Stesura Seveso Archivio Italiano di Urologia e Andrologia 2024; 96(1):12186 1 ORIGINAL PAPER World Health Organization (WHO) ejaculate analysis labo- ratory guidelines 2021, each standard ejaculate analysis test takes about 60 minutes, so the timing to test fructose is extended, which may affect the measurement of the level of fructose in ejaculate (3). In fact, when fructose is tested in this way, the fructose level obtained will differ from the real fructose level because the fructose in the ejaculate will undergo fructolysis. Nonspecific and sensitive test results will inevitably influence the diagnosis. There are several methods to test for fructose, including colorimetric, enzymatic, and chromatographic methods (4). WHO used Karvonen and Malm's modified methods to test the amount of fructose in ejaculation (3). The man- ual fructose testing method has disadvantages, such as a complicated procedure with a long waiting time. Since 1981, the use of semiautomatic or automatic machines for testing fructose began to develop (5). The semiauto- matic machine can shorten the time to test the fructose level. The advantages of the semiautomatic fructose method include easy reagent preparation, minimal reagent use, minimal error, reduced human resources, and easy calibration and quality control processes (6). There is no standardized time and method for semi-auto- mated fructose testing, so the researchers felt it was nec- essary to research to find the best timing for testing fruc- tose in order to obtain results close to real fructose levels. In this study, we carried out the process using a BTS-350 semiautomatic machine. The method used was an enzy- matic method using hexokinase and phosphoglucoiso- merase enzymes and then switching to a colorimetric method. The method used in BTS-350 semiautomatic machines makes the processing time much faster. METHODS This was an observational analytic study using the ejacu- lates of infertile men who visited the Policlinic Andrology Outpatient General Hospital, Dr. Soetomo Surabaya. The study was approved by the ethics committee of RSUD Dr. Soetomo with no. 0669/KEPK/V/2023. Based on the sample calculation, 13 ejaculates from dif- ferent men were included in this study. Objective: Various factors, such as obstruc- tive azoospermia, cause infertility in men. Biochemical examination of ejaculate, especially measurement of fructose, can be an additional investigation that can be used for this diagnosis in reproductive health. Examination of fruc- tose is carried out after routine ejaculate analysis, resulting in prolonging the examination time so that it will affect the meas- urement of fructose level in the ejaculate and the accuracy of the diagnosis. This study aims to determine the best timing and pro- cedure for measurement of fructose using a semiautomatic method. Methods: This research is an analytic observational study con- ducted at Dr. Soetomo General Hospital, Surabaya. A total of 13 ejaculate samples from infertile male patients who met the inclusion criteria were evaluated. Each ejaculate was divided into eight aliquots that were examined for fructose using a semi- automated method after different intervals of time and centrifu- gation modalities. Results: This study showed a significant difference in fructose levels when aliquots were centrifuged and examined immediate- ly or after different interval of time (p = 0.036). In addition, aliquots left standing for more than 60 minutes (p = 0.012) and 120 minutes (p < 0.001) before centrifugation, showed signifi- cantly lower levels compared to aliquots that were centrifuged and then immediately examined. Conclusions: We suggest that measuring fructose immediately after centrifugation is more reliable than measuring fructose left standing before or after centrifugation. Leaving the ejaculate standing will reduce the fructose level so that it does not resem- ble its real level. KEY WORDS: Infertile; Fructose; Semiautomatic; Time; Centrifugation; Reproductive health. Submitted 13 December 2023; Accepted 4 January 2024 INTRODUCTION The fructose examination is one of the biochemical tests used to diagnose the cause of male infertility (1). It is not a routine test, but it is a useful supplemental test to provide information on specific clinical conditions, that is done after routine testing ejaculate analysis (2). According to Examination of ejaculate fructose levels on male infertility patients at various times and centrifugation using semiautomatic method Hermansyah Hermansyah 1, 3*, Muhammad Fadhli Abdullah 1, 3*, Cennikon Pakpahan 1, 2, 3*, Reny I’tishom 1, 2, Supardi Supardi 1, 3, Ilhamsyah Ilhamsyah 1, 3 1 Andrology Study Program, Faculty of Medicine, Universitas Airlangga, Surabaya, Indonesia; 2 Department of Biomedical Sciences, Faculty of Medicine, Universitas Airlangga, Surabaya, Indonesia; 3 Andrology Outpatient Clinic, General Academic Dr. Soetomo Hospital, Surabaya, Indonesia. * These authors contributed equally to this paper. DOI: 10.4081/aiua.2024.12186 Summary Archivio Italiano di Urologia e Andrologia 2024; 96(1):12186 H. Hermansyah, M. Fadhli Abdullah, C. Pakpahan, et al. 2 Inclusion criteria were samples from men aged 18-50 years old, with ejaculate volume > 2 ml, and willing to participate in the study by signing informed consent; we exclude men with anejaculation, haematospermia, hyper- viscosity, and men with reproductive infection. Each man who meets the criteria was explained about