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Alcohol consumption is internationally recognized as one of the compelling risk factors for breast cancer, but it does not necessarily correlate with the prognosis of breast cancer patients. Alcohol consumption in breast cancer patients was addressed in the 2022 Breast Cancer Clinical Practice Guidelines. A systematic review and meta-analysis of epidemiological studies on alcohol consumption and breast cancer recurrence, breast cancer-related mortality, all-cause mortality, and cardiovascular disease mortality in breast cancer patients was performed. The PubMed, Cochrane Library, and Ichushi-Web databases were searched for relevant publications reporting cohort or case–control studies published until March 2021. A total of 33 studies (32 cohort studies and 1 case–control study) met the eligibility criteria; 4638 cases of recurrence, 12,209 cases of breast cancer-specific mortality, and 21,945 cases of all-cause mortality were observed. With regard to breast cancer recurrence, 7 studies assessed pre-diagnosis alcohol consumption (relative risk (RR) 1.02, 95% confidence interval (95% CI) 0.77–1.37, p = 0.88) and 3 studies assessed post-diagnosis alcohol consumption (RR 0.96, 95% CI 0.85–1.10, p = 0.57), and no significant increase or decrease in risk was observed. With regard to breast cancer-related mortality, 19 studies assessed pre-diagnosis alcohol consumption (RR 1.02, 95% CI 0.93–1.11, p = 0.69), 9 studies assessed post-diagnosis alcohol consumption (RR 0.96, 95% CI 0.77–1.19, p = 0.70), and no significant increase or decrease in risk was observed. With regard to all-cause mortality, 18 studies assessed pre-diagnosis alcohol consumption (RR 0.90, 95% CI 0.82–0.99, p = 0.02), 8 studies assessed post-diagnosis alcohol consumption (RR 0.88, 95% CI 0.74–1.02, p = 0.08), and pre-diagnosis alcohol consumption was associated with a significantly decreased risk. With regard to cardiovascular disease mortality and alcohol consumption, 2 studies assessed it, and the RRwas 0.47 (95% CI 0.28–0.79, p = 0.005), showing that alcohol consumption was associated with a significantly decreased risk. The limitations of this study are that drinking status was mainly based on a questionnaire survey, which is somewhat inaccurate and has many confounding factors, and the cut-off value for the maximum alcohol intake in many studies was low, and it is possible that the actual intake was only an appropriate amount. In many countries, a standard drinking amount is set, and wise decisions are required.

Takumi Kurabayashi,1 Yuki Ideno,2 Kazue Nagai,3 Naho Maruoka,3 Kiyoshi Takamatsu,4 Toshiyuki Yasui,5 Kunihiko Hayashi3 1Department of Obstetrics and Gynecology, Niigata City General Hospital, Niigata, Japan; 2Gunma University Center for Mathematics and Data Science, Maebashi, Gunma, Japan; 3Department of International/Community Health Laboratory Sciences, Graduate School of Health Sciences, Gunma University, Maebashi, Gunma, Japan; 4Department of Obstetrics and Gynecology, Tokyo Dental College Ichikawa General Hospital, Chiba, Japan; 5Department of Reproductive and Menopausal Medicine, Institute of Biomedical Science, Tokushima University Graduate School, Tokushima, JapanCorrespondence: Takumi KurabayashiDepartment of Obstetrics & Gynecology, Niigata City General Hospital, 463-7, Shumoku, Chuo-ku, Niigata City, Niigata Prefecture, 950-1197, JapanTel +81-25-281-5151Fax +81-25-281-5169Email takumi@hosp.niigata.niigata.jpPurpose: Although the validity of self-reported osteoporosis is often questioned, validation studies are lacking. This study was performed to investigate how well self-reported diagnoses of osteoporosis agreed with validated clinical information in young and middle-aged women in the Japan Nurses’ Health Study (JNHS), a nationwide prospective cohort study of nursing professionals.Patients and Methods: Data were reviewed for 15,717 subjects from the combined cohorts of the JNHS and a preceding pilot study (Gunma Nurses’ Health Study). The subjects’ mean age at the baseline (BL) survey was 41.6 ± 8.3 years, and the mean follow-up period was 11.5 ± 4.4 years. Participating nurses were mailed a follow-up questionnaire every 2 years. Respondents who self-reported a positive osteoporosis diagnosis during the study period were sent an additional confirmation questionnaire to corroborate the details.Results: The number (proportion) of women with osteoporosis was 884 (5.6%) [primary osteoporosis, 812 (5.2%); secondary osteoporosis, 72 (0.5%)]. The cumulative incidence of osteoporosis at the age of 40, 50, 60, 70, and 80 years was estimated to be 0.1% (95% confidence interval, 0.1– 0.2), 1.1% (0.9– 1.3), 7.7% (7.0– 8.4), 23.6% (21.6– 25.7), and 54.2% (40.2– 68.1), respectively. For BL and regular follow-up + expert review versus BL and regular follow-up + confirmation questionnaire + expert review, the positive predictive value (PPV) was 61.3% versus 85.6% and the negative predictive value (NPV) was 98.9% versus 98.2%, respectively.Conclusion: Self-reporting was associated with a high NPV for the incidence of osteoporosis. Although the PPV was slightly lower, additional corroborations by confirmation questionnaire might improve the PPV.Keywords: validation, self-reported diagnosis, osteoporosis, prospective cohort study, Japan Nurses’ Health Study

