Non-mosaic Klinefelter Syndrome Patients Research Articles

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

P-097 Embryological and Clinical Outcomes of Microdissection testicular sperm extraction combined with Intracytoplasmic sperm injection (microTESE-ICSI) in patients with and without Klinefelter syndrome

Abstract Study question This retrospective study aims to identify the embryo development and clinical outcomes in non-mosaic Klinefelter syndrome patients treated with microTESE-ICSI Summary answer Non-mosaic Klinefelter patients can retrieve their sperm using microTESE and can have their biological offsprings by combining microTESE with ICSI What is known already Klinefelter syndrome patients are classified into two main groups, namely non-mosaic Klinefelter (47, XXY) and mosaic Klinefelter (46, XY/47, XXY). To retrieve sperm from non-mosaic Klinefelter, microTESE is a crucial treatment that yields a successful rate of approximately 50%. Recent research reports that microTESE-ICSI is a treatment regime that brings a higher in-vitro fertilization (IVF) success rate for those with non-mosaic Klinefelter syndrome. However, most of the previous studies on this group focuses on sperm retrieval rate and male factors. Few studies have reported embryo development and clinical outcomes, especially, under the adjustment for female partner’s age and oocyte quantity Study design, size, duration This retrospective study was conducted to evaluate spermatozoa retrieval, embryo development, and clinical outcomes in 931 patients with microTESE treatment at Andrology and Fertility Hospital of Hanoi from 6/2019 to 9/2022. Patients were divided into two groups including 118 patients with non-mosaic Klinefelter syndrome (KS) and 813 patients without Klinefelter syndrome (non-KS). Participants/materials, setting, methods In 931 patients, 63 KS and 417 non-KS had spermatozoa retrieved from microTESE were carried out ICSI. In ICSI group, 59 KS and 319 non-KS patients whose partners were under 35 years old were selected to compare embryological and clinical outcomes. Outcomes included fertilization rate, day 3 usable embryo rate, usable blastocyst rate, pregnancy rate (positive beta-hCG test), biochemical pregnancy rate, ongoing pregnancy, and live-birth rate (calculated on the total number of IVF cycles). Main results and the role of chance Spermatozoa retrieval rate by microTESE in the KS group was 53.4% (63/118), while this rate in the non-KS group was 51.2% (417/813) (p > 0.05). The mean age of female partners was significantly lower in the KS group than in non-KS group (26.6 ± 4.0 vs. 28.0 ± 3.5; p = 0.006). The mean of mature oocytes between these two groups was similar (15.7 ± 8.1 vs 14.5 ± 7.2; p > 0.05). Fertilization rates in KS and non-KS groups were 62.7% and 65.3%, respectively. Day 3 usable embryo rate (embryo with over 6 blastomeres at day 3) in two groups was not significantly different (69% vs 69.5%, p > 0.05). Usable blastocyst rate (embryo quality is over 2BB, Gardner grading system) in the KS group (19.9%, n = 54) was significantly lower than in non-KS group (41.1%, n = 281) (p < 0.001). Pregnancy rate and biochemical pregnancy rate were 75.9% and 6.9% in KS group (n = 58), respectively. Those rates were 81.5% and 3.2% in non-KS group (n = 313), respectively. There was no significant difference in ongoing pregnancy rate between these two groups (61.1% vs 74.5%; p > 0.05). Live-birth rate in the KS group was 43.2% (16/37), while this rate in the non-KS group was 46.5% (67/145). Limitations, reasons for caution This research had limitations of a retrospective study. Male hormonal factors and baby health could not be thoroughly measured because of difficulties in contact with patients at the time of the study. Besides, day 3 top-quality embryos were directed to freeze, so usable blastocyst rate was low in this study. Wider implications of the findings These data confirmed that microTESE provides opportunities for non-mosaic Klinefelter syndrome patients to retrieve their own sperm. Combination of microTESE and ICSI is an effective treatment regimen that helps these patients to have their own usable embryos and babies. Trial registration number not applicable

P–038 Clinical outcomes of 77 testicular sperm extraction treatment cycles in non-mosaic Klinefelter syndrome patients

