Stesura Seveso Archivio Italiano di Urologia e Andrologia 2023; 95(4):11906 1 ORIGINAL PAPER semen analysis (2). Defective sperm motility in the form of decreased progressive motility or absence of motility is defined as asthenozoospermia which is usually accompa- nied by oligozoospermia or teratozoospermia (3). Harbouring above 85% morphologically abnormal sperms in semen is considered teratozoospermia (TS). TS is classified into monomorphic and polymorphic. In monomorphic TS, all sperms have the same morphologi- cal abnormality while in polymorphic type, there are dif- ferent varieties of abnormal sperm morphologies (4). Calbindin 2 (CALB 2; Calretinin; 29 kDa Calbindin) is a calcium binding protein that is mostly secreted in the nervous system as well as the ovary, the adrenal glands, and the testis. The main function of CALB 2 is to buffer intracellular calcium ion to stop Ca2+ overload as well as a Ca2+ receptor (5). Ca2+, which is also a second mes- senger in the cytoplasm, plays an essential role in differ- ent physiological functions such as cell proliferation and apoptosis (6). Additionally, it regulates the synthesis of reproductive hormones (7). The primary studies revealed that the highest CALB 2 was secreted in the cytoplasm of Leydig cells of adult rats in synchrony with androgen level so postulating that CALB 2 might prompt steroido- genesis (8). Increment in viability and proliferation of Leydig cells by CALB 2 was attributed to inhibition of mitochondrial related apoptotic pathway through induc- ing ERK1/2 and AKT pathways as well as supressing cell apoptosis (9). Interestingly, CALB 2 can be deployed as a marker of nor- mal and neoplastic Leydig cells of the testis as well as diagnosing atypical Leydig cell tumor (10). Non obstruc- tive azoospermia (NOA) and OAT of unknown causes are common and are noticed in a high sector of infertile men. However, the underlying molecular mechanisms of these conditions remain unknown (11). We aimed to assess seminal CALB 2 expression in men with different semen parameters as well as to investigate potential correlations between seminal CALB 2 and different semen parameters in OAT patients. Furthermore, we aimed to find any cor- relation between seminal CALB 2 and reproductive hor- mones in NOA patients. Objectives: We aimed to assess seminal cal- bindin 2 (CALB 2) expression in men with different semen parameters as well as its correlation with repro- ductive hormones in azoospermic patients and different semen parameters in oligoasthenoteratozoospermic patients. CALB 2 is also known as calretinin and 29 kDa calbindin. Materials and methods: This prospective study was performed on 96 cases from the andrology outpatient clinic divided into 3 groups as follows: group 1 including 32 non obstructive azoospermic (NOA) patients, group 2 including 32 patients with oligoasthenoteratozoospermia (OAT), and Group 3 including normozoospermic individuals as controls. Semen analysis and estimation of seminal CALB 2 concentrations by enzyme linked immunosorbent assay (ELISA) technique were performed for all participants. Reproductive hormones were measured in non- obstructive NOA patients. Results: The mean seminal CALB 2 level was higher in OAT patients compared to NOA patients and controls (7.8 ± 1.30 ng/ml, 7.3 ± 0.80 and 7.4 ± 1.0, respectively). Furthermore, the study had shown strong positive correlations between CALB 2 and sperm normal forms in controls and OAT patients. In con- trast, there was no significant correlation between seminal CALB 2 and any of the reproductive hormones measured in NOA patients. Conclusions: Seminal CALB 2 may play a role in increasing the abnormal forms in OAT patients. KEY WORDS: Non obstructive azoospermia; Oligoasthenoteratozoospermia; normozoospermia; Seminal CALB 2 (Calretinin & 29 kDa Calbindin); Reproductive hormones. Submitted 2 October 2023; Accepted 9 October 2023 INTRODUCTION Abnormalities in the form of oligozoospermia, astheno- zoospermia, teratozoospermia and high percentage of sperm DNA fragmentation are associated with decreased probability to conceive (1). Oligozoospermia is classical- ly defined when sperm count is below 10M/mL. 15 M is the 5% percentile of the new 2021 WHO manual for Seminal calbindin 2 level in azoospermia and oligoasthenoteratozoospermia and its correlation with seminal and hormonal parameters Sameh Fayek GamalEl Din 1, Noha Abdelhafeez Abdelkader 2, Mohamed Yousry El-Amir 1, Asmaa Anter Sayed Ahmed 3, Hesham Fouad Abdel-latif 1, Mohamed Farag Azmy 4 1 Department of Andrology, Sexology and STDs, Kasr Al-Ainy Faculty of Medicine, Cairo University, Cairo, Egypt; 2 Department of Clinical and Chemical Pathology, Faculty of Medicine, Beni-Suef University, Beni-Suef, Egypt; 3 Egypt Ministry of Health & Population, Cairo, Egypt; 4 Department of Andrology, Sexology