Objective: The aim of this study was to find out whether ThinPrep Integrated Imager (Hologic Inc.) screening is non-inferior to manual screening in the detection of cervical lesion. Study Design: For a total of 4,011 ThinPrep Pap test specimens stained by ThinPrep staining, manual screening (Manual arm) and ThinPrep Integrated Imager screening (Imager arm) were performed so as not to be screened by the same cytotechnologist, and the sensitivity and specificity in the detection of cervical lesion were compared using McNemar’s test. Results: The sensitivity to detect CIN1 or more squamous cell abnormalities or glandular abnormalities was 91.67% (= 374/408, 95% confidence interval [CI]: 88.44–94.08%) for the Manual arm and 92.40% (= 377/408, 95% CI: 89.28–94.70%) for the Imager arm, and the specificity was 88.87% (= 3,113/3,503, 95% CI: 87.77–89.88%) for the Manual arm and 89.55% (= 3,137/3,503, 95% CI: 88.48–90.54%) for the Imager arm. The differences in sensitivity and in specificity, respectively, were 0.74% (95% CI: −3.14–4.61%, McNemar’s test, p = 0.8041) and 0.69% (95% CI: −0.13–1.50%, McNemar’s test, p = 0.1125). About the equality of sensitivity and specificity between the 2 methods, 95% CIs of the difference between sensitivity and specificity are in the clinical equivalence range of ±5%, so the Imager arm is non-inferior to the Manual arm. Conclusion: The Imager arm was confirmed to have an equivalent and non-inferior capacity in the detection of cervical lesions compared with the Manual arm, suggesting that its practical application in cervical cytology tests is highly possible.

Pregnancy after conization is associated with a high risk of preterm delivery. However, because risk factors for preterm delivery after conization remain unknown, we conducted a multicenter observational study to investigate risk factors associated with preterm delivery. We selected patients who had previously undergone conization and reviewed medical records from 18 hospitals in cooperation with Keio University School of Medicine between January 2013 and December 2019. Women were classified as nulliparous and primiparous, and a multiple logistic regression analysis was performed to evaluate the relative contributions of the various maternal risk factors for preterm delivery (i.e. delivery before 37 gestational weeks). Among 409 pregnant women after conization, 68 women delivered preterm (17%). The incidence of nulliparity (p = .014) was higher and a history of preterm delivery (p = .0010) was more common in the preterm delivery group than in the term delivery group. Furthermore, the proportion of women diagnosed with adenocarcinoma in situ (AIS) and cervical cancer in the preterm delivery group was higher than that in the term delivery group (p = .0099 and .0004, respectively). In multiple regression models in nulliparous women, cervical cancer or AIS (Odds ratio [OR]: 4.16, 95% CI: 1.26–13.68, p = .019) and a short cervix in the second trimester (OR: 13.41, 95% CI: 3.88–46.42, p < .0001) increased the risk of preterm delivery. Furthermore, a history of preterm delivery (OR: 7.35, 95% CI: 1.55–34.86, p = .012), cervical cancer or AIS (OR: 5.07, 95% CI: 1.24–20.73, p = .024), and a short cervix in the second trimester (OR: 4.29, 95% CI: 1.11–16.62, p = .035) increased the risk of preterm delivery in the multiple regression models in primiparous women. Pregnant women who previously underwent conization are at risk for preterm delivery. The histological type of AIS and cervical cancer was evaluated as a risk factor for preterm delivery. KEY MESSAGESPrior preterm delivery, presence of a short cervix, and cervical cancer or AIS were predictors of preterm delivery after conization.The depth of conization in cervical cancer or AIS group was significantly larger than that in the CIN group. Prior preterm delivery, presence of a short cervix, and cervical cancer or AIS were predictors of preterm delivery after conization. The depth of conization in cervical cancer or AIS group was significantly larger than that in the CIN group.

