Tested Research Articles

Employee Loyalty and Performance of Taiwan's Semiconductor Industry: Analysis of TSMC's Business Alliance

Taiwan's semiconductor industry plays a critical role in the global economy with its technological innovation, efficient manufacturing, and market leadership, profoundly shaping the global semiconductor landscape. It explores whether there is a positive correlation between employee loyalty and corporate performance and further examines if the parent-subsidiary relationship or business alliance with TSMC or UMC influences the relationship between employee loyalty and corporate performance. The study compiles data from Taiwan’s semiconductor industry, with the upstream comprising IP design and IC design, the midstream including IC manufacturing, wafer fabrication, related testing equipment, reticle masks, and chemicals, and the downstream covering IC packaging and testing, IC modules, and IC distribution. Data from 2005 to 2020, comprising 1015 valid data, were used. Correlation and regression analyses were conducted to observe the relationship between employee loyalty and corporate performance. The study aims to deeply investigate the impact of employee loyalty on the performance of Taiwanese semiconductor companies, especially considering the scenarios of consortium and business alliance cooperation. Through literature review and empirical analysis, the study assesses how employee loyalty affects the performance indicators of semiconductor companies and explores the moderating role of consortium and business alliance cooperation in this impact. By delving into employee loyalty in Taiwan’s semiconductor sector, this study aims to provide practical recommendations for human resource management strategies and consortium cooperation decisions to promote the sustainable development of Taiwanese semiconductor companies. The findings reveal a negative correlation between the average seniority and turnover rate of employees and performance variables, while employee growth rate shows a positive correlation in upstream and midstream companies but a negative correlation overall. The results vary slightly across the upstream, midstream, and downstream sectors. In terms of business alliance effects, companies with business ties to TSMC exhibit a moderating effect on the relationship between employee loyalty and performance variables.

Precocious Puberty in Boys with NR0B1 Variants

Precocious puberty (PP) requires appropriate management to prevent short adult height, psychosocial issues, and other adverse outcomes. Genetic diagnosis potentially improves the management of PP. Pathogenic NR0B1 variants, which typically cause X-linked adrenal hypoplasia congenita, can also affect gonadal function. While boys with NR0B1 variants usually exhibit hypogonadotropic hypogonadism during adolescence, previous reports have suggested that minipuberty, a physiological transient activation of the hypothalamic–pituitary–gonadal axis during infancy, occurs in these patients and can persist beyond a typical duration. In rare cases, NR0B1 variants cause PP. PP associated with NR0B1 variants has unique features such as early onset and high serum testosterone levels that are often disproportionate to testicular size. Three underlying mechanisms have been proposed for the association between PP and NR0B1 variants: (1) adrenocorticotropic hormone (ACTH)-dependent, (2) gonadotropin-dependent, and (3) ACTH- and gonadotropin-independent mechanisms. The factors contributing to PP vary among cases. Determining the underlying mechanisms is crucial for adopting appropriate therapeutic strategies to control PP. However, as the detailed molecular networks mediating these mechanisms are largely unclear, further research is needed to pave the way for a more effective and personalized management of patients with PP associated with NR0B1 variants.

Bassa statura non sindromica e ritardo puberale: non solo ritardo costituzionale di crescita e pubertà

A 14-year-old boy with suspected constitutional delay of growth and puberty (CDGP) presented with short stature, absence of secondary sex characteristics, bone age delayed by one year and family history of CDGP in his mother. The external genitalia were normal. Re-evaluated at the age of 17 years and 7 months (as he missed several visits), he presented with a short near-final height (bone age consistent with chronological age) below the target height, signs of progressed virilization, but insufficient testicular volume. The prevalence of nonsyndromic 46,XX testicular disorder is estimated at 1:20,000 males. In 80% of cases the condition is due to the presence of the SRY gene on the X chromosome, resulting from abnormal paternal meiosis. It is characterized by short stature, hypergonadotropic hypogonadism and sterility. The need for routine screening of gonadal cancer is still a matter of debate. Treatment of this condition consists of testos-terone replacement from the pubertal age. Psychological support should also be warranted since the communication of the diagnosis. The case suggests that non-syndromic 46,XX testicular disorder should be considered when evaluating a boy with hypogonadism and short stature, especially in patients with suspected CDGP who have progressive virilization and advancement of bone age, despite no increase in testicular volume.

Zinc Deficiency Leads to Reproductive Impairment in Male Mice Through Imbalance of Zinc Homeostasis and Inflammatory Response.

