At what age should we attempt to retrieve sperm from males with Klinefelter syndromeShanta Shepherd and Robert Oates
Department of Urology, Boston University School of Medicine, Boston, MA, USA;
Department of Urology, Boston Medical Center, Boston, MA, USA
Correspondence to: Robert Oates, MD. Professor of Urology, Boston University School of Medicine, Boston, MA, USA; Vice-Chair, Department of Urology, Boston Medical Center, 725 Albany Street, Shapiro 3B, Boston, MA, USA.
Although there are numerous aspects of Klinefelter’s Syndrome worthy of discussion, this contribution will focus specifically on the controversial, and as yet unresolved, issue of whether it is advantageous to harvest testis tissue from peri-pubertal or adolescent boys with Klinefelter’s Syndrome in a heroic effort to preserve that child’s chances of reproduction in his future adult life. What would be the rationale for that, how does the biology of spermatogenesis in the Klinefelter testis impact that decision, and what does the data show? The answer, assembled from a selection of seemingly disparate sources and directions, appears to be “No”. We do not have to advocate for an aggressive approach, we do not have to pre-emptively preserve future fertility. We can justifiably wait until adulthood with equivalent chances of success.
Klinefelter’s occurs in approximately 1 in 600 males. It is not identified in all, however, as many men proceed along in life undiagnosed. The genotype is characteristically 47,XXY, the additional X chromosome arising from either a mitotic (early embryo) or maternal/paternal germ cell meiotic mishap. Diagnosis may be made as a fetus based on results of amniocentesis, chorionic villous sampling or maternal cell-free DNA testing. There are no tell-tale signs in infants, other than perhaps cryptorchidism, but occasionally a karyotype done for other reasons may reveal a 47,XXY aneuploidy. Learning difficulties in children may trigger a paediatrician’s thought of KS as a possible etiology; while very small testis size, taller than expected height, or failure to virilise (an uncommon presentation) may also ignite a genetic diagnostic search. Finally, a 47,XXY chromosomal constitution may be discovered at the time of evaluation of male infertility and non-obstructive azoospermia or severe oligospermia. While there is no singular and consistent presentation, what is common to all KS males are very small testes with compromises in both functions of the testis—sperm and androgen production.
What is the natural history of the KS testis—why is it so exceedingly small? It is believed that in the first year of life, there may be a gradual decline in the number of 47,XXY spermatogonia but not nearly enough to explain fully the diminutive adult KS testis volume (5cc or so). Johannsen et al. demonstrated that the mini-puberty surge in gonadotropins, and the consequent rise in testosterone, was normal in 13 infants with KS as compared to control male infants. It is during the pubertal years, however, when the events that will determine the ultimate size (and function) of the KS testis begin to play out. The vast majority of 47,XXY spermatogonia which populate the vast majority of the seminiferous tubules in each testis cannot complete the meiotic process and become apoptotic [normal X-Y pairing is mechanistically reviewed in the mouse model by Kauppi et al ]. Whether this is specific to the germ cells themselves (sex chromosomal trisomy), whether it is malfunction and failure of the Sertoli cells in their nutritive and supporting role (overexpression and resultant functional “toxicity” of X-linked, testis-expressed genes), or a combination of both is not fully understood. Tubules populated by these apoptotic germ cells wither and become largely fibrotic. A few remain with surviving 47,XXY spermatogonia but are devoid of complete spermatogenesis. On average, then, each tubule is either shrivelled with few remaining germ and Sertoli cells or shrunken and devoid of all cells—each then possessing a girth only a fraction of normal. The totality of all of those minute tubules and/or remnants leads to an overall testis mass far from the typical, common length and width. So, where do the spermatozoa come from that are found upon TESE in approximately 50% of KS cases—an insightful question indeed.
