Inhibition of WNT/ß-catenin signalling during sex-specific gonadal differentiation is essential for normal human fetal testis development.

Sex-specific gonadal differentiation is directed by complex signalling promoting development in either male or female direction, while simultaneously inhibiting the opposite pathway. In mice, the WNT/ß-catenin pathway promotes ovarian development and the importance of actively inhibiting this pathway to ensure normal testis development has been recognised. However, the implications of alterations in the tightly regulated WNT/ß-catenin signalling during human fetal gonad development has not yet been examined in detail. Thus, the aim of this study was to examine the consequences of dysregulating the WNT/ß-catenin signalling pathway in the supporting cell lineage during sex-specific human fetal gonad development using an established and extensively validated ex vivo culture model. Inhibition of WNT/ß-catenin signalling in human fetal ovary cultures resulted in only minor effects, including reduced secretion of RSPO1 and reduced cell proliferation although this was not consistently found in all treatment groups. In contrast, promotion of WNT/ß-catenin signalling in testes severely affected development and function. This included disrupted seminiferous cord structures, reduced cell proliferation, reduced expression of SOX9/AMH, reduced secretion of Inhibin B and AMH as well as loss of the germ cell population. Additionally, Leydig cell function was markedly impaired with reduced secretion of testosterone, androstenedione and INSL3. Together, this study suggests that dysregulated WNT/ß-catenin signalling during human fetal gonad development severely impairs testicular development and function. Importantly, our study highlights the notion that sufficient inhibition of the opposite pathway during sex-specific gonadal differentiation is essential to ensure normal development and function also applies to human fetal gonads.

Perimenopausal abnormal uterine bleeding.

Abnormal uterine bleeding is a frequent symptom in the perimenopause. Causes are numerous, ranging from physiological reactions due to decreasing/unstable ovarian function to premalignant and malignant conditions. Benign findings such as endometrial polyps and myomas increase with age, leading to more abnormal uterine bleeding in the perimenopause. Cervical and vaginal causes of abnormal uterine bleeding should be excluded by speculum examination. Sexually transmitted diseases or pregnancy should be ruled out. Measurement of haemoglobin and iron levels, human chorion gonadotropin and thyroid hormones are relevant in selected cases. Transvaginal ultrasound is an ideal first step for the evaluation of perimenopausal abnormal uterine bleeding. Saline or gel contrast sonohysterography improves the diagnostic accuracy. Based on the ultrasound findings, invasive procedures such as endometrial biopsy or hysteroscopy can be planned. Once premalignant and malignant causes are excluded, the necessity for treatment can be evaluated in collaboration with the patient. Heavy menstrual bleeding causing anaemia will need immediate treatment. In less severe cases and in intermenstrual bleeding, expectant management can be considered. Hormonal treatment, such as oral progestogens, combined oral contraceptives or insertion of the levonorgestrel intrauterine system, may be a possibility if anovulatory bleeding is interfering with quality of life. The amount of bleeding can be reduced both by antifibrinolytic and non-steroidal anti-inflammatory drugs, progestogens and the levonorgestrel intrauterine system. Focal intrauterine lesions such as endometrial polyps or submucous myomas may require operative hysteroscopic procedures. Endometrial ablation or endometrial resection are good choices in selected cases, but some women will need a hysterectomy to treat their abnormal uterine bleeding in perimenopause.

Dexamethasone affects human fetal adrenal steroidogenesis and subsequent ACTH response in an ex vivo culture model.

