Infertility in Patients With Klinefelter Syndrome: Optimal Timing for Sperm and Testicular Tissue Cryopreservation

Management Update

ManageMent Update Infertility in Patients With Klinefelter Syndrome: Optimal Timing for Sperm and Testicular Tissue Cryopreservation Dorota J. Hawksworth, MD, MBA,1 April A. Szafran, MD, PhD,1 Philip W. Jordan, PhD,2 Adrian S. Dobs, MD,3 Amin S. Herati, MD1 1Department of Urology, Johns Hopkins Medical Institutions, Baltimore, MD; 2Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD; 3Department of Endocrinology, Johns Hopkins Medical Institutions, Baltimore, MD Male factor infertility is a complex issue presenting many diagnostic and management challenges. It is responsible for about 50% of all causes of infertility and thus carries significant medical, financial, and psychological implications for the couples struggling with conception. Klinefelter syndrome is the most common chromosomal male anomaly associated with male infertility. This review focuses specifically on non-obstructive azoospermia secondary to Klinefelter syndrome and discusses controversies surrounding fertility management in patients with this genetic disorder. [Rev Urol. 2018;20(2):56–62 doi: 10.3909/riu0790] ® © 2018 MedReviews , LLC KEY WORDS Klinefelter syndrome • Non-obstructive azoospermia • Sperm retrieval I nfertility, defined as failure to conceive after 1 year of unprotected sexual intercourse, is estimated to affect up to 15% of couples worldwide with a male factor implicated in approximately 50% of cases.1-3 Male infertility can manifest from numerous etiologies ranging from obstruction of the vasa deferentia to non-obstructive etiologies, such as genetic anomalies resulting in testicular dysfunction. An endocrine evaluation, consisting of a serum testosterone and follicle-stimulating hormone (FSH) level, is part of the standard evaluation of the infertile male as it can assist in establishing an underlying diagnosis and can also guide medical and surgical therapy. With male infertility, FSH level values inversely reflect the quality of spermatogenesis. An FSH value over 7.6 IU/mL has been shown to be a strong predictor of spermatogenic failure, whereas a normal FSH value is predictive of normal spermatogenesis. Schoor and colleagues showed that 91% of men with azoospermia and FSH value less 56 • Vol. 20 No. 2 • 2018 • Reviews in Urology 4170018_02_RIU0790_V2_ptg01.indd 56 9/11/18 5:15 PM Infertility in Patients With Klinefelter Syndrome than 7.6 IU/mL had an obstructive etiology explaining their infertility. Similarly, elevated FSH values also correlate with a lower probability of sperm retrieval rates with testicular sperm extraction (TESE) procedures.4,5 Although decreasing semen parameters, such as sperm concentration, and rising FSH values can be used as an indication of progressive spermatogenic failure, it is not a perfect biomarker of spermatogenesis. Ramasamy and colleagues showed that among men with nonobstructive azoospermia (NOA), sperm retrieval rates using microsurgical testicular sperm extraction (mTESE) were higher among men with an FSH value of .15 IU/mL than men with an FSH ,15 IU/mL.6 In this study, three men with FSH values .90 IU/mL had successful sperm extraction. These findings demonstrate the limited utility of FSH and the importance of microsurgical examination in men who are actively seeking fertility. In this review, we focus specifically on non-obstructive azoospermia secondary to Klinefelter syndrome (KS), which is characterized by a high FSH level, and discuss the optimal timing of sperm retrieval in these patients, many of whom are not actively seeking fertility. Genetic Basis for Male Infertility and Klinefelter Syndrome Overall, genetic and genomic abnormalities may contribute up to 50% of male factor infertility and infertile men have up to 10-fold higher prevalence of chromosomal abnormalities when compared with fertile men.7 Aneuploidy is the most common chromosomal error identified in infertile men and the most common of those are KS, XYY syndrome, XX male syndrome, mixed gonadal dysgenesis, autosomal translocations, and Y-chromosome microdeletions.8 KS is the most common chromosomal male anomaly, the most common sex chromosome disorder of infertile men, and as such, it specifically results in NOA. A comprehensive hormonal evaluation of the patients with NOA sub-classifies them into two groups: those with hypogonadotropic hypogonadism (HH) and those with hypergonadotropic hypogonadism. Hypergonadotropic hypogonadism is caused by an intrinsic testicular dysfunction and its causes include genetic defects (aneuploidy, Y-chromosome