Highlights of the 14th Japan Bioanalysis Forum Symposium.

The 14th Japan Bioanalysis Forum Symposium was held at Tower Hall Funabori, Japan from 1-3 March 2023. The conference theme, 'Bringing Together - the Expertise of Bioanalysis', aimed to enable people from various fields to gather, learn and collaborate together for the common goal of delivering medicines to patients faster. Approximately 360 participants from various fields, including pharmaceutical industries, contractors, academia and regulatory authorities, gathered at an in-person symposium which had an online participation option, for the first time in 4 years. The symposium offered a wide range of topics including ICH M10, new modalities, biomarkers, immunogenicity, electronization and patient-centric sampling. The latest research results were provided from domestic and overseas scientists. This report summarizes the major topics.

Highlights of the 13th Japan Bioanalysis Forum Symposium.

Approximately 280 people from pharmaceutical industries, contractors, academic institutions and regulatory authorities attended the 13th Japan Bioanalysis Forum Symposium. The symposium was held via web to prevent the spread of COVID-19 from the 28 February to 2 March 2022. The theme of the symposium was 'All for One Goal', and the event has provided an opportunity for open discussion among researchers with different backgrounds but who share a common goal: "to deliver more effective and safe pharmaceuticals to patients as quickly as possible". The speakers focused on hot topics in bioanalysis, including chromatography, biomarker analysis, cell and gene therapy, COVID-19 and antidrug antibody. This symposium provided a great opportunity for the participants to have meaningful discussions, even though 'on the web' was a limited space.

Perturbation by Antimicrobial Bacteria of the Epidermal Bacterial Flora of Rainbow Trout in Flow-Through Aquaculture.

The bacterial flora of the epidermal mucus of fish is closely associated with the host's health and susceptibility to pathogenic infections. In this study, we analyzed the epidermal mucus bacteria of rainbow trout (Oncorhynchus mykiss) reared in flow-through aquaculture under environmental perturbations. Over ~2 years, the bacteria present in the skin mucus and water were analyzed based on the 16S rDNA sequences. The composition of the mucus bacterial community showed significant monthly fluctuations, with frequent changes in the dominant bacterial species. Analysis of the beta- and alpha-diversity of the mucus bacterial flora showed the fluctuations of the composition of the flora were caused by the genera Pseudomonas, Yersinia, and Flavobacterium, and some species of Pseudomonas and Yersinia in the mucus were identified as antimicrobial bacteria. Examination of the antimicrobial bacteria in the lab aquarium showed that the natural presence of antimicrobial bacteria in the mucus and water, or the purposeful addition of them to the rearing water, caused a transition in the mucus bacteria community composition. These results demonstrate that specific antimicrobial bacteria in the water or in epidermal mucus comprise one of the causes of changes in fish epidermal mucus microflora.

Long-term conservative management of symptomatic bladder endometriosis: A case series of 17 patients.

OBJECTIVES: This study aimed to evaluate the course of long-term conservative management of bladder endometriosis (BE). MATERIALS AND METHODS: We retrospectively reviewed 17 cases of BE conservatively managed without surgery in our facility. The following factors were analyzed: age, medical history, lesion size, symptoms, hormonal treatment, and follow-up outcomes. RESULTS: In this study, 15 patients received hormonal therapy and 2 did not. Oral contraceptive (OC), dienogest (DNG), and gonadotropin-releasing hormone agonist (GnRHa) were administered as the first regimen in 7, 5, and 3 patients, respectively. Of the 7 patients, OC administration was effective in alleviating urinary symptoms in all but 2 patients. Of 3 patients who received GnRHa, 2 switched to OC and then DNG, and 1 patient discontinued the treatment because of adverse effects. Of 5 patients who received DNG, all experienced symptom relief. DNG, OC, and GnRHa administration were effective and tolerable in 9 of 10 patients (90.0%), in 5 of 9 patients (55.6%), and in 2 of 3 patients (66.7%), respectively. In particular, 3 patients completed DNG treatment until menopause. The size of the BE lesion significantly decreased after 3 months of DNG administration, and the reduction effect was maintained until 48 months thereafter. CONCLUSION: This study proposed that hormonal therapy for BE is an effective option for those who are not planning to conceive or to undergo surgery. Specifically, DNG may be suitable for patients refusing surgery, considering the effectiveness and tolerance for long-term use.

Elevated phosphorylation of estrogen receptor α at serine-118 in ovarian endometrioma.