Abstract Study question What are the clinical results of patients with azoospermia and nonmosaic Klinefelter syndrome, using fresh and cryopreserved sperm? Summary answer The results showed a recovery rate of testicular sperm in the order of 40% and a life newborn rate of 52% when using fresh sperm What is known already In Klinefelter syndrome (KS), the rates of successful testicular sperm retrieval were shown to be similar either using conventional TESE or micro-TESE (Corona et al., 2017), which highlights that the variability observed between studies is due to differences in patient characteristics. There are a few works with a large number of KS patients elucidating the clinical outcomes using fresh and cryopreserved testicular sperm. However, these studies revealed contradictory outcomes, either revealing better (Greco et al., 2013; Vicdan et al., 2016) or worst (Madureira et al., 2014) results with cryopreserved testicular sperm, or finding no differences (Chen et al, 2019). Study design, size, duration This study includes all patients up to 2019 presenting azoospermia due to non-mosaic Klinefelter syndrome (n = 76) that went for infertility consultations in a private fertility clinic. Patients were evaluated by the same Urologist. The genetic analysis of the patients was performed at an academic institution. At examination patients did not refer other complaints besides infertility, and referred to have not received any hormone replacement therapy in the past. Participants/materials, setting, methods The 76 azoospermic patients with non-mosaic Klinefelter syndrome (KS) were treated by testicular sperm extraction (TESE) followed by intracytoplasmic sperm injection (ICSI), using fresh and cryopreserved testicular sperm. Most patients used fresh testicular sperm, where others preferred to postpone ICSI treatment cycles and used cryopreserved testicular sperm. Aneuploidy screening in children was performed by prenatal diagnosis and MLPA (Multiplex ligation-dependent probe amplification). Full embryological, clinical and newborn outcomes are provided. Main results and the role of chance Of the 76 patients with non-mosaic Klinefelter syndrome, one repeated the testicular sperm extraction (TESE) procedure. Testicular sperm were recovered in 31/77 (40.3%) of the cases. Comparisons between the 31 cases with successful sperm recover (group–1) and the 46 cases without a successful TESE (group–2) revealed no significant differences regarding age, time of infertility, testicular volume, serum levels of FSH, LH and testosterone, total number of testicular fragments analyzed, and time of search in samples. The mean male age was 34 years. In most of the cases, the testicular volume was reduced (96.1%), the levels of FSH (98.3%) and LH (94.1%) were increased, and the levels of testosterone were normal (77.6%). There were 25 intracytoplasmic sperm injection (ICSI) treatment cycles using fresh testicular sperm and 22 ICSI treatment cycles using frozen testicular sperm. The rates of fertilization (63.5% fresh sperm vs 41.6% frozen sperm), implantation (37% fresh sperm vs 13.2% frozen sperm), clinical pregnancy (60.9% fresh sperm vs 19% frozen sperm), live birth delivery (52.2% fresh sperm vs 19% frozen sperm) and newborn (65.2% fresh sperm vs 23.8% frozen sperm) were higher in the group using fresh testicular sperm. Chromosome analysis of the 21 newborn was normal. Limitations, reasons for caution Although presenting a high number of cases with azoospermic non-mosaic Klinefelter syndrome treated with testicular sperm extraction and intracytoplasmic sperm injection, future studies are needed with a higher number of cycles using frozen testicular sperm, in order to confirm or rebut that the freezing methodology affects negatively the clinical outcomes. Wider implications of the findings: Data adds further information regarding testicular sperm retrieval rates and use of fresh or frozen testicular sperm in Klinefelter syndrome (KS) patients. High newborn rates were obtained only with fresh testicular sperm. Results also reassure KS patients about the safety relative to any abnormal chromosomal transmission to the born children. Trial registration number Not applicable

Clinical outcomes of 77 TESE treatment cycles in non-mosaic Klinefelter syndrome patients.

