Skip to main content

Epigenetics, cryptorchidism, and infertility



Cryptorchid boys with defective mini-puberty and impaired differentiation of Ad spermatogonia (high infertility risk) have altered expression of several genes encoding histone methyltransferases compared to patients with intact differentiation of gonocytes into Ad spermatogonia (low infertility risk).


High infertility risk cryptorchid boys display hypogonadotropic hypogonadism, which, together with the diminished expression of histone deacetylases and increased expression of HDAC8 decrotonylase, indicates altered histone marks and, thus, a perturbed histone code. Curative GnRHa treatment induces normalization of histone methyltransferase, chromatin remodeling, and histone deacetylase gene expression. As a result, histone changes induce differentiation of Ad spermatogonia from their precursors and, thus, fertility. In this short report, we describe key functions of histone lysine methyltransferases, chromatin remodeling proteins, and long-noncoding RNAs, and discuss their potential roles in processes leading to infertility.


Our findings suggest that epigenetic mechanisms are critical to better understanding the root causes underlying male infertility related to cryptorchidism and its possible transgenerational transmission.



Chez les garçons cryptorchides qui présentent une minipuberté défectueuse et une différenciation altérée des spermatogonies Ad (risque élevé d'infertilité), l'expression de plusieurs gènes codant pour les histone méthyltransférases est altérée par rapport aux garçons présentant une différenciation intacte des gonocytes en spermatogonie Ad (faible risque d'infertilité).


Les garçons cryptorchides à risque élevé d'infertilité présentent un hypogonadisme hypogonadotrope, qui, avec la diminution de l'expression des histone désacétylases et l’augmentation de l'expression de la décrotonyase HDAC8, indiquent une altération des marques d'histones et, par conséquent, un code d'histones perturbé. Le traitement curatif par la GnRHa induit une normalisation de l'histone-méthyltransférase, du remodelage de la chromatine et l’expression du gène de l'histone-désacétylase. En conséquence, les changements d'histones induisent la différenciation des spermatogonies Ad à partir de leurs précurseurs, et donc la fertilité. Dans le court rapport qui suit, nous décrivons les fonctions clés des histones lysine méthyltransférases, des protéines de remodelage de la chromatine et des ARN longs non codants; leur rôles potentiels dans les processus menant à l'infertilité sont discutés.


Nos résultats suggèrent que les mécanismes épigénétiques sont un élément critique pour une meilleure compréhension des causes sous-jacentes de l'infertilité masculine liée à la cryptorchidie et sa transmission transgénérationnelle.


Two major goals in the field of male reproductive biology are to elucidate the molecular mechanisms that underlie cryptorchidism and to develop an effective treatment to prevent infertility. Male-specific epigenetic information involves global re-organization of and localized changes in the chromatin structure during different stages of the male germ cell differentiation [1]. Multiple studies have demonstrated that histone lysine methyltransferases regulate gene transcription, thereby influencing cell proliferation, cell differentiation, cell migration, and tissue invasion [1]. In humans, inherited alterations in nuclear maturity contributing to subfertility have been found in spermatozoids of adult males who had grandfathers diagnosed with cryptorchidism [2]. This may indicate an epigenetic mode of transmission underlying the disorder.

Using the model for artificially induced cryptorchidism, Nishio et al. found that Kdm5a (lysine demethylase 5a) expression is significantly higher in undescended testes than in descended testes. Kdm5a over-expression led to increased expression of Esr2, Neurog3, Pou5f1, Ret, and Thy1. Nishio et al. concluded that Kdm5a is likely involved in the transformation of gonocytes into spermatogonial stem cells by transcriptionally regulating specific genes via H3K4 histone modification [3].

Cryptorchidism could also be caused by prenatal exposure to external disruptors of normal embryogenesis. Any effect observed on male reproductive functions is probably due to altered epigenetic modifications following disruption of DNA methyltransferases and histone marks in the neonatal and/or adult testis [4,5,6,7].

In this short report, we present research that extends our previously published work, describe the key functions of histone lysine methyltransferases and chromatin remodeling, and summarize their role in infertility.

Patients and methods

The patients, biopsy samples, histological analyses, and RNA sequencing protocol were described in detail in the previous study [8]. The high infertility risk (HIR) group was defined by the presence of Ad spermatogonia (< 0.005 Ad spermatogonia per tubular cross section), whereas the low infertility risk (LIR) group had a normal distribution of Ad spermatogonia [8]. Here, we interpreted the gene expression patterns observed in different prepubertal testicular cell types using our own RNA profiling data, and single-cell RNA sequencing data for adult testis (Table 1) provided by the Human Protein Atlas ( [9]. We analyzed the HIR and LIR groups and the HIR group before and after GnRHa treatment. Analyzed testes were not from boys with syndromic or familial cryptorchidism.