Zinc is an essential trace element crucial for growth and development and plays a significant role in male reproductive function. The aim of this study was explore the mechanism of male reproductive damage caused by different degrees of zinc deficiency. Thirty male ICR mice were randomly assigned to three groups: zinc-normal diet group (ZN, n = 10, Zn content = 30mg/kg), low zinc-deficiency diet group (LZD, n = 10, Zn content = 15mg/kg), and high zinc-deficiency diet group (HZD, n = 10, Zn content = 7.5mg/kg). The mice were maintained for 8weeks. At the end of the experiment, they were sacrificed, and their blood, testicular, and epididymal tissues were collected for further study. Zinc-deficient diet led to weight loss, testicular structural disorder, decreased semen quality, imbalance of zinc homeostasis, and inflammatory damage in mice. Semen quality, testosterone, serum Zn, testicular tissue Zn, testicular free Zn ions, Zrt-, Irt-like protein8 (ZIP8), Zrt-, Irt-like protein5 (ZIP5), and interleukin-10 (IL-10) were significantly decreased; zinc transporter 4(ZnT4), NF-κB p65, P-NF-κB p65, NLRP3, Caspase-8, and Caspase-3 were significantly increased in both LZD and HZD group mice. While compared with the LZD group, Zrt-, Irt-like protein13 (ZIP13), TNF-α, NF-κB p65, P-NF-κB p65, NLRP3, Caspase-1, and GSDMD were significantly increased in the HZD group. Both low and high zinc-deficiency diets can disrupt zinc homeostasis in mice, leading to heightened inflammatory responses, the activation of the NF-κB pathway, and increased apoptosis in testicular cells. Notably, a high zinc-deficiency diet led to an up-regulation of ZIP13 expression, exacerbated inflammation, and induced testicular pyroptosis, resulting in more severe reproductive damage in male mice.

Analysis of Molding Defection in IC Packaging and Testing Process

Molding injects a molding compound into a mold to form a protective shell around the wafer. During the injection process, overflow may occur, leading to mold flash, which reduces yield and causes significant manufacturing cost losses. This paper proposes a deep-learning-based method for detecting and predicting the occurrence of mold flash probability to address this issue. First, the paper conducts random forest importance analysis and correlation analysis to identify the key parameters that significantly impact mold flash. This paper uses these key parameters as input signals for the prediction model. The paper introduces an HLGA Transformer to construct an ensemble meta-learning model that predicts the probability of molding defects, achieving a prediction accuracy of 98.16%. The ensemble meta-learning approach proposed in this paper outperforms other methods in terms of performance. The model predictions can be communicated to the system in real time, allowing it to promptly adjust critical machine operation parameters, thereby significantly improving the molding process yield and reducing substantial manufacturing cost losses.

Microscopic Testicular Sperm Extraction in Patients with Klinefelter Syndrome: Long-Term Outcomes from a Single Center

Objective: Klinefelter syndrome (KS) represents a sex chromosome anomaly observed in approximately 1 in 500–600 phenotypic males. It is observed in 3% of infertile males and up to 11.9% of azoospermic males. KS manifests in either non-mosaic (47, XXY) or mosaic (47, XXY/46, XY) forms, with 85% of cases presenting as the non-mosaic 47, XXY karyotype. The average rate of surgical sperm retrieval in patients with KS is around 50%, ranging from 28% to 69%. In this study, we aimed to present the outcomes of microscopic testicular sperm extraction (micro-TESE) in patients with non-mosaic KS. Materials and Methods: The results of 61 patients diagnosed with KS, who presented to the Harran University Urology Clinic with azoospermia between 2017 and 2024, were retrospectively reviewed. Hormonal assessments, including follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol, and total testosterone (TT), were conducted for all patients, and their partners underwent gynecological evaluations for infertility. Testicular dimensions were recorded via scrotal ultrasonography. Patients were categorized into TESE-positive and TESE-negative groups, and parameters were compared between these groups. Results: The mean age of the patients was 29.0±5.1 years, and their mean infertility duration was 5.9±4.1 years. The sperm retrieval rate was 29.5% (n=18). Mean levels of FSH, LH, prolactin, estradiol, and TT were 44.9 IU/L, 23.3 IU/L, 10 nmol/L, 31.4 pmol/dL, and 219 ng/dL, respectively. Sperm was retrieved in 18 patients (29.5%), while no sperm was obtained in 43 (70.5%). No significant correlation was observed between patient age, testicular size, serum levels of FSH, LH, prolactin, estradiol, and TT, and sperm retrieval rates when comparing the TESE-positive and TESE-negative groups (P>0.005). Conclusion: In patients with non-mosaic KS, hormonal parameters, age, and infertility duration were not found to be significant predictors of the success of micro-TESE in sperm retrieval.

Longitudinal trends in testicular volume z scores from puberty to adulthood, sperm quality, and paternity outcomes after childhood cancer.

