Klinefelter Syndrome Patients Research Articles

Gender identity in Klinefelter Syndrome: a patient-centered approach to treatment

Introduction There is increasing evidence that gender dysphoria (GD) is more prevalent in the Klinefelter Syndrome (KS) population than in males in the general population; however, the exact incidence is uncertain. The aim of this study was to further explore the prevalence of gender-related issues, the role that physical characteristics play in gender identity, and the issues surrounding Hormone Replacement Therapy (HRT) in KS. Methods As part of a registered Quality Improvement Project (QIP), one online 23-point questionnaire on KS patient attitudes toward gender identity was shared with members of the Klinefelter Syndrome Association (KSA). In total, 139 anonymous responses were collected between December 2021 and January 2023. The questionnaire was developed with the guidance of multiple clinicians (including gender psychiatrists, urologists, psychosexual medicine specialists, and endocrinologists) and patient Delphi rounds. Data was reviewed and analyzed by 4 independent researchers within the QIP team. Results Only 53% of KS patients responding to this survey fully identified as male and 19% stated that they did not enjoy living as the sex on their birth certificate, with 43% considering changing aspects of their physical appearance to better match their gender. Regarding HRT, 67% of respondents were receiving Testosterone Replacement Therapy (TRT). 63% wanted TRT and 17% wanted estrogen, including 6% of TRT users who would prefer estrogen instead. 36% that were currently receiving TRT did not identify as male, and 3 participants stated that they have GD. Conclusion These results indicate that a significant proportion of KS patients do not fully identify with the male gender and are unhappy living as the sex on their birth certificate. Although TRT worked for most, its use should be discussed carefully with those with gender identity concerns.

7341 Establishing a Multidisciplinary Care Program for patients with Klinefelter Syndrome and other Aneuploidy X and Y Variations

Abstract Disclosure: C.J. Romero: None. N.R. Malhotra: None. A. Norris-Brilliant: None. C. Mintz: None. Objectives: Klinefelter syndrome (KS) is one of the most common genetic aneuploidies denoted by a XXY karyotype and affects up to 1 in 600 patients. Most patients are not diagnosed until adolescent years or adulthood. KS is one of the leading causes of male infertility and testosterone insufficiency. KS is also associated with developmental delays, particularly speech and language delay. Advances in genetics screenings has led to early identification of KS and other aneuploidy X and Y variations, access to neurodevelopmental screening and fertility preservation resources during adolescence and young adulthood. We believe a multidisciplinary program for KS can provide the necessary resources for patients and families at any age from infancy to young adulthood. Methods: A group of faculty from the divisions of pediatric endocrinology, genetics, urology and psychiatry assembled in 2023 to establish a multidisciplinary clinic (MDC) to provide comprehensive care to patients/parents; this ranged from prenatal diagnosis to young adulthood. Two major challenges of care are access to neuro-psychological evaluations and fertility preservation options. Testosterone supplementation requires appropriate referral to endocrinology and timing of treatment remains controversial. Our goals include: expertise guidance and planning after prenatal diagnosis or later in age; access to cost effective neuro-psychological testing during early childhood years; urological consultation beginning in early puberty to educate and prepare patients for choices of surgical and non-surgical options of fertility preservation. Results: Our current center cares for 12 patents ranging from ages 3 months to 21 years. Of this cohort, 3 (25%) of the patients had initial consultations with the parents during pregnancy. 7 (58%) of the patients were of pubertal age that one would consider testosterone replacement, but only 3 (43%) were taking testosterone. Only 1 patient of school age reported neuro-psychological evaluation that was completed at a different institution. No patients received testosterone supplementation during year one of life. Conclusions: A multidisciplinary team can offer simultaneous assessment, anticipatory guidance and management for KS patients and their families. It is a comprehensive resource to our community. Prenatal genetic screenings bring awareness to this diagnosis and therefore timely education toward understanding the lifelong management of KS. Identifying developmental delays early in childhood will help with scholastic success. Monitoring of pubertal development will ensure appropriate pubertal progression in addition to education and management of fertility preservation. Undoubtedly future urological advances will assist patients more effectively. We believe our model provides the access to resources that allow our patients and family to successfully thrive. Presentation: 6/2/2024

Impact of sex chromosome abnormalities in male chronic myeloid leukemia patients

IntroductionChronic Myeloid Leukemia (CML) is a myeloproliferative disorder characterized by the Philadelphia chromosome (Ph) resulting from the reciprocal translocation t(9;22)(q34;q11). Sex chromosomal abnormalities are found rarely in CML patients. Materials and methodsConventional and molecular cytogenetic analysis (Fluorescent In Situ Hybridization (FISH)) were performed to reveal the presence of Ph and additional chromosomal abnormalities (ACA). ResultsThree patients with abnormalities within sex chromosomes, one Klinefelter syndrome (KS) patient and two cases with LOY (Loss of Y) diagnosed with CML CP were included in the study. All three patients initially responded to the targeted therapy, became resistant to their treatment course and switched to 2nd line therapies. In the 15th month, the KS patient abruptly turned into BC and showed clonal evolution with four karyotypic patterns. According to Mitelman databases, this is the first KS-CML patient who showed three major route abnormalities, +8, i(17) and + der(22) in a single clone. Survival analysis showed one patient with LOY chromosome expired on the 48th month of diagnosis. ConclusionThe application of cytogenetic techniques in identifying the constitutional abnormality of KS, ACA at initial CML diagnosis, revealing of extra Ph during the treatment course and clonal evolution in BC (Blast Crisis) assist the proper monitoring of disease transformation and serves as a major diagnostic tool for CML patients thereby switching the treatment protocols. A systematic stratification of patients according to the chromosomal abnormalities is needed in CML patients.

