Dried Ginger Extract Restores the T Helper Type 1/T Helper Type 2 Balance and Antibody Production in Cyclophosphamide-Induced Immunocompromised Mice after Flu Vaccination.

Dried ginger (Zingiberis Processum Rhizoma (ZR)) is frequently used to prevent or treat common cold and flu. This study aimed to investigate the influence of ZR extracts on influenza-specific antibody production in cyclophosphamide (Cy)-induced immunocompromised mice. Female BALB/c mice were injected three times with saline or Cy. To investigate the effect of ZR, either distilled water or ZR was administered orally to mice daily for 10 days after Cy injection. After ZR administration, the mice were immunized with the 2017/2018 influenza vaccine. Pretreatment with ZR extracts enhanced influenza-specific antibody production in Cy-induced immunocompromised mice after flu vaccination and restored the influenza antigen-specific T helper (Th) type 1/Th2 balance to the normal state. Further, ZR suppressed the eosinophil enrichment caused by Cy injection in the spleen. We demonstrated that ZR can be used to increase antibody production in immunocompromised individuals before vaccination.

Acupuncture for Poor Ovarian Response: A Randomized Controlled Trial.

Acupuncture is believed to improve ovarian reserve and reproductive outcomes in women undergoing in vitro fertilization (IVF). This study was conducted to evaluate the effect of network-optimized acupuncture followed by IVF on the oocyte yield in women showing a poor ovarian response. This study was an exploratory randomized controlled trial conducted from June 2017 to January 2020 at the Pusan National University Hospital. Women diagnosed with poor ovarian response were enrolled and randomly divided into two groups: IVF alone and Ac + IVF groups (16 acupuncture sessions before IVF treatment). Eight acupoints with high degree centrality and betweenness centrality were selected using network analysis. Among the participants, compared with the IVF treatment alone, the acupuncture + IVF treatment significantly increased the number of retrieved mature oocytes in women aged more than 37 years and in those undergoing more than one controlled ovarian hyperstimulation cycle. The negative correlation between the number of retrieved mature oocytes and consecutive controlled ovarian hyperstimulation cycles was not observed in the Ac + IVF group irrespective of the maternal age. These findings suggest that physicians can consider acupuncture for the treatment of women with poor ovarian response and aged > 37 years or undergoing multiple IVF cycles.

Effect of samul-tang on female fertility via RAS signaling pathway in ovaries of aged mice.

Samul-tang (SM), a traditional herbal medicine, is used to treat age-related human conditions, such as infertility and menstrual irregularities. The mechanism underlying the role of SM in ovary function needs elucidation. In this study, the influence of SM administration on the ovarian reserve of aged mice was investigated. Female BALB/c mice (8 and 40 weeks-old) were administered with distilled water (young or old group) or SM for 4 weeks. SM administration prevented age-related ovarian follicle loss in mice. Quality of oocytes and blastocysts were enhanced in SM-administrated mice compared to those of non-treated old mice. Further, SM administration increased the pregnancy rate and number of litters. SM triggered changes in aging-related genes that are linked to the RAS-mediated pathway. Thus, we demonstrate that SM can be used to increase the oocyte yield in aged women, potentially improving age-related cognitive decline in the ovarian reserve.

Samul-tang ameliorates oocyte damage due to cyclophosphamide-induced chronic ovarian dysfunction in mice.

Samul-tang (SM), a traditional herbal medicine, has been used to treat menstrual irregularities and infertility in women. However, the cellular and molecular mechanisms underlying the effects of SM remain elusive. We investigated the potential protective effect of SM against chronic ovarian dysfunction and used bioinformatics analysis to identify its underlying mechanism in a mouse model of cyclophosphamide (CP)-induced diminished ovarian reserve. Female C57BL/6 mice were intraperitoneally injected with CP three times a week, followed by oral administration of distilled water (CP group) or SM (CP + SM group) for 4 weeks. Four weeks later, the effect of SM was assessed by ovarian tissue histological analysis, steroid hormone measurement, oocyte quality, and mRNA and microRNA microarray analysis in the ovaries. Although SM administration did not prevent CP-induced follicle loss in mice, the quality of oocytes was better in CP + SM mice than in CP mice. Gene expression analysis revealed that the expression of fertilisation- and ovarian follicle development-related genes was altered by CP treatment but normalized after SM administration. Further bioinformatics analysis showed possible interactions between differentially expressed mRNAs and microRNAs. Therefore, we demonstrated the protective effects of SM on ovarian function and oocyte maturation against CP-induced damage via multiple epigenetic mechanisms.