OBJECTIVE: To evaluate the phosphorylation of estrogen receptor α at serine-118 (phospho-ERα S118) in the endometrium, ovarian endometrioma, and deep infiltrating endometriosis (DIE). DESIGN: Experimental study. SETTING: University-affiliated hospital and academic research laboratory. PATIENT(S): Twenty-five patients underwent a hysterectomy, 18 patients underwent surgical removal of ovarian endometrioma, and 6 patients underwent DIE. INTERVENTION(S): Tissue samples were obtained from patients who underwent surgical procedures. MAIN OUTCOME MEASURE(S): Immunostaining for phospho-ERα S118, ERα, or phosphorylated p44/42 mitogen-activated protein kinase (phospho-p44/42 MAPK) was performed to evaluate the endometrium with or without endometriosis, ovarian endometrioma, and DIE. For in vitro analysis, endometrial epithelial cells (Ishikawa cells) were stimulated with estradiol (E2) or tumor necrosis factor alpha (TNFα), and the expression levels of phospho-ERα S118 and phospho-p44/42 MAPK were evaluated via Western blotting. RESULT(S): First, phospho-ERα S118 level was significantly higher in the glands and stroma of ovarian endometriosis samples than in those of endometrial and DIE samples. Second, colocalization of phospho-p44/42 MAPK and phospho-ERα S118 was observed in the glands of ovarian endometrioma. The proportions of cells strongly expressing phospho-p44/42 and phospho-ERα were 87% in phosphor-p44/42 MAPK-positive cells and 79% in phosphor-ERα-positive cells. Third, E2 stimulation significantly enhanced phospho-ERα S118 after 15 and 30 minutes in in vitro analysis using endometrial epithelial cells. Fourth, TNFα stimulation modestly but significantly enhanced phospho-ERα S118 after 15 and 30 minutes. Fifth, in Ishikawa cells, treatment with a p44/42 inhibitor (PD98059) significantly reduced phospho-ERα S118 by TNFα but not by E2. CONCLUSION(S): ERα-S118 phosphorylation was increased in ovarian endometriosis. Our findings may provide a new perspective for understanding the mechanism of increased ERα action in the pathophysiology of endometriosis.

Highlights of the 12th Japan Bioanalysis Forum Symposium.

Approximately 300 people associated with pharmaceutical industries, contractors, academic institutions and regulatory authorities attended the 12th Japan Bioanalysis Forum Symposium. The webinar was conducted from 9 to 11 March 2021. The theme of the symposium was 'for the next generation', and the event provided 'an opportunity for young researchers in bioanalysis (including students)' and 'an opportunity to discuss new frontiers of bioanalysis'. The speakers focused on hot topics of bioanalysis, including biomarker analysis, patient centric sampling, virtual clinical trials, gene therapy, cancer genome medicine and therapeutic middle molecules. The symposium presented a platform for the discussion of the prospects and challenges facing bioanalysts working in the field of pharmacokinetics. This report presents the key issues discussed.

IFITM1 is a Novel, Highly Sensitive Marker for Endometriotic Stromal Cells in Ovarian and Extragenital Endometriosis.

When the presence of endometriotic lesions are not evident by hematoxylin and eosin staining, CD10 is used to highlight and confirm the presence of endometriotic stroma. However, CD10 is not specific only to the endometrial stroma but is also expressed in many other cells. Recently, interferon-inducible transmembrane protein 1 (IFITM1) was reported as a highly specific immunohistochemical marker of normal endometrial stroma and endometrial stromal neoplasm. In this study, we examined the expression of IFITM1 and CD10 in 18 cases of ovarian endometriosis and 44 cases of extragenital endometriosis. Among the 62 patients, 62 (100.0%) were positive for IFITM1 and 60 (96.8%) for CD10, and CD10 was negative in 2 cases that were positive for IFITM1. Additionally, we found that IFITM1 sensitivity was unaffected by the presence or absence of hormonal therapy. To the best of our knowledge, this represents the first demonstration of IFITM1 as a highly sensitive stromal marker of ovarian and extragenital endometriosis.

PAX8: A Highly Sensitive Marker for the Glands in Extragenital Endometriosis.