Klinefelter syndrome in a patient with double Y-autosomal translocation

Klinefelter syndrome is one of the most common chromosomal abnormalities and the most common genetic cause of male infertility. About 85 % of patients have 47,XXY karyotype, other patients have other non-mosaic and mosaic Klinefelter syndrome variants. We report a unique clinical case — Klinefelter syndrome patient with double Y autosomal translocation. The proband is a 15-year-old male patient (height 180 cm, weight 50 kg, normal IQ) who was admitted for cytogenetic examination and genetic counseling due to delayed puberty. He was diagnosed with testicular hypoplasia, hypergonadotropic hypogonadism, pituitary microadenoma and left-sided varicocele. The proband was born in a nonconsanguineous marriage after in vitro fertilization due to paternal male factor infertility. Cytogenetic analysis was performed on cultured peripheral blood lymphocytes using standard chromosome analysis with GTG staining and FISH analysis. Molecular analysis of the Y chromosome was performed by multiplex PCR. Complex cytogenetic examination revealed a 46,XX,der(Y) t(Y;15)(q12;q11.1),der(13)t(Y;13)(q12;p11.2),-15 karyotype in the proband. Molecular analysis showed that the proband is SRY positive; no microdeletion of the Y chromosome was found. The detected double Y autosomal translocation is a chromosomal abnormality independent of KS. The father of the proband is an oligozoospermic man with robertsonian translocation 13;15 — 45,XY,der(13;15)(q10;q10), the mother has a normal karyotype 46,XX. Apparently, the der(13) and der(Y) chromosomes result from abnormal meiotic recombination in paternal meiosis between the heterochromatic region Yq12 and the centromeric/pericentromeric heterochromatin of chromosomes 13 and 15 involved in the paternal robertsonian translocation, and Klinefelter syndrome is due to the presence of two X chromosomes in the karyotype in the presence of a derivative Y chromosome. The detected double Y-autosomal translocation is a chromosomal abnormality unrelated to Klinefelter syndrome, arising on the background of the paternal robertsonian translocation.

Testicular mosaicism in non-mosaic postpubertal Klinefelter patients with focal spermatogenesis and in non-mosaic prepubertal Klinefelter boys.

Do testis-specific cells have a normal karyotype in non-mosaic postpubertal Klinefelter syndrome (KS) patients with focal spermatogenesis and in non-mosaic prepubertal KS boys? Spermatogonia have a 46, XY karyotype, and Sertoli cells surrounding these spermatogonia in postpubertal patients also have a 46, XY karyotype, whereas, in prepubertal KS boys, Sertoli cells surrounding the spermatogonia still have a 47, XXY karyotype. A significant proportion of patients with non-mosaic KS can have children by using assisted reproductive techniques thanks to focal spermatogenesis. However, the karyotype of the cells that are able to support focal spermatogenesis has not been revealed. Testicular biopsy samples from non-mosaic KS patients were included in the study. Karyotyping for sex chromosomes in testis-specific cells was performed by immunohistochemical analysis of inactive X (Xi) chromosome and/or fluorescent in situ hybridization (FISH) analysis of chromosomes 18, X, and Y. A total of 22 KS patients (17 postpubertal and 5 prepubertal) who were non-mosaic according to lymphocyte karyotype analysis, were included in the study. After tissue processing, paraffin embedding, and sectioning, the following primary antibodies were used for cell-specific analysis and Xi detection; one section was stained with MAGE A4 for spermatogonia, SOX9 for Sertoli cells, and H3K27me3 for Xi; the other one was stained with CYP17A1 for Leydig cells, ACTA2 for peritubular myoid cells, and H3K27me3 for Xi. Xi negative (Xi-) somatic cells (i.e. Sertoli cells, Leydig cells, and peritubular myoid cells) were evaluated as having the 46, XY karyotype; Xi positive (Xi+) somatic cells were evaluated as having the 47, XXY. FISH stain for chromosomes 18, X, and Y was performed on the same sections to investigate the karyotype of spermatogonia and to validate the immunohistochemistry results for somatic cells. According to our data, all spermatogonia in both postpubertal and prepubertal non-mosaic KS patients seem to have 46, XY karyotype. However, while the Sertoli cells surrounding spermatogonia in postpubertal samples also had a 46, XY karyotype, the Sertoli cells surrounding spermatogonia in prepubertal samples had a 47, XXY karyotype. In addition, while the Sertoli cells in some of the Sertoli cell-only tubules had 46, XY karyotype, the Sertoli cells in some of the other Sertoli cell-only tubules had 47, XXY karyotype in postpubertal samples. In contrast to the postpubertal samples, Sertoli cells in all tubules in the prepubertal samples had the 47, XXY karyotype. Our data also suggest that germ cells lose the extra X chromosome during embryonic, fetal, or neonatal life, while Sertoli cells lose it around puberty. Peritubular myoid cells and Leydig cells may also be mosaic in both postpubertal patients and prepubertal boys, but it requires further investigation. The number of prepubertal testicle samples containing spermatogonia is limited, so more samples are needed for a definitive conclusion. The fact that not all the cell nuclei coincide with the section plane limits the accurate detection of X chromosomes by immunohistochemistry and FISH in some cells. To overcome this limitation, X chromosome analysis could be performed by different techniques on intact cells isolated from fresh tissue. Additionally, there is no evidence that X chromosome inactivation reoccurs after activation of the Xi during germ cell migration during embryogenesis, limiting the prediction of X chromosome content in germ cells by H3K27me3. Our findings will lay the groundwork for new clinically important studies on exactly when and by which mechanism an extra X chromosome is lost in spermatogonia and Sertoli cells. This study was funded by The Scientific and Technological Research Council of Türkiye (TUBITAK) (2219 - International Postdoctoral Research Fellowship Program for Turkish Citizens) and the Strategic Research Program (SRP89) from the Vrije Universiteit Brussel. The authors declare no competing interests. N/A.

