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. 2022 Aug 19;54(10):e14558. doi: 10.1111/and.14558

Determining the expression levels of CSF‐1 and OCT4 , CREM‐1 , and protamine in testicular biopsies of adult Klinefelter patients: Their possible correlation with spermatogenesis

Maram Abofoul‐Azab 1,2,3, Eitan Lunenfeld 4, Sandra Kleiman 5,6,7, Shimi Barda 5, Ron Hauser 5,6,7, Mahmoud Huleihel 1,2,3,✉
PMCID: PMC9786270  PMID: 36177809

Abstract

Klinefelter syndrome (KS) is the most prevalent genetic disorder of infertile males. This study aimed to determine in Klinefelter patients (KS) the expression levels of spermatogenic markers and testicular growth factors that might predict spermatogenesis based on conventional testicular sperm extraction (TESE). The expression levels of the pre‐meiotic (OCT4, CD9, GFR‐α1, α‐6‐INTEGRIN, SALL4, C‐KIT), meiotic (CREM‐1), and post‐meiotic (protamine) markers, as well as the colony stimulating factor‐1 (CSF‐1) were examined in testicular biopsies with and without mature sperm of KS and normal karyotype of azoospermic patients (AZO) with complete spermatogenesis. In the biopsies of AZO, the expression levels (fold of expression compared to the PPI of the same sample) of OCT4 were 9.68± 7.93, CREM 42.78± 28.22, CSF‐1 3.07 ± 3.19, and protamine 78498.12 ± 73214.40. Biopsies from KS included 7 with sperm and 17 without sperm. Among the biopsies with sperm, the expression levels of OCT4 were 7.27± 9.29, CREM 3.13± 7.89, CSF‐1 35.5 ± 48.01, and protamine 902.97 ± 2365.92. In 14 biopsies without sperm, we found low expression levels of OCT4, CREM and CSF‐1, and no expression of protamine. However, in three of the biopsies without sperm that highly expressed OCT4 and CSF‐1, the expression levels of CREM‐1 and protamine were high. These results may be used for further consulting with patients considering repeating conventional TESE or micro TESE and cryopreservation for possible future in‐vitro spermatogenesis.

Keywords: Klinefelter patients, male infertility, NOA patients, OCT4, spermatogenesis

1. INTRODUCTION

Klinefelter syndrome (KS) is the most prevalent genetic disorder, occurring in about 0.2% of males, 3%–4% of those who are barren (infertile), and consists of 11% of azoospermic (AZO) cases (Forti et al., 2010; Jacobs & Strong, 1959; Klinefelter et al., 1942; Morris et al., 2008; Nielsen & Wohlert, 1991). It is caused by non‐disjunction at the meiotic or mitotic stage. The karyotype of KS patients is non‐mosaic (47, XXY) in about 90% of the cases. More critical aneuploidies (48, XXYY; 48, XXXY; 49, XXXXY), in addition to the mosaicism (46, XY/47, XXY), comprise approximately 10% of cases (Kim et al., 2017; Lanfranco et al., 2004; Rajender & Oates, 2018).

The phenotypic characterizations of KS patients vary, but usually include small testes, hypogonadism, high gonadotrophins levels, relatively low testosterone (T) and AZO (Bonomi et al., 2017; Groth et al., 2013; Kamischke et al., 2003; Kim et al., 2017; Lanfranco et al., 2004). The levels of testosterone reflect the number and function of Leydig cells. Aksglaede et al., 2007 reported that around 66% of KS patients between 20 and 40 years old had normal testosterone levels.

Throughout puberty, the testes of KS patients start to deteriorate, changing to abnormal seminiferous tubules that show hyalinization and fibrosis. This process leads to Leydig cell hyperplasia and severe germ cell diminution (or depletion) in 95% of KS patients (Aksglaede et al., 2007; Gordon et al., 1972; Groth et al., 2013; Klinefelter et al., 1942; Rohayem et al., 2015; Wikström et al., 2004; Wikström & Dunkel, 2011). Nevertheless, spermatogenesis spots can be found in the seminiferous tubules of KS patients, enabling the presence of sperm in the ejaculate in around 8.4% of patients and the presence of sperm in testicular sperm extraction (TESE) in around 40% of KS patients (Corona et al., 2017; Lanfranco et al., 2004; Van Saen et al., 2018; Wikström & Dunkel, 2008). However, factors predicting successful TESE are still controversial (Corona et al., 2017). Also, arguments still exist concerning the aetiology and mechanism of impaired spermatogenesis, which includes genetic, epigenetic, and environmental testicular factors (Franik et al., 2016; Gies et al., 2016).