the procedures and objectives of the study and signed an informed consent if he was willing to participate to the research with his sample. Then, the participant was asked to masturbate and collect ejaculate into a non-toxic con- tainer. After that, each ejaculate was divided into eight aliquots which were managed in different ways after ejaculate liq- uefaction. K1: the aliquot was immediately examined for fructose content; K2: the aliquot was centrifuged, then the supernatant was taken, and fructose content was examined; K3: the aliquot was centrifuged and allowed to stand for 30 minutes, then the supernatant was taken and examined for fructose; K4: the aliquot was centrifuged, then was left standing for 60 minutes and examined for fructose; K5: the aliquot was centrifuged, left for 120 minutes and then the supernatant was taken and exam- ined for fructose; K6: the aliquot was allowed to stand for 30 minutes, then centrifuged, and the supernatant was examined for fructose content; K7: the aliquot was allowed to stand for 60 minutes, then centrifuged, and supernatant examined for fructose content; K8: the aliquot after standing for 120 minutes was centrifuged and supernatant examined for fructose content. Fructose was examined using a Semi-Automated Method. The procedure of Fructose Test with Semi-Automated Method Using the BTS-350 semiautomatic machine, a colorimet- ric procedure followed an enzymatic process involving phosphoglucoisomerase and hexokinase enzymes. A spectrophotometer was used to detect NADPH. To measure fructose with the BTS 350 semiautomatic device, the reagent mixture Reagent A (PIPES 70 mmol/l, NADP+ 1.2 mmol/l, hexokinase > 15 U/ml, phosphoglucose iso- merase > 10 U/ml, preservative, pH 7), Reagent B (ATP > 15 mmol/l, glucose-6-phosphate dehydrogenase > 10 U/ml, preservative, pH 9), and fructose standard (D-fruc- tose 75 mg/dL equivalent to 375 mg/dL or 28 mmol/l fructose according to the sample dilution factor) were used. Samples were added to reagents and put in a unique tube made of Teflon to be sucked automatically by the machine until, within a few minutes, the value of fructose content will appear. After that, the fructose con- centration needs to be calculated manually. Statistical analysis The data obtained were analyzed statistically with the Graph Pad Prism 10 software package. Data normality test was conducted with the Shapiro Wilk test, and com- parative analysis between variables was tested with the Wilcoxon Sign Rank Test for non-normally distributed data, and paired t-test for normally distributed data. RESULTS The results of this study were obtained from samples of 13 subjects divided into eight aliquots. The general char- acteristics of the study participants and observational data of macroscopic analysis of ejaculate are shown in Table 1. In this study, the results of ejaculate ejaculate of infertile men with azoospermia criteria were 8%, severe oligo- zoospermia 23%, oligoteratozoospermia 23%, asthne- zoospermia 31%, and teratozoospermia 15%. Table 2 shows no significant difference between fructose levels measured in the aliquot that was immediately examined after centrifugation compared to fructose levels measured in aliquots that were left standing after cen- trifugation for 30 (p = 0.100) and 60 (p = 0.133) minutes. However, there was a significant difference between the level measured immediately after centrifugation com- pared to the level measured in the aliquot left for 120 minutes (p = 0.036). These results suggest that 60 min- utes may be the recommended most extended time limit for measurement of fructose after centrifugation, as after that time, fructose levels may drop not resembling real levels. In the comparison between fructose levels that are left standing before centrifugation, there was a significant dif- ference between immediate measurement and measure- ments aliquots that are left standing for 60 minutes (p = 0.012) and 120 minutes (p < 0.001) before centrifuga- tion. The decrease in fructose levels between direct exam- ination and 120 minutes was highly significant. This find- ing indicates that leaving samples for more than 60 min- utes before centrifugation significantly decreases the measurement of the level of fructose content (Table 3). The results of the measurements of fructose in the differ- ent eight aliquots were plotted on a graph, showing that aliquots that were left standing for a period of time and Table 1. The general characteristics of the study participants and observational data of macroscopic analysis of ejaculate. General characteristics of participants Mean ± SD Median and results of macroscopic ejaculate analysis (min-max) Age (years old) 32.69 ± 4.76 - Abstinence (day) - 4 (2-7) Volume (ml) 3.39 ± 1.31 - Liquefaction (minute) - - pH - 7.5 (7-8,1) Viscosity (cm) - < 2 Table 2. Comparison of fructose concentrations (Mean ± SD) in seminal plasma standing for 0, 30, 60, or 120 minutes after centrifugation of ejaculate. Aliquot N Fructose (mmol/L) 0 minutes 13 15.77 ± 4.81 30 minutes 13 15.18 ± 5.34 a 60 minutes 13 14.87 ± 5.47 b 120 minutes 13 14.23 ± 5.76 c a p = 0.100; b p = 0.133; c p = 0.036. Archivio Italiano di Urologia e Andrologia 2024; 96(1):12186 3 Semen fructose levels and semiautomatic method then centrifuged showed a more significant decrease in fructose levels than measurement in aliquots that were centrifuged and then left standing for a period of time. Our findings suggest that the best