Guideline for gynecological practice in Japan: Japan Society of Obstetrics and Gynecology and Japan Association of Obstetricians and Gynecologists 2023 edition.

Twelve years after the first edition of The Guideline for Gynecological Practice, which was jointly edited by The Japan Society of Obstetrics and Gynecology and The Japan Association of Obstetricians and Gynecologists, the 5th Revised Edition was published in 2023. The 2023 Guidelines includes 5 additional clinical questions (CQs), which brings the total to 103 CQ (12 on infectious disease, 30 on oncology and benign tumors, 29 on endocrinology and infertility and 32 on healthcare for women). Currently, a consensus has been reached on the Guidelines, and therefore, the objective of this report is to present the general policies regarding diagnostic and treatment methods used in standard gynecological outpatient care that are considered appropriate. At the end of each answer, the corresponding Recommendation Level (A, B, C) is indicated.

Short-term efficacy and safety of early medical abortion in Japan: A multicenter prospective study.

Purpose: To evaluate the short-term efficacy and safety of a combined mifepristone-misoprostol regimen in individuals seeking medical abortion at up to 63 days of gestational age. Methods: This open-label, multicenter, prospective study evaluated the short-term efficacy and safety of medical abortion, with the primary outcome being the abortion success rate 24 h after misoprostol administration. The participants received 200 mg of mifepristone orally and 800 µg of misoprostol buccally in the hospital/clinic 36-48 h later. Bleeding and lower abdominal pain, which are the main symptoms associated with medical abortion, were recorded. Results: The abortion success rate was 93.3% (95% confidence interval [CI]: 87.3-97.1%) within 24 h of misoprostol administration, 63.3% (95% CI: 54.05-71.94%) within 4 h, and 90.0% (95% CI: 83.18-94.73%) within 8 h. The median time from misoprostol administration to a successful abortion was 3.93 h. Bleeding was most commonly observed 0-4 h prior to the confirmation of gestational sac (GS) expulsion. The most intense lower abdominal pain occurred 0-1 h before the confirmation of GS expulsion. Conclusion: The combined regimen of mifepristone and buccal misoprostol for medical abortion showed short-term efficacy and a favorable safety profile.

Clinical usefulness of cell-free and concentrated ascites reinfusion therapy (CART) in combination with chemotherapy for malignant ascites: a post-marketing surveillance study.

BACKGROUND: Cell-free and concentrated ascites reinfusion therapy (CART) has been suggested to be able to treat malignant ascites more safely and effectively with chemotherapy because of its ability to retain serum protein and albumin. Although the characteristics of cancer types and CART and the clinical implications of combination therapy with antitumor agents are becoming widespread, there are limited reports on its efficacy and complications. METHODS: In this prospective observational national post-marketing study, 128 patients with malignancies received 300 CART sessions at 22 centers. After excluding other malignancies, the patients were divided into four groups: gynecological malignancies with chemotherapy (GYC+; 18 cases and 36 times) and without chemotherapy (GYC-; 35 cases and 52 times), and gastrointestinal malignancies with chemotherapy (GIC+; 8 cases and 16 times) and without chemotherapy (20 cases and 58 times). RESULTS: There were significant reductions in the body weight in all groups and significant reductions in abdominal circumference and significant improvements in the diet and Eastern Cooperative Oncology Group performance status only in the GYC+ group. The total serum protein and albumin increased significantly in all groups, except for the GIC+ group, before and after CART. There was no significant difference in the presence or absence of antitumor medication. CONCLUSION: With CART, there were differences in the improvement of the clinical symptoms between malignancy groups. The combination of CART and antineoplastic agents may be as safe as CART alone in cases of exudative malignant ascites.

Guideline for Gynecological Practice in Japan: Japan Society of Obstetrics and Gynecology and Japan Association of Obstetricians and Gynecologists 2020 edition.

Nine years after the first edition of The Guideline for Gynecological Practice, which was jointly edited by The Japan Society of Obstetrics and Gynecology and The Japan Association of Obstetricians and Gynecologists, the 4th Revised Edition was published in 2020. The 2020 Guidelines includes 4 additional clinical questions (CQ), which brings the total to 99 CQ (12 on infectious disease, 29 on oncology and benign tumors, 29 on endocrinology and infertility and 29 on healthcare for women). Currently, a consensus has been reached on the Guidelines, and therefore, the objective of this report is to present the general policies regarding diagnostic and treatment methods used in standard gynecological outpatient care that are considered appropriate. At the end of each answer, the corresponding Recommendation Level (A, B, C) is indicated.