Childhood cancer therapy may cause subfertility. This study correlated cancer therapy exposures with testicular volumes from puberty to adulthood, spermatogenesis, and paternity outcomes in adulthood. The study population comprised 255 male childhood cancer survivors (CCS) (survival ≥5 years, diagnosed in 1964-2000 at the Helsinki Children's Hospital) whose testicular volume was measured at ages 12 years (n=38), 14 years (n=57), 16 years (n=63), 18 years (n=105), and in adulthood (n=43; median age, 27 years). Testicular volumes were converted to age-specific z scores. In addition, 92 CCS provided semen sample in adulthood (median age, 25.2 years); and paternity was evaluated through national register data (mean age at assessment, 37.6 years; n=252). Compared with age-specific reference values, CCS generally exhibited low testicular volume z scores at ages 12-18 years. Testicular volume z scores in CCS treated exclusively with chemotherapy returned to the reference range in adulthood. In contrast, patients exposed to testicular radiation ≥1 gray (Gy) (median dose, 12 Gy) showed no late recovery in testicular size. Testicular radiation ≥1 Gy and a cyclophosphamide equivalent dose ≥12 g/m2 were identified as risk factors for azoospermia in adulthood. Patients exposed to testicular radiation ≥1 Gy and a cyclophosphamide equivalent dose ≥4 g/m2 had lower paternity rates. Testicular volume growth after prolonged follow-up suggests a potential late recovery of spermatogenesis in CCS treated exclusively with chemotherapy. However, alkylating agents increased the risk of having prolonged azoospermia and nonpaternity. High-dose testicular radiation causes long-term depletion of spermatogonia and was the strongest risk factor for azoospermia and nonpaternity.

Self-puberty staging in endocrine encounters during the COVID pandemic.

Evaluation of pubertal development is crucial in Endocrinology. The rise in telemedicine during COVID-19 has made conduct of physical examinations more challenging, especially for pubertal assessment. Previous studies on validity of pubertal self-staging versus clinical examination have yielded mixed results. This study aimed to determine validity and reliability of self-staging of puberty, with potential application during telemedicine visits. The present study is the first to assess pediatric self-rated pubertal staging during the pandemic. The study included patients aged 7-22 years referred to Pediatric Endocrinology for specialty care, including pubertal staging. At clinic check-in, patients received a packet with study description, an option to "opt in" or "opt out", sex-specific self-staging instructions, and Tanner (T) stage illustrations. Males received materials for pubic hair (PH) stages T1-T5; females received materials for PH and breast (BR) stages T1-T5. Patients who opted in had 10 minutes to select the image(s) that best matched their bodies, which they sealed in an envelope. This was followed by a clinic visit, where a board-certified pediatric endocrinologist conducted a physical examination, including breast staging (females), testicular size measurement (males), and pubic hair staging (both sexes). Pubertal stage Kappa statistics with 95% CI were calculated for each body part by sex, with Kappa ≥ 0.60 indicating significant agreement between self-assessment and physician assessment (0.40-0.60 moderate; 0.20-0.40 fair). Of 516 distributed packets, 243 self-assessments (125 females) were returned, with 81% (94 females/102 males) being complete (including pediatric endocrinologist staging). Mean age of participants was 12.8 years. Mean BMI was 22.2 kg/m² (males) and 23.7 kg/m² (females). Hypothyroidism was the most common endocrine diagnosis. For females, kappa was highest for BR and PH in T1 (BR 0.65, PH 0.57) and T5 (BR 0.57, PH 0.65). For males, kappa was highest in T1 (0.73) and T2 (0.58). Grouping Tanner stages into prepuberty (T1), early to mid-puberty (T2-T3), and late puberty (T4-T5) showed greater agreement. Patients can reliably distinguish between "puberty" and "no puberty" using self-staging, though differentiating between later pubertal stages is more challenging. These findings help define the utility and limitations of self-staging during telemedicine visits.

Ovarian and testicular ripening process, reproductive season and size at maturity in Octopus hubbsorum (Cephalopoda: Octopodidae)

ABSTRACT This paper contributes to the knowledge of the reproductive biology of Octopus hubbsorum Berry 1953. Its reproductive season in Bahía Magdalena, Baja California Sur ranges from May to October. The increase of gonadosomatic index in females coupled with the decline in Guerra’s index and the increase in oocyte diameter in May and September were indicative of spawning. The total sex ratio was 1:1; males were larger than females. Females reach sexual maturity at 119.7 mm of dorsal mantle length and 628.6 g of total weight. This paper also addressed different aspects on reproductive studies, allowing the identification of the type of gonadal development as a group-synchronous type and the transition from physiological to functional maturity, in both qualitative and quantitative terms (proportion of oocyte stages). Detailed descriptions of the oogenesis/folliculogenesis stages of O. hubbsorum are presented with detailed photographs (in Supplemental online material).

Improved diagnostic confidence using Super Resolution CEUS imaging in testicular lesions.

Ultrasound is the most used interdisciplinary non-ionizing imaging technique in clinical pathologies of the testis. The testis may be affected by a plethora of different disorders such as vasculopathies, trauma, infections and manifestations of primary and secondary malignant masses. Conventional ultrasound represents the basic imaging modality of choice to assess scrotal disorders. Contrast-enhanced ultrasound (CEUS) can provide further information to distinguish between benign and malignant testicular mass lesions. The recent introduction of Super Resolution CEUS Micro-Vascular Imaging (MVI SR) and Time of Arrival (TOA SR) parametric mapping compliments the information provided by conventional CEUS, since these two new post-processing techniques improve the visualization of microvascular structures with slow blood flow and provide high-resolution images of the peak contrast enhancement and temporal perfusion patterns. This paper gives a comprehensive overview of differential diagnoses of the testicular disorder and their corresponding sono-morphologic correlates based on representative cases of the Interdisciplinary Ultrasound Center of the University Hospital Munich.

