Stesura Seveso Archivio Italiano di Urologia e Andrologia 2023; 95(3):11462 1 ORIGINAL PAPER INTRODUCTION Non-obstructive azoospermia (NOA) is one of the most important causes of male infertility. It characterized by the absence of sperm in semen repeatedly (1). At present, its pathogenesis is complex and can be categorized into primary and secondary testicular failure (1). About 10-15% of people of childbearing age are infertile in the world, of which male infertility accounts for about 50% (2). NOA is a type of male infertility caused by spermato- genic dysfunction of testicular tissue. Patients with NOA cannot produce sperm or can only produce a very small amount of sperm. In patients with NOA, the structure of the seminiferous tubules in the testis is disordered, while the maturation of spermatogenic cells is blocked, and the meiosis of spermatogenic cells is arrested (3). Galectins are a family of soluble carbohydrate-binding proteins that reg- ulate cell phenotype and function in development and dis- ease (4). Galectin-1 (Gal-1) and Galectin-3 (Gal-3) are expressed by many immune cells and receive considerable attention in the context of immunity (5, 6). Gal-1 was the first member of the lectin family, reported more than 3 decades before, as a +15 kDa protein existing in a non- covalent homodimer form that was previously known as electrolectin, b-galactosidebinding lectin, galaptin or L-14 (4). Different organs and tissues secrete it including thy- mus (7), spleen (8), smooth muscle (9), colon (10), ovary (11) and also the nervous system (12). It is an endogenous Introduction: Galectin-1 (Gal-1) and galectin-3 (Gal-3) are expressed by many immune cells and receive considerable attention in the context of immunity. We aimed to compare between seminal plasma and serum levels of Gal-1 and Gal-3 in azoospermic patients and fertile men. Materials and methods: This cross-sectional study was conduct- ed at the andrology outpatient clinic from January (2022) to September (2022). A total of 90 participants were enrolled and divided into two equal groups: azoospermic and normal group. Semen analysis was done for all participants. Hormonal profile including FSH, LH, serum prolactin, total testosterone and estradiol was performed as well as assessment of serum and seminal levels of Gal-1 and Gal-3 by ELISA commercial kits. Finally, scrotal Duplex was done in standing and supine position. Results: Serum and seminal levels of Gal-1 and Gal-3 were sta- tistically significant higher in azoospermic patients compared with normal individuals (p < 0.001 for all). In addition, in healthy individuals there were statistically significant positive correlations between serum levels of Gal-1 and age, FSH, LH levels (r = 0.296, p = 0.005; r = 0.333, p = < 0.001; r = 0.312, p = 0.003, respectively) and serum levels of Gal-2 and FSH and LH (r = 0.436, p < 0.001; r = 0.350, p < 0.001, respectively), whereas serum Gal-3 showed a borderline positive correlation with age (r = 0.2, p = 0.059). Additionally, statistically signifi- cant positive correlations between seminal levels of Gal-1 and Gal-3 and free testosterone in healthy individuals were reported (r = 0.205, p = 0.053; r = 0.219, p = 0.038, respectively). On the other hand, there were negative correlations between serum and seminal levels of Gal-1 and Gal-3, total and progres- sive sperm motility, sperm count and abnormal sperm forms in healthy individuals (r = -0.382, p < 0.001; r = -0.405, p < 0.001; r = -0.376, p < 0.001; r = -0.364, p < 0.001) (r = -0.394, p < 0.001; r = -0.467, p < 0.001; r = -0.413, p < 0.001; r = -0.433, p < 0.001); (r = -0.372, p < 0.001; r = -0.377, p < 0.001; r = -0.317, p = 0.002; r = -0.311, p = 0.003)(r = -0.445, p < 0.001; r = -0.498, p < 0.001; r = -0.453, p < 0.001; r = -0.463, p < 0.001, respectively). Furthermore, statistically significant positive correlations between serum levels of Gal-1 and Gal-3 and age in azoosper- mic patients were reported (r = 0.511, p < 0.001; r = 0.390, p = 0.008, respectively). On the other hand, there were negative correlations between seminal Gal-1 and estradiol (E2) and semi- nal Gal-3 and FSH and LH in azoospermic patients Galectin-1 (Gal-1) and Galectin-3 (Gal-3) levels in seminal plasma and serum in azoospermic patients versus fertile men: A cross-sectional study Sameh Fayek GamalEl