Evaluation of cellular adequacy in endometrial liquid-based cytology.

OBJECTIVE: This study evaluated cellular adequacy in endometrial liquid-based cytology (LBC) specimens. METHODS: In total, 1267 cases were obtained and the rate of unsatisfactory specimen and diagnostic accuracy for malignancy were assessed. If ≥10 cellular clusters composed of ≤30 endometrial cells were found per specimen, then the sample was provisionally considered adequate. RESULTS: The unsatisfactory rate (with fewer than 10 clusters) was 15.4%. Diagnostic accuracy in specimens with ≥10 clusters was significantly higher (90.5% vs 36.4%) than that in specimens with fewer than10 clusters. Moreover, the unsatisfactory rate in patients aged ≥60 years was significantly higher (33.8% vs 13.2%) than that in patients younger than 60 years. Although the unsatisfactory rate was decreased, significant differences were not found between cases with fewer than five clusters (22.6%) and fewer than 10 clusters (33.8%) in patients aged ≥60 years. Diagnostic accuracy in cases with five or more clusters was significantly higher (90.3% vs 0%) than that in cases with fewer than five clusters. CONCLUSIONS: We propose that ≥10 clusters with ≥30 endometrial cells per cluster could be used as a specimen adequacy criterion for endometrial LBC. If ≥10 clusters cannot be found in patients aged ≥60 years, then the use of the alternative criterion of five or more clusters may yield satisfactory specimen adequacy.

Guidelines for office gynecology in Japan: Japan Society of Obstetrics and Gynecology (JSOG) and Japan Association of Obstetricians and Gynecologists (JAOG) 2017 edition.

Six years after the first edition of The Guideline for Gynecological Practice, which was jointly edited by The Japan Society of Obstetrics and Gynecology and The Japan Association of Obstetricians and Gynecologists, the third revised edition was published in 2017. The 2017 Guidelines includes 10 additional clinical questions (CQ), which brings the total to 95 CQ (12 on infectious disease, 28 on oncology and benign tumors, 27 on endocrinology and infertility and 28 on healthcare for women). Currently a consensus has been reached on the Guidelines and therefore the objective of this report is to present the general policies regarding diagnostic and treatment methods used in standard gynecological outpatient care that are considered appropriate. At the end of each answer, the corresponding recommendation level (A, B, C) is indicated.

Possible Application of Ascites-infiltrating Gamma-delta T Cells for Adoptive Immunotherapy.

BACKGROUND/AIM: Malignant ascites contain many tumour-infiltrating lymphocytes. γδ T cells with antitumour activity have attracted attention as effector cells in cancer immunotherapy. Vδ2+ T cells were cultured from peripheral blood mononuclear cells (PBMCs) and ascites-infiltrating lymphocytes (AILs) to compare the differences in response to 2-methyl-3-butenyl-1-pyrophosphate (2M3B1-PP) and zoledronate (Zol) as antigens in vitro. MATERIALS AND METHODS: To expand Vδ2+ T cells from PBMCs and AILs from 29 patients with cancer, these cells were cultured and subjected to analysis. RESULTS: The proliferation rate of Vδ2+ T cells was higher in both PBMCs and AILs when cultured with Zol than with 2M3B1-PP. Although Vδ2+ T cells show a higher rate of expansion in AILs compared to PBMCs, the number of mixed tumour cells in ascites was decreased when cultured with Zol. CONCLUSION: Vδ2+ T cells in AILs are cytotoxic to tumour cells in ascites and may be considered in adoptive immunotherapy.

Endometrial regeneration using cell sheet transplantation techniques in rats facilitates successful fertilization and pregnancy.

