Reducing 3D Hydrogel Stiffness, Addition of Oestradiol in a Physiological Concentration and Increasing FSH Concentration Improve In Vitro Growth of Murine Preantral Follicles.

This study aimed to optimise culture conditions for murine preantral follicles to improve their growth and survival. Preantral follicles (diameter 100-130 µm) were isolated from prepubertal NMRI mice and individually cultured within alginate beads for 12 days. Three conditions were evaluated: (1) follicle re-encapsulation on day 6 of culture-reducing alginate concentration (0.5% to 0.25% w/v), (2) the presence of oestradiol (E2), and (3) increased follicle-stimulating hormone (FSH) concentration in the culture medium (from 10 to 100 mIU/mL FSH). Follicle morphology and growth, as well as anti-Müllerian hormone (AMH) production, were evaluated. From day 8, re-embedded follicles had a larger average diameter compared to follicles without alginate re-encapsulation (0.5% and 0.25% groups, p < 0.05). Oestradiol (1 µM) had a significantly positive effect on the mean follicular diameter and antrum formation (p < 0.001). Moreover, follicles cultured with 100 mIU/mL FSH showed faster growth (p < 0.05) and significantly higher antrum formation (p < 0.05) compared to the low FSH group. Nevertheless, AMH production was not affected by any of the culture conditions. In conclusion, the growth and survival of mouse preantral follicles during a 12-day period were improved by altering the alginate concentration midways during culture and adding E2 and FSH to the culture medium.

Association between serum ß-oestradiol and health-related quality of life in postmenopausal women with degenerative lumbar scoliosis.

OBJECTIVES: Degenerative lumbar scoliosis (DLS) is an age-related spinal disease. It is an important cause of low back pain, lower limb pain and intermittent claudication, which seriously affects the quality of life of middle-aged and elderly people. DESIGN: This article aims to study the changes in serum oestrogen levels in postmenopausal women with DLS and its relationship. PATIENTS: One hundred and sixty-eight postmenopausal women diagnosed with DLS (DLS group) and 140 healthy postmenopausal women (control group) were recruited. MEASUREMENTS: Lumbar spinal bone mineral density (LSBMD) was measured by dual-energy X-ray absorptiometry and a chemiluminescence immunoassay analyser was used to detect serum ß-oestradiol (E2) levels. The severity of lower back pain was assessed by the visual analogue scale score and dysfunction was evaluated by Oswestry Disability Index (ODI). The quality of life was evaluated by Medical Outcomes Study 36-item Short Form Health Survey (SF-36). Diagnostic efficiency was evaluated by the receiver-operating characteristics curve (ROC). RESULTS: LSBMD and the level of E2 in the serum in DLS patients were significantly reduced when compared with the control group. The levels of E2 in the serum of postmenopausal women are reliable for predicting DLS revealed by ROC (p < .001). Serum E2 levels were negatively correlated with Cobb angle, VAS and ODI and were positively correlated with LSBMD and SF-36 scores. CONCLUSIONS: In postmenopausal women, serum E2 levels in DLS patients are significantly reduced and low levels of E2 are associated with lower bone density and poorer quality of life.

Corticotroph adenoma and pituitary fungal infection: a rare association.

SUMMARY: Pituitary infections, particularly with fungus, are rare disorders that usually occur in immunocompromised patients. Cushing's syndrome predisposes patients to infectious diseases due to their immunosuppression status. We report the case of a 55-year-old woman, working as a poultry farmer, who developed intense headache, palpebral ptosis, anisocoria, prostration and psychomotor agitation 9 months after initial diabetes mellitus diagnosis. Cranioencephalic CT scan showed a pituitary lesion with bleeding, suggesting pituitary apoplexy. Patient underwent transsphenoidal surgery and the neuropathologic study indicated a corticotroph adenoma with apoplexy and fungal infection. Patient had no preoperative Cushing's syndrome diagnosis. She was evaluated by a multidisciplinary team who decided not to administer anti-fungal treatment. The reported case shows a rare association between a corticotroph adenoma and a pituitary fungal infection. The possible contributing factors were hypercortisolism, uncontrolled diabetes and professional activity. Transsphenoidal surgery is advocated in these infections; however, anti-fungal therapy is still controversial. LEARNING POINTS: Pituitary infections are rare disorders caused by bacterial, viral, fungal and parasitic infections. Pituitary fungal infections usually occur in immunocompromised patients. Cushing's syndrome, as immunosuppression factor, predisposes patients to infectious diseases, including fungal infections. Diagnosis of pituitary fungal infection is often achieved during histopathological investigation. Treatment with systemic anti-fungal drugs is controversial. Endocrine evaluation is recommended at the time of initial presentation of pituitary manifestations.