ObjectiveThe current study aimed to present the clinical outcomes of 76 azoospermic patients with non-mosaic Klinefelter syndrome (KS), treated with testicular spermatozoa extraction (TESE) followed by intracytoplasmic sperm injection (ICSI) using either fresh or cryopreserved testicular spermatozoa.MethodsWe retrospectively evaluated 76 patients with non-mosaic KS belonging to a special group of cases that besides infertility did not present the classical signs and symptoms of testosterone deficiency. One of the patients repeated the TESE procedure (76 patients, 77 TESE cycles). Sixty of these 76 patients accepted to undergo TESE associated with ovarian stimulation, while 16 patients underwent TESE followed by testicular spermatozoa cryopreservation. Aneuploidy screening of the offspring was performed by Multiplex ligation-dependent probe amplification and by amniotic fluid karyotyping. Statistical analysis used the Chi-Squared Test, Fisher's Exact Test, 2-sided, for rates, and the Independent Samples T-test for equality of means, 2-sided.ResultsTesticular spermatozoa were recovered in 31 (40.3%) of the attempts. The patients underwent 47 ICSI cycles, 25 with fresh testicular spermatozoa and 22 with cryopreserved testicular spermatozoa. Fertilization (63.5% vs. 41.6%, p=0.000), implantation (37% vs. 13.2%, p=0.014), clinical pregnancy (60.9% vs. 19%, p=0.005) and live birth (65.2% vs. 23.8%, p=0.006) rates were higher with fresh testicular spermatozoa. Chromosome analysis of the 21 newborns was normal.ConclusionsThe present data adds further information regarding the recovery rate of spermatozoa after TESE and the embryological and clinical outcomes with fresh and cryopreserved testicular spermatozoa, besides reassuring the safety concerning chromosomal transmission of KS from parents to their offspring.

Generation of an iPSC cohort of isogenic iPSC lines (46-XY and 47-XXY) from a non-mosaic Klinefelter Syndrome patient (47-XXY) (KAUSTi008-A, KAUSTi008-B, KAUSTi008-C, KAUSTi008-D, KAUSTi008-E, KAUSTi008-F, KAUSTi008-G).

Klinefelter Syndrome (KS) is the most commonXchromosome aneuploidy in males characterized by highly heterogeneous clinical manifestations including a subtle cognitive impairment and multisystemic disorders such as infertility, metabolic syndrome, gynecomastia and cardiovascular diseases. To date dosage-dependent correlation studies of X-linked genes and low- and high-grade KS clinical phenotypes have not been performed. Here we generated multiple isogenic 47-XXY and 46-XY iPSC lines from one 47-XXY patient. Leveraging on a fully matched genetic background, our cohort represents a highly informative tool to study the impact ofXchromosome dosage on KS pathophysiology.

Role of treatment with human chorionic gonadotropin and clinical parameters on testicular sperm recovery with microdissection testicular sperm extraction and intracytoplasmic sperm injection outcomes in 184 Klinefelter syndrome patients

To investigate whether preoperative human chorionic gonadotropin (hCG) treatment can help predict the outcomes of microdissection testicular sperm extraction (micro-TESE) and affect fertility outcomes in non-mosaic Klinefelter syndrome (KS) patients. Retrospective cohort study. University-affiliated fertility center. A total of 184 non-mosaic KS patients who underwent micro-TESE with or without preoperative hCG treatment from January 2016 to July2019. None. Sperm retrieval rate (SRR) with and without hCG treatment, logistic models analysis. Eighty KS patients (43.5%) had successful sperm retrievals after micro-TESE. There was no statistically significant difference in the SRR between the group who received hCG treatment and the group that did not (44.0% vs. 43.3%). Logistic regression analyses demonstrated that the hCG treatment had no statistically significant effect on successful sperm retrieval. However, higher preoperative testosterone (T) levels seemed to be associated with a higher probability of successful sperm retrieval (multivariate adjusted odds ratio 1.09; 95% confidence interval [CI], 1.04-1.16). The prediction model for SRR on KS patients had an area under the curve of 67.3% (95% CI, 59.3-75.3%). In the hCG treatment group, the data indicated that the three parameters of testicular volume, pretreatment T level, and alterations of T were associated with the probability of successful sperm retrieval. Moreover, hCG therapy did not affect intracytoplasmic sperm injection (ICSI) outcomes. No differences in the pregnancy rate or live-birth rate were observed between the two groups. Therapy with hCG does not affect SRR or ICSI outcomes of non-mosaic KS patients. However, preoperative T levels, whether treated with hCG or not, can predict the chance of sperm retrieval with micro-TESE.

