Klinefelter syndrome (KS), the most common sex chromosomal abnormality, was first described by Harry F. Klinefelter (1) in 1942 as a clinical entity presenting with features of primary hypogonadism, and the disorder, confirmed by Jacobs (2) through cytogenetics, was demonstrated to be caused by one or more X chromosomes inherited from the paternal or maternal chromosome. The classical clinical manifestations of KS are small firm testes, eunuchoidism stature, sexual dysfunction, infertility and gynecomastia, sometimes accompanied by hyperglycemia, obesity, osteoporosis, cognitive dysfunction and psychological problems, but the phenotype can vary from the typical one with androgen deficiency to a normally virilized group (3, 4). The prevalence of the syndrome, ranging between 0.1% and 0.2% in newborn male infants, increases to approximately 3% in infertile males and to 10–12% among azoospermic patients, and the incidence tends to increase yearly according to a recent study (4, 5).

Nevertheless, due to the variability of the phenotype or to the lack of typical symptoms in prepuberty, approximately two-thirds of Klinefelter patients are not confirmed during their whole life, and the rest can sometimes be diagnosed mainly because of infertility in the reproductive domain (3). Assisted reproductive technology, especially intracytoplasmic sperm injection (ICSI) combined with microdissection testicular sperm extraction (mTESE), is the core therapy for KS patients who suffer from azoospermia to seize the hope of having their own offspring (6). Unfortunately, there are still many problems in the existing diagnosis and treatment of KS. On the one hand, the failure of generative cell function starts from puberty, but the low diagnostic rate may deprive us of the best timeline for testicular sperm extraction (TESE) and fertility preservation action (3, 7, 8). On the other hand, although the fluctuation of serum hormones such as testosterone, follicle-stimulating hormone and luteinizing hormone is helpful in evaluating spermatogenesis, there are discrepant data and opinions on whether these indexes are capable of accurately predicting the success of mTESE in KS (9, 10). From the aspect of histopathology, KS is characterized by progressive hyalinization of the seminiferous tubules of the testis and loss of spermatogonia, accompanied by the hyperplasia of Leydig cells, which is contradictory to the absence of androgen levels in adult patients with KS (11, 12). The majority of previous studies of the various cell types that exist in the testis have focused on the degeneration of germ cells and Sertoli cells in the field of KS (7, 13–15). Although the disturbed maturation of Leydig cells has attracted further attention in recent research on KS, there is still little conclusive evidence of the pathophysiological mechanism and treatment methods based on Leydig cells (16, 17). An in-depth understanding of the mechanisms of testicular cell failure and its timeline is supposed to help better inform the constant debate regarding the related factors affecting therapeutic outcome in KS and to help further understand the clinical heterogeneity in different age groups of KS.

Taken together, a comprehensive assessment of fertility in KS patients is essential for patient counseling, treatment decision-making and therapeutic benefits. In this study, we preliminarily summarized the emerging trends and the cluster of studies on Leydig cells in patients with KS using a bibliometric analysis of the scientific landscape, which is a qualitative and quantitative tool frequently applied to systematically study the characteristics of global scientific literature and to build the knowledge frame in specific fields (18, 19). The interesting results of the visual landscape include numerous randomized controlled trials (RCTs), in which recruited participants meet the specific inclusion criteria and are monitored with predefined protocols.

Therefore, a real-world study was then conducted to consistently demonstrate whether the results could be extended to an actual clinical environment. In real-world clinical settings, global multidisciplinary clinicians have been actively exploring multiple factors and seeking means to predict the testicular function of patients with KS at different ages. At present, there is a lack of specific predictive methods for the occurrence of focal spermatogenesis in the testicles of KS patients. However, in clinical practice, it has been found that the probability of focal spermatogenesis varies among KS patients at different ages. Leydig cells constitute the microenvironment of spermatogenesis, and we hypothesized that metabolic function may have potential value in these individuals’ focal spermatogenesis, and the sign of Leydig cell metabolism disorder or advanced failure in the testis among different age groups could help better understand the dynamic progression of KS.