Spermatogenesis is a complicated process involving spermatogonial stem cells (SSCs) that proliferate and differentiate to generate mature sperm (De Rooij & Grootegoed, 1998; Fayomi & Orwig, 2018; Phillips et al., 2010; Tagelenbosch & de Rooij, 1993). The number of SSCs is very low (Phillips et al., 2010; Tagelenbosch & de Rooij, 1993). Under normal conditions, SSCs proliferate, and their progeny are highly proliferative cells that differentiate to generate meiotic and post‐meiotic cells, and thereafter by spermiogenesis, develop millions of mature sperm daily (De Rooij & Grootegoed, 1998; Fayomi & Orwig, 2018; La & Hobbs, 2019; Phillips et al., 2010; Tagelenbosch & de Rooij, 1993). This process is under the regulation of endocrine/autocrine/paracrine factors produced mainly by the Sertoli cells (SCs), other somatic cells in the testes, and other endocrine organs. Sertoli cells are considered to be the major cellular component in the seminiferous tubules that provides a suitable niche for spermatogonial stem cell (SSC) development (Meng et al., 2000). They produce crucial growth factors sustaining SSCs division and maturation. One of these factors is glial cell line‐derived neurotropic factor (GDNF), which regulates SSCs proliferation. In addition, SCs and Leydig cells produce colony‐stimulating factor‐1 (CSF‐1). SCs also produce fibroblast growth factor 2 (FGF2) and other factors that are suggested to be essential for SSCs self‐renewal. In addition, SCs secrete factors important for SSCs differentiation, such as stem cell factor (SCF) and others (De Rooij, 2009; Meng et al., 2000; Oatley et al., 2009; Oliver & Stukenborg, 2020; Takashima et al., 2015).

Spermatogonial cells express specific molecular markers such as glial cell line‐derived nerve factor family receptor alpha 1 (GFR‐α1), promyelocytic leukaemia zinc finger protein (PLZF), octamer‐binding transcription factor 4 (OCT4), cluster of differentiation 9 (CD9), α6‐integrin, b1‐integrin, G protein‐coupled receptor 125 (GPR125), c‐KIT, CD133, SSEA4, VASA, DAZL, and MAGEA4. Meiotic and post‐meiotic cells express different markers including BOULE, CREM, acrosin, protamine, and others (Conrad et al., 2008; Dym et al., 2009; Fayomi & Orwig, 2018; Hermann et al., 2010; Huleihel et al., 2015; Zohni et al., 2012).

However, knowledge about the presence of different spermatogonial cells in the testes of KS patients is still limited (Braye et al., 2019; Deebel et al., 2020; Gies et al., 2012; Goossens et al., 2013; Van Saen et al., 2012; Wikström et al., 2004). Van Saen et al., 2018 reported the detection of spermatogonia in testicular tissues of some KS patients. The estimation of their presence was based on the expression of OCT4 and MAGE‐A4 markers. Moreover, it was suggested that the spermatogenic arrest that occurs at the spermatogonium or primary spermatocyte level in KS boys, undergoes an apoptotic process at puberty instead of development (Gies et al., 2012; Wikström & Dunkel, 2008). In a study by Kleiman et al., 2016 of KS patients in which some had positive sperm retrieval and some did not, they showed that the expression levels of spermatogenic markers DAZ, RBM, BOLL, were identified in 45% of the KS patients, while CDYI, which is a post‐meiotic spermatogenic marker, was identified in only 19%. In a systematic review and meta‐analysis, Deebel et al., 2020 reported around 49% of spermatogonia were positive among adult KS patients in whom sperm was not retrieved. It is expected that in those KS patients in whom sperm was retrieved using TESE, premeiotic/miotic and post‐miotic markers will be present.

Difficulty in having biological offspring is a very important issue among KS patients and their partners, as shown by Maiburg et al., 2011. Thus, among those in whom no sperm was found in testicular sperm extraction, some will consider re‐TESE or micro TESE even without having good prognostic markers. Moreover, cryopreservation of these tissues for future in‐vitro maturation is also a dilemma in these cases. In the present study, we examined the expression levels of different markers of premeiotic spermatogonial cells and cells of meiotic and post‐meiotic stages, and the growth factor CSF‐1 in testicular biopsies with and without the sperm of KS patients, as well as several cases with normal karyotype and complete spermatogenesis, in order to detect markers that may help to predict hidden spermatogenesis.

