The frequency and pathogenicity of BRCA1 and BRCA2 variants in the general Japanese population.

Hereditary breast and ovarian cancer syndrome (HBOC) resulting from pathogenic variants of BRCA1 or BRCA2 is the most common and well-documented hereditary tumor. Although founder variants have been identified in population-based surveys in various countries, the types of variants are not uniform across races and regions. Recently, the Tohoku Medical Megabank Organization (ToMMo) released whole-genome sequence data including approximately 54,000 individuals from the general population of the Tohoku area in Japan. We analyzed these data and comprehensively identified the prevalence of BRCA1/2 pathogenic and truncating variants. We believe that an accurate understanding of the unique distribution and characteristics of pathogenic BRCA1/2 variants in Japan through this analysis will enable better surveillance and intervention for HBOC patients, not only in Japan but also worldwide.

Endocrinological and Metabolic Heterogeneity Is Low in Japanese Women With Polycystic Ovary Syndrome.

OBJECTIVES: This study aims to evaluate the endocrine differences among polycystic ovary syndrome (PCOS) phenotypes in Japanese women. METHODS: 118 Japanese women that we diagnosed with PCOS agreed to be included in the study. The study group was classified into the following 4 phenotypes: (A) hyperandrogenism (HA); ovulatory disorder (OvD) and polycystic ovary morphology (PCOM); (B) HA and OvD; (C) HA and PCOM; and (D) OvD and PCOM. We also recruited 66 healthy Japanese women to the study as control participants. Age, body mass index, androgens, luteinizing hormone, follicle-stimulating hormone, and insulin resistance (IR) index were evaluated and compared. RESULTS: The proportions of phenotypes A, B, C, and D were 57/120 (47.5%), 4/120 (3.3%), 13/120 (10.8%), and 46/120 (38.3%), respectively. The proportion of phenotype B was too small; therefore, phenotypes A and B were grouped as classical PCOS for intergroup comparisons. The luteinizing hormone/follicle-stimulating hormone ratio in the classical PCOS group was higher than that in the phenotype D group (P < 0.001). Androgen concentrations in the phenotype D group were significantly lower than those in the other groups (P < 0.01). Phenotype D was more common in lean women with PCOS. The surrogate marker of IR (homeostasis model assessment of IR) was not different irrespective of PCOS and its phenotypes. CONCLUSIONS: Except for androgens, endocrine differences by PCOS phenotype are not evident, suggesting that diversity among patients with PCOS is relatively low in Japanese women.

Pregnancy-specific beta-1-glycoprotein 6 is a potential novel diagnostic biomarker of placenta accreta spectrum.

Early diagnosis is essential for the safer perinatal management of placenta accreta spectrum (PAS). We used transcriptome analysis to investigate diagnostic maternal serum biomarkers and the mechanisms of PAS development. We analyzed eight formalin-fixed paraffin-embedded placental specimens from two placenta increta and three placenta percreta cases who underwent cesarean hysterectomy at Sapporo Medical University Hospital between 2013 and 2019. Invaded placental regions were isolated from the uterine myometrium and RNA was extracted. The transcriptome difference between normal placenta and PAS was analyzed by microarray analysis. The PAS group showed markedly decreased expression of placenta-specific genes such as LGALS13 and the pregnancy-specific beta-1-glycoprotein (PSG) family. Term enrichment analysis revealed changes in genes related to cellular protein catabolic process, female pregnancy, autophagy, and metabolism of lipids. From the highly dysregulated genes in the PAS group, we investigated the expression of PSG family members, which are secreted into the intervillous space and can be detected in maternal serum from the early stage of pregnancy. The gene expression level of PSG6 in particular was progressively decreased from placenta increta to percreta. The PSG family, especially PSG6, is a potential biomarker for PAS diagnosis.

Breakpoints in complex chromosomal rearrangements correspond to transposase-accessible regions of DNA from mature sperm.