Background Many women experience various symptoms during the period of menopausal transition, including complaints of reduced cognitive functioning. However, these complaints are not necessarily recognized as core menopausal symptoms. In this study, we sought to characterize subjective complaints of reduced cognitive functioning by analyzing cross-sectional data from the Japan Nurses’ Health Study (JNHS). Methods The JNHS 4-year follow-up questionnaire containing a 21-item climacteric symptom checklist, which included a question about “poor memory or forgetfulness”, was mailed between 2005 and 2011 to all JNHS participants, regardless of their age at the time of the survey. We estimated the prevalence of slight and severe complaints in 5-year age-groups. We used principal component analysis to explore the underlying factors among the 21 symptoms during the menopausal transition period in women aged 45–54 years at the time of the survey. We also examined risk factors for complaints using multivariable modified Poisson regression analysis. Results In total, 12,507 women responded to the 4-year survey. The mean age at the time of the 4-year survey was 46.5 years (range 27–82). “Poor memory or forgetfulness” showed a peak prevalence of 81.7% (severe 27.9%; slight 53.8%) at 50–54 years, and gradually decreased after 55 years. Principal component analysis indicated that “poor memory or forgetfulness” belonged to somatic symptoms and was close to psychological symptoms in women aged 45–54 years. In women aged 45–54 years, the complaint was also significantly associated with hot flashes and sweats. Multivariable modified Poisson regression analysis showed that menopausal status (uncertain and postmenopausal), less sleep (sleep of Conclusions We found that prevalence of “poor memory or forgetfulness” was highest during the menopausal transition period and among perimenopausal women. This subjective complaint was associated with somatic, psychological complaints and VMS. It may be useful for women with cognitive problems in the transition period to consider management of comorbid menopausal symptoms.

The World Conference of the International Society of Psychosomatic Obstetrics and Gynecology held in The Hague, Netherlands in October 2019 ran a dedicated session debating the global research prio...

Background Perimenopausal women experience a wide variety of systemic symptoms: hot flashes, sweating, mental health concerns and various oral sensory complaints (OSC). OSC in perimenopausal women include xerostomia, taste disturbance and burning mouth. However, the factors associated with these OSC have not been identified. The purpose of this investigation was to elucidate the factors associated with OSC in perimenopausal women. Methods The study cohort comprised 43 perimenopausal women aged 45–55 years. Data on medical history, medications, menstrual status, menopausal symptoms, quality of life, xerostomia, taste disturbance and burning mouth were collected. Volumes of unstimulated and stimulated saliva were measured. Tongue coating was evaluated according to a tongue coating index. Univariate analysis was performed to identify factors significantly associated with having xerostomia, taste disturbance, burning mouth and more than two OSC (2OSC). Next, the factors strongly associated with these symptoms were examined by logistic regression analysis. Results The number of menopausal symptoms was significantly higher, and volume of unstimulated saliva was significantly lower in participants with xerostomia, taste disturbance, burning mouth or 2OSC than in those without these characteristics. Agents targeting the central nervous system were more frequently taken by participants with burning mouth and 2OSC than by those without these characteristics. According to logistic regression analysis, the number of menopausal symptoms was an explanatory variable for xerostomia, taste disturbance, burning mouth and 2OSC. Conclusions Our findings suggested that OSC associated with the number of menopausal symptoms. Management of menopausal symptoms may decrease OSC, leading to improved quality of life of perimenopausal women.

Objective. Kampo medicine, a traditional Japanese medicine, is widely used in Japan, especially in the field of menopause medicine. However, few studies have shown evidence-based effects. This study aimed to confirm the effects of kamishoyosan on menopausal symptoms with a randomized, placebo-controlled, double-blind clinical trial. Methods. Subjects were randomly allocated to groups that received either kamishoyosan (n = 101) or a placebo resembling kamishoyosan (n = 104). The primary outcomes were the change in the number of hot flashes, depression scores, improvements of anxiety, quality of life (QOL), and menopausal symptoms before and 4 and 8 weeks after initiation of treatment with the study drug. The secondary outcome was drug safety. Results. After 8 weeks, the number of hot flashes decreased after treatment in both groups, but there was no significant difference between the two groups. The changes in SDS scores showed the same results. Moreover, no significant differences were observed between the two groups in assessments with the STAI, SF-36, and JSOG menopausal index. No serious adverse effect was reported. Conclusions. This first placebo-controlled double-blind randomized trial with kamishoyosan demonstrated that it was safe and had some effects on climacteric symptoms, but not significant compared with placebo. Some problems, such as placebo effects, in the study of Kampo therapy for menopausal symptoms, were revealed. This trial is registered with the trial registration number. UMIN 000006042.