Current clinical practice status of Korean medicine for managing female infertility: A cross-sectional survey.

BACKGROUND: This survey aimed to investigate clinicians' perceptions and current practice patterns of Korean medicine (KM) treatment for female infertility. METHODS: A questionnaire on clinical practice patterns of KM treatment for female infertility was constructed and distributed to 703 KM doctors (KMDs). RESULTS: A total of 20.5% of physicians (n = 144/703) completed the survey and delivered integrative care for female infertility patients. Integrative care mainly consisted of acupuncture, herbal medicine, and moxibustion. The participants largely relied on radiological findings on ultrasonography and hormone tests for diagnosis. The most frequently prescribed herbal medication was Jogyeongjongok-tang (Tiaojing Zhongyu decoction), and the most frequently applied acupoints were SP6, CV4 and ST36. CONCLUSIONS: This study provides expert opinions and information on actual clinical practice patterns of KM for treating female infertility. However, we cannot completely discount the possibility that biased selection of subjects and the low response rate limit the interpretations of the study results.

Acupuncture for in vitro fertilization in women with poor ovarian response: a systematic review.

BACKGROUND: Poor ovarian response (POR) is one reason for infertility. In vitro fertilization (IVF) is frequently used to help achieve pregnancy, and performing acupuncture before IVF may promote ovulation and reduce egg retrieval pain. The purpose of this systematic review was to evaluate the effectiveness of acupuncture on clinical pregnancy rates (CPR) after IVF in women with POR. METHODS: Eight electronic databases were searched in January 2020, and reference lists of retrieved articles and previous review articles were hand-searched. Randomized controlled trials (RCTs) using any type of acupuncture for women with POR undergoing IVF were considered. Risk of bias was assessed using the Cochrane risk of bias standards. RESULTS: Three RCTs were included in this review. CPR and the number of retrieved oocytes were measured in two studies, while the values of anti-Mullerian hormone (AMH) and antral follicle count (AFC) were only reported in one study. In two studies, CPR was higher in the intervention group than the control group [37.8 % vs 24.3 %]. We did not conduct a meta-analysis, as there was a high level of heterogeneity in interventions among the included trials. CONCLUSIONS: This study suggests that acupuncture may improve CPR, AMH, AFC and the number of retrieved oocytes in women with POR undergoing IVF. However it is difficult to conclude that acupuncture is more effective than conventional treatment. Additionally, more clinical trials are needed to evaluate the effectiveness of acupuncture on CPR and other outcomes of POR. STUDY REGISTRATION: PROSPERO CRD42018087813; https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42018087813.

Korean medicines for poor ovarian reserve in infertility: A protocol for a multicenter observational study.

INTRODUCTION: Many infertile patients have used Korean medicines (KMs) as a primary or adjuvant therapeutic method to improve in vitro fertilization success rates. The aim of this multicenter observational study is to investigate the effects of KMs on poor ovarian reserve (POR) in infertile patients. METHODS AND ANALYSIS: We will perform a prospective multicenter observational study. This study will recruit 50 women between 25 and 44 years of age with infertility caused by POR from among patients who visit the KM clinic. All participants will visit the KM clinic on the 2nd or 3rd day of menstruation to receive the KMs. KM treatment will be delivered for 3 menstrual cycles (3 months) and will include herbal decoction, acupuncture, or moxibustion on demand. Every participant will be assessed based on KM clinical symptoms, a quality of life questionnaire, and ovarian reserve test results. ETHICS AND DISSEMINATION: The study was approved by Institutional Review Board of Semyung University (SMU-IM-190501). The results will be published in a peer-reviewed journal and will be disseminated electronically and in print. TRIAL REGISTRATION NUMBER: Clinical Research Information Service: KCT0004209.

Four Subgroups of Blood Stasis Syndrome Are Identified by Manifestation Cluster Analysis in Males.