Din 1, Olfat Gamil Shaker 2, Ahmad Fawzy Megawer 1, Mohamed Ahmed Abdel Salam 1, Abdelhalim Nabil Abdelhalim 1, Ahmed Adel 1 1 Department of Andrology, Sexology and STDs, Kasr AlAiny Faculty of Medicine, Cairo University, Egypt; 2 Department of Medical Biochemistry and Molecular Biology, Kasr AlAiny Faculty of Medicine, Cairo University, Egypt. DOI: 10.4081/aiua.2023.11462 Summary (r= -0.318, p = 0.033; r = -0.322, p = 0.031; r = -0.477, p < 0.001, respectively). Also, negative correlations between serum Gal-3 and total and free testosterone in azoospermic patients were detected (r = -0.396, p = 0.007; r = -0.375, p = 0.011, respectively). Conclusions: Elevated serum and seminal levels of Gal-1 and Gal-3 have detrimental effects on spermatogenesis. Furthermore, the current study demonstrated potential regula- tory effects of reproductive hormones on Gal-1 and Gal-3. Thus, future studies are needed to confirm such findings. KEY WORDS: Gal-1; Gal-3; Azoospermia; Normal spermatogenesis. Submitted 11 May 2023; Accepted 20 June 2023 Archivio Italiano di Urologia e Andrologia 2023; 95(3):11462 GamalEl Din S.F., Shaker O.G., Fawzy Megawer A., et al. 2 protein that might play a key role in Leydig cell biology as well as potential control of the development of normal Leydig cells through autocrine and paracrine mechanisms (13). However, cells such as Sertoli or peritubular cells might be responsible for Gal-1 regulation in Leydig cell functions (13). In contrast, Gal-3 is the most commonly reported type (14). Gal-3 is approximately 30kDa protein that acts several roles in cell to cell interaction, cancer pro- gression, pathogenesis of infections, and immunomodula- tion (15). In male reproductive tract, Gal-3 is present in testes, epididymis, vas deferens, prostate, seminal vesicles and in semen (15). Early reports demonstrated that extracellular Gal-3 sig- nals apoptosis via cytochrome c-release and caspase-3 activation independent of caspase-8 activation (16), with more recent data suggesting that Gal-3 activates caspase- 9 upstream of caspase-3 through phosphorylation of extracellular signal-regulated kinase (ERK) (17). Differences in the pro-apoptotic signaling pathways acti- vated by extracellular Gal-1 and Gal-3 may arise because they recognize different cell surface glycoproteins by way of their selectivity for different oligosaccharide ligands (18). Gal-3 expression has been identified in human Sertoli cells where it is under follicle stimulating hormone (FSH) control (19, 20). A potential role of Gal-3 in germ cell survival/regeneration is suggested based on its increased expression one month after a transient germ cell death process (20). Although luteinizing hormone (LH)/testosterone and FSH potentially exert their control on spermatogenesis via identified components, there are still other hormonally regulated Sertoli cell factors which remain unknown (20). We aimed in the current study to compare between seminal plasma and serum levels of Gal-1 and Gal-3 in azoospermic patients and fertile men. Also, we aimed to find out the potential relationships between reproductive hormones and Gal-1 and Gal-3. METHODS This cross-sectional study was conducted at the andrology outpatient clinic from January 2022 to September 2022. A total of 90 participants were enrolled and divided into two equal groups as follows: azoospermic and normal group. All participants signed an informed consent. The ethical committee approved the study that conforms to Helsinki declaration (2013) (21) (MS-197-2022). Inclusion criteria Any azoospermic patient or fertile individual aged 20-50 years old was included. Exclusion criteria Any azoospermic patient with abnormal karyotyping was excluded. Also, any participant with chronic medical condition was excluded. All participants were evaluated by history taking as well as general and local examinations. Testicular volume (V) was calculated from measurements of length (L) and width (W) according to the formula, (V = pi/6 X L X W2) using a plastic ruler or caliper. Two semen analyses with an interval of 1 month were obtained. Hormonal profiles including: FSH, LH, serum prolactin, total testosterone, and estradiol were performed. Scrotal Duplex in standing and supine position was done. Gal-1 and Gal-3 were assessed in seminal plasma and serum. Serum and semen were