Table 1 Gene expression profiles in testicular cells


Different methyltransferase and chromatin remodeling genes were found to be preferentially expressed in the prepubertal testis. Relevant genes were grouped into classes based on expression in Leydig and Sertoli cells (class 1), in all testicular cells (class 2), and in germ cells (class 3; Table 1).

Chromatin regulators show distinct testicular expression patterns

Class 1

KDM6A and TET1 are two demethylase genes that are mostly expressed in Leydig/Sertoli cells in HIR testes (Table 1). KDM6A plays a critical role in the differentiation of embryonic stem cells [10]. TET1 is predominately expressed in Leydig cells and their precursors and is highly methylated in the pluripotent state (Table 1) [11]. Its expression results in reduced cell proliferation [12].

Class 2

Eighteen genes encoding modifiers of histone marks were primarily expressed in all three testicular cell types (Leydig, Sertoli, and germ cells, Fig. 1). Except for the three histone deacetylase genes, all 15 genes had increased expression in the HIR group (Table 1). ARID4A and ARID5B function as transcriptional coactivators for androgen receptor and play an integral role in androgen receptor signaling pathways [13]. Methyltransferase ATRX is an ATP-dependent chromatin remodeling factor with high homology to SWI/SNF. The protein is important for genome stability, DNA damage repair, and heterochromatin formation, and functions as a transcriptional repressor [14]. The histone acetyltransferase EPC1 is a component of the NuA4 histone acetyltransferase complex and can act as both a transcriptional activator and repressor [15]. Catalytically active methyl transferase DNMT3A is particularly active during germ cell development [16].

Fig. 1
figure 1

Class 2 epigenetic factors. Genes are grouped together according to their broad activities in the indicated epigenetic control mechanisms

A putative regulatory component of the chromatin remodeling INO80D complex is involved in transcriptional regulation, DNA replication, and cell cycle control [17]. INO80D and KDM4A expression, and thus likely signaling, was enhanced in the HIR group (Table 1). Histone demethylase KDM4A participates in transcriptional repression and plays a central role in histone code modification [18]. KMT2E protein associates with chromatin regions downstream of transcriptional start sites of actively expressed genes and regulates DNA repair and apoptosis [18]. The chromatin remodeler PBRM1 acts as a negative regulator of cell proliferation, whereas the arginine methyltransferase PRMT2 is involved in histone methylation, regulation of androgen receptor signaling, and the regulation of transcription [19]. The histone lysine methyltransferase SETD7 is involved in DNA repair [20].

SMARCA genes belong to the SWI1/SNF1 family and are responsible for chromatin remodeling and DNA repair [21]. Chromatin regulators SMARCA1 and SMARCA2 act as negative regulators of chromatin remodelers by forming inactive complexes (Table 1). TSPYL4 is thought to possess chromatin and histone binding activity (see for references) and is involved in cholesterol metabolism [22].

Histone deacetylases (HDACs) catalyze the removal of acetyl groups from lysine residues in histones and other proteins, often in association with transcriptional repression. We found that testicular HDAC2 mRNA is down-regulated in the HIR group (Table 1). Expression of the decrotonylase gene HDAC8 was increased in HIR samples (Table 1). HDAC8 protein mediates decrotonylation of histones, inducing global transcriptional regression. It acts independently, without forming any co-complexes to exert this activity [23].

Class 3

Six genes had increased gene expression predominately in germ cells (spermatogonia and spermatocytes, Table 1). Chromatin remodeler ARID2 is involved in transcriptional activation and repression of its target genes by chromatin remodeling, which is defined as an alteration of the DNA-nucleosome topology ( for reference). Methyltransferase ASH1L is required for efficient expression and H3K4 methylation of HOXA10 [24]. Members of the BAZ2B gene family encode proteins that are integral components of chromatin remodeling complexes ( for reference) (Table 1). Histone transcriptional repressor SCML2 works with PRC1 and promotes RNF2-dependent ubiquitination of H2A, thereby marking somatic/progenitor genes on autosomes for repression [25]. SETD2 is a histone methyltransferase and represents the main enzyme generating H3K36me3, a specific mark associated with transcriptional activity that plays an essential role in the maintenance of a heterochromatic state by recruiting DNA methyltransferase DNMT3A [26]. TRDMT1 is an arginine methyltransferase, one of a group of enzymes that catalyze the transfer of methyl groups from S-adenosylmethionine to the arginine residues on histones and other proteins. This gene participates in DNA damage repair [27].