Constitutional complex chromosomal rearrangements (CCRs) are rare cytogenetic aberrations arising in the germline via an unknown mechanism. Here we analyzed the breakpoint junctions of microscopically three-way or more complex translocations using comprehensive genomic and epigenomic analyses. All of these translocation junctions showed submicroscopic genomic complexity reminiscent of chromothripsis. The breakpoints were clustered within small genomic domains with junctions showing microhomology or microinsertions. Notably, all of the de novo cases were of paternal origin. The breakpoint distributions corresponded specifically to the ATAC-seq (assay for transposase-accessible chromatin with sequencing) read data peak of mature sperm and not to other chromatin markers or tissues. We propose that DNA breaks in CCRs may develop in an accessible region of densely packaged chromatin during post-meiotic spermiogenesis.

Clinical application of long-read nanopore sequencing in a preimplantation genetic testing pre-clinical workup to identify the junction for complex Xq chromosome rearrangement-related disease.

OBJECTIVE: Xq chromosome duplication with complex rearrangements is generally acknowledged to be associated with neurodevelopmental disorders, such as Pelizaeus-Merzbacher disease (PMD) and MECP2 duplication syndrome. For couples who required a PGT-M (pre-implantation genetic testing for monogenic disease) for these disorders, junction-specific PCR is useful to directly detect pathogenic variants. Therefore, pre-clinical workup for PGT-M requires the identification of the junction of duplicated segments in PMD and MECP2 duplication syndrome, which is generally difficult. METHODS: In this report, we used nanopore long-read sequencing targeting the X chromosome using an adaptive sampling method to identify breakpoint junctions in disease-causing triplications. RESULTS: By long-read sequencing, we successfully identified breakpoint junctions in one PMD case with PLP1 triplication and in another MECP2 triplication case in a single sequencing run. Surprisingly, the duplicated region involving MECP2 was inserted 45 Mb proximal to the original position. This inserted region was confirmed by FISH analysis. With the help of precise mapping of the pathogenic variant, we successfully re-established STR haplotyping for PGT-M and avoided any potential misinterpretation of the pathogenic allele due to recombination. CONCLUSION: Long-read sequencing with adaptive sampling in a PGT-M pre-clinical workup is a beneficial method for identifying junctions of chromosomal complex structural rearrangements.

Changes in preterm and extremely preterm birth rates in Japan after the introduction of obstetrical practice guidelines in 2008.

AIM: Obstetrical guidelines were established in Japan in 2008, and obstetrical diagnoses and treatments were subsequently standardized nationally. We examined changes in the preterm birth rate (PTBR) and extremely preterm birth rate (EPTBR) following the introduction of such guidelines. METHODS: Information on 50 706 432 live births in Japan between 1979 and 2021, including Japanese reproductive medicine, the childbearing age of pregnant women, and the employment status of reproductive-age women between 2007 and 2020, were obtained from the Japanese government and academic societies. Regression analysis was used to compare chronological changes nationally and those of eight Japanese regions. Regional and national average PTBRs and EPTBRs from 2007 to 2020 were compared by using a repeated measures analysis of variance. RESULTS: From 1979 to 2007, PTBRs and EPTBRs in Japan increased significantly. However, from 2008, the national PTBR and EPTBR decreased until 2020 (p < 0.001) and 2019 (p = 0.02), respectively. From 2007 to 2020, overall PTBR and EPTBR were 5.68% and 0.255%, respectively. A significant difference in the PTBR and EPTBR existed between the eight Japanese regions. During this period, the number of pregnancies using assisted reproductive technology increased from 19 595 to 60 381, pregnant women became older, the employment rate of those of reproductive age increased, and nonregular employment was 54%, which was 2.5 times higher than for men. CONCLUSIONS: In Japan, after obstetrical guidelines were enacted in 2008, PTRBs decreased significantly even under the pressure of increasing preterm births. Countermeasures may be necessary for regions showing high PTBRs.

Target enrichment long-read sequencing with adaptive sampling can determine the structure of the small supernumerary marker chromosomes.