In cases of extragenital endometriosis or microscopic endometriosis lesions, pathological diagnosis can be challenging because endometriotic stroma and glands represent only a minor component of fibrotic endometriotic lesions. For better accuracy of diagnosis, the development of a sensitive and specific epithelial marker is beneficial. Previous studies showed that PAX8 is a highly sensitive and specific marker for primary and metastatic Mullerian epithelial tumors. Therefore, we sought to examine whether PAX8 is a highly sensitive marker for glands in extragenital endometriosis. Eight and 47 samples of ovarian endometrioma and extragenital endometriosis, respectively, were evaluated in this study. We calculated the percentage of samples positively immunostained for PAX8, CD10, estrogen receptor (ER), and progesterone receptor (PR). PAX8 was positive for endometriotic epithelial cells in 95.7% (45/47) of extragenital endometrioses and in 100% (8/8) of ovarian endometrioses. CD10 was positive for endometriotic stromal cells in 97.9% (46/47) of extragenital endometrioses. PAX8 was strongly positive for glands, even in a CD10-negative case. The expression of PAX8, CD10, and PR was not affected by preoperative hormonal therapy, and the positive rate of ER staining was significantly reduced by preoperative hormonal therapy. In conclusion, PAX8 is a highly sensitive epithelial marker for extragenital endometriosis. This specific expression was maintained under hormonal therapy. It is noteworthy that extragenital endometriosis maintains the expression of this lineage marker, although it occurs at various sites, and its cause and mechanism of development might be different. PAX8 nuclear expression can be useful in detecting extragenital endometriosis in clinical practice.

Expression of ALDH1A Isozymes in Human Endometrium with and without Endometriosis and in Ovarian Endometrioma.

Human endometrium is a highly regenerative and dynamic tissue that undergoes cyclic changes during menstrual cycle. It has been reported that endometrial epithelium contains a population of progenitor/stem cells. Increasing amount of evidence indicates that progenitor/stem cells are involved in the pathogenesis of endometriosis. Proteins belonging to the aldehyde dehydrogenase 1 (ALDH1A) family have been reported to be markers of normal tissue stem cells and cancer stem cells. In this study, by using immunohistochemistry, we examined the expression of ALDH1A isozymes in human endometrial tissue, including that affected by endometriosis, and in ovarian endometrioma. Positive staining for ALDH1A isozymes was observed in the stroma of the endometrium and in endometriotic ovarian tissue. In the glands, expression patterns were distinct for different ALDH1A isozymes. ALDH1A1 and ALDH1A3 were highly expressed in the epithelium of stratum basalis of the endometrium and in the epithelium of ovarian endometrioma irrespective of the menstrual cycle, whereas ALDH1A2 was highly expressed only in the epithelium of endometrioma. Furthermore, ALDH1A1 co-localized with N-cadherin, which is a marker of endometrial epithelial progenitor cells, in the glands of stratum basalis. These findings support and reinforce the notion about the presence of progenitor/stem cells in endometrial glands in stratum basalis and in endometriotic glands, suggesting that these cells are involved in the physiology of the endometrium and in the pathology of endometriosis.

A survey of public attitudes toward uterus transplantation, surrogacy, and adoption in Japan.

This study aimed to evaluate the attitudes of male and female members of the public toward uterus transplantation (UTx), surrogacy, and adoption in Japan via a web-based survey. One thousand six hundred participants were recruited with equal segregation of age (20s, 30s, 40s, and 50s) and gender. We assessed the association between ethical view and gender, age, infertility, and the knowledge level of UTx, using a questionnaire. The findings were as follows. First, 36.5% and 31.0% of respondents agreed that UTx and gestational surrogacy should be approved, respectively. Second, the respondents would potentially choose to receive UTx (34.4%), gestational surrogacy (31.9%), and adoption (40.3%), if they or their partners experienced absolute uterine factor infertility. Third, 10.1%, 5.8%, and 14.3% of the respondents chose UTx, gestational surrogacy, and adoption as the most favorable option, respectively. Fourth, if their daughters suffered from absolute uterine factor infertility, 32.3% of female respondents might want to be donors, and 36.7% of male respondents might ask their wives to be donors. These data were affected by age, gender, infertility, or the knowledge level of UTx. UTx was a more acceptable option than gestational surrogacy and adoption. The effects of gender, age, infertility, and the level of knowledge of UTx are important in understanding the attitude toward UTx. On the other hand, there were concerns about the safety of UTx for recipients, donors, and babies. It is important to continue to understand public attitudes to inform the development and safety of UTx, which will enhance the discussion on the ethical consensus on UTx.

Clinical aspects and management of inguinal endometriosis: A case series of 20 patients.