P-075 A new clinically relevant classification of seminiferous tubules in Klinefelter Syndrome patients based on the karyotype of the Sertoli cells

Abstract Study question Can a new seminiferous tubule classification be made that indicates the chance of sperm retrieval by TESE in Klinefelter Syndrome (KS) patients? Summary answer Spermatogonia are only present in tubules with euploid Sertoli cells. Classification of tubules based on Sertoli cell ploidy may predict sperm retrieval success with TESE. What is known already KS patients can have children thanks to focal spermatogenesis. However, this can only happen if sperm is produced. Histological classification of seminiferous tubules in these patients has been made based on the maturity of the Sertoli cells (type A and type B seminiferous tubules). However, so far there is no classification predicting the presence of germ cells. In case two X chromosomes are present, one is inactivated. The histone-3-lysine-27-tri-methylation (H3K27me3) protein is involved in X chromosome inactivation and its immunohistochemical staining may be used for a new seminiferous tubule classification based on the karyotype of Sertoli cells. Study design, size, duration Inactive X (Xi) chromosomes were immunohistochemically stained with H3K27me3 and seminiferous tubules were classified as Xi+ tubules (presence of inactive X in Sertoli cells) or Xi- tubules (absence of inactive X in Sertoli cells). Testicular biopsies were used from 17 non-mosaic KS adult patients. Participants/materials, setting, methods After tissue fixation, processing, and paraffin embedding, sections were cut at 5 µm. Immunohistochemical staining was applied with antibodies H3K27me3 (for inactive X chromosome), MAGE-A4 (for spermatogonia), and SOX9 (for Sertoli cells). Seminiferous tubules in testicular sections were classified as Xi+ or Xi- tubules and analyzed for the presence of spermatogonia or focal spermatogenesis. X chromosome number in spermatogonia and Sertoli cells was confirmed by the FISH method using chromosome 18, Y, and X probes. Main results and the role of chance A total of 311 (18±16) seminiferous tubule sections were analyzed in testicular tissue of 17 KS men. Of the 311 tubules analyzed, 253 (14±16) of the tubules were Xi- tubules and 58 (3±4) were Xi+ tubules. Spermatogonia were present in 41 of the Xi- tubules. As an exception, only one patient had spermatogonia in two Xi+ tubules, but these tubules were immature and spermatogenesis was not observed. Ongoing spermatogenesis was observed in two of Xi- tubules in one of the patients. Sixteen of the patients had both Xi- tubules and Xi+ tubules in their testicular sections. In 13 of these 16 patients with Xi- tubules, sperm was either obtained as a result of TESE or spermatogonia were observed in the Xi- tubules. Limitations, reasons for caution In Klinefelter patients, there are few seminiferous tubules due to widespread testicular fibrosis and degeneration of seminiferous tubules. Studies including more patient samples are needed for more definitive and comprehensive conclusions. Wider implications of the findings Spermatogonia are only present in tubules with euploid Sertoli cells. Therefore, we hypothesize that focal spermatogenesis only can occur in tubules with euploid Sertoli cells. Xi+ and Xi- seminiferous tubule classification based on H3K27me3 staining may thus be used to predict sperm production in KS patients. Trial registration number Not applicable

P-007 Surgical sperm retrieval rate in men with Klinefelter syndrome is associated with testicular volume - but not with body constitution or Sertoli cell markers

Abstract Study question Do sperm retrieval and pregnancy rates in men with Klinefelter syndrome (KS) depend upon sperm retrieval method, age, testicular volume, body weight, and hormone concentrations? Summary answer Successful sperm retrieval was significantly associated with total testicular volume but not with sperm retrieval method, age, body weight, BMI, FSH, inhibin-B or AMH. What is known already Testicular sperm can be retrieved from approximately 40% of men with KS, resulting in an average pregnancy rate of 16%. The sperm retrieval rate seems to be unrelated to the age of the man and to preoperative FSH-levels. A decrease in testosterone concentration is usually observed 3 months after surgery. Possible negative effects of testosterone substitution therapy prior to surgery and the predictive value of body constitution, testicular volume and the Sertoli cell markers, AMH and inhibin-B, for sperm retrieval success in KS men are only partly known. Study design, size, duration A total of 14 KS patients underwent subcapsular orchiectomy and 34 KS patients underwent m-TESE at Centre of Andrology & Fertility Clinic, Odense University Hospital. Pre-surgery examination included ultrasonography of the testicles, genetic analyses (standard chromosome analysis and screening for Y microdeletions and CFTR variants) and hormone analyses (including FSH, LH, testosterone, estradiol, prolactin, TSH, AMH, inhibin-B). Testosterone, LH, and FSH were also measured 3 months after surgery. Participants/materials, setting, methods Forty-three men had a 47,XXY karyotype, while 5 (2 from the subcapsular group and 3 from the m-TESE group) had a mosaic karyotype including 47,XXY. Subcapsular orchiectomy was performed unilaterally, while m-TESE was usually performed bilaterally. To avoid freezing the sperm retrieved, oocytes from the female partner was aspirated and vitrified after FSH-stimulation according to our usual IVF protocols. Oocytes were thawed on the day of sperm retrieval and embryos transferred in estrogen-progesterone stimulated cycles. Main results and the role of chance Sperm were obtained in 19 (40%) of the 48 men with KS. In 10 couples (21%) pregnancy was obtained and 7 women (15%) gave birth to at least one child resulting in ten children (6 girls and 4 boys), while one pregnancy is still ongoing. In the group who had a subcapsular orchiectomy, 5 (36%) had sperm retrieved, and pregnancy was obtained in 3 (20%) cases. Two gave birth to a child, while the third woman have had several abortions, most likely due to large fibromyomas of the uterus. In the m-TESE group 14 (41%) had sperm retrieved, and pregnancy was obtained in 7 (21%) cases. Five women (15%) gave birth to 8 children, while 1 pregnancy is still ongoing and the seventh experienced an early pregnancy loss. Furthermore, 3 couples have still not embryos transferred. Sperm were found in 3 men (60%) with mosaic karyotype. Sperm retrieval success was significantly associated with testicular volume (mean total testicular volumes of 3.4 mL vs. 2.3 mL; p < 0.01) but not with age, height, body weight, BMI or preoperative FSH, inhibin-B or AMH levels. Testosterone levels measured 3 months after the surgery, did not differ between men irrespective of surgical method. Limitations, reasons for caution The numbers of patients are limited and randomized studies with sufficient power are difficult to perform. AMH and inhibin-B, were only measured in the m-TESE group. Wider implications of the findings In KS patients, sperm retrieval and pregnancy rates were similar irrespective of the sperm retrieval method. Also serum testosterone 3 months postoperatively and cosmetic results experienced by patients were similar. Multicenter studies and larger studies are needed to provide more detailed information of the potential predictors for successful sperm retrieval. Trial registration number Not applicable

