence on Serum FSH, E2, and LH Levels of Patients.

Objective: To explore the clinical efficacy of assisted reproductive technology (ART) combined with progesterone capsules in the treatment of infertility caused by the diminished ovarian reserve (DOR) and its influence on serum FSH, E2, and LH levels of patients. Methods: In the manner of retrospective study, the data of 120 patients with infertility caused by DOR admitted to our hospital (February 2019-February 2020) were retrospectively analyzed, and the patients were equally divided into the experimental group and the control group according to the order of admission. All patients underwent in vitro fertilization and embryo transfer (IVF-ET), and the experimental group was received progesterone capsules at the same time. Ovarian-related indexes, follicular development, serum hormone levels, and pregnancy outcomes were compared between both groups. Results: After treatment, compared with the control group, ovarian-related indexes and follicular development in the experimental group were conspicuously better (P < 0.001). In the experimental group, the FSH level was (5.99 ± 1.20) U/L, the E2 level was (540.12 ± 3.54) ng/L, and the LH level was (3.10 ± 0.35) U/L after treatment, which was significantly better than those of the control group (P < 0.001). After treatment, compared with the control group, the clinical pregnancy rate in the experimental group was conspicuously higher (P < 0.05), and the abortion rate in the experimental group was conspicuously lower (P < 0.05). No obvious difference was observed in multiple births rate between the two groups (P > 0.05). Conclusion: ART combined with progesterone capsules can improve serum hormone levels, ovarian function, follicular development, and clinical pregnancy rate for patients with infertility caused by DOR, which should be applied in practice.

Preterm Birth Is Correlated With Increased Oral Originated Microbiome in the Gut.

Background: Preterm birth is one of the leading causes of perinatal morbidity and mortality. Gut microbiome dysbiosis is closely related to adverse pregnancy outcomes. However, the role of the gut microbiome in the pathogenesis of preterm birth remains poorly studied. Method: We collected fecal samples from 41 women (cases presenting with threatened preterm labor =19, 11 of which delivered preterm; gestational age-matched no-labor controls, all of which delivered at term = 22) were recruited for the study. We performed 16S rRNA amplicon sequencing to compare the composition of the gut microbiome in threatened preterm labor cases and controls and among women who delivered preterm and at term. By annotating taxonomic biomarkers with the Human Oral Microbiome Database, we observed an increased abundance of potential oral-to-gut bacteria in preterm patients. Results: Patients with preterm birth showed a distinct gut microbiome dysbiosis compared with those who delivered at term. Opportunistic pathogens, particularly Porphyromonas, Streptococcus, Fusobacterium, and Veillonella, were enriched, whereas Coprococcus and Gemmiger were markedly depleted in the preterm group. Most of the enriched bacteria were annotated oral bacteria using the Human Oral Microbiome Database. These potential oral-to-gut bacteria were correlated with clinical parameters that reflected maternal and fetal status. Conclusions: This study suggests that patients who deliver preterm demonstrate altered gut microbiome that may contain higher common oral bacteria.

IL-1ß promotes cervical cancer through activating NF-κB/CCL-2.

Cervical cancer is a malignancy with high morbidity and mortality among women. Interleukin (IL)-1ß, chemokine (C-C motif) ligand 2 (CCL-2), and activation of NF-κB have been proven to be closely related to the progression of various tumors. However, their role in cervical cancer remains unclear. Cell proliferation, migration, and invasion were detected using MTT, wound healing, and transwell assays. Western blotting and qRT-PCR were used to measure expression of target genes. IL-1ß greatly promoted the release of CCL-2 from HeLa cells. Activation of NF-κB and phosphorylated NF-κB (p65) nuclear translocation were accelerated by IL-1ß. TPCA-1, a blocker of NF-κB, significantly inhibited the release of CCL-2 from HeLa cells. TPCA-1 markedly reversed the promotional effect of IL-1ß on viability of HeLa cells. IL-1ß increased the cell migration, proliferation, and invasion of HeLa cells through targeting the NF-κB/CCL-2 pathway. IL-1ß/NF-κB/CCL-2 might be a promising treatment target for cervical cancer treatment and prevention.

miR-613 inhibits the proliferation of human ovarian granulosa cells by arresting cell cycle progression via the targeting of IGF-1.