Structural analysis of small supernumerary marker chromosomes (sSMCs) has revealed that many have complex structures. Structural analysis of sSMCs by whole genome sequencing using short-read sequencers is challenging however because most present with a low level of mosaicism and consist of a small region of the involved chromosome. In this present study, we applied adaptive sampling using nanopore long-read sequencing technology to enrich the target region and thereby attempted to determine the structure of two sSMCs with complex structural rearrangements previously revealed by cytogenetic microarray. In adaptive sampling, simple specification of the target region in the FASTA file enables to identify whether or not the sequencing DNA is included in the target, thus promoting efficient long-read sequencing. To evaluate the target enrichment efficiency, we performed conventional pair-end short-read sequencing in parallel. Sequencing with adaptive sampling achieved a target enrichment at about a 11.0- to 11.5-fold higher coverage rate than conventional pair-end sequencing. This enabled us to quickly identify all breakpoint junctions and determine the exact sSMC structure as a ring chromosome. In addition to the microhomology and microinsertion at the junctions, we identified inverted repeat structure in both sSMCs, suggesting the common generation mechanism involving replication impairment. Adaptive sampling is thus an easy and beneficial method of determining the structures of complex chromosomal rearrangements.

Increased cervical Chlamydia trachomatis and syphilis infections in Japanese females of reproductive age in the late 2010s: Possible cause.

From 2000 to 2019, Japan's reproductive-age population gradually declined by 24%. In comparison, the Chlamydia trachomatis infection rate increased from 2016, with the syphilis infection rate increasing more sharply from 2014. Since 2013, the numbers of foreign tourists to Japan have also increased. From 2011 to 2018, the rate of increase in tourists was 5.02 times, while the rate of increase in syphilis patients was higher at 22.4 times. The lack of a one-to-one relationship between foreign tourists and syphilis cases suggests that cases of syphilis were transmitted to others. Although the prevalence of syphilis in the tourists' home countries (Korea in 2014 and China in 2013) was 20-30 times higher than that in Japan, the Japanese sex industry did not discriminate against foreign tourists, leading to increased STI infections in Japanese female sex workers. Indeed, from 2017 to 2018, a history of working in the sex industry for six months was identified as a risk factor for syphilis. The rise in Chlamydia trachomatis infections has lagged behind that of syphilis by two years, with the rate of increase lower. We suspect the difference in increasing rates of syphilis and chlamydial infections is due to the different methods of infection: syphilis can be transmitted by light physical contact, such as a kiss, whereas chlamydia requires close sexual contact, such as oral sex or sexual intercourse. Regardless, examinations and infection control are necessary to prevent the spread of STIs in Japan due to inbound tourists.

Common iliac artery dissection as a complication of common iliac artery balloon occlusion for placenta percreta: A case report.

A 37-year-old pregnant woman who had undergone three previous cesarean sections was diagnosed as having placenta percreta. We decided to perform cesarean hysterectomy with bilateral common iliac artery balloon occlusion (CIABO). The duration of surgery was 2 h and 2 min and total estimated blood loss was 2600 mL. Surgery was completed without any surgical complications, but the pulse oximeter waveform of the left leg became undetectable during surgery. We immediately performed angiography after closure of laparotomy and found abnormal pooling of contrast media at the left common iliac artery in the region in which the balloon was positioned. We made a diagnosis of left common iliac artery dissection caused by CIABO. We performed emergent revascularization by intravascular stenting. We conclude that CIABO can cause common iliac artery dissection by mechanical stimulation of the inflated balloon. Careful intraoperative evaluation of limb ischemia and preparation of intravascular treatment is needed for a safe procedure.

Less correlation between mismatch repair proteins deficiency and decreased expression of HLA class I molecules in endometrial carcinoma: a different propensity from colorectal cancer.