As the most common sexual chromosome aneuploidy abnormality in men, the cause of KS has always been a complex matter. Although there have been many epidemiological studies since the discovery of KS for more than 60 years, no definitive conclusions have been reached. Currently, there are many opinions that the occurrence of KS is the result of gene mutation, but what causes the mutation? However, this is not yet known. An increasing number of studies have confirmed that changes in the Earth’s environment also affect changes in human genetic material, resulting in a large number of chromosomal aneuploidies. As a special type of sex chromosome aneuploidy, the main reason for its occurrence may have a potential relationship with changes in the Earth’s environment. For the first time in our study, we analyzed the distribution of birthplaces of KS patients. Although our number of cases is small, they come from various regions in China and exhibit different distribution patterns. In particular, the distribution of patients in Guangdong Province is also very uneven. We compared the gross domestic product (GDP) of the cities where these patients reside in 2021 (Data from http://stats.gd.gov.cn/fsjdgnsczz/content/post_3813633.html) and found that the distribution of KS patients in Guangzhou and Dongguan with higher GDP was also higher. Although we cannot trace whether the GDP of these regions was at a high level when these patients were born, from the perspective of Jieyang and Yangjiang, where the industrialization process is relatively slow, their KS distribution is also higher. At the time of the birth of these patients, these two cities were at the beginning of the industrialization process. Therefore, we speculate that there may be some inherent links between the industrialization process and the occurrence of KS. In future research, we may pay closer attention to the relationship between the occurrence of KS and industrialization.

By means of the bibliometric analysis of 105 documents included and the mapping of the scientific landscape of KS and Leydig cells, we analyzed the countries, journals and authors contributing to this domain and identified some focus topics worth continuous and in-depth exploration for our follow-up studies. From the perspective of the general trend of publications, research on KS and Leydig cells, with erratic growth in annual output, has gradually emerged among academic fields since the 1990s. In particular, the annual publication output reached its peak in the past decade, which suggested that the topic had come into the spotlight with certain research heat and depth over time. The reason for the explosion might be that thanks to the improvement in the diagnostic rate of KS, researchers have an urge to further determine the related internal pathogenesis and mechanisms of germ cell development through pathological histochemistry or burgeoning technologies such as single-cell transcriptomic analysis.

As the geographic visualization and data analysis showed, international cooperation has been primarily conducted in the countries with the largest output of relevant publications, although the regional distribution is dispersed. In the analysis of clustering, temporal and density distribution of keywords in cooperation network, we determined the distribution and internal relationship of hotspots in the field. The keywords with the highest frequency are “KS”, “Leydig cells”, “testosterone”, “inferiority”, “Sertoli cells” and “boys and men”, which indicates that the function of testicular somatic cells is the center of research in this field and that there is a highly connected interaction network between KS and Leydig cells. Whether disordered Leydig cell maturation or later pathological hyperplasia might be associated with testicular failure, ultimately leading to the general clinical characteristic of infertility in KS patients.

Combined with the temporal network clustered by average publication date, we found that the age of KS patients and serological biomarkers have gradually attracted increasing attention from scholars over time. Numerous studies have valued the variation in testosterone, luteinizing hormone (LH) and insulin-like factor 3 (INSL3, regarded as a promising biomarker of Leydig cell maturation) in KS patients of different age cohorts and aimed to use the changing pattern of these indexes as a reflection of the functional status of somatic cells in seminiferous tissue, as well as the prediction of testicular spermatogenesis degeneration (35, 36). For instance, these biomarkers might assist clinicians in choosing the appropriate opportunity for micro sperm extraction and judging the clinical outcome of hormone therapy (37). These might be the most recent topics that suggest future trends in the domain.

Overall, we used the rigorous method of bibliometric analysis to obtain the above valuable information and systematically perused the literature that has a significant impact on the research status and trends in the field. According to the influence of countries and authors, the cited situation of the articles and the frequency of the keywords in the studies within the past three decades (1992-2022), we found that the unstable annual publication output, scattered regional distribution and unsatisfactory scholar cooperation might lead to a lack of an intact theoretical framework in this field, but it has been gradually acknowledged and emphasized that there is a strong link between KS and Leydig cells, which will be one of the core topics for KS. Morphological proliferation, insufficiency of function or disorder of apoptosis regulation in Leydig cells might be tightly related to testicular function at different ages of KS and ultimately show changes in serum markers such as testosterone, LH and INSL3. Eventually, this bibliometric analysis provides suggestions and approaches for the following subgroup analysis and real world research, by which we expect to determine a reasonable and considered method for fertility optimization and preservation of KS patients.