Blood stasis syndrome (BSS) is an important pathological condition in traditional East Asian medicine and is associated with ischemic heart disease, cerebral vascular accident, diabetes mellitus, chronic renal failure, severe traumatic injury, and dysmenorrhea. However, previous studies have been unable to reveal the clinical and biological characteristics or biological markers of BSS. We hypothesized that the heterogeneity among the manifestations of BSS or non-BSS could interfere with an analysis to describe the characteristics of BSS. In this study, male participants based on the severity of BSS-associated symptoms and signs were clustered and classified into four subgroups: BSS subgroups (1), (2), (3), and (4). Non-BSS core subgroup was redefined using manifestation cluster analysis. Biological characteristics of subgroups BSS(1) and BSS(2) belong to the range of the non-BSS core subgroup (1), whereas that of subgroups BSS(3) and BSS(4) are characterized by different biological parameters such as systemic inflammatory conditions and elevated D-dimer level. Our results suggested that patients in subgroups of BSS(3) and BSS(4) are more likely to be exposed in an inflammatory state than other BSS subgroups. We found the heterogeneity among the manifestations which could mask the characteristics of BSS and identified the clinical and biological profiles of the four BSS subgroups through comparisons of the redefined non-BSS and BSS subgroups. This finding could provide accurate diagnostic criteria and new approaches for BSS treatments in different subgroups.

A systematic review of herbal medicines for the treatment of cancer cachexia in animal models.

OBJECTIVE: The aim of this study is to summarize preclinical studies on herbal medicines used to treat cancer cachexia and its underlying mechanisms. METHODS: We searched four representing databases, including PubMed, EMBASE, the Allied and Complementary Medicine Database, and the Web of Science up to December 2016. Randomized animal studies were included if the effects of any herbal medicine were tested on cancer cachexia. The methodological quality was evaluated by the Collaborative Approach to Meta-Analysis and Review of Animal Data from Experimental Studies (CAMARADE) checklist. RESULTS: A total of fourteen herbal medicines and their compounds were identified, including Coptidis Rhizoma, berberine, Bing De Ling, curcumin, Qing-Shu-Yi-Qi-Tang, Scutellaria baicalensis, Hochuekkito, Rikkunshito, hesperidin, atractylodin, Sipjeondaebo-tang, Sosiho-tang, Anemarrhena Rhizoma, and Phellodendri Cortex. All the herbal medicines, except curcumin, have been shown to ameliorate the symptoms of cancer cachexia through anti-inflammation, regulation of the neuroendocrine pathway, and modulation of the ubiquitin proteasome system or protein synthesis. CONCLUSIONS: This study showed that herbal medicines might be a useful approach for treating cancer cachexia. However, more detailed experimental studies on the molecular mechanisms and active compounds are needed.

A randomized, open phase IV exploratory clinical trial to evaluate the efficacy and safety of acupuncture on the outcome of induction of ovulation in women with poor ovarian response: A study protocol for a randomized controlled trial.

INTRODUCTION: Women with infertility who have a poor ovarian responder (POR), characterized by a low number of retrieved oocytes after ovulation induction, often have a significantly reduced pregnancy rate after in vitro fertilization-embryo transfer (IVF-ET), due to the few transferred embryos. Acupuncture is a form of Korean Traditional Medicine. It involves the insertion of a microscopic needle at a specific point in the body, known as an acupuncture point or an acupoint. In this study, our purpose is to investigate how acupuncture affects the retrieval of mature oocytes after ovulation induction in patients with POR. METHODS AND ANALYSIS: This study will be a randomized clinical trial comprising an IVF-ET trial and an IVF-ET trial after acupuncture. Seventy patients will by enrolled and randomly assigned to either of the 2 groups. The study subjects will be required to be diagnosed as having POR. Participants will be divided into 2 groups: IVF-ET single treatment group, and acupuncture and IVF-ET combined treatment group. The study subjects will be required to participate in a 15-week trial involving 16 acupuncture treatments over a period of approximately 2 months before ovulation induction for oocyte retrieval. The primary assessment of all participants will be comparing the number of oocytes. RESULT: This treatment will be a therapeutic model for POR. DISCUSSION: Our results will provide patients with POR as well as complementary and alternative medicine professionals, such as Korean medicine doctors, about the potential role of acupuncture in the treatment of POR. This will improve the quality of life in women with infertility and provide an important treatment option for patients with POR. Further studies can be performed to determine the optimal treatment for POR.