P-022 Klinefelter syndrome couples are expected higher live birth rates than unexplained NOA couples after ICSI with testicular spermatozoa

Abstract Study question What are sperm retrieval rates (SRR) by microdissection TESE (micro TESE), fertilization rates, and pre- and post- implantation development in Klinefelter syndrome (KS) couples? Summary answer In KS couples, SRR, clinical pregnancy rates (CPR) per embryo transfer (ET), and live birth rates (LBR) per patient were higher than unexplained NOA couples. What is known already Micro TESE, in combination with ICSI, is presently used to treat infertility in cases of NOA including KS, which is the most common sex-chromosome disorder among infertile males, with a prevalence of 1 in 660 men and is a frequent cause of hypogonadism and infertility. There were few reports regarding ICSI outcomes in the couples of KS. The aim of this study is to assess the prevalence and the significance including SRR by micro TESE of ICSI outcomes with embryonic development in KS couples. Study design, size, duration A retrospective study was conducted in 212 patients with non-mosaic KS and 963 unexplained NOA patients with 46, XY without past history (unexplained NOA; not including after orchidopexy, KS, cryptozoospermia, post chemotherapy, mumps orchitis, etc) who underwent micro TESE and ICSI with fresh or cryopreserved testicular spermatozoa for their wives between September 2013 to November 2023. Participants/materials, setting, methods A total of 2204 azoospermic patients were examined for chromosomal analysis. We evaluated SRR of microdissection TESE, two pronuclei (2PN) rates, blastocyst development rates, good-quality blastocyst (Grade 3BB and above on day 5 by the Gardner scoring) rates, CPR per ET, and LBR per couple patient who underwent micro TESE. We did not undergo preoperative hormonal therapy for KS patients. Statistical analysis was performed using unpaired t-tests and chi-squared tests. Main results and the role of chance We identified 212 KS out of 2204 azoospermic patients (9.6%). SRR of the first attempt micro TESE in KS (91/175=52.0%) was significantly higher than unexplained NOA (166/768=21.6%) (p < 0.001). 2PN, blastocysts development, and good-quality blastocysts rates were 49.2%, 42.9%, 16.9% in KS and 50.8, 45.8%, 21.0% in unexplained NOA respectively. Only good-quality blastocysts rate of KS was significantly lower than that of unexplained NOA (p < 0.01). Taking into account deflection of wives age, each result was calculated at their age 38 or under, but they were the same. CPR and LBR per ET in KS (42.6%, 30.4%) were significantly higher than unexplained NOA (33.1%, 23.7%) (p < 0.01, p < 0.05). The couples were 26.4% (56/212) in KS and 12.0% (116/963) in unexplained NOA (p < 0.001) who have given birth or ongoing pregnancy up to now after underwent micro TESE. That is, KS couples were significantly easier to give birth than unexplained NOA. Rates of congenital disorders had no significant difference between KS and unexplained NOA. Limitations, reasons for caution We did not show the data using ejaculated sperm with KS. The natal outcomes and development of these children has not been fully investigated, long-term follow-up of babies of the KS couples is needed. Wider implications of the findings Micro TESE is particularly helpful for KS cases, but embryonic development depend on the quality of spermatozoa. However, if a blastocyst can be obtained, the expectation of having a child is considered to be high. These results were shown to be useful in informing azoospermic couples undergoing micro TESE. Trial registration number not applicable

Sperm extraction in nonmosaic Klinefelter syndrome patients: A case series and literature review of sperm extraction in Klinefelter syndrome patients.

Klinefelter syndrome (KF) is a group of chromosomal disorders with at least one extra X chromosome in male individuals that leads to infertility and diminished hair growth in affected males. In this study, we present a case series of 16 nonmosaic KF and an extensive literature review. This is a retrospective study including 16 nonmosaic Klinefelter Syndrome patients that underwent micro-testicular sperm extraction (m-TESE) at our center between January 2016 and December 2022. Frequencies and percentages were used to present categorical variables, whereas continuous variables were presented as the median and interquartile range (IQR). The sperm retrieval rate (SRR) was assessed using a one-sample proportions test with continuity correction. Fisher's exact test was to assess the differences between patients with negative and positive retrieval in terms of the categorical variables. A Wilcoxon rank-sum test was applied to explore the between-group differences in the numerical variables. A literature search was performed for additional publications of discussing m-TESE among KF patients. The median (IQR) age of patients was 40.0 years (34.5-47.0). All of the patients had nonobstructive azoospermia, and the majority of them (93.8%) had primary infertility. The most common histopathological findings were atrophic tubules (57.1%), followed by Sertoli cell-only (28.6%). Sperm retrieval was positive for two patients with a rate of 12.5% (95% confidence interval 2.2 to 39.6). Patients with positive sperm retrieval were significantly younger than their peers with negative retrieval (median = 28.0, IQR = 27.5 to 28.5 vs. median = 41.5, IQR = 35.8 to 47.0, P = 0.031). The successful conception rate was 100% (n = 2) using intracytoplasmic sperm injection with a birth rate of 100% (n = 2). Our observed SRR among nonmosaic KF patients was marginally lower than the reported literature. Younger-age patients were significantly more likely to benefit from the procedure.

Klinefelter syndrome: etiology and clinical considerations in male infertility†.