Laparoscopic myomectomy may become a cause to increase intraoperative hemorrhage. The purpose of this investigation was to study the relationship between clinical features, MR imaging findings and intraoperative hemorrhage during laparoscopic myomectomy. This study included 126 patients clinically and pathologically diagnosed as uterine leiomyoma between 2010 and 2015. These findings were statistically compared between significant intraoperative hemorrhage group (greater than 300 ml) and nonsignificant intraoperative hemorrhage group (less than 300 ml). Correlation between the maximum diameter of resected leiomyomas and the maximum diameter leiomyomas on MR imaging were also evaluated. Significant hemorrhage group showed larger maximum diameter of resected leiomyoma (p = 0.01), larger maximum diameter of leiomyoma on MR imaging (p = 0.01) and have cellular leiomyomas on MR imaging more frequently (p = 0.03) than nonsignificant intraoperative hemorrhage group. The maximum diameter of resected leiomyomas significantly correlated with that of leiomyomas on MR imaging (Spearman’s rho = 0.797, p

Hereditary breast and ovarian cancer syndrome (HBOC) is a hereditary tumor that can be definitively diagnosed by detection of germline mutation of the BRCA1 or BRCA2 gene. The HBOC Public Awareness and Management Sub-committee of the Tumor Committee, Japan Society of Obstetrics and Gynecology carried out a fact-finding survey on the status of treatment of HBOC in Japan. The directors of medical specialty teaching facilities were notified of the questionnaire by post, with the request for one respondent per facility. The response period was from 8 July 2014 to 31 March 2015. Of the 678 facilities that were asked to compete the questionnaire, 341 (50.3%) responded. The responses are shown in the respective tables. For questions with free responses, similar answers have been grouped together, and the written answers have been freely translated. Based on these results, the Japan Society of Obstetrics and Gynecology considers that the 14 conditions, including consultations by specialist staff, must be met before risk-reducing salpingo-oophorectomy is carried out.

Objective. The KOSMOS study, a multicenter, randomized, double-blind, placebo-controlled trial, investigated the effects and safety of kamishoyosan (TJ-24), a traditional Japanese medicine, in the treatment of climacteric disorder. Methods. Japanese women with climacteric disorder were administered a placebo during a 4-week run-in period, after which they were classified as placebo responders (R group) if their score on the modified Questionnaire for the Assessment of Climacteric Symptoms in Japanese Women (m-QACS) with excitability and irritability as the primary outcome improved by ≥ 3 points and as placebo nonresponders (NR group) otherwise. Members of the NR group were randomly allocated to receive either TJ-24 or placebo. After 12 weeks, their m-QACS scores, anxiety and depression, sleep, and overall quality of life (QOL) were compared. Results. The TJ-24 and placebo arms in the NR group included 20 patients each. The change in the m-QACS scores of members of the NR group for excitability and irritability at 12 weeks versus baseline was –3.1 ± 1.7 in the TJ-24 arm, a significant decrease, but compared with –2.7 ± 2.2 in the placebo arm, no significant difference was between two arms. However, the proportion of participants whose score improved by ≥3 points was significantly higher in the TJ-24 arm. In the subgroup analysis of premenopausal women, the changes in the score for excitability and irritability were significantly larger in the TJ-24 arm. The incidence of adverse drug reactions or adverse events did not differ between the two arms, and no serious events were reported. Conclusion. Although no significant difference was identified for the primary outcome, a significantly higher proportion of patients who received TJ-24 displayed improvement. Its high level of safety and effects on excitability and irritability in premenopausal women suggest that TJ-24 may be a useful treatment.

Remarkable technological advances have been made in the field of medicine in recent years, one result of which is that a prenatal diagnosis of cleft lip and/or cleft palate (CL/P) is now possible. In this situation, it is extremely important to provide the parents with mental care from the moment they are informed. Here, we describe cases of CL/P treated at our hospital and how such a diagnosis and prenatal counseling are handled. A survey was carried out on 4 cases seen at our department between April 2013 and March 2014. Patients are referred to our department from local or our own obstetrics clinics on a prenatal diagnosis of CL/P based on findings from ultrasonography. If the case is a referral from outside, the patient will first be seen at our own obstetrics department. Our department may then be subsequently requested to provide the parents with prenatal counseling. Effort is made to reassure the parents that postnatal support will be provided, right from the start. Next, the multidisciplinary nature of the treatment process is explained. However, only the essential outline is given at first so as to avoid inducing unnecessary anxiety. A response is also given to any questions the parents may have. Our experience of giving such care leads us to believe that improvements are required in the way that explanations and assistance are provided. The number of cases in which prenatal counseling is required is expected to increase in future.

Background: Patients with malignancy (n = 130) participated in the sperm cryopreservation program.