Misdiagnosis of Mullerian agenesis in a patient with 46, XX gonadal dysgenesis: a missed opportunity for prevention of osteoporosis.

SUMMARY: Primary amenorrhea could be caused by disorders of four parts: disorders of the outflow tract, disorders of the ovary, disorders of the anterior pituitary, and disorders of hypothalamus. Delay in diagnosis and hormone substitution therapy causes secondary osteoporosis. Herein, we report a case of a 23-year-old phenotypical female who presented with primary amenorrhea from 46, XX gonadal dysgenesis but had been misdiagnosed as Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome or Mullerian agenesis. The coexistence of gonadal dysgenesis and MRKH was suspected after laboratory and imaging investigations. However, the vanishing uterus reappeared after 18 months of hormone replacement therapy. Therefore, hormone profiles and karyotype should be thoroughly investigated to distinguish MRKH syndrome from other disorders of sex development (DSD). Double diagnosis of DSD is extremely rare and periodic evaluation should be reassessed. This case highlights the presence of estrogen deficiency state, the uterus may remain invisible until adequate exposure to exogenous estrogen. LEARNING POINTS: An early diagnosis of disorders of sex development (DSD) is extremely important in order to promptly begin treatment, provide emotional support to the patient and reduce the risks of associated complications. Hormone profiles and karyotype should be investigated in all cases of the presumptive diagnosis of Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome or Mullerian agenesis. The association between 46, XX gonadal dysgenesis and Mullerian agenesis has been occasionally reported as a co-incidental event; however, reassessment of the presence of uterus should be done again after administration of exogenous estrogen replacement for at least 6-12 months. A multidisciplinary approach is necessary for patients presenting with DSD to ensure appropriate treatments and follow-up across the lifespan of individuals with DSD.

Clinical heterogeneity of hypophysitis secondary to PD-1/PD-L1 blockade: insights from four cases.

SUMMARY: Programmed cell death protein 1/programmed cell death protein ligand 1 (PD-1/PD-L1) and cytotoxic T-lymphocyte antigen 4/B7 (CTLA-4/B7) pathways are key regulators in T-cell activation and tolerance. Nivolumab, pembrolizumab (PD-1 inhibitors), atezolizumab (PD-L1 inhibitor) and ipilimumab (CTLA-4 inhibitor) are monoclonal antibodies approved for treatment of several advanced cancers. Immune checkpoint inhibitors (ICIs)-related hypophysitis is described more frequently in patients treated with anti-CTLA-4; however, recent studies reported an increasing prevalence of anti-PD-1/PD-L1-induced hypophysitis which also exhibits slightly different clinical features. We report our experience on hypophysitis induced by anti-PD-1/anti-PD-L1 treatment. We present four cases, diagnosed in the past 12 months, of hypophysitis occurring in two patients receiving anti-PD-1, in one patient receiving anti-PD-1 and anti-CTLA-4 combined therapy and in one patient receiving anti-PD-L1. In this case series, timing, clinical presentation and association with other immune-related adverse events appeared to be extremely variable; central hypoadrenalism and hyponatremia were constantly detected although sellar magnetic resonance imaging did not reveal specific signs of pituitary inflammation. These differences highlight the complexity of ICI-related hypophysitis and the existence of different mechanisms of action leading to heterogeneity of clinical presentation in patients receiving immunotherapy. LEARNING POINTS: PD-1/PD-L1 blockade can induce hypophysitis with a different clinical presentation when compared to CTLA-4 blockade. Diagnosis of PD-1/PD-L1 induced hypophysitis is mainly made on clinical grounds and sellar MRI does not show radiological abnormalities. Hyponatremia due to acute secondary adrenal insufficiency is often the principal sign of PD-1/PD-L1-induced hypophysitis and can be masked by other symptoms due to oncologic disease. PD-1/PD-L1-induced hypophysitis can present as an isolated manifestation of irAEs or be in association with other autoimmune diseases.