Klinefelter syndrome (KS) is the most prevalent chromosomal disorder occurring in males. It is defined by an additional X chromosome, 47,XXY, resulting from errors in chromosomal segregation during parental gametogenesis. A major phenotype is impaired reproductive function, in the form of low testosterone and infertility. This review comprehensively examines the genetic and physiological factors contributing to infertility in KS, in addition to emergent assisted reproductive technologies, and the unique ethical challenges KS patients face when seeking infertility treatment. The pathology underlying KS is increased susceptibility for meiotic errors during spermatogenesis, resulting in aneuploid or even polyploid gametes. Specific genetic elements potentiating this susceptibility include polymorphisms in checkpoint genes regulating chromosomal synapsis and segregation. Physiologically, the additional sex chromosome also alters testicular endocrinology and metabolism by dysregulating interstitial and Sertoli cell function, collectively impairing normal sperm development. Additionally, epigenetic modifications like aberrant DNA methylation are being increasingly implicated in these disruptions. We also discuss assisted reproductive approaches leveraged in infertility management for KS patients. Application of assisted reproductive approaches, along with deep comprehension of the meiotic and endocrine disturbances precipitated by supernumerary X chromosomes, shows promise in enabling biological parenthood for KS individuals. This will require continued multidisciplinary collaboration between experts with background of genetics, physiology, ethics, and clinical reproductive medicine.

Genes and Pathways Underpinning Klinefelter Syndrome at Bulk and Single-Cell Levels.

Klinefelter syndrome (KS) is the most frequent genetic anomaly in infertile men. Given its unclear mechanism, we aim to investigate critical genes and pathways in the pathogenesis of KS based on three bulk and one single-cell transcriptome data sets from Gene Expression Omnibus. We merged two data sets (GSE42331 and GSE47584) with human KS whole blood samples. When comparing the control and KS samples, five hub genes, including defensin alpha 4 (DEFA4), bactericidal permeability increasing protein (BPI), myeloperoxidase (MPO), intelectin 1 (ITLN1), and Xg Glycoprotein (XG), were identified. Besides, infiltrated degree of certain immune cells such as CD56bright NK cell were positively associated with the expression of ITLN1 and XG. Kyoto Encyclopedia of Genes and Genomes analysis identified upregulated phosphatidylinositol 3-kinase (PI3K)/AKT pathway in KS. Gene set enrichment analysis followed by gene set variation analysis confirmed the upregulation of G2M checkpoint and heme metabolism in KS. Thereafter, the GSE200680 data set was used for external validation of the expression variation of hub genes from healthy to KS testicular samples, and each hub gene yielded excellent discriminatory capability for KS without exception. At the single-cell level, the GSE136353 data set was utilized to evaluate intercellular communication between different cell types in KS patient, and strong correlations were detected between macrophages/ dendritic cells/ NK cells and the other cell types. Collectively, we provided hub genes, pathways, immune cell infiltration degree, and cell-cell communication in KS, warranting novel insights into the pathogenesis of this disease.

Serum Lipocalin-2 Levels as a Biomarker in Pre- and Post-Pubertal Klinefelter Syndrome Patients: A Pilot Study.

Klinefelter syndrome (KS) is a male genetic disease caused by the presence of an extra X chromosome, causing endocrine disorders mainly responsible for a high rate of infertility and metabolic disorders in adulthood. Scientific research is interested in identifying new biomarkers that can be predictive or prognostic of alterations strictly connected to KS. Lipocalin-2 (LCN-2, also known as NGAL) is a small protein initially identified within neutrophils as a protein related to innate immunity. Serum LCN-2 estimation seems to be a useful tool in predicting the metabolic complications caused by several pathological conditions. However, little is known about its potential role in infertility conditions. The present pilot study aims to investigate the presence of LCN-2 in the serum of a group of pre-pubertal and post-pubertal children affected by KS, compared to healthy controls. We demonstrated for the first time the presence of elevated levels of LCN-2 in the serum of KS patients, compared to controls. This increase was accompanied, in pre-pubertal KS patients, by the loss of correlation with LH and HDL, which instead was present in the healthy individuals. Moreover, in all KS individuals, a positive correlation between LCN-2 and inhibin B serum concentration was found. Despite the limited size of the sample analyzed, our preliminary data encourage further studies to confirm the findings and to extend the study to KS adult patients, to verify the predictive/prognostic value of LCN-2 as new biomarker for metabolic diseases and infertility associated with the pathology.

Generation of iPSC Cell Lines from Patients with Sex Chromosome Aneuploidies.

Somatic cell reprogramming allows the generation of human induced pluripotent stem cells (iPSCs) from patient's cells. The derived iPSCs provide an unlimited source of patient-specific cells that can be virtually differentiated in any cell of the human body. The generation of iPSCs has important implications for all human medicine fields, as they can be used for drug discovery, regenerative medicine, and developmental studies. Klinefelter Syndrome (KS) is the most common chromosome aneuploidy in males. KS is typically characterized by a 47,XXY karyotype, representing 80-90% of KS patients. In rare cases, high-grade sex chromosome aneuploidies (SCAs), 48,XXXY; 48,XXYY; 49,XXXXY, are also observed in males. Since the advent of the reprogramming technique, a few KS-iPSCs have been described. Here, we detail the methodology for generating primary fibroblasts from patients' skin biopsies and the subsequent derivation of iPSCs using an efficient integrative-free mRNA-based somatic reprogramming approach.

Leydig cell metabolic disorder act as a new mechanism affecting for focal spermatogenesis in Klinefelter syndrome patients: a real world cross-sectional study base on the age.

Klinefelter's syndrome (KS) was once considered infertile due to congenital chromosomal abnormalities, but the presence of focal spermatozoa changed this. The key to predict and promote spermatogenesis is to find targets that regulate focal spermatogenesis. To explore the trend of fertility changes in KS patients at different ages and identify potential therapeutic targets. Bibliometric analysis was used to collect clinical research data on KS from the Web of Science Core Collection (WoSCC) from 1992 to 2022.A cross-sectional study was conducted on 75 KS patients who underwent microscopic testicular sperm extraction (mTESE) from 2017 to 2022 in the real world. The reproductive hormones, testicular histopathology, androgen receptors, insulin-like factor 3 (INSL3) receptors and sperm recovery rate (SRR) were analyzed. Male infertility, dysplasia, Sertoli cells, Leydig cells, testosterone and spermatogenesis were the research focuses related to KS. Luteinizing hormone (LH), testosterone, and INSL3 were evaluation indicators of Leydig cell function that fluctuate with age. Testosterone and LH peaked at ages 13-19 and 30-45, while INSL3 only peaked at ages 13-19. 27 patients (27/75) recovered sperm through mTESE and experienced SRR peaks at the ages of 20, 28, 34, and 37. The SRR of fibrosis patients was 46.15%, fatty degeneration was 7.14%, and melanosis was 40.00%. The INSL3 and androgen receptors were highly expressed and roughly balanced in focal spermatogenesis. Abnormal metabolism of Leydig cells led to imbalanced expression of INSL3 and androgen receptors, which might be a potential target for spermatogenesis in KS.