microdeletions), varicocele, exposure to gonadotoxins, orchitis, prior surgery/trauma, or testicular torsion.9 Of these causes, KS is the most common aneuploidy in men resulting in male factor infertility and is characterized by a male karyotype with one or more additional X chromosomes. The disease affects 1 in 600 newborn males and typically manifests in adolescence or early adulthood with characteristic findings of hypergonadotropic hypogonadism and primary infertility.10,11 On physical examination, patients usually have normal or tall stature, gynecomastia, and small testes. Additionally, these patients may have mild cognitive impairment. Due to a wide variation in clinical presentation, many patients may go undiagnosed. The diagnosis, when made, depends on a combination of history, physical examination, semen analysis (SA), and, ultimately, karyotype testing. With increasing utilization of prenatal or other genetic testing, the detection of KS is likely to increase. The majority of patients carry a 47XXY karyotype, whereas the remaining 10% to 20% are mosaics (46,XY/47,XXY), have higher grade aneuploidy (48,XXXY, 49,XXXXY), or possess partial fragments of supranumery X chromosomes (eg, 47,X,iXq,Y).10,12 Whereas some mosaic patients present with less severe infertility phenotypes and possess reduced concentrations of sperm on SA (oligozoospermia), most men with KS are azoospermic and for paternity reasons require assisted reproductive technologies (ART). Natural History of KS Patients may be diagnosed with KS during different stages of their lives, ranging from the prenatal period via amniocentesis to adulthood. Most patients undergo chromosomal evaluation in their adolescence or adulthood, when delayed or incomplete puberty or infertility arise. However, an increasing number of KS patients are detected prenatally secondary to their parents delaying reproduction due to socioeconomic factors and gender roles changes in the work force.13 As a consequence of increasing maternal age, more amniocenteses and chorionic villi biopsies are performed thus increasing prenatal diagnoses of KS. In addition to chromosomal analysis, all men with KS should undergo a thorough reproductive workup with hormonal and SA evaluations. Levels of testosterone, luteinizing hormone (LH), FSH, estradiol (E2), prolactin, sex hormone binding globulin (SHBG), and inhibin B should be measured. It has been documented that prepubertal males have normal levels of testosterone, LH and FSH, whereas at puberty testosterone levels start to decline and FSH and LH rise. In addition to a hormonal evaluation, at least two semen samples should be analyzed. Testicular function in KS presents a story of progressive degeneration. Ultimately, this degeneration leads to infertility as the normal testicular Vol. 20 No. 2 • 2018 • Reviews in Urology • 57 4170018_02_RIU0790_V2_ptg01.indd 57 9/11/18 5:15 PM Infertility in Patients With Klinefelter Syndrome continued A – Aromatase ABP – Androgen Binding Protein E2 – Estradiol FSH – Follicle Stimulating Hormone GnRH – Gonadotropin Releasing Hormone LH – Luteinizing Hormone T - Testosterone HYPOTHALAMUS GnRH PITUITARY FSH LH LEYDIG CELL SERTOLI CELL INHIBIN A T T E2 ABP Figure 1. Representation of hypothalamus-pituitary-gonadal axis. architecture is replaced initially by either tubular atrophy, sclerosis, or maturation arrest and ultimately degenerates to fibrosis and hyalinized tissue.14 Numerous studies demonstrate already reduced numbers of germ cells in biopsies of 47XXY fetal testes evaluated during the prenatal period, between 18 and 22 weeks of gestation.15,16 This deficit is further augmented in non-descended testes. In the neonatal period, based on lower levels of serum testosterone during the initial months of life in non-mosaic 47XXY patients, Leydig cell dysfunction is postulated to play a significant role.17 Sertoli cells, however, appear to be histologically intact in both the fetal and neonatal periods in subjects with a 47XXY genotype. These subtle changes may create a platform for the later testicular failure that ensues in adolescence. The transition to rapid deterioration in production of both germ cell lines as well as in the histological composition of the testes in KS patients occurs during puberty. Wilkstrom and colleagues demonstrated that prepubertal KS patients with bilateral descended testes retained germ cells on biopsy, though at lower levels than normal children.18 The subjects who had undergone puberty at the time of biopsy had no germ cells present and had concomitant degeneration of Sertoli cells and hyalinization of the seminiferous tubules.18 Therefore, activation of the