Lipophilic Statins Eliminate Senescent Endothelial Cells by inducing Anoikis-Related Cell Death.

Pre-clinical studies from the recent past have indicated that senescent cells can negatively affect health and contribute to premature aging. Targeted eradication of these cells has been shown to improve the health of aged experimental animals, leading to a clinical interest in finding compounds that selectively eliminate senescent cells while sparing non-senescent ones. In our study, we identified a senolytic capacity of statins, which are lipid-lowering drugs prescribed to patients at high risk of cardiovascular events. Using two different models of senescence in human vascular endothelial cells (HUVECs), we found that statins preferentially eliminated senescent cells, while leaving non-senescent cells unharmed. We observed that the senolytic effect of statins could be negated with the co-administration of mevalonic acid and that statins induced cell detachment leading to anoikis-like apoptosis, as evidenced by real-time visualization of caspase-3/7 activation. Our findings suggest that statins possess a senolytic property, possibly also contributing to their described beneficial cardiovascular effects. Further studies are needed to explore the potential of short-term, high-dose statin treatment as a candidate senolytic therapy.

The effect of micronutrient supplementation on serum anti-Mullerian hormone levels: a retrospective pilot study.

BACKGROUND: The anti-Müllerian-hormone (AMH) is secreted by the granulosa cells of the oocytes and can be used as a marker of the ovarian reserve; helpful to estimate female fertility or the menopause onset. Although various factors may influence AMH levels, the correlation with nutritional factors needs more research. OBJECTIVE: To evaluate the effect of a micronutrient supplementation on female AMH levels. METHODS: This retrospective analysis includes a total of 244 women, who attended the Karl Landsteiner Institute, Korneuburg, Austria from January 2013 to June 2019 due to an unfulfilled desire for a child. All women were treated with an oral micronutrient preparation consisting the dosage of one soft capsule and one tablet per day for 3 months. The soft capsule contains omega-3 fatty acids and the tablet is a standardized combination of coenzyme Q10, vitamin E, folic acid, selenium, catechins from green tea extract, and glycyrrhizin from licorice extract. Serum AMH levels before and after 3 months were compared. In addition, available clinical data such as ovulation frequency, endometrium thickness, and luteal phase duration were analyzed. RESULTS: The mean age of the women was 37.3 ± 1.8 years, the mean body mass index of 24.3 ± 4.6 k/m2. The mean serum AMH levels and endometrial thickness values were significantly higher after micronutrient supplementation as compared to baseline (1.42 ± 0.86 versus 1.86 ± 0.82 ng/mL and 6.10 ± 1.76 versus 7.29 ± 1.65 mm, respectively). In addition, ovulation frequency and luteal phase duration significantly improved in more than 60%. CONCLUSION: Proposed micronutrient supplementation had a positive effect on serum AMH levels, endometrial thickness, ovulation frequency, and luteal phase duration. It could be a simple, risk-free therapeutic option to improve female fertility. More research is warranted to prove this effect.

The effect of micronutrient supplementation on spermatozoa DNA integrity in subfertile men and subsequent pregnancy rate.