OR04-03 Eunuchoid Skeletal Proportions In Male Hypogonadism: A Comparative Analysis Of Anthropometric Measures Between Men With Congenital Hypogonadotropic Hypogonadism (CHH) And Klinefelter Syndrome (KS)

Abstract Disclosure: S. De Vincentis: None. A. Bellelli: None. R. Corleto: None. L. Zirilli: None. A. Granata: None. V. Rochira: None. BACKGROUND: Patients with congenital hypogonatropic hypogonadism (CHH) and Klinefelter syndrome (KS) have eunuchoid body proportions of the skeleton compared to normal male subjects, characterized by tall stature, and reduced upper-to-lower segment ratio (U/L). Vice versa, steroids exposure deeply differs between CHH and KS at puberty, with both testosterone and estradiol being very low only in CHH compared to KS. At present, a comparison of body skeletal proportions between CHH and KS is not available.AIM: To compare anthropometric measurements of adult male CHH patients to adult KS patients.METHODS: A prospective, cross-sectional, observational study was carried out. CHH patients were subdivided into 2 subgroups according to the timing of treatment start (testosterone replacement therapy [TRT] or gonadotropins): CHH1) CHH patients who started treatment late after 18 years; CHH2) CHH patients who started treatment on time before 18. All KS patients did not start TRT before 18 since KS do not usually delay puberty. The following anthropometric measurements were collected by using a digital scale and stadiometer (Seca gmbh&co®): height, weight, sitting height,total arm span. Legs length was obtained by subtracting sitting height from stature; U/L was calculated dividing sitting height for legs length.RESULTS: A total of 70 CHH and 45 KS age-matched patients were enrolled (mean age 33.7±13.7 and 35.3±13.7 years, respectively). CHH1 showed a longer arm span compared to CHH2 (p=0.001) and KS (p=0.003), and a shorter sitting height compared to KS (p=0.008). On the contrary, legslength was shorter in CHH2 compared to CHH1 (p<0.001) and KS (p=0.011). Accordingly, U/L and upper-to-height ratios were lower in CHH1 compared to CHH2 (p<0.001) and KS (p=0.001). Furthermore, the arm span-to-height ratio was higher in CHH1 compared to CHH2 (p<0.001) and KS (p=0.008).CONCLUSIONS: Under the same definition of eunuchoid body proportions, the traditional hallmark of male hypogonadism, more fine differences are observed comparing adult CHH to KS patients. CHH patients who delayed treatment showed longer arms length and lower U/L in comparison to CHH patients receiving treatment on time at pubertal age and KS. This suggests adifferent mechanism involved in the eunuchoid skeleton development between CHH and KS confirming a major role for estrogen/androgen deficiency in the former (leading to disproportional growth of both legs and arms due to delay in epiphyseal closure that could benefit from on timereplacement treatment) and a possible role of genetic supernumerary X in the latter, displaying a disproportional growth only at the legs site since infancy. Presentation: Thursday, June 15, 2023

Evaluation of Predicting Factors Affecting Sperm Retrieval in Patients with Klinefelter Syndrome: A Prospective Study.

This study aimed to evaluate the predicting factors affecting sperm retrieval. We prospectively assessed the relationship between sonographic and microdissection testicular sperm extraction (mTESE) findings in Klinefelter syndrome (KS). In this prospective study, 44 azoospermic men with 47, XXY karyotypes participated in this study. In order to evaluate the amount of blood supply in different parts of testicular tissue, a doppler ultra-sonographic was performed. Also, for the detection of sperm in this group mTESE technique was performed. The age average of positive mTESE and negative mTESE groups was 29.4 and 33.6 years, respectively. By comparing the testicle volume (based on the data obtained from the clinical examinations conducted by the urologist) it was determined that there is no significant difference between mTESE positive and negative groups. Folliclestimulating hormone (FSH) levels in men with negative mTESE (P=0.03) and testosterone levels in men with positive mTESE significantly increased (P=0.017). The overall rate of testis vascularity was significantly higher in the positive mTESE group than in the negative mTESE group. The clinical pregnancy rate in positive mTESE men was 9% per cycle, 16.6% per embryos were transferred (ET), and 12.5% per cycle. Totally, our observation indicated that there is not a significant relationship between sonographic and mTESE results in KS patients. However, more investigations with bigger sample Size can be useful to validate our results.

O-139 Testicular mosaicism in non-mosaic Klinefelter Syndrome patients with focal spermatogenesis