used for determination of Gal-1 using ELISA kit provided by Bioassay Technology Laboratory with Cat. No E2989Hu (Zhejiang. China). Serum and semen were used for determination of Gal-3 using ELISA kit provided by Bioassay Technology Laboratory with Cat. No E3449Hu (Zhejiang. China). Serum separator tubes (SST) were used, and samples were allowed to clot for 30 min at room temperature before centrifugation for 15 min at 1000 x g. Serum was removed and assayed immediate- ly or divided into aliquot and stored at ≤ -20°C. Repeated freeze-thaw cycles were avoided. The ejaculates were obtained after 4 days of sexual abstinence into sterile con- tainers for immediate analysis. Semen was examined according to 5th guidelines WHO guidelines (2010) (22). Seminal plasma was centrifuged for 15 min at 1000 x g within 30 min of collection. Next, it was assayed imme- diately or divided into aliquot and stored at ≤ -20°C. Repeated freeze-thaw cycles were avoided. Statistical analysis Recorded data were analyzed using the statistical package for social sciences, version 23.0 (SPSS Inc., Chicago, Illinois, USA). The quantitative data were presented as mean± standard deviation and ranges. Qualitative vari- ables were presented as number and percentages. Data were explored for normality using Kolmogorov-Smirnov and Shapiro-Wilk Test. Independent-samples t-test of significance was used when comparing between two means. Mann Whitney U test was used for two-group comparisons in non-parametric data. Chi-square (x2) test of significance was used in order to compare proportions between qualitative parameters. Spearman's rank correla- tion coefficient (rs) was used to assess the degree of asso- ciation between two sets of variables if one or both of them was skewed. Values of two variables are plotted along two axes as scatter plots showing the presence of correlations. The confidence interval was set to 95% and the margin of error accepted was set to 5%. RESULTS The current study did not reveal any statistically signifi- cant difference among participants regarding age. There were statistically significant higher means of Gal-1 and Gal-3 in azoospermic patients compared to healthy indi- viduals (p < 0.001 for all) (Table 1). In addition, in healthy individuals there were statistically significant positive correlations between serum levels of Gal-1 and age, FSH, LH levels (r = 0.296, p = 0.005; r = 0.333, p = < 0.001; r = 0.312, p = 0.003, respectively) and serum levels of Gal-2 and FSH and LH (r = 0.436, p < 0.001; r = 0.350, p < 0.001, respectively), whereas serum Gal-3 showed a borderline positive correlation with age (r = 0.2, p = 0.059) (Table 2). Additionally, statistically significant positive correlations between seminal levels of Gal-1 and Gal-3 and free testos- terone in healthy individuals were observed (r = 0.205, p = 0.053; r = 0.219, p = 0.038, respectively) (Table 2). On the other hand, there were negative correlations Archivio Italiano di Urologia e Andrologia 2023; 95(3):11462 3 Gal-1 and Gal-3 and spermatogenesis and reproductive hormones between serum and seminal levels of Gal-1 and Gal-3, total and progressive sperm motility, sperm count and abnormal sperm forms in healthy individuals (r = -0.382, p < 0.001; r = -0.405, p < 0.001; r = -0.376, p < 0.001; r = -0.364, p < 0.001=) (r = -0.394, p<0.001; r = -0.467, p < 0.001; r = -0.413, p < 0.001; r = -0.433, p < 0.001) (r = -0.372, p < 0.001; r = -0.377, p < 0.001; r = -0.317, p = 0.002; r = -0.311, p = 0.003)(r = -0.445, p < 0.001; r = -0.498, p < 0.001; r = -0.453, p < 0.001; r = -0.463, p < 0.001, respectively) (Table 2). Furthermore, statisti- cally significant positive correlations between serum levels of Gal-1 and Gal-3 and age in azoospermic patients were reported (r = 0.511, p < 0.001; r = 0.390, p = 0.008, respec- tively) (Table 3). On the other hand, there were negative correlations between seminal Gal-1 and estradiol (E2) and seminal Gal-3 and FSH and LH in azoospermic patients (r = -0.318, p = 0.033; r = -0.322, p = 0.031; r = -0.477, p < 0.001, respectively) (Table 3). Also, negative correlations between serum Gal-3 and total and free testosterone in azoospermic patients were detected (r = -0.396, p = 0.007; r = -0.375, p = 0.011, respectively) (Table 3). DISCUSSION The current cross-sectional study was conducted at andrology