A rare cause of postpartum acute hyponatremia.

A previously healthy 24-year-old female underwent an emergent caesarean section without a major bleeding described. During the first post-operative days (POD) she complained of fatigue, headache and a failure to lactate with no specific and conclusive findings on head CT. On the following days, fever rose with a suspicion of an obstetric surgery-related infection, again with no evidence to support the diagnosis. On POD5 a new-onset hyponatremia was documented. The urine analysis suggested SIADH, and following a treatment failure, further investigation was performed and demonstrated both central hypothyroidism and adrenal insufficiency. The patient was immediately treated with hydrocortisone followed by levothyroxine with a rapid resolution of symptoms and hyponatremia. Further laboratory investigation demonstrated anterior hypopituitarism. The main differential diagnosis was Sheehan's syndrome vs lymphocytic hypophysitis. Brain MRI was performed as soon as it was available and findings consistent with Sheehan's syndrome confirmed the diagnosis. Lifelong hormonal replacement therapy was initiated. Further complaints on polyuria and polydipsia have led to a water deprivation testing and the diagnosis of partial central insipidus and appropriate treatment with DDAVP. Learning points: Sheehan's syndrome can occur, though rarely, without an obvious major post-partum hemorrhage. The syndrome may resemble lymphocytic hypophysitis clinically and imaging studies may be crucial in order to differentiate both conditions. Hypopituitarism presentation may be variable and depends on the specific hormone deficit. Euvolemic hyponatremia workup must include thyroid function test and 08:00 AM cortisol levels.

Restoration of fertility in a woman with giant prolactinoma in response to cabergoline treatment.

SUMMARY: A 32-year-old woman presented with primary amenorrhoea, prolactin (PRL) level of 154 150 mIU/L and was diagnosed with a giant pituitary adenoma measuring maximum 6.2 cm. Cabergoline (CAB) treatment at a dose of 0.5 mg/week was prescribed to the patient. The treatment decreased the tumour size after 3 months (MRI scans of the brain) and brought back to normal the level of the PRL (345 mIU/L) after 6 months of CAB treatment. After 7 months of CAB treatment, menarche was achieved, and after 12 months, the patient became pregnant. She discontinued taking CAB at 4-week gestation. The pregnancy resulted in a missed miscarriage at 6-7 weeks; an abortion was conducted by the vacuum aspiration method. The MRI scans of the brain did not show any tumour enlargement. After 18 months from the start of the treatment the patient got pregnant for the second time. At 25-week gestation an MRI scan of the brain was conducted which did not show any increase in the tumour size. At 38 weeks the patient delivered a healthy full-term girl via C-section. The patient chose not to breastfeed and resumed CAB therapy after the delivery. During the treatment, the PRL level returned to the normal range and the menstrual cycle was restored. After 3 years the patient got pregnant for the third time. The patient did not receive CAB during the pregnancies; the examination did not show any tumour enlargement. Further MRI scans did not show any tumour growth. CAB therapy was effective in normalization of the PRL level, tumour shrinkage, menarche and pregnancy-induction which led to the birth of healthy children in a woman with primary amenorrhoea and a giant prolactinoma invading the skull base bones. LEARNING POINTS: Giant prolactinomas are very rarely found in women. Cabergoline therapy can be effective in the normalization of the PRL level, tumour shrinkage, menarche induction in a woman with primary amenorrhoea, and giant prolactinoma. Cabergoline therapy can be effective in pregnancy induction which leads to the birth of children in a woman with giant prolactinoma. Cabergoline discontinuation did not trigger tumour enlargement during pregnancy.