BACKGROUND: Male infertility has been related to an increased sperm DNA fragmentation index (DFI). Nutritional factors may improve sperm nuclear DNA integrity and thus pregnancy rates. Objective: To evaluate the effect of micronutrient supplementation on sperm DNA integrity in subfertile men and subsequent pregnancy rates. METHODS: In this retrospective comparative study 339 subfertile males were included on whom a sperm chromatin dispersion test (SCD) was performed as a method to detect DNA fragmentation, as well as an initial semen analysis. Of all, n = 162 received a nutritional management program for three months, consisting of two daily capsules of a standardized combined micronutrient formulation together with a guidance to diet modification and to lifestyle changes (study group). Each capsule contained L-carnitine, L-arginine, coenzyme Q10, zinc, vitamin E, folic acid, glutathione, and selenium. The control group consisted of those patients who did not receive the active treatment (n = 177), yet were instructed to engage in a healthy lifestyle, including a modification of their regular diet. The SCD test was repeated for both groups after three months. As part of the routine follow up, pregnancy rate was assessed six months after the second SCD test. Males with complete follow up and healthy female partners (aged 18 to 40 years) where included. RESULTS: Data of men with an initial mean DFI of >15% were analyzed first (n = 81;46 study and 35 control patients). After three months, both groups displayed a significant decrease of mean DFI values; however, the mean percent difference was higher in the study group (10.46 ± 1.20 % vs. 5.29 ± 0.57 %; p < .001). Then, the entire population was considered (n = 339). After three months, only the study group displayed a significant decrease of mean DFI initial values (10.48 ± 7.76 % to 6.51 ± 4.61%; p < .001); and the percent difference was higher in the study group (3.97 ± 0.28 % vs. 0.91 ± 0.28 %; p < .001). At six months follow-up, pregnancy rate was higher in the study group (27.78% vs. 15.25%, p = .002). CONCLUSION: Both regimes significantly reduced sperm DNA fragmentation among subfertile men with a DFI >15%; however, when any baseline DFI value was considered, only micronutrient supplementation achieved a better result on DFI and thus pregnancy rate was higher.

[Case Report: Heterotopic Retransplantation of Cryopreserved Ovarian Tissue after Adenocarcinoma of the Uterine Cervix]. / Fallbericht: heterotope Retransplantation von kryokonserviertem Ovarialgewebe nach Adenokarzinom der Cervix uteri.

Introduction: Retransplantation of cryopreserved ovarian tissue has become an established method of restoring autologous hormone production and fertility after radiotherapy and/or chemotherapy for underlying oncological disease in women of reproductive age and has so far led to more than 170 births worldwide. Case presentation and course: In 2013, the 31-year-old patient developed adenocarcinoma of the uterine cervix, pT1b1V0L0. In January 2014, an extended hysterectomy with lymph node dissection and bilateral adnexectomy were performed. At the patient's request, ovarian tissue was cryopreserved 2 days previously. In November 2019, the retransplantation of two ovarian tissue pieces along the brachial fascia of the left forearm was performed, with no recurrence for 5 years under ongoing hormone replacement therapy (HRT). At 1 month following retransplantation, the patient stopped taking HRT, and 3 months later proper function of the retransplanted tissue could be demonstrated by checking gonadotropins and E2 levels. There was a clear swelling in the area of the retransplantation site, and three vital follicles could be visualized during an ultrasound examination in May 2020. Conclusion: This is the first successful retransplantation of cryopreserved ovarian tissue to restore autologous hormone production in a cervical cancer patient in Austria. Based on blood, ultrasound and cytological examinations, not to mention the patient's personal well-being, functionality of the retransplanted tissue could be demonstrated even at 6 months after the procedure. Finally, the authors would like to highlight the importance of informing and consulting young patients with tumor diseases on the various possibilities of fertility preservation.

Premature senescence of endothelial cells upon chronic exposure to TNFα can be prevented by N-acetyl cysteine and plumericin.

Cellular senescence is characterized by a permanent cell-cycle arrest and a pro-inflammatory secretory phenotype, and can be induced by a variety of stimuli, including ionizing radiation, oxidative stress, and inflammation. In endothelial cells, this phenomenon might contribute to vascular disease. Plasma levels of the inflammatory cytokine tumor necrosis factor alpha (TNFα) are increased in age-related and chronic conditions such as atherosclerosis, rheumatoid arthritis, psoriasis, and Crohn's disease. Although TNFα is a known activator of the central inflammatory mediator NF-κB, and can induce the intracellular generation of reactive oxygen species (ROS), the question whether TNFα can induce senescence has not been answered conclusively. Here, we investigated the effect of prolonged TNFα exposure on the fate of endothelial cells and found that such treatment induced premature senescence. Induction of endothelial senescence was prevented by the anti-oxidant N-acetyl cysteine, as well as by plumericin and PHA-408, inhibitors of the NF-κB pathway. Our results indicated that prolonged TNFα exposure could have detrimental consequences to endothelial cells by causing senescence and, therefore, chronically increased TNFα levels might possibly contribute to the pathology of chronic inflammatory diseases by driving premature endothelial senescence.