In a reverse twist of genetic fate, a spermatogonial germ cell, during a mitotic replication, might randomly and sporadically lose the supernumerary X chromosome and revert to a 46,XY spermatogonial germ cell with all of the proper machinery for normal mitosis, meiosis and spermiogenesis and with none of the Xtra baggage that was constraining to those processes. It is these euploid cells that are thought to be the progenitors of the haploid spermatozoa unearthed during TESE. They initially and comfortably reside on the internal side of a lucky seminiferous tubule basement membrane as per usual, surrounded and enveloped by the nurturing Sertoli cells until they are roused from their pre-pubertal sleep and the spermatogenic sequence is initiated, the spawn of which are capable and competent haploid 23,X or 23,Y spermatozoa. This most likely of scenarios also explains why the reported babies born have been 46,XY or 46,XX, confirming studies demonstrating that the preponderance of spermatozoa subjected to chromosomal analysis are indeed haploid. If 47,XXY spermatogonia cannot complete the meiotic cascade [although some argue they might rarely be able to ] and, therefore, will not be the ancestors of any sperm found in the testis at any age (adolescent to adult), is there any reason to “cryopreserve” them by performing TESE at an early age, or at least well before the KS male is ready to have a child? At the present moment, there is no in-vitro technology or culture system that can drive the expulsion of that extra X chromosome from all or even a fraction of the 47,XXY spermatogonia and then, in addition, assist them in manufacturing functional, genetically safe haploid gametes. The answer to the query of whether we should be aggressive and surgically extract testis tissue from the pre-pubertal, peri-pubertal or adolescent Klinefelter testis in order to freeze all existing 47,XXY early germ cells in that morsel of tissue would appear to be a resounding “No”. Indeed, in the review by Gies et al., they prudently offer this piece of advice, “banking testicular tissue from pre-pubertal KS boys should only be performed in a research framework”. But will this still be true tomorrow, or next year, or in a decade or two. Makar and Sasaki review the present science and state-of-the-art technology being used to study in vitro gametogenesis. Will their hopeful conclusion, “In vitro gametogenesis will constitute an emergent new field in human reproductive medicine in the near future” be applicable for the 47,XXY Klinefelter male—perhaps one day we will be able to generate haploid spermatozoa from 47,XXY cells.
If we are not going to freeze pre-adult 47,XXY spermatogonia for future use to insure fertility because of the limitations of our present technology, a new question arises. Should we be offering, even advising, that TESE with cryopreservation of any spermatozoa found be carried out in the adolescent or young adult male prior to the time at which the individual is trying to conceive with a partner. There are two intersecting lines of thought that generate this question. The first depends on Leydig cell testosterone production and what level is seen typically and what is the “lifespan” of that level. That is, do all KS young males require or benefit from testosterone supplementation and, whatever the level is at puberty, even if adequate, does it remain so? Will it drop precipitously through the teenage and early adult years such that all KS males will ultimately need testosterone supplementation or replacement and TESE might as well be carried out sooner than later since exogenous testosterone may/will decrease spermatogenesis—a result unwelcome if there is little sperm production capacity to begin with? The second driver behind early TESE is the thought that spermatozoal production is quantitatively maximal just after puberty and falls dramatically in the ensuing years. If this were true, then any delay in TESE would compromise future biologic paternity. That is, not intervening immediately and aggressively upon diagnosis would be an opportunity missed to preserve future chances of genetically-linked fatherhood. What a tragedy that would be—but does this supposed decline actually occur? Let’s look at both of these arguments for early TESE and see if they are supported by the evidence.