Molecular changes following GnRHa treatment

Genes that are involved in transcriptional repression or DNA damage repair and/or negative regulation of chromatin exhibit lower expression levels after GnRHa treatment (Table 2 and 3). We found no significant differences in gene expression between the LIR group and hormone-treated HIR group (Table 2 and 3). Importantly, a previous study found no significant differences in gene expression between their LIR group and a control group [28]. Thus, lower gene expression may be interpreted as a normalization of signaling induced by testosterone stimulation during GnRHa treatment.

Table 2 Gene expression profiles in testicular cells before and after GnRHa treatment
Table 3 Gene expression profiles following GnRHa treatment in HIR samples, compared to LIR

Long noncoding RNA and chromatin

Long non-coding RNAs (lncRNAs) are critical for modulating chromatin during development [29]. One lncRNA downregulated in the HIR group was TINCR, which produces a spliced long noncoding RNA to bind SEDT7, ARID5B, KDM5A, and LINC00222. TINCR is a key lncRNA required for somatic tissue differentiation, which occurs through lncRNA binding to differentiation mRNAs to ensure their expression [30]. Another lncRNA, HOX antisense intergenic RNA (HOTAIR), coordinates with chromatin-modifying enzymes, regulates gene silencing, and is transcriptionally induced by estradiol [31]. We found that LINC00261 stimulates the expression of HOTAIR and HOTTIP together with FOXA1 [8]. HOTTIP and HOTAIR expression was downregulated in the HIR group and positively responded to GnRHa treatment [8]. Similarly, FOXA1 had decreased RNA expression in the HIR group (-1.53 log2FC; FDR 0.006) and reacted positively to GnRHa treatment (1.15 log2FC; FDR 0.03).


Post-translational modification of histone proteins and their interpretation by specific binding proteins, the so-called histone code, represents a fundamental regulatory mechanism that has an impact on most chromatin-templated processes, with far-reaching consequences for cell fate decisions and both normal and pathological development [32]. This epigenetic phenomenon is likely altered in HIR samples. The observed increase in gene signaling in boys with HIR may be interpreted as compensation for disturbed acetylation. GnRHa treatment induced LH and testosterone secretion, which normalizes the expression of the most methyl transferases and chromatin remodeler mRNA levels (Table 1). This is different from the results obtained with experimentally induced cryptorchidism.

In artificially induced cryptorchidism, Kdm5a overexpression led to increased stimulation of five testicular development genes [3]. In contrast, in the HIR group, developmental genes NEUROG3, POU5F1, and RET (-2.1 log2FC; 0.0001 FDR) were downregulated and ESR2 and THY1 were not increased compared to the LIR group [28, 33]. In addition, no differences were found in the gene expression between the LIR group and a control group [28, 33]. Therefore, the differences between the HIR group and control group would be expected to be identical to the differences in gene expression between the LIR and HIR groups. Furthermore, we could not confirm overexpression of KDM5A.

Of particular interest is that, in contrast to artificial cryptorchidism, GnRHa treatment led to normalization of KDM5A mRNA levels from 31.7 to 20.0 RPKM (-0.66 log2FC; 0.008 FDR). Thus, transformation of gonocytes into spermatogonial stem cells in humans is not a result of overexpression of Kdm5a, but involves an intact hypothalamus-pituitary–gonadal axis [28, 33]. We have described three different expression patterns of methyltransferase, deacetylase, and acetylase proteins and chromatin remodelers in prepubertal testes. It seems that chromatin formation in testicular cells requires different sets of these enzymes in different cell types.