OBJECTIVE: To regenerate functional endometrium tissue using "cell sheet" techniques as a regenerative medicine approach to address endometrial disorders causing female factor infertility. DESIGN: In vivo experimental study. SETTING: Preclinical surgical and biomedical research laboratories. ANIMAL(S): Green fluorescent protein (GFP) transgenic rats [SD-Tg (CAG-EGFP) rats] and nude rats (F344/NJcl-rnu/rnu). INTERVENTION(S): GFP-positive rat uterine-derived cells as cell sheets were transplanted into resected rat uterine endometrial sites. Transplanted cell sheet areas were then analyzed using macroscopic observations and histological analysis including immunohistochemistry. Subsequently, crossbreeding was performed to establish fertility and confirm pregnancy in the rat-regenerated uterus. MAIN OUTCOME MEASURE(S): Morphologic and biochemical markers of regenerated endometrium and establishment of pregnancy in otherwise sterile animals. RESULT(S): After cell sheet transplantation, regenerated endometrium was confirmed as GFP-positive tissue engraftment both visually and under histological analysis. After crossbreeding, GFP-positive tissue areas and living fetuses were observed in the transplantation group. CONCLUSION(S): Cell sheet transplantation can regenerate endometrial tissue with histological structure and physiological function supporting pregnancy similar to normal endometrial tissue. Translation of this endometrial cell sheet transplantation method to human patients with endometrial disorders could yield a novel therapy for uterine infertility.

Safety and efficacy of cell-free and concentrated ascites reinfusion therapy (CART) in refractory ascites: Post-marketing surveillance results.

We performed post-marketing surveillance to evaluate the safety and efficacy of cell-free and concentrated ascites reinfusion therapy (CART). In total, 356 CART sessions in 147 patients at 22 centers were performed. The most common primary disease was cancer (128 cases, 300 sessions). Mean amount of ascites collected was 3.7 L, and mean concentration ratio was 9.2. Mean amount of reinfused protein was 67.8 g (recovery rate, 72.0%). Performance status, dietary intake, urine volume, body weight and abdominal circumference were significantly improved after CART. Body temperature increased significantly, by 0.3°C on average. Concomitant steroids and/or NSAIDs use before reinfusion was significantly and negatively associated with increases in body temperature. Most adverse events were fever and chills. This study examined a large number of patients compared with previous studies, and showed that CART is an effective and relatively safe treatment for refractory ascites, such as malignant ascites.

Liquid-Based Endometrial Cytology Using SurePath™ Is Not Inferior to Suction Endometrial Tissue Biopsy in Clinical Performance for Detecting Endometrial Cancer Including Atypical Endometrial Hyperplasia.

OBJECTIVE: We evaluated the clinical performance of liquid-based endometrial cytology (SurePath™) for detecting endometrial malignancies by comparison with the performance of suction endometrial tissue biopsy. STUDY DESIGN: From November 2011 to May 2013, we consecutively collected 1,118 liquid-based endometrial cytology specimens and 674 suction endometrial tissue biopsy specimens. RESULTS: The rate of nonpositive final histology in nonpositive liquid-based endometrial cytology (98.2%) was higher than the rate of nonpositive final histology in nonpositive suction endometrial tissue biopsy (97.0%). None of the clinical performance values of liquid-based endometrial cytology for detecting the endometrial malignancies were statistically inferior to those of the suction endometrial tissue biopsy. When the positivity threshold was more than "atypical endometrial cells of undetermined significance," the rate of positive liquid-based endometrial cytology from cases with a positive final histology (84.5%) was higher than the rate of positive suction endometrial tissue biopsy from cases with a positive final histology (69.8%). However, there were still no significant differences among all the performance values. CONCLUSIONS: Our liquid-based endometrial cytology would be more appropriate in various clinical situations as the initial detection tool for endometrial malignancies, rather than suction endometrial tissue biopsy. In addition, it could be used in screening for endometrial malignancies on a broader scale.

Gorlin syndrome with an ovarian leiomyoma associated with a PTCH1 second hit.

We describe a Gorlin syndrome (GS) case with two different second hit mutations of PTCH1, one in a keratocystic odontogenic tumor (KCOT) and the other in an ovarian leiomyoma. GS is a rare genetic condition manifesting as multiple basal cell nevi associated with other features such as medulloblastomas, skeletal abnormalities, and ovarian fibromas. A 21-year-old Japanese woman with a history of two KCOTs was diagnosed with GS according to clinical criteria. A PTCH1 mutation, c.1427del T, was detected in peripheral blood. A novel PTCH1 mutation, c.264_265insAATA, had been found in the maxillary KCOT as a second hit mutation. More recently, the ovarian tumor was detected during a gynecological examination. Laparoscopic adnexectomy was performed, and the pathological diagnosis of the ovarian tumor was leiomyoma. Interestingly, another novel mutation, loss of heterozygosity spanning from 9q22.32 to 9q31.2, including PTCH1 and 89 other genes, was detected in this ovarian tumor, providing evidence of a second hit mutation. This is the first report describing a GS-associated ovarian tumor carrying a second hit in the PTCH1 region. We anticipate that accumulation of more cases will clarify the importance of second hit mutations in ovarian tumor formation in GS.