2. MATERIALS AND METHODS

2.1. Testicular biopsies of patients

The present study was approved by the local institutional ethical committees [Tel‐Aviv Medical Center (TLV‐01‐262)]. All patients signed informed consent documentation to use their testicular biopsy for research (normal AZO were from 2006 to 2008. XXY patients from 2002–2012). Testicular biopsies were obtained from 5 AZO with normal karyotype (two patients agreed to perform analysis for AZF, and no microdeletions were found; while the others did not agree to this analysis) who had undergone testicular sperm extraction, and histology revealed complete spermatogenesis (average of ≥15 sperm cells per tubule) and from 24 non‐mosaic KS (47, XXY) men who were referred for conventional TESE due to absence of sperm in their ejaculate after centrifugation and a meticulous search of sperm cells. In seven of them, sperm cells were retrieved from testicular tissue to be used in an IVF‐ intracytoplasmic sperm injection (ICSI) program. One to three samples, depending on testicular size, were taken from each testis under general anaesthesia (Hauser et al., 1998). The age of the patients ranged from 28 to 40 years. The TESE specimens were histologically characterized and identified as described in Tables 1, 2, 3. Testicular biopsies were divided for histological evaluation and for RNA extraction (Kleiman et al., 2011; Kleiman et al., 2016). None of the patients underwent surgery or chemotherapy/radiotherapy prior to the retrieval procedure. All patients underwent karyotype analysis.

TABLE 1.

Hormone levels from AZO with complete spermatogenesis (CS)

Pt# Age (Years) FSH (IU/L) LH (IU/L) Total T (nmol/L) Histology
1 33 NA NA NA CS
2 24 11.3 3.56 14.1 CS
3 35 18.5 6.7 NA CS
4 30 4 3 17.6 CS
5 30 3.3 3 22 CS
Mean ± SD 30.4 ± _4.16 9.27 ± 7.13 4.06 ± 1.77 17.9 ± 3.96
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TABLE 2.

Hormone levels from KS patients with mature sperm on TESE

Pt# Age (years) FSH (IU/L) LH (IU/L) Total, T (nmol/L) Histology
1 32 31.0 14.0 NA Mixed of focal spermatogenesis and hyalinized tubules (MFSHT)
2 33 30.6 10.5 7.8 MFSHT
3 31 24.4 17.0 15.4 MFSHT
4 31 27.3 17.3 NA MFSHT
5 37 36.0 13.5 17.5 MFSHT
6 36 15.9 8.9 9.0 MFSHT
7 31 33.0 27.8 14.2 MFSHT
Mean ± SD 33.0± 2.5 28.3 ± 6.6 15.6 ± 6.2 12.8 ± 4.2
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TABLE 3.

Hormone levels from KS patients without mature sperm on TESE

Pt# Age FSH (mIU/ml) LH mIU/ml) Total testosterone (nmol/L) Histology
1 28 15.9 12.5 8.5 SCO
2 35 26.1 17.8 13.58 SCO
3 35 26.2 20.1 NA SCO
4 40 33.7 16.4 NA SCO
5 30 43.2 32 16.30 SCO
6 39 36 23 15 SCO
7 33 60 33.8 9.7 SCO
8 28 49.6 18.6 9.7 SCO
9 28 24 NA 13.0 SCO
10 37 14.3 14.9 18.35 SCO
11 31 40.9 26 NA SCO
12 a 34 15.9 NA NA SCO
13 35 19.3 18.2 8.3 SCO
14 31 27.4 29.9 10.82 SCO
15 31 41.5 23.7 12.9 SCO
16 28 25.6 16.50 11 SCO
17 35 48.52 27.7 13.4 SCO
Mean ± SD 32.8 ± 3.9 32.2 ± 12.6 22.8 ± 5.8 11.4± 4.1
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Note: The levels of follicle stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (T) were examined in the blood of KS patients (without sperm in their testicular biopsies) by radioimmunoassay.

a

Under hCG treatment; NA, not available; SCO, Sertoli cell only.

