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Context Thyroid autoantibody positivity has been associated with an increased rate of obstetrical complications. Objective We aimed to evaluate the role of thyroid autoantibodies in adverse pregnancy outcomes. Methods This prospective study was conducted in the Endocrinology Unit of Pisa Hospital. A total of 975 pregnant women were studied from 2012 to 2021; 572 (59%) were diagnosed with autoimmune thyroid (AT) diseases; 403 (41%) served as controls. Levothyroxine (LT4) treatment was introduced when TSH was > 2.5 mIU/L in the AT group and when TSH was > 4 mIU/L in the controls. Rates of obstetrical complications in each group were measured. Results Although the frequency of miscarriage in the AT group was greater (4.8%) than in the controls (2.9%), no significant differences were detected (P = 0.181). There were no differences between the 2 groups concerning the other pregnancy complications, and no association with the titer of thyroid antibodies was observed. The frequency of congenital malformations was greater in the AT group than in the controls (P = 0.019), but no correlation with major congenital malformations was detected (P = 0.872). Given that thyroid hormone concentrations were strictly controlled in our population, we documented a tendency (not significant) toward an increase in miscarriage and preterm birth among women with TSH > 4 mIU/L. Conclusion If thyroid function is adequately controlled, the presence and titer of thyroid autoantibodies does not negatively influence gestation. Although not significant, suboptimal thyroid hormone status seems to affect pregnancy outcomes more than thyroid autoimmunity.

Background Physiologically regulated insulin secretion and euglycemia are achievable in type 1 diabetes (T1D) by islet or pancreas transplantation. However, pancreas transplant alone (PTA) remains a debated approach, with uncertainties on its relative benefits and risks. We determined the actual long-term (10 years) efficacy and safety of PTA in carefully characterized T1D subjects. Methods This is a single-centre, cohort study in 66 consecutive T1D subjects who received a PTA between April 2001 and December 2007, and were then all followed until 10 years since transplant. Main features evaluated were patient survival, pancreas graft function, C-peptide levels, glycemic parameters, and the function of the native kidneys. Results Ten-year actual patient survival was 92.4%. Optimal (insulin-independence) or good (minimal insulin requirement) graft function was observed in 57.4 and 3.2% of patients, respectively. Six (9.0%) patients developed stage 5 or 4 chronic kidney disease. In the remaining individuals bearing a successful PTA, eGFR decline per year was -2.29±2.69 ml/min/1.73m. Reduction of eGFR at 1 year post-PTA was higher in those with pre-PTA hyperfiltration and higher HbA1c concentrations; eGFR changes afterwards significantly correlated with diabetes duration. In recipients with normoglycemia at 10 years, 74% of normo- or micro-albuminuric subjects pre-PTA remained stable, and 26% progressed towards a worse stage; conversely, in 62.5% of the macro-albuminuric individuals albuminuria severity regressed. Conclusions These long-term effects of PTA on patient survival, graft function and the native kidneys support PTA as a suitable approach to treat diabetes in selected T1D patients.

Background Premature ovarian insufficiency (POI) is an ovarian defect characterized by primary or secondary amenorrhea, hypergonadotropism and hypoestrogenism which occurs before the age of 40 years with a major genetic component. In this study we performed clinical evaluation and genetic analysis of a group of 18 patients with POI. The study involved 18 consecutive women with POI. Karyotiping and genetic analysis for research of mutations in GDF9 (Growth Differentation Factor 9) and BMP15 (Bone morphogentic protein 15) genes and FMR1 (Fragile X Mental Retardation 1) premutation were carried out. In vitro functional study of the novel BMP15 mutation was performed using COV434 (Human ovarian granulosa tumour cells 434) cells of ovarian granulosa, which consistently express BMP responsive element, and luciferase reporter assay. Results Three patients (17%) had a family history of POI. Ten patients (56%) had a family history of autoimmune diseases and nine patients (50%) showed a personal history of one or more autoimmune diseases. Of patients for whom morphological assessment was available, almost half (44%) had poor follicle assets or small ovaries’s size at pelvic US. Two patients (13%) showed reduced bone density at DEXA (Dual Energy X-ray Absorptiometry). All the women had normal female kariotype and no mutations in the GDF-9 gene or FMR1 premutations were found. A novel heterozygous mutation c.406G > C (V136L) of BMP15 gene was identified in one patient. After transfection in COV434 cells, BMP15 variant showed a significantly reduced luciferase activity compared to wild type. Conclusions POI is a multifactorial disease with several health implications. Autoimmunity and genetics represent the most common aetiology. We identified and characterized a novel BMP15 mutation, providing an additional elucidation of molecular basis of this complex disorder.