We have developed a determination method for alcohol using high-performance liquid chromatography by using a diamond electrode-electrochemical detector. At this time, 4 alcohols (methanol, ethanol, 2-propanol and 1-butanol) were separated on a Shodex® ODP2 HP-4E column with an acetonitrile-25 mM disodium hydrogen phosphate buffer (pH 11.0, 1 : 99, v/v) as the mobile phase. The electrochemical detector, equipped with a diamond electrode, was set at 2000 mV (applied voltage). The detection limits of the four alcohols were 20-25 ng/mL. The proposed liquid chromatography was fifty-times more sensitive compared to reported gas chromatography – flame ionization method, and was found to be suitable for large-volume aqueous sample solutions compared to gas chromatography. In the case of poor absorbance compounds, an easy-to-set additional detector in wide using the liquid chromatography – ultraviolet detection system. It was meaningful economical work to use an existing apparatus in the laboratory. With this method, one could determine volatile alcohols, e.g., ethanol, which could not be determined by other universal-type detectors (corona-CAD and ELSD). The proposed method was applied to the alcohol in detergents (contains ethanol and 2-propanol) and alcoholic beverages. Good relationships were obtained compared to the enzymatic method.

In the present study, a novel system for the hypoxic culture of individual tissues was established for the subculturing of cell lines for research as well as for clinical culture of primary cells. To provide a hypoxic environment throughout the process of tissue handling and culture, we designed a clean bench with CO2 gas circulation and a hypoxic culture incubator containing disposable capsules. The bench top was covered with an acrylic chamber, and an atmosphere of 5.0% CO2-air was maintained using a sensor control. The cleanliness class of the chamber could easily be improved to 1 within 5 min of circulative filtration, even though it was found to be 105 before the unit was operated. Gas buffer solution (220 ml of 20 mM HEPES, 25 mM NaHCO3, pH 7.4) placed in a 500-ml plastic capsule in the unit stabilized the culture environment by functioning as a heat storage and gas pool. The inflow of air that occurred by the cap of the capsule was opened was excluded by the infusion of purging gas (5.0% CO2 and 95% N2); the O2 level returned to 2.0% within 4 min, after which the gas supply automatically switched to the culture gas (2.0% O2, 5.0% CO2, and 93% N2). If this purging process was omitted, restoration of the O2 level required 120 min, even though the inner volume was only 280 ml.

Improvement of the sensitivity and specificity of a simultaneous stress-free screening method for catechol estrogens as a potential prostate cancer marker in urine has been accomplished by HPLC with a diamond-electrode electrochemical detector and a fluorescence detector. Since taking urine samples generates less stress (or pain) than the drawing of blood, the method can readily be applied to almost any patient, and will also assist in improving the sensitivity and specificity of the prostatic specific antigen test. Catechol estrogens (2-hydroxyestrone, 4-hydroxyestrone, 2-methoxyestrone, 2-hydroxyestradiol, 4-hydroxyestradiol, 2-methoxyestradiol, and 2-hydroxyestriol) and estrogens (estrone, estradiol, estriol) were separated on an Inertsil ODS-II column with acetonitrile-potassium dihydrogen phosphate (pH 3.0). The diamond-electrode electrochemical detector used had the great advantage of being a maintenance-free system, and could sequentially analyze hundreds of samples. Fluorescence detection improved the sensitivity 10-500 times (e. g., the LOD of 2-hydroxyestriol was improved 250 times) compared to previous electrochemical detection reports, and dual detection improved peak identification in the urine samples. The proposed method was applied to the simultaneous determination of catechol estrogens in spiked urine in a preliminary study on estrogens and PSA values in biopsy and prostate cancer patients.

Background Patients with malignancy (n = 130) participated in the sperm cryopreservation program.

Purpose: Color Doppler imaging permits the accurate localization of vessels and high-frequency pulsed Doppler ultrasonography has improved the resolution of flow velocity waveforms. In this study, intraovarian arterial blood flow before and after follicle rupture in the natural cycle was examined using transvaginal color flow Doppler imaging and changes in intraovarian arterial resistance in relation to the outcome of infertility treatment was analyzed. Methods: In a prospective study, 227 spontaneous cycles in 118 infertile patients who were undergoing infertility treatment at the division of Reproductive Medicine in our center were recruited in this study. The impedance to flow in intraovarian vessels was measured by means of transvaginal color flow Doppler imaging during the periovulatory period in the natural cycle of all patients. The pulsatility index (PI) of intraovarian arterial blood flow and pregnancy rate was evaluated. Results: On the basis of PI values before and after follicular rupture, 227 cycles were classified into severely decreased (113 cycles) and not-severely decreased groups (114 cycles). The pregnancy rate per cycle in the severely decreased group was 18.6% (21/113), significantly higher than that in the not-severely decreased group (7/114; 6.1%, p = 0.004). The miscarriage rate was similar in the two groups. Conclusions: A reduction in intraovarian blood vessel resistance is necessary to achieve pregnancy in a natural cycle.