hypothalamicpituitary-gonadal (HPG) axis (Figure 1) and stimulation of the gonadal tissue appears to accelerate testicular demise in puberty. This is thought to arise from aneuploidyinduced non-homologous recombination and subsequent activation of apoptosis-related genes within the spermatogonial cell line as spermatogonia differentiate into primary spermatocytes and progress through meiosis.12,19 While the molecular mechanisms underlying spermatogenic failure are poorly understood, recent investigations into the transcriptome have highlighted many candidate genes that are dysregulated in KS spermatogonial cells. D’Aurora and colleagues analyzed the transcriptome of testicular biopsies obtained from three men with KS and compared them with the transcriptome of three controls.12 Differential expression was observed in 1050 genes, with 747 genes downregulated and 303 up-regulated genes. One-third of the genes upregulated were linked to apoptosis. Gene cluster and pathway analysis showed four possible mechanisms responsible for hypospermatogenesis in KS patients: impaired development of spermatogonia to mature spermatozoa, defects in the testis architecture, pathophysiology of the testis environment, and apoptosis of the germinal and somatic cells.20 Of all the dysregulated genes, four genes mapped to the X chromosome including solute carrier family 25 member 5 gene (SLC25A5) on the Par1 region, phosphoribosyl pyrophosphate synthetase 1 (PRPS1), TSC22 domain family member 3 (TSC22D3), and A-kinase anchoring protein 4 (AKAP4). Other important dysregulated genes include downregulation of the cAMP responsive element modulator (CREM) gene, which is an important transcription factor for spermatogenesis, the HORMA domain containing 2 (HORMAD2) gene, which surveils the synaptic events during prophase of meiosis, and the cyclin A1 (CCNA1) gene, which is required for spermatocyte passage into the first meiotic division.21,22 Compared with controls, the majority of downregulated genes were those essential for spermatogenesis, whereas apoptotic genes were common among those up-regulated. The spermatogonia of the testis can possess significant heterogeneity, even among patients with sex chromosome trisomy (SCT). Recent 58 • Vol. 20 No. 2 • 2018 • Reviews in Urology 4170018_02_RIU0790_V2_ptg01.indd 58 9/11/18 5:15 PM Infertility in Patients With Klinefelter Syndrome 47XXY iPSC iPSC PASSAGE 2-6 FIBROBLAST– 47XXY PGCLC PGCLC PGCLC iPSC DIFFERENTIATION TO EPIBLAST-LIKE STATE 46XY/46XX TR A NS PL AN T iPSC iPSC iPSC 46XY/46XX SEMINIFEROUS TUBULE Figure 2. Trisomic chromosome loss and development of spermatozoa from euploid primordial germ cell–like cells (PGCLC) from induced pluripotent stem cells (iPSC). Methodology adapted from Hirota T et al.23 data from Hirota and colleagues demonstrate a mechanism for spermatogonia to escape the massive wave of apoptosis that occurs during puberty in KS patients (Figure 2).23 To demonstrate this phenomenon, fibroblasts derived from control and sex chromosome trisomy mice were dedifferentiated to form-induced pluripotent stem cells (iPSCs). Fluorescent in situ hybridization performed on iPSCs derived from SCT fibroblasts showed a propensity for sex chromosome loss over autosomal chromosome loss, returning the iPSC cells to a euploid state. This concept of trisomic chromosome loss has been similarly observed in human trisomic cell cultures obtained and reprogrammed into iPSC from the fibroblasts of patients with Down syndrome.23,24 Hirota and colleagues provide an important proof of concept for the mechanism by which KS patients may retain the ability to preserve spermatogenesis into later years of life.23 Pros and Cons of Early Fertility Management It is well established that men with KS are born with spermatogonia and that the onset of puberty is associated with increased rates of progressive testicular germ cell depletion and subsequent decline in testicular function. It is also widely accepted that small, patchy distribution of spermatogenesis exists even in the adult men’s testes, as spermatozoa have been found both in the testicular tissue and occasionally in the ejaculate. At present, thanks to the advances in testicular sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI) techniques, approximately 50% of men with KS will have sperm detected with TESE/microTESE, of which a 50% pregnancy and live birth rate can be expected.25 Based on prior data indicating that younger age is a major positive predictive factor for successful sperm retrieval, it has been advocated that fertility preservation should be offered to prepubertal and adolescent boys with KS. Because the testicular function