Comparative and evolutionary analysis of RIP kinases in immune responses.

The group of receptor-interacting protein (RIP) kinases has seven members (RIPK1-7), with one homologous kinase domain but distinct non-kinase regions. Although RIPK1-3 have emerged as key modulators of inflammation and cell death, few studies have connected RIPK4-7 to immune responses. The divergence in domain structures and paralogue information in the Ensembl database have raised question about the phylogeny of RIPK1-7. In this study, phylogenetic trees of RIPK1-7 and paralogues constructed using full-length amino acid sequences or Kinase domain demonstrate that RIPK6 and RIPK7 are distinct from RIPK1-5 and paralogues shown in the Ensembl database are inaccurate. Comparative and evolutionary analyses were subsequently performed to gain new clues about the potential functions of RIPK3-7. RIPK3 gene loss in birds and animals that undergo torpor, a common physiological phenomenon in cold environments, implies that RIPK3 may be involved in ischemia-reperfusion injury and/or high metabolic rate. The negligible expression of RIPK4 and RIPK5 in immune cells is likely responsible for the lack of studies on the direct role of these members in immunity; RIPK6 and RIPK7 are conserved among plants, invertebrates and vertebrates, and dominantly expressed in innate immune cells, indicating their roles in innate immunity. Overall, our results provide insights into the multifaceted and conserved biochemical functions of RIP kinases.

Effects of Septum Resection for Secondary Infertility on Subsequent Reproductive Outcomes of in vitro Fertilization-Intracytoplasmic Sperm Injection.

OBJECTIVE: To assess the effect of uterine septum resection on reproductive outcomes of in vitro fertilization (IVF) / intracytoplasmic sperm injection (ICSI) in patients with secondary infertility complicated with uterine septum. METHODS: A retrospective cohort study included 269 patients. Surgical group included 169 patients with secondary infertility complicated with uterine septum, who underwent 252 embryo-transfer (ET) cycles following septum resection. Control group consisted of 100 patients with secondary infertility and uterine septum, who underwent 178 ET cycles. Cumulative pregnancy rate and cumulative live birth rate after one complete assisted reproductive technology (ART) cycle were the primary outcomes. RESULTS: The results showed that the cumulative pregnancy rate was higher in the surgery group, and statistically significant difference was observed in the cumulative pregnancy rate between the two groups (71.0 vs. 59%, P = 0.044). In fresh ET cycle, no statistically significant difference between the two groups was evident (54.9 vs. 40.6%, P = 0.061). Statistical analysis of other results of the fresh ET cycle did not differ significantly between the two groups. In terms of frozen embryo transfer (FET) cycle outcomes, the clinical pregnancy rate and delivery rate in surgery group were 52.7 and 38.2%, respectively, which were significantly higher than those in the control group (38.2 and 22.5%, respectively) (P = 0.028 and P = 0.011). CONCLUSION: The reproductive outcomes of IVF/ICSI after septum resection in patients with secondary infertility were better than that in the untreated group, suggesting that uterine septum resection can be performed in patients with uterine septum combined with infertility to improve their reproductive outcomes.

Effect of hysteroscopic septum resection on subsequent in vitro fertilization-intracytoplasmic sperm injection outcomes in cases of primary infertility.

OBJECTIVE: Women with septate uteri are at risk for subfertility, recurrent miscarriage, and preterm birth. It is not clear if hysteroscopic septum resection is beneficial to subsequent in vitro fertilization-intracytoplasmic sperm injection o (IVF/ICSI) outcomes in women with primary infertility. STUDY DESIGN: We analyzed all 278 women with uterine septum and primary infertility between January 2011 and January 2019. In this retrospective cohort study, the patients were divided into a surgery group and an expectant (non-surgery) group. RESULTS: Among them, 87 had a complete and 191 a partial septate uterus. The IVF-ET characteristics of the two groups showed no significant differences in the patients' age, body mass index, or basal follicle-stimulating hormone, luteinizing hormone, and estradiol levels (P>0.05). The miscarriage rate in those who underwent hysteroscopic septum resection, however, was significantly reduced (5.1% vs. 12.9%, P = 0.035). In contrast, the live birth rate between the two groups revealed no significant difference (51.4% vs. 43.6%, P = 0.1771), nor did the obstetric and neonatal outcomes (P>0.05). CONCLUSIONS: Hysteroscopic septum resection can be recommended prior to IVF/ICSI.