The production and apoptosis of cells are fundamental to the cell cycle. As the basis of human reproduction, sperm also follow the principles of all things. Research shows that the natural production and apoptosis cycle of human sperm is approximately 3 months (38, 39). Bhanmeecho et al. conducted a group study of 55 dogs of different ages and found that the degree of testicular interstitial fibrosis in elderly dogs was significantly higher than that in other age groups. With increasing age, there was a significant difference in the degree of spermatogenic cell degeneration. Age was positively correlated with interstitial fibrosis and positively correlated with the degree of testicular tubular atrophy (40). Therefore, age is considered to be one of the most critical factors regulating spermatogenesis.

Research has found that aging of the gonads of men over the age of 40 can significantly reduce their fertility, including not only changes in sperm quality and sperm function but also a reduction in the ability to combine sperm and testis (41). As the center of the male reproductive system, testicular dysfunction, oxidative stress damage, apoptosis, inflammation, and decreased immune function all contribute to the cessation of spermatogenesis (42). However, there were also opposing views. Mularoni et al. found that with age, the number of Leydig cells in the testis decreased, but the peripheral serum androgen concentration did not significantly decrease (43). Research has shown that there are two different types of Leydig cells in mammalian testes: fetal Leydig cells (FLCs) and adult Leydig cells (ALCs), which appear before and after birth, respectively. Among them, FLCs contain a large number of lipid droplets, while ALCs contain a small amount of lipid droplets. When FLCs fail to degrade normally after birth and still exist in the adult testicles, it can affect the function of ALCs in late adolescence (44). Therefore, to clarify the relationship between statue of Leydig cells disorder and spermatogenesis in patients with KS at different ages might be helpful in changing the fertility of patients with KS, We suspect that the proportion of FLCs may be significantly higher than that of ALCs in KS patients with transitional steatosis of the testicular interstitium, resulting in decreased local spermatogenic function, which may also be the main reason for their low mTESE sperm acquisition rate.

Unlike previous scholars, we found significant differences in reproductive hormone indicators among patients with KS at different ages, with FSH having a peak at 26, 32, and 37 years; LH having a peak at 19, 26, 33, and 37 years; T having a peak at 19, 28, 32, and 37 years; and E₂ having a peak at 19, 20, 36, and 39 years. There were also differences in whether there was focal spermatogenesis in the testicles of KS patients at different ages, with the probability of recovering sperm through mTESE reaching a peak at the ages of 20, 28, 34, and 37. The LH of patients with sperm acquisition was significantly lower than that of patients without sperm acquisition, and the T was significantly higher than that of patients without sperm acquisition. There were also significant differences in E₂ among patients of different ages.

This phenomenon is greatly different from the view of other scholars that the younger a KS patient is, the easier it is to obtain sperm (45, 46). Our analysis may have the following reasons: (1) Our small sample size for each age group may cause bias in the results. (2) Our research population was mainly older adults, not adolescents, so the population composition may have some interference with the results. (3) Although we may have the first two types of interference, from the perspective of the probability of mTESE sperm recovery in men of childbearing age, at least the viewpoint of scholars in the early and middle stages of childbearing age is not applicable, which also suggests that the spermatogenesis of men of childbearing age does not develop according to the rule of age, and there may be other regulatory factors in the spermatogenesis of patients of KS childbearing age, and such factors can be interfered with. This may also be a breakthrough in solving the bottleneck in the treatment of KS spermatogenesis and provide some inspiration for our further research.

In the past, the histopathological Johnson score has been considered the gold standard for evaluating testicular spermatogenesis (48). However, with the development of microscopic testicular sperm extraction (mTESE), an increasing number of clinical practices have confirmed the presence of focal spermatogenesis in the testicles of patients with nonobstructive azoospermia (49, 50). In previous studies related to mTESE, we have also found that the sperm recovery rate of patients with nonobstructive azoospermia undergoing mTESE surgery can reach 38.7% (51). KS is a typical nonobstructive azoospermia with congenital chromosomal aneuploidy defects, but spermatogenesis may still exist in its testicles (52, 53). Similar to studies by other scholars, we also found focal spermatogenesis in infertile patients with KS, with a mTESE sperm recovery rate of 36.0%. Although this number is lower than that reported by other scholars, this is accurate data, which may be related to the patient’s sample size, age, and previous treatment history. The sample size of this study is small, the age is concentrated in the reproductive age period, and all patients have not received any treatment three months before surgery, or a 3-month washout period is ensured after receiving treatment. Therefore, the mTESE sperm recovery rate in this study will be low.