AIM: This study aimed to describe the clinical presentation, diagnostic evaluation, and operative or medical management of inguinal endometriosis. METHODS: In this study, we retrospectively reviewed 20 cases of inguinal endometriosis in our facility, particularly on the clinical characteristics, diagnosis, and surgical and medical treatment. RESULTS: We retrospectively investigated the following items for each patient: age at diagnosis, surgical history, presence of extragenital endometriosis, symptoms, imaging modalities, surgical therapy, hormonal therapy, follow-up period, postoperative recurrence and time to recurrence. We identified 20 cases of inguinal endometriosis in our facility. First, 75% of the patients had right inguinal endometriosis. Second, T1-weighted or fat-saturated T1-weighted images showed hyperintensity in the lesions in 17 patients (17/18 patients, 94.4%). Third, in 5 of 6 patients who underwent surgical therapy, we performed radical surgery to excise the inguinal lesion including the round ligament. One patient had disease relapse. Fourth, in 6 of 7 cases, dienogest effectively improved pain without significant adverse effects, but oral contraceptive was effective in 1 of 4 patients without significant adverse effects. CONCLUSION: We retrospectively reviewed 20 patients with inguinal endometriosis in our facility. We have shown that magnetic resonance imaging can be a useful imaging modality to obtain a specific diagnosis of this disease. In addition, inguinal endometriosis can be managed with radical surgery to resect lesions including the round ligament and with hormonal treatment. In particular, dienogest ameliorated symptoms, which can be an option for patients who do not want surgery.

The 10th Japan Bioanalysis Forum Symposium conference: a report.

The Japan Bioanalysis Forum Symposium was held on 12-14 February 2019 (Yokohama, Japan), in celebration of its 10th anniversary, and over 370 participants from pharmaceutical industries, contractors, academia and regulatory authorities from home and abroad came together in Yokohama. The 3-day symposium particularly aimed to foster collaboration with the scientists surrounding bioanalysts, according to the theme 'Open to the Public.' The symposium also included a broad range of pioneering programs, such as lectures by speakers from DMPK/metabolomics fields, discussions of future bioanalysis and poster presentations by publicly offered presenters as well as the regular ones we had organized. This report summarizes the major topics as a conference report.

Contralateral ovarian endometrioma recurrence after unilateral salpingo-oophorectomy.

BACKGROUND: The recurrence rate after unilateral salpingo-oophorectomy (USO) for unilateral endometrioma has not been reported. We evaluated the rate of and risk factors for endometrioma recurrence after USO. METHODS: In this retrospective observational study, we enrolled 110 women (age, 35-45 years) who underwent laparoscopic USO (n = 50) or cystectomy (n = 60) for unilateral ovarian endometrioma from January 2010 through December 2012. We compared patients' characteristics between patients who underwent USO and those who underwent cystectomy. We also compared patients with and without an endometrioma recurrence after USO using univariate and multivariate stepwise logistic regression models to identify recurrence risk factors. Endometrioma recurrence was defined as an ovarian cyst (> 2 cm) with features typical of an endometrioma identified by postoperative transvaginal sonography. RESULTS: Endometrioma recurred in 8 (16%) patients after USO (mean follow-up, 46.0 ± 12.9 months [range, 15-73]). The post-USO cumulative recurrence rates at 12, 24, 36, and 60 months were 8.0, 10.2, 12.7, and 24.7%, respectively (Kaplan-Meier analysis). In logistic regression analysis, a contralateral side adhesion score ≥ 4 was an independent risk factor for endometrioma recurrence after USO (odds ratio, 19.48, 95% confidence interval, 1.59-237.72). The post-USO cumulative recurrence rates at 12, 24, 36, and 57 months were 19.5, 24.1, 31.0, and 54.0%, respectively, in cases with contralateral side adhesion scores ≥4, and 0.0, 0.0, 0.0, and 5.9%, respectively, in cases with scores < 4 (log-rank test, P = 0.0023). CONCLUSIONS: To our knowledge, this is the first report on the recurrence rate and risk factors associated with recurrence after USO. Endometrioma recurrence rates were 24.7% during the first 5 years after USO. The post-USO recurrence rate increased significantly in cases with contralateral side adhesions. Our findings could improve the planning of USO and patient selection for postoperative hormonal therapy.

IFITM1 is a Novel, Highly Sensitive Marker for Endometriotic Stromal Cells in Ovarian and Extragenital Endometriosis.