Lung neoplasm mimicking as ectopic pregnancy due to paraneoplastic secretion of human chorionic gonadotropin: a case report and literature review.

PURPOSE: The present systematic review aimed to examine the relationship between lung neoplasm and human chorionic gonadotropin (HCG). Especially, women with lung neoplasm mimicking as ectopic pregnancy were explored. METHODS: A rare case of lung neoplasm with high serum ß-HCG, which was initially thought to be ectopic pregnancy, was reported. A literature search was performed of the US National Library of Medicine (MEDLINE), EMBASE, PubMed, and the Cochrane Database of Systematic Reviews using appropriate keywords and subject headings to February 2020. RESULTS: Studies assessed lung neoplasm patients with positive HCG were included. Twenty studies, including 24 patients, were included. These cases illustrate the importance of considering the possibility of paraneoplastic secretion of ß-HCG in patients who have a positive pregnancy test. This may prevent a delay in the diagnosis and treatment of malignancy in young women. Of the 24 cases, only 7 (29.17%) were managed surgically; others were managed conservatively or with chemotherapy or radiation. CONCLUSION: The present systematic review shows the need to re-awaken awareness and high index of suspicion to lung neoplasm diagnosis in patients with positive pregnancy test.

Effect of Recurrence of Hydrosalpinx after Tubal Ligation on the Outcome of In vitro Fertilization Treatment: A Retrospective Cohort Study.

OBJECTIVE: The objective of the study was to evaluate the effects of recurrent hydrosalpinx after proximal tubal ligation and distal salpingostomy on the outcomes of in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) treatment. MATERIALS AND METHODS: Seven hundred and twenty-six patients with hydrosalpinx undergoing laparoscopic surgery before IVF were enrolled in the study. Five hundred and sixty-two patients treated with proximal tubal ligation and distal salpingostomy were included in Group A. One hundred and sixty-four cases managed with salpingectomy were grouped into Group B. Group A were further divided into two subgroups. One hundred and forty-six patients in Group A1 had a recurrence of hydrosalpinx. Four hundred and sixteen patients in Group A2 had no repetition of hydrosalpinx. We compared the pregnancy outcomes of their subsequent fresh embryo transfer cycles among the three groups. RESULTS: There were no significant differences among the three groups in terms of age, body mass index (23.56 ± 3.27 vs. 23.13 ± 3.42 vs. 23.63 ± 3.73, P = 0.195), basal hormone level (7.03 ± 1.75 vs. 7.08 ± 2.26 vs. 7.44 ± 2.93, P = 0.195), antral follicle count (12.25 ± 5.92 vs. 12.63 ± 5.71 vs. 11.70 ± 4.98, P = 0.188), duration of gonadotropin (Gn) (11.19 ± 2.1 vs. 10.93 ± 1.84 vs. 10.79 ± 2.03, P = 0.182), consumption of Gn (2136.73 ± 855.65 vs. 1997.15 ± 724.72 vs. 2069.05±765.12 , P = 0.14), endometrial thickness (1.1 ± 0.27 vs. 1.1 ± 0.24 vs. 1.1 ± 0.17, P = 0.352), base follicle-stimulating hormone (6.21 ± 3.43 vs. 6.52 ± 3.20 vs. 5.89 ± 3.10, P = 0.1), number of embryos transferred (1.87 ± 0.36 vs. 1.83 ± 0.42 vs. 1.88 ± 0.37, P = 0.224), and number of high-grade embryos (3.77 ± 2.42 vs. 4.01 ± 2.72 vs. 4.17 ± 2.74, P = 0.41). No differences were detected in clinical pregnancy rate (50% vs. 54.8% vs. 50%, P = 0.439), the live birth rate (86.3% vs. 82.0% vs. 87.8%, P = 0.398), fertilization rate (64.1% vs. 64.4% vs. 64.7%, P = 0.928), and biochemical pregnancy rate (4% vs. 4.5% vs. 7%, P = 0.332) among the three groups. CONCLUSION: The recurrence of hydrosalpinx after tubal ligation does not affect the outcomes of IVF/ICSI. It is not necessary to worry about the effect of recurrent hydrosalpinx on pregnancy outcomes of IVF/ICSI that may due to the spread of inflammation through lymphatic circulation or blood circulation.