Detection of low-grade mosaicism and its correlation with hormonal profile, testicular volume, and semen quality in a cohort of Egyptian Klinefelter and Klinefelter-like patients

Klinefelter syndrome (KS) is the most common chromosomal syndrome, causing infertility in men and leading to non-obstructive azoospermia. Previous studies on mosaicism have shown contradictory results on its correlation with both serum hormone levels and the presence of spermatozoa in the ejaculate of KS, KS-like, and non-KS-like infertile patients. So, the present study was designed to detect low-grade mosaicism in the peripheral blood lymphocytes and buccal mucosa cells of 14 KS and 8 KS-like patients by using fluorescence in situ hybridization (FISH) and to investigate its correlation with luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone (T) levels, testicular volume, and semen analysis compared with 10 normal healthy fertile men. Our results indicated that mosaicism was only found in 42.9 % of the KS patients and completely absent in all KS-like patients. Moreover, mosaicism has led to complete azoospermia and non-significant differences in both hormone levels and testicular volume between mosaic and non-mosaic KS patients. All KS patients demonstrated significant differences in both hormone levels and testicular volume compared with normal men. Conversely, they revealed non-significant differences in hormone levels and significant differences in testicular volume compared with KS-like patients. Additionally, the KS-like patients exhibited non-significant variations in both LH and FSH levels and significant variations in T level and testicular volume compared with normal men. Moreover, all KS-like patients had azoospermia, except for one patient who showed oligozoospermia. Therefore, no correlations were found either between mosaicism and serum hormone levels or with testicular volume and semen analysis.

Spermatological characteristics of mosaic and non-mosaic forms of Klinefelter syndrome

The study objectiveis to compare of ejaculate parameters in mosaic, non-mosaic Klinefelter syndrome (KS).Materials and methods. Eighty-five patients with KS were examined. The group 1 included 75 patients between the ages of 17 and 39 with non-mosaic KS (47,XXY), the group 2 included 10 males between the ages of 22 and 57 years with mosaic KS: 47,XXY/46,XY (n = 9), 48,XXY,der(X)/47,XXY/46,XY (n = 1).Results. KS patients semen volume was 1.9 ± 1.3 (0.1–5.5) ml in non-mosaic KS patients (47,XXY) and 1.5 ± 1.2 (0.05–4.00) ml in patients with a mosaic form of a KS, respectively, рН semen – 7.8 ± 0.5 (6.5–9.0) and 7.8 ± 0.2 (7.5–8.1), sperm count – 0.27 ± 1.42 (0.00–12.50) and 0.12 ± 0.28 (0.00–0.90) million/ml, respectively. The viscosity was increased (>20 mm) at 41 % non-mosaic KS (group 1) and 22 % of mosaic KS (group 2) patients. The ejaculate sediment was investigated by quantitative karyological analysis of immature germ cells. The germ cells in 42 % samples of the ejaculate of the patients with a classical form of a KS and in 20 % samples of the ejaculate of the patients with a mosaic form was found. That indicates a partial preservation of spermatogenesis.Conclusion. The degree of spermatogenesis depletion in KS patients widely varied, ejaculate and germ cell parameters in the ejaculate sediment weren’t significantly different. Presence of few sperms (cryptozoospermia) and immature cells in the ejaculate sediment point to partial preservation of spermatogenesis.

AMH is a predictive factor for successful sperm retrieval in non-mosaic Klinefelter syndrome patients

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

What should be the minimum frequency of micro testicular sperm extraction (m-TESE) in patients with klinefelter syndrome?