Thyroidectomy Induced Toxicity of Testis and Possible Amelioration by Chia (Salvia hispanica) Seeds Extract

Background: Thyroidectomy or thyroid surgery induces hypothyroidism and causes thyroid and testicular hormone deficiency. The current Study Aimed to examine the therapeutic effect of chia seeds extract against thyroidectomy-induced testicular toxicity, oxidative stress, DNA damage, proliferation in rats. Methods: 40 male rats were equally divided into 4 groups [Gp1, control ; Gp2, CSE ; Gp3, thyroidectomies rats; Gp4, treated thyroidectomies rats with chia seeds extract (Thy+CSE )]. Result: Following a thyroidectomy, significant reductions (P less than 0.05) were observed in The Levels of triiodothyronine (T3), thyroxine (T4), sexual hormones (Testosterone (tt), Follicle-Stimulating Hormone and Luteinizing Hormone And prolactin), count of sperm, index of morphology, vitality, progressive motility, total motility (PR+NP), Glutathione, Superoxide Dismutase, Glutathione S-Transferase and PCNA expressions compared to the control and CSE after three weeks. Additionally, thyroidectomy leads to significant (P less than 0.05) increases in the serum of thyrotropin (TSH), alanine transaminase, alkaline phosphatase, non-progressive sperms, immotile sperms, malondialdehyde, DNA damage and injury of testis and significant (P less than 0.05) decreases in blood. Thy+CSE treatment helped to modulate most of these changes and recover testicular injury when compared To The Control and CSE. Rats treated with CSE after thyroidectomy Showed gradual improvements in testicular functions, histology and spermatogenesis due to its beneficial affection in free radicals’ scavenger.

Integration of long-read sequencing, DNA methylation and gene expression reveals heterogeneity in Y chromosome segment lengths in phenotypic males with 46,XX testicular disorder/difference of sex development.

46,XX testicular disorder/difference of sex development (46,XX DSD) is a rare congenital condition, characterized by a combination of the typical female sex chromosome constitution, 46,XX, and a variable male phenotype. In the majority of individuals with 46,XX DSD, a Y chromosome segment containing the sex-determining region gene (SRY) has been translocated to the paternal X chromosome. However, the precise genomic content of the translocated segment and the genome-wide effects remain elusive. We performed long-read DNA sequencing, RNA sequencing and DNA methylation analyses on blood samples from 46,XX DSD (n = 11), male controls (46,XY; variable cohort sizes) and female controls (46,XX; variable cohort sizes), in addition to RNA sequencing and DNA methylation analysis on blood samples from males with Klinefelter syndrome (47,XXY, n = 22). We also performed clinical measurements on all 46,XX DSD and a subset of 46,XY (n = 10). We identified variation in the translocated Y chromosome segments, enabling subcategorization into 46,XX DSD (1) lacking Y chromosome material (n = 1), (2) with short Yp arms (breakpoint at 2.7-2.8Mb, n = 2), (3) with medium Yp arms (breakpoint at 7.3Mb, n = 1), and (4) with long Yp arms (n = 7), including deletions of AMELY, TBLY1 and in some cases PRKY. We also identified variable expression of the X-Y homologues PRKY and PRKX. The Y-chromosomal transcriptome and methylome reflected the Y chromosome segment lengths, while changes to autosomal and X-chromosomal regions indicated global effects. Furthermore, transcriptional changes tentatively correlated with phenotypic traits of 46,XX DSD, including reduced height, lean mass and testicular size. This study refines our understanding of the genetic composition in 46,XX DSD, describing the translocated Y chromosome segment in more detail than previously and linking variability herein to genome-wide changes in the transcriptome and methylome.

Successful Neoadjuvant Chemotherapy and Surgical Removal of a Nonmetastatic Testicular Round Cell Tumor in a Solomon Island Eclectus Parrot (Eclectus roratus solomonensis).

An intracoelomic mass was palpated on an annual exam of a 24-year-old male Solomon Island eclectus parrot (Eclectus roratus solomonensis). The initial diagnostic workup included a complete blood count, plasma biochemistry panel, and coelomic ultrasound. Computed tomography was highly suggestive of a testicular mass. Tamoxifen and the gonadotropin-releasing hormone agonists leuprolide and deslorelin were administered as neoadjunctive endocrine therapies. Biopsy and histologic examination confirmed a testicular mass consistent with a round cell tumor. Four doses of carboplatin 15 mg/kg IV were administered as neoadjunctive chemotherapy, and testicular size decreased by approximately 95%. The remaining gross tumor was removed via orchidectomy with clean but narrow margins. Seven months following surgery, a contrast CT scan did not show any evidence of recurrence of or metastasis from the original mass. This is the first report of successful treatment of a testicular tumor in a psittacine with neoadjuvant chemotherapy and orchidectomy.