outpatient clinic. The current study had shown that serum and seminal levels of Gal-1 and Gal-3 were statistically significant higher in azoospermic cases com- pared to fertile individuals. This finding is attributed to the fact that Gal-3 expression and/or its subcellular local- ization could be modified in the human infertile testes as Gal-3 immunostaining appears more intense in the infer- tile testes with an absence of germ cells (Sertoli cell-only syndrome) (20). Furthermore, animal studies on rat testes had revealed that Gal3 levels are increased in severely damaged spermatogenesis (20). These findings could be seen in agreement with GamalEl Din et al. who evaluated seminal plasma and serum levels of Gal-1 in NOA patients (23). The aforementioned case- control study that included in total 48 NOA patients and 50 age matched healthy controls demonstrated that semi- nal plasma levels of Gal-1 were higher in NOA men ver- sus healthy controls (23). Consistently, Gal-3 levels are increased in oligozoospermic cases (24). In addition, there Table 1. Shows levels of seminal plasma and serum Gal-1 and Gal-3 in azoospermic cases and healthy individuals. Azoospermic cases (n = 45) Healthy individuals (n = 45) Mean SD range Mean SD range p-value Serum Galectin 1 (ng/ml) 13.79 ± 10.14 6.7-56 7.35 ± 1.12 5.2-9.3 < 0.001 Seminal plasma Gal-1 (ng/ml) 12.11 ± 6.24 3.6-27.4 7.25 ± 0.90 5.7-8.7 < 0.001 Serum Gal-3 (pg/ml) 402.56 ± 295.59 153.8-1300 230.11 ± 59.33 120-312.3 < 0.001 Seminal plasma Gal-3 (pg/ml) 404.15 ± 300.11 114.6-1230 152.77 ± 22.50 120.6-198.2 < 0.001 P value was calculated using Mann-Whitney test. Table 2. Shows correlation between Gal-1 (ng/ml) and Gal-3 (pg/ml) with age and different sperm parameters and reproductive hormones. Parameters Gal-1 (ng/ml) Gal-3 (pg/ml) Serum Seminal plasma Serum Seminal plasma r p-value r p-value r p-value r p-value Age (years) 0.296 0.005 0.013 0.904 0.200 0.059 -0.092 0.387 Sperm count -0.382 < 0.001 -0.394 < 0.001 -0.372 < 0.001 -0.445 < 0.001 Total sperm motility -0.405 < 0.001 -0.467 < 0.001 -0.377 < 0.001 -0.498 < 0.001 Progressive sperm motility -0.376 < 0.001 -0.413 < 0.001 -0.317 0.002 -0.453 < 0.001 Abnormal sperm forms -0.364 < 0.001 -0.433 < 0.001 -0.311 0.003 -0.463 < 0.001 FSH 0.333 < 0.001 0.319 0.002 0.436 < 0.001 0.151 0.155 LH 0.312 0.003 0.337 < 0.001 0.350 < 0.001 0.007 0.951 Total testosterone -0.139 0.191 0.046 0.666 -0.184 0.082 0.054 0.614 Free testosterone -0.111 0.298 0.205 0.053 -0.197 0.063 0.219 0.038 PRL 0.033 0.757 0.120 0.260 0.032 0.765 0.008 0.940 E2 0.053 0.622 -0.151 0.155 0.079 0.460 -0.049 0.645 P value calculated using Spearman's rank correlation coefficient. Table 3. Shows correlation between Gal-1 (ng/ml) and Gal-3 (pg/ml) with age and reproductive hormones among azoospermic patients. Parameters Serum Gal-1 Seminal plasma Gal-1 Serum Gal-3 Seminal plasma Gal-3 (ng/ml) (ng/ml) (pg/ml) (pg/ml) r p-value r p-value r p-value r p-value Age (years) 0.511 < 0.001 0.064 0.674 0.390 0.008 -0.102 0.505 FSH 0.077 0.615 -0.024 0.874 0.265 0.078 -0.322 0.031 LH 0.093 0.546 0.053 0.731 0.152 0.320 -0.477 < 0.001 Total testosterone -0.236 0.118 0.045 0.769 -0.396 0.007 0.127 0.405 Free testosterone -0.207 0.173 0.224 0.138 -0.375 0.011 0.293 0.051 PRL 0.077 0.613 0.279 0.064 0.032 0.834 0.046 0.765 E2 -0.011 0.945 -0.318 0.033 0.004 0.980 -0.173 0.256 P value calculated using Spearman's rank correlation coefficient. Archivio Italiano di Urologia e Andrologia 2023; 95(3):11462 GamalEl Din S.F., Shaker O.G., Fawzy Megawer A., et al. 4 were statistically significant positive correlations between serum and seminal levels of Gal-1 and Gal-3, age, FSH, LH and abnormal sperm forms. Similarly, it should be noted that FSH enhances Gal-3 expression probably through the classical cAMP/PKA/CREB transducing path- way (20). Furthermore, CAMP responsive element (CRE) and activator protein complex (AP1) have been detected in gal-3 promoter, a finding which agrees with the poten- tial direct stimulatory actions of FSH and EGF, respective- ly. On the contrary, Gal-1 has been detected in interstitial cells in mouse testis (25) where it might modulate Leydig cell growth through its multivalent binding and cross- linking properties as well as its ability to interact with extra cellular matrix causing changes in cell adhesivity (26). In the same