Estrogen effect

17-β-estradiol and its receptors are key regulators of gene transcription by binding to estrogen-responsive elements in the genome. Its receptors are important regulators of passive and active DNA demethylation [34]. Furthermore, estrogen receptor bound to estradiol recruits histone acetyl transferases (HATs), altering the balance of HATs and HDACs. The HDACs are confronted with greater amounts of acetylated histone substrates, requiring a longer time to deacetylate the acetylated histones [35, 36]. We observed increased methyltransferase gene expression and decreased deacetylase gene expression in HIR samples, indicating impaired acetylation. In pregnant women, viral infections trigger an immune response that leads to an increased concentration of 17-β-estradiol in the syncytiotrophoblast. Elevated estradiol in syncytiotrophoblasts from women who have given birth to cryptorchid boys are indicative of increased estradiol levels in the fetus [37]. Thus, hypogonadotropic hypogonadism and cryptorchidism have been hypothesized to be the result of elevated fetal estradiol levels caused by viral infection during pregnancy [37]. In a prospective study, we showed that the placentas of cryptorchid newborns had significantly higher levels of estradiol compared with control placentas of boys with bilateral descended testes [38]. Furthermore, analyzed testes were from idiopathic, non-syndromic, and non-familial cryptorchid boys who had Leydig cell atrophy, retarded tubular development implicating Sertoli cell maldevelopment, and decreased number of germ cells [38]. Testicular histology resembled that observed by Niestal et al., which was described as estrogen-induced pathological changes [39]. A negative effect of estrogen was found in the fractions of spermatozoa from infertile men with shortened ano-genital distance. This fraction is more likely to contain transposable elements harboring an estrogen receptor response element and their sperm shows substantial hypomethylation in estrogenic Alu sequences [40]. In summary, our observations are consistent with a postulated role of estrogen in modulating the expression of enzymes that modify histones, impacting the histone code.

LncRNAs have emerged as a critical layer of epigenetic regulation in which different lncRNAs are associated with distinctive epigenetic states but share a common mechanism; they physically associate with chromatin-modifying and chromatin-remodeling complexes and guide them to specific genomic loci that are crucial for proper cellular function [41]. A good example is HOTAIR, an lncRNA that coordinates with chromatin-modifying enzymes, regulates gene silencing, and is transcriptionally induced by estradiol [42]. HOTAIR expression is negatively regulated by estrogen, positively regulated by FOXA1, and inversely correlated with estrogen receptor expression [43].

Almost all estrogen receptor-chromatin interactions and gene expression changes are dependent on the presence of FOXA1. As such, FOXA1 is a major determinant of estrogen-estrogen receptor activity [43]. Fendrr regulates Foxa1 and other genes via a Polycomb-dependent epigenetic mechanism [44]. FENDRR expression increased substantially following curative GnRHa treatment (2.05 log2FC; FDR 7.21E-05).

Possible transgenerational effect of estrogen

Transgenerational epigenetic inheritance in humans has been challenged and dismissed because of difficulties ruling out the possibility that epimutation induction depends on genetic variants [45]. By generating DNA methylation-edited mice, Takahashi et al. showed that acquired methylation of CGIs can be transmitted to offspring through the parental germ line in subsequent generations of mice [45]. Moreover, they found that the CGIs with heritable DNA methylation can be demethylated in primordial germ cells. This suggests that DNA methylation memory, elicited by as yet unidentified factors, is transmitted to the next generation in mammals instead of inheritance of epigenetic information [45]. Given the commonalities in biological systems between humans and mice, Takahashi et al.’s findings may support the hypothesis that transgenerational inheritance of CGI methylation can occur in humans and, thus, could contribute to heritable susceptibility to cancer and obesity [45], as well as cryptorchidism and infertility. Reports suggest that prenatal exposure to endocrine disruptors may induce transgenerational effects on male reproductive functions, probably due to altered epigenetic modification following disruption of DNMTs and histone marks in the neonatal and/or adult testis [5, 6]. In humans, alterations of nuclear maturity able to contribute to the subfertility have been found in the spermatozoids of adult males whose grandfathers had cryptorchidism [2]. Given that histones transfer genetic material to the next generation after being transformed into protamines, abrogated histone code in HIR samples due to hypogonadotropic hypogonadism may contribute to the observed transgenerational effect in cryptorchid men. Persistent effects over several generations occur due to changes in the level of expression of master regulator genes, such as the key pluripotency gene POU5F1, which could contribute to propagating the epigenetic effects [46]. Pou5f1 could directly alter the expression of up to 400 genes, which in turn would modulate many downstream target genes, ultimately affecting the global transcriptional network [46, 47]. The observed POU5F1 master regulator gene downregulation in HIR samples may contribute to propagating epigenetic effects [28].

Can orchidopexy alone rescue fertility in high infertility risk group?

Current treatment recommendations are early orchidopexy without hormonal treatment with the expectation that successful surgery will be sufficient to protect from infertility [48,49,50]. However, early and successful orchidopexy fails to induce transformation of gonocytes into Ad spermatogonia in cryptorchid boys with HIR [51]. Failure to develop Ad spermatogonia results in infertility despite successful orchidopexy [51]. The incidence of HIR estimated with semi-thin sections of testicular biopsies ranges from 50 to 70% [51, 52]. Therefore, hormonal therapy provides a better chance of obtaining adequate sperm quality in adulthood [53,54,55].