Introduction: Administration of dexamethasone (DEX) has been used experimentally to suppress androgenization of external genitalia in 46,XX fetuses with congenital adrenal hyperplasia. Despite this, the prenatal biological mechanism-of-action of DEX on fetal development is not known. This study aimed to examine direct effects of DEX on human fetal adrenal (HFA) steroidogenic activity including possible effects on the subsequent response to ACTH-stimulation. Methods: Human fetal adrenal (HFA) tissue from 30 fetuses (1st trimester) were cultured ex vivo with A) DEX (10 µm) for 14 days, or B) DEX (10 µm) for 10 days followed by ACTH (1 nM) for 4 days. DEX-mediated effects on HFA morphology, viability, and apoptosis (immunohistochemistry), gene expression (quantitative PCR), and steroid hormone secretion (LC-MS/MS) were investigated. Results: DEX-treatment caused decreased androstenedione (p<0.05) and increased cortisol (p<0.01) secretion suggesting that direct effects on the adrenal gland may contribute to the negative feedback on the hypothalamic-pituitary-adrenal axis in vivo. An altered response to ACTH stimulation in HFA pre-treated with DEX included increased androgen (p<0.05) and reduced cortisol production (p<0.05), supporting clinical observations of a temporary decreased ACTH-response following prenatal DEX-treatment. Additionally, the secretion of corticosterone was decreased (p<0.0001) following ACTH-stimulation in the initially DEX-treated HFAs. Discussion: The observed effects suggest that prenatal DEX-treatment can cause direct effects on HFA steroidogenesis and in the subsequent response to ACTH-stimulation. This may indicate a requirement for careful monitoring of adrenal function in prenatally DEX-treated neonates, with particular focus on their mineralocorticoid levels.

Identification of a window of androgen sensitivity for somatic cell function in human fetal testis cultured ex vivo.

BACKGROUND: Reduced androgen action during early fetal development has been suggested as the origin of reproductive disorders comprised within the testicular dysgenesis syndrome (TDS). This hypothesis has been supported by studies in rats demonstrating that normal male development and adult reproductive function depend on sufficient androgen exposure during a sensitive fetal period, called the masculinization programming window (MPW). The main aim of this study was therefore to examine the effects of manipulating androgen production during different timepoints during early human fetal testis development to identify the existence and timing of a possible window of androgen sensitivity resembling the MPW in rats. METHODS: The effects of experimentally reduced androgen exposure during different periods of human fetal testis development and function were examined using an established and validated human ex vivo tissue culture model. The androgen production was reduced by treatment with ketoconazole and validated by treatment with flutamide which blocks the androgen receptor. Testicular hormone production ex vivo was measured by liquid chromatography-tandem mass spectrometry or ELISA assays, and selected protein markers were assessed by immunohistochemistry. RESULTS: Ketoconazole reduced androgen production in testes from gestational weeks (GW) 7-21, which were subsequently divided into four age groups: GW 7-10, 10-12, 12-16 and 16-21. Additionally, reduced secretion of testicular hormones INSL3, AMH and Inhibin B was observed, but only in the age groups GW 7-10 and 10-12, while a decrease in the total density of germ cells and OCT4+ gonocytes was found in the GW 7-10 age group. Flutamide treatment in specimens aged GW 7-12 did not alter androgen production, but the secretion of INSL3, AMH and Inhibin B was reduced, and a reduced number of pre-spermatogonia was observed. CONCLUSIONS: This study showed that reduced androgen action during early development affects the function and density of several cell types in the human fetal testis, with similar effects observed after ketoconazole and flutamide treatment. The effects were only observed within the GW 7-14 period-thereby indicating the presence of a window of androgen sensitivity in the human fetal testis.

Reduced Complications Following Implementation of Laparoscopic Hysterectomy: A Danish Population-based Cohort Study of Minimally Invasive Benign Gynecologic Surgery between 2004 and 2018.