背景:悪性褐色細胞腫の骨盤内再発はきわめてまれである. 今回われわれは, 原発腫瘍摘出11年後にダグラス窩に再発した悪性褐色細胞腫の1例を経験したので報告する.症例:48歳, 女性. 右副腎褐色細胞腫にて右副腎摘出術施行. 11年後に肝転移を認め肝部分切除術が施行されたが, カテコールアミン高値が持続し, 画像診断にてダグラス窩に腫瘤をめたため, 腫瘤摘出術を施行した. 摘出術後, 血圧, カテコールアミン値はすみやかに低下した. 摘出腫瘤の捺印標本には大小不同の目立つ異型細胞が多数みられ, 細胞質は微細顆粒状であった. 核クロマチンは増量し, 大型の核小体が1ないし数個認められた. 病理組織学的検索では, 付属器表面とダグラス窩に限局した転移と, 子宮筋層内への浸潤を認めた.結論:細胞診による褐色細胞腫の良・悪判定はきわめて困難で, 良性腫瘍であってもその異型性の強さから癌や肉腫と誤診されることもまれではない. 本症例は, 原発巣摘出11年後に起こったダグラス窩転移であったが, 細胞学的にも褐色細胞腫の特徴を示していた.

We developed a detection method for circulating tumor cells (CTCs) using the telomerase-specific adenovirus OBP-401. This recombinant virus has a telomerase promoter at the 5′-end of the viral genome and GFP at the 3′-end. To date, CTC enumeration using OBP-401 has shown prognostic impact for gastric and small cell lung cancer patients. In the present study, peripheral blood samples from patients with eight types of cancer, including some cancers previously untested with OBP-401 (i.e., esophagus, pancreas, and prostate cancers) were subjected to this method in order to evaluate its versatility. It was recently discovered that some white blood cells (WBCs) false-positively react with OBP-401. Although anti-CD45 antibodies can absorb these adverse cells from peripheral blood, the simplicity of the OBP-401 method would be diminished by the introduction of antibody treatment. Therefore, we evaluated another approach to minimize the false positivity of WBCs. Seven anti-CD antibodies were employed to stain the species of WBCs that false-positively reacted with OBP-401. We revealed that the false-positively reacted WBCs were monocytes in the peripheral blood of both healthy subjects and cancer patients. Based on a size distribution analysis of the GFP-positive monocytes, the size criterion for CTCs using OBP-401 was defined to be a cellular diameter >8.4 μm. In total, 43% of 86 cancer patients examined in the present study were CTC-positive using this definition. CTCs were enumerated from peripheral blood samples collected from patients with each of the eight types of cancer; the detectability of CTCs for esophagus, pancreas and prostate cancers by the OBP-401 method was confirmed for the first time in the present study. However, no clear correlation between CTC positivity and the clinical characteristics of patients with any type of cancer was observed because of the small number of patients with each type of cancer. An additional clinical study will be conducted to confirm the clinical meaning of CTCs enumerated by OBP-401.

Throughout the past decade, the neutral and alkaline comet assays, which lyses cells with high salt in the absence of proteolysis, have been the most widely used methods to observe single- or double-strand breaks (SSB, DSB) in a single nucleus. These methods evaluate the degree of DNA damage based on the amount of granular fragments discharged from the origin, the so-called “comet tail,” but not of long chain DNA fibers. Meanwhile, a novel, single-cell pulsed-field gel electrophoresis (SCPFGE) method, which digested cells with trypsin, observed a different course of DNA fragmentation: first, a few large fibrous fragments were derived from a bundle of long chain fibers, then the cleavages were advanced until finally almost all the DNA was shredded to granular fragments. Some nuclear DNA binding components were tolerant for high salt and high alkalinity, but were degraded by trypsin digestion. A lack of trypsin digestion causes false negative results in both SCPFGE and comet assays. Also, most DNA fibers were still fixed with components, and the comet tail did not reflect a total amount of granular fragments, but rather those that released components. Repair of DSB is intrinsically difficult as compared to other DNA lesions, and the critical threshold is extremely low.DNA fibers that have already been shredded to numerous granular fragments may be irreparable as a result. Although 100 - 300 mmol/L NaOH was commonly used in the alkaline comet assay, the naked DNA fibers persisted in 10 mmol/L NaOH after trypsin digestion, but were shredded to granular fragments by 30 mmol/L. Neogenesis of the granular fragments by high pH did not clarify the mechanism of such a result; that is, it was unknown whether it was due to dissociation of hydrogen bonds, strand breaks through alkaline labile sites, artifactual DSB, or a combination of actions. Optimum conditions for the comet assay need to be defined to achieve quantitative measurements. DNA instability analyses by means SCPFGE is likely to serve as a fundamental step in single-cell genomics to determine the competence of the cell population provided for DNA amplification methods and is likely to play an important role in ensuring the safety of clinical regenerative medicine.