This question is an important one because if testosterone levels did indeed decay steadily through the first few years following puberty to symptomatically low levels, an argument could be made to perform TESE before this actually happens. If it did, then is that a signal that spermatogenic capability is also deteriorating inexorably and simultaneously? The evidence suggests not. In 1985, Salbenblatt looked at the testosterone levels of 40 individuals with KS and noted that all entered puberty spontaneously between the ages of 11-14, all developed normal secondary sexual characteristics and that testosterone levels rose to the low-middle of the “normal range” and plateaued there into adulthood. Wikström et al. followed 14 Finnish 47,XXY boys and concluded that both onset and progression of puberty did not deviate from the pattern seen in 46,XY control boys, that there was no difference in skeletal maturation between 47,XXY and 46,XY boys, testosterone levels of the KS boys fell within the normal range, and that SHBG and PSA levels were similar to the control boys. They state that, “we found no phenotypic evidence for androgen deficiency in boys with KS during early and mid-puberty”. Finally, they conclude as well, “no indisputable androgen deficiency appeared in KS boys, and thus they would require no androgen supplementation during early puberty”. Aksglaede et al. reported on 166 boys, adolescents and adults with non-mosaic KS and found the same pattern: a rise at the onset of puberty to low-normal testosterone levels and a levelling off at those values such that there was no obvious and predictable diminution in testosterone output from the span of adolescence to adulthood, “the serum concentration is most often in the lower half of the reference range of healthy males, and rarely below the reference range”.
Clearly the value of testosterone is adequate in these boys/men, but are there other reasons that an individual may benefit from higher levels? The evidence is not conclusive but is ever evolving. One area that has received attention recently is bone health—is bone health optimized by supplementation or full replacement testosterone therapy in the adolescent and adult male with KS? If so, it may be a reason to begin treatment and, again, provide a push for TESE prior to the institution of that therapy. However, the data does not support this contention. Stagi et al., in their study involving 40 KS children and adolescents, as well as 80 age-matched healthy subjects, noted that the KS patients had impaired bone mineral status, higher PTH levels, and reduced 25-OH-D and bone formation markers. What is of interest here is that these impairments were already discernible in pre-pubertal KS boys. In terms of bone health in the KS boy, it may not be all about testosterone. As Tahani et al. state after treating 15 KS men with testosterone, “In untreated hypogonadal men with KS, lumbar and femoral BMD (bone mineral density) was reduced, and femoral bone quality was impaired… However, TRT (testosterone replacement therapy) failed to remedy these negative effects on bone”. Their conclusion was in agreement with that of Shanbhogue et al. who stated that while the indices of bone structure, bone strength, and bone biomarkers that they measured were compromised in the adult KS patients as compared to a control group, there was no significant difference in these indices in the 21 KS patients on long-term testosterone therapy compared to the 11 KS men not on long-term testosterone therapy. So at this time, it is not at all clear that the magic solution to any reduction in bone parameters that may be found in a KS man is testosterone replacement. It certainly is more convoluted than that simplistic linear relationship, perhaps involving other less well-known and studied proteins such as INSL3 and Sclerostin.
Are there other compelling reasons to prescribe testosterone replacement that would oblige a preemptive TESE? Increased height is found in many, but not all KS boys, and was thought to be a reflection of a hypogonadal state and, if indeed it were, would be an indicator of the need for testosterone therapy. However, increased stature in these boys is already recognized well before puberty begins when testosterone levels are minimal. This is most likely due to an increased dosage effect of the short stature homeobox-containing gene (SHOX) which is located on the pseudoautosomal region of both the X and Y chromosome. In the KS boy, 3 copies exist, not the normal 2 and increased linear growth may be the result. Therefore, when a KS boy is taller than his peers, it does not necessarily signal a hypogonadal state as may occur in a teenager with idiopathic hypogonadotropic hypogonadism.