Preventive effect of oral mucosal epithelial cell sheets on intrauterine adhesions.

STUDY QUESTION: Can regenerative-medicine techniques using oral mucosal epithelial cell sheets (OMECS) provide a new treatment method for intrauterine adhesions (IUA) which cause female infertility? SUMMARY ANSWER: Transplantation of OMECS was confirmed to be effective in preventing IUA after endometrial damage in rats. WHAT IS KNOWN ALREADY: Uterine disorders such as IUA, commonly known as Asherman's syndrome, are one factor that can result in infertility. Clinical therapy for this kind of disease is targeted at the prevention of re-adhesion by surgical synechiotomy, administration of hormones after the operation, and the use of intrauterine devices. Recently, a new approach called 'cell-sheet engineering', which harvests confluent culture cells as a contiguous cell sheet having intact cell-cell junctions and an extracellular matrix, without having to use enzymatic treatment, has been developed for tissue regeneration. STUDY DESIGN, SIZE, DURATION: OMECS were prepared from rat oral mucosal tissues. An IUA model was made in rat uteri, and OMECS were transplanted into the model. Uteri transplanted with OMECS were compared with the non-transplanted control uteri by histological analysis at 1, 2 and 8 days after surgery (n = 3). PARTICIPANTS/MATERIALS, SETTING, METHODS: Oral mucosal tissues were resected from neonatal rats, and oral mucosal epithelial cells were collected with enzymatic treatment. An isolated cell suspension was seeded on a temperature-responsive cell culture-insert and incubated. After being detached from the insert, a cell sheet was transplanted onto the endometrium defect. At 1, 2 and 8 days after surgery, uteri were resected and examined. MAIN RESULTS AND THE ROLE OF CHANCE: Histological examination of the non-treated specimens at 1, 2 and 8 days after surgery did not show any uterine cavities typically caused by IUA. In contrast, the histology of uteri transplanted with OMECS immediately after endometrial damage showed the presence of uterine cavities, and furthermore, stratified squamous epithelial cells on the luminal surface (n = 3). LIMITATIONS, REASONS FOR CAUTION: The results of this study are difficult to apply directly to humans, because the structure and function of rat uteri are different from those of human. WIDER IMPLICATIONS OF THE FINDINGS: Transplantation of OMECS offers a reliable method not only to protect the woman's fertility from intrauterine re-adhesion after synechiotomy for IUA or uterine lumen adhesion but also to prevent adhesion after any intrauterine surgery in clinical cases.

Reconstruction of functional endometrium-like tissue in vitro and in vivo using cell sheet engineering.

Uterus is a female specific reproductive organ and plays critical roles in allowing embryo to grow. Therefore, the endometrial disorders lead to female infertility. Hence, the regeneration of endometrium allowing fertilized ovum to implant might be valuable in the field of fertility treatment. Recently, cell sheet engineering using a temperature-responsive culture dish has advanced in regenerative medicine. With this technology, endometrial cells were harvested as a contiguous cell sheet by reducing temperature. Firstly, mouse endometrial cell sheets were re-cultured for 3 days to evaluate the function. Histological analyses revealed that endometrial epithelial cell-specific cytokeratin 18 and female-specific hormone receptors, estrogen receptor ß and progesterone receptor, were expressed. Furthermore, endometrial epithelial cells constructed epithelial layer at the apical side. Then, endometrial cell sheets from green-fluorescent-protein rat cells were transplanted onto the buttock muscle of nude rat for evaluating the function in vivo. Histological analyses showed that endometrial cell sheets reconstructed endometrium-like tissue, which was found to form uterus-specific endometrial glands having hormonal receptor to estrogen. In this study, endometrial cell sheets were speculated to contribute to the regeneration of functional endometrium as a new therapy.

An SNP in CYP39A1 is associated with severe neutropenia induced by docetaxel.