Polycystic ovary syndrome (PCOS) is a prevalent endocrine disorder, and microRNA (miRNA) molecules have been implicated in the pathological process of PCOS. The aim of the present study was to elucidate the regulatory effects of miR-613 and insulin-like growth factor-1 (IGF-1) on the pathological process of polycystic ovary syndrome (PCOS). The targeting of IGF-1 by miR-613 was investigated by dual-luciferase reporter assay. The regulatory effect of miR-613 on the mRNA and protein levels of IGF1 was determined by reverse transcription-quantitative PCR and western blot analysis. The regulatory effects of miR-613 and IGF-1 on the proliferation and cell cycle progression of KGN cells were evaluated by colony formation assay and flow cytometric analysis. The results revealed that miR-613 targeted IGF-1 and reduced its translational level. In KGN cells, miR-613 arrested cell cycle progression in the G2/M phase and downregulated the expression of cyclin D1 and CDK1. The overexpression of IGF-1 attenuated the inhibitory effects of miR-613 on cell cycle arrest, cyclin D1 and CDK1 expression, and the proliferation of KGN cells. In conclusion, the present study demonstrated that miR-613 targets IGF-1 and thus suppresses its translation. It arrests cell cycle progression and attenuates the proliferation of KGN cells via the targeting of IGF-1. Therefore, it is suggested that miR-613 and IGF-1 could potentially be diagnostic biomarkers and therapeutic targets for PCOS.

MicroRNA­145­5p suppresses fascin to inhibit the invasion and migration of cervical carcinoma cells.

MicroRNAs (miRs) can affect the progression of cervical cancer (CC). The present study investigated the function of miR­145­5p in CC and demonstrated its association with fascin (FSCN1). The expression levels of miR­145­5p in CC tissues and cell lines were analyzed using reverse transcription­quantitative PCR, and its direct targets were explored using a luciferase reporter assay. The viability, migration and invasion of HeLa cells transfected with small interfering FSCN1 or with miR­145­5p mimics and inhibitors were analyzed using Cell Counting Kit­8 and Transwell assays. The expression levels of FSCN1 mRNA and protein were investigated using reverse transcription PCR and western blotting. miR­145­5p was downregulated in CC tissues and cell lines. Moreover, overexpression of miR­145­5p inhibited the migration, invasion and viability of HeLa cells. miR­145­5p directly targeted FSCN1, which regulated the suppressive functions of miR­145­5p in CC cells. Overall, miR­145­5p is a tumor suppressor gene and a promising target for CC treatment.

[Silencing long non-coding RNA HIF1A-AS2 inhibits proliferation, invasion and migration of cervical cancer cells in vitro].

OBJECTIVE: To explore the inhibitory effects of silencing long non-coding RNA (LncRNA) HIF1A-AS2 on epithelialmesenchymal transition (EMT) and tumor stem cell-like phenotype in cervical cancer cells. METHODS: We designed 3 shRNA constructs for silencing HIF1A-AS2 in CaSki cells, and the shRNA with the strongest interference effect was selected for subsequent experiment. CaSki cells were transfected with shRNA-NC or Sh-HIF1A-AS2, and the changes in cell viability, invasion ability, EMT, expressions of EMT-related proteins, formation of cell spheres and expressions of stem cell markers were detected. RESULTS: Transfection with shRNA-NC and Sh-HIF1A-AS2 did not significantly affected the viability of CaSki cells (P > 0.05). Compared with the cells transfected with shRNA-NC, the cells transfected with Sh- HIF1A-AS2 showed significantly reduced invasion ability, expressions of vimentin N-cadherin, and cell sphere formation ability. HIF1A-AS2 silencing obviously lowered the rate of ABCG2-positive cells, significantly reduced the mRNA and protein expressions of Nanog, OCT4, and SOX2, and strongly enhanced the expression of E-cadherin in CaSki cells (P < 0.05). CONCLUSIONS: Silencing HIF1A-AS2 can inhibit proliferation, invasion and migration of cervical cancer cells in vitro.