2.2. RNA evaluation

Evaluation of RNA for spermatogenic markers and growth factors was performed. The expression of the premeiotic markers OCT4, CD9, GFR‐α, α‐6‐INTEGRIN, SALL4, C‐KIT, the meiotic marker CREM, and the post‐meiotic marker protamine were examined. The expression levels of the growth factor CSF‐1 were also examined. The examination was performed using reverse transcriptase with poly‐dT oligonucleotides (Kleiman et al., 2016). 1.5 μl of the cDNA product was assessed by a quantitative real‐time polymerase chain reaction (qPCR), as described by Kleiman et al., 2011. Briefly, total RNA was extracted from testicular tissue samples (8–15 mg) and stored at −80°C until used. RNA was extracted from tissue using TRI Reagent (Sigma, MO, USA) except for five tissue samples that were extracted with a GeneEluteTM Mammalian Total RNA Kit (Sigma, MO). Reverse transcription (RT) was performed in 30 μl aliquots containing 0.7 μg RNA and oligo(dT)15 using a RevertAidTM First strand cDNA Synthesis Kit (Fermentas, Burlington, CA). The designed primers were tested by RT‐PCR, gel polymerase, and Real Time PCR. They were checked for genomic DNA, and the RT negative control (DDW was used) was always negative in all of the steps above. Gene expression was normalized with PPI (as a reference gene) of the same sample.

Gene amplification of the markers and growth factors was handled using the specific oligonucleotide sequences of the primers, as presented in Table S1.

3. RESULTS

3.1. Testicular findings of patient biopsies

The histology of five AZO with normal karyotypes who had undergone testicular sperm revealed complete spermatogenesis (Table 1). In seven testicular biopsies of KS men, sperm cells were detected in the cytological search, but the histological findings were similar to those without sperm cells. With few tubules with SCs and many obliterated ones, as well as hyperplasia and hyalinization of the interstitial tissue. Those with mature sperm cells were classified as mixed atrophy (Table 2). Testicular biopsies of the KS men (n = 17), who were without sperm following meticulous cytological search by biologist technicians in the andrology and IVF lab, presented concordant histological findings of Sertoli Cell Only (SCO) with severe atrophy of the tubules, extensive hyalinization, and hyperplasia in eosin (H&E) staining of testicular sections, and were classified as SCs only (Table 3).

3.2. Hormone levels of patients

The hormone levels of FSH, LH and testosterone (T) were examined in the blood of KS patients, as well as among the AZO in whom histology revealed complete spermatogenesis (Tables 1, 2, 3). The FSH and LH levels of all KS men with sperm (Table 2) and without sperm (Table 3) were highly abnormal with mean and standard deviation levels 28.2 ± 6.4, 32.2 ± 12.6, and 16.0± 6.0, 22.8 ± 5.8 mIU/ml respectively. Mean T levels were12.8± 4.2 and 11.4 ± 4.1 nmol/ml respectively. Only one KS patient without sperm was under hCG treatment (Table 3; #12). Among the AZO in whom histology revealed complete spermatogenesis, two had normal LH and FSH levels, and two had elevated FSH levels and normal LH levels with mean and standard deviation levels of FSH 9.3 ± 7.1, and LH 4.1 ± 1.8 mIU/ml. All four had normal testosterone levels with mean levels of 17.9± 4.0.

3.3. Expression of spermatogenic markers and growth factors in testicular biopsies

3.3.1. Spermatogenic markers

Testicular biopsies from AZO with normal karyotypes, in which histology revealed complete spermatogenesis, at different levels, expressed the examined premeiotic cells markers OCT4 (5/5), CD9 (5/5), GFR‐α (5/5), α‐6‐INTEGRIN (5/5), SALL4 (5/5), and C‐KIT (5/5) (Table 4). The meiotic marker CREM‐1 was expressed in all 5/5 and the post‐meiotic marker protamine was expressed only in 4/5 (Table 4).

TABLE 4.

Expression levels of specific pre‐meiotic markers is correlated with the expression levels of meiotic and post‐meiotic markers in biopsies with complete spermatogenesis.