On the other hand, the Johnson score of testicular tissue in all patients with KS in this study remained between 1-2 points, while only one of 75 patients had a score of 4. According to the Johnson scoring standard, the lower the score, the poorer the development of testicular seminiferous tubules and the lower the rate of spermatogenesis. Therefore, this may also be one of the main factors that caused the sperm acquisition rate in this study to be lower than that of other scholars. Our comparison of Johnson scores between patients with and without sperm also confirms this phenomenon, and the Johnson scores of patients with sperm are significantly higher than those of patients without sperm.

However, from a single case of histopathological Johnson score, we cannot obtain a valuable predictive conclusion on the presence or absence of focal spermatogenesis from a patient’s Johnson score. Therefore, we believe that conducting testicular biopsy in patients with KS before mTESE is meaningless, as it can lead to damage to the testicular microenvironment and is not conducive to the development of focal spermatogenesis.

The hypothalamus-pituitary-testicular gonadal axis is considered to be the center for regulating spermatogenesis (54, 55). Our previous studies have also found that the concentration of reproductive hormones in the testicular environment also plays a key role in regulating spermatogenesis (51). Therefore, reproductive hormones should have a crucial role in spermatogenesis in patients with KS. Due to the presence of X-chromosome aneuploidy, patients with KS are clinically characterized by high levels of follicle stimulating hormone and luteinizing hormone, accompanied by low or normal testosterone. Their follicle-stimulating hormone and luteinizing hormone levels can exceed the normal range by 2-3 times or even higher (56). It has been reported that lowering FSH and LH can promote spermatogenesis in patients with nonobstructive azoospermia (57), but the effect on KS patients is unclear. Therefore, exploring the rules of reproductive hormones in patients with KS has a guiding role in regulating spermatogenesis.

Previous studies have found that Leydig cells appear and can secrete androgens at the 8th week of a male embryo. At the 9th to 14th week of the embryo, Leydig cells turn into differentiated cells. At the 15th to 18th week of the embryo, Leydig cells mature and have a strong androgen secretion ability, forming a peak of androgen secretion during the embryonic period. Thereafter, they gradually degenerate until puberty before increasing and increasing the secretion of androgens again (58–60). Currently, it is believed that there are a large number of LH receptors on the surface of Leydig cells, which, after binding with LH, initiate the transformation process of neutral fatty acids and cholesterol lipids in Leydig cells, promote the conversion of cholesterol into testosterone, keep the epithelium of spermatogenic tubules close to the lumen in a long-term high concentration of testosterone, promote the differentiation of late spermatocytes, and complete the spermatogenesis process (61, 62). There may be a local “short loop” regulatory pattern between the functions of mesenchymal cells and Sertoli cells, and estradiol secreted by Sertoli cells may interfere with the expression of receptors on the surface of mesenchymal cells (63, 64). Therefore, analyzing the function of Leydig cells is crucial for promoting spermatogenesis.

In the subgroup analysis following the bibliometric analysis, we extracted the indicators testosterone, LH, and INSL3 related to Leydig cells and found that the levels of these hormone or hormone-like indicators in patients with KS fluctuated with age, especially when the levels of testosterone, LH, and INSL3 in men with KS were observed to be different at various age groups, and the peaks also occurred at different ages. The change trend of serum testosterone and LH is generally similar, reaching a peak at the ages of 13-19 and 30-45, respectively. Comparatively, INSL3 is relatively stable, peaking only at the age of 13-19, and the peaks of testosterone and INSL3 significantly lag behind those of LH. Correspondingly, in our cross-sectional study of men of childbearing age, we found that the peak ages of testosterone and LH were also similarly reached at the ages of 19, 26-28, 32-33, and 37. The age cohort with higher success rates in sperm extraction was also concentrated at 20 and 37 years old. In the same age group, lower LH and higher testosterone may be associated with positive spermatogenic function. This indicates that the fluctuations of hormones in patients with KS at childbearing age follow a certain rule, which might be related to the development status of testicular spermatogenic tissue, the maturity of somatic cells adjacent to germ cells, and sensitivity to the hormone-stimulating effect at different ages.