Abstract Study question Do testis-specific cells have a normal karyotype in regions with spermatogonia (SPG) and ongoing spermatogenesis in non-mosaic Klinefelter Syndrome (KS) patients? Summary answer While SPG and Sertoli cells (SC) have 46,XY karyotype in regions with spermatogenesis, most Leydig cells (LC) and peritubular myoid cells (PTMC) have 47,XXY karyotype. What is known already Although the majority of non-mosaic KS patients is azoospermic, some patients with focal spermatogenesis (FS) can have children with artificial reproductive technologies. However, the genetic origin of FS is unknown. Independent of FS, dimorphism in sex chromatin has been reported amongst the seminiferous tubules of KS men. However, the karyotype of testis-specific cells in FS regions and other regions has not been clearly revealed so far. The histone-3-lysine-27-tri-methylation (H3K27me3) modification is involved in suppression of the extra X chromosome, and its immunohistochemical staining allows the analysis of the X chromosome aneuploidy. Study design, size, duration Testicular biopsies were taken from 7 patients in whom spermatozoa were found by testicular sperm extraction (TESE). Muscle cells of female colon samples were used as positive control. Participants/materials, setting, methods After tissue processing and paraffine embedding, sections were cut at 5 µm. Immunohistochemical staining was performed with antibodies for H3K27me3 (inactive X chromosome), and MAGE-A4 (SPG), SOX9 (SC) and CYP17A1 (LC). PMTC was evaluated according to their position around the tubules and elongated shape. Inactive X (Xi) positive (Xi+) and Xi negative (Xi-) cells were counted and, Xi+ cells were evaluated as having the 47,XXY karyotype; Xi- cells were evaluated as having the 46,XY karyotype. Main results and the role of chance A total of 25 SPG+ tubule sections were detected in 4 of 7 samples. Both SPG and SC were Xi-, i.e. 46,XY karyotype, in all of these tubules. In some of the Sertoli cell only (SCO) tubules, SCs were Xi+ (i.e. 47,XXY), while in others they were Xi- (i.e. 46,XY). The rate of Xi+ SC in SCO tubules containing Xi+ SCs was 31.6%. Similarly, the Xi+ ratio in muscle cells in the positive control was 31.0%. PTMCs surrounding tubules with SPG present, or ongoing FS were Xi+ (i.e. 47,XXY). LCs were Xi+ (i.e. 47,XXY) around all tubules. However, the percentage of Xi+ PTMCs (21.4%) and LC (24.6%) was low compared to the female control (31.0%), suggesting mosaicism for these cells as well. Limitations, reasons for caution The fact that not all of the cell nuclei coincide with the section plane and the cells are in the mitotic stage limit the detection of Xi with H3K27me3. To overcome this limitation, X chromosome analysis could be performed by different techniques on intact cells isolated from fresh tissue. Wider implications of the findings FS occurs only in tubules with 46,XY SPGs and 46,XY SCs. Non-mosaic 47,XXY (diagnosed on blood cells) KS patients may present with testicular mosaicism, increasing their chances for sperm retrieval. Trial registration number Not applicable

O-138 Factors influencing sperm retrieval rate of microdissection testicular sperm extraction in nonobstructive azoospermia patients with different etiologies: a retrospective study of 3104 patients

Abstract Study question What are predictors of successful sperm retrieval (SRR) with microdissection testicular sperm extraction (mTESE) in men with non-obstructive azoospermia (NOA) according to different etiologies? Summary answer Predictors for SRR were different in patients with various etiologies and effects of age on SRR in idiopathic NOA and KS patients were opposite. What is known already NOA patients due to spermatogenic dysfunction, accounting for about 60 percent of the total azoospermia cases, may have opportunities to obtain sperm by mTESE and overall SRR of mTESE in NOA patients is about 50%. There were some of predictive factors for SRR at mTESE including age, testis volume, serum follicle stimulating hormone (FSH), testosterone (T), inhibin B, anti-Mullerian hormone and testicular histopathology, while the predictive factors of SRR remains underappreciated, especially in accordance with different type of etiology. Most notably, there are some controversies about the relationship between SSR and age at mTESE surgery. Study design, size, duration This retrospective study involved 3104 NOA patients with different etiologies treated with the first mTESE at the Reproductive Medical Centre of Peking University Third Hospital from March 2012 to December 2022. Participants/materials, setting, methods 3104 NOA patients were classified into seven groups according to etiologies including 1530 males with idiopathic NOA (iNOA), 763 males with Klinefelter syndrome (KS), 345 males with microdeletion of the AZFc, 177 cases with the history of cryptorchidism, 131 with a history of mumps orchitis, 89 males with cryptozoospermia and 69 cases with other causes. The end-point was the presence of one or more sperm. Multi-variable logistic regression was used to analyze sperm retrieval outcome. Main results and the role of chance Overall SRR was 43.46%, and there were different SRR in NOA patients with various etiologies with highest SRR of 89.31% in patients with a history of mumps orchitis and lowest SRR of 29.35% in iNOA patients. For patients with a history of mumps orchitis, there was a negative relationship between T and SRR (0.805 [0.654, 0.991] p = 0.041). For cryptozoospermia patients, shorter infertility duration was predictive for successful SRR (0.837 [0.704, 0.995], p = 0.044). Males with bigger testes had more likelihood of positive SRR (1.077 [1.005; 1.153], p = 0.035) in patients with cryptorchidism. BMI was an independent factor of SRR in AZFc-deleted patients (1.084 [1.013, 1.161], p = 0.020). For KS patients, lower male age (0.938 [0.898, 0.979], p = 0.004) and bigger testes (1.214[1.099, 1.342], p＜0.001) were predictive for successful SRR. Lower levels of FSH (0.984[0.970, 0.998] p = 0.025) and higher male age(1.079[1.050, 1.110], p＜0.001) were predictive for successful sperm retrieval in iNOA patients. So, the optimum age range of iNOA male undergoing mTESE should be 30 to 35 years old with the SRR of 29.13% when considering low SRR of iNOA males aged＜30 years (23.96%) and the negative effect of female age on ICSI outcome. Limitations, reasons for caution Surgeon performing mTESE maybe another predictor for SRR which is a potential selection bias. Inhibin B and Anti-Müllerian hormone weren’t included due to limited information in our database. We couldn’t verify the conclusion in more population from multicenter despite the current study with the large sample size so far. Wider implications of the findings Our results provide valuable information for NOA patients who want to counsel surgeons about their treatments to help patients and surgeons to perform a shared decision-making for optimal therapy methods, especially about optimal age treated with mTESE in iNOA and KS patients. Trial registration number not applicable

O-140 studying the effect of mosaicism on the outcome of micro-TESE among Klinefelter syndrome patients