Mismatch repair protein deficiency (dMMR) is a favorable prognostic factor in colorectal cancer. It is also associated with aberrant expression of HLA class I molecules, which are required for cytotoxic T lymphocyte-mediated cancer immunotherapy. Because dMMR is frequently also found in endometrial cancers (ECs), we retrospectively investigated the expression of mismatch repair proteins and HLA class I molecules in 127 EC patients. In this study, EC patients being treated in our hospital were recruited from 2005 to 2009 and observed until December 2017. Lesion specimens were evaluated via immunohistochemistry for MSH6 and PMS2 (mismatch repair proteins) and HLA class I molecules. Expression of these molecules was statistically related to clinical and pathological factors and prognosis. dMMR was detected in 33 patients and did not correlate with the expression level of HLA class I molecules (P = 0.60). On the other hand, unexpectedly, multivariate analysis revealed that intact expression of HLA class I molecules was associated with p53 overexpression (P = 0.004). Neither dMMR nor decreased expression of HLA class I molecules were prognostic factors. These results are inconsistent with previous findings for colorectal cancer. A distinctive local tissue immune microenvironment would underlie the discrepancy in the results between EC and colorectal cancer.

Next-generation sequencing of 16S rRNA for identification of invasive bacterial pathogens in a formalin-fixed paraffin-embedded placental specimen: a case report of perinatal fulminant Streptococcus pyogenes infection.

Intrauterine infection is one of the most important causes of maternal death. In perinatal emergency, we often miss an opportunity to obtain culture specimens. In this study, we tried to examine whether we investigated whether bacteria causing infection can be detected from a formalin-fixed paraffin-embedded (FFPE) placental specimen. We examined the placenta from a maternal invasive infection that resulted in infectious abortion at 18 weeks of gestation. The case was diagnosed by acute fever and abdominal pain, and the patient was cured after 3 weeks of intensive antimicrobial treatment. Four Streptococcus pyogenes strains were isolated from vaginal fluid and blood cultures of the patient. All of the strain types were emm1/ST28. We amplified the V1-V2 region of 16S rRNA from an FFPE placental specimen and sequencing was performed using a next-generation sequencer (NGS). Taxonomic analysis was then performed for sequenced data. We succeeded in detecting causative pathogens from the FFPE placenta: 69.1% of the predominantly identified bacteria were S. pyogenes and other small populations of bacteria were detected. Our results revealed the utility of NGS for 16S rRNA analysis of an FFPE placenta. This method may reveal previous perinatal invasive infections of unknown origin retrospectively.

Pregnancies after vaginal radical trachelectomy (RT) in patients with early invasive uterine cervical cancer: results from a single institute.

BACKGROUND: Radical tracheletomy (RT) with pelvic lymphadenectomy has become an option for young patients with early invasive uterine cervical cancer who desire to maintain their fertility. However, this operative method entails a high risk for the following pregnancy due to its radicality. METHODS: We have performed vaginal RT for 71 patients and have experienced 28 pregnancies in 21 patients. They were followed up carefully according to the follow-up methods we reported previously. Their pregnancy courses and prognoses after the pregnancy were retrospectively reviewed. RESULTS: All the vaginal RTs were performed safely without serious complications, including 6 patients who underwent the operation during pregnancy. The median time to be pregnant after RT was 29.5 months. 13 patients (46%) became pregnant without artificial insemination by husband or assisted reproductive technology. Cesarean section was performed for all of them. The median time of pregnancy was 34 weeks, and emergent cesarean section was performed for 7 pregnancies (25%). The median birth weight was 2156 g. Four patients had trouble with cervical cerclage, and they suffered from sudden premature preterm rupture of the membrane (pPROM) during the second trimester of pregnancy. We underwent transabdominal cerclage (TAC) for all of them and careful management for the prevention of uterine infection was performed. One patient had a recurrence of cancer during pregnancy. CONCLUSIONS: Both the obstetrical prognosis and oncological prognosis after vaginal RT have become favorable for pregnant patients after vaginal RT.

Clonal analysis revealed functional heterogeneity in cancer stem-like cell phenotypes in uterine endometrioid adenocarcinoma.