PURPOSE: Docetaxel is one of the most widely used chemotherapy drugs for gynecological cancers. A dose-limiting factor of docetaxel is severe neutropenia, and previous reports showed that grade 4 neutropenia was observed in approximately 70% of Japanese patients treated with docetaxel. In order to elucidate a valid biomarker for docetaxel-induced neutropenia, we analyzed 42 Japanese patients with gynecological cancers such as ovarian cancer and endometrial cancer of the uterus. METHODS: As a first step, AUC of docetaxel was examined in 10 patients and 1,936 SNPs of 225 genes were genotyped using DMET Plus™ genotyping systems. RESULTS: The first screening revealed that 28 SNPs were associated with the AUC (P < 0.05), and we analyzed the associations between the 28 SNPs and neutrophil counts in the other 32 patients, with the result that CYP39A1 (rs7761731) was found to be the only SNP significantly associated (P = 0.049 OR = 9.0) with the incidence of grade 4 neutropenia among 28 SNPs. CONCLUSIONS: This SNP in CYP39A1 may be a useful biomarker for predicting the risk of docetaxel-induced neutropenia.

[A case of malignant peritoneal mesothelioma successfully treated with concurrent chemoradiotherapy].

Malignant peritoneal mesothelioma is extremely rare, and its prognosis is poor. The median survival period is said to be approximately one year after diagnosis. We report a case of recurrent malignant peritoneal mesothelioma treated with concurrent chemoradiotherapy (CCRT). The patient has been alive for six years without recurrence. This report seems to be the first that indicates CCRT to be useful for peritoneal mesothelioma. The patient was a 21-year-old woman who underwent emergency surgery of the in acute abdomen at another hospital. The resected tumor was 18 cm in size and pathological examination revealed that it was a malignant mesothelioma of the epithelioid type. CAP therapy (cyclophosphamide+adriamycine+cisplatin)+CPT-11 administration was given only one course, and the patient was then transferred to our hospital. She underwent resection of the residual disease and six courses of TC therapy (paclitaxel+carboplatin) as adjuvant chemotherapy. Twelve months after chemotherapy, pelvic recurrence occurred. We attempted surgery but only biopsy could be performed because of a pelvic wall invasion. The patient underwent CCRT with weekly cisplatin. The tumor was reduced by irradiation of 50. 4 Gy and disappeared after 6 months. No recurrence has been found six years since the last treatment. CCRT might be effective against malignant peritoneal mesothelioma of the epithelioid type.

Novel virtual cytological analysis for the detection of endometrial cancer cells using autoscan fluoromicroscopy.

The current medical examinations for detecting endometrial cancer can sometimes be stressful and inconvenient for examinees and examiners. Therefore, we attempted to develop an autoscan-virtual cytology system for detecting endometrial cancer without relying on judgment by the human eye. Exfoliated cells from the uterus were retrieved using a tampon inserted for 3 h. More than 100 monoclonal antibodies (mAb) developed by us were screened in three steps of immunohistochemistry to find mAb sets that would enable the cancer and normal endometrium to be perfectly distinguished. The exfoliated cells provided by 30 endometrial cancer patients and a total of 37 samples of 14 non-malignant volunteers including the menstrual cycle were analyzed using imaging cytometry. All samples contained epithelial cells and dysplasia cells, but the pathologist could not definitively diagnose all of them as endometrial cancer cells because most cells had degenerated. Twenty-two of 28 endometrial cancer tissues (79%) were positive with four mAb sets, CRELD1, GRK5, SLC25A27 and STC2, and 22 of 22 normal endometriums (100%) were negative. Our newly developed autoscan-virtual cytology for exfoliated endometrial cells showed overall sensitivity for endometrial cancer patients and overall specificity for volunteers of 50% (15/30) and 95% (35/37), respectively. Our autoscan-virtual cytology combined with cancer-specific mAb and imaging cytometry could be useful for endometrial cancer detection. Autoscan-virtual cytology for endometrial cancer deserves further evaluation for future endometrial cancer screening.

Familial correlation of bone mineral density, birth data and lifestyle factors among adolescent daughters, mothers and grandmothers.

This study aimed to clarify the relationship between skeletal or lifestyle factors among Japanese daughter-mother, mother-grandmother and daughter-grandmother pairs. We performed a cross-sectional study in a cohort of Japanese adolescent daughters (12-18 years of age), their mothers (339 pairs) and grandmothers on their mothers' side (34 pairs). Gestational age, birth weight, age at menarche and presence of menarche or menopause were surveyed in the participants. Height, body weight and lumbar 2-4 bone mineral density (BMD) were measured. Dietary intake and current physical activity were assessed by using questionnaires. Gestational age and age at menarche were significantly correlated among daughters, mothers and grandmothers (P < 0.001). BMD was significantly correlated between daughters and mothers (P < 0.001), while it was not significantly correlated between daughters and grandmothers or between mothers and grandmothers. Dietary intake of calcium and vitamin D, and the frequency, duration and intensity of current physical activity were significantly correlated between daughters and mothers (P < 0.05), although no significant correlation was found between daughters and grandmothers, or between mothers and grandmothers. The parameters for exercise indicated a positive correlation for BMD in the daughters and the mothers, but not in the grandmothers. The results suggested that estrogen deficiency decreases familial correlation for BMD after menopause. Achieving high BMD through exercise may be important for prevention of postmenopausal osteoporosis in premenopausal low-height mothers.