A New Hysteroscopic Scoring System for Diagnosing Chronic Endometritis.

STUDY OBJECTIVE: To develop a new hysteroscopic morphologic scoring system to diagnose chronic endometritis (CE). DESIGN: Prospective study. SETTING: Medical hysteroscopy office. PATIENTS: In total, 320 patients underwent hysteroscopy, dilation and curettage, and endometrial biopsies from February 2017 to June 2018 with the intention of undergoing assisted reproductive technology treatment because of infertility or recurrent miscarriage. INTERVENTIONS: All patients underwent hysteroscopy, dilation and curettage, and endometrial biopsies for histologic examination and were classified according to the new hysteroscopic morphologic scoring system. MEASUREMENTS AND MAIN RESULTS: Of the 320 patients, 164 received a diagnosis of CE by histology (group A), whereas 156 patients were found not to have CE (group B). A total of 116 patients were diagnosed by our hysteroscopy scoring system to have CE, and 204 patients did not have CE. The scoring system showed a sensitivity and specificity of 62.8% and 91.7%, respectively. The positive predictive values and negative predictive values were 88.8% and 70.1%, respectively. Receiver operating characteristic analysis showed a cutoff value of >2 and an area under the curve of 0.823. Hysteroscopic and histologic grading showed moderate agreement (κ index = 0.529). CONCLUSION: Our hysteroscopic scoring system has a high sensitivity and specificity for CE; it is hoped that its use can reduce interobserver variability. Future clinical studies are warranted to confirm the validity and clinical applicability of the proposed hysteroscopic morphologic scoring system for CE.

Management strategies of heterotopic pregnancy following in vitro fertilization-embryo transfer.

OBJECTIVE: The purpose of this study was to evaluate the efficiency and safety of different treatment modalities for heterotopic pregnancy (HP) in vitro fertilization-embryo transfer (IVF-ET) cycles to avoid influence on intrauterine pregnancy (IUP). MATERIALS AND METHODS: Cases of HP (n = 90) were from the IVF/ICSI registry database at the Reproductive Hospital Affiliated to Shandong University. An additional 360 women were randomly selected as controls. The primary outcome to examine the risk factors, diagnostic modalities and the impact of different treatment modalities for HP. RESULTS: Our results showed that surgical treatment had a certain effect on improving the live-birth rate, although the effect was not statistically significant (87.9% vs. 70.8%, P = 0.055). The risk factors for HP included previous tubal surgery and hydrosalpinx. Fourteen days after embryo transfer, the serum levels of ß-human chorionic gonadotropin (ß-hCG) and estradiol (E2) were lower in the HP group than in the IUP group (P < 0.05). Furthermore, age and endometrial thickness showed a significant difference between the early abortion and the live-birth groups of HP. CONCLUSIONS: In our retrospective study, we supported early surgical laparoscopic intervention to minimize the incidence of abortion of IUP, which resulted in a better live-birth rate. A history of ectopic pregnancy and previous tubal surgery may increase the risk of HP. Low levels of serum ß-hCG and E2 on the 14th day after embryo transfer could indicate the incidence of HP.

Huaier extract synergizes with tamoxifen to induce autophagy and apoptosis in ER-positive breast cancer cells.

Tamoxifen (TAM) is the most widely used endocrine therapy for estrogen receptor (ER)-positive breast cancer patients, but side effects and the gradual development of insensitivity limit its application. We investigated whether Huaier extract, a traditional Chinese medicine, in combination with TAM would improve treatment efficacy in ER-positive breast cancers. MTT, colony formation, and invasion and migration assays revealed that the combined treatment had stronger anticancer effects than either treatment alone. Huaier extract enhanced TAM-induced autophagy, apoptosis, and G0/G1 cell cycle arrest, as measured by acidic vesicular organelle (AVO) staining, TUNEL, flow cytometry, and western blot. Additionally, combined treatment inhibited tumorigenesis and metastasis by suppressing the AKT/mTOR signaling pathway. Huaier extract also enhanced the inhibitory effects of TAM on tumor growth in vivo in a xenograft mouse model. These results show that Huaier extract synergizes with TAM to induce autophagy and apoptosis in ER-positive breast cancer cells by suppressing the AKT/mTOR pathway.