Subcapsular renal hematoma (SRH) is a challenging condition, which may jeopardize kidney function or constitute a life-threatening event. This is particularly true in single-kidney patients, such as kidney-transplant recipients. SRH may exert an excessive pressure on the surrounding parenchyma, thus resulting in hypoperfusion and ischemia, with high risk of acute kidney failure and graft loss. Moreover, SRH may precede an overt renal rupture with subsequent hemorrhage and hemodynamic instability. The indication to an interventional management for this condition is still a matter of debate, with some authors advocating the high possibilities of spontaneous resolution and others advocating the high-risk of graft loss and even internal bleeding in case of overt renal rupture. Herein, we report the case of a 51-year-old simultaneous pancreas-kidney transplantation recipient who presented a SRH following a mild trauma. The therapeutic choices were carefully balanced on the specific case, and the conservative management proved successful.

Purpose: Control of thyroid function in hyperthyroid women during pregnancy is based on antithyroid drugs (ATD) [propylthiouracil (PTU) and methimazole (MMI)]. While a teratogenic effect has been suggested for MMI and, more recently, for PTU, a clear demonstration is still lacking. Aim of this study was to assess the safety of ATD during pregnancy. Methods: A total of 379 pregnancies were retrospectively recruited in eight Italian Departments of Endocrinology and divided in five groups: (1) MMI-treated and euthyroid throughout pregnancy (n = 89); (2) MMI-treated and hyperthyroid on at least two occasions (n = 35); (3) PTU-treated women and euthyroid throughout pregnancy (n = 32); (4) PTU-treated women and hyperthyroid on at least two occasions (n = 20); and (5) non-ATD-treated (n = 203). Data on maternal thyroid function, miscarriages, type of delivery, neonatal weight, length and TSH, perinatal complications and congenital malformation were analyzed. Results: The gestational age at delivery, the rate of vaginal delivery, neonatal weight, length and neonatal TSH did not significantly differ among groups. In all groups, the rates of spontaneous miscarriage and of major congenital malformations were not higher than in the general population. No newborns were born with a phenotype similar to those described in the "MMI embryopathy". Conclusions: While a clear demonstration of a teratogenic effect of MMI is currently lacking, it seems reasonable to follow the current guidelines and advice for PTU treatment in hyperthyroid women during the first trimester of pregnancy. Further, large and prospective worldwide studies will be needed to fully clarify the issue of ATD safety during pregnancy. © 2015 Italian Society of Endocrinology (SIE).

Neurokinin B (NKB) and its G-protein-coupled receptor, NK3R, have been implicated in the neuroendocrine control of GnRH release; however, little is known about the structure-function relationship of this ligand-receptor pair. Moreover, loss-of-function NK3R mutations cause GnRH deficiency in humans. Using missense mutations in NK3R we previously identified in patients with GnRH deficiency, we demonstrate that Y256H and Y315C NK3R mutations in the fifth and sixth transmembrane domains (TM5 and TM6), resulted in reduced whole-cell (79.3±7.2%) or plasma membrane (67.3±7.3%) levels, respectively, compared with wild-type (WT) NK3R, with near complete loss of inositol phosphate (IP) signaling, implicating these domains in receptor trafficking, processing, and/or stability. We further demonstrate in a FRET-based assay that R295S NK3R, in the third intracellular loop (IL3), bound NKB but impaired dissociation of Gq-protein subunits from the receptor compared with WT NK3R, which showed a 10.0 ± 1.3% reduction in FRET ratios following ligand binding, indicating activation of Gq-protein signaling. Interestingly, R295S NK3R, identified in the heterozygous state in a GnRH-deficient patient, also interfered with dissociation of G proteins and IP signaling from wild-type NK3R, indicative of dominant-negative effects. Collectively, our data illustrate roles for TM5 and TM6 in NK3R trafficking and ligand binding and for IL3 in NK3R signaling.—Noel, S. D., Abreu, A. P., Xu, S., Muyide, T., Gianetti, E., Tusset, C., Carroll, J., Latronico, A. C., Seminara, S. B., Carroll, R. S., Kaiser, U. B. TACR3 mutations disrupt NK3R function through distinct mechanisms in GnRH-deficient patients.