STUDY OBJECTIVE: To monitor and report nationwide changes in the rates of and complications after different methods for benign hysterectomy, operative hysteroscopy, myomectomy, and embolization in Denmark. To report the national mortality after benign hysterectomy DESIGN: National prospective, observational cohort study. SETTING: The Danish Hysterectomy and Hysteroscopy Database. PATIENTS: Women undergoing surgery for benign gynecologic diseases: 64 818 hysterectomies, 84 175 hysteroscopies, 4016 myomectomies, and 1209 embolizations in Denmark between 2004 and 2018. INTERVENTIONS: National meetings with representatives from all departments, annual working reports of institutional complication rates, workshops, and national guideline initiative to improve minimally invasive surgical methods. MEASUREMENTS AND MAIN RESULTS: Rates of the different methods and complications after each method with follow-up to 5 years as recorded by the database directly in the National Patient Registry. Nationwide, a decline in the use of hysterectomy, myomectomy, embolizations, and endometrial ablation. The total short-term complications were 9.8%, 7.5%, 8.9%, and 2.7% respectively, however, with a persistent risk of approximately 20% for recurrent operations within 5 years after endometrial ablation. Initially, we urged for increased use of vaginal hysterectomy, but only reached 36%. From 2010, we urged for reducing abdominal hysterectomies by implementing laparoscopic hysterectomy and reached 72% laparoscopic and robotic procedures. Since 2015, we used coring or contained morcellation for removal of large uterus at laparoscopic hysterectomy. The major and minor complication rates (modified Clavien-Dindo classification) were reduced significantly from 8.1% to 4.1% and 9.9% to 5.7% respectively. Mortality after benign hysterectomy was 0.27‰. The odds ratio for major complications after abdominal hysterectomy was 1.66 (1.52-1.81) compared to minimally invasive hysterectomy independent of the length of stay, high-volume departments, indications, comorbidity, age, and calendar year. CONCLUSION: Fifteen years with a national database has resulted in a marked quality improvement. Denmark has 85% minimally invasive hysterectomies and has reduced the number of major complications by 50%.

Asherman's syndrome: current perspectives on diagnosis and management.

Intrauterine adhesions with symptoms like hypomenorrhea or infertility are known under the term Asherman's syndrome. Although the syndrome has been widely investigated, evidence of both prevention of the syndrome and the ideal treatment are missing. Understanding the pathogenesis of intrauterine adherences is necessary for the prevention of the formation of intrauterine scarring. Intrauterine adhesions can develop from lesion of the basal layer of the endometrium caused by curettage of the newly pregnant uterus. The syndrome may also occur after hysteroscopic surgery, uterine artery embolization or uterine tuberculosis. For initial diagnosis the less invasive contrast sonohysterography or hysterosalpingography is useful. The final diagnosis is based on hysteroscopy. Magnetic resonance imaging is required in cases with totally obliterated uterine cavity. Intrauterine adherences are classified in accordance with different classification systems based on the hysteroscopic diagnosis of severity and localization of adherences. Classification is necessary for the planning of surgery, information on prognosis and scientific purposes. Surgery is performed in symptomatic patients with either infertility or with painful periods. Intrauterine adherences are divided with a hysteroscope using scissors or a power instrument working from the central part of the uterus to the periphery. Peroperative ultrasonography is useful in an outpatient setting for the prevention of complications. Hysteroscopy with fluoroscopy is a solution in difficult cases. Use of intrauterine devices like balloon catheters or intrauterine contraceptive devices seems to be the preferred methods for the prevention of re-occurrence of adhesions after treatment. Both primary prevention after hysteroscopic surgery or curettage and secondary prevention of new adhesions after adhesiolysis have been investigated. The aim of this review was to summarize the literature on diagnosis, classification, treatment and prevention, based on a literature search with a wide range of search terms.

Up or down? A randomized trial comparing image orientations during transvaginal ultrasound training.

