Fibroids and unexplained infertility treatment with epigallocatechin gallate: a natural compound in green tea (FRIEND) - protocol for a randomised placebo-controlled US multicentre clinical trial of EGCG to improve fertility in women with uterine fibroids.

INTRODUCTION: Uterine fibroids affect 30%-77% of reproductive-age women and are a significant cause of infertility. Surgical myomectomies can restore fertility, but they often have limited and temporary benefits, with postoperative complications such as adhesions negatively impacting fertility. Existing medical therapies, such as oral contraceptives, gonadotropin hormone-releasing hormone (GnRH) analogues and GnRH antagonists, can manage fibroid symptoms but are not fertility friendly. This study addresses the pressing need for non-hormonal, non-surgical treatment options for women with fibroids desiring pregnancy. Previous preclinical and clinical studies have shown that epigallocatechin gallate (EGCG) effectively reduces uterine fibroid size. We hypothesise that EGCG from green tea extract will shrink fibroids, enhance endometrial quality and increase pregnancy likelihood. To investigate this hypothesis, we initiated a National Institute of Child Health and Human Development Confirm-funded trial to assess EGCG's efficacy in treating women with fibroids and unexplained infertility. METHODS AND ANALYSIS: This multicentre, prospective, interventional, randomised, double-blinded clinical trial aims to enrol 200 participants with fibroids and unexplained infertility undergoing intrauterine insemination (IUI). Participants will be randomly assigned in a 3:1 ratio to two groups: green tea extract (1650 mg daily) or a matched placebo, combined with clomiphene citrate-induced ovarian stimulation and timed IUI for up to four cycles. EGCG constitutes approximately 45% of the green tea extract. The primary outcome is the cumulative live birth rate, with secondary outcomes including conception rate, time to conception, miscarriage rate, change in fibroid volume and symptom severity scores and health-related quality of life questionnaire scores. ETHICS AND DISSEMINATION: The FRIEND trial received approval from the Food and Drug adminstration (FDA) (investigational new drug number 150951), the central Institutional Review Board (IRB) at Johns Hopkins University and FRIEND-collaborative site local IRBs. The data will be disseminated at major conferences, published in peer-reviewed journals and support a large-scale clinical trial. TRIAL REGISTRATION NUMBER: NCT05364008.

Probiotics alleviate constipation and inflammation in late gestating and lactating sows.

Constipation and systemic inflammation are common in late pregnant and lactating sows, which cause health problems like uteritis, mastitis, dystocia, or even stillbirth, further influencing piglets' survival and growth. Probiotic supplementation can improve such issues, but the beneficial mechanism of relieving constipation and enhancing gut motility remains underexplored. This study aimed to investigate the effects and mechanism of probiotic supplementation in drinking water to late pregnant sows on constipation, inflammation, and piglets' growth performance. Seventy-four sows were randomly allocated to probiotic (n = 36) and control (n = 38) groups. Probiotic treatment significantly relieved sow constipation, enhanced serum IL-4 and IL-10 levels while reducing serum IL-1ß, IL-12p40, and TNF-α levels, and increased piglet daily gain and weaning weight. Furthermore, probiotic administration reshaped the sow gut bacteriome and phageome structure/diversity, accompanied by increases in some potentially beneficial bacteria. At 113 days of gestation, the probiotic group was enriched in several gut microbial bioactive metabolites, multiple carbohydrate-active enzymes that degrade pectin and starch, fecal butyrate and acetate, and some serum metabolites involved in vitamin and amino acid metabolism. Our integrated correlation network analysis revealed that the alleviation of constipation and inflammation was associated with changes in the sow gut bacteriome, phageome, bioactive metabolic potential, and metabolism.

Probio-X Relieves Symptoms of Hyperlipidemia by Regulating Patients' Gut Microbiome, Blood Lipid Metabolism, and Lifestyle Habits.