Huaier extract suppresses breast cancer via regulating tumor-associated macrophages.

Macrophages in tumor microenvironment are mostly M2-polarized - and have been reported to promote tumorigenesis, which are also defined as tumor-associated macrophages (TAMs). Here, we examined the regulatory effects of Huaier extract on TAMs using RAW264.7 murine macrophage cell line. Our data demonstrated that Huaier extract could inhibit the infiltration of macrophages into tumor microenvironment in a dose-dependent manner. By performing RT-PCR, immunofluorescence and phagocytosis assay, we were able to find that Huaier extract could regulate the polarization of macrophages, with decreased M2-polarization and increased phagocytosis of RAW264.7 cells. Moreover, we identified that Huaier extract could suppress macrophages-induced angiogenesis by using HUVEC migration assay, tube formation and chorioallantoic membrane assay. Additionally, western blotting showed decreased expression of MMP2, MMP9 and VEGF with the use of Huaier extract. Finally, we found that Huaier extract could inhibit M2-macrophages infiltration and angiogenesis through treating 4T1 tumor bearing mice with Huaier extract. Our study revealed a novel mechanism of the anti-tumor effect of Huaier extract which inhibited angiogenesis by targeting TAMs. These findings provided that Huaier was a promising drug for clinical treatment of breast cancer.

MicroRNA-99a inhibits tumor aggressive phenotypes through regulating HOXA1 in breast cancer cells.

MicroRNAs (miRNAs) are key regulators of tumor progression. Based on microarray data, we identified miR-99a as a potential tumor suppressor in breast cancer. Expression of miR-99a is frequently down-regulated in breast cancer tissues relative to normal breast tissues. Reduced miR-99a expression was highly associated with lymph node metastasis and shorter overall survival of patients with breast cancer. Gain- and loss-of-function studies revealed that, miR-99a significantly inhibits breast cancer cell proliferation, migration, and invasion. An integrated bioinformatics analysis identified HOXA1 mRNA as the direct functional target of miR-99a, and this regulation was confirmed by luciferase reporter assay. Furthermore, we showed for the first time that HOXA1 expression is elevated in breast cancer tissues. Knockdown of HOXA1 significantly inhibited breast cancer cell proliferation, migration and invasion, and restoration of HOXA1 partially rescued the inhibitory effect of miR-99a in breast cancer cells. Collectively, our data indicate that miR-99a plays a tumor-suppressor role in the development of breast cancer, and could serve as a potential therapeutic target for breast cancer treatment.

Huaier Extract Induces Autophagic Cell Death by Inhibiting the mTOR/S6K Pathway in Breast Cancer Cells.

Huaier extract is attracting increased attention due to its biological activities, including antitumor, anti-parasite and immunomodulatory effects. Here, we investigated the role of autophagy in Huaier-induced cytotoxicity in MDA-MB-231, MDA-MB-468 and MCF7 breast cancer cells. Huaier treatment inhibited cell viability in all three cell lines and induced various large membranous vacuoles in the cytoplasm. In addition, electron microscopy, MDC staining, accumulated expression of autophagy markers and flow cytometry revealed that Huaier extract triggered autophagy. Inhibition of autophagy attenuated Huaier-induced cell death. Furthermore, Huaier extract inhibited the mammalian target of the rapamycin (mTOR)/S6K pathway in breast cancer cells. After implanting MDA-MB-231 cells subcutaneously into the right flank of BALB/c nu/nu mice, Huaier extract induced autophagy and effectively inhibited xenograft tumor growth. This study is the first to show that Huaier-induced cytotoxicity is partially mediated through autophagic cell death in breast cancer cells through suppression of the mTOR/S6K pathway.

Autophagy facilitates the development of resistance to the tumor necrosis factor superfamily member TRAIL in breast cancer.