Previous studies (9, 32, 35) have shown that during childhood and even early puberty of KS, pituitary-gonadal function is normal, and testicular biopsies of prepubertal KS boys show that the number of germ cells is reduced, but the seminiferous tubules are preserved and Sertoli cells and Leydig cells still work regularly, which indicates that hormone synthesis and secretion may be normal. From the middle of puberty, the seminiferous tubules of patients have rapidly accelerated degradation, with varying degrees of fibrosis, transparency, and interstitial proliferation, as well as maturation disorders and functional deficiencies of Sertoli cells and Leydig cells. However, in this study, LH, FSH, and other hormones in patients with KS still fluctuated during childbearing age and did not directly increase to high levels of gonadotropins, while testosterone levels dropped to low or subnormal levels after an initial increase and were still able to reach a new peak. The reason for these phenomena may be related to the residual spermatogenic tissue in the testis, and it might also be because the stimulation response of LH-testosterone may still exist at a specific childbearing age and stimulates the spermatogenesis of the remaining spermatogenic tissue, which shows that KS at childbearing age still has the potential to produce sperm. In other words, not all somatic cells decline irreversibly with age, and the remaining cells may have the potential to perform compensatory functions, but this needs to be verified in further research. Thus, we believe that the evolution of the morphology and function of somatic cells such as Leydig cells in the testicles of KS of childbearing age and the generated hormone fluctuations are of great significance for research related to the pathogenesis and progression of KS, which also provides a novel explanation for the controversial topic of the relationship between testicular spermatogenic function and hormones.

In our study, we found that LH in sperm recipients was significantly lower than that in nonsperm recipients, and T was significantly higher than that in nonsperm recipients. There were also significant differences in E₂ among patients of different ages. The differences among the three have once again confirmed the relationship between spermatogenesis and the LH——T——E₂ regulatory axis. We believe that LH activation of testicular Leydig cells to secrete sufficient T is the basis for stimulating spermatogenesis, but the balance of transformation between T and E₂ is also a necessary condition for ensuring spermatogenesis, and none of the three is indispensable. We have also confirmed through immunofluorescence single and double labeling that the proportion of androgen receptors and INSL3 receptors is relatively coordinated in the testicular tissue around the seminiferous tubules with normal spermatogenesis, while in the testicular tissue around the seminiferous tubules without spermatogenesis, there is a serious imbalance in the proportion of androgen receptors and INSL3 receptors. This phenomenon is the first time that it has been discovered. Unlike previous studies, we found that androgen receptors and INSL3 receptors are more intensively distributed in the wall and inside the seminiferous tubules. These two receptors are jointly expressed on the surface of supporting cells. In local areas of transitional proliferation of interstitial tissue, there is a high concentration of androgen receptors, while INSL3 receptors are not widely distributed. These areas are also the areas where interstitial tissue degeneration is most severe, and there is even the possibility of mesenchymal neoplasia. Therefore, we believe that the ratio of INSL3 combined with T may be more suitable as an accurate indicator for predicting spermatogenesis in patients with KS.

There are significant differences in LH, T, and E₂ levels between KS patients with focal spermatogenesis at different ages and those without spermatogenesis. The age points of KS patients with easy access to sperm are 20, 28, 34, and 37 years old. The INSL3 receptor and androgen receptor are centrally expressed in the walls and inside the spermatogenic tubules, and a balanced of these metabolites is an important basis for ensuring spermatogenesis. Abnormal metabolism of Leydig cells led to imbalanced expression of INSL3 and androgen receptors, which might be a potential target for spermatogenesis in KS.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

The studies involving humans were approved by Ethics Committee of Guangdong Provincial Reproductive Science Institute (Guangdong Provincial Fertility Hospital) (Approval No.2021(08)). The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants’ legal guardians/next of kin.

HuL: Funding acquisition, Methodology, Project administration, Writing – original draft, Writing – review & editing. ZHZ: Data curation, Formal Analysis, Investigation, Methodology, Software, Writing – original draft. YG: Conceptualization, Investigation, Resources, Validation, Writing – original draft. HaL: Data curation, Formal Analysis, Investigation, Methodology, Resources, Writing – original draft. ZYZ: Conceptualization, Investigation, Methodology, Writing – original draft. HZ: Conceptualization, Investigation, Methodology, Writing – original draft. WY: Writing – original draft. ZL: Conceptualization, Investigation, Methodology, Writing – original draft. ZQ: Conceptualization, Investigation, Methodology, Writing – original draft. QX: Conceptualization, Investigation, Methodology, Writing – original draft. LH: Investigation, Writing – review & editing. YZ: Methodology, Writing – review & editing. XZ: Methodology, Writing – review & editing.