8320 Integration of long-read sequencing, DNA methylation and gene expression reveals heterogeneity in Y-chromosome segment lengths in phenotypic males with 46,XX testicular disorder of sex development

Abstract Disclosure: E.B. Johannsen: None. A. Berglund: None. A. Skakkebæk: None. S. Chang: None. J. Rohayem: None. S. Laurentino: None. A. Hørlyck: None. S.O. Drue: None. E.N. Bak: None. J. Fedder: None. F. Tuttelmann: None. J. Gromoll: None. J. Just: None. C.H. Gravholt: None. Background: 46,XX testicular disorder of sex development is a rare congenital condition affecting approximately three per 100,000 newborn males. It is characterized by a combination of the typical female sex chromosome constitution, 46,XX, and a variable male phenotype. In the majority of XX males, a Y chromosome segment containing the sex-determining region gene (SRY) has been translocated to the paternal X chromosome. However, the precise genomic content of the translocated segment, breakpoints, genome-wide effects, and the effect on other biological pathways essential for the phenotype remain unknown. Methods: We performed long-read DNA sequencing (Oxford Nanopore), RNA sequencing (paired-end, 100 bp) and DNA methylation (Infinium EPIC) analysis on blood samples from males with 46,XX testicular disorder of sex development (XX males; n = 11), male controls (46,XY; n = 12 for long-read DNA sequencing, n = 26 for RNA sequencing and DNA methylation) and female controls (46,XX; n = 12 for long-read DNA sequencing, n = 32 for RNA sequencing and DNA methylation), in addition to RNA sequencing and DNA methylation analysis on blood samples from males with Klinefelter syndrome (47,XXY, n = 22). We also performed clinical measures on all XX males and a subset of male controls (n = 10). Results: We identified variation in the translocated Y-chromosome segment of our XX male cohort, allowing for subcategorization into 1) XX males without Y chromosome material (n = 1), 2) those with short Yp arms (breakpoint at 2.7-2.8 Mbp, n = 2), 3) those with medium Yp arms (breakpoint at 7.3 Mbp, n = 1) and 4) those with long Yp arms, including AMELY, TBLY1 deletions, and in some cases PRKY deletions (n = 7). We also identified variable expression of the X-Y homologues PRKY and PRKX, appeared to have balanced expression level resulting in overall equilibrium. The Y-chromosomal transcriptome and methylome were affected, reflecting the Y-chromosome segment length. Genomic changes induced by the translocated Yp segment were evident in the transcriptome and methylome of autosomal and X-chromosomal regions, indicating global effects. Furthermore, the transcriptional changes in XX males correlated with phenotypic traits of the XX males, including lower height, decreased lean mass and smaller testicular size. Conclusion: Integration of long-read DNA sequencing, DNA methylation, and RNA sequencing analysis allowed for the identification of a heterogenous translocation process in XX males that exerted widespread effects on methylation and transcription. Presentation: 6/2/2024

8786 Novel TGFbeta Ligand Regulated Genes in WT and FSTL3 KO Testis Identified by ChIP-Seq Analysis: Implications for Signalling Cross-talk

Abstract Disclosure: R. Ballesteros: None. A. Mukherjee: None. Folistatin Like 3 (FSTL3) is a known antagonist of activin and myostatin action that when deleted from the mouse genome displayed a set of phenotypic changes that are directly linked to glucose metabolism and reproductive function. Although our previous work showed that β-cell hyperplasia, increased pancreatic islet number and size and reduced visceral fat seen in FSTL3 knockout mice (FSTL3 KO) could be explained by enhanced activin and myostatin action, the molecular bases of the changes in the reproductive function seen in this mouse model remain elusive. In contrast to testicular degeneration produced by INHBA (activin A) transgenic overexpression in mice, deletion of FSTL3 results in increased testicular size, with increased Sertoli cell numbers and early meiotic entry of the first spermatogenic wave. This suggests a possibility that the phenotype in FSTL3 deleted mice might involve alteration of additional transcripts other than those induced by activin alone in the testis. To identify transcripts induced in FSTL3 deleted testis as a consequence of induced TGFβ ligand action we performed sequencing of Smad4 Chromatin immunoprecipitation (ChIP-Seq) from postnatal (3d) and post-pubertal (56d) whole testis. Our results show high activity of Smad4 signalling during postnatal stages compared to post-pubertal stages in both genotypes (Fstl3-KO vs wild-type). Interestingly, postnatal testis from FSTL3 KO contains significantly less activity from Smad4 signalling (69 peaks) compared to wild type (775 peaks). By contrast, on the whole, Smad4 signalling in post-pubertal stages is less active with 34 peaks in wild-type and 29 peaks in Fstl3-KO of which 8 genes are SMAD activated in both genotypes. To identify whether SMAD-dependent signalling produces significant differences in RNA expression, we then used the list of genes that generated peaks in the Smad4-ChIP-Seq experiment to filter our RNA-Seq data generated in a parallel experiment followed by differential gene expression analysis of GO terms by GSEA in postnatal testis from both genotypes. We found that Smad4-interacting genes in posnatal FSTL3 KO testis generate enrichment of 13 pathways including the androgen receptor signalling pathway. Taken together so far, our findings reveal a novel catalogue of SMAD-regulated genes in the testis, identifies a set of genes specifically upregulated in the testis in a SMAD-dependent manner upon FSTL3 deletion and also identify possible cross-talk between TGFβ and 13 other signalling pathways. Currently we are further analysing our data to identify whether there are SMAD regulated genes and pathways common in postnatal and postpubertal testes. We are also undertaking experiments to investigate expression pattern in FSTL3 KO testis to identify which pathways might specifically affect Sertoli cell proliferation and activity. Presentation: 6/2/2024