Impact of dietary intake, education, and physical activity on bone mineral density among North Indian women.

The aim of the study was to determine the relationship of dietary nutrients and bone mineral density (BMD) in North Indian women. This cross-sectional study was conducted from April 2006 to March 2008. Subjects included 255 healthy women, aged 20-69 years, who were relatives of patients being admitted in the hospital. Various demographic characteristics including socioeconomic status and serum parameters in relationship to BMD were evaluated. In addition, the daily dietary intake of energy, protein, fat, and calcium and the amount of physical activity were assessed. BMD at the lumbar spine, femoral neck, and Ward's triangle was measured by dual-energy X-ray absorptiometry (DXA). Body mass index (BMI), physical activity, and educational level were positively correlated with BMD. The daily intakes of energy (1563.4 +/- 267.2 kcal) and protein (48.7 +/- 8.7 g) were below the recommended dietary allowance. Daily dietary energy, protein, and calcium intakes were correlated with BMD at the lumbar spine. Stepwise multiple linear regression analyses showed that age, BMI, and physical activity were significant predictors for BMD at all sites. In addition, energy intake was also a predictor for BMD at the lumbar spine. The protein intake was associated with BMD at the spine (P = 0.02 and beta = 0.163) even after making adjustments for energy intake. Thus, dietary pattern coupled with higher education levels and greater physical activity favored bone health.

[Usefulness of FRAX and future issues in women's health].

With the recent development of diverse therapeutic modalities for osteoporosis, there is an increasingly recognized need to develop cost-effective strategies to determine which patients will benefit most from pharmacologic treatment. The World Health Organization (WHO) has developed a 10-year osteoporotic fracture risk assessment tool (FRAX) based on clinical risk factors combined with bone mineral density measured at the femoral neck. In postmenopausal women, age and prior history of fragility fracture were found to be major contributing factors to osteoporotic fracture risk as assessed by FRAX. In the US, the National Osteoporosis Foundation (NOF) guidelines recommend treatment in patients with osteopenia when the 10-year probability of major osteoporotic fracture is = 20% or the hip fracture is = 3%. On the other hand, in the UK, the National Osteoporosis Guideline Group (NOGG) has developed a case finding approach for individuals whose 10-year probability of major osteoporotic fracture exceeds age-specific thresholds. The FRAX algorithms may be worth incorporating in a cost-effectiveness model for socioeconomic analyses to determine the pharmacological intervention threshold for osteoporosis treatment. FRAX represents a significant advance for practitioners in women's health as an easy-to-use calculator of osteoporotic fracture probability. However, it should be noted that FRAX is intended as a guideline for clinical decision making only and does not force the decision to treat on physicians.

Variations in circulating osteoprotegerin and soluble RANKL during diurnal and menstrual cycles in young women.

BACKGROUND/AIMS: Physiological bone turnover shows diurnal variations and changes within the menstrual cycle. The aim of this study was to assess the variability of osteoprotegerin (OPG) and soluble RANKL (sRANKL) serum levels during diurnal and menstrual cycles. METHOD: Blood was collected from 15 young women at 6-hour intervals between 08.00 and 20.00 h during the follicular phase. Moreover, to compare the follicular and luteal phases, blood was also collected at 14.00 h during the luteal phase. Serum bone-specific alkaline phosphatase (BAP), N-telopeptide of type I collagen (NTX), OPG and free sRANKL were measured. RESULTS: No diurnal variations in BAP, OPG, sRANKL and sRANKL/OPG ratio were detected. NTX was significantly higher in the morning than in the afternoon and at night (p = 0.02). There were no menstrual variations in either. CONCLUSIONS: The consistent absence of diurnal variations in circulating OPG and sRANKL levels may reflect the absence of diurnal variation in their expression in the bone microenvironment. In this case, the nocturnal rise and the fall in bone resorption in the luteal phase should be accounted for by other factors than RANKL/OPG-mediated factors. Timing of sampling is unlikely to influence the results of circulating OPG and sRANKL measurement.