The extra X chromosome in KS men causes germ cell degeneration and adverse effects to somatic cells, including Leydig and Sertoli cells, resulting in infertility. As a matter of fact, germ cell degeneration take place early in the fetal period, whereas the density and number of seminiferous tubules and mesenchymal tissues remain normal, without obvious pattern change (21). The hormone levels are unaffected until the onset of puberty, which is when the germ cell depletion, Sertoli cell degeneration, Leydig cell hyperplasia and seminiferous tubule hyalinization initiate and accelerate (22). Accelerated degeneration of the Sertoli cell is also evidenced by the sign of aberrant nuclear expression of the α-subunit in the Sertoli cells in older teenage KS boys (23). Based on the concept of early fertility deterioration, sperm banking at a younger age is recommended at puberty, according to their Tanner stage. The results, however, were not promising with regards to KS adolescent semen quality. Sperm in the ejaculate can only be detected in limited cases (8).
If sperm cannot be traced in the semen, using mTESE for testicular sperm retrieval associated with assisted reproductive technology such as ICSI is a recommended option with a satisfactory outcome, and offers these patients the opportunity to father biological children. As shown in several studies, age is a predictive factor for successful sperm retrieval in KS men receiving mTESE; In adult men younger than 35 years old, sperm retrieval rate (SRR) is reported to range from 30% to 77%, with a higher SRR than subjects > 35 years. Consequently, experts recommend performing mTESE under the critical age of 35 or even 30 years to yield a better outcome (18, 24). However, age as a predictive factor for sperm retrieval was not unanimously proved in other literatures (25, 26). In our study, age was not a predictive factor for sperm retrieval, and SRR was comparable for KS men under or above the age of 35 (33.3% vs 37.5%, respectively). The mean age of our study population is 36.4 years; only two KS men (age 25 and 27 years) under the age of 30 was evaluated, which will limit the ability to evaluate the influence of age on successful sperm retrieval.
Our Klinefelter cases with older age may be explained by the fact that a majority of cases coming to our center are either referral or consultation for infertility other than delayed puberty, which also implies that most of the Taiwanese KS men remained undiagnosed before marriage. According to Taiwanese government statistics, the average age of marriage for men has increased in the last decade and was 34.2 years in 2015.
gr/gr deletion, the most common form of partial AZFc deletion, has been demonstrated a correlation with male infertility in many meta-analysis studies (27, 28). However, debates rage over whether gr/gr deletion and copy number reduction of AZFc genes influence spermatogenesis. The diverse phenotypes in gr/gr deletion prevent researchers from drawing definite conclusions on this topic, which is best shown in the Japanese population with 33.9% gr/gr deletion men in the control group, and 23.9% in the infertility group, although the controls have unknown fertility status (29). Based on ethnicity and region, Caucasians (OR=3.721), people from Europe (OR=2.465), and people from South Asia (OR=2.523) with gr/gr deletion had a higher risk of male infertility in comparison to those without deletions (28). But published literatures within the same ethnicity have drawn opposite conclusions. In China, Wu et al. pointed out that b2/b3 deletion was significantly associated with idiopathic male infertility (30), while in contrast, Yang et al. recorded gr/gr deletion, instead of b2/b3 deletion, serve as a more important anomaly in determining the susceptibility to spermatogenic failure (31).
As a matter of fact, the results may vary without contradiction. Two study populations that were 1500 km apart belonged to different Y chromosome haplogroup, representing distinct paternal lineage. As demonstrated previously, Y lineages as defined by different haplotypes could influence the phenotypical expression of identical gr/gr deletion genotype (32). Gr/gr deletions and b2/b3 have been demonstrated fixed in haplogroup Q1 and haplogroup N; the prevalence of partial deletion will likely mask actual influence from other haplogroups (14, 27, 33, 34). In the Taiwanese population, Lin et al. found that 14% of men have deletions and duplications of AZFc and that the frequency of deletion vary between different Y haplogroups, ranging from 2.9% in O3e to 100% in N and Q (14). AZFc partial duplication other than partial deletion increases the risk of spermatogenic failure. Although, in our study, we did not perform quantitative PCR analysis to evaluate DAZ (Deleted in AZoospermia) dosage and duplication in our patients, with regard of the presence of AZFc, partial deletion did not seem to affect SRR.
In Danish and Chinese populations, specific Y haplogroup has been suggested to be an “at risk Y haplogroup” by their over-presentation in the spermatogenic impairment population (31, 35). Until now, no study has looked into the relationship between different Y haplogroup and SRR in azoospermic cases. Hence, it would be interesting to understand the possible influence of Y haplogroup not only on semen parameters but also on spermatogenesis in the testes.
We examined Y chromosome microdeletions in KS men using 10 sets of primers and detected 9 cases with partial AZFc deletion, including 4 men with possible gr/gr deletion, 4 with possible b2/b3 deletion, and one with sY1206 deletion KS. Our study did not detect complete AZFa, AZFb or AZFc deletion in KS men, which is compatible with the Lin et al. study, suggesting a low percentage of complete deletion pattern in Han Chinese in Taiwan (14). The incidence of AZFc partial deletion did not differ between the KS men and the fertility control men; the result is consistent with a recent study investigating AZF microdeletion in KS men (36). However, in a study of Han Chinese KS men in China, the proportion of partial AZFc deletion is higher than our population – 27 (24.3%) of 111 KS men and 11 (11.7%) of the 94 controls compared to our population of 13.6% in KS men and 6.5% in the control (36). The outcome of our study indicates that the possible gr/gr and b2/b3 deletions are not correlated with KS men in the Taiwan Han-Chinese population, suggesting that although formation of sperm with an extra copy of X chromosome by non-disjunction during meiosis I contributed to paternal error, it did not increase the chance of de novo Y chromosome partial microdeletion.
Previous studies on the preoperative hCG or aromatase inhibitor on KS men have demonstrated varying results, therefore our KS cases were not routinely given hCG or aromatase inhibitors before mTESE. (17, 24, 26). Prior experience with mTESE in KS men with non-obstructive azoospermia reported SRR range from 16%-70% and cumulative SRR per TESE cycle of 44% (37). The SRR for KS men above 35 years of age in 3 series were 25%, 30% and 50%; it was 37.5% in our study (17, 18, 24).
To our knowledge this is the first article looking into Y chromosome deletion in Klinefelter patients for SRR evaluation. Our results demonstrate that AZFc partial deletion, age, and endocrine variables do not predict sperm retrieval in KS men. Sperm retrieval rate is comparable for men below and above 35 years, and we should not discourage AZFc partial deletion men or men over the age of 35 from accepting mTESE for possible sperm retrieval.