Rosiglitazone ameliorates palmitic acid-induced endoplasmic reticulum stress and steroidogenic capacity in granulosa cells.

Granulosa cells play essential roles in follicular development, oocyte maturation and sex hormone secretion. The exposure of granulosa cells to palmitic acid (PA), the main component of dietary saturated fat, inhibits cell viability. However, the mechanism underlying PA-induced cytotoxicity in granulosa cells has not been deeply investigated. Rosiglitazone (RSG) is a member of the thiazolidinedione family and is reported to protect cells from cytotoxicity and endoplasmic reticulum (ER) stress in other cell types, but whether RSG protects granulosa cells remain unknown. In this study, KGN cell line and primary granulosa cells were used as models of granulosa cells to explore the effects of PA and RSG and the underlying mechanisms. The results showed that PA inhibits cell viability and estradiol secretion through inducing ER stress and cAMP/PKA/CREB pathway. CCAAT/enhancer-binding protein homologous protein (CHOP), an ER stress marker, was demonstrated to participate in PA-induced cytotoxicity. RSG treatment rescued granulosa cells from PA-induced cell death and ER stress. Moreover, RSG was identified to ameliorate ER stress induced by tunicamycin in granulosa cells. In addition, RSG treatment rescued granulosa cells from PA-induced decrease of estrogen secretion by cAMP/PKA/CREB pathway. In conclusion, RSG can protect granulosa cells against PA-induced cytotoxicity by inhibiting ER stress, and can recover steroidogenic capacity, indicating a potential use of RSG in the treatment of granulosa cell dysfunction.

Clinical significance of RAD51C and its contribution to ovarian carcinogenesis.

AIM: The underlying mechanisms of chemoresistance-induced recurrence of ovarian carcinoma are largely unknown. The purpose of this study was to investigate the clinical significance of RAD51C and its role in ovarian tumorigenesis and progression. METHODS: 60 cases of ovarian epithelial tumors (30 benign and 30 malignant tumors, respectively) were enrolled from 2014 to 2016. Immunohistochemistry was used to evaluate RAD51C expression in tumor tissues, and RT-PCR was employed to test RAD51C mRNA levels in SKOV3, A2780, and CAOV3 cell lines. Targeted knockdown of RAD51C was achieved with siRNA to explore the changes of cell proliferation, migration, and apoptosis. RESULTS: RAD51C protein level in carcinoma tissues, especially in the high-grade group (P<0.001), was significantly higher than that of benign tumors and associated with pathological type, stage, and overall survival (P<0.05). Downregulation of RAD51C promoted apoptosis and decreased cell survival rate and migration. CONCLUSIONS: Our results supported that RAD51C contributes to the progression of ovarian carcinoma, suggesting its promising application as an independent prognostic marker for diagnosis and treatment.

Effect of lanthanum chloride on tumor growth and apoptosis in human ovarian cancer cells and xenograft animal models.