A clinically functioning gonadotroph adenoma presenting with abdominal pain, ovarian hyperstimulation and fibromatosis.

Pituitary adenomas can be classified as functioning or non-functioning adenomas. Approximately 64% of clinically non-functioning pituitary adenomas are found to be gonadotroph adenomas on immunohistochemistry. There are reported cases of gonadotroph adenomas causing clinical symptoms, but this is unusual. We present the case of a 36-year-old female with abdominal pain. Multiple large ovarian cysts were identified on ultrasound requiring bilateral cystectomy. Despite this, the cysts recurred resulting in further abdominal pain, ovarian torsion and right oophorectomy and salpingectomy. On her 3rd admission with abdominal pain, she was found to have a rectus sheath mass which was resected and histologically confirmed to be fibromatosis. Endocrine investigations revealed elevated oestradiol, follicle-stimulating hormone (FSH) at the upper limit of the normal range and a suppressed luteinising hormone (LH). Prolactin was mildly elevated. A diagnosis of an FSH-secreting pituitary adenoma was considered and a pituitary MRI revealed a 1.5 cm macroadenoma. She underwent transphenoidal surgery which led to resolution of her symptoms and normalisation of her biochemistry. Subsequent pelvic ultrasound showed normal ovarian follicular development. Clinically functioning gonadotroph adenomas are rare, but should be considered in women presenting with menstrual irregularities, large or recurrent ovarian cysts, ovarian hyperstimulation syndrome and fibromatosis. Transphenoidal surgery is the first-line treatment with the aim of achieving complete remission. Learning points: Pituitary gonadotroph adenomas are usually clinically non-functioning, but in rare cases can cause clinical symptoms. A diagnosis of a functioning gonadotroph adenoma should be considered in women presenting with un-explained ovarian hyperstimulation and/or fibromatosis. In women with functioning gonadotroph adenomas, the main biochemical finding is elevated oestradiol levels. Serum FSH levels can be normal or mildly elevated. Serum LH levels are usually suppressed. Transphenoidal surgery is the first-line treatment for patients with functioning gonadotroph adenomas, with the aim of achieving complete remission.

Utilisation of gonadotrophin-releasing hormone (GnRH) analogue to differentiate ovarian from adrenal hyperandrogenism in postmenopausal women.

Postmenopausal hyperandrogenism is a relatively rare diagnosis resulting from excess androgen production from the adrenals or ovaries. The exclusion of malignant causes is a priority. Laboratory tests and imaging are utilised to help differentiate the source of excess androgens. We report two cases of postmenopausal hyperandrogenism in women aged 75 and 67 years. Both cases presented with clinical features suggestive of hyperandrogenism which had developed gradually over the previous 2 years. Laboratory investigations confirmed a significant elevation in their serum testosterone levels. In both cases, imaging did not reveal any abnormality of the adrenals or ovaries. To help differentiate an adrenal vs ovarian source a single-dose GnRH analogue was given with measurement of testosterone and gonadotrophin levels pre and post. The reduction in gonadotrophins achieved by the GnRH analogue resulted in suppression of testosterone levels which suggested an ovarian source. Both patients proceeded to bilateral oophorectomy. Histology revealed a benign hilus cell tumour in one case and a benign Leydig cell tumour in the other. Learning points: A key part of the work-up of postmenopausal hyperandrogenism is to differentiate between an adrenal or an ovarian source of excess androgens; Imaging may not identify small ovarian tumours or hyperthecosis and may also identify incidental adrenal masses which are non-functioning; Current guidelines suggest ovarian and adrenal venous sampling when imaging is inconclusive but this requires technical expertise and has a high failure rate; GnRH analogue use can successfully confirm ovarian source and should be considered as a diagnostic tool in this setting.

Partial androgen insensitivity syndrome presenting as pubertal gynecomastia: clinical and hormonal findings and a novel mutation in the androgen receptor gene.