Paljung-San, a traditional herbal medicine, attenuates benign prostatic hyperplasia in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Paljung-san is a traditional herbal medicine used widely for the treatment of urogenital diseases in East Asia. However, scientific evidence of the efficacy of Paljung-san and its mechanisms of action against benign prostatic hyperplasia (BPH) is not clearly established. AIM OF THE STUDY: We investigated the inhibitory effect of Paljung-san water extract (PSWE) and its mechanisms against BPH in vitro and in vivo. MATERIALS AND METHODS: Active compounds of PSWE were analyzed quantitatively by High-performance liquid chromatography (HPLC). For in vitro study, PSWE treated BPH-1 cells were used to perform western blot analysis, cell cycle analysis and enzyme-linked immunosorbent assay. For in vivo BPH model, male rats were subcutaneously injected with 10 mg/kg of testosterone propionate (TP) every day for four weeks. 200 and 500 mg/kg of PSWE was administrated daily by oral gavage with s.c. injection of TP, respectively. RESULTS: HPLC revealed that PSWE contains 1.21, 1.18, 2.27, 3.56, 4.23, 3.00, 6.78, and 0.004 mg/g of gallic acid, 5-caffeoylquinic acid, chlorogenic acid, geniposide, liquiritin apioside, liquiritin, glycyrrhizin, and chrysophanol components, respectively. In human BPH-1 cells, PSWE treatment reduced cell proliferation through arresting the cell cycle in the DNA synthesis phase. Moreover, PSWE suppressed prostaglandin E2 production with reduced cyclooxygenase-2 expression. In TP -induced BPH rat model, PSWE administration showed reduced prostate weights and dihydrotestosterone levels and led to a restoration of normal prostate morphology. PSWE also decreased TP-induced Ki-67 and cyclin D1 protein levels in the prostatic tissues. Decreased glutathione reductase activity and increased malondialdehyde levels in the BPH groups were reversed by PSWE administration. CONCLUSION: PSWE attenuates the progression of BPH through anti-proliferative, anti-inflammatory and anti-oxidant activities in vitro and in vivo. Therefore, these data provide the scientific evidence of pharmacological efficacy of PSWE against BPH.

Anti-adipogenic effects of the traditional herbal formula Dohongsamul-tang in 3T3-L1 adipocytes.

BACKGROUND: Blood stasis syndrome (BSS) is a general pattern identification and refers to pathological stagnation of blood circulation, dysfunction of endothelial cells or metabolic disorder in traditional Korean medicine (TKM). Dohongsamul-Tang (DHSMT) is a well-known traditional herbal formula which used for treatment and prevention of BSS by promoting blood circulation in TKM. METHODS: Cytotoxicity of DHSMT was examined by cell counting kit-8 (CCK-8). We also investigated the anti-adipogenesis effect of DHSMT by using Oil Red O staining, intracellular triglyceride assay leptin ELISA and western blot analysis in 3T3-L1 adipocytes. In addition, the accumulation of adiponectin, resistin and plasminogen activator inhibitor-1 (PAI-1) were measured by magnetic bead panel kit. RESULTS: Oil Red O staining showed that DHSMT markedly reduced fat accumulation without affecting cell cytotoxicity. DHSMT also significantly decreased accumulation of triglyceride and adipokines such as leptin, adiponectin, resistin and PAI-1 compared with fully differentiated adipocytes. Furthermore, our results found that DHSMT significantly suppressed the adipocyte differentiation by downregulating adipogenic-specific transcriptional factors such as peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer binding proteins alpha (C/EBPα) and fatty acid binding protein 4 (FABP4) in adipocytes. CONCLUSIONS: Taken together, our findings provide that DHSMT has potential for treatment and prevention of obesity or MS related to BSS.

Gyejibongneyong-hwan, a herbal medicine for the treatment of dysmenorrhoea with uterine fibroids: a protocol for a randomised controlled trial.