context, the same study had reported that Gal-1 induces changes in Leydig cell morphology and reduces cell viability and testosterone production (26). Furthermore, this is the first time that an endogenous pro- tein, Gal-1, is shown to possess apoptosis-inducing activ- ity on Leydig cells. Also, there were negative correlations between Gal-1, Gal-3, total and progressive sperm motili- ty and sperm count. We agreed with Menteşoğlu and col- leagues (2021) who assessed the correlation between semen parameters and galectin-3 levels of infertile men (24). Moreover, the possible role of Gal-3 and sperm motility in the current study is also supported by the neg- ative correlation between Gal-3 levels and total progres- sive motile sperm. Consistently, Gal-3 levels were found to be negatively cor- related with total progressive sperm count in oligo- zoospermic patients (24). Furthermore, there was a high- ly statistically significant positive correlation between serum Gal-1 and Gal-3 and age in azoospermic patients that could be seen contradictory to the study conducted by GamalEl Din et al who failed to demonstrate any cor- relation between serum Gal-1 and age in NOA patients (23). Furthermore, there was a statistically significant neg- ative correlation between seminal plasma Gal-1 and estra- diol (E2) in azoospermic patients. Similarly, Perzelova et al. demonstrated an inverse relation between Gal-1 and estrogen as they showed that the pharmacological activa- tion of estrogen receptor-b led to a significant alteration in the pattern of differentiation and the proliferation activity of keratinocytes including Gal-1 (27). Interestingly, there were statistically significant negative correlations between serum Gal-3, total testosterone and free testosterone in azoospermic patients. These findings could be seen con- tradictory to a recent study that was conducted on rats and revealed the favorable effect of testosterone administration to halt the progression of cavernosal fibrosis by decreasing Gal-1 through enhancing the expression of miR-22-3p (28). Furthermore, there were statistically significant neg- ative correlations between seminal plasma Gal-3, FSH and LH in azoospermic patients. Remarkably, there are several points of strength of the current study that can be summarized as follows. It clear- ly demonstrates the detrimental effects of Gal-1 and Gal- 3 on spermatogenesis. Also, it highlights the horizons for a potential regulatory effect of reproductive hormones on these proteins. Admittedly, lack of immunohistochem- istry can be seen as the major limitation of the current study. Also, small sample size can be added as another limitation. Finally, five cases of the healthy individuals suffering from hypothyroidism and diabetes mellitus and hypertension and disc prolapsed were included. CONCLUSIONS Elevated serum and seminal levels of Gal-1 and Gal-3 have detrimental effects on spermatogenesis. Furthermore, the current study demonstrated potential regulatory effects of reproductive hormones on Gal-1 and Gal-3. Thus, future studies are needed to confirm such findings. REFERENCES 1. Wu X, Lin D, Sun F, Cheng CY. Male Infertility in Humans: An Update on Non-obstructive Azoospermia (NOA) and Obstructive Azoospermia (OA). Adv Exp Med Biol. 2021; 1288:161-173.. 2. Gifford JA. The role of WNT signaling in adult ovarian folliculo- genesis. Reproduction. 2015; 150:R137-R148. 3. Kohn TP, Pastuszak AW. Non-obstructive azoospermia and short- ened leukocyte telomere length: further evidence linking poor health and infertility. Fertil Steril. 2018; 110:629-630. 4. Cummings RD, Liu FT. Galectins. 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Correspondence Sameh Fayek GamalEl Din, MD (Corresponding Author) samehfayek@kasralainy.edu.eg Department of Andrology and STDs Kasr Al-Ainy, Faculty of Medicine Cairo University, Al-Saray Street, El Manial, Cairo, 11956, Egypt Ahmad Fawzy Megawer, MD Mohamed Ahmed Abdel Salam, MD Abdelhalim Nabil Abdelhalim, MBBCH Ahmed Adel, MD Department of Andrology, Sexology and STDs, Kasr AlAiny Faculty of Medicine, Cairo University, Cairo, Egypt Olfat Gamil Shaker, MD Department of Medical Biochemistry and Molecular Biology, Kasr AlAiny Faculty of Medicine, Cairo University, cairo, Egypt Conflict of interest: The authors declare no potential conflict of interest.