and STDs, Faculty of Medicine, Beni-Suef University, Beni-Suef, Egypt. DOI: 10.4081/aiua.2023.11906 Summary Archivio Italiano di Urologia e Andrologia 2023; 95(4):11906 S. Fayek GamalEl Din, N. Abdelhafeez Abdelkader, M. Yousry El-Amir, et al. 2 PATIENTS AND METHODS The current prospective study was conducted in the andrology outpatient clinic during the period from April (2021) till January (2022). Approval by the local research and ethical committee was obtained that conforms to Helsiniki declaration (2013) (FMBUREC/09052021) (12). Written informed consents were obtained from the patients who were involved in the study. The study was performed on 96 randomized patients using simple numbering method. They were divided into 3 groups as follows: group (1) comprised of 32 NOA patients. Group (2) comprised of 32 patients with OAT. Group (3) comprised of 32 patients with normal semen parameters as controls. Inclusion criteria of the patients Any infertile patient with normal testicular volume was included. Exclusion criteria of the patients Any patient with varicocele, smoker, leukocytospermia, abnormal karyotyping and finally evidence of severe uncon- trolled medical diseases was excluded from the study. Inclusion criteria of the controls Any healthy age matched individual was recruited in group 3. Sample size was calculated using G power. At least 89 par- ticipants should be included in the three groups using F tests - ANCOVA: Fixed effects, main effects and interac- tions as well as a priori analysis (13). The participants were asked about any relevant medical history and were sub- jected to clinical examination. Also, 5 cc blood was with- drawn from each participant for hormones investigation [FSH, LH, total testosterone, estradiol, prolactin (PRL)] and semen analysis. Furthermore, all participants were asked about a history of hernia repair, scrotal surgery, pelvic sur- gery, endoscopic urethral instrumentation, or genitouri- nary infection. Testicular volume of all participants was routinely determined by Prader’s orchidometer. Furthermore, scrotal duplex was done for all participants to exclude varicocele. Semen analysis Semen samples were collected by masturbation following abstinence for 3-4 days. A special wide-mouth container was used to collect semen and incubated at 37°C until semen was liquefied. Semen analysis was then performed within 1 hour following the WHO manual criteria (5th edition, 2010) (14). Duplicate semen analyses were per- formed twice at the beginning and 3 months after initiat- ing the study and the average of the two values was used for analysis. The same investigator performed all semen analyses to optimize repeatability (15). Seminal analysis was carried out within 30 minutes after liquefaction. The volume, viscosity, pH and appearance of semen were evaluated together with sperm concentration, progressive and total motility and morphology. Sperm concentration was evaluated using a Makler count- ing chamber (Irvine Scientific, Santa Ana, CA, USA) under an optical microscope (Nikon, Nikon Europe B.V., Amsterdam, The Netherlands) at 200x magnification. Sperm morphology was evaluated by using pre-coloured glasses (Testsimplets) and the eosin Y test was applied to evaluate sperm vitality. Determination of CALB 2 Human CALB 2 was determined using Calretinin ELISA Kits supplied by SinoGeneClon Biotech Co., Ltd. Cat. No.: SG- 00383. This kit uses the Sandwich-ELISA principle with sensitivity 0.01 ng/mL and detection range 0.06-4 ng/mL. The microtiter plate of the Calretinin ELISA is coated with a capture antibody. The diluted sample was added and any antigen present bound to capture antibody. After a wash- ing step, the detecting antibody (biotinylated anti-calre- tinin antibody) was added and bound to antigen. After another washing step the enzyme conjugate streptavidin- peroxidase was added and bound to detect the antibody. The following substrate TMB/peroxidase reaction was monitored at 450 nm (reference wavelength at 620 nm). Determination of reproductive hormones in NOA patients Blood samples were obtained from azoospermic patients, and then samples were left to clot for 1 hour at room tem- perature or overnight at 2-8°C before centrifugation for 20 min at 1000×g at 2-8°C. The supernatant was collect- ed to carry out the assay. Follicle stimulating hormone (FSH) was determined using ELISA Kits supplied by Elabscience Biotechnology, Inc, United States. Cat. No.: E-EL- H1143. This kit used the sandwich-ELISA principle with sensitivity 0.94 mIU/mL and detection range 1.56-100 mIU/mL. Luteinizing hormone (LH) was determined using ELISA Kits supplied by Elabscience Biotechnology, Inc, United States. Cat. No.: E-EL-H6019. This kit used the sandwich-ELISA principle with sensitivity: 0.1mIU/mL and detection range 0.16-10mIU/mL. Serum prolactin was measured according to human prolactin (PRL) OneStep ELISA Kit (Boster Biological Technology, Pleasanton CA, USA, Catalog # EK7006) with analytical sensitivity 11.7 pg/mL and assay range 15.6-1000 pg/mL. Testosterone was determined using ELISA Kits supplied by MyBioSource, Inc., San Diego, USA. Cat. No.: MBS580035. Estradiol was determined using ELISA Kits supplied by Thermo Fisher Scientific, Inc. Third Avenue Waltham, MA USA. Cat. No.: KAQ0621. This kit used the sandwich-ELISA principle with sensitivity: 5 pg/mL and detection range 13-935 pg/mL. Statistical analysis Analysis of data was performed using SPSS v. 23 (Statistical Package for Social science) for Windows. Mean, standard deviation (SD), minimum and maximum were used to describe quantitative variables whereas number (No.) and percentages (%) were used to describe qualitative variables. Shapiro/Kolomogrov tests of normality were utilized to test for normality. The Chi square test was used to determine the statistical difference of the categorical data between the two groups. Pearson correlation was used to test the corre- lation between different quantitative variables. RESULTS There was no significant difference between the studied par- ticipants regarding their baseline characteristics (Table 1). Archivio Italiano di Urologia e Andrologia 2023; 95(4):11906 3 Seminal CALB 2 and azoospermia and OAT Furthermore, one case had atrophic right testis and 3 cases presented with small testis in NOA patients. There were three cases with absent right epididymis and 4 cases with absent vas deferens in the same group. Additionally, the medians of FSH, LH, Testosterone, E2, and prolactin in NOA patients were 5.40, 6.35, 4.50, 42.00 and 7.50, respectively. Furthermore, there was no significant difference between NOA, OAT and nor- mozoospermic participants regarding seminal CALB 2 level (7.3 ± 0.80 ng/ml; 7.8 ± 1.40 ng/ml; 7.4 ± 1.0 ng/ml, p = 0.15, respectively). Although seminal CALB 2 levels in NOA and OAT groups were not significantly different to levels of normozoosper- mic participants (p > 0.05), yet, there was a higher predic- tive role of seminal CALB 2 levels in predicting OAT cases compared to azoospermic cases (Table 2, Figures 1-2). Moreover, there were significant linear strong positive cor- relations between sperm normal forms and seminal CALB 2 levels in normozoospermic and OAT cases (Tables 3-4). Table 2. Cutoff values, area under the curve (AUC), sensitivity, specificity, positive predictive values and negative predictive values of seminal CALB 2 in azoospermic and oligoasthenoteratozoospermic (OAT) cases. Azoospermic cases OAT cases P-value 0.92 0.15 Cut off 7.2500 7.7500 AUC 0.493 0.609 Sensitivity 60 % 50% Specificity 46% 66% Positive predictive value 60% 50% Negative predictive value 45% 65% Table 3. Correlation between seminal calbindin 2 (CALB 2) and age and semen parameters in normozoospermic participants. Normozoospermic cases Seminal CALB 2 (ng/ml) Age Pearson correlation (r) 0.055 p-value 0.77 Sperm concentration (106/ml) Pearson correlation (r) -0.072 p-value 0.71 Sperm total motility (%) Pearson correlation (r) 0.285 p-value 0.13 Sperm normal forms (%) Pearson correlation (r) 0.709 p-value 0.00 P value was calculated using Pearson correlation. Table 4. Correlation between seminal calbindin 2 (CALB 2) and age and semen parameters in oligoasthenoteratozoospermic (OAT) cases. OAT cases Seminal CALB 2 (ng/ml) Age Pearson correlation (r) -0.596 p-value 0.00 Sperm concentration (106/ml) Pearson correlation (r) -0.088 p-value 0.64 Sperm total motility (%) Pearson correlation (r) 0.347 p-value 0.06 Sperm normal forms (%) Pearson correlation (r) 0.763 p-value 0.00 P value was calculated using Pearson correlation. Figure 1. Receiver Operating characteristic curve for prediction of non obstructive azoospermia (compared with normozoospermia) using seminal calretinin level. Figure 2. Receiver Operating characteristic curve for prediction of oligoathenoteratospermia (compared with normozoospermia) using seminal calretinin level. Table 1. Sociodemographic characteristics of the participants. Group (1) Group (2) Group (3) P value azoospermic patients oligoasthenoteratozoospermic normozoospermia (n = 30) (OAT) (n = 30) (n = 30) Mean SD Mean SD Mean SD Age (years) 33.3 ± 10.4 30.4 ± 8.1 29 ± 6.7 0.15 Marital status N % N % N % Single 5 16.7 - Married without offspring 16 53.3 30 100 Married with offspring 9 30.0 30 100 P value was calculated using Chi-square test. Archivio Italiano di Urologia e Andrologia 2023; 95(4):11906 S. Fayek GamalEl Din, N. Abdelhafeez Abdelkader, M. Yousry El-Amir, et al. 4 However, there was a significant linear strong negative cor- relation between age and seminal CALB 2 levels in OAT cases (Table 4). Finally, there was no significant linear cor- relation between seminal