Pt. # Markers of spermatogenic stages Growth factor
Pre‐meiotic Meiotic Post‐meiotic
OCT4 CD9 GFR‐α α‐6‐INTEGRIN SALL4 C‐KIT CREM PROTAMINE CSF‐1
1 3.627 17.862 2.343 0.363 22.144 0.317 36.856 59428.040 3.928
2 5.116 161.888 19.012 0.286 41.179 3.326 48.632 22597.196 ND
3 5.183 16.840 4.450 0.489 36.729 2.343 0.550 0 7.694
4 11.784 25 2.156 0.935 40.895 1.584 78.730 152542.972 3.614
5 22.687 16.898 2.987 2.956 69.737 0.010 49.141 157922.388 0.095
Mean 9.679 47.697 6.189 1.005 42.136 1.516 42.781 78498.12 3.832
STD 7.926 63.924 7.224 1.118 17.271 1.384 28.220 73214.40 3.105
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Most of the testicular biopsies from KS patients with sperm expressed the examined pre‐meiotic cells markers [OCT4 (6/7), CD9 (7/7), GFR‐α (7/7), α‐6‐INTEGRIN (7/7), SALL4 (7/7), and C‐KIT (7/7)] at different levels (Table 5). Only part of these biopsies expressed the meiotic marker CREM (2/7 biopsies) and the post‐meiotic marker protamine (3/7) (Table 5).

TABLE 5.

Expression levels of specific pre‐meiotic markers is correlated with the expression levels of meiotic and post‐meiotic markers in biopsies with sperm from KS patients.

Pt. # Markers of spermatogenic stages Growth factor
Pre‐meiotic Meiotic Post‐meiotic
OCT4 CD9 GFR‐α1 α‐6‐INTEGRIN SALL4 C‐KIT CREM PROTAMINE CSF‐1
1 0.0 0.2152 0.173 0.278 27.547 0.000 0 19.75 0.71393082
2 1.04 212.137 3.781 1.651 2.618 0.142 0 32.75 4.70389609
3 2.52 440.762 10.438 4.239 16.666 0.701 0 0 19.4791145
4 3.0 232.946 4.404 0.083 2.393 0.099 0.92 0 0.59207678
5 4.18 103.526 2.127 6.163 8.391 0.254 0 0 13.3971683
6 15.17 177.768 6.121 19.888 19.479 2.171 0 6268.28 97.9420298
7 25 247.941 15.712 3.280 1.369 0.670 21 0 111.728714
Mean 7.272 202.185 6.108 5.083 11.209 0.576 3.131 902.968 35.508
STD 9.298 136.124 5.328 6.882 10.176 0.754 7.886 2365.915 48.013
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Variable number of testicular biopsies from KS patients without sperm expressed the examined pre‐meiotic cells markers [OCT4 (10/17), CD9 (17/17), GFR‐α (16/17), α‐6‐INTEGRIN (17/17), SALL4 (17/17) and C‐KIT (14/17)], and at remarkably different levels (Table 6). The meiotic marker CREM‐1 was expressed only in 6/17 testicular biopsies of KS patients, and the post‐meiotic marker protamine was expressed only in 3/17 (Table 6). It should be noted that all testicular biopsies that did not express OCT4 (patients 1–7; Table 6), also did not express the meiotic and post‐meiotic markers (CREM and protamine, respectively). The highest expression levels of OCT4 were associated with high expression levels of CREM and protamine (Table 6). The highest levels of GFR‐α and α‐6‐INTEGRIN in all examined biopsies (1–17) were positively associated with the highest OCT4 levels and the meiotic and post‐meiotic markers (CREM and protamine, respectively) (Table 6).

TABLE 6.

Expression levels of specific pre‐meiotic markers is correlated with the expression levels of meiotic and post‐meiotic markers in biopsies without sperm from KS patients.