7640 The first description of an MC4R variant in a patient with Kallmann syndrome and obesity

Abstract Disclosure: A.A. Aslam: None. S. Lim: None. R. Willemsen: None. K. Koysombat: None. M. Young: None. W.S. Dhillo: None. A. Abbara: None. S. Howard: None. E.F. Gevers: None. Introduction: Pathogenic MC4R gene variants result in hyperphagia and early onset obesity but puberty is not usually affected. We describe an MC4R variant in a patient with Kallmann syndrome and obesity. A 16 year old male with repaired Tetralogy of Fallot, anosmia, autism and anxiety, was referred with obesity and delayed puberty. His birth weight was 3360 g (-0.26 SDS). Height was -1.31 SDS, BMI 30.7 kg/m2. He had high arched palate, normal skin and hair colour, mild hypertelorism and upward slanting palpebral fissures. Pubertal staging was A2P1G1 5ml testes bilaterally. Results: LHRH test showed borderline low peaks (LH 5.0 U/L, FSH 2.6 U/L) and inhibin B 108 pg/mL (25-325 pg/mL). MRI brain showed hypoplastic olfactory bulbs, absent olfactory sulci and normal pituitary anatomy. CGH microarray, karyotype and the UK hypogonadism gene panel were normal. He underwent whole genome sequencing in the Genetic Factors Affecting the Timing of Puberty Study; no abnormalities in 52 known genes associated with GnRH deficiency were found but a previously described heterozygous variant was detected, MC4R c.542G>A, p.Gly181Asp, classified pathogenic and absent in control databases. Management: He was commenced on Testosterone but showed progression of testicular size to 10 mL and testosterone was stopped but required restarting. Repeat investigations off treatment, aged 22 years (height 178 cm, BMI 42 kg/m2, testes 15 mL): inhibin B 66 pg/mL, testosterone 2 nmol/L, baseline LH 2.4 U/L, stimulated peak 24.9 U/L, baseline FSH 1.2 U/L, stimulated peak 4.2 U/L. Cortisol and IGF1 were in the normal range. He also developed autoimmune hypothyroidism with raised TPO antibodies (TSH 24.7 mU/L, FT4 5.6 pmol/L). Discussion: Previous in vitro work has shown complete loss of function of MC4R p.Gly181Asp due to reduced cell surface expression and reduced binding to a-MSH. Heterozygous p.Gly181Asp variants have been described in several children/adults with obesity; but hypogonadism in heterozygous carriers has not. Homozygous MC4R p.Gly181Asp was found in a male with obesity and partial HH thought to be due to abnormal GnRH production but with normal olfactory bulbs. We are the first to describe a heterozygous MC4R p.Gly181Asp variant in a patient with partial hypogonadism and anosmia with hypoplastic bulbs in addition to obesity. Interaction between POMC-MC-leptin circuits and Kisspeptin-GnRH circuits is recognised although not completely understood. Previous work has shown that a subset of kisspeptin neurons express MC4R and that KNDy neurones receive synaptic input from POMC neurons. In addition, inhibition of MC4R/MC3R results in delayed onset of puberty in rats. Our results support a role for MC4R in GnRH secretion and potentially olfactory /GnRH neuronal migration. Assessment of spontaneous gonadotrophin production and response of gonadotrophin production to kisspeptin stimulation is currently in progress. Presentation: 6/1/2024