procedure for fructose examination is immediate centrifugation with measure- ment within 60 minutes. Levels of fructose in samples left standing before centrifugation tend to be lower than real levels (Figure 1). DISCUSSION The results showed that there was a significant difference in fructose levels in the aliquots that were immediately centrifuged after liquefaction and then immediately checked compared to the aliquots that, after centrifuga- tion, were allowed to stand for 120 minutes before meas- urement (p = 0.036). There is a significant difference in fructose levels in the aliquots immediately checked com- pared to those allowed to stand 60 minutes and 120 min- utes after liquefaction, then centrifuged and checked (p = 0.012 and p < 0.001). The difference in results in the present study for the aliquots that were allowed to stand 120 minutes after centrifugation compared to the results of Lu et al. may be due to different centrifugation speeds and the number of samples measured. The present study used a centrifuga- tion speed of 4000 rpm for 30 minutes, while the research conducted by Lu et al. used a centrifugation speed of 3000 x g for 15 minutes. Although research con- ducted by Lu et al. showed that different centrifugation speeds have little effect on fructose levels, remaining spermatozoa or non-cellular components (7, 8), includ- ing zinc, alpha-glucosidase, citric acid may mildly affect the fructose level (9). The difference may also be due to differences in sample size being the sample size in the present study was 13 people, while the study research by Lu et al. included 20 subjects. The data from the aliquots examined directly after cen- trifugation compared to those examined after a standing period before centrifugation showed no significant differ- ence after a 30-minute standing (15.78 mmol/L and 15.30 mmol/L), but after 60- and 120-minute standing the fructose levels decreased significantly (14.25 mmol/L and 10.51 mmol/L). The finding of no difference of fruc- tose level after 30 minutes standing align with the research of Elzanaty and Malm although they found no significant difference between ejaculate fructose levels examined also 60 minutes, and 90 minutes after ejacula- tion (10). The fructose levels of the aliquots that were allowed to stand for 60 and 120 minutes before centrifugation were significantly lower than those allowed to stand for only 30 minutes. This is in line with research conducted by Lu et al., although the method used is different. Lu et al. used the resorcinol method, while this study used the enzymatic method. They reported a difference in the fructose content of ejaculate that was allowed to stand for 2 hours compared to those directly examined, and fructose levels significantly lower after 4 hours with Table 3. Comparison of fructose concentrations (Mean ± SD) in seminal plasma obtained from centrifuged ejaculate after standing for 0, 30, 60, or 120 minutes. Aliquot N Fructose (mmol/L) 0 minutes 13 15.77 ± 4.81 30 minutes 13 15.30 ± 5.64 a 60 minutes 13 14.25 ± 5.84 b 120 minutes 13 10.51 ± 7.21 c a p = 0.294; b p = 0.012; c p = < 0.001. Figure 1. Comparison of mean fructose levels measured in aliquots immediately centrifugated and then allowed to stand compared to levels measured in aliquots allowed to stand before centrifugation. Archivio Italiano di Urologia e Andrologia 2024; 96(1):12186 H. Hermansyah, M. Fadhli Abdullah, C. Pakpahan, et al. 4 respect to 0 or 2 hours. In other words, the fructose con- centration decreased with the length of standing time (7). The results in this study are also in line with the research conducted by Andrade-Rocha, in which fructose concentration was significantly lower in the samples examined after 120 minutes of liquefaction (11). The process of fructolysis causes a decrease in fructose levels in ejaculate. This process is influenced by many factors such as concentration, motility and metabolism of spermatozoa, temperature, pH, and other substances in the ejaculate (9). An increase in spermatozoa concen- tration requires more fructose, which decreases fructose levels in ejaculate and vice versa (12). Increased fructose levels due to decreased fructose utilization can be caused by reduced spermatozoa, abnormal spermatozoa morphology, and decreased spermatozoa activity. Low fructose levels can be caused by good spermatozoa motility (13). There is a positive correlation between the motility rate of spermatozoa and the fructolysis rate in human ejaculation (9). Very low temperatures can stop all spermatozoa metabolic activity, affecting fructose utilization and resulting in a slow decline in fructose levels (14). REFERENCES 1. Trang NT. Seminal Fructose Concentration in Man Infertility and the Fructose Test’s Meaning In Diagnosis Reason of Azoospermia Man. 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Srivastava N, Pande M. Protocols in semen biology (comparing assays). Springer, Singapore, 2017; p. 1-288. Correspondence Hermansyah Hermansyah drherman09@gmail.com Muhammad Fadhli Abdullah muhammad.fadhli.abdullah-2021@fk.unair.ac.id Cennikon Pakpahan cennikon.pakpahan@fk.unair.ac.id Reny I’tishom (Corresponding Author) ritishom@fk.unair.ac.id Department of Biomedical Sciences, Faculty of Medicine, Universitas Airlangga, Surabaya, Indonesia Supardi Supardi supardi.unair@gmail.com Ilhamsyah Ilhamsyah ilhamsyah-2021@fk.unair.ac.id Conflict of interest: The authors declare no potential conflict of interest.