Aim: To investigate whether a second or even a third surgery is required for sperm retrieval in non-mosaic Klinefelter syndrome patients who underwent a successful/failed micro-testicular sperm extraction (m-TESE).Material and Methods: The patients underwent physical examination, genetic analyses, pathological screening between 2008 and 2018. In the patients, sperm retrieval rates, pregnancy after intracytoplasmic sperm injection (ICSI) and baby take-home rates were examined.Results: M-TESE was repeated for the second time in thirty-five patients in total, with twenty of that underwent their first m-TESE with a negative result and fifteen with a positive result. In =6/20 patients (30%) who had a negative result with the first m-TESE and in n=9/15 patients (60%) who had a positive result with the first m-TESE, sperm was detected the second time. Pregnancy was achieved in n=2/6 patients (33%) who were negative the first time and positive the second time. Baby take-home was achieved in one patient (16.6%). Conclusion: In addition to patients from whom sperm could be retrieved previously, sperm could be detected especially in the second and even third redo m-TESE in patients from whom sperm could not be retrieved. Pregnancy occurred and baby take-home was achieved. Despite the presence of a very limited testicular tissue, it is recommended for this procedure to be performed by expert practitioners upon discussing very openly the complications and achievements, to decide accordingly and repeat m-TESE in these patients where possible.

A history of undescended testes in young men with Klinefelter syndrome does not reduce the chances for successful microsurgical testicular sperm extraction

Klinefelter syndrome (KS) and undescended testes (UDT) are known etiologies for non-obstructive azoospermia (NOA), and coexistence of both etiologies is not uncommon. Patients with both KS and a history of UDT are therefore considered to have extremely reduced chances for paternity. We aimed to analyze the impact of previous surgically corrected unilateral or bilateral UDT on sperm retrieval rates (SRRs) by microsurgical testicular sperm extraction (mTESE) in azoospermic men with KS. Age, testicular volumes, and hypothalamo-pituitary-gonadal axis function were investigated in relation to SRRs in 29 non-mosaic KS patients (47,XXY) with a history of UDT (group 1) who underwent mTESE between 2008 and 2016 in our center and compared to the data of age- and serum testosterone-matched non-mosaic KS controls with eutopic testes at birth (group 2), and to those of 51 men with NOA and a normal male karyotype (46,XY), but previous UDT (group 3). SRRs in KS patients with surgically corrected UDT during childhood were comparable to SRRs of KS patients with eutopic testes at birth: 31% (35% in unilateral and 22% in bilateral UDT) vs. 38% (p=0.581). SRRs and Leydig cell function in group 1 were negatively correlated with age. Significantly higher SRRs (66%) were found in euploid azoospermic men with surgically corrected UDT (p<0.001). A history of UDT does not preclude chances for future fatherhood in young azoospermic males with KS. In one of three men with previous unilateral UDT and in one of 4-5 in those with previous bilateral UDT, spermatozoa can be harvested by mTESE during late adolescence or young adulthood for immediate or future use in assisted reproduction.

Decreased miRNA expression in Klinefelter syndrome

The widelyvariable phenotypic spectrum and the different severity of symptoms in men with Klinefelter syndrome (KS) suggest a role for epigenetic mediators. Therefore, the aim of this study is to evaluate the possible involvement of miRNAs in the clinical manifestations of KS. To accomplish this, we performed a transcriptome analysis in peripheral blood mononuclear cells (PBMCs) of 10 non-mosaic KS patients, 10 aged-matched healthy men and 10 aged-matched healthy female controls with normal karyotype. After RNA extraction from PBMC and the preparation of RNA libraries, the samples were sequenced using next generation high-throughput sequencing technology. Expression profiling analysis revealed a significant differential expression of 2 miRNAs in KS compared to male controls. In particular, MIR3648 resulted significantly (q-value < 0.0001) down-regulated by −19.084- fold, while MIR3687was strongly down-regulated (q-value < 0.0001) considering KS patients. These results were confirmed by qRT-PCR. The functional analysis of the two transcripts showed that they seem to play a role in breast cancer, hemopoietic abnormalities, immune defects and adipocyte differentiation and fat cell maturation. Therefore, we speculate that both miRNAs may play a role in the immune and metabolic disorders and in the risk of breast cancer development in men with KS.

Is there any clinical relevant difference between non mosaic Klinefelter Syndrome patients with or without Androgen Receptor variations?