CALB 2 levels and any of the reproductive hormones measured in NOA cases (Table 5). DISCUSSION The present study is a case-control study conducted com- paring seminal CALB 2 expression in men with normal semen parameters, OAT and NOA. The mean seminal CALB 2 level was higher in OAT cases compared to azoospermic cases and controls. Although, the values of seminal CALB 2 levels in OAT and azoospermic cases were not significantly different compared to normo- zoospermic participants, there was a higher predictive role of seminal CALB 2 levels in predicting OAT cases com- pared to NOA cases. Moreover, there was no significant linear correlation between seminal CALB 2 level and any of the reproductive hormones measured in NOA cases. On the other hand, there were significant linear strong posi- tive correlations between sperm normal forms and semi- nal CALB 2 levels in normozoospermic and OAT cases. There was a high significant linear negative correlation between age and seminal CALB 2 level in OAT cases. Bar-Shira Maymon et al. (16) demonstrated that the expres- sion of CALB 2 in abnormal Sertoli cells in non-obstructive azoospermia contributes to the multifactorial etiology of spermatogenic failure. In the same context, GamalEl Din et al. (17) demonstrated a negative impact of seminal CALB 2 on sperm normal forms in patients with varicocele (17). Furthermore, OAT patients showed the highest median seminal CALB 2 compared to the patients of other 2 groups in the current study. This can be explained by the fact that OAT patients would have more immature Sertoli cells compared to azoospermic patients and controls. Thus, they have increased levels of CALB 2 expression by the seminif- erous epithelium and consequently have increased levels of seminal CALB 2. This agrees with GamalEl Din et al. (2023) who demonstrated that patients with bilateral varicocele had higher seminal CALB 2 compared to unilateral varico- cele (17). Interestingly, the current study did not show any correlation between CALB 2 and any of the reproductive hormones measured in NOA cases. On the contrary, sever- al studies have demonstrated a potential link between CALB 2 and steroidogenesis and reproductive hormones. Firstly, steroidogenesis in Leydig cells can be enhanced by increased free Ca2+ (18). In the same context, Altobelli et al. (2017) had detected CALB 2 immunoreactivity in human fetal testis, testis Leydig cells, seminiferous epithelium and epididymal epithelial cells (13). They assumed that CALB 2 is involved in the processes of production and/or secre- tion of hormones as well as in all calcium dependent dif- ferentiation processes that occur during gonadal develop- ment suggesting its involvement in steroidogenesis and spermatogenesis (13). However, the role played by CALB 2 cannot be precisely determined similarly to other calcium- binding proteins that behave differently in the presence of this ion to the extent that they are classified as “Ca-buffer” or “Ca sensor” proteins (19). This uncertainty of their exact role is due to the possible exposure of their hydrophobic residues after binding with calcium (19). Admittedly, our study is no free from limitations. Firstly, the small sample size can be considered as a major limi- tation. Moreover, the inability to use immunohistochem- istry in the azoospermic cases to properly localize the site of production of CALB 2 can be regarded as another lim- itation. Also, not including cases of obstructive azoosper- mia could be seen as an additional limitation. Besides, another limitation was including one case with atrophic right testis and 3 cases with small testis in NOA patients. Finally, we were unable to evaluate reproductive hor- mones in all participants. CONCLUSIONS In brief, seminal CALB 2 may play a role in increasing the abnormal forms in OAT cases. REFERENCES 1. Milachich T, Dyulgerova-Nikolova D. The Sperm: Parameters and Evaluation. In Innovations In Assisted Reproduction Technology. IntechOpen; 2020. 2. 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Correspondence Sameh Fayek GamalEl Din, MD (Corresponding Author) samehfayek@kasralainy.edu.eg Mohamed El-Amir, MD yousr82@kasralainy.edu.eg Hisham Foaad, MD hisham.foaad@kasralainy.edu.eg Department of Andrology and STDs Kasr Al-Ainy, Faculty of Medicine Cairo University, Al-Saray Street, El Manial, Cairo, 11956, Egypt Noha Abdel Kader, MD noha.abdelkader@med.bsu.edu.eg Department of Clinical and Chemical Pathology, Faculty of Medicine, Beni-Suef University, Beni-Suef, Egypt Asmaa Anter, MD allahtaim2@gmail.com Egypt Ministry of Health & Population, Cairo, Egypt Mohamed Azmy, MD mohamed.Azmy@med.bsu.edu.eg Department of Andrology, Sexology and STDs, Faculty of Medicine - Beni-Suef University, Beni-Suef, Egypt Conflict of interest: The authors declare no potential conflict of interest.