Ovarian cancer is the leading cause of mortality resulting from gynecologic cancer. A common anti-ovarian tumor drug is cisplatin; however, repeated use of cisplatin causes severe resistance and leads to poor long-term survival rate in ovarian cancer patients. Recently, it was reported that lanthanum chloride (LaCl3) may inhibit tumor growth and induce apoptosis in certain cancer cells. In the present study, the effect of LaCl3 on ovarian cancer was determined in vivo and in vitro. A cisplatin-sensitive human ovarian cancer cell line, COC1, was used in the current study. A xenograft animal model of ovarian cancer was established injecting COC1 or cisplatin-resistant COC1 cells (COC1/DDP) cells into mice. A TUNEL assay was used to determine the apoptosis of the COC1 or COC1/DDP cells and a immunohistochemical assay was conducted to measure the expression of B-cell lymphoma-2, Ki67, breast cancer 1 (BRCA)1, BRCA2 and excision repair cross-complementation group 1 in COC1 or COC1/DDP cells. It was observed that LaCl3 promoted apoptosis in COC1 and COC1/DDP cells. In addition, LaCl3 plus cisplatin led to further increase in the expression levels of tumor suppressor genes and decrease in the expression of oncogenes. Furthermore, application of LaCl3 and cisplatin inhibited tumor growth in vivo in a xenograft animal model. These results indicated the synergistic role of LaCl3 on cisplatin-induced inhibition of cancer cell proliferation and tumor growth, providing a potential and effective candidate for the treatment of ovarian tumors.

Capn4 overexpression indicates poor prognosis of ovarian cancer patients.

Recent studies have shown a close correlation between Capn4 expression and the prognosis of patients with solid tumors. This study aimed to investigate clinical role of Capn4 in ovarian cancer. The expression of Capn4 in 113 ovarian cancer and 35 non-tumor tissue samples were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Capn4 expression was significantly upregulated in ovarian cancer tissues compared with non-tumor tissues (p < 0.01), and was positively correlated to FIGO stage, tumor grade and distant metastasis of ovarian cancer. Kaplan-Meier analysis indicated that patients with high Capn4 expression had shorter overall survival (HR = 1.929, 95%CI: 1.210-3.077, P= 0.006) and progress-free survival (PFS) (HR = 2.043, 95%CI: 1.276-3.271, P= 0.003). Moreover, univariate Cox regression analysis demonstrated that Capn4 overexpression was an unfavorable prognostic factor for ovarian cancer (HR = 2.819, 95%CI: 1.365-3.645, P = 0.003). After the adjustment with age, histological type and tumor size, multivariate Cox regression analysis showed that Capn4 expression level (HR = 2.157,95%CI: 1.091-3.138, P = 0.014), distant metastasis (HR = 1.576, 95%CI: 1.025-3.012, P = 0.028), tumor grade (HR = 1.408, 95%CI: 0.687-2.884, P = 0.037), and FIGO stage (HR = 1.791, 95%CI: 1.016-3.158, P=0.036) were independent poor prognostic indicators for ovarian cancer. In conclusion, Capn4 has the potential as a new prognostic marker for patients with ovarian cancer.

[Quantitative Analysis of Immuno-fluorescence of Nuclear Factor-κB Activation].

Immuno-fluorescence technique can qualitatively determine certain nuclear translocation, of which NF-κB/ p65 implicates the activation of NF-κB signal pathways. Immuno-fluorescence analysis software with independent property rights is able to quantitatively analyze dynamic location of NF-κB/p65 by computing relative fluorescence units in nuclei and cytoplasm. We verified the quantitative analysis by Western Blot. When we applied the software to analysis of nuclear translocation in lipopolysaccharide (LPS) induced (0. 5 h, 1 h, 2 h, 4 h) primary human umbilical vein endothelial cells (HUVECs) , we found that nuclear translocation peak showed up at 2h as with calculated Western blot verification results, indicating that the inventive immuno-fluorescence analysis software can be applied to the quantitative analysis of immuno-fluorescence.

The effects of lanthanum chloride on proliferation and apoptosis of cervical cancer cells: involvement of let-7a and miR-34a microRNAs.