Pt. # Markers of spermatogenic stages Growth factor
Pre‐meiotic Meiotic Post‐meiotic
OCT4 CD9 GFR‐α α‐6‐INTEGRIN SALL4 C‐KIT CREM PROTAMINE CSF‐1
1 0 24.741 0.492 2.119 0.533 0.001 0 0 0
2 0 0.386 0.117 1.782 25.436 0.004 0 0 1.530
3 0 1.801 0.590 11.033 12.543 0.00 0 0 2.700
4 0 0.229 0.088 0.404 21.991 0.003 0 0 0.200
5 0 0.127 0 0.137 4.343 0 0 0 0.296
6 0 9.773 0.330 1.02E‐10 0.421 0 0 0 0.680
7 0 53.403 0.286 0.179 0.296 0 0 0 0.099
8 0.33 100.695 1.964 0.199 0.270 0.019 0 0 0.689
9 0.63 46.009 5.831 1.897 5.111 0.123 0 0 9.340
10 0.861 63.287 3.491 0.955 6.983 0.126 0.600 0 17.920
11 1.728 137.078 7.748 0.961 1.692 0.045 0 0 8.400
12 2.393 56.448 6.886 1.651 17.253 0.033 0 0 1.277
13 2.538 252.275 2.555 0.091 1.364 2.401 2.451 0 0.830
14 5.076 155.832 5.771 0.243 17.862 1.417 5.953 0 9.940
15 28.817 718.505 11.149 4.736 10.584 2.040 50 1364.216 162.45
16 38.689 581.589 8.391 3.467 3.201 0.668 21.315 19.614 86.450
17 61.132 783.536 20.518 6.886 20.096 0.475 28.126 465.893 88.270
Mean 8.364 175.630 4.482 2.161 8.822 0.432 6.379 108.807 23.004
STD 17.530 259.057 5.432 2.961 8.684 0.768 13.909 342.531 45.586
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Note: The RNA expression levels of the spermatogenic cell markers and the growth factor CSF‐1 were examined by qPCR analysis as described in material and methods section. The levels are expressed as fold of increase compare to the PPI of the same sample.

3.3.2. Growth factors

CSF‐1 showed low levels of expression in parallel to undetectable or low levels of OCT4, CREM, and protamine (patients 1–14; Table 6), and high expression levels in parallel to high expression levels of CREM and protamine (patients 15–17; Table 6).

Thus, our results show a positive correlation between the expression levels of the pre‐meiotic marker OCT4, CD9, GFR‐α, the growth factor CSF‐1, and the meiotic and post‐meiotic markers CREM and protamine respectively.

In all five patients with complete spermatogenesis (Table 7, Group A) OCT4 was detected and in 4/5, CSF‐1 was also detected. In 4/5 of patients, protamine was detected at high levels (>22,000), and in only in one patient, protamine was undetectable (Table 7, Group A).

TABLE 7.

Mean age, endocrine and paracrine/autocrine factors expression levels are in correlation with the mean expression levels of premeiotic and postmeiotic markers in biopsies with and without sperm from KS patients and from AZO with complete spermatogenesis.

N Subgroup Karyotype Sperm search Protamine (fold) OCT4 (fold) CSF‐1 (fold) Age (years) FSH (mIU/ml) LH (mIU/ml) Testosterone (nmol/ml)
5 A 46XY + 78498.12 ± 732 9.679 ± 7.92 3.070 ± 3.19 30.4 ± 4.2 9.3 ± 7.1 4.1 ± 1.8 17.9 ± 4.0
4 B1 47XXY + Undetectable 10.6 ± 12.5 48.2 ± 55.1 33.0 ± 3.5 30.8 ± 5.6 19.4 ± 7.4 15.7 ± 1/7
2 B2 47XXY + 19.7 Undetectable 0.7 32 31.0 14.0 NA
1 B3 47XXY + 3150.5 ± 4409.2 8.1 ± 10.0 4.7 ± 97.9 34.5 ± 2.1 23.3 ± 10.4 11.4 ± 4.6 8.4 ± 0.8
14 C1 47XXY − Undetectable 0.968 ± 1.49 3.85 ± 5.42 33.1 ± 3.97 30.8 ± 13.7 21.9 ± 6.99 12.3 ± 3.46
3 C2 47XXY − 616.6 ± 559.2 42.9 ± 13.5 112.4 ± 35.4 31.3 ± 3.5 38.5 ± 9.6 22.0 ± 4.6 12.4 ± 4.5
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Note: The levels of hormones and the RNA expression levels of OCT4 and CSF‐1 were examined as described in Section 2.

In 6/7 of the KS patients in whom mature sperm cells were retrieved (Table 7, Group B1, 2, 3), the expression of OCT4 was detected, and CSF‐1 was observed in 7 out of 7 patients (Table 7, Group B1, 2, 3). The expression of protamine (a post‐meiotic marker) was observed at variable extent, 3 with expression levels and 4 with undetectable levels (Table 7, Group B1, 2, 3).

In only 3 of the 17 KS patients in whom no sperm was found (Table 7, Group C2), the expression levels of OCT4 detected were relatively high (42.9 ± 13.5), and in 14, the expression was rather low (0.968 ± 1.49) (Table 7, Group C1). The expression levels of CSF‐1 were variably detected in this group of patients (Table 7, Group C1, 2). Among the 17 KS patients in whom no mature sperm was retrieved, but in whom OCT 4 was relatively high, 3 patients (Table 7, Group C2) exhibited distinct expression of protamine (616.6 ± 559.2). In the other 14 with relatively low OCT4, protamine expression was undetectable (Table 7, Group C1).