When the presence of endometriotic lesions are not evident by hematoxylin and eosin staining, CD10 is used to highlight and confirm the presence of endometriotic stroma. However, CD10 is not specific only to the endometrial stroma but is also expressed in many other cells. Recently, interferon-inducible transmembrane protein 1 (IFITM1) was reported as a highly specific immunohistochemical marker of normal endometrial stroma and endometrial stromal neoplasm. In this study, we examined the expression of IFITM1 and CD10 in 18 cases of ovarian endometriosis and 44 cases of extragenital endometriosis. Among the 62 patients, 62 (100.0%) were positive for IFITM1 and 60 (96.8%) for CD10, and CD10 was negative in 2 cases that were positive for IFITM1. Additionally, we found that IFITM1 sensitivity was unaffected by the presence or absence of hormonal therapy. To the best of our knowledge, this represents the first demonstration of IFITM1 as a highly sensitive stromal marker of ovarian and extragenital endometriosis.

PAX8: A Highly Sensitive Marker for the Glands in Extragenital Endometriosis.

In cases of extragenital endometriosis or microscopic endometriosis lesions, pathological diagnosis can be challenging because endometriotic stroma and glands represent only a minor component of fibrotic endometriotic lesions. For better accuracy of diagnosis, the development of a sensitive and specific epithelial marker is beneficial. Previous studies showed that PAX8 is a highly sensitive and specific marker for primary and metastatic Mullerian epithelial tumors. Therefore, we sought to examine whether PAX8 is a highly sensitive marker for glands in extragenital endometriosis. Eight and 47 samples of ovarian endometrioma and extragenital endometriosis, respectively, were evaluated in this study. We calculated the percentage of samples positively immunostained for PAX8, CD10, estrogen receptor (ER), and progesterone receptor (PR). PAX8 was positive for endometriotic epithelial cells in 95.7% (45/47) of extragenital endometrioses and in 100% (8/8) of ovarian endometrioses. CD10 was positive for endometriotic stromal cells in 97.9% (46/47) of extragenital endometrioses. PAX8 was strongly positive for glands, even in a CD10-negative case. The expression of PAX8, CD10, and PR was not affected by preoperative hormonal therapy, and the positive rate of ER staining was significantly reduced by preoperative hormonal therapy. In conclusion, PAX8 is a highly sensitive epithelial marker for extragenital endometriosis. This specific expression was maintained under hormonal therapy. It is noteworthy that extragenital endometriosis maintains the expression of this lineage marker, although it occurs at various sites, and its cause and mechanism of development might be different. PAX8 nuclear expression can be useful in detecting extragenital endometriosis in clinical practice.

A survey of public attitudes towards third-party reproduction in Japan in 2014.

OBJECTIVE: The objective of this study was to examine public attitudes towards third-party reproduction and the disclosure of conception through third-party reproduction. METHODS: We conducted the web-based survey for the public attitude towards third-party reproduction in February 2014. Twenty-five hundred people were recruited with equal segregation of age (20s, 30s, 40s, and 50s) and gender. We analyzed the association between gender, age, infertility, and ethical view using a questionnaire regarding donor sperm, donor oocyte, donor embryo, gestational surrogacy, and disclosure to offspring. RESULTS: Of the respondents, 36.2% approved and 26.6% disapproved of gamete or embryo donation. The frequency of those who approved was lowest in females in the 50-59 year age group, and was significantly higher in males or females with infertility. Secondly, 40.9% approved and 21.8% disapproved of gestational surrogacy. The frequency of those who approved gestational surrogacy was higher in males or females with infertility. Thirdly, 46.3% of respondents agreed and 20.4% disagreed with "offspring have the right to know their origin". Those who disagreed were primarily in the 50-59 year age group of both genders, and disagreement was significantly higher in the infertility group compared with non-infertility group. CONCLUSION: In this study, public attitudes were affected by gender, age, and experience of infertility. These study findings are important in understanding the attitude towards third-party reproduction and disclosure to the offspring. Respondents having indecisive attitudes were >30%, which might indicate an increased requirement for information and education to enhance the discussion on the ethical consensus on third-party reproduction in Japan.

Draft Genome Sequencing of Ascomycetes Yeast Pichia membranifaciens KS47-1, Which Shows High Acetate Resistance in Lignocellulosic Feedstock Hydrolysate.

Pichia membranifaciens KS47-1 is capable of growing on hydrolysate containing high concentrations of acetate and other growth inhibitors. To reveal the acetate-resistant associate genes of strain KS47-1, we present the 11.4-Mb draft genome sequence.