A broad spectrum of GnRH-deficient phenotypes has been identified in individuals with both mono- and biallelic GNRHR mutations.The objective of the study was to determine the correlation between the severity of the reproductive phenotype(s) and the number and functional severity of rare sequence variants in GNRHR.Eight hundred sixty-three probands with different forms of GnRH deficiency, 46 family members and 422 controls were screened for GNRHR mutations. The 70 subjects (32 patients and 38 family members) harboring mutations were divided into four groups (G1-G4) based on the functional severity of the mutations (complete or partial loss of function) and the number of affected alleles (monoallelic or biallelic) with mutations, and these classes were mapped on their clinical phenotypes.The prevalence of heterozygous rare sequence variants in GNRHR was significantly higher in probands vs. controls (P0.01). Among the G1-G3 groups (homozygous subjects with successively decreasing severity and number of mutations), the hypogonadotropic phenotype related to their genetic load. In contrast, subjects in G4, with only monoallelic mutations, demonstrated a greater diversity of clinical phenotypes.In patients with GnRH deficiency and biallelic mutations in GNRHR, genetic burden defined by severity and dose is associated with clinical phenotype. In contrast, for patients with monoallelic GNRHR mutations this correlation does not hold. Taken together, these data indicate that as-yet-unidentified genetic and/or environmental factors may combine with singly mutated GNRHR alleles to produce reproductive phenotypes.

In 2003, three groups around the world simultaneously discovered that KISS1R (GPR54) is a key gatekeeper of sexual maturation in both mice and men. Developmental changes in the expression of the ligand for KISS1R, kisspeptin, support its critical role in the pubertal transition. In addition, kisspeptin, a powerful stimulus of GNRH-induced gonadotropin secretion and may modulate both positive and negative sex steroid feedback effects at the hypothalamic level. Genetic studies in humans have revealed both loss-of-function and gain-of-function mutations in patients with idiopathic hypogonadotropic hypogonadism and precocious puberty respectively. This review examines the kisspeptin/KISS1R pathway in the reproductive system.

Objective Premature ovarian insufficiency (POI) is defined as a primary ovarian defect characterized by absent menarche (primary amenorrhea) or premature depletion of ovarian follicles before the age of 40 (secondary amenorrhea) with hypergonadotropism and hypoestrogenism. Methods We studied the clinical, biological, and genetic data related to 50 POI patients with a mean age of menopause of 29 years (94% with secondary amenorrhea, 6% with primary amenorrhea and 15% with a family history of POI). Seventeen patients were affected by endocrine autoimmune diseases, antral follicles were observed in 31 patients by ultrasonography. Results Karyotype analysis did not show any abnormality of the X chromosome. No mutation in FSH receptor and GDF-9 genes was reported, while in one patient a variant of BMP-15 gene (A180T) was found. Four patients had fragile X mental retardation 1 gene (FMR1) premutation and one an intermediate sized CGG repeats of the same gene. Two patients with FMR1 premutation were sister and developed secondary amenorrhea at the age of 34 and 37 years. The other two patients presented with oligoamenorrhea at the age of 39 and 34 years. The patient harboured the intermediate sized CGG repeats developed secondary amenorrhea at the age of 33 years. Conclusions The genetic analysis performed on a cohort of patients with POI revealed that 8% had FMR1 premutation and only one patient a previously known variant of BMP-15 gene. No alteration of the karyotype and FSH receptor and GDF-9 genes was evidenced.