decline begins in puberty and worsens in adulthood, intervention prior to or at the beginning of this decline should yield the most successful sperm retrieval. Sperm has been identified in 70% of ejaculated semen specimens in adolescents with KS aging 12 to 20 years.26 Therefore, if younger patients are able and willing to provide an ejaculated specimen, they may ultimately avoid more invasive surgical interventions in their future. Testicular dissection for sperm harvesting has well documented negative effects and those may result in irreversible scarring and atrophy, potential testicular injury or loss, as well as further decline in testicular function and resulting decrease in testosterone levels.27 The need for chronic hormonal therapy (HT) in these patients further complicates their fertility potential. HT is often initiated in boys with KS at around 12 years of age, especially if they exhibit evidence of hypergonadotropic hypogonadism. Androgen replacement at the time of puberty supports normal development of secondary sexual characteristics, body habitus, and results in overall improvement in energy levels. Furthermore, longterm HT prevents development of significant medical issues, to include osteoporosis, diabetes, obesity, and depression. Excess extratesticular androgens, however, further suppress already impaired spermatogenesis in patients with KS. It has been postulated that sperm harvest at time of puberty, or prior to initiation of HT provides best chance of success.26 Although new approaches to medical management of these patients’ hypogonadism allows successful sperm harvest, despite long-term androgen supplementation, sperm cryopreservation should be offered to all adolescents with KS irrespective of their hormonal status, particularly those who are either considering or receiving HT. Currently, there are no established guidelines for appropriate timing or and harvesting technique choices, and only sperm cryopreservation is considered accepted standard of care. An important consideration in determining the Vol. 20 No. 2 • 2018 • Reviews in Urology • 59 4170018_02_RIU0790_V2_ptg01.indd 59 9/11/18 5:15 PM Infertility in Patients With Klinefelter Syndrome continued optimal timing of microTESE in KS patients is whether fresh or cryopreserved-thawed testicular sperm yields different outcomes with in vitro fertilization (IVF) and ICSI. Although few studies have compared ICSI outcomes between fresh and cryopreserved-thawed testicular spermatozoa from KS patients, the available studies show comparable outcomes. In one study by Friedler and colleagues, fresh testicular spermatozoa resulted in improved two pronuclear fertilization rate (66% vs 58%), embryo cleavage rate (98% vs 90%), and embryo implantation rate (33.3% vs 21.4%) over cryopreserved-thawed testicular spermatozoa; however, this difference was not statistically significant.28 These findings are consistent with those of a 2017 meta-analysis by Corona and colleagues, who showed no difference in pregnancy and live birth rates between fresh and cryopreservedthawed testicular spermatozoa using data extracted from 1248 KS patients from 37 studies, and a 2014 meta-analysis by Ohlander and coworkers, who observed no statistically significant difference between fertilization and clinical pregnancy rates using the two types of spermatozoa in men with NOA.25,29 Based on these limited studies, we believe that cryopreserved-thawed testicular sperm is a viable option for KS patients who are not actively planning for conception but wish to retain their sperm for future use. Other, more experimental approaches, such as testicular tissue or spermatogonial stem cell cryopreservation with subsequent goal of transplantation, can be offered to patients only as part of an institutional research protocol. We anticipate that pre-pubescent fertility management will gain in importance as more KS patients are detected prenatally via amniocentesis or chorionic villi biopsy. Alternative strategies may also become available to prevent the activation of spermatogonia and their subsequent apoptosis. These approaches to fertility preservation in young adolescent males are also laden with significant technical challenges and ethical controversy. Young patients may not be emotionally mature to consider future fertility issues, may not be able or willing to provide an ejaculated semen sample, and may be too afraid of any invasive surgical interventions. Specimen storage fees may also carry a significant long-term financial burden on both the patients and their parents. At this point, fertility and hormonal management should be offered to KS boys as early as 12 