6694 A Case of an Extraneous Organ: A Uterus Found in a Genotypic and Phenotypic Male

Abstract Disclosure: R.A. Zielinski: None. S. Shteyman: None. A.G. Samat: None. Background: Persistent Mullerian Duct Syndrome (PMDS) is a rare entity that occurs when a phenotypic male is found to have female reproductive organs. In our case we describe an XY male who was found to have an abdominal mass consistent with a uterus on pathology. Clinical Case: Our patient is a 58-year-old male with hypertension, obesity, and obstructive sleep apnea who presented to the ED with 3 weeks of left upper quadrant pain. Abdominal CT showed two homogeneous enhancing oval lesions in the pelvis superior to the urinary bladder; 8.3 x 4.6 cm on the right and 3.6 x 3 cm on the left. Exploratory laparotomy was performed, and intraoperatively the mass was firm with a left satellite lesion and two fibrous bands that appeared similar in structure to round ligaments. Microscopically the tissue contained mullerian-type epithelium and smooth muscle with cystic degeneration. It was CK7 positive, estrogen receptor variable positive, and CK20 negative. Genetic testing confirmed the patient’s karyotype to be XY without genetic mosaicism. Our patient had two children spontaneously, regular spontaneous erections, and occasional hot flashes. Physical exam revealed an obese male (BMI 51) with bilateral gynecomastia and normal testicular size. He had no history of intra-abdominal or undescended testes requiring surgery. Labs showed increased total estrogen (283.7 pg/mL, n 60-190 pg/mL) and estradiol (39 pg/mL, n <= 29 pg/mL), decreased total testosterone (86 ng/dL, n 250-1,100 ng/dL), and normal free testosterone when corrected for SHBG (140 ng/dL, n 110-574 ng/dL). Testicular ultrasound was unrevealing. 3 years postoperatively his estradiol level normalized (34 pg/mL) and his total testosterone improved from 86 ng/dL to 223 ng/dL after losing 40 pounds on semaglutide. Discussion and Conclusion: Although the exact incidence of PMDS is unknown, it is a rare phenomenon. Only 200 cases between 1964 and 2000 and 34 cases between 2013 and 2017 have been reported. Mullerian ducts are embryologic structures present in both XY and XX fetuses until week 8 of life, when XY fetuses produce anti-mullerian hormone (AMH). In PMDS, the XY fetus either fails to produce AMH, or there is a defect in the AMH receptors, leading to the persistence of mullerian ducts. These ducts can then differentiate into a uterus, fallopian tubes, and/or the upper vagina. Our patient is a genotypical and phenotypical male with a uterus-like organ. The mild elevation in his estrogen level is likely secondary to his obesity. The estrogen could have increased the size of the uterus, causing the patient’s initial symptoms of abdominal pain which prompted his presentation to the hospital.

8786 Novel TGFbeta Ligand Regulated Genes in WT and FSTL3 KO Testis Identified by ChIP-Seq Analysis: Implications for Signalling Cross-talk

Abstract Disclosure: R. Ballesteros: None. A. Mukherjee: None. Folistatin Like 3 (FSTL3) is a known antagonist of activin and myostatin action that when deleted from the mouse genome displayed a set of phenotypic changes that are directly linked to glucose metabolism and reproductive function. Although our previous work showed that β-cell hyperplasia, increased pancreatic islet number and size and reduced visceral fat seen in FSTL3 knockout mice (FSTL3 KO) could be explained by enhanced activin and myostatin action, the molecular bases of the changes in the reproductive function seen in this mouse model remain elusive. In contrast to testicular degeneration produced by INHBA (activin A) transgenic overexpression in mice, deletion of FSTL3 results in increased testicular size, with increased Sertoli cell numbers and early meiotic entry of the first spermatogenic wave. This suggests a possibility that the phenotype in FSTL3 deleted mice might involve alteration of additional transcripts other than those induced by activin alone in the testis. To identify transcripts induced in FSTL3 deleted testis as a consequence of induced TGFβ ligand action we performed sequencing of Smad4 Chromatin immunoprecipitation (ChIP-Seq) from postnatal (3d) and post-pubertal (56d) whole testis. Our results show high activity of Smad4 signalling during postnatal stages compared to post-pubertal stages in both genotypes (Fstl3-KO vs wild-type). Interestingly, postnatal testis from FSTL3 KO contains significantly less activity from Smad4 signalling (69 peaks) compared to wild type (775 peaks). By contrast, on the whole, Smad4 signalling in post-pubertal stages is less active with 34 peaks in wild-type and 29 peaks in Fstl3-KO of which 8 genes are SMAD activated in both genotypes. To identify whether SMAD-dependent signalling produces significant differences in RNA expression, we then used the list of genes that generated peaks in the Smad4-ChIP-Seq experiment to filter our RNA-Seq data generated in a parallel experiment followed by differential gene expression analysis of GO terms by GSEA in postnatal testis from both genotypes. We found that Smad4-interacting genes in posnatal FSTL3 KO testis generate enrichment of 13 pathways including the androgen receptor signalling pathway. Taken together so far, our findings reveal a novel catalogue of SMAD-regulated genes in the testis, identifies a set of genes specifically upregulated in the testis in a SMAD-dependent manner upon FSTL3 deletion and also identify possible cross-talk between TGFβ and 13 other signalling pathways. Currently we are further analysing our data to identify whether there are SMAD regulated genes and pathways common in postnatal and postpubertal testes. We are also undertaking experiments to investigate expression pattern in FSTL3 KO testis to identify which pathways might specifically affect Sertoli cell proliferation and activity. Presentation: 6/2/2024