Limitations of the study

A critical issue, especially when working with human samples, is the number of cases that are included in a given analysis. First, the number of replicates affects the statistical confidence level. Second, human tissue samples exhibit intrinsic variability that needs to be controlled for. In this exploratory RNA profiling study, we included seven patients taken sequentially from a large ongoing study based on randomized patient samples. Their inclusion in the cohorts to be treated or to remain untreated was completely unbiased by any parameter other than undescended testes, which were surgically corrected. This sample size, though small, is sufficient for an initial transcriptome study as we present here. Furthermore, the current study lacks validation experiments for RNA profiling data. However, we previously validated the transcriptome data via qPCR [28].

In conclusion, we found impaired chromatin remodeling due to diminished expression of histone deacetylase and increased expression of methyltransferase and HDAC8 decrotonylase in HIR testes. Assuming that lncRNAs can cooperate with chromatin-modifying enzymes to promote epigenetic regulation of genes, GnRHa treatment may act as a surrogate for mini-puberty by triggering the differentiation of Ad spermatogonia via lncRNA-mediated epigenetic effects. Our observations indicate that Linc00261, FENDRR, HOTAIR, and FOXA1 participate in the alternate pathway for curative GnRHa treatment to rescue impaired fertility. It is unlikely that the described epigenetic changes could be corrected by early orchidopexy. In this regard, appropriate guidance needs to be adopted for cryptorchidism treatment.

Availability of data and materials

Raw data files were deposited at the Database of Genotypes and Phenotypes (dbGaP) under accession number phs001275.v1.p1.



A dark spermatogonia


AT-rich interaction domain 2


AT-rich interaction domain 4a


AT-rich interaction domain 5b


ASH1 like histone lysine methyltransferase


ATRX chromatin remodeler


Bromodomain adjacent to zinc finger domain 2B


Claudin 3


DNA methyltransferase 3A


Enhancer of polycomb homolog 1


FOXF1 adjacent non-coding developmental regulatory RNA


Forkhead box A1


Gonadotropin releasing hormone agonist


Histone deacetylase 1


Histone deacetylase 2


Histone deacetylase 3


Histone deacetylase 8


HOX transcript antisense RNA


HOXA distal transcript antisense RNA


INO80 complex subunit D


Lysine demethylase 4A


Lysine demethylase 6A


Lysine methyltransferase 2E (inactive)


Neurogenin 3


Polybromo 1


POU class 5 homeobox 1


Protein arginine methyltransferase 2


Ret proto-oncogene


Scm polycomb group protein like 2


SET domain containing 2, histone lysine methyltransferase


SET domain containing 7, histone lysine methyltransferase


SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 1


SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 2


Tet methylcytosine dioxygenase 1


Thy-1 cell surface antigen


TINCR ubiquitin domain containing


TRNA aspartic acid methyltransferase 1


TSPY-like 4


  1. Rousseaux S, Caron C, Govin J, Lestrat C, Faure AK, Khochbin S. Establishment of male-specific epigenetic information. Gene. 2005;345(2):139–53.

    Article  CAS  PubMed  Google Scholar 

  2. Fernández Valadés R, Hortas Nieto ML, Castilla JA, López de la Torre Casares M, Valladares Mendias JC, Alaminos Mingorance M, et al. Alteraciones de la madurez nuclear en espermatozoides de varones con antecedentes de criptorquidia [Alterations of nuclear maturity in spermatozoids of males with antecedents of cryptorchidism]. Cir Pediatr. 2001;14(3):95–7.

  3. Nishio H, Hayashi Y, Moritoki Y, Kamisawa H, Mizuno K, Kojima Y, et al. Distinctive changes in histone H3K4 modification mediated via Kdm5a expression in spermatogonial stem cells of cryptorchid testes. J Urol. 2014;191(5 Suppl):1564–72.

    Article  CAS  PubMed  Google Scholar 

  4. Manfo FP, Jubendradass R, Nantia EA, Moundipa PF, Mathur PP. Adverse effects of bisphenol A on male reproductive function. Rev Environ Contam Toxicol. 2014;228:57-82.

  5. Stenz L, Escoffier J, Rahban R, Nef S, Paoloni-Giacobino A. Testicular dysgenesis syndrome and long-lasting epigenetic silencing of mouse sperm genes involved in the reproductive system after prenatal exposure to DEHP. PLoS One. 2017;12(1):e0170441.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Shi M, Whorton AE, Sekulovski N, MacLean JA, Hayashi K. Prenatal exposure to bisphenol A, E, and S induces transgenerational effects on male reproductive functions in Mice. Toxicol Sci. 2019;172(2):303–15.