Comparison of the effect of a combination of eight micronutrients versus a standard mono preparation on sperm parameters.

BACKGROUND: There are reports showing that l-carnitine alone or in combination with other micronutrients improve sperm parameters. However, comparative studies are still lacking. This study was carried out to compare the short term effects of a combination of eight micronutrients including l-carnitine vs. a mono-substance (l-carnitine alone) on sperm parameters. METHODS: This was a prospective, open-labelled, nonrandomized study that included male subjects (20 to 60 years) with at least 1 year of subfertility and at least one pathological semen analysis who received 3 months treatment with a mono-substance (500 mg l-carnitine/twice a day, n = 156) or a combined compound (440 mg l-carnitine + 250 mg l-arginine + 40 mg zinc + 120 mg vitamin E + 80 mg glutathione + 60 µg selenium + 15 mg coenzyme Q10 + 800 µg folic acid/once a day, n = 143) for the same time period. Sperm parameters were analyzed before and after treatment and groups comparisons performed. RESULTS: Baseline characteristics were similar among studied groups (age and body mass indices). Semen parameters (volume, density, overall progressive motility [including slow and fast motility]) and percentage of sperm with normal morphology improved after 3 months in both groups as compared to baseline. However, relative change (expressed as % increase of absolute values) for sperm density and overall progressive motility (including fast motility) was found to be higher for the combined micronutrient treatment group as compared to the mono-treatment using l-carnitine alone. CONCLUSION: Both analyzed groups displayed a positive short term effect on all sperm parameters; however effect on density and motility was significantly better for the combined formulation. There is need for more research in this matter that includes long term outcome data. TRIAL REGISTRATION: Retrospectively registered at ISRCTN (7th October 2016). Study ID: ISRCTN48594239.

Red clover isoflavone metabolite bioavailability is decreased after fructooligosaccharide supplementation.

BACKGROUND: Red clover is an important source of isoflavones; which has been made commercially available as dietary supplements for the treatment of menopausal symptoms. Bioavailability and metabolism of these red clover isoflavones (RCI) have not been studied in detail. Fructooligosaccharides (FOS) stimulate the growth of intestinal bacteria and play an important role in the formation of certain isoflavone metabolites, such as equol and O-desmethylangolensin. OBJECTIVE: To determine the bioavailability of RCI metabolites and analyse whether FOS supplementation could influence their bioavailability. METHODS: Seventeen healthy adults were enrolled in the study carried out in two periods. In the first, compound bioavailability was determined after consumption of 80 mg of RCI (MF11RCE). In the second, a 6-day supplementation of 2×3000 mg/day of FOS was administered before isoflavone consumption. RESULTS: Biochanin A and formononetin were rapidly absorbed and both reached maximum concentrations at an average of 5-7h. Demethylation was a major reaction in the metabolic pathway. Daidzein serum level peaked after about 12.6h. Supplementation with FOS led to a significant decrease in the bioavailability of daidzein, dihydroformononetin, dihydrogenistein and dihydrodaidzein. An increase in equol production was also observed which did not reach statistical significance (p>0.05). CONCLUSION: This study is the first to provide detailed data on RCI bioavailability in humans and determine no influence of FOS yet a trend toward increased equol production. More research is warranted involving a greater sample size.

The effect of red clover isoflavone supplementation over vasomotor and menopausal symptoms in postmenopausal women.