INTRODUCTION: There is no international consensus on the orientation of transvaginal ultrasound images and no evidence exists to support the superiority of one image orientation over the other. The aim of this study was to compare learning curves and skills transfer in a group of novices randomized to top-down or bottom-up image orientation, and to determine whether individual preferences for image orientation affect learning and skills transfer. MATERIAL AND METHODS: 60 senior medical students, with no prior ultrasound experience, were randomized to orient the image top-down or bottom-up during training on an ultrasound simulator until attaining expert levels of performance. Participants then completed a transfer test involving a systematic ultrasound examination on a physical mannequin using real ultrasound equipment. Performance was assessed during the transfer test by two independent raters using the objective structured assessment of ultrasound skills (OSAUS) score and a global rating score. RESULTS: The bottom-up group reached the expert level with significantly fewer attempts than did the top-down group [median ± interquartile range: 4 ± 2 vs 5 ± 3] (U = 285.5, P = 0.014). The bottom-up group used less time to achieve the expert level (median ± interquartile range: 3 h 2 m ± 1 h 14 m vs 3 h 28 m ± 2 h 21 m) (U = 301.5, P = 0.029). The two groups performed similarly during the transfer test with respect to their OSAUS scores (top-down 56.7% vs bottom-up 53.2%, P = 0.13). The global rating scores were higher in the top-down group (top-down 57.1% vs bottom-up 50.0%, P = 0.02). CONCLUSIONS: Orientation of the images bottom-up rather than top-down, led to a steeper learning curve, but had little or no impact on the subsequent transfer of skills.

Müllerian duct anomalies diagnosed by saline contrast sonohysterography: prevalence in a general population.

OBJECTIVE: To estimate the prevalence of uterine müllerian duct anomalies in a general population. DESIGN: Cross-sectional study. SETTING: University hospital in collaboration with the Danish Civil Registry. PATIENT(S): A total of 1,654 women randomly recruited from a general population; 686 women were eligible and accepted inclusion (429 pre- and 257 postmenopausal). Saline contrast sonohysterography (SCSH) was finally performed in 622 women (aged 20-74 years) (the procedure was impossible owing to cervical stenosis in 58, contraindicated in 2, other patient-related factors in 4). INTERVENTION(S): The shape of the uterus was dynamically evaluated in the transversal and longitudinal planes during SCSH and classified in accordance with American Fertility Society as normal, arcuate, septate (partial, complete), bicorn (partial, complete), or unicorn. History of previous miscarriage and menstrual cycle was obtained by a questionnaire. MAIN OUTCOME MEASURE(S): Prevalence of müllerian anomalies, miscarriage, and oligomenorrhea. RESULT(S): The overall prevalence of müllerian anomalies was 9.8% (61 of 622) (95% confidence interval [CI] 7.5-12.1). The majority had arcuate uteri (n=42, 6.8%), 17 partial septate (2.7%), 1 complete septate (1.6%), and 1 unicorn uterus (1.6%). Müllerian anomalies were significantly more frequently diagnosed in nulliparous (20% [26 of 128]) compared with parous women (7% [35 of 494]). Müllerian anomalies were more frequent in women with oligomenorrhea compared with women with normal menstrual periods (19% [15 of 79] vs. 10% [34 of 339]). One first-trimester miscarriage or multiple miscarriages (more than one) were not significantly more frequent in premenopausal women with müllerian anomalies compared with women with normal-shaped uteri (24% [6 of 25] vs. 22% [57 of 265]). CONCLUSION(S): In a general population examined by SCSH the prevalence of Müllerian anomalies is estimated at 9.8%. Müllerian anomalies were more frequent in nulliparous women. Both impaired fertility and a pregnancy-associated modulation of the uterine corpus are among explanations. Müllerian anomalies were associated with oligomenorrhea in premenopausal women. In this general population there was no association between miscarriage and müllerian anomalies; however, the number of cases was limited.

EMAS clinical guide: assessment of the endometrium in peri and postmenopausal women.