Hyperlipidemia is a key risk factor for cardiovascular disease, and it is associated with lipid metabolic disorders and gut microbiota dysbiosis. Here, we aimed to investigate the beneficial effects of 3-month intake of a mixed probiotic formulation in hyperlipidemic patients (n = 27 and 29 in placebo and probiotic groups, respectively). The blood lipid indexes, lipid metabolome, and fecal microbiome before and after the intervention were monitored. Our results showed that probiotic intervention could significantly decrease the serum levels of total cholesterol, triglyceride, and low-density lipoprotein cholesterol (P < 0.05), while increasing the levels of high-density lipoprotein cholesterol (P < 0.05) in patients with hyperlipidemia. Probiotic recipients showing improved blood lipid profile also exhibited significant differences in their lifestyle habits after the 3-month intervention, with an increase in daily intake of vegetable and dairy products, as well as weekly exercise time (P < 0.05). Moreover, two blood lipid metabolites (namely, acetyl-carnitine and free carnitine) significantly increased after probiotic supplementation cholesterol (P < 0.05). In addition, probiotic-driven mitigation of hyperlipidemic symptoms were accompanied by increases in beneficial bacteria like Bifidobacterium animalis subsp. lactis and Lactiplantibacillus plantarum in patients' fecal microbiota. These results supported that mixed probiotic application could regulate host gut microbiota balance, lipid metabolism, and lifestyle habits, through which hyperlipidemic symptoms could be alleviated. The findings of this study urge further research and development of probiotics into nutraceuticals for managing hyperlipidemia. IMPORTANCE The human gut microbiota have a potential effect on the lipid metabolism and are closely related to the disease hyperlipidemia. Our trial has demonstrated that 3-month intake of a mixed probiotic formulation alleviates hyperlipidemic symptoms, possibly by modulation of gut microbes and host lipid metabolism. The findings of the present study provide new insights into the treatment of hyperlipidemia, mechanisms of novel therapeutic strategies, and application of probiotics-based therapy.

Lacticaseibacillus rhamnosus Probio-M9 enhanced the antitumor response to anti-PD-1 therapy by modulating intestinal metabolites.

BACKGROUND: Probiotics have been increasingly proposed for enhancing immune checkpoint blockade (ICB) treatments against cancer. However, its causal relationship with immunotherapeutic efficacy remains unclear, which promoted us to explore if and how probiotic Lacticaseibacillus rhamnosus Probio-M9 manipulates gut microbiome for expected outcomes. METHODS: We evaluated the effects of Probio-M9 on the anti-PD-1 treatment against colorectal cancer in mice via a multi-omics approach. We defined the mechanisms of Probio-M9-mediated antitumor immunity by comprehensive analyses of metagenome and metabolites of commensal gut microbes as well as the immunologic factors and serum metabolome of the host. FINDINGS: The results indicated that Probio-M9 intervention strengthened the anti-PD-1-based tumor inhibition. Both prophylactic and therapeutic administration of Probio-M9 showed conspicuous performance in controlling tumor growth with ICB treatment. The supplement of Probio-M9 modulated enhanced immunotherapy response through promoting beneficial microbes (e.g., Lactobacillus and Bifidobacterium animalis), producing beneficial metabolites including butyric acids in the gut, and accumulating blood-derived α-ketoglutaric acid, N-acetyl-l-glutamic acid and pyridoxine in particular, which promoted the infiltration and activation of cytotoxic T lymphocytes (CTLs) and suppressing the function of regulatory T cells (Tregs) in the tumor microenvironment (TME). Subsequently, we found that enhanced immunotherapeutic response was transmissible by transplanting either post-probiotic-treatment gut microbes or intestinal metabolites to new tumor-bearing mice. INTERPRETATION: This study offered valuable insight into the causal role of Probio-M9 in correcting the defects in gut microbiota that compromised anti-PD-1 therapeutic efficacy, which can be used as an alternative synergetic agent with ICB for clinical cancer treatment. FUNDING: This research was supported by Research Fund for the National Key R&D Program of China (2022YFD2100702), Inner Mongolia Science and Technology Major Projects (2021ZD0014), and China Agriculture Research System of MOF and MARA.

Assessing the Hepatic Safety of Epigallocatechin Gallate (EGCG) in Reproductive-Aged Women.