years of age. With proper counseling, education and multidisciplinary approach to these patients’ complex issues, their future reproductive and overall health can be successfully managed long term. Strategies to Optimize Sperm Retrieval Rates Unsuccessful sperm recovery has negative impact on patients and their partners from an emotional and a financial standpoint. Literature indicates that surgical sperm retrieval rates (SRR) in men with KS are estimated to be approximately 51%, ranging from 28% to 70% with a pregnancy and live birth rate of 50%.25 Recent surgical advances with introduction of surgical microscope and micro-TESE, optimization of ART techniques, improvements in medical management of hypogonadism, as well as more proactive early approach to management of these patients all contribute to improved SRR and ultimately fertility outcomes. The use of high-power surgical microscope (magnification of 20×-25×) and development of micro-TESE has reduced the amount of testicular tissue needed and has minimized the damage to the testicular blood supply and resulted in much higher overall SRR. Schlegel and colleagues reported a significant statistical difference in the overall SRR when comparing patients undergoing standard TESE to those undergoing micro-TESE (45% vs 63%) and further demonstrated much higher spermatozoa yield from smaller, micro-dissected samples (64,000 vs 160,000).30 Moreover, sperm retrieval rates in patients with KS have been demonstrated to be equivalent to those men who have NOA secondary to other reasons.31 Unfortunately, microTESE requires highly specialized microsurgical training and close cooperation with the reproductive endocrinologist and the ART team. As such, patients requiring micro-TESE are usually referred to high-volume, specialized infertility centers. Normal testosterone levels have been found to be an independent factor in improving SRR. Currently, the primary goal of medical management in men with hypergonadotropic hypogonadism, in addition to correcting their hypogonadism, is to improve the quantity and quality of the retrieved sperm. Antiestrogens (clomiphene citrate, tamoxifen), aromatase inhibitors (testolactone, anastrozole), and gonadotropins (recombinant FSH and hCG) have been evaluated in patients with NOA and KS. Non-steroidal antiestrogens block the feedback inhibition of estrogen on the pituitary, resulting in increased levels of LH and FSH and subsequent rise in testosterone. Clomiphene citrate has been used in severely oligozoospermic men and thus far, only one series evaluated its use in NOA patients.32 Clomiphene citrate enabled the 60 • Vol. 20 No. 2 • 2018 • Reviews in Urology 4170018_02_RIU0790_V2_ptg01.indd 60 9/11/18 5:15 PM Infertility in Patients With Klinefelter Syndrome detection of ejaculated sperm in two-thirds of men who were originally NOA and diagnosed with either maturation arrest or hypospermatogenesis on initial testicular biopsy. However, it is important to emphasize that KS men often have elevated FSH values and are not candidates for clomiphene citrate therapy. Exogenous gonadotropins decrease the endogenous gonadotropin levels and in turn “re-set” FSH and LH receptors in the Sertoli and Leydig cells, respectively, ultimately resulting in their improved function. Ramasamy and colleagues reported improved TESE outcomes in patients with NOA and KS who received gonadotropin therapy.6 Testosterone and other androgens are converted into E2 by aromatase, an enzyme present in the liver, adipose tissue, and testes. Elevated E2 levels further suppress LH and FSH secretion from the pituitary and inhibit testosterone biosynthesis. Aromatase inhibitors, at doses of 1 mg anastrozole daily, are designed to block the conversion of androgens to E2 and thus further re-establishing a testosterone and E2 (T/E) balance. Although significant improvements in sperm counts were noted in men with severe oligozoospermia, men with NOA had no such benefit. The experts argue, however, that use of non-steroidal aromatase inhibitor (anastrozole) specifically, results in improved intra-testicular testosterone levels that further improve SRR over a period of 3 months. In conclusion, use of any of the non–testosterone-based formulations may be considered in KS men planning on surgical sperm extraction. The selection of this type of therapy and the decision to start it should be made on individual basis, following appropriate patient counseling, especially because current clinical evidence for this indication is not well supported by large randomized, placebo-controlled studies. Conclusions KS results in infertility in all affected men. Early fertility preservation, although currently not standard of care, is recommended, as sperm retrieval rates have been higher in younger patients. Complex, multidisciplinary care should be provided to these patients to optimize their overall health status in addition to their ability to father children. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. Jarow J, Sigman M, Kolettis PN, et al. The optimal evaluation of the infertile male: AUA Best Practice Statement. Linthicum, MD: American Urological Association, Inc.®; 2010. Templeton A, Fraser C, Thompson B. The epidemiology of infertility in Aberdeen. BMJ. 1990;301:148-152. World Health Organization. Programme of Maternal and Child Health and Family Planning Unit. Infertility : a tabulation of available data on prevalence of primary and secondary infertility. Geneva: World Health Organization; 1991. http://www.who.int/iris/ handle/10665/59769. Schoor RA, Elhanbly S, Niederberger CS, Ross LS. The role of testicular biopsy in the modern management of male infertility. J Urol. 2002;167:197-200. Bromage SJ, Falconer DA, Lieberman BA, et al. Sperm retrieval rates in subgroups of primary azoospermic males. Eur Urol. 2007; 51:534-539; discussion 539-540. Ramasamy R, Lin K, Gosden LV, et al. High serum FSH levels in men with nonobstructive azoospermia does not affect success of microdissection testicular sperm extraction. Fertil Steril. 2009;92:590-593. Yatsenko AN, Yatsenko SA, Weedin JW, et al. Comprehensive 5-year study of cytogenetic aberrations in 668 infertile men. J Urol. 2010;183:1636-1642. O’Flynn O’Brien KL, Varghese AC, Agarwal A. The genetic causes of male factor infertility: a review. Fertil Steril. 2010;93:1-12. Kumar R. Medical management of non-obstructive azoospermia. Clinics (Sao Paulo). 2013;68(suppl 1):75-79. Foresta C, Galeazzi C, Bettella A, et al., Analysis of meiosis in intratesticular germ cells from subjects affected by classic Klinefelter’s syndrome. J Clin Endocrinol Metab. 1999;84:3807-3810. Klinefelter HF, Reifenstein EC, Albright F. Syndrome characterized by gynecomastia, aspermatogenesis without aledyigism and increased secretion of follicle-stimulating hormone. J Clin Endocrinol Metab. 1942;2:615-627. D’Aurora M, Ferlin A, Garolla A, et al. Testis transcriptome modulation in Klinefelter patients with hypospermatogenesis. Sci Rep. 2017;7:45729. United States Census Bureau. 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Main Points • Klinefelter syndrome (KS) is the most common chromosomal disorder in men and is associated with hypergonadotropic hypogonadism and infertility. • Early hormonal therapy is recommended for patients with KS to assure normal puberty and prevent long-term consequences of hypogonadism. • Cryopreservation of ejaculated samples or testicular tissue samples should be offered to all young, postpubescent KS men who are starting or considering androgen replacement therapy. • Improvements in microsurgical sperm retrieval and assisted reproductive techniques, in addition to management of patients’ hypogonadism with non–testosterone-based formulations and interventions offered in adolescence all contribute to significant improvements in sperm retrieval rates and provide maximum future fertility potential. 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Hum Reprod Update. 2017;23:265-275. Mehta A, Paduch DA. Klinefelter syndrome: an argument for early aggressive hormonal and fertility management. Fertil Steril. 2012;98:274-283. Ramasamy R, Yagan N, Schlegel PN. Structural and functional changes to the testis after conventional versus microdissection testicular sperm extraction. Urology. 2005;65:1190-1194. 28. 29. 30. 31. 32. Friedler S, Raziel A, Strassburger D, et al. Outcome of ICSI using fresh and cryopreserved-thawed testicular spermatozoa in patients with non-mosaic Klinefelter’s syndrome. Hum Reprod. 2001;16:2616-2620. Ohlander S, Hotaling J, Kirshenbaum E, et al. Impact of fresh versus cryopreserved testicular sperm upon intracytoplasmic sperm injection pregnancy outcomes in men with azoospermia due to spermatogenic dysfunction: a meta-analysis. Fertil Steril. 2014;101:344-349. Schlegel PN. Testicular sperm extraction: microdissection improves sperm yield with minimal tissue excision. Hum Reprod. 1999;14:131-135. Schlegel PN. Nonobstructive azoospermia: a revolutionary surgical approach and results. Semin Reprod Med. 2009;27:165-170. Hussein A, Ozgok Y, Ross L, Niederberger C. Clomiphene administration for cases of nonobstructive azoospermia: a multicenter study. J Androl. 2005;26: 787-791; discussion 792-793. 62 • Vol. 20 No. 2 • 2018 • Reviews in Urology 4170018_02_RIU0790_V2_ptg01.indd 62 9/11/18 5:15 PM