Lanthanide elements have been documented to possess various biologic effects, and their compounds have been studied intensely for their anti-cancer potential. However, the underlying mechanisms remain largely unknown. In the present study, we propose that the levels of proliferation and apoptosis related microRNAs (miRNAs), let-7a and miR-34a, which mediate the apoptosis of cervical cancer cells, can be affected by the lanthanum ion. Our data showed that LaCl3 inhibited the proliferation and induced the apoptosis of cervical cancer cells both in vivo and in vitro by regulating let-7a, miR-34a and their downstream genes. This study provides novel evidence demonstrating that the anticancer mechanism of lanthanum chloride is partially attributed to miRNAs regulation and establishes an experimental basis for the clinical application of lanthanum chloride as an anti-cancer drug.

Preparation and characterization of luteinising-hormone releasing hormone nanoliposomal microbubbles specifically targeting ovarian cancer cells in vitro.

The aim of the present study was to prepare luteinizing-hormone releasing hormone (LHRH) nanoliposomal microbubbles specifically targeting ovarian cancer cells. The lyophilization/sonication method was used to prepare non-targeting nanoliposomal microbubbles (N-N-Mbs). Using the biotin-avidin bridge method, conjugated LHRH antibodies to N-N-Mbs generated LHRH nanoliposomal microbubbles (LHRH-N-Mbs) specifically targeting ovarian cancer cells. The morphology and physicochemical properties of the microbubbles was detected using an optical microscope and zeta detector. The binding affinity between the secondary antibody and LHRH-N-Mbs or N-N-Mbs was determined by flow cytometry. The binding of LHRH-N-Mb to human ovarian cancer cells (OVCAR-3) was detected by light microscopy. The rounded and uniformly distributed N-N-Mbs and LHRH-N-Mbs were successfully generated. The particle size ranged from 295-468 nm with a mean of 360 nm for N-N-Mbs or 369-618 nm with a mean of 508 nm for LHRH-N-Mbs. There was a significant difference in size between the two groups (P<0.05), although the surface potential of the two microbubbles remained the same (-14.6 mV). Following being kept at room temperature for 14 days, no significant difference in the physicochemical properties of the LHRH-N-Mbs was detected compared with that of freshly prepared microbubbles. The secondary antibody binding rate of LHRH-N-Mbs and N-N-Mbs was 75.6 and 0.83%, respectively. Furthermore, the formation of a rosette-like structure surrounding OVCAR-3 cells was observed after the cells were incubated with LHRH-N-Mbs, whereas pre-incubation with LHRH antibody blocked this rosette formation. In conclusion, LHRH-N-Mbs specifically targeting ovarian cancer cells were successfully prepared through biotin-avidin mediation and the lyophilization/sonication method. The key feature of LHRH-N-Mbs is their small size, stability and high efficiency in targeting human OVCAR-3 cells in vitro.

Survey of the situation of infertile women seeking in vitro fertilization treatment in China.

BACKGROUND: In previous studies, people's knowledge of reproductive health and infertile women's psychological states was surveyed in several countries. However, there has been limited information concerning the psychological states of infertile women seeking treatment and the outcomes of in vitro fertilization (IVF) in China. METHODS: Infertile women were asked to complete short questionnaires on the day that their oocytes were retrieved; these questionnaires covered the durations of their infertility, levels of education, sources of pressure, and psychological states. Data concerning IVF outcomes were provided by embryologists and clinicians. The correlations between the duration of infertility and educational level, psychological state and education level, and psychological state and outcome of IVF were analyzed in the cohort study. RESULTS: The duration of infertility in more than half of the females was longer than 5 years. Compared with less-educated women, women with higher levels of education sought treatment earlier and their rates of depressive symptoms were lower. There is an association between negative emotions and outcome of IVF. CONCLUSIONS: The survey of the situations of infertile women seeking IVF treatment in China indicates the importance of popularizing knowledge concerning reproductive health. Improving medical conditions, reducing the costs of treatment, and developing social culture will aid in relieving the stress of infertile women and improving assisted reproductive treatment.

Patients' attitudes towards the surplus frozen embryos in China.