    Article  CAS  PubMed  Google Scholar 

  7. Loaeza-Loaeza J, Beltran AS, Hernández-Sotelo D. DNMTs and impact of CpG content, transcription factors, consensus motifs, lncRNAs, and histone marks on DNA methylation. Genes (Basel). 2020;11(11):1336.

    Article  CAS  PubMed  Google Scholar 

  8. Hadziselimovic F, Verkauskas G, Vincel B, Stadler MB. Testicular expression of long non-coding RNAs is affected by curative GnRHa treatment of cryptorchidism. Basic Clin Androl. 2019;29:18.

    Article  PubMed  PubMed Central  Google Scholar 

  9. Regev A; Teichmann SA; Lander ES; Amit I; Benoist C; Birney E; et al. The human cell atlas. Elife. 2017. PMC: PMC5762154

  10. Tran N, Broun A, Ge K. Lysine demethylase KDM6A in differentiation, development, and cancer. Mol Cell Biol. 2020;40(20):e00341-e420.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Mellén M, Ayata P, Heintz N. 5-Hydroxymethylcytosine accumulation in postmitotic neurons results in functional demethylation of expressed genes. Proc Natl Acad Sci U S A. 2017;114(37):E7812–21.

    Article  PubMed  PubMed Central  Google Scholar 

  12. Charlton J, Jung EJ, Mattei AL, Bailly N, Liao J, Martin EJ, et al. TETs compete with DNMT3 activity in pluripotent cells at thousands of methylated somatic enhancers. Nat Genet. 2020;52(8):819–27.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Wu RC, Jiang M, Beaudet AL, Wu MY. ARID4A and ARID4B regulate male fertility, a functional link to the AR and RB pathways. Proc Natl Acad Sci U S A. 2013;110(12):4616–21.

    Article  PubMed  PubMed Central  Google Scholar 

  14. Gibbons RJ, Higgs DR. Molecular-clinical spectrum of the ATR-X syndrome. Am J Med Genet. 2000;97(3):204–12.

    Article  CAS  PubMed  Google Scholar 

  15. Tezel G, Shimono Y, Murakumo Y, Kawai K, Fukuda T, Iwahashi N, et al. Role for O-glycosylation of RFP in the interaction with enhancer of polycomb. Biochem Biophys Res Commun. 2002;290(1):409–14.

    Article  CAS  PubMed  Google Scholar 

  16. Gao L, Emperle M, Guo Y, Grimm SA, Ren W, Adam S, et al. Comprehensive structure-function characterization of DNMT3B and DNMT3A reveals distinctive de novo DNA methylation mechanisms. Nat Commun. 2020;11(1):3355.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Yoo S, Lee EJ, Thang NX, La H, Lee H, Park C, et al. INO80 is required for the cell cycle control, survival, and differentiation of mouse ESCs by transcriptional regulation. Int J Mol Sci. 2022;23(23):15402.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Young NL, Dere R. Mechanistic insights into KDM4A driven genomic instability. Biochem Soc Trans. 2021;49(1):93–105.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Bissinger EM, Heinke R, Spannhoff A, Eberlin A, Metzger E, Cura V, et al. Acyl derivatives of p-aminosulfonamides and dapsone as new inhibitors of the arginine methyltransferase hPRMT1. Bioorg Med Chem. 2011;19(12):3717–31.

    Article  CAS  PubMed  Google Scholar 

  20. Kim SH, Park J, Park JW, Hahm JY, Yoon S, Hwang IJ, et al. SET7-mediated TIP60 methylation is essential for DNA double-strand break repair. BMB Rep. 2022;55(11):541–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Chetty R, Serra S. SMARCA family of genes. J Clin Pathol. 2020;73(5):257–60.