Human sperm often contain vacuoles, and it is therefore necessary to determine whether vacuoles in the chromosomal region are responsible for DNA deterioration. To that end, the present study developed a novel clinical examination technique, translucent staining, for the visualization of vacuoles. Highly diluted Reactive Blue 2 (RB2, 100 pmol/L) faintly stained the sperm head to reveal a translucent bluish body with toneless spots. Differential interference contrast showed the vacuoles as shaded-relief images. In the same, field of view the localization and the shape of the toneless spots were coincided with the shaded-relief images. Dual staining with RB2 and DNA fluorescent dye suggested that the vacuoles might include vacant or lowdensity DNA. The present results revealed important issues in clinical Assisted Reproductive Technology (ART), that is, a variety of vacuoles were involved regardless of the normalcy of outline or the motility. Features were classified into three categories: zero or one small vacuole; multiple sporadic vacuoles of various sizes; and one large vacuole. Moreover, in vitro processing steps such as density gradient centrifugation and subsequent swim up could not isolate sperm without vacuoles. Since it is still unknown whether the vacuoles merely push out DNA to the surroundings without damaging it, or if the empty region is a consequence of DNA degradation, the translucent staining for simultaneous visualization of the outline and vacuoles constitutes a major step for pre-ART cyto-diagnosis to determine whether the sperm population is competent for use in clinical ICSI.

Background Because the primary aim of infertility treatment is to achieve pregnancy, mental health care during this treatment is often neglected. However, the inability to conceive children is stressful for couples throughout the world. Thus, the purpose of this study was to investigate factors related to the anxiety and depression of female infertility patients. Methods Participants included 83 Japanese women who initially visited the Reproduction Center of the Tokyo Dental College Ichikawa General Hospital to undergo testing and receive infertility treatment between February and April 2008. We administered two psychological tests, the Self-rating Depression Scale (SDS) test and the Hospital Anxiety and Depression Scale (HADS) test. We then examined the association of the test results with age, pregnancy and delivery history, employment status, duration of infertility, infertility treatment history, and male infertility. Results As patient age increased, total HADS and depression scores also increased. No correlation was observed between duration of infertility and SDS or HADS scores. Results were similar when the presence and absence of delivery history was compared. Patients who underwent infertility treatment were more likely to have high HADS depression scores compared to patients who had not undergone treatment. Additionally, patients whose husbands were infertile had significantly lower total HADS and anxiety scores than those whose husbands were not infertile. Conclusions Age and male infertility are factors that influence the presence of anxiety and depression in female infertility patients.

The objective of this clinical trial was to examine the efficacy of a supplement containing natural S-(-)equol, a daidzein metabolite, in reducing menopausal symptoms.In this multicenter, double-blind placebo-controlled trial, 160 equol nonproducing, postmenopausal Japanese women who experienced at least 1 hot flush/day were randomly assigned to consume 10 mg/day S-(-)equol (n=77 women) or placebo (n=83 women) for 12 weeks. Participants completed a standardized menopausal symptom checklist and rated five common menopause symptoms by a visual analog scale at baseline, week 12, and week 18 (6-week postintervention). Physical, blood, and urine examinations were conducted. One hundred twenty-six women completed the study.At baseline, daily hot flush frequency was 2.9±2.1 for the S-(-)equol group and 3.2±2.4 for the placebo group. After the 12-week intervention, the S-(-)equol group had a greater decrease from baseline in hot flush frequency compared with the placebo group (-1.9±1.8/day, -58.7%, vs. -1.0±2.0/day, -34.5%, p=0.009). The severity of hot flushes and neck or shoulder muscle stiffness significantly decreased in the S-(-)equol group compared with the placebo group. No changes in clinical parameters or serious adverse effects were reported.This is the first trial to show beneficial effects of a 10-mg natural S-(-)equol supplement consumed daily for 12 weeks on major menopausal symptoms, specifically, hot flushes and neck or shoulder muscle stiffness, in postmenopausal Japanese women. This supplement offers a promising alternative for management of menopausal symptoms.