INTRODUCTION: Gyejibongneyong-hwan (GBH), or the Guizhi Fuling Formula in Chinese, is widely used to treat uterine fibroids in East Asian countries including Korea, China and Japan. This study will assess the efficacy and safety of the GBH formula for the treatment of dysmenorrhoea. METHODS AND ANALYSIS: This study will be a randomised double-blind controlled trial with two parallel arms: the GBH group and the placebo group. This trial will recruit 38 women between 18 and 45 years of age with secondary dysmenorrhoea with uterine fibroids. The investigational drugs, either GBH or placebo, will be administered to the participants three times per day for two menstrual periods (8 weeks). The participants will be followed up for three menstrual cycles after administration of the drugs. The primary outcome will be the Numeric Rating Scale score of average menstrual pain. All analyses will be performed with SAS (V.9.1.3; SAS Institute, Cary, North Carolina, USA) by a statistician blinded to the allocation of the groups. Statistical analysis will be undertaken on the intent-to-treat (ITT) basis with a 95% CI using the last observation carried forward for missing values. The ITT analysis will include all randomised patients. ETHICS AND DISSEMINATION: This research protocol has been reviewed and approved by the institutional review boards of the trial centre (number WSOH IRB 1606-03). Written informed consent will be obtained from all study participants prior to enrolment in the study. The results will be published in a peer-reviewed journal and will be disseminated electronically and in print. TRIAL REGISTRATION NUMBER: KCT0001967.

The use of maca (Lepidium meyenii) to improve semen quality: A systematic review.

The aim of this review was to assess the evidence for the effectiveness of maca (Lepidium meyenii) in improving semen quality. We searched 11 databases from their inception to March 2016 and included all clinical trials on the improvement of semen quality parameters in infertile and healthy men, regardless of the study design or the type of maca. The risk of bias for each study was assessed using the Cochrane criteria. The selection of studies, data extraction, and validation were performed independently by the first two authors. Discrepancies were resolved through discussion by the same two authors. Five studies - 3 randomized clinical trials (RCTs) and 2 uncontrolled observational studies (UOSs) - met all of the inclusion criteria. One RCT found favorable effects of maca on sperm mobility in infertile men. The two other RCTs showed positive effects of maca on several semen quality parameters in healthy men. The two UOSs also suggested favorable effects of maca on semen quality. The results of our systematic review provide suggestive evidence for the effectiveness of maca in improving semen quality. However, the total number of trials, the total sample size, and the risk of bias of the included studies prevent the drawing firm conclusions. More rigorous studies are warranted.

Standardization and future directions in pattern identification research: International brainstorming session.

An international brainstorming session on standardizing pattern identification (PI) was held at the Korea Institute of Oriental Medicine on October 1, 2013 in Daejeon, South Korea. This brainstorming session was convened to gather insights from international traditional East Asian medicine specialists regarding PI standardization. With eight presentations and discussion sessions, the meeting allowed participants to discuss research methods and diagnostic systems used in traditional medicine for PI. One speaker presented a talk titled "The diagnostic criteria for blood stasis syndrome: implications for standardization of PI". Four speakers presented on future strategies and objective measurement tools that could be used in PI research. Later, participants shared information and methodology for accurate diagnosis and PI. They also discussed the necessity for standardizing PI and methods for international collaborations in pattern research.

Expert opinions on the concept of blood stasis in China: An interview study.

OBJECTIVE: To study various experts' opinions on the defifinition and diagnosis of blood stasis in China. METHODS: We e-mailed the selected experts to explain the purpose of the study and to invite them to participate and asked them to name a time for the investigator to call them. Eight experts and fifive organizations were interviewed in the research community investigating blood stasis in China. RESULTS: Six main categories emerged from the interviews: (1) blood stasis concepts; (2) blood stasis-related biomarkers; (3) methods of diagnosing blood stasis; (4) drugs for promoting blood circulation and dissipating stasis; (5) blood stasis-related diseases; and (6) blood stasis-related societies. The consensus among the interviewed experts was that the defifinition of blood stasis is rather complicated and that there is no gold standard marker for detecting blood stasis. CONCLUSIONS: This paper acquired experts' opinions on the defifinition and diagnosis of blood stasis in order to establish a modern concept of blood stasis. This paper also developed a diagnostic tool and diagnostic indices for blood stasis and identifified biological indices and pathologic mechanisms related to blood stasis, which might be of great reference value in future blood stasis standardization research.

Current research and future directions in pattern identification: Results of an international symposium.

