OBJECTIVE: NR5A1 is a key regulator of sex differentiation and has been implicated in spleen development through transcription activation of TLX1. Concerns exist about hypo- or asplenism in individuals who have a difference of sex development (DSD) due to an NR5A1 disease-causing variant. We aimed to assess spleen anatomy and function in a clinical cohort of such individuals and in their asymptomatic family member carriers.
METHODS: Cross-sectional assessment in 22 patients with a DSD or primary ovarian insufficiency and 5 asymptomatic carriers from 18 families, harboring 14 different NR5A1 variants.
METHODS: Spleen anatomy was assessed by ultrasound, spleen function by peripheral blood cell count, white blood cell differentiation, percentage of nonswitched memory B cells, specific pneumococcal antibody response, % pitted red blood cells, and Howell-Jolly bodies.
RESULTS: Patients and asymptomatic heterozygous individuals had significantly decreased nonswitched memory B cells compared to healthy controls, but higher than asplenic patients. Thrombocytosis and spleen hypoplasia were present in 50% of heterozygous individuals. Four out of 5 individuals homozygous for the previously described p.(Arg103Gln) variant had asplenia.
CONCLUSIONS: Individuals harboring a heterozygous NR5A1 variant that may cause DSD have a considerable risk for functional hyposplenism, irrespective of their gonadal phenotype. Splenic function should be assessed in these individuals, and if affected or unknown, prophylaxis is recommended to prevent invasive encapsulated bacterial infections. The splenic phenotype associated with NR5A1 variants is more severe in homozygous individuals and is, at least for the p.(Arg103Gln) variant, associated with asplenism.

OBJECTIVE: This study aimed to present the clinical features and gene mutation characteristics of a child with 46,XY disorders of sex development (DSD) caused by a novel heterozygous mutation in the NR5A1 gene to determine the potential association between this heterozygous mutation and the pathogenesis of 46,XY DSD.
METHODS: We present the case of a Chinese child with ambiguous genitalia at birth but a normal adrenal gland. Targeted next-generation sequencing, comprising 163 candidate genes involved in sexual differentiation and development, was performed, followed by the functional evaluation of the novel NR5A1 mutation.
RESULTS: The patient had a novel heterozygous mutation in the NR5A1 gene, c.630delG (p.Y211Tfs*85). Results revealed that overexpression of p.Y211Tfs*85 impaired steroidogenic factor-1 (SF-1) protein synthesis. Immunofluorescence analysis revealed that both SF-1 wild-type and p.Y211Tfs*85 mutation proteins were localized in the cell nucleus. Furthermore, dual-luciferase reporter assay results revealed that the p.Y211Tfs*85 mutation could effectively downregulate the transcriptional activation of anti-Müllerian hormone and steroidogenic acute regulatory protein genes (P < 0.01). Additionally, the p.Y211Tfs*85 mutation changed three-dimensional conformation of SF-1, and three conformations could be constructed with the mutated amino acid sequences. Therefore, the novel frameshift mutation could result in decreased protein expression of SF-1.
CONCLUSIONS: We described a novel mutation in NR5A1 and showed that it might affect protein structure, thereby seriously compromising the role of SF-1 in regulating gonadal development. The novel p.Y211Tfs*85 mutation in the NR5A1 gene enriches the boy of information available regarding the mutation spectrum of this gene in the Chinese population.

Hypercortisolism is one of the most commonly diagnosed endocrinopathies in dogs, and new targeted medical treatment options are desirable. Steroidogenic factor-1 (SF-1), an orphan nuclear hormone receptor, is a key regulator of adrenal steroidogenesis, development, and growth. In pituitary-dependent hypercortisolism (PDH), high plasma ACTH concentrations increase the transcriptional activity of SF-1. In adrenal-dependent hypercortisolism, SF-1 expression is significantly greater in dogs with recurrence after adrenalectomy than in those without recurrence. Inhibition of SF-1 could therefore be an interesting treatment option in canine spontaneous hypercortisolism. We determined the effects of 3 SF-1 inverse agonists, compounds IsoQ A, #31, and #32, on cortisol production, on the messenger RNA (mRNA) expression of steroidogenic enzymes and SFs, and on cell viability, in primary adrenocortical cell cultures of 8 normal adrenal glands and of 3 cortisol-secreting adrenocortical tumors (ATs). To mimic PDH, the normal adrenocortical cell cultures were stimulated with ACTH. The results show that only compound #31 inhibited cortisol production and SF-1 target gene expression in non-ACTH-stimulated and ACTH-stimulated normal adrenocortical cells but did not affect cell viability. In the AT cell cultures, the effects of #31 on cortisol production and target gene expression were variable, possibly caused by a difference in the SF-1 mRNA expressions of the primary tumors. In conclusion, inhibition of SF-1 activity shows much promise as a future treatment for canine hypercortisolism.

OBJECTIVE: Silent corticotroph adenomas (SCAs) are clinically silent and non-secreting, but exhibit positive adrenocorticotropic hormone (ACTH) immunostaining. We characterized a single center cohort of SCA patients, compared the SCAs to silent gonadotroph adenomas (SGAs), identified predictors of recurrence, and reviewed and compared the cohort to previously published SCAs cases.
METHODS: Retrospective review of SCA and SGA surgically resected patients over 10 years and 6 years, respectively. Definitions; SCA-no clinical or biochemical evidence of Cushing\'s syndrome and ACTH positive immunostaining, and SGA-steroidogenic factor (SF-1) positive immunostaining. A systematic literature search was undertaken using Pubmed and Scopus.
RESULTS: Review revealed 814 pituitary surgeries, 39 (4.8%) were SCAs. Mean follow-up was 6.4 years (range 0.5-23.8 years). Pre-operative magnetic resonance imaging demonstrated sphenoid and/or cavernous sinus invasion in 44%, 33% were > 50% cystic, and 28% had high ACTH levels pre-operatively. Compared to SGAs (n = 70), SCAs were of similar size and invasiveness (2.5 vs. 2.9 cm, p = 0.2; 44 vs. 41%, p = 0.8, respectively), but recurrence rate was higher (36 vs. 10%, p = 0.001) and more patients received radiation therapy (18 vs. 3%, p = 0.006). Less cystic tumors (0 vs. 50%, p < 0.001) and higher pre-operative ACTH levels (54 vs. 28 pg/ml, p = 0.04) were predictors of recurrence for SCAs.
CONCLUSIONS: This review is unique; a strict definition of SCA was used, and single center SCAs were compared with SGAs and with SCAs literature reviewed cases. We show that SCAs are aggressive and identify predictors of recurrence. Accurate initial diagnosis, close imaging and biochemical follow up are warranted.

Steroidogenic factor 1 (SF-1) belongs to a small group of the transcription factors that bind DNA only as a monomer. Three different approaches-Sitecon, SiteGA, and oPWM-constructed using the same training sample of experimentally confirmed SF-1 binding sites have been used to recognize these sites. The appropriate prediction thresholds for recognition models have been selected. Namely, the thresholds concordant by false positive or negative rates for various methods were used to optimize the discrimination of steroidogenic gene promoters from the datasets of non-specific promoters. After experimental verification, the models were used to analyze the ChIP-seq data for SF-1. It has been shown that the sets of sites recognized by different models overlap only partially and that an integration of these models allows for identification of SF-1 sites in up to 80% of the ChIP-seq loci. The structures of the sites detected using the three recognition models in the ChIP-seq peaks falling within the [-5000, +5000] region relative to the transcription start sites (TSS) extracted from the FANTOM5 project have been analyzed. The MATLIGN classified the frequency matrices for the sites predicted by oPWM, Sitecon, and SiteGA into two groups. The first group is described by oPWM/Sitecon and the second, by SiteGA. Gene ontology (GO) analysis has been used to clarify the differences between the sets of genes carrying different variants of SF-1 binding sites. Although this analysis in general revealed a considerable overlap in GO terms for the genes carrying the binding sites predicted by oPWM, Sitecon, or SiteGA, only the last method elicited notable trend to terms related to negative regulation and apoptosis. The results suggest that the SF-1 binding sites are different in both their structure and the functional annotation of the set of target genes correspond to the predictions by oPWM+Sitecon and SiteGA. Further application of Homer software for de novo identification of enriched motifs in ChIP-Seq data for SF-1ChIP-seq dataset gave the data similar to oPWM+Sitecon.

DAX1 is an orphan nuclear receptor that has a key role in the development and function of the adrenal and reproductive axes. Mutations in NR0B1, the gene encoding DAX1, result in X-linked adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HHG). A Chinese pedigree with X-linked AHC and HHG was investigated in the present study. Sequence analysis identified a novel small indel variant, c.195_207delinsTG, in the NR0B1 gene. To determine the effect of this variant on DAX1 expression, reverse‑transcription quantitative PCR and western blot assays were performed. The mRNA expression levels in carriers of mutant NR0B1 were significantly reduced (62% decrease) compared to those in individuals with wild-type NR0B1 (WT). The c.195_207delinsTG mutation was demonstrated to lead to various truncated DAX1 proteins, including the C‑terminal truncated DAX1, which was only detected in the cytoplasm, and the N‑terminal truncated DAX1, which was present in the cytoplasm and nucleus. A luciferase assay was then performed to assess the repressor function of DAX1 in modulating steroidogenic factor 1 (SF‑1)‑mediated transactivation. WT DAX1 significantly suppressed the SF‑1‑mediated promoter activity of the steroidogenic acute regulatory protein by 35.5±1.9%. In contrast to other known pathogenic mutations which abolish the repressor function of DAX1, the c.195_207delinsTG mutant proxkduced a higher repressor activity, demonstrating a 49.9±2.6% reduction of promoter activity. These findings suggested that the mutation of NR0B1 in X‑linked AHC with HHG enhanced the function of DAX1 to repress SF-1 activation, while DAX1 is expected to have additional roles in the pathological mechanism.

OBJECTIVE: The expression of aromatase (via CYP19 and the CYP19 PII promoter) and the orphan nuclear receptor family members, liver receptor homologue-1 (LRH-1) and steroidogenic factor-1 (SF-1) in cultured luteinized granulosa cells from women with endometriosis were investigated.
METHODS: Luteinized granulosa cells from patients undergoing in vitro fertilization (16 patients with endometriosis and 28 controls) were examined for messenger ribonucleic acid (mRNA) expression of CYP19, CYP19 PII, LRH-1 and SF-1, determined by quantitative reverse transcription-polymerase chain reaction. Student\'s t-test, the Mann-Whitney U test and Spearman\'s rank correlation were used for statistical analysis.
RESULTS: The number of high quality embryos in the endometriosis group was significantly lower than in the control group. The mRNA expression levels of CYP19, CYP19 PII, LRH-1 and SF-1 in granulosa-lutein cells were decreased in women with endometriosis compared to the control group. The simultaneous down-regulation expression of LRH-1, SF-1 and CYP19 PII in endometriotic granulosa cells indicated their positive correlation.
CONCLUSIONS: Our results demonstrate aberrant expressions of SF-1 and LRH-1 in endometriotic granulosa-lutein cells. This finding may be helpful in understanding infertility associated with endometriosis and reduced P450 aromatase activity in endometriotic granulosa cells.

Steroidogenic factor 1 (NR5A1/SF-1) mutations usually manifest in 46,XY individuals with variable degrees of disordered sex development and in 46,XX women with ovarian insufficiency. So far, there is no genotype-phenotype correlation. The broad spectrum of phenotype with NR5A1 mutations may be due to a second hit in a gene with similar function to NR5A1/SF-1. Liver receptor homologue-1 (LRH-1/NR5A2) might be a good candidate. We performed in vitro studies for the interplay between SF-1, LRH-1 and DAX-1, expression profiles in human steroidogenic tissues, and NR5A2 genetic studies in a cohort (11 patients, 8 relatives, 11 families) harboring heterozygote NR5A1/SF-1 mutations. LRH-1 isoforms transactivate the CYP17A1 and HSD3B2 promoters similarly to SF-1 and compensate for SF-1 deficiency. DAX-1 inhibits SF-1- and LRH-1-mediated transactivation. LRH-1 is found expressed in human adult and fetal adrenals and testes. However, no NR5A2/LRH-1 mutations were detected in 14 individuals with heterozygote NR5A1/SF-1 mutations. These findings demonstrate that in vitro LRH-1 can act like SF-1 and compensate for its deficiency. Expression of LRH-1 in fetal testis suggests a role in male gonadal development. However, as we found no NR5A2/LRH-1 mutations, the \'second genetic hit\' in SF-1 patients explaining the broad phenotypic variability remains elusive.

Adrenal rest is uncommon in adults, and usually represents a small lesion incidentally detected during surgery or autopsy. The adrenal rest can be detected anywhere along the path of embryonic migration of adrenal cortex, including celiac axis, genitals and broad ligament, and may be formed with the separation of cortical fragments by the migration of medullary elements from the sympathochromaffin tissue into the preformed cortical primordium. In addition, even primary adrenocortical carcinoma is a rare tumor with incidence 0.5-2 per million annually; therefore, adrenocortical carcinoma arising in adrenal rests is extremely rare. We encountered a patient with non-functioning ectopic adrenocortical carcinoma in retroperitoneum. A 34-year-old female presented with an incidentally discovered retroperitoneal mass revealed by abdominal ultrasound in her regular health examinations. She did not have any clinical abnormalities and underwent hand-assisted laparoscopic resection of the tumor. A dark-brown tumor, measuring 65 × 56 × 45 mm, was identified in the retroperitoneal space between lower pole of right kidney and inferior vena cava. Histologically, the tumor was predominantly composed of compact eosinophilic cells forming nest-like arrangements and diffusely positive for the steroidogenic factor-1. The tumor met four of the criteria of Weiss used in histological diagnosis of adrenocortical carcinoma (eosinophillic cytoplasm, nuclear atypia, atypical mitosis, and sinusoidal invasion). The tumor cells were immunohistochemically positive for 17α-hydroxylase, dehydroepiandrosterone sulfotransferase and 3β-hydroxysteroid dehydrogenase, each of which is involved in the synthesis of adrenocortical steroids. Therefore, based on these findings, we diagnosed this tumor as ectopic adrenocortical carcinoma arising in adrenal rest of retroperitoneum.

This study aimed to identify an immunohistochemical panel to aid in the differential diagnosis for tumors with clear cell morphology. Twenty-five clear cell renal cell carcinomas (CCRCCs), 19 clear cell ovarian carcinoma (CCOCs), 20 cases of adrenal cortical carcinomas(ACCs), and 10 chordomas were stained for renal cell carcinoma marker (RCC Ma), Pax8, brachyury, and steroidogenic factor 1 (SF-1). The extent of stains was scored as focal (<25%), nonfocal (25%-50%), and diffuse (>50%). The intensity was scored as weak, moderate, and strong. Twenty-two CCRCCs were positive for RCC Ma (88%) and Pax8 (88%), respectively. The RCC Ma cytoplasmic staining was largely diffuse (76%) and strong (76%). The nuclear Pax8 staining was usually diffuse (76%) and moderate (64%) to strong (8%). All of CCRCCs were negative for brachyury and SF-1. All of 19 CCOCs were positive for Pax8 nuclear staining. The staining was diffuse, moderate (21%) to strong (79%). All of CCOCs were negative for RCC Ma, brachyury, and SF-1. All of 20 ACCs were positive for SF-1 nuclear staining. The staining was largely diffuse (95%), moderate (55%) to strong (15%). All of ACC were negative for RCC Ma, Pax8, and brachyury. All of 10 chordomas were positive for brachyury nuclear staining. The staining was diffuse and strong. All of chordomas were negative for RCC Ma, Pax8, and SF-1. In summary, the panel of RCC Ma, Pax8, brachyury, and SF-1 is useful in the differential diagnosis of tumors with clear morphology.