In human cervical epithelium and uterine endometrium, globo-series neutral glycosphingolipids with N-alpha-hydroxy fatty acyl phytosphingosine (4-D-hydroxysphinganine) as the ceramide and sulfatide (I3SO3-GalCer), which were contained in trace amount at the follicular phase, significantly increased in concentration at the luteal phase, comprising about 20% of the individual neutral glycosphingolipids and about 15% of the total acidic glycosphingolipids, respectively. However, in the mucosa of fallopian tube, neutral glycosphingolipids with the same polarity as those in the cervical epithelium and uterine endometrium at the luteal phase and sulfatide remained at a constant and higher level independently of the menstrual cycle. The structures of neutral glycosphingolipids in the fallopian tube, having the same polarity as that of N-alpha-hydroxy fatty acyl phytosphingosine-containing molecules appeared in the cervical epithelium and uterine endometrium at the luteal phase, were determined to be N-alpha-hydroxy palmitoyl 4-sphingenine-containing ones by negative-ion FABMS. Also, laminin, but not collagen type IV, was found to be contained in the concentration correlated well with that of sulfatide in the genital tract, when determined by western blotting with monoclonal anti-laminin and anti-collagen type IV antibodies, indicating a possible function of sulfatide as a receptor for laminin in the human female genital tract.

Changes in CA602 and CA546, two carbohydrate-related antigens newly established in our institute, were evaluated during the course of in-vitro fertilization and embryo transfer. The concentration of CA602, an antigen with characteristics similar to those of CA125, did not change substantially until embryo transfer. In patients with clinical evidence of ovarian hyperstimulation syndrome (OHSS), a significant increase in serum concentrations of CA602 was observed 10 days after embryo transfer (P < 0.01). There were no significant changes in the patients without OHSS. A significant elevation of oestradiol concentration in OHSS patients was also observed, with the variation before oocyte retrieval being greater than that seen with CA602. The concentration of CA546, which had lower false-positive rates in benign disease, did not differ significantly in the OHSS and non-OHSS patients. Our data demonstrate that serum concentrations of CA602 are increased even in early stages of OHSS, and suggest a possible role for this antigen as a marker for OHSS.

Single-cell pulsed-field gel electrophoresis (SCPFGE) with dual electrode pairs was developed to detect the early stage of DNA fragmentation in human sperm. The motile sperm were purified by the commonly used density-gradient centrifugation technique and subsequent swim-up. The sperm were embedded in a thin film of agarose containing bovine trypsin (20 µg/mL) and were then lysed. Prior to SCPFGE, proteolysis of DNA-binding components, such as protamine and the nuclear matrix was essential to separate the long chain fibers from the fibrous and granular fragments derived from a single nucleus. The overall electrophoretic profiles elucidated the course of DNA fragmentation. A few large fibrous fragments were observed at the beginning of the process, however, as the fragmentation advanced, the long chain fibers decreased and shortened, and, conversely, the granular fragments increased until finally almost all the DNA was shredded. Although the ejaculate contained sperm with heterogeneous stages, the purified motile sperm exhibited several dozens of uniformly elongated fibers arising from the tangled DNA at the origin, whereas a part of these fibers gave rise to fibrous fragments beyond the tip of the elongated fibers, and their numbers and sizes varied among the sperm. Conventional intra-cytoplasmic sperm injection (ICSI) usually depends on intra-operative light microscopic observation to select a sperm for injection. The present results revealed that sperm motility could not give full assurance of DNA integrity. SCPFGE is likely to serve an important role in the preoperative differential diagnosis to determine the competence of the sperm population provided for injection.

Background Granulosa cell tumours (GCTs) are frequently seen in menopausal women and are relatively indolent. Although the physiological properties of normal granulosa cells have been studied extensively, little is known about the molecular mechanism of GCT progression. Here, we characterise the unique behavioural properties of a granulosa tumour cell line, KGN cells, for the molecular analysis of GCT progression. Methods Population doubling was carried out to examine the proliferation capacity of KGN cells. Moreover, the invasive capacity of these cells was determined using the in vitro invasion assay. The expression level of tumour markers in KGN cells at different passages was then determined by Western blot analysis. Finally, the growth and metastasis of KGN cells injected subcutaneously (s.c.) into nude mice was observed 3 months after injection. Results During in vitro culture, the advanced passage KGN cells grew 2-fold faster than the early passage cells, as determined by the population doubling assay. Moreover, we found that the advanced passage cells were 2-fold more invasive than the early passage cells. The expression pattern of tumour markers, such as p53, osteopontin, BAX and BAG-1, supported the notion that with passage, KGN cells became more aggressive. Strikingly, KGN cells at both early and advanced passages metastasized to the bowel when injected s.c. into nude mice. In addition, more tumour nodules were formed when the advanced passage cells were implanted. Conclusion KGN cells cultured in vitro acquire an aggressive phenotype, which was confirmed by the analysis of cellular activities and the expression of biomarkers. Interestingly, KGN cells injected s.c. are metastatic with nodule formation occurring mostly in the bowel. Thus, this cell line is a good model for analysing GCT progression and the mechanism of metastasis in vivo.