What about the metabolic syndrome, in general, or specific conditions such as diabetes mellitus type 2 (T2DM) which is found in approximately 13% of Klinefelter men. Is T2DM reversible or mitigated with testosterone therapy, even when started as an infant? Probably not as O’Connor et al. point out, “observational data suggest that testosterone replacement is not associated with lower rates of diabetes or improved glycemic control”. Høst et al. are in agreement that while testosterone replacement may not improve glycemic control, it may help decrease total body and abdominal fat (indices of body composition). There are also ongoing debates about whether an improvement in cognitive phenotype and brain development in KS infants/boys/teenagers/adults can be realized with timely testosterone treatment. The data is unclear. So for now, there is little consensus in regards to absolute indications for tactical testosterone therapy and as Gravholt et al. conclude in their comprehensive review of the genetics, neuropsychology and endocrinology in KS, “Although hypogonadism is among the classic characteristics of KS, the effects of testosterone replacement therapy are not well studied, and many questions concerning timing, dose, and route of administration remain to be answered”. Finally, as this clinical conundrum continues to be clarified through prospective and focused research, we may be seeing an off-road that would be a compromise, in a way, between the polar ends of the spectrum: no testosterone therapy of any type at all before TESE versus unabashed testosterone treatment regardless of the timing of present or future TESE. This path may be illuminated by consideration of the type of testosterone therapy prescribed and the level of hypothalamic-pituitary suppression it causes. That is, as Garolla et al. document, the sperm retrieval rate (SRR) during TESE was similar (34%) in those men on testosterone replacement therapy (TRT) and those men not on TRT, but only if there was minimal, if any, reduction in pituitary gonadotropin output, especially FSH. This is in line with data in Plotton et al. and Mehta et al. that also suggests that topical testosterone therapy may not be as suppressive as would be assumed. Perhaps, there is some middle ground.
However, we must realize that in terms of the entire scope of phenotypic variation in the 47,XXY KS individual seen as compared to the 46,XY male, as discussed above, it is much more complex than just testosterone levels and androgen replacement. Quoting from Skakkebæk et al., “Characterization of the methylome as well as the transcriptome of both coding and non-coding genes identified a unique epigenetic and genetic landscape of both autosomal chromosomes as well as the X chromosome in KS. A subset of genes show significant correlation between methylation values and expression values”. Panula et al. echoed and elaborated on those thoughts by looking at human induced pluripotent stem cells from two KS males and showed that the pattern of X chromosomal inactivation of the second X in the KS males was similar in many ways to the pattern of X chromosome inactivation of the second X in female pluripotent stem cells. In addition, they demonstrated that the differentially expressed genes between the 47,XXY KS men and two 46,XY healthy males showed “enrichment in gene ontology terms associated with fertility, cardiovascular development, ossification, and brain development, all associated with KS genotype-related clinical features”. The genesis of many of the phenotypic variations known to occur in the 47,XXY male most probably has a rich and multifaceted genetic/epigenetic basis, one not influenced nor ameliorated by testosterone or androgen replacement, respectively. In summary, there is little compelling and persuasive evidence to support a global approach to the KS adolescent in which all are automatically prescribed TRT. However, individualized treatment plans and strategies are always reasonable.
The anxiety surrounding the concept of proactive TESE in the adolescent (and even adult) 47,XXY Klinefelter male seems unwarranted for both clinicians, patients and their families. There is no compelling evidence that a 16-year-old KS patient will quickly and unavoidably lose some presumed high level of sperm production over the next several years such that if TESE is not strongly recommended, forever lost will be his chance of biological and genetic fatherhood. Indeed, the same holds true for the adult male with KS. As Oates has opined, the same individual human Klinefelter male who has sperm present in the testis at age 16 will be the same individual human Klinefelter male who will have sperm present at age 30. Conversely, if no sperm are present at 16, no sperm will be found at age 30. Where is the rush? In addition, there seems to be little data suggestive of an absolute need for testosterone replacement in all KS boys that would complicate the decision of whether a TESE should be performed or not prior to the institution of that therapy. Of course, there will indeed be a small population of KS boys who are not virilizing at all (the extreme end of the endocrinological spectrum) and will benefit from TRT. However, there is an increased awareness of the phenotypic issues common to the KS male and newer research avenues are being explored and the results disseminated. For now, we should have a more relaxed position vis-à-vis the timing of TESE.