INTRODUCTION: Invasive as well as non-invasive methods are available for assessment of the endometrium. AIMS: The purpose of this clinical guide is to provide evidence-based advice on endometrial assessment in peri and postmenopausal women. MATERIAL AND METHODS: Literature review and consensus of expert opinion. RESULTS AND CONCLUSIONS: Presuming speculum examination and cervical cytology are assessed, transvaginal ultrasound should be undertaken initially as it is non-invasive and will not only measure endometrial thickness, but will also detect other pelvic pathology such as leiomyomas and ovarian tumours. The main indication for invasive methods is to obtain endometrial tissue to diagnose or exclude the presence of endometrial cancer or pre-malignancies. Biopsy is mainly undertaken as an outpatient procedure, but sampling is 'blind'. Hysteroscopy is used when focal lesions affecting the uterine cavity are suspected such as endometrial polyps or sub-mucous fibroids. None of the available methods are perfect. Ultrasound evaluation is dependent on the experience of the examiner, the equipment and the quality of visualization. Hysteroscopy too is dependent on the examiner and fibroids may obstruct visualization. Blind endometrial biopsy procedures often miss focal lesions. Thus re-examination is necessary when symptoms persist and no explanation for these has been identified. This clinical guide will evaluate the different methods of endometrial assessment, their indications and limitations. Guidance is also given about dealing with inconclusive investigations and persistent symptoms.

Endometrial polyps and associated factors in Danish women aged 36-74 years.

OBJECTIVE: To identify factors associated with endometrial polyps. STUDY DESIGN: Case-control study of 140 women with endometrial polyps and 367 controls. Information on potentially associated factors was obtained by a validated questionnaire. RESULTS: In an age-adjusted logistic regression model, the following was positively associated with endometrial polyps: current use of hormone therapy (odds ratio, 2.81; 95% CI, 1.29-6.13) and being overweight (body mass index > 25 kg/m(2)) (odds ratio, 2.06; 95% CI, 1.12-3.79) (postmenopausal women). Negatively associated was use of oral contraceptive pills (odds ratio, 0.20; 95% CI, 0.06-0.66). Histopathology diagnosed benign endometrial polyps (n = 137), polyp with premalignant disease (n = 3), and benign polyp with concomitant complex hyperplasia/endometrial cancer (n = 3). CONCLUSION: Being overweight and current use of hormone therapy in postmenopausal women were positively associated, whereas the use of oral contraceptive pills was negatively associated with endometrial polyps. Hypertension and cervical polyps were not associated with endometrial polyps. Endometrial polyps were infrequently related to premalignant and malignant disease.

Outcome of repeated hysteroscopic resection of the endometrium.

STUDY OBJECTIVE: To evaluate the efficacy and safety of repeated endometrial resection. DESIGN: Retrospective study (Canadian Task Force classification II-3). SETTING: University teaching hospital. PATIENTS: Women with a failed primary endometrial resection. INTERVENTION: Repeated endometrial resection, roller-ball coagulation, or both. MEASUREMENTS AND MAIN RESULTS: In all, 65 women had a repeated endometrial resection during the period from January 1995 through December 2002. Data were collected from standardized data sheets, hospital records, and the National Patient Registry of the National Board of Health. The primary outcome measurement was subsequent hysterectomy, and secondary, perioperative complications. The median follow-up time was 56 months (range 40-110 months). In all, 28 (43%) women required a hysterectomy eventually. Six (9%) women had operative complications. CONCLUSION: Repeat endometrial resection is a relatively safe, minimally invasive operation and approximately 60% of women operated on will avoid hysterectomy. The operation should be performed by an experienced hysteroscopic surgeon.

[Use of dipsticks and microscope in the diagnosis of hematuria in general practice]. / Brug af urinstiks og mikroskop ved haematuridiagnostik i almen praksis.

INTRODUCTION: Haematuria is a serious symptom and is often detected by dipstick analysis. We examined the use of dipsticks in general practice. MATERIALS AND METHODS: An anonymous questionnaire was sent to all GPs in a county in Denmark in April 2000. RESULTS: The response rate was 92%. All GPs used dipsticks for the detection of haematuria. Answers to the questionnaire were compared with the guidelines of the dipstick manufacturers, and we found that 80% handled the dipstick incorrectly. A microscope was available in 65% of practices, and 44% performed urine microscopy in the case of dipstick haematuria. DISCUSSION: The validity of dipsticks in the detection of haematuria is discussed, and we conclude that dipsticks must be handled strictly in accordance with the instructions of the manufacturer, so as to maintain a high rate of specificity and sensitivity. A positive dipstick should be followed up immediately with microscopy of the same urine sample.