OBJECTIVE: To evaluate the effect of red clover isoflavone supplementation over vasomotor and overall menopausal symptoms in postmenopausal women. METHODS: One hundred and nine postmenopausal women aged 40 or more were assigned to randomly receive either two daily capsules of the active compound (80 mg red clover isoflavones, Group A) or placebo of equal appearance (Group B) for a 90-day period. After a washout period of 7 days, medication was crossed over and taken for 90 days more. Daily hot flush and night sweat frequency and overall menopausal symptom intensity (Kupperman Index) were measured at baseline, 90, 97 and 187 days. RESULTS: Daily hot flush/night sweat frequency and Kupperman Index values were similar in both studied groups at baseline. All indices significantly decreased after red clover phase in Group A, corresponding, respectively to a 73.5%, 72.2% and 75.4% average decrement. These decrements were significantly higher than those observed for Group B after placebo phase (8.2%, 0.9% and 6.7% respectively). In Group A, after washout and placebo phases all values significantly increased. In Group B, all indices remained similar after placebo and washout phases, however significantly dropping after red clover treatment. These values were also significantly lower than those observed in Group A after placebo phase. No side effects were encountered after treatment with the active compound or placebo. CONCLUSION: Red clover isoflavone supplementation was more effective than placebo in reducing daily vasomotor frequency and overall menopausal intensity in postmenopausal women.

Effect of Red Clover Isoflavones over Skin, Appendages, and Mucosal Status in Postmenopausal Women.

Objective. Evaluate in postmenopausal women the effect of red clover extract (RCE) isoflavones over subjective status of skin, appendages, and several mucosal sites. Method. Postmenopausal women (n = 109) were randomly assigned to receive either two daily capsules of the active compound (80 mg RCE, Group A) or placebo of equal appearance (Group B) for a 90-day period. After a washout period of 7 days, medication was crossed over and taken for 90 days more. Subjective improvement of skin, appendages, and several mucosal site status was assessed for each studied group at 90 and 187 days using a visual analogue scale (VAS). In addition, libido, tiredness, and urinary, sleep, and mood complaints were also evaluated. Results. Women after RCE intervention (both groups) reported better subjective improvement of scalp hair and skin status, libido, mood, sleep, and tiredness. Improvement of urinary complaints, nail, body hair, and mucosa (oral, nasal, and ocular) status did not differ between treatment phases (intra- and intergroup). Overall satisfaction with treatment was reported higher after RCE intervention (both groups) as compared to placebo. Conclusion. RCE supplementation exerted a subject improvement of scalp hair and skin status as well as libido, mood, sleep, and tiredness in postmenopausal women.

Laparoscopic ovarian tissue harvesting for cryopreservation: an effective and safe procedure for fertility preservation.

OBJECTIVE: To critically review our experience with the method of laparoscopic ovarian tissue harvesting for ovarian cryopreservation. STUDY DESIGN: In a retrospective cohort study 85 patients undergoing surgery for cryopreservation of ovarian tissue were included. One random ovarian cortical slice was histologically examined in order to determine the presence of primordial follicles and to detect possible malignant cells. Selective microbiological culture techniques from swabs were taken from all slices immediately after excision. Intra- and postoperative courses, histological and microbiological findings were evaluated. RESULTS: Eighty-five patients underwent cryopreservation of ovarian tissue, mostly for malignant diseases (78/85, 91.8%). Sixty patients (70.6%) underwent laparoscopy for ovarian tissue harvesting only, without any additional surgical procedure. The median operating time was 30 min (range 10-75 min). The intraoperative course was uneventful in these patients. In two patients slight postoperative increases in C-reactive protein levels were found. Microbiological examination revealed no contamination apart from one case revealing sporadic Propionibacterium acnes. Histological examination revealed intact ovarian tissue with primordial follicles in 81/85 patients (95.3%). CONCLUSION: Laparoscopy is a safe and effective procedure for ovarian tissue harvesting. We suggest microbiological and histological testing of ovarian tissue as mandatory tools to guarantee safety regarding ovarian tissue transplantation.

Improvement of postmenopausal depressive and anxiety symptoms after treatment with isoflavones derived from red clover extracts.

OBJECTIVE: To evaluate the effect of isoflavones derived from red clover extracts (MF11RCE) over anxiety and depressive symptoms among postmenopausal women. METHODS: One hundred and nine postmenopausal women aged 40 or more were randomly assigned to receive two daily capsules of MF11RCE (80mg red clover isoflavones, Group A) or placebo of equal appearance (Group B) for a 90-day period. After a washout period of 7 days, medication was crossed over and taken for 90 days more. Anxiety and depressive symptoms were measured at baseline, 90 and 187 days with the Hospital Anxiety and Depression Scale (HADS) and Zung's Self Rating Depression Scale (SDS). RESULTS: After receiving the MF11RCE compound the total HADS (anxiety and depression subscale scores also) and the total SDS scores decreased significantly. This effect was equivalent to a 76.9% reduction in the total HADS score (76% for anxiety and 78.3% for depression) and an 80.6% reduction in the total SDS score. After placebo, total HADS (anxiety and depression subscale also) and total SDS scores also decreased significantly in comparison to baseline but only equivalent to an average 21.7% decline. CONCLUSION: Red clover derived isoflavones (MF11RCE) were effective in reducing depressive and anxiety symptoms among postmenopausal women.

Ovarian tissue cryopreservation for non-malignant indications.

BACKGROUND: Ovarian tissue cryopreservation (OTC) is not restricted to patients suffering from malignancies. Benign haematopoietic diseases such as autoimmune diseases and others have been mentioned to be feasible indications. The aim of our study was to demonstrate that surgery for OTC is a safe procedure for fertility preservation in patients with non-malignant diseases. METHODS: In a retrospective cohort study intra- and postoperative courses were evaluated. Histological examination of one random ovarian cortical slice was performed to determine the presence of primordial follicles. Immediately after the excision, microbiological culture techniques from swabs were taken from all slices. RESULTS: Seven of 85 patients (8.2%) have undergone surgery for OTC for non-malignant indications. All patients had an uncomplicated intraoperative course. A median number of 12 cortical stripes were excised and cryopreserved (range 7-17). Only for one patient an adverse event was found which was not related to laparoscopy for OTC. All patients having undergone routine laparoscopy for OTC only could be released from hospital on the first postoperative day. Selective microbiological tests showed negative results in all cases. Histological examinations revealed intact ovarian tissue with primordial follicles in 6/7 patients. CONCLUSION: Whether surgery for OTC in patients with non-malignant indications is feasible from an ethical point of view remains open. However, laparoscopy for ovarian tissue harvesting is a safe procedure for patients undergoing cytotoxic treatment for benign diseases.

Immunological and ultrastructural characterization of endothelial cell cultures differentiated from human cord blood derived endothelial progenitor cells.

The replacement of endothelium by endothelial progenitor cells (EPCs) for therapeutic use in order to ameliorate the vascular status of ischemic organs is now in the focus of vascular research. The aim of our studies was to investigate whether EPCs derived from peripheral blood mononuclear cells (PBMNCs-derived EPCs) or EPCs propagated from CD34(+) hematopoietic stem cells (HSCs-derived EPCs), both isolated from human cord blood, are able to differentiate into early mature endothelial cells (ECs) under certain in vitro conditions. We characterized both cell populations by flow cytometry, phase contrast microscopy, fluorescence microscopy and confocal laser scanning microscopy as well as ultrastructurally using transmission and scanning electron microscopy. While PBMNCs gave rise to clusters of spindle-like EPCs after few days but did not further mature under in vitro conditions, mature ECs could only be successfully propagated from a starting population of isolated HSCs. Both, PBMNCs- and HSCs-derived EPCs, took up Dil-labeled acetylated low density lipoprotein (Dil-Ac-LDL) and could be positively stained for CD31, CD105, the vascular endothelial growth factor receptor 2 (VEGFR-2, KDR) and ulex europaeus agglutinin 1 (UEA-1) at the cell surface. EPC showed surface expression of CD54 and CD106. However, only a small portion of HSCs-derived EPCs was positive for CD54 but negative for CD106. Intracellular staining for von Willebrand factor (vWF) provided a homogenous stain in PBMNC-derived EPCs while in HSCs-derived EPCs, during cultivation for 2-3 weeks, more and more a typical punctuated staining pattern related to Weibel-Palade bodies (WPBs) was visible. By phase contrast and scanning electron microscopy, an arrangement of PBMNCs-derived EPCs in cord-like structures could be demonstrated. In these formations, cells showed parallel alignment but exhibited only few cell contacts. Well-developed WPBs could never be found in PBMNCs-derived EPCs. In contrast, differentiating HSCs-derived EPCs developed adherence junctions, interdigitating junctions as well as syndesmos. During maturation, spindle-like cell types appeared with abundant WPBs as well as cobblestone-like cell types with a fewer content of these organelles. WPBs, in the spindle-like cell types displayed conspicuous shapes and were concentrated in close proximity to mitochondria-rich areas. HSCs-derived EPCs exhibited signs of high synthetic activity such as a well-developed rough endoplasmic reticulum (RER) and multiple Golgi complexes. In the trans-Golgi network (TGN), close to the Golgi complex, a new formation of WPBs could be observed. These morphological features correlated well with a high growing capacity. Although it was not possible to demonstrate the complete differentiation line from HSCs to early matured ECs by immunologic markers because of the limited number of cells available for such investigations, distinct morphologic maturation stages could be shown at light and electron microscopical levels. In conclusion, the study presented here characterizes not only the different cell populations involved in the differentiation of early EPCs into mature ECs but also the transition stage where the maturation step takes place by demonstration of the new formation of WPBs. In this respect, these investigations provide new insights into the in vitro differentiation which could have some in vivo correlation.

Orthotopic microvascular reanastomosis of whole cryopreserved ovine ovaries resulting in pregnancy and live birth.

OBJECTIVE: To evaluate the feasibility of contralateral autotransplantation of cryopreserved whole ovaries with microanastomosis of the ovarian vascular pedicle. DESIGN: Animal study. SETTING: Department of Biomedical Sciences, General Hospital of Vienna, Austria. ANIMAL(S): Nine ewes, six month of age. INTERVENTION(S): Laparotomic unilateral oophorectomy was performed. Ovaries were frozen using a controlled-rate freezing system. After frozen storage, contralateral laparotomic oophorectomy was performed, and the thawed ovaries were returned to the contralateral orthotopic site with microsurgical vascular anastomosis. MAIN OUTCOME MEASURE(S): Histologic examination and serum follicle-stimulating hormone and progesterone levels. RESULT(S): Four sheep showed postoperative luteal function. One sheep conceived after spontaneous intercourse and delivered a healthy lamb 545 days after transplantation. Histologic examination of the ovaries 18-19 months after transplantation showed that the structural integrity of the ovarian stroma had largely been retained in six out of nine animals. Follicular survival rate in the grafted ovaries was 1.7%-7.6%. CONCLUSION(S): Microvascular anastomosis of whole ovaries and orthotopic transplantation after cryopreservation is technically feasible and a promising procedure in ovarian tissue banking.

Effects of a red clover extract (MF11RCE) on endometrium and sex hormones in postmenopausal women.

OBJECTIVE: To evaluate the effects of a non-prescription red clover extract (MF11 RCE, Melbrosin International, Vienna, Austria) on selected sex hormones and endometrium in postmenopausal women. PATIENTS AND METHODS: One-hundred and nine postmenopausal women with an age > or =40 years were randomly assigned to one of two groups either two capsules of MF11RCE (80mg isoflavone) per day for a 90 day period, or placebo of equal design. After a 7 day washout period, medication was crossed-over for another 90 days. RESULTS: Combined evaluation demonstrated that supplementation with MF11RCE (verum), in contrast to placebo, significantly increased plasma testosterone levels and decreased endometrial thickness. CONCLUSION: MF11RCE exerts a moderate effect on testosterone levels in postmenopausal women, while estradiol levels remained unchanged. The observed reduction of endometrial thickness provides further support for a safe role for isoflavones in terms of endometrial hyperplasia.