8796 Increased Sertoli Cell Proliferation in FSTL3 Deleted Mouse Testis

Abstract Disclosure: R. Ballesteros: None. A. Mukherjee: None. Activin, a TGFβ ligand, plays key roles in Sertoli cell proliferation. Follistatin like 3 (FSTL3) is a glycoprotein inhibitor of activin expressed strongly in the testis. We have shown that FSTL3 deletion (FSTL3 KO) in mice increases testicular size, likely caused by increased spermatogenesis supported by the increased Sertoli cell numbers seen in FSTL3 KO mouse testes. It is not clear, however, whether there is increased proliferation of Sertoli cells postnatally. We, therefore, investigated Sertoli cell proliferation, and molecular pathways that might be activated upon FSTL3 deletion that lead to increased Sertoli cell proliferation in mice. Paired testes weight comparison indicates that there is significant increase in testicular mass in FSTL3 KO between 28d and 56d coinciding with the onset of continued spermatogenesis. Immunofluorescence studies using PCNA as marker for proliferation and SOX9 to identify Sertoli cells, however, found no significant differences between proliferation of Sertoli cells in WT and FSTL3 KO. BrdU injection of mice followed by immunofluorescence labelling of proliferating Sertoli cells also showed no difference between the two genotypes by immunofluorescence. We then proceeded to assess whether there is an alteration of differentiation in FSTL3 KO testes to test the possibility that instead of proliferation there is an increased differentiation of a pre-Sertoli like cell in FSTL3 KO mice using WT1 and SOX9 as markers for early or late Sertoli cells. This experiment revealed that there was no difference between the ratio of WT1 and SOX9 expression in the two genotypes. Flow cytometric analyses using testicular dispersates from mice using Sox9 and Ki67 antibodies to identify proliferating Sertoli cells, however, revealed an approximately two-fold increase in proliferating Sertoli cells in FSTL3 KO compared to WT at 7d. At 56d there were very few proliferating Sertoli cells in either genotype and there was no difference in this number in FSTL3 deleted mice compared to WT. mRNA sequence analysis showed that approximately 1000 genes are differentially regulated between WT and FSTL3 KO at 3d. Gene ontology and pathway analysis revealed that a constellation of meiosis related genes and activin signalling pathway, among others, are enriched in FSTL3 KO testis at this stage. Data presented here demonstrates that FSTL3 deletion induces activin receptor signalling pathway, genes crucial for meiosis and therefore the spermatogenic process, and Sertoli cell proliferation. This opens the possibility that the early arrest of Sertoli cell proliferation seen in mammals including mice and humans might be circumvented by inactivating FSTL3. This could then lead to a therapeutic angle in male infertility that are based on paucity of Sertoli cells. It will be important to address why FSTL3 deletion induces Sertoli cell proliferation only postnatally but not post pubertally. Presentation: 6/1/2024

8320 Integration of long-read sequencing, DNA methylation and gene expression reveals heterogeneity in Y-chromosome segment lengths in phenotypic males with 46,XX testicular disorder of sex development

Abstract Disclosure: E.B. Johannsen: None. A. Berglund: None. A. Skakkebæk: None. S. Chang: None. J. Rohayem: None. S. Laurentino: None. A. Hørlyck: None. S.O. Drue: None. E.N. Bak: None. J. Fedder: None. F. Tuttelmann: None. J. Gromoll: None. J. Just: None. C.H. Gravholt: None. Background: 46,XX testicular disorder of sex development is a rare congenital condition affecting approximately three per 100,000 newborn males. It is characterized by a combination of the typical female sex chromosome constitution, 46,XX, and a variable male phenotype. In the majority of XX males, a Y chromosome segment containing the sex-determining region gene (SRY) has been translocated to the paternal X chromosome. However, the precise genomic content of the translocated segment, breakpoints, genome-wide effects, and the effect on other biological pathways essential for the phenotype remain unknown. Methods: We performed long-read DNA sequencing (Oxford Nanopore), RNA sequencing (paired-end, 100 bp) and DNA methylation (Infinium EPIC) analysis on blood samples from males with 46,XX testicular disorder of sex development (XX males; n = 11), male controls (46,XY; n = 12 for long-read DNA sequencing, n = 26 for RNA sequencing and DNA methylation) and female controls (46,XX; n = 12 for long-read DNA sequencing, n = 32 for RNA sequencing and DNA methylation), in addition to RNA sequencing and DNA methylation analysis on blood samples from males with Klinefelter syndrome (47,XXY, n = 22). We also performed clinical measures on all XX males and a subset of male controls (n = 10). Results: We identified variation in the translocated Y-chromosome segment of our XX male cohort, allowing for subcategorization into 1) XX males without Y chromosome material (n = 1), 2) those with short Yp arms (breakpoint at 2.7-2.8 Mbp, n = 2), 3) those with medium Yp arms (breakpoint at 7.3 Mbp, n = 1) and 4) those with long Yp arms, including AMELY, TBLY1 deletions, and in some cases PRKY deletions (n = 7). We also identified variable expression of the X-Y homologues PRKY and PRKX, appeared to have balanced expression level resulting in overall equilibrium. The Y-chromosomal transcriptome and methylome were affected, reflecting the Y-chromosome segment length. Genomic changes induced by the translocated Yp segment were evident in the transcriptome and methylome of autosomal and X-chromosomal regions, indicating global effects. Furthermore, the transcriptional changes in XX males correlated with phenotypic traits of the XX males, including lower height, decreased lean mass and smaller testicular size. Conclusion: Integration of long-read DNA sequencing, DNA methylation, and RNA sequencing analysis allowed for the identification of a heterogenous translocation process in XX males that exerted widespread effects on methylation and transcription. Presentation: 6/2/2024