Klinefelter Syndrome (KS) is the most common chromosomal disorder in men leading to non-obstructive azoospermia. Spermatozoa can be found by TESE in about 50% of adults with KS despite severe testicular degeneration. We evaluated AR variations and polymorphism length in 135 non-mosaic KS patients, aimed to find possible correlation with clinical features, sex hormones and sperm retrieval. Among 135 KS patients we found AR variations in eight subjects (5.9%). All variations but one caused a single amino acid substitution. Four variations P392S, Q58L, L548F, A475V found in six patients had been previously described to be associated with different degrees of androgen insensitivity. Moreover we observed in two patients Y359F and D732D novel variations representing respectively a missense variation and a synonymous variation not leading to amino acid substitution. All the Klinefelter patients with AR gene variations were azoospermic. Spermatozoa were retrieved with TESE for two men (40%), sperm retrieval was unsuccessful in other 3 patients. This is the only study reporting AR variations in KS patients. Relevant clinical differences not emerged between AR mutated and not AR mutated KS patients, but does each variation play an important role in the trasmission to the offspring obtained by ART in this patients?

Hereditary risk in icsi with sperm from non-mosaic Klinefelter syndrome patients

To verify the actual risk of hereditary Klinefelter Syndrome (KS) or incidental aneuploidy in ICSI treatment of KS patients, which has been warned in previous cytogenetic studies using testicular cells from KS patients.

Ploidy of spermatogenic cells of men with non-mosaic Klinefelter's syndrome as measured by a computerized cell scanning system.

This study aims to characterize the origin of testicular post-meiotic cells in non-mosaic Klinefelter's syndrome (KS). The study included testicular tissue specimens from 11 non-mosaic KS patients, with (6 positive) and without (5 negative) spermatozoa presence. The obtained testicular cells were affixed and stained for morphology followed by fluorescence in situ hybridization (FISH) for centromeric probes X, Y, and 18. We used a computerized automated cell scanning system that enables simultaneous viewing of morphology and FISH in the same cell. A total of 12,387 cells from the positive cases, 11,991 cells from the negative cases, and 1,711 cells from the controls were analyzed. The majority of spermatogonia were 47, XXY in both the positive and negative KS cases (88.9 ± 4.76 % and 90.6 ± 4.58 %) as were primary spermatocytes (76.8 ± 8.14 % and 79.6 ± 7.30 %). The respective rates of secondary spermatocytes and post-meiotic cells (round, elongating spermatids and sperm cells) were 1.1 ± 1.39 % in the positive cases, 2.9 ± 3.33 % in the negative cases, compared to 67.6 ± 6.22 % in the controls (P < 0.02). Pairing of both 18 and XY homologous chromosomes in 46,XY primary spermatocytes was 2.5 ± 2.31 % and 3.4 ± 2.39 %, respectively, compared to 19.8 ± 8.95 % in the control group (P < 0.02) and in 47,XXY primary spermatocytes in 2.4 ± 3.8 % in the positive group and 3.2 ± 2.26 % in the negative group. This study presents data to indicate that the majority of primary spermatocytes in the testes of non-mosaic KS patients are 47,XXY and could possibly develop into post-meiotic cells.

Role of vitamin D levels and vitamin D supplementation on bone mineral density in Klinefelter syndrome.

This manuscript describes the role of low vitamin D in bone metabolism of Klinefelter subjects. Low vitamin D is frequent in this condition and seems to be more important than testosterone in inducing low bone mineral density (BMD) and osteoporosis. Supplementation with vitamin D restores BMD after 2 years of treatment, whereas testosterone alone seems to be ineffective. Decreased bone mineral density (BMD) in Klinefelter syndrome (KS) is frequent, and it has been traditionally related to low testosterone (T) levels. However, low BMD can be observed also in patients with normal T levels and T replacement therapy does not necessarily increase bone mass in these patients. Nothing is known about vitamin D levels and supplementation in KS. In this study, we determine vitamin D status and bone mass in KS subjects and compare the efficacy of T therapy and vitamin D supplementation on BMD. A total of 127 non-mosaic KS patients and 60 age-matched male controls were evaluated with reproductive hormones, 25-hydroxyvitamin D, PTH, and bone densitometry by dual-energy X-ray absorptiometry (DEXA). Patients with hypogonadism and/or 25-hydroxyvitamin D deficiency were treated with T-gel 2% and/or calcifediol and re-evaluated after 24 months of treatment. 25-hydroxyvitamin D levels were significantly lower in KS patients with respect to controls, and they had significantly lower lumbar and femoral BMD. The percentage of osteopenia/osteoporosis in subjects with 25-hydroxyvitamin D deficiency was higher with respect to subjects with normal 25-hydroxyvitamin D and was not related to the presence/absence of low T levels. Subjects treated with calcifediol or T + calcifediol had a significant increase in lumbar BMD after treatment. No difference was found in T-treated group. These data highlight that low 25-hydroxyvitamin D levels seem to have a more critical role than low T levels in inducing low BMD in KS subjects. Furthermore, vitamin D supplementation seems to be more effective than T replacement therapy alone in increasing BMD.