Pubertal gynecomastia is common, can be seen in 65% of the adolescent boys and is considered physiological. It is thought to be due to transient imbalance between the ratio of testosterone and estradiol in the early stages of puberty. It resolves in 1-2 years and requires no treatment. However, more persistent and severe pubertal gynecomastia is less common and can be associated with pathological disorders. These can be due to diminished androgen production, increased estrogen production or androgen resistance. We report a case of persistent pubertal gynecomastia due to partial androgen insensitivity syndrome (PAIS), classical hormone findings and a novel mutation in the androgen receptor (AR) gene. Learning points: Laboratory testing of follicle-stimulating hormone (FSH), leutinizing hormone (LH) and testosterone for pubertal gynecomastia is most helpful in the setting of undervirization. The hormonal finding of very high testosterone, elevated LH and estradiol and relatively normal FSH are classical findings of PAIS. Gynecomastia due to PAIS will not resolve and surgery for breast reduction should be recommended.

Osteoprotective effect of combination therapy of low-dose oestradiol with G15, a specific antagonist of GPR30/GPER in ovariectomy-induced osteoporotic rats.

Identified and cloned in 1996 for the first time, G protein-coupled oestrogen receptor (ER) 30 (GPR30/GPER) has been a hot spot in the field of sex hormone research till now. In the present study, we examined the effects of low-dose oestradiol (E2) combined with G15, a specific antagonist of GPR30 on ovariectomy (OVX)-induced osteoporosis in rats. Female Sprague-Dawley (SD) rats undergoing OVX were used to evaluate the osteoprotective effect of the drugs. Administration of E2 [35 µg/kg, intraperitoneally (ip), three times/week) combining G15 (160 µg/kg, ip, three times/week) for 6 weeks was found to have prevented OVX-induced effects, including increase in bone turnover rate, decrease in bone mineral content (BMC) and bone mineral density (BMD), damage of bone structure and the aggravation in biomechanical properties of bone. The therapeutic effect of these two drugs in combination was better than that of E2 alone. Meanwhile, the administration of G15 prevented body weight increase or endometrium proliferation in the rats. In conclusion, administration of low-dose E2 combining G15 had a satisfactory bone protective effect for OVX rats, without significant influence on body weight or the uterus. This combination therapy may be an effective supplement of drugs in prevention and treatment for postmenopausal osteoporosis.

MicroRNA-203 up-regulates nitric oxide expression in temporomandibular joint chondrocytes via targeting TRPV4.

OBJECTIVE: MicroRNAs (miRNAs) are recognised as important regulators of a variety of fundamental biologic processes. Our study was undertaken to examine the role of MicroRNA-203 (miR-203) in modulating nitric oxide (NO) expression in female Sprague-Dawley rat mandibular condylar chondrocytes (MCCs) via targeting transient receptor potential vanilloid 4 (TRPV4) and to demonstrate the possible mechanism of NO inhibition by chondroprotective factor 17ß-oestradiol (E2). METHODS: The expression of TRPV4 in mandibular condylar cartilage tissue and MCCs was detected by immunohistochemistry, immunofluorescence (IF), RT-PCR and Western blot, respectively. Primary SD rat MCCs were exposed to lipopolysaccharide (LPS), plus Ruthenium Red, 4α-phorbol 12,13-didecanoate (4αPDD), over-expressed miR-203 or E2 (10(-9) to 10(-6)M), the cellular supernatants were used for NO assay, miR-203 levels were measured by quantitative RT-PCR while TRPV4 expression changes were analysed by Western blot. The dual luciferase activity assay was performed to identify the target gene of miR-203. RESULTS: TRPV4 and miR-203 were stably expressed in MCCs. The MCCs' expression of NO evoked by LPS could be enhanced or depressed by Ruthenium Red or 4αPDD. The dual luciferase assay suggested that TRPV4 was the direct target gene of miR-203. Over-expression of miR-203 inhibited the expression of TRPV4 and increased NO expression in MCCs. E2 inhibited NO expression by inhibition of miR-203, which was concurrent with the up-regulation of TRPV4 expression level in MCCs. CONCLUSION: Our findings first suggested that miR-203 could up-regulate NO expression in female rat MCCs via targeting TRPV4. Moreover, the inhibition of NO by E2 might be at least in part through this mechanism.