Autophagy, an important homeostatic cellular recycling mechanism, has emerged as a novel cytoprotective mechanism to increase tumor cell survival through escaping chemotherapy­induced cell death. To explore whether autophagy plays a protective role in the resistance to the tumor necrosis factor­related apoptosis­inducing ligand (TRAIL), we evaluated the autophagy levels in TRAIL­sensitive MDA­MB­231 breast cancer cell lines and in TRAIL­refractory MDA­MB­231 cells before and after TRAIL treatment. After treatment with 40 ng/ml TRAIL, TRAIL­sensitized MDA­MB­231 parental cells expressed higher level of LC3B protein and accumulated more autophagic vacuoles. Compared with TRAIL­sensitive MDA­MB­231, MDA­MB­231 TRAIL­refractory cells showed higher levels of the lipidated form of LC3B and decreased p62/SQSTM1 protein expression, characterizing the occurrence of increased autophagic flux in TRAIL­refractory cells. Electron microscopy and monodansylcadaverine (MDC) autophagy­specific fluorescence staining analyses also revealed that the accumulation of autophagic vacuoles was drastically higher in TRAIL­refractory MDA­MB­231 parental cells. We demonstrated that chloroquine (CQ) and 2­(4­morpholinyl)­8­phenylchromone (LY294002) could effectively reduce TRAIL­refractory breast cancer cell viability. Combination of TRAIL with CQ could effectively reverse the resistance of MDA­MB­231 TRAIL­refractory cells to TRAIL. Knockdown of light chain 3 (LC3) expression via small interfering RNA (siRNA) similarly resulted in reduced TRAIL­refractory cell proliferation and re­sensitizing to TRAIL. This is the first report showing that breast cancer cells chronically exposed to TRAIL exhibit upregulation of the autophagic activity, indicating that autophagy efficiently protects breast cancer cells from TRAIL. Therapeutic targeting of autophagosome formation could be a novel molecular avenue to reduce the resistance of TRAIL in breast cancer.

Nitidine chloride induces apoptosis, cell cycle arrest, and synergistic cytotoxicity with doxorubicin in breast cancer cells.

Medicinal plant extracts have been widely used for cancer treatment. Nitidine chloride (NC) is a natural bioactive alkaloid that has recently been reported to have diverse anticancer properties. We aimed to investigate the cytotoxic effects of NC and the effectiveness of combinatorial treatment including NC and doxorubicin in breast cancer cells. Using MTT and flowcytometry assays, we found that NC induced cell growth inhibition and G2/M cell cycle arrest in a time- and dose-dependent manner both in MCF-7 and MDA-MB-231 breast cancer cell lines. Cancer cell growth inhibition was associated with increased levels of the p53 and p21 proteins. Apoptosis induction by NC treatment was confirmed by JC-1 mitochondrial membrane potential, annexin V-positive cell, and TUNEL staining. Using western blot analysis, we found that NC upregulated the pro-apoptotic proteins Bax, cleaved caspase-9 and -3 and cleaved PARP and that it downregulated the anti-apoptotic proteins Bcl-2 and PARP. By using the PI3K/Akt inhibitor LY294002, we further demonstrated that NC-induced apoptosis might be Akt-specific or dependent. In addition, NC exhibited a synergistic effect with doxorubicin on the growth inhibition of the human breast cancer cell lines MCF-7 and MDA-MB-231. Our study demonstrated the anticancer effect of NC on breast cancer and highlighted the potential clinical application of NC.

Huaier aqueous extract suppresses human breast cancer cell proliferation through inhibition of estrogen receptor α signaling.

Estrogen receptor α (ERα) has been reported to play a critical role in promoting the growth of breast tumor cells. In the present study, we explored the effect of Huaier extract on estrogen receptor α signaling in breast cancer cell lines. Our data demonstrated that Huaier extract effectively inhibited the proliferation of the MCF-7, T47D and ZR-75-1 human breast cancer cell lines. For the mechanism analysis, we demonstrated that Huaier extract significantly reduced the mRNA and protein levels of ERα in all three ERα-positive cell lines. The downregulation of ERα protein levels was correlated with activation of the proteasomes. We demonstrated that Huaier extract markedly decreased the expression of both ERα and its downstream genes, inhibited the estrogen-stimulated proliferation and reversed the estrogen-induced activation of the nuclear factor κB (NFκB) pathway. Our study provides evidence that Huaier extract is a novel estrogen receptor modulator and is a promising drug for the prevention and treatment of ERα-positive human breast cancers.