BACKGROUND: Assisted reproductive techniques have been used in China for more than 20 years. This study investigates the attitudes of surplus embryo holders towards embryos storage and donation for medical research. METHODS: A total of 363 couples who had completed in vitro fertilization (IVF) treatment and had already had biological children but who still had frozen embryos in storage were invited to participate. Interviews were conducted by clinics in a narrative style. RESULTS: Family size was the major reason for participants' (dis)continuation of embryo storage; moreover, the moral status of embryos was an important factor for couples choosing embryo storage, while the storage fee was an important factor for couples choosing embryo disposal. Most couples discontinued the storage of their embryos once their children were older than 3 years. In our study, 58.8% of the couples preferred to dispose of surplus embryos rather than donate them to research, citing a lack of information and distrust in science as significant reasons for their decision. CONCLUSIONS: Interviews regarding frozen embryos, including patients' expectations for embryo storage and information to assist them with decisions regarding embryo disposal, are beneficial for policies addressing embryo disposition and embryo donation in China.

[Effects of lanthanum chloride on proliferation and migration of human cervical cancer cell line HeLa cells].

OBJECTIVE: To investigate the effects of lanthanum chloride on proliferation and migration activity of human cervical cancer cells in vitro which may be a new anti-cervical cancer drug and provide experimental data for cervical cancer treatment. METHODS: HeLa cells cultured in vitro were divided into two groups: experimental group and control group. In experimental group, the cells were respectively treated with lanthanum chloride at different concentrations, 5, 50 and 100 µmol/L, while the cells in the control group were not treated with lanthanum chloride. The cell growth was observed by inverted microscope and the morphology changes of the cells were observed by the laser scanning confocal microscope (LSCM). Proliferation of HeLa cells in the two groups was detected by methyl thiazolyl tetrazolium (MTT) test; apoptosis rate was analyzed by flow cytometry (FCM). Cell migration test was applied to observe the effect of lanthanum chloride on migration. Reverse transcription (RT)-PCR was employed to evaluate the effects of lanthanum chloride on proliferation gene (cyclinD1), anti-apoptosis gene (zinc finger protein A20) and migration-related gene (matrix metalloproteinase 9, MMP-9). RESULTS: The status of cell growth was observed under the inverted microscope: with the increased of the lanthanum chloride concentrations, the cell density of reduced, the granule in cytoplasm increased, color intensifying and intercellular space enlarged; some cells became rounding and dead, floating in the culture media; the exfoliated cells increased gradually in the experimental groups. While In the control group, the cells grew adherently, with clear morphology and plump cytoplasm, and adjacent cell grew in lamellar. Observed with LSCM: the nuclear chromatin condensated and marginated with the volume of nuclear decreased in experimental groups. With the increase of the lanthanum chloride concentrations, nuclei in the experimental groups became pyknotic and then underwent karyorrhexis. However, the nuclear of the cells in control group were inact. The growth inhibition rates of lanthanum chloride groups (5, 50, 100 µmol/L) were 24%, 51% and 78%, respectively, in which each was significantly higher than that of the control group (P < 0.05); the apoptosis rates of lanthanum chloride group were (4.91 ± 0.39)%, (7.30 ± 0.71)% and (13.48 ± 0.92)%, respectively, which were all significantly higher than that of the control group [(0.89 ± 0.11)%, P < 0.01]. The migration ability of the cells was also decreased by the treatment of lanthanum chloride, the number of migrated cells in lanthanum chloride groups were 22.2 ± 4.3, 12.0 ± 3.2 and 7.8 ± 2.6 respectively, which were all significantly lower than that of the control group (41.2 ± 5.4, P < 0.01). The expression of genes of cyclinD1, A20 and MMP-9, were all decreased by the treatment of lanthanum chloride in a dose-dependent manner. CONCLUSION: Lanthanum chloride can inhibit the proliferation and migration of cervical cancer cells, and induce apoptosis by down-regulating cyclinD1, A20, and MMP-9 expressions in vitro.