Uterine endometrial carcinoma is one of the common cancers in females. Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are a small subpopulation of cancer cells that are tumorigenic and are resistant to treatments, thus they are focused as treatment targets. However, the heterogeneity of CSCs/CICs is still elusive, and we therefore analyzed CSCs/CICs at the clonal level. We previously established sphere-cultured CSCs/CICs from primary human uterine endometrial carcinoma, and we isolated several clones from CSCs/CICs in this study. Interestingly, we established two types of clones based on the growth pattern. The clones were termed sphere clones (S clones) and leukemia-like clones (LL clones). Functional analysis revealed that S clones are resistant to chemotherapy, whereas LL clones are sensitive to chemotherapy. On the other hand, S clones are less tumorigenic, while LL clones are highly tumorigenic. Transcriptome analysis using serial analysis of gene expression sequencing (SAGE-Seq) revealed distinctive gene expression profiles in S clone cells and LL clone cells. The results indicate that CSCs/CICs are composed of functionally heterogenic subpopulations including highly tumorigenic clones and treatment-resistant clones and that the characteristics of CSCs/CICs might be determined by the characteristics of different clones that compose CSCs/CICs.

Obstetrical prognosis of patients who underwent vaginal radical trachelectomy during pregnancy.

AIM: Radical trachelectomy (RT) with pelvic lymphadenectomy has become an option for young patients with early invasive uterine cervical cancer who decide to maintain their fertility. However, this operative method entails a high risk for the following pregnancy due to its radicality. Therefore, RT for pregnant patients can be a challenge both for gynecologic oncologists and obstetricians. METHODS: We have performed vaginal RT for five pregnant patients with uterine cervical cancer stage 1B1 according to the method of Dargent et al. The operations were performed between 16 and 26 weeks of pregnancy, and the patients were followed up carefully according to the follow-up methods we reported previously. RESULTS: Vaginal RT was performed for five patients without any troubles. Four of the patients continued their pregnancies until almost 34 weeks or longer under our previously published follow-up schedule. The pregnancy of one patient was terminated at 26 weeks due to recurrence of the cancer. CONCLUSION: Expansion of vaginal RT for pregnant patients with uterine cervical cancer could be a practical option for pregnant patients with early invasive uterine cervical cancer.

Virological and cytological clearance in laser vaporization and conization for cervical intra-epithelial neoplasia grade 3.

AIM: Cervical intra-epithelial neoplasia (CIN) is the precancerous stage of cervical cancer. Standard treatment for high-grade CIN is conization of the cervix. The risk of preterm birth following conization has been discussed recently. In contrast, laser vaporization is believed not to affect perinatal outcome, but the long-term effectiveness of each surgical procedure is still unclear. The aim of this prospective unmatched-cohort study was therefore to compare virological and cytological clearance and recurrence risk between conization and vaporization for CIN3. METHODS: Subject consisted of CIN3 patients treated at the present hospital between 2007 to 2011 and followed up until December 2014. One hundred and one patients were treated with laser conization, and 137 with vaporization. The surgical procedure was selected on the basis of colposcopy, pathological grade and patient's hope for pregnancy. RESULTS: There were no significant differences in cure rate, human papilloma virus (HPV) clearance rate or recurrence rates between the conization and vaporization groups. Risk ratio of recurrence for each surgical procedure adjusted for age and HPV persistence status were analyzed on Cox proportional hazards modeling. Recurrence risk ratio for patients treated by vaporization was 6.21 (95%CI: 0.65-59.19; P = 0.111) compared with conization and there were no significant differences. No adverse pregnancy outcome was observed in the vaporization group compared with conization. CONCLUSIONS: Laser vaporization is useful for young patients with CIN3 who hope for pregnancy in the future.

Fibroblasts induce expression of FGF4 in ovarian cancer stem-like cells/cancer-initiating cells and upregulate their tumor initiation capacity.

Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are defined as a small population of cells within cancer that contribute to cancer initiation and progression. Cancer-associated fibroblasts (CAFs) are stromal fibroblasts surrounding tumor cells, and they have important roles in tumor growth and tumor progression. It has been suggested that stromal fibroblasts and CSCs/CICs might mutually cooperate to enhance their growth and tumorigenic capacity. In this study, we investigated the effects of fibroblasts on tumor-initiating capacity and stem-like properties of ovarian CSCs/CICs. CSCs/CICs were isolated from the ovarian carcinoma cell line HTBoA as aldehyde dehydrogenase 1 high (ALDH1(high)) population by the ALDEFLUOR assay. Histological examination of tumor tissues derived from ALDH1(high) cells revealed few fibrous stroma, whereas those derived from fibroblast-mixed ALDH1(high) cells showed abundant fibrous stroma formation. In vivo tumor-initiating capacity and in vitro sphere-forming capacity of ALDH1(high) cells were enhanced in the presence of fibroblasts. Gene expression analysis revealed that fibroblast-mixed ALDH1(high) cells had enhanced expression of fibroblast growth factor 4 (FGF4) as well as stemness-associated genes such as SOX2 and POU5F1. Sphere-forming capacity of ALDH1(high) cells was suppressed by small-interfering RNA (siRNA)-mediated knockdown of FGFR2, the receptor for FGF4 which was expressed preferentially in ALDH1(high) cells. Taken together, the results indicate that interaction of fibroblasts with ovarian CSCs/CICs enhanced tumor-initiating capacity and stem-like properties through autocrine and paracrine FGF4-FGFR2 signaling.

BAG3 upregulates Mcl-1 through downregulation of miR-29b to induce anticancer drug resistance in ovarian cancer.

OBJECTIVE: Ovarian cancer is the leading cause of death from gynecologic cancer, reflecting its often late diagnosis and its chemoresistance. We identified a set of microRNAs whose expression is altered upon BAG3 knockdown. Our primary objective was to examine the relationships between BAG3, miR-29b and Mcl-1, an antiapoptotic Bcl-2 family protein, in ovarian cancer cells. METHODS: Ovarian cancer cells were cultured and their responsiveness to paclitaxel was tested. Microarray analysis was performed to identify microRNAs differentially expressed in ES2 BAG3 knockdown ovarian cancer cells and their control cells. Primary ovarian cancer tissues were obtained from 56 patients operated on for ovarian cancer. The patients' clinical and pathological data were obtained from their medical records. RESULTS: BAG3 knockdown increased the chemosensitivity to paclitaxel of ES2 ovarian clear cell carcinoma cells to a greater degree than AMOC2 serous adenocarcinoma cells. qRT-PCR analysis showed that miR-29b expression was significantly upregulated in primary cancer tissue expressing low levels of BAG3, as compared to tissue expressing high levels. Moreover, levels of miR-29b correlated significantly with progression-free survival. Upregulation of miR-29b also reduced levels of Mcl-1 and sensitized ES2 cells to low-dose paclitaxel. CONCLUSIONS: BAG3 knockdown appears to downregulate expression of Mcl-1 through upregulation of miR-29b, thereby increasing the chemosensitivity of ovarian clear cell carcinoma cells. This suggests that BAG3 is a key determinant of the responsiveness of ovarian cancer cells, especially clear cell carcinoma, to paclitaxel and that BAG3 may be a useful therapeutic target for the treatment of ovarian cancer.

Placenta Percreta Progression to Resistance Against Uterine Artery Embolization and Penetration Into the Bladder.

A 31-year-old female sought termination of pregnancy due to a fetal body stalk anomaly diagnosed at 18 weeks of gestation. Despite an anterior placenta previa, successful vaginal delivery occurred. However, placental adhesion over a previous cesarean scar occurred, and part of the placenta could not be removed. Immediate postpartum bleeding prompted imaging studies, revealing extravasation from adherent placental remnants. Uterine artery embolization (UAE) provided initial hemostasis, but recurrent bleeding necessitated re-embolization. Although conservative treatment was initially pursued, significant hematuria prompted reevaluation, revealing extensive uterine wall and bladder penetration. Surgical intervention with total hysterectomy and partial bladder resection was performed, leading to the successful recovery of bladder function following surgical repair. While this case achieved a positive outcome, there is a potential for permanent urinary dysfunction if lesions are more extensive. While achieving a conservative cure is ideal, it is essential to assess the timing for opting for surgical intervention.