Clinical characteristics of men with non-mosaic Klinefelter syndrome in northeastern China: implications for genetic counseling.

Klinefelter syndrome (KS) is the most common genetic cause of male infertility. Widespread development in assisted reproductive technology has provided non-mosaic KS patients with the opportunity of having biological children. Testosterone replacement therapy and micro-dissection testicular sperm extraction are effective sperm retrieval techniques for KS patients. Despite the success of sperm retrieval and intracytoplasmic sperm injection (ICSI), some areas of early aggressive hormonal spermatogenesis and appropriate management of KS remain controversial. Androgenotherapy, a common treatment for KS, carries a risk of decreasing focal spermatogenesis by lowering the gonadotropin content. Inadequately treated hypogonadism increases psychosocial morbidity in KS patients. Preventive care must be provided from the time of diagnosis, preferentially through a multidisciplinary approach. This indicates the need for improved genetic counseling of KS patients. The aim of this study was to report the prevalence of non-mosaic KS in a Chinese infertile male population. The rate of early diagnosis was lower in KS patients; most of these were diagnosed after rising concerns of reproductive capacity. The mean age of patients with sperm or germ cells was significantly lower, while the semen volume of these patients was significantly higher. However, the semen volume was negatively correlated with the age and ratio of luteinizing hormone/testosterone content in KS patients. Therefore, genetic counseling of KS patients should focus on early diagnosis and timely treatment, in addition to improving the quality of life of all KS patients. The use of testosterone replacement therapy and/ or micro-dissection testicular sperm extraction should be preferentially considered for fertility preservation.

Microdissection testicular sperm extraction for non-mosaic klinefelter syndrome patients

OBJECTIVE: Fertilization and pregnancies can be obtained with spermatozoa recovered not only from the ejaculate, but seminiferous tubules (testicular sperm extraction, TESE). Microdissection (Micro)TESE, in combination with intracytoplasmic sperm injection (ICSI), presently is used to treat infertility in cases of NOA including Klinefelter syndrome (KS). KS 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. The aim of this study is to assess the prevalence and the significance including sperm retrieval rate (SRR) by micro-TESE in non-obstructive azoospermic patients including KS. DESIGN: Retrospective clinical analysis. MATERIALSANDMETHODS: The records were retrospectively evaluated for 1028 NOA patients (including 123 non-mosaic KS patients) who were underwent micro-TESE. Chromosomal analysis was performed on all patients on cultured lymphocytes from peripheral blood. Micro-TESE was used in which seminiferous tubule are directly examined throughout the testicle using an operating microscope and selectively biopsied for all of the NOA patients in modified Schlegel’s method. We did not undergo preoperative hormonal therapy for KS patients. In our study, KS patients did not have microdeletion of Y chromosomes. RESULTS: Testicular sperm were successfully retrieved by microTESE in 69 of 123 (56.1%) non-mosaic KS and 380 of 905 (42.0%) not KS NOA patients. Of these, 48 (69.6%) KS and 344 (90.5%) not KS NOA patinets had sperm identified through the initial wide incision alone. For patients with KS, the chance of sperm retrieval on the contralateral side after a negative unilateral microdissection was 28.0% (21/75) and significant higher than not KS NOA patients (36/5611⁄46.4%) (p <0.001). In almost all patients in whom micro-TESE was successful we could identify focal spermatogenesis in dilated and opaque seminiferous tubules surrounded by shrunken tubules or fibrous tissue. No correlation was found between serum FSH, LH, and T level with the success of sperm retrieval. Testicular volume also did not affect the SRR for micro-TESE. Patient age in successful micro-TESE for cases of KS is significantly younger (33.5 4.2 years) than that in failed cases (37.4 3.9 years) (p <0.05). CONCLUSION: Micro-TESE is particularly helpful for successful sperm retrieval in KS cases. In NOA, the absence of uniformity in testicular tissue is a critical key to succeed and rationale in microTESE.