Abstract Study question Does mosaicism affect sperm retrieval rate in Klinefelter patients? Summary answer Patients with mosaic Klinefelter syndrome (KS) have a higher success rate of sperm retrieval than those with non-mosaic KS. What is known already Klinefelter syndrome is characterised by the presence of an extra X chromosome in male individuals leading to a 47XXY karyotype and rarely to 46XY/47XXY Mosaicism; it is the most common sex chromosomal aneuploidy and the most common genetic cause of Azoospermia (Kanakis GA et al., 2018 and Gravholt CH et al.,2018). It accounts for 3–4% of male infertility cases and 10–12% of men with Azoospermia (Winters SJ,2018). Study design, size, duration After approval of the ethical committee of the faculty of medicine at Cairo University, This prospective study was conducted where 70 men who complained of primary infertility and azoospermia and were diagnosed with Klinefelter Syndrome were recruited for this study between 1/11/2021 and 30/11/2022. Patients with previous testicular disease (torsion, trauma) or who had a history of undescended testes or received chemotherapy or radiotherapy were excluded. Participants/materials, setting, methods Seventy patients (n = 70) aged between 20 and 50 who fit the inclusion criteria were recruited and underwent micro_TESE. Semen analysis (Azoospermia), hormonal profile, scrotal colour doppler ultrasonography, Karyotyping and FISH for Y and X chromosomes were performed. Statistical analysis was conducted using SPSS 22nd edition. Main results and the role of chance Our study groups comprised 14 patients with mosaic KS and 56 with non-mosaic KS. The clinical characteristics were not significantly different statistically between the two groups. Micro_TESE was performed in all 70 patients; in 26 patients (37.1%), mature sperm were found in the wet preparation. No statistically significant correlation was found between the presence of spermatozoa in the extraction specimen and age, serum follicle-stimulating hormone, serum testosterone, prolactin or oestradiol. The statistically significant predictor variable for sperm extraction was mosaicism, luteinising hormone, testicular volume and FISH. The sperm retrieval rate of the patients with mosaic and non-mosaic KS was 57.1% and 32.1%, respectively. Limitations, reasons for caution 1) Larger (n) number of patients was needed. 2) Despite our valuable findings, further studies should be performed to illuminate the relation between FISH for X and Y chromosomes and sperm retrieval rate success. Wider implications of the findings Klinefelter patients with mosaicism should be offered high sperm retrieval rate during IVF counselling. Trial registration number n/a

O-147 Factors influencing ICSI outcome after sperm retrieval in nonobstructive azoospermia patients with different types of etiologies: a retrospective study of 1157 patients

Abstract Study question What are predictors of fertilization, clinical pregnancy, miscarriage and livebirth delivery outcomes after microdissection testicular sperm extraction (mTESE)-ICSI in NOA patients with different etiologies? Summary answer Predictors for ICSI outcome were different in patients with various etiologies and type of NOA are associated with ICSI outcome in these patients. What is known already NOA patients due to spermatogenic dysfunction, accounting for about 60 percent of the total azoospermia cases, may have opportunities to be fathers with their own biological children by mTESE combined with ICSI. For them, there were some of predictive factors for ICSI outcome including sperm motility, sperm morphology, testis volume and levels of reproductive hormone including follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone (T), while the predictive factors of ICSI outcome remains underappreciated, especially about the relationship between ICSI outcome and male factors after reviewing the large scale clinical data. Study design, size, duration This retrospective study involved 1157 NOA patients with treated 1534 complete ICSI cycles at the Reproductive Medical Centre of Peking University Third Hospital from March 2012 to October 2021. Participants/materials, setting, methods 1534 ICSI cycles included 558, 359, 243, 155, 128, 56 and 35 cycles performed in males with idiopathic NOA (iNOA), Klinefelter syndrome (KS), AZFc microdeletions, cryptorchidism, a history of mumps orchitis, cryptozoospermia and with other causes separately. Primary outcome was livebirth delivery per ICSI cycle (LBR) and secondary outcomes were clinical pregnancy rate per ICSI cycle (PR) and fertilization rate (FR). Multi-variable logistic and linear regression was used to analyze ICSI outcome. Main results and the role of chance Overall LBR, PR and FR were 37.35%, 44.98%, 49.34% separately. Patients with mumps orchitis showed the highest LBR, PR and FR of 53.13%, 60.94% and 59.52% respectively, and AZFc-deleted patients displayed the lowest LBR, PR and FR of 25.93%, 30.04% and 32.87% separately. Lower female age (0.947 [0.904;0.993], p = 0.024), more transferred embryos (1.345 [1.225;1.477], p＜0.001), higher AFC (1.033[1.011;1.056], p = 0.003) were related with higher LBR, and higher male BMI (0.974[0.950; 0.999], p = 0.046) and lower AFC (1.057[1.034; 1.080], p＜0.001) were related with lower PR. Comparing outcome of mumps orchitis group, patients with cryptorchidism were associate with lower LBR(0.467[0.281; 0.778], p = 0.003), PR (0.440[0.268;0.721], p = 0.001) and FR(-0.169 [-0.226; -0.11], p＜0.001) separately. AZFc-deleted patients had a relationship with lower LBR (0.426[0.260; 0.697], p = 0.001), (0.273[0.170; 0.437], p＜0.001) and FR (-0.282 [-0.336; -0.228], p＜0.001) respectively. KS patients were related with lower LBR (0.455[0.288; 0.720], p = 0.001), PR (0.477 [0.306; 0.745], p = 0.001) and FR (-0.073 [-0.126; -0.020], p = 0.007) separately. iNOA patients were associated with lower PR (0.616 [0.409; 0.928], p = 0.020) and FR (-0.076 [-0.124; -0.029], p = 0.002) respectively. Furthermore, patients with cryptozoospermia were related with lower FR (-0.141 [-0.220; -0.061], p = 0.001). Limitations, reasons for caution Sperm characteristics maybe predictors for ICSI outcome which is a potential selection bias. Pituitary prolactin and Anti-Müllerian hormone weren’t included as covariates due to limited information in our database. We couldn’t verify the conclusion in more population from multicenter despite the current study with the large sample size so far. Wider implications of the findings Our results provide valuable information for NOA patients who want to counsel surgeons about their ICSI outcome to help patients and surgeons to perform a shared decision-making for optimal therapy methods, especially for AZFc-deleted patients with worse ICSI outcome who may consider donor semen to get better ICSI outcome. Trial registration number not applicable