A symposium on pattern identification (PI) was held at the Korea Institute of Oriental Medicine (KIOM) on October 2, 2013, in Daejeon, South Korea. This symposium was convened to provide information on the current research in PI as well as suggest future research directions. The participants discussed the nature of PI, possible research questions, strategies and future international collaborations in pattern research. With eight presentations and an extensive panel discussion, the symposium allowed participants to discuss research methods in traditional medicine for PI. One speaker presented the topic, 'Clinical pattern differentiation and contemporary research in PI.' Two speakers presented current trends in research on blood stasis while the remaining five other delegates discussed the research methods and future directions of PI research. The participants engaged in in-depth discussions regarding the nature of PI, potential research questions, strategies and future international collaborations in pattern research.

Accelerated RBC senescence as a novel pathologic mechanism of blood stasis syndrome in traditional East Asian medicine.

Blood stasis syndrome (BSS) is an important pathologic condition in traditional East Asian medicine, characterized by multiple signs and symptoms, including sublingual varicosis, angiotelectasis, slow and choppy pulse, local fixed pain, nyctalgia, menstrual cramps, dark-purple tongue and infra-orbital darkness. However, recent studies have been restricted to the circulatory disorder and could not suggest the pathologic core to explain all of the characteristics of BSS. Here, we review the current research on the senescence of red blood cells (RBCs), focusing on the correlation between the pathologic properties of senescent RBCs and BSS-specific manifestations. The accumulation of senescent RBCs and their products induce pathological conditions that affect blood flow resistance and cause thrombosis, vasoconstriction and methemoglobinemia. These pathological alterations are identical to the characteristics of BSS, therefore supporting the hypothesis that accelerated RBC aging could be considered as a novel pathologic mechanism of BSS.

Korean studies on blood stasis: an overview.

Blood stasis is one of the important pathological concepts in Korean medicine. We analyzed the Korean studies concerning blood stasis. We searched for articles in eight electronic databases from their inception to September, 2014. We included reviews, clinical studies, and preclinical studies that had studied blood stasis and excluded articles in which blood stasis was not mentioned or in which the original authors had not explained blood stasis. Of 211 total included studies, 19 were reviews, 52 were clinical studies, and 140 were preclinical articles. "Stagnant blood within the body" was the most frequently mentioned phrase of the traditional concept of blood stasis. Traumatic injury was the most frequently studied disease/condition in the clinical studies. In the preclinical studies, coagulopathy was studied most frequently, followed by hyperviscosity, hyperlipidemia, inflammation, neoplasm, ischemic brain injury, and atherosclerosis. Hyeolbuchukeo-tang and Angelicae Gigantis Radix were the most frequent formula and single herb, respectively, used in the blood stasis researches. The results showed that blood stasis was mainly recognized as disorder of circulation and many studies showed the effectiveness of activating blood circulating herbs for diseases and pathologies such as traumatic injury or coagulopathy. Further studies are needed in the pathologic mechanisms and various diseases of blood stasis.

Perception of pattern identification in traditional medicine: a survey of Korean medical practitioners.

OBJECTIVE: To survey assesses the perception of pattern identification (PI) diagnosis of Traditional Medicine (TM) by Korean medical doctors (KMDs). METHODS: A total of 14,485 KMDs affiliated with the Association of Korean Medicine were sent surveys via email, and 1646 (11.1%) responded to the questionnaire on their perception of PI diagnosis. RESULTS: Of the 1646 respondents, more than ninety percent (1562, 94.9%) reported that they treated patients using PI. The most critical problem with PI diagnosis was the lack of objective diagnostic indicators (561, 34.1%). Ninety percent had issues diagnosing patients because of different diagnoses between KMDs (1491, 90.5% ). The majority of respondents thought herbal medicine was most related to PI (1528, 92.8%). Half of the respondents answered that PI of Ba Gang was the most commonly used PI system. Participants reported that it was most important to study standardisation of PI diagnosis and to develop standardised PI diagnoses using the classification system of the Korean Standard Classification of Diseases. The foremost PI type that physicians thought should be included in standardisation and objectification of PI ofTKM was the PI of Ba Gang. CONCLUSION: Our data suggest that we should focus on the standardisation of PI diagnosis and PI of Ba Gang in future research on PI diagnosis in TM. However, we cannot completely discount the possibility that a biased selection of subjects and a low response rate limit the generalisability of the findings.