    Article  CAS  PubMed  Google Scholar 

  22. Zhu X, Gao H, Qin S, Liu D, Cairns J, Gu Y, et al. Testis-specific Y-encoded-like protein 1 and cholesterol metabolism: regulation of CYP1B1 expression through Wnt signaling. Front Pharmacol. 2022;13:1047318.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Fontana A, Cursaro I, Carullo G, Gemma S, Butini S, Campiani G. A therapeutic perspective of HDAC8 in different diseases: an overview of selective inhibitors. Int J Mol Sci. 2022;23(17):10014.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Gregory GD, Vakoc CR, Rozovskaia T, Zheng X, Patel S, Nakamura T, et al. Mammalian ASH1L is a histone methyltransferase that occupies the transcribed region of active genes. Mol Cell Biol. 2007;27(24):8466–79.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Hasegawa K, Sin HS, Maezawa S, Broering TJ, Kartashov AV, Alavattam KG, et al. SCML2 establishes the male germline epigenome through regulation of histone H2A ubiquitination. Dev Cell. 2015;32(5):574–88.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Yoh SM, Lucas JS, Jones KA. The Iws1:Spt6:CTD complex controls cotranscriptional mRNA biosynthesis and HYPB/Setd2-mediated histone H3K36 methylation. Genes Dev. 2008;22(24):3422–34.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Sha C, Chen L, Lin L, Li T, Wei H, Yang M, et al. TRDMT1 participates in the DNA damage repair of granulosa cells in premature ovarian failure. Aging (Albany NY). 2021;13(11):15193–213.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Hadziselimovic F, Hadziselimovic NO, Demougin P, Krey G, Hoecht B, Oakeley EJ. EGR4 is a master gene responsible for fertility in cryptorchidism. Sex Dev. 2009;3(5):253–63.

    Article  CAS  PubMed  Google Scholar 

  29. Rinn JL. lncRNAs: linking RNA to chromatin. Cold Spring Harb Perspect Biol. 2014;6(8): a018614.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Kretz M, Siprashvili Z, Chu C, Webster DE, Zehnder A, Qu K, et al. Control of somatic tissue differentiation by the long non-coding RNA. Nature. 2013;493(7431):231–5.

    Article  CAS  PubMed  Google Scholar 

  31. Bhan A, Mandal SS. Estradiol-induced transcriptional regulation of long non-coding RNA. HOTAIR Methods Mol Biol. 2016;1366:395–412.

    Article  CAS  PubMed  Google Scholar 

  32. Jenuwein T, Allis CD. Translating the histone code. Science. 2001;293(5532):1074–80.

    Article  CAS  PubMed  Google Scholar 

  33. Gegenschatz-Schmid K, Verkauskas G, Demougin P, Bilius V, Dasevicius D, Stadler MB, et al. DMRTC2, PAX7, BRACHYURY/T and TERT are implicated in male germ cell development following curative hormone treatment for cryptorchidism-induced infertility. Genes (Basel). 2017;8(10):267.

    Article  PubMed  Google Scholar 

  34. Kovács T, Szabó-Meleg E, Ábrahám IM. Estradiol-induced epigenetically mediated mechanisms and regulation of gene expression. Int J Mol Sci. 2020;21(9):3177.

    Article  PubMed  PubMed Central  Google Scholar 

  35. Sun JM, Chen HY, Davie JR. Effect of estradiol on histone acetylation dynamics in human breast cancer cells. J Biol Chem. 2001;276(52):49435–42.

    Article  CAS  PubMed  Google Scholar 

  36. Pasqualini JR, Mercat P, Giambiagi N. Histone acetylation decreased by estradiol in the MCF-7 human mammary cancer cell line. Breast Cancer Res Treat. 1989;14(1):101–5.

    Article  CAS  PubMed  Google Scholar 

  37. Hadziselimovic F. Viral infections that alter estrogen levels during pregnancy may contribute to the etiology of cryptorchidism. Basic Clin Androl. 2021;31(1):16.

    Article  PubMed  PubMed Central  Google Scholar 

  38. Hadziselimović F, Geneto R, Emmons LR. Elevated placental estradiol: a possible etiological factor of human cryptorchidism. J Urol. 2000;164:1694–5.

    Article  PubMed  Google Scholar 

  39. Nistal M, Gonzalez-Peramato P, De Miguel MP. Sertoli cell dedifferentiation in human cryptorchidism and gender reassignment shows similarities between fetal environmental and adult medical treatment estrogen and antiandrogen exposure. Reprod Toxicol. 2013;42:172–9.

    Article  CAS  PubMed  Google Scholar 

  40. Stenz L, Beyens M, Gill ME, Paoloni-Giacobino A, De Geyter C. Altered DNA methylation in estrogen-responsive repetitive sequences of spermatozoa of infertile men with shortened anogenital distance. Clin Epigenetics. 2022;14(1):185.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Choo SW, Zhong Y, Sendler E, Goustin AS, Cai J, Ju D, et al. Estrogen distinctly regulates transcription and translation of lncRNAs and pseudogenes in breast cancer cells. Genomics. 2022;114(4): 110421.