4. DISCUSSION

In the present study, we showed that some testicular biopsies without sperm from KS patients expressed markers of the premeiotic stage such as OCT4, CD9, GFR‐α, α‐6‐INTEGRIN, and the differentiating marker C‐KIT even though their pathological findings were of SCO. In some testicular biopsies without sperm, we also found the expression of the meiotic marker CREM and the post‐meiotic marker protamine. A very interesting observation was the positive association between high expression levels of OCT4, and the high expression levels of CREM and protamine, as well as with CSF‐1 in patients without sperm in their testicular tissue. Previous studies showed that the presence of OCT4 and MAGE‐4 correlates with positive germ cells in testicular biopsies of KS patients, as well as DAZ, RBM, BOLL and CDY1 (Kleiman et al., 2016). It is suggested that, at birth, KS patients have a normal number of germ cells in their testes, but the period in which germ cells diminution occurs is still under discussion. Recent studies suggest germ cells loss occurs before the age of 4, and even prenatally (Braye et al., 2019; Deebel, et al., 2020; Gies et al., 2012; Goossens et al., 2013; Van Saen et al., 2012, 2018; Wikström et al., 2004). In a study from D'Aurora et al. (2015), a KS testis transcriptome analysis showed overexpression of SOCS3, suggesting that the development of protective mechanisms against inflammatory response is likely taking place in the testis of KS patients.

According to different authors, the testicular biopsies in around 50%–60% of KS patients who undergo TESE or micro TESE will have no mature sperm that could be used in an IVF‐ICSI program (Aksglaede & Juul, 2013; Corona et al., 2017). Due to the fact that the seminiferous tubules in KS patients are not homogenous and the desire of couples to have their own biological child, many of these couples will consider re‐TESE or micro TESE rather than sperm donation. Consultation with these cases is rather difficult since there are no good predictors of mature sperm cells presence yet established.

Controversy still exists whether young age could influence sperm retrieval rates in KS patients (Ferhi et al., 2009; Plotton et al., 2015; Ramasamy et al., 2009). Deebel et al., 2020 in their meta‐analysis could not determine a correlation between age and the presence of spermatogonia. However, they could demonstrate that about 49% of postpubertal adults without mature testicular sperm had spermatogonial cells, while prepubertal boys had 83% spermatogonia cells present in their testicular tissue. Our group of patients included only adult KS patients, with no age difference between sub‐groups.

Hormone patterns were also suggested as prognostic factors for sperm retrieval in KS patients (Cissen et al., 2016). In a study by Rohayem et al., 2015, it was suggested that KS patients with LH levels below 17.5 and testosterone levels above 7.5 nmol/L have higher sperm retrieval rates. Deebel et al., 2020 in their systematic review on testicular tissue from KS, showed that those without spermatogonia had higher FSH, LH and testosterone levels than those who did have spermatogonia in their testicular tissue; however, there were no significant differences in Inhibin B levels found between biopsies with and without spermatogonia (Deebel et al., 2020). The authors themselves and others (Aksglæde et al., 2011) suggest that none of the hormone levels are a good predictor for mature sperm retrieval or for the presence of spermatogonia in the unsuccessful TESE group. Our study also showed no correlation between FSH, LH or testosterone levels to the expression of premeiotic and post‐meiotic markers.

In the present study, we looked at different premeiotic, meiotic, and post‐meiotic markers and found OCT4 and the testicular growth factor CSF‐1 as possible predictors of the presence of meiotic/post‐meiotic cells in biopsies in which mature sperm cells were not retrieved. Assessment of premeiotic markers OCT4 and CSF‐1, in conjunction with post‐meiotic protamine, might further improve the prediction of mature sperm cells beyond conventional histological analysis.

In the present study, we used only conventional TESE with multiple biopsies. It is possible to suggest a repeated sperm retrieval with the micro TESE technique, but it is still controversial whether this is advantageous (Corona et al., 2017). In a recent study by Corona et al., 2017, they were unable to demonstrate any advantage of micro TESE compared to conventional TESE. This might be due to the low testicular volume among KS patients (Corona et al., 2017). In the study by Haliloglu et al., 2014, in only 3 out of 18 KS patients (16.7%) who failed sperm retrieval using micro TESE was repeated micro TESE successful.

Since no predictors exist concerning re‐TESE or microTESE, and medical treatment has not been proven effective among KS patients, we suggest evaluating RNA expression using RT‐multiplex PCR analysis in order to better classify those KS patients in whom no sperm cells were retrieved. According to our study, if OCT4 is not expressed or showed very low expression, the chance of finding post‐meiotic germ cells is negligible; therefore, the chance of retrieving sperm in re‐TESE or micro TESE is also probably low. On the other hand, if protamine, the post‐meiotic marker or CSF‐1 growth factor is expressed at high levels, we would assume that there would be a good chance of successful sperm retrieval in a second TESE or micro TESE. This assumption, of course, must be tested in future studies.

The differentiation of spermatogonial cells in a 3D‐culture system has demonstrated by us and others (Abu Elhija et al., 2012; Huleihel et al., 2015; Khajavi et al., 2014) in murine and monkey models, as well as recently by us (Abofoul‐Azab et al., 2018) in prepubertal cancer patients. However, due to the fact that only very low numbers of spermatogonia exist in KS patients, we will probably have to proliferate them in vitro before differentiation to post‐meiotic cells. Sadri‐Ardekani et al. (2009) demonstrated their ability to increase the number of human spermatogonia in culture up to 18,450‐fold (Sadri‐Ardekani et al., 2009). Development of expanding the number of spermatogonia and further differentiating them to post‐meiotic cells might be used by ICSI to produce healthy babies from KS patients with negative mature sperm retrieval. This future option should be kept in mind and might also be discussed with KS patients with negative sperm retrieval and the presence of OCT4, a premeiotic marker.

In conclusion, we suggest that larger scale studies are needed to verify pathological results using OCT4 and CSF‐1 markers to try to increase the precision diagnosis among NOA patients in whom sperm was not retrieved. These results may be used for further consulting patients considering re‐TESE or re‐micro TESE and cryopreservation of testicular tissue for possible future use.

AUTHOR CONTRIBUTIONS

Mahmoud Huleihel and Eitan Lunenfeld made substantial contributions to the conception and design of the study. Mahmoud Huleihel, Maram Abofoul‐Azab, Sandra Kleiman and Shimi Barda designed the experiments. Maram Abofoul‐Azab, Sandra Kleiman and Shimi Barda performed the experiments and analyses. Mahmoud Huleihel, Eitan Lunenfeld and Maram Abofoul‐Azab wrote the manuscript. Mahmoud Huleihel, Eitan Lunenfeld, Maram Abofoul‐Azab, Sandra Kleiman, Shimi Barda and Ron Hauser participated in interpreting the data and their presentation, and critically revising the paper for key intellectual content. All of the authors read and approved the final manuscript.

FUNDING INFORMATION

This study was partially supported by the Natural Science Foundation of China (NSFC) – Israel Science Foundation (ISF) (NSFC‐ISF; #3425/20) (MH) and Hub Reproduction (M.H. and E.L.), Faculty of Health Sciences, Ben‐Gurion University of the Negev, Beer‐Sheva, Israel.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

Supporting information

TABLE S1: Primers and their base pairs (bps) used in qPCR analysis for the examined spermatogenic markers (pre‐meiotic, meiotic and post‐meiotic) and the growth factor (colony stimulating factor‐1; CSF‐1).

Click here for additional data file. (19.5KB, docx)

Abofoul‐Azab, M. , Lunenfeld, E. , Kleiman, S. , Barda, S. , Hauser, R. , & Huleihel, M. (2022). Determining the expression levels of CSF‐1 and OCT4 , CREM‐1 , and protamine in testicular biopsies of adult Klinefelter patients: Their possible correlation with spermatogenesis. Andrologia, 54(10), e14558. 10.1111/and.14558

Funding information Ben‐Gurion University of the Negev; Natural Science Foundation of China (NSFC) – Israel Science Foundation (ISF), Grant/Award Number: 3425/20

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

TABLE S1: Primers and their base pairs (bps) used in qPCR analysis for the examined spermatogenic markers (pre‐meiotic, meiotic and post‐meiotic) and the growth factor (colony stimulating factor‐1; CSF‐1).

Click here for additional data file. (19.5KB, docx)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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