Context: Mutations in TAC3 and TACR3 (encoding neurokinin B and its receptor) have been identified in Turkish patients with idiopathic hypogonadotropic hypogonadism (IHH), but broader populations have not yet been tested and genotype-phenotype correlations have not been established. Objective: A broad cohort of normosmic IHH probands was screened for mutations in TAC3/TACR3 to evaluate the prevalence of such mutations and define the genotype/phenotype relationships. Design and Setting: The study consisted of sequencing of TAC3/TACR3, in vitro functional assays, and neuroendocrine phenotyping conducted in tertiary care centers worldwide. Patients or Other Participants: 345 probands, 18 family members, and 292 controls were studied. Intervention: Reproductive phenotypes throughout reproductive life and before and after therapy were examined. Main Outcome Measure: Rare sequence variants in TAC3/TACR3 were detected. Results: In TACR3, 19 probands harbored 13 distinct coding sequence rare nucleotide variants [three nonsense mutations, six nonsynonymous, four synonymous (one predicted to affect splicing)]. In TAC3, one homozygous single base pair deletion was identified, resulting in complete loss of the neurokinin B decapeptide. Phenotypic information was available on 16 males and seven females with coding sequence variants in TACR3/TAC3. Of the 16 males, 15 had microphallus; none of the females had spontaneous thelarche. Seven of the 16 males and five of the seven females were assessed after discontinuation of therapy; six of the seven males and four of the five females demonstrated evidence for reversibility of their hypogonadotropism. Conclusions: Mutations in the neurokinin B pathway are relatively common as causes of hypogonadism. Although the neurokinin B pathway appears essential during early sexual development, its importance in sustaining the integrity of the hypothalamic-pituitary-gonadal axis appears attenuated over time.

Summary Objective Genetic analysis of the TSH receptor gene in seven subjects with subclinical hypothyroidism (SH), in whom the diagnosis of autoimmune thyroid disease had been excluded by laboratory and instrumental techniques currently available. Patients Three families where different members (2 children and 5 adults) affected by SH were studied. Genetic analysis Genomic DNA was extracted from peripheral lymphocytes and the entire coding sequence of the TSHr gene was sequenced. pSVL-TSHr construct harbouring a Q8fsX62 insertion was obtained by site-directed mutagenesis. COS-7 cells transfected with wild-type and mutant receptor were used for binding studies, flow cytometry, and cyclic AMP (cAMP) determination. Results A four base pair (bp) duplication in position 41 (41TGCAins), leading to a premature stop of translation at codon 62 (Q8fsX62), was found to be heterozygous in the proband, the father and the sister in Family 1. In Family 2 the proband and the sister were heterozygous for the mutation D410N. In Family 3 the proband and the father were heterozygous for the mutation P162A. After transfection in COS-7 cells, the mutant receptor Q8fsX62 displayed a low expression at the cell surface, and a reduced response to bovine TSH (bTSH) in terms of cAMP production. Conclusions We identified TSH receptor mutations in seven members of three families with subclinical hypothyroidism.

This study was designed to assess the relationship between mutations in the FSH receptor (FSHr) gene and polycystic ovary syndrome (PCOS) in Italian women. The study population included 50 patients with PCOS and 50 age- and body mass index (BMI)-matched controls. A complete anthropometrical, hormonal and pelvic ultrasonographic evaluation was performed in all subjects. Genomic DNA was extracted from peripheral lymphocytes and then each exon of the FSHr gene was amplified by PCR. The mutation identified was cloned and the functional properties were studied after transient expression in COS-7 cells. Direct sequencing of exons 1–10 of the FSHr gene revealed the presence of a heterozygous AAT/ATT mutation affecting the isoleucine residue at position 411, which was replaced by an asparagine, in the second transmembrane segment (I411N). This mutation was only found in one woman with PCOS and not in her parents. This mutation was not present in 50 age and BMI controls and in another 150 women not affected by PCOS. The functional study after transient expression in COS-7 cells revealed that this I411N had similar functional characteristics with respect to the wild type FSHr (wtFSHr). Genetic analyses of polymorphisms in the human FSHr gene were also performed. All 50 women with PCOS harbored the A307T polymorphic variant, 56% harbored N680S, 30% S680S and 14% N680N polymorphisms. In conclusion, the present study demonstrates that mutations of the FSHr gene are rare in Italian women. The only mutation that we found does not appear to have any pathophysiological significance in PCOS.

Summary background Premature ovarian failure (POF) is defined by cessation of ovarian function after puberty and before the age of 40. The syndrome is characterized by amenorrhoea, oestrogen deficiency and elevated levels of gonadotrophins. Autoimmunity has been proposed as a mechanism for some cases of destruction or malfunction of ovarian follicles. POF is often associated with type I and type II polyglandular autoimmune syndromes. It has also been postulated that receptors such as the LH and FSH receptors might become targets for blocking antibodies and such antibodies could be a cause of ovarian failure. patients and methods Sixty-nine patients with POF isolated or associated with other endocrine autoimmune diseases (autoimmune thyroid diseases, Addison's disease, type 1 diabetes mellitus, multiple sclerosis, myasthenia gravis) were studied. All the patients had secondary amenorrhoea. The patient group had a median age of 33·1 years (range 15–57). Ovarian failure had been diagnosed at a median age of 29 years (range 15–39). The median time since diagnosis was almost 1 year but in six patients gonadal insufficiency had appeared 10–30 years earlier. All had a normal chromosomal karyotype (46, XX). Patients with POF were characterized by duration of amenorrhoea > 1 year, with elevated FSH and LH levels and undetectable or low oestrogen levels. Cell lines stably expressing recombinant human LH (CHO-LHr) and FSH (CHO-FSHr) receptors were prepared and used to search for antibodies able to inhibit LH- or FSH-stimulated cAMP production. Immunoglobulins extracted from sera of patients with POF were incubated with CHO-LHr and CHO-FSHr in the presence of human recombinant CG and FSH, respectively. results and conclusions None of the immunoglobulin G (IgG) preparations from patients with POF was able to inhibit the activity of the FSH- and CG-stimulated cAMP production.

Background The type 2 iodothyronine deiodinase (D2) converts the pro-hormone thyroxine into T3 within target tissues. D2 is essential for a full thermogenic response of brown adipose tissue (BAT), and mice with a disrupted Dio2 gene (D2KO) have an impaired response to cold. BAT is also activated by overfeeding. Methodology/Principal Findings After 6-weeks of HFD feeding D2KO mice gained 5.6% more body weight and had 28% more adipose tissue. Oxygen consumption (V02) was not different between genotypes, but D2KO mice had an increased respiratory exchange ratio (RER), suggesting preferential use of carbohydrates. Consistent with this, serum free fatty acids and β-hydroxybutyrate were lower in D2KO mice on a HFD, while hepatic triglycerides were increased and glycogen content decreased. Neither genotype showed glucose intolerance, but D2KO mice had significantly higher insulin levels during GTT independent of diet. Accordingly, during ITT testing D2KO mice had a significantly reduced glucose uptake, consistent with insulin resistance. Gene expression levels in liver, muscle, and brown and white adipose tissue showed no differences that could account for the increased weight gain in D2KO mice. However, D2KO mice have higher PEPCK mRNA in liver suggesting increased gluconeogenesis, which could also contribute to their apparent insulin resistance. Conclusions/Significance We conclude that the loss of the Dio2 gene has significant metabolic consequences. D2KO mice gain more weight on a HFD, suggesting a role for D2 in protection from diet-induced obesity. Further, D2KO mice appear to have a greater reliance on carbohydrates as a fuel source, and limited ability to mobilize and to burn fat. This results in increased fat storage in adipose tissue, hepatic steatosis, and depletion of liver glycogen in spite of increased gluconeogenesis. D2KO mice are also less responsive to insulin, independent of diet-induced obesity.