Risk level of intracytoplasmic sperm/spermatid injection for 115 non-mosaic klinefelter syndrome patients

It has been reported that the sex chromosome abnormality incidence rate for ICSI children increased when Klinefelter syndrome patient sperm had been used. We analyzed clinical data of spermatid injections for 115 non-mosaic Klinefelter syndrome patients to clarify whether the report was correct or not.

Birth of 16 healthy children after ICSI in cases of nonmosaic Klinefelter syndrome

Does the health status of infants fathered by nonmosaic Klinefelter syndrome (KS) patients whose partners underwent ICSI with sperm obtained from testicular dissection reveal any genetic risk for the offspring?. KS patients undergoing testicular sperm extraction (TESE) are capable of conceiving healthy children. Paternity has been successfully achieved in nonmosaic KS patients (47,XXY karyotype) by ICSI using either ejaculated or testicular spermatozoa. A crucial concern is the potential transmission of genetic abnormalities to the offspring. Some studies reported that 47,XXY spermatogonia are capable of completing spermatogenesis leading to the production of mature spermatozoa with increased aneuploidies. Other authors showed that where focal spermatogenesis is present in nonmosaic KS males, it originates from euploid germ cells and, therefore, produces normal mature gametes. In support of this finding, at present, the great majority of children born from nonmosaic KS patients are chromosomally normal. From April 2004 to June 2010, 38 azoospermic patients with nonmosaic KS were examined for the presence of testicular spermatozoa. Spermatozoa were retrieved from 15 patients and 26 ICSI cycles were done (16 with cryopreserved sperm). There were 15 pregnancies leading to the birth of 16 babies who were karyotyped at amniocentesis and after birth. Participants were recruited from couples attending the European Hospital, Rome, and Clinica MAR&Gen, Granada, for infertility treatment. Both the European Hospital and Clinica MAR&Gen are private clinics. Testicular tissue was extracted with TESE or micro-TESE. After retrieval, fresh sperm was used for ICSI or it was cryopreserved for future use. Spermatozoa were retrieved from 15 patients (14 TESE and 1 micro-TESE) out of 38 (39.5%). A total of 26 ICSI cycles were performed: 10 with fresh and 16 with cryopreserved-thawed sperm. Mean ages (y) of patients with positive and negative sperm retrieval were, respectively, 34.8 ± 1.72 and 35.6 ± 4.08 (NS, nonsignificant). Comparing ICSI cycles performed with fresh sperm (n = 10) to those performed with frozen-thawed sperm (n = 16): Fertilization rates per injected oocyte were 53.0% (44 of 83) and 47.8% (32 of 67), respectively (NS). The cleavage rate per injected oocyte was 90.6% (29 of 32) versus 68.2% (30 of 44); P = 0.026. Clinical outcomes were not significantly different between the fresh and the frozen-thawed sperm group: clinical pregnancy rates were 7 of 10 (70.0%) and 8 of 16 (50.0%); implanted embryos (per transferred embryo) were 8 of 23 (34.8%) and 8 of 29 (27.6%); delivery rates were 6 of 10 (60.0%) and 5 of 16 (31.3%). Sixteen babies were born, all of them are healthy with a normal karyotype, eight from the fresh sperm group and eight from the frozen-thawed sperm group. The small numbers available for study mean that only common problems can be excluded. This study provides further reassurance that KS men can father healthy children and that pre-implantation genetic diagnosis on embryos conceived with their sperm is not strongly indicated. However, until conclusive information is available, such couples should be offered extensive genetic counseling. No external funding was obtained for the present study. None of the authors has any conflict of interest to declare. Not applicable.