A similar abstract of the interim analysis was previously published in Fertility and Sterility. EPIGALLOCATECHIN GALLATE (EGCG) FOR TREATMENT OF UNEXPLAINED INFERTILITY ASSOCIATED WITH UTERINE FIBROIDS (PRE-FRIEND TRIAL): EARLY SAFETY ASSESSMENT. Uterine fibroids are the most common cause of unexplained infertility in reproductive-aged women. Epigallocatechin gallate (EGCG), a green tea catechin, has demonstrated its ability to shrink uterine fibroids in prior preclinical and clinical studies. Hence, we developed an NICHD Confirm-funded trial to evaluate the use of EGCG for treating women with fibroids and unexplained infertility (FRIEND trial). Prior to embarking on that trial, we here conducted the pre-FRIEND study (NCT04177693) to evaluate the safety of EGCG in premenopausal women. Specifically, our aim was to assess any adverse effects of EGCG alone or in combination with an ovarian stimulator on serum liver function tests (LFTs) and folate level. In this randomized, open-label prospective cohort, participants were recruited from the FRIEND-collaborative clinical sites: Johns Hopkins University, University of Chicago, University of Illinois at Chicago, and Yale University. Thirty-nine women, ages ≥18 to ≤40 years, with/without uterine fibroids, were enrolled and randomized to one of three treatment arms: 800 mg of EGCG daily alone, 800 mg of EGCG daily with clomiphene citrate 100 mg for 5 days, or 800 mg of EGCG daily with Letrozole 5 mg for 5 days. No subject demonstrated signs of drug induced liver injury and no subject showed serum folate level outside the normal range. Hence, our data suggests that a daily dose of 800 mg of EGCG alone or in combination with clomiphene citrate or letrozole (for 5 days) is well-tolerated and is not associated with liver toxicity or folate deficiency in reproductive-aged women.

Bifidobacterium lactis Probio-M8 improves bone metabolism in patients with postmenopausal osteoporosis, possibly by modulating the gut microbiota.

PURPOSE: Postmenopausal osteoporosis (PMO) is usually managed by conventional drug treatment. However, prolonged use of these drugs cause side effects. Gut microbiota may be a potential target for treatment of PMO. This work was a three-month intervention trial aiming to evaluate the added effect of probiotics as adjunctive treatment for PMO. METHODS: Forty patients with PMO were randomized into probiotic (n = 20; received Bifidobacterium animalis subsp. lactis Probio-M8 [Probio-M8], calcium, calcitriol) and placebo (n = 20; received placebo material, calcium, calcitriol) groups. The bone mineral density of patients was measured at month 0 (0 M; baseline) and month 3 (3 M; after three-month intervention). Blood and fecal samples were collected 0 M and 3 M. Only 15 and 12 patients from Probio-M8 and placebo groups, respectively, provided complete fecal samples for gut microbiota analysis. RESULTS: No significant change was observed in the bone mineral density of patients at 3 M. Co-administering Probio-M8 improved the bone metabolism, reflected by an increased vitamin D3 level and decreased PTH and procalcitonin levels in serum at 3 M. Fecal metagenomic analysis revealed modest changes in the gut microbiome in both groups at 3 M. Interestingly, Probio-M8 co-administration affected the gut microbial interactive correlation network, particularly the short-chain fatty acid-producing bacteria. Probio-M8 co-administration significantly increased genes encoding some carbohydrate metabolism pathways (including ABC transporters, the phosphotransferase system, and fructose and mannose metabolism) and a choline-phosphate cytidylyltransferase. CONCLUSIONS: Co-administering Probio-M8 with conventional drugs/supplements was more efficacious than conventional drugs/supplements alone in managing PMO. Our study shed insights into the beneficial mechanism of probiotic adjunctive treatment. REGISTRATION NUMBER OF CLINICAL TRIAL: Chinese Clinical Trial Registry (identifier number: ChiCTR1800019268).

Senegenin Rescues PC12 Cells with Oxidative Damage Through Inhibition of Ferroptosis.

Oxidative stress is one of the pathological mechanisms of Alzheimer's disease (AD), and ferroptosis has been determined to be involved in neurodegenerative diseases such as AD. Senegenin (Sen) prevents oxidative damage in nerve cells via a mechanism that may be highly related to ferroptosis. However, the mechanism of ferroptosis pathway involvement in AD is unclear. In this study, we established a model of PC12 cytotoxic injury induced by Aß25-35, and we detected the level of oxidative damage, MMP, and ferroptosis-related protein expression. The results showed that, compared with control group, the level of ROS increased, GPX activities decreased, and MDA levels increased in Aß25-35 group. Aß25-35 could induce mitochondrial depolarization in PC12 cells and Fer-1 could not reverse this damage. WB revealed that Aß25-35 group had increased ACSL4 and PEBP1 proteins, and decreased GPX4 protein. After adding Sen in the model, the level of oxidative damage was reduced, and mitochondrial depolarization was reversed compared with Aß25-35 group. WB suggested that the expression of ACSL4 and PEBP1 proteins decreased, and the expression of GPX4 protein increased by Sen treatment. In conclusion, we found that Sen exhibits strong neuroprotective activity against Aß25-35 induced oxidative damage and lipid metabolic associated with ferroptosis. Inhibiting nerve cell ferroptosis might facilitate the future development of strategies to AD.

Effects of Lactobacillus plantarum P9 Probiotics on Defecation and Quality of Life of Individuals with Chronic Constipation: Protocol for a Randomized, Double-Blind, Placebo-Controlled Clinical Trial.

Background: Although probiotics have been shown to improve constipation-related symptoms, a clear consensus on the use of probiotics as a constipation-relieving agent has not been reached, which is attributed to the limited available evidence and inconsistent protocols used in existing studies. Method: A randomized, double-blind, placebo-controlled clinical trial is designed to study the efficiency and possible mechanism of action of probiotics for chronic constipation, in which 200 eligible volunteers with chronic constipation will be randomly assigned to a probiotic group (oral Lactobacillus plantarum P9 probiotic powder, 100 billion colony-forming units (CFUs)/day) or a placebo group. Volunteers, treatment distributors, data collectors, and data analysts will be blinded. The primary outcome is the weekly mean frequency of complete spontaneous bowel movements (CSBMs), and secondary outcomes include weekly mean frequency of CSBMs ≥3, weekly mean frequency of spontaneous bowel movements (SBMs), weekly mean stool appearance score, weekly mean difficulty of passing stool score, weekly percentage of volunteers who use auxiliary measures to assist with defecation (WPUAMA), quality-of-life (QOL) score, emotional status score, gut microbiome, and faecal metabolome. Each outcome measure will be assessed at the time points of preadministration (day 0), administration (day 14 and/or 28), and postadministration (day 42) to identify inter- and intragroup differences. Adverse events will be recorded to evaluate the safety of L. plantarum P9. Discussion. The protocol will provide methodological guidance for other similar studies, avoiding methodological bias and ultimately facilitating the formulation of consensus on the use of probiotics as a constipation-relieving agent. In addition, the results are more comprehensive than those of existing studies and may objectively and scientifically reflect the effectiveness of L. plantarum P9 on constipation. If the expected study findings are obtained, L. plantarum P9, taken as a probiotic, may become a complementary choice for chronically constipated patients. This trial is registered with Chinese Clinical Trial Registry (ChiCTR) (no. ChiCTR2000038396) registered on November 22, 2020, https://www.chictr.org.cn/showproj.aspx?proj=54024.

Bifidobacterium lactis Probio-M8 Adjuvant Treatment Confers Added Benefits to Patients with Coronary Artery Disease via Target Modulation of the Gut-Heart/-Brain Axes.

Accumulating evidence suggests that gut dysbiosis may play a role in cardiovascular problems like coronary artery disease (CAD). Thus, target steering the gut microbiota/metabolome via probiotic administration could be a promising way to protect against CAD. A 6-month randomized, double-blind, placebo-controlled clinical trial was conducted to investigate the added benefits and mechanism of the probiotic strain, Bifidobacterium lactis Probio-M8, in alleviating CAD when given together with a conventional regimen. Sixty patients with CAD were randomly divided into a probiotic group (n = 36; received Probio-M8, atorvastatin, and metoprolol) and placebo group (n = 24; placebo, atorvastatin, and metoprolol). Conventional treatment significantly improved the Seattle Angina Questionnaire (SAQ) scores of the placebo group after the intervention. However, the probiotic group achieved even better SAQ scores at day 180 compared with the placebo group (P < 0.0001). Moreover, Probio-M8 treatment was more conducive to alleviating depression and anxiety in patients (P < 0.0001 versus the placebo group, day 180), with significantly lower serum levels of interleukin-6 and low-density lipoprotein cholesterol (P < 0.005 and P < 0.001, respectively). In-depth metagenomic analysis showed that, at day 180, significantly more species-level genome bins (SGBs) of Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, and Butyricicoccus porcorum were detected in the probiotic group compared with the placebo group, while the abundances of SGBs representing Flavonifractor plautii and Parabacteroides johnsonii decreased significantly among the Probio-M8 receivers (P < 0.05). Furthermore, significantly more microbial bioactive metabolites (e.g., methylxanthine and malonate) but less trimethylamine-N-oxide and proatherogenic amino acids were detected in the probiotic group than placebo group during/after intervention (P < 0.05). Collectively, we showed that coadministering Probio-M8 synergized with a conventional regimen to improve the clinical efficacy in CAD management. The mechanism of the added benefits was likely achieved via probiotic-driven modulation of the host's gut microbiota and metabolome, consequently improving the microbial metabolic potential and serum metabolite profile. This study highlighted the significance of regulating the gut-heart/-brain axes in CAD treatment. IMPORTANCE Despite recent advances in therapeutic strategies and drug treatments (e.g., statins) for coronary artery disease (CAD), CAD-related mortality and morbidity remain high. Active bidirectional interactions between the gut microbiota and the heart implicate that probiotic application could be a novel therapeutic strategy for CAD. This study hypothesized that coadministration of atorvastatin and probiotics could synergistically protect against CAD. Our results demonstrated that coadministering Probio-M8 with a conventional regimen offered added benefits to patients with CAD compared with conventional treatment alone. Our findings have provided a wide and integrative view of the pathogenesis and novel management options for CAD and CAD-related diseases.

Effects of the Zishen Yutai Pill Compared With Placebo on Live Births Among Women in a Fresh Embryo Transfer Cycle: A Randomized Controlled Trial.

OBJECTIVE: To assess the efficacy of the Zishen Yutai Pill compared with placebo on live birth rates among women after fresh embryo transfer cycles. METHODS: We conducted a double-blind, multicenter, placebo-controlled, randomized trial to investigate whether administration of the Zishen Yutai Pill would improve pregnancy outcomes among women undergoing fresh embryo transfer after in vitro fertilization or intracytoplasmic sperm injection. The primary outcome was live birth rate. Secondary outcomes were rates of implantation, biochemical pregnancy, clinical pregnancy, pregnancy loss, cycle cancellation, and maternal, fetal, and neonatal complications. A total sample size of 2,265 women (1:1 in two groups) was used to detect a live birth rate difference between the Zishen Yutai Pill and placebo. Participants were enrolled and randomized to receive 5 g of the Zishen Yutai Pill or placebo orally, three times per day during the study. RESULTS: Recruitment was completed between April 2014 and June 2017, with 2,580 patients screened. Two thousand two hundred sixty-five patients were randomized: 1,131 to the Zishen Yutai Pill and 1,134 to placebo. Characteristics were similar between groups. In intention-to-treat analysis, the rates of live birth in the Zishen Yutai Pill (ZYP) group and placebo group were 26.8% and 23.0% (rate ratio [RR], 1.16; 95% CI 1.01-1.34; P=.038), respectively. The implantation rates were 36.8% and 32.6% in the ZYP and placebo groups, respectively (RR 1.13; 95% CI 1.01-1.25; P=.027). The biochemical pregnancy rate for the ZYP group was 35.5% compared with 31.1% in the placebo group (RR 1.14; 95% CI 1.02-1.28; P=.026). The rates of clinical pregnancy in the ZYP and placebo groups were 31.2% compared with 27.3%, respectively (RR 1.14; 95% CI 1.00-1.30; P=.043). There were no significant between-group differences in the rates of pregnancy loss, maternal, or neonatal complications (all P>.05). CONCLUSION: The Zishen Yutai Pill increased the rate of live birth after fresh embryo transfer compared with placebo. CLINICAL TRIAL REGISTRATION: Chictr.org.cn, Chictr-TRC-14004494.

Male vitamin D status and male factor infertility.

OBJECTIVE: To determine the association between vitamin D levels in the male partner and fertility outcomes in couples with mild male factor infertility. DESIGN: Secondary analysis of a randomized, controlled trial. SETTING: Nine fertility centers in the United States. PATIENT(S): Men (n = 154) with sperm concentration between 5 and 15 million/mL, motility ≤40%, or normal morphology ≤4% were eligible. Female partners were ovulatory, ≤40 years old, and had documented tubal patency. INTERVENTION(S): Men provided semen and blood at baseline for semen analysis and 25-hydroxyvitamin D (25(OH)D) levels. They were randomly assigned to receive a vitamin formulation including vitamin D 2,000 IU daily or placebo for up to 6 months. Couples attempted to conceive naturally during the first 3 months and with clomiphene citrate with intrauterine insemination of the female partner in months 4 through 6. MAIN OUTCOME MEASURE(S): Primary: sperm concentration, motility, morphology, and DNA fragmentation at baseline. Secondary: cumulative pregnancy, miscarriage, and live birth rates. RESULT(S): Semen parameters and sperm DNA fragmentation were not statistically significantly different between men with vitamin D deficiency and men with 25(OH)D levels ≥20 ng/mL. In addition, clinical pregnancy and live birth rates were similar. Male 25(OH)D level <20 ng/mL was associated with a higher rate of pregnancy loss (adjusted odds ratio 9.0; 95% confidence interval 1.3 to 61.3). CONCLUSION(S): Vitamin D deficiency in the male partner did not significantly impact semen parameters or treatment outcomes. Further study is warranted to better characterize the rate of miscarriage in couples with male vitamin D deficiency.

Probiotic consumption relieved human stress and anxiety symptoms possibly via modulating the neuroactive potential of the gut microbiota.

Stress has been shown to disturb the balance of human intestinal microbiota and subsequently causes mental health problems like anxiety and depression. Our previous study showed that ingesting the probiotic strain, Lactobacillus (L.) plantarum P-8, for 12 weeks could alleviate stress and anxiety of stressed adults. The current study was a follow-up work aiming to investigate the functional role of the gut metagenomes in the observed beneficial effects. The fecal metagenomes of the probiotic (n = 43) and placebo (n = 36) receivers were analyzed in depth. The gut microbiomes of the placebo group at weeks 0 and 12 showed a significantly greater Aitchison distance (P < 0.001) compared with the probiotic group. Meanwhile, the Shannon diversity index of the placebo group (P < 0.05) but not the probiotic group decreased significantly at week 12. Additionally, significantly more species-level genome bins (SGBs) of Bifidobacterium adolescentis, Bifidobacterium longum, and Fecalibacterium prausnitzii (P < 0.01) were identified in the fecal metagenomes of the probiotic group, while the abundances of SGBs representing the species Roseburia faecis and Fusicatenibacter saccharivorans decreased significantly (P < 0.05). Furthermore, the 12-week probiotic supplementation enhanced the diversity of neurotransmitter-synthesizing/consuming SGBs and the levels of some predicted microbial neuroactive metabolites (e.g., short-chain fatty acids, gamma-aminobutyric acid, arachidonic acid, and sphingomyelin). Our results showed a potential link between probiotic-induced gut microbiota modulation and stress/anxiety alleviation in stressed adults, supporting that the gut-brain axis was involved in relieving stress-related symptoms. The beneficial effect relied not only on microbial diversity changes but more importantly gut metagenome modulations at the SGB and functional gene levels.

Characterization of potentially probiotic lactic acid bacteria and bifidobacteria isolated from human colostrum.

Breast milk is the main source of nutrition for infants; it contains considerable microflora that can be transmitted to the infant endogenously or by breastfeeding, and it plays an important role in the maturation and development of the immune system. In this study, we isolated and identified lactic acid bacteria (LAB) from human colostrum, and screened 2 strains with probiotic potential. The LAB isolated from 40 human colostrum samples belonged to 5 genera: Lactobacillus, Bifidobacterium, Streptococcus, Enterococcus, and Staphylococcus. We also isolated Propionibacterium and Actinomyces. We identified a total of 197 strains of LAB derived from human colostrum based on their morphology and 16S rRNA sequence, among them 8 strains of Bifidobacterium and 10 strains of Lactobacillus, including 3 Bifidobacterium species and 4 Lactobacillus species. The physiological and biochemical characteristics of strains with good probiotic characteristics were evaluated. The tolerances of some of the Bifidobacterium and Lactobacillus strains to gastrointestinal fluid and bile salts were evaluated in vitro, using the probiotic strains Bifidobacterium lactis BB12 and Lactobacillus rhamnosus GG as controls. Among them, B. lactis Probio-M8 and L. rhamnosus Probio-M9 showed survival rates of 97.25 and 78.33% after digestion for 11 h in artificial gastrointestinal juice, and they exhibited growth delays of 0.95 and 1.87 h, respectively, in 0.3% bile salts. These two strains have the potential for application as probiotics and will facilitate functional studies of probiotics in breast milk and the development of human milk-derived probiotics.

Subcellular distribution and chemical forms involved in manganese accumulation and detoxification for Xanthium strumarium L.

Xanthium strumarium L. is a candidate species for manganese (Mn)-phyto-remediation. To reveal the mechanism of this species adaptive to Mn stress, the growth, Mn subcellular distribution, chemical forms, as well as micro-structure and ultra-structure responses of the mining ecotype (ME) of X. strumarium to Mn stress were studied with the non-mining ecotype (NME) as the reference by a hydroponic experiment. The results showed the ME demonstrated a higher tolerance to Mn stress with a superior growth and a higher tolerance index (TI) when compared with the NME. The concentrations of Mn in leaves, stems, and roots of the ME were 1.1-1.8, 1.2-1.9, and 1.3-1.9 times higher than those in the corresponding organs of the NME, respectively. The micro-structure and ultra-structure showed abnormal alterations, such as shrunken ducts and sieve canals, round-shaped chloroplasts, increased starch and osmiophilic granules, as well as expanded and non-compact granum thylakoids in the NME, compared to the ME. More than 83% of Mn was localized in cell wall and soluble fraction, while the Mn concentration in all fractions had a direct linear relationship with Mn treatment in the ME. The proportions of pectates and protein integrated-Mn, phosphate-Mn, and oxalate-Mn forms were dominant in leaves and stems of the ME, whereas, in the NME the relative proportions of inorganic Mn and water-soluble Mn forms in the roots was higher than the other forms. Altogether, the combination of preferential distribution of Mn in the cell wall and soluble fraction, and storage of Mn in low toxicity forms, such as phosphate-Mn, pectates and protein-bound Mn, and oxalate-Mn, might be responsible for alleviating Mn toxicity in the ME.

Effects of manganese stress on phenology and biomass allocation in Xanthium strumarium from metalliferous and non-metalliferous sites.

Xanthium strumarium is an annual pseudometallophyte. To reveal the mechanisms of this species to adapt to metallicolous environmental conditions, phenological traits and biomass allocation of metallicolous and non-metallicolous populations of X. strumarium under six Mn2+ concentrations by pot culture experiments were performed. The results showed that both time to bolting and time to fruit setting in the metallicolous population were earlier than those in the non-metallicolous population. The number of flowers, fruits, seeds and 1000-seed weight in the metallicolous population were higher than those in the non-metallicolous population under Mn stress. Reproductive allocation and harvest index in the metallicolous population were higher than those in the non-metallicolous population. Furthermore, all the Mn concentrations in leaves, stems, roots, and fruits of the metallicolous population were higher than the counterparts of non-metallicolous population. These results suggested that metallicolous population had higher tolerance to Mn stress than non-metallicolous population, the earlier flowering and fruiting, and the enhancement in reproductive allocation may contribute to plant tolerance to Mn toxicity for X. strumarium.

Morphophysiological responses and tolerance mechanisms of Xanthium strumarium to manganese stress.

Effective phytoremediation of manganese (Mn) requires the careful selection of a species that has a relatively high manganese tolerance. Exploring the physiological mechanisms related to Mn stress responses is crucial for identifying and employing species for Mn phytoremediation. Xanthium strumarium is a species that can accumulate high levels of Mn, thus it is a candidate species for Mn-phytoremediation. To reveal the tolerance mechanisms of this species to manage Mn stress, the morphological, physiological, and biochemical responses of seedlings grown in water cultures under six different Mn concentrations were analyzed. The results showed that X. strumarium can accumulate high levels of Mn, even as plant growth was inhibited by rising Mn concentrations. Malondialdehyde (MDA) content increased and catalase (CAT) activity decreased along with the increased Mn concentrations, while soluble protein and proline content, as well as the superoxide dismutase (SOD) and peroxidase (POD) enzymes, all increased initially and then declined. The highest value of POD, SOD, soluble protein and proline all occurred at 5000 µM of Mn stress, which means that X. strumarium can adapt to low concentration of Mn stress. The net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO2 concentration (Ci) and transpiration rate (Tr) decreased, and the stomatal limitation (Ls) increased in response to Mn stress. Furthermore, water use efficiency (WUE) and intrinsic water use efficiency (WUEi) increased first under low concentration of Mn, and then reduced as the concentration of Mn increased. The maximum quantum efficiency of PSII photochemistry (Fv/Fm), efficiency of excitation capture by open PSII reaction centers (Fv'/Fm'), electron transport rate (ETR) declined as Mn concentration increased. In conclusion, the above results showed that X. strumarium can be effectively used for phytoremediation of Mn-contaminated soils.

Effect of Acupuncture and Clomiphene in Chinese Women With Polycystic Ovary Syndrome: A Randomized Clinical Trial.

Importance: Acupuncture is used to induce ovulation in some women with polycystic ovary syndrome, without supporting clinical evidence. Objective: To assess whether active acupuncture, either alone or combined with clomiphene, increases the likelihood of live births among women with polycystic ovary syndrome. Design, Setting, and Participants: A double-blind (clomiphene vs placebo), single-blind (active vs control acupuncture) factorial trial was conducted at 21 sites (27 hospitals) in mainland China between July 6, 2012, and November 18, 2014, with 10 months of pregnancy follow-up until October 7, 2015. Chinese women with polycystic ovary syndrome were randomized in a 1:1:1:1 ratio to 4 groups. Interventions: Active or control acupuncture administered twice a week for 30 minutes per treatment and clomiphene or placebo administered for 5 days per cycle, for up to 4 cycles. The active acupuncture group received deep needle insertion with combined manual and low-frequency electrical stimulation; the control acupuncture group received superficial needle insertion, no manual stimulation, and mock electricity. Main Outcomes and Measures: The primary outcome was live birth. Secondary outcomes included adverse events. Results: Among the 1000 randomized women (mean [SD] age, 27.9 [3.3] years; mean [SD] body mass index, 24.2 [4.3]), 250 were randomized to each group; a total of 926 women (92.6%) completed the trial. Live births occurred in 69 of 235 women (29.4%) in the active acupuncture plus clomiphene group, 66 of 236 (28.0%) in the control acupuncture plus clomiphene group, 31 of 223 (13.9%) in the active acupuncture plus placebo group, and 39 of 232 (16.8%) in the control acupuncture plus placebo group. There was no significant interaction between active acupuncture and clomiphene (P = .39), so main effects were evaluated. The live birth rate was significantly higher in the women treated with clomiphene than with placebo (135 of 471 [28.7%] vs 70 of 455 [15.4%], respectively; difference, 13.3%; 95% CI, 8.0% to 18.5%) and not significantly different between women treated with active vs control acupuncture (100 of 458 [21.8%] vs 105 of 468 [22.4%], respectively; difference, -0.6%; 95% CI, -5.9% to 4.7%). Diarrhea and bruising were more common in patients receiving active acupuncture than control acupuncture (diarrhea: 25 of 500 [5.0%] vs 8 of 500 [1.6%], respectively; difference, 3.4%; 95% CI, 1.2% to 5.6%; bruising: 37 of 500 [7.4%] vs 9 of 500 [1.8%], respectively; difference, 5.6%; 95% CI, 3.0% to 8.2%). Conclusions and Relevance: Among Chinese women with polycystic ovary syndrome, the use of acupuncture with or without clomiphene, compared with control acupuncture and placebo, did not increase live births. This finding does not support acupuncture as an infertility treatment in such women. Trial Registration: clinicaltrials.gov Identifier: NCT01573858.

A novel Lactobacillus plantarum strain P-8 activates beneficial immune response of broiler chickens.

To investigate whether Lactobacillus plantarum P-8 may be used as an alternative to antibiotics in the broiler chicken diet, we compared P-8 and antibiotics for their immunobiotic properties and their effect on growth performance of broiler chickens in a 42-day trial. The results showed that P-8 provided similar benefits in weight gain, feed intake and feed efficiency as antibiotics did. Importantly, P-8 activated protective immune responses of the broilers while antibiotics lacked this effect. P-8 induced higher fecal secretory IgA (sIgA) levels on day 42 (P≤0.027) and IgA(+) lymphocytes in the jejunum and Peyer's patches (PP) (P<0.001) compared to antibiotic treatment. Antibiotics reduced the IgA(+) lymphocytes in jejunum and PP on day 42 compared to the control. P-8 increased CD3(+) T cells in the small intestinal tissues in most test situations whereas antibiotics had fewer CD3(+) cells in PP and cecal tonsil compared with the control broilers at the end of the trial. In addition, P-8 increased CD4(+) T cells significantly in the intestinal tissues compared to both antibiotics and the control (P<0.0052). Both Th1 and Th2 cytokine expression were enhanced by P-8 on day 14, consistent with the clinical trial results showing probiotic benefits in diseases. Antibiotics up- and down-regulated interleukin (IL)-2, IL-4 and IL-10 transcripts in an age-dependent manner, and showed anti-inflammatory potential. These data indicate that P-8 may provide protective immune response to broilers while maintaining similar growth performance and may be a potential alternative to antibiotics supplemented in chicken feeds.