    Article  CAS  PubMed  Google Scholar 

  42. Milevskiy MJ, Al-Ejeh F, Saunus JM, Northwood KS, Bailey PJ, Betts JA, et al. Long-range regulators of the lncRNA HOTAIR enhance its prognostic potential in breast cancer. Hum Mol Genet. 2016;25(15):3269–83.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Hurtado A, Holmes KA, Ross-Innes CS, Schmidt D, Carroll JS. FOXA1 is a key determinant of estrogen receptor function and endocrine response. Nat Genet. 2011;43(1):27–33.

    Article  CAS  PubMed  Google Scholar 

  44. Grote P, Herrmann BG. The long non-coding RNA Fendrr links epigenetic control mechanisms to gene regulatory networks in mammalian embryogenesis. RNA Biol. 2013;10(10):1579–85.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Takahashi Y, Morales Valencia M, Yu Y, Ouchi Y, Takahashi K, Shokhirev MN, et al. Transgenerational inheritance of acquired epigenetic signatures at CpG islands in mice. Cell. 2023;186(4):715-731.e19.

    Article  CAS  PubMed  Google Scholar 

  46. Legoff L, D’Cruz SC, Tevosian S, Primig M, Smagulova F. Transgenerational inheritance of environmentally induced epigenetic alterations during mammalian development. Cells. 2019;8(12):1559.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Hill PWS, Leitch HG, Requena CE, Sun Z, Amouroux R, Roman-Trufero M, et al. Epigenetic reprogramming enables the transition from primordial germ cell to gonocyte. Nature. 2018;555:392–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Ritzén EM, Bergh A, Bjerknes R, Christiansen P, Cortes D, Haugen SE, et al. Nordic consensus on treatment of undescended testes. Acta Paediatr. 2007;96(5):638–43.

    Article  PubMed  Google Scholar 

  49. Kolon TF, Herndon CD, Baker LA, Baskin LS, Baxter CG, Cheng EY, et al. Evaluation and treatment of cryptorchidism: AUA guideline. J Urol. 2014;192(2):337–45.

    Article  PubMed  Google Scholar 

  50. Radmayr C. Management of undescended testes: European Association of Urology/European Society for Paediatric Urology guidelines. J Pediatr Urol. 2017;13(5):550.

    Article  PubMed  Google Scholar 

  51. Hadziselimovic F, Hoecht B. Testicular histology related to fertility outcome and postpubertal hormone status in cryptorchidism. Klin Padiatr. 2008;220(5):302–7.

    Article  CAS  PubMed  Google Scholar 

  52. Bilius V, Verkauskas G, Dasevicius D, Kazlauskas V, Malcius D, Hadziselimovic F. Incidence of high infertility risk among unilateral cryptorchid boys. Urol Int. 2015;95(2):142–5.

    Article  PubMed  Google Scholar 

  53. Hadziselimovic F. Successful treatment of unilateral cryptorchid boys risking infertility with LH-RH analogue. Int Braz J Urol. 2008;34(3):319–26; discussion 327–8.

  54. Bogaert G, Vanhoyland M, Hadziselimovic F. Low-dose every-second-day LHRH treatment following bilateral orchidopexy in children with bilateral cryptorchidism may improve their fertility outcome. J Pediatr Urol. 2023;19(1):128.e1-7.

    Article  PubMed  Google Scholar 

  55. Bartoletti R, Pastore AL, Fabris FM, Di Vico T, Morganti R, Mogorovich A, et al. 16 years follow-up evaluation of immediate vs. delayed vs. combined hormonal therapy on fertility of patients with cryptorchidism: results of a longitudinal cohort study. Reprod Biol Endocrinol. 2022;20(1):102.

Download references


This study was supported in part by the European Social Fund under the Global Grant measure. Work in the Stadler group is supported by funding from the MetastasiX project of

Author information

Authors and Affiliations



FH conceived and designed the study, interpreted the data, and organized and wrote the manuscript. GV performed experiments, analyzed the data, and read the paper. MBS analyzed and interpreted the data, contributed analysis tools, and read the paper.

Corresponding author

Correspondence to Faruk Hadziselimovic.

Ethics declarations

Ethics approval and consent to participate

Investigations were carried out in accordance with the Declaration of Helsinki of 1975 (revised in 2008). The study was approved by the Institutional Review Board and the Independent Ethics Committee of Vilnius University (Vilnius Regional Biomedical Research Ethics Committee, No. 158200-580-PPI-17, 11 June 2013).

Consent for publication

Written informed consent was obtained from the patients’ guardians after approval by the ethical committee.

Competing interests

The authors declare no conflicts of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Hadziselimovic, F., Verkauskas, G. & Stadler, M.B. Epigenetics, cryptorchidism, and infertility. Basic Clin. Androl. 33, 24 (2023).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: