Dithiothreitol effects on the viscosity and quality of human semen*

Transcription

1 FERTILITY AND STERILITY Copyright 1994 The American Fertility Society Vol. 62, No.6, December 1994 Printed on acid-free paper in U S. A. Dithiothreitol effects on the viscosity and quality of human semen* Juan Alberto Gonzalez-Estrella, M.D. t PonJola Coney, M.D.:j: Kristie Ostash, B.Sc. David Karabinus, Ph.D. Reproductive Endocrinology and Infertility Section, Department of Obstetrics and Gynecology, Arizona Health Science Center, University Medical Center, University of Arizona, Tucson, Arizona Objective: To determine the effects of treatment with dithiothreitol (DTT) on seminal viscosity, consistency, and sperm motility, motion characteristics, and morphology. Design: Prospective, in vitro study. Setting: University teaching hospital. Patients: Semen donors and patients of the infertility program. Experiment 1, n = 34; experiment 2, n = 82. Sufficient seminal volume for use was the only selection criterion. Interventions: Experiment 1: semen was combined with 5.9 mm DTT solution 1:1 (vol/vol), combined with distilled water 1:1 (vol/vol), or untreated. Experiment 2: semen was combined with 5.9 mm DTT solution 1:1 (vol/vol) or untreated. Main Outcome Measures: Seminal consistency and viscosity, percentage of motile sperm, sperm motion characteristics (straight line velocity, curvilinear velocity, mean linearity, and angle of lateral head displacement), sperm concentration, and sperm morphology. Results: Treatment of semen with DTT reduced seminal consistency and viscosity. Treatment with DTT had no effect on sperm concentration or percentage motile sperm. Sperm velocity was reduced by DTT treatment, particularly in semen that had normal initial consistency. Morphology of sperm in semen exhibiting abnormal initial consistency was unaffected by DTT. Conclusion: Dithiothreitol effectively induced liquefaction of nonliquefied semen in vitro and had minimal effects on the sperm motility, motion characteristics, and morphology. Evaluation of DTT effects on the chromatin and on other functional traits of human sperm must be conducted before its use can be advocated. Fertil Steril 1994;62: Key Words: Dithiothreitol, human semen, liquefaction, semen consistency, semen viscosity, and semen quality Received January 24, 1994; revised and accepted June 24, *Presented in part at the 48th Annual Meeting of The American Fertility Society, New Orleans, Louisiana, October 31 to November 5, t Present address: Consultorio #23, Hermosillo, Sonora, Mexico. :j: Reprint requests: Ponjola Coney, M.D., Arizona Health Sciences Center, Department of Obstetrics and Gynecology, 1501 North Campbell Avenue, Tucson, Arizona (FAX: ). Under normal conditions, the fluidity of human semen changes with time, being ejaculated in the liquid state and immediately coagulating (1, 2). Human semen normally undergoes liquefaction within 20 minutes of ejaculation (1, 3, 4) because of the action of a protease secreted by the prostate, prostate-specific antigen (4, 5). Prostate-specific antigen induces proteolytic fragmentation of semenogelin, the major protein constituent of the seminal coagulum (6) that is produced and secreted by the seminal vesicles ( 7, 8). The incidence of nonliquefaction of human semen in vitro has been reported to be 11.3% (9). It is not known if coagulated semen persists after deposition in the female reproductive tract or what effect persistently coagulated semen has in vivo. In vitro, nonliquefied semen renders sperm recovery diffi Gonzalez-Estrella et al. Dithiothreitol and human semen quality Fertility and Sterility

2 cult for use in fertility-enhancing procedures such as lui and IVF (10). The resultant effect on fertility is not known. Dithiothreitol (DTT), a redq.cing agent that converts disulfides to sulfhydryls (11, 12), is effective in liquefying sputum (13) by reducing mucoprotein disulfide bonds (14). The in vitro treatment of semen with a preparation containing DTT was shown to induce liquefaction and to preserve sperm motility as determined by qualitative or subjective methods (14). If results of objective evaluation supported previously reported (14) subjective results, DTT treatment would be indicated as a potentially useful method of manipulating unliquefied semen. However, the effects of DTT on seminal fluidity, sperm motility, and motion characteristics, as determined by objective methods, have not been reported. The purposes of this initial study were to evaluate the efficacy of DTT in changing human seminal consistency and viscosity and to determine the effect of DTT on human sperm concentration, motility, motion characteristics, and morphology. MATERIALS AND METHODS Experiment 1 Semen samples (n = 34), obtained from male patients and sperm donors enrolled in the infertility program at the Arizona Health Sciences Center, Tucson, Arizona, were evaluated to differentiate the effects of dilution from those of the action of DTT. Semen was collected by masturbation into sterile specimen containers and allowed to liquefy at room temperature for 90 minutes. Initial seminal consistency (a qualitative assessment of fluidity) and viscosity (a quantitative assessment of fluidity) were determined as described in the Seminal Consistency and Seminal Viscosity sections, respectively. Three aliquots of each ejaculate were treated as follows: untreated; diluted 1:1 (vol/vol) with distilled water; and diluted 1: 1 (vol/vol) with 5.9 mm DTT in water (Sputolysin; Behring Diagnostic Inc., Somerville, NJ) to yield a final DTT concentration of 2.95 mm. Application of treatments was ordered to avoid time effects during evaluation. After treatment, aliquots were allowed to stand for 15 minutes at room temperature, with periodic gentle mixing, and then re-evaluated for seminal consistency and viscosity. Experiment 2 This experiment was conducted to determine if the DTT treatment used in experiment 1 affected Vol. 62, No.6, December 1994 Figure 1 Detail of viscometer with sample chamber removed, exposing flattened cone at the base of the vertical spindle. Resistance to the rotation of the flattened cone, exerted by the specimen in the sample chamber, was measured and results presented in units of centipoise (cps). Tubing connects sample chamber inlet and outlet to a waterbath to maintain constant chamber temperature. L-shaped clip holds sample chamber in place at the base of the viscometer during measurement. sperm motility, morphology, and seminal fluidity. Semen samples (n = 82) were obtained and allowed to liquefy as described in experiment 1. Two aliquots were taken from each semen sample; one aliquot was mixed 1: 1 (vol/vol) with 5.9 mm DTT in water (Sputolysin; Behring Diagnostic Inc.) to yield a final DTT concentration of 2.95 mm. The second aliquot remained untreated. Application of treatments was ordered to avoid time effects during evaluation. After 15 minutes at room temperature with periodic gentle mixing, both aliquots were evaluated for consistency, viscosity, sperm motility, and sperm morphology. Seminal Viscosity Quantitative measurement of seminal fluidity was performed using a Brookfield Cone/Plate Digital Viscometer, model DV -II (Brookfield Engineering Laboratories, Inc., Stoughton, MA) equipped with a CP-40 spindle and operating at 12 rpm. Slight modifications of the Brookfield Cone/Plate Digital Viscometer, model DV -II, (Fig. 1), but using the same principle, have been demonstrated to accurately measure viscosity in biological fluids having non -Newtonian or anomalous flow properties, such as blood and respiratory tract secretions (15-17). ~ flattened cone is attached to a vertical spindle (Fig. 1) that, in turn, is connected to a torque (shear rate)-sensing element via a calibrated berylliumcopper spring. The principle of operation involves Gonzalez-Estrella et al. Dithiothreitol and human semen quality 1239

3 ---,. the rotation of a flat cone upon, and perpendicular to, a planar surface (plate) at different, selectable speeds of rotation. The rotation creates constant and uniform rates of shear. The sample, placed in the bottom of the sample chamber (Fig. 1, plate), lies between the cone and the plate and offers resistance to the rotation of the cone. The torque necessary to overcome the resistance to the rotation of the flat cone exerted by the sample is a function of the sample's viscosity and is measured in absolute values. These values are provided in continuous readouts of percent full scale(%), centipoise (cps), and shear stress (dyn/cm 2 ) by means of an integrated three-digital LED display. For the present study, results were recorded in cps. A precision synchronous motor powered the viscometer, assuring exact speed of rotation for eight selectable speeds (0.3, 0.6, 1.5, 3, 6, 12, 30, and 60 rpm). Sample chamber temperature was maintained by circulating 25 C water from a constant (±0.03 C) temperature bath (model TC-500; Brookfield Engineering Laboratories, Inc.) through the sample chamber water jacket (Fig. 1). Calibration was performed daily using two viscosity standards (Brookfield Engineering Laboratories, Inc.) certified at 47.5 and 101 cps, respectively. In addition, water was measured daily and served as a consistent, low viscosity standard (0.900 to cps). For measurement of semen samples, a 0.5-mL volume of each aliquot was placed in the bottom of the sample cup and evenly spread. Highly viscous samples were placed in the center of the bottom of sample cup and spread as completely and evenly as possible. All samples were measured in triplicate. Viscosity values obtained at 12 rpm have an error factor of or approximately 1% of the value. Seminal Consistency Qualitative evaluation of seminal fluidity followed World Health Organization (WHO) (18) guidelines for measuring seminal consistency. Semen was gently pushed from a syringe through a 21-gauge injection needle. A consistency score of 1 (normal) was assigned when the sample exited the needle in discrete drops or when a thread :::;; 2 em in length formed between the drop and needle tip. A consistency score of 2 (abnormal) was assigned when a thread of >2 em was formed. Semen Quality Sperm motility, motion characteristics, and sperm concentration were determined for treated and untreated aliquots using a computer-aided semen analysis (CASA) system (Motion Analysis, Santa Rosa, CA). A preconstructed counting chamber (#l-cell; Fertility Technologies, Inc., Natick, MA), 12 #lm in depth, was loaded with 5 #LL semen and placed on a phase-contrast microscope equipped with a heated (37 C) stage. A CCD video camera (model TI-23A; NEC Corporation, Broadcast Equipment Department, Irving, TX) attached to the microscope transmitted images from the microscope to the CASA system for evaluation. When sperm concentrations exceeded 40 X 10 6 sperm per ml, semen was diluted with Dulbecco's phosphatebuffered saline (GIBCO, Grand Island, NY) before analysis, as per the CASA system manufacturer's instructions. The CASA motility and motion characteristics results were recorded as percentage motile, straight line velocity (VSL), curvilinear velocity (VCL), mean linearity (LIN), and lateral head displacement (ALH). Sperm Morphology Sperm morphology was evaluated from semen smears that were stained following the simplified Papanicolaou stain technique for sperm (18). Stained smears were coded and then evaluated using brightfield microscopy at X1,000 total magnification under oil. One hundred sperm on each smear were evaluated for head, midpiece, tail, and cytoplasmic droplet morphology based on WHO (18) guidelines. Statistical Analysis Data were analyzed by least-squares methods using the general linear models procedure of the Statistical Analysis System (19). Results are reported as least-squares means ± SEM unless otherwise stated. Experiment 1 RESULTS Semen samples were assigned to two groups based on evaluation of initial consistency (90 minutes after collection), using WHO (18) criteria. Normal initial consistency was evident in 70.6% (n = 24) of the specimens; the remainder (n = 10; 29.4%) exhibited abnormal initial consistency. For semen samples exhibiting normal initial consistency, the viscosity did not exceed 10.6 cps. Seminal I I 1240 Gonzalez-Estrella et al. Dithiothreitol and human semen quality Fertility and Sterility

4 Table 1 Consistency and Viscosity of Untreated, Diluted, and OTT-Treated Human Semen of Normal and Abnormal Initial Consistency (Experiment 1) Normal initial consistency (n = 24)* Abnormal initial consistency (n = 10)* Untreated Dilution DTT Untreated Dilution DTT Consistency* Viscosity (cps) 7.4 ± 0.2:j: 5.2 ± ± ± ± ± ± 1.1 8t 4.8 ± 1.1 *World Health Organization (18) criteria. t Dithiothreitol treatment differs significantly from Untreated and dilution (P < 0.001). :j: All treatments differ significantly (P < 0.05). Untreated differs significantly from Dilution and DTT treatment (P < 0.001). viscosity in this experiment ranged from 3.4 to 23.7 cps. The effects of dilution and DTT treatment on semen viscosity and consistency are shown in Table 1. Overall, dilution and DTT treatment each resulted in significantly reduced viscosity compared with untreated samples. For semen having normal initial consistency, viscosity was progressively reduced (P < 0.05) by dilution and DTT treatment, respectively. For semen exhibiting abnormal initial consistency, a significant reduction in viscosity was evident in diluted versus untreated samples. Treatment with DTT resulted in a further reduction in viscosity that approached significance (P = 0.08). Seminal consistency, on the other hand, did not differ for untreated and diluted samples, whereas it was significantly reduced after treatment with DTT. Experiment 2 The viscosity of semen used in this experiment ranged from 3.4 to 22.8 cps. None of the samples exhibiting normal initial consistency had a viscosity value exceeding 14.6 cps. Samples were assigned to two groups based on initial consistency as in experiment 1. Normal initial consistency was evident in 79.3% (n = 65) of the specimens; the remainder (n = 17; 20.7%) exhibited abnormal initial consistency. Seminal consistency and viscosity results for this experiment are summarized in Table 2. For semen with normal initial consistency, DTT treatment had no effect on consistency. However, for semen with abnormal initial consistency, treatment with DTT caused a significant reduction in consistency to normal. Viscosity, on the other hand, was significantly reduced by DTT treatment, regardless of initial consistency. There were no significant differences between treated and untreated samples for sperm concentration or percentage of motile sperm (Table 2). However, for sperm motion characteristics, samples with normal initial consistency had lower (P < 0.001) VSL and VCL in DTT-treated samples when compared with untreated samples (Table 2). Dithiothreitol did not affect LIN or ALH. For semen having abnormal initial consistency, DTT treatment did not affect VSL, LIN, or ALH; only VCL was significantly reduced. The results of sperm morphology evaluation are summarized in Table 3. Compared with untreated semen, DTT had no effect on the morphology of sperm in semen having abnormal initial consistency. For semen having normal consistency, only tail abnormalities, primarily bent, folded, and coiled tails (data not shown) were elevated (P < 0.01) in DTT-treated versus untreated samples. DISCUSSION Upadhyaya et al. (14), showed that treatment of semen with a commercially available preparation containing DTT resulted in improved seminal fluidity, as measured by qualitative means. The results of the present study are consistent with those earlier results. They show that when the same product was used to achieve a final DTT concentration of 2.95 mm, seminal consistency and viscosity were reduced (Tables 1 and 2). The effect of dilution and the action of DTT both contributed to these reductions (Table 1) and had measurable effects on semen of normal or abnormal initial consistency. The present results show that seminal consistency evaluation using WHO (18) guidelines, a simple but somewhat subjective and nonquantitative test, detected changes in seminal fluidity only for semen having abnormal initial consistency (Tables 1 and 2). In comparison, viscosity measurement, a quantitative, objective evaluation, was considerably more sensitive than evaluating seminal consistency Vol. 62, No.6, December 1994 Gonzalez-Estrella et al. Dithiothreitol and human semen quality 1241

5 Table 2 Consistency, Viscosity, Sperm Concentration, Sperm Motility, and Motion Characteristics Results for Untreated and DTT-Treated Human Semen of Normal and Abnormal Initial Consistency (Experiment 2) Normal initial consistency (n = 65)* Abnormal initial consistency (n = 17)* Untreated Treated Untreated Treated Consistency* Viscosity (cps) Concentration (X10 6 /ml) Motility(%) VSL (llm/s) VCL (llm/s) LIN ALH (11m) 7.7 ± ± ± ± ± ± ± ± 0.1t 74.3 ± ± ± 0.9t 53.7±l.lt 51.2 ± ± ± ± ± ± ± ± ± ± ± O.ot 5.0 ± 0.9t 81.8 ± ± ± ± 2.4:j: 54.9 ± ± 0.1 *World Health Organization (18) criteria. t Treated differs significantly from Untreated (P < 0.001). :j: Treated differs significantly from Untreated (P < 0.05). for detecting the effects of treatment for semen having normal initial consistency (Tables 1 and 2). Viscid human semen presents clinical and laboratory challenges because its fluidity is not ideal for recovering sperm or for evaluating semen. Some evidence indicates that nonliquefied semen may impair sperm motility, as manifested by sluggish sperm movement (1) but not affect the percentage of motile sperm. This suggests that the matrix of the seminal coagulum may restrict sperm movement but not the percentage of moving sperm. Likewise, the matrix may interfere with even distribution of sperm when thorough mixing is attempted before evaluation. The present results show, however, that sperm motion characteristics were comparable between untreated semen of normal versus abnormal initial consistency and that no differences existed between untreated and DTT-treated semen, regardless of initial consistency, for sperm concentration or percentage motile sperm (Table Table 3 Morphology Results for Sperm in Untreated and DTT-Treated Human Semen of Normal and Abnormal Initial Consistency (Experiment 2) Normal Abnormalities Head Midpiece Tail Normal initial consistency (n = 65)* Abnormal initial consistency (n = 17)* Untreated Treated Untreated Treated % 40.1 ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.5t 5.0 ± ± 0.8 *World Health Organization (18) criteria. t Untreated is significantly different from Treated (P < 0.05). 2). Therefore, the liquefaction status of these specimens did not affect the percentage of motile sperm or the distribution of sperm in the semen sample. Uniform distribution of sperm may have occurred either after ejaculation but before coagulation or as a result of mixing of semen, or both. It should be noted that gentle but thorough mixing was attempted for all specimens, liquefied and nonliquefied alike, before sampling. Contrary to the sperm motility results, the sperm motion characteristics were affected by DTT treatment, particularly for sperm in semen exhibiting normal initial consistency. Both VSL and VCL were reduced in DTT-treated semen that had normal initial consistency, whereas only VCL was reduced by DTT treatment in semen having abnormal initial consistency (Table 2). The reduced velocity may have been a result of DTT action on the sperm tail that is known to contain large amounts of sulfhydryl and disulfide material (12). Sperm velocity appeared to be more affected in semen of normal (VSL and VCL) versus abnormal initial consistency (VCL only). This suggests that for semen having abnormal initial consistency, less DTT may have been available to act on the sperm, possibly because of a greater amount of seminal plasma disulfides competing for DTT in that semen. The treatment of semen with DTT generally had little effect on sperm morphology (Table 3). The significantly elevated proportion of tail abnormalities in DTT-treated semen exhibiting normal initial consistency indicate that the sperm tails were adversely affected by DTT treatment. These results are consistent with the sperm motion characteristic results of this experiment that also indi Gonzalez-Estrella et al. Dithiothreitol and human semen quality Fertility and Sterility

6 cated an adverse effect of DTT on sperm tails. However, these results should be interpreted with caution; because the incidence of tail abnormalities tended to be low, the biological relevance is unclear. Semen liquefaction can be induced in vitro using mucolytic agents, mechanical disruption, or dilution. Pipetting through a small bore instrument can improve semen fluidity (1), but irregular results and potential physical trauma to sperm limit its applications. The use of dilution or mucolytic agents such as Avelar, a-amylase, a-chymotrypsin, pancreatic dornase, bromelin, and DTT have been used to improve semen fluidity (3). The present study demonstrated, through objective methods, that DTT was effective in changing seminal fluidity while having minimal effects on sperm motility, motion characteristics, and morphology. Adverse effects of DTT on the sperm were mainly evident when DTT was used under conditions when it would normally not be used, that is, when semen had undergone normal liquefaction. Based on the results of the present study, we conclude that DTT at a final concentration of 2.95 mm was effective in reducing the consistency and viscosity of nonliquefied semen. Although this treatment did not meaningfully affect sperm motility, motion characteristics, or morphology, the effects of DTT on other sperm functional traits must be investigated before its use can be advocated. Although the effects of DTT on other sperm functional traits of human sperm are not known, the present results would suggest that traits relying on motility or viability may not be meaningfully affected. On the other hand, reducing agents such as DTT may likely affect the intermolecular and intramolecular disulfide bonds that stabilize the protamine-dna complex of sperm chromatin (20) and may result in chromatin destabilization, premature nuclear decondensation, or DNA breaks. Similarly, exposure for longer periods than the present experimental conditions may adversely affect the sulfhydryl and disulfide material (12) in the sperm tail. Effects of DTT on the acrosome are not known. Studies are currently being conducted to investigate these aspects. REFERENCES 1. Amelar RD. Coagulation, liquefaction and viscosity of human semen. J Urol 1962;87: Acosta AA, Swanson RJ, Ackerman SB, Kruger TF, van Zyl JA, Menkeveld R. Human spermatozoa in assisted reproduction: laboratory procedures: review and background. Baltimore: Williams and Wilkins, 1990: Bunge RG, Sherman JK. Liquefaction of human semen by alpha-amylase. Fertil Steril1954;5: Finney A, Fukuda A, Breuel K, Thatcher SS. Coagulation and liquefaction of seminal plasma. Asst Rep Rev 1992; 2: Lundwall A, Lilja H. Molecular cloning of human prostate specific antigen edna. FEBS Lett 1987;214: Lilja H, Oldbring J, Rannevik G, Laurel! CB. Seminal vesicle-secreted proteins and their reactions during gelation and liquefaction of human semen. J Clin Invest 1987;80: Lilja H, Abrahamsson PA, Lundwall A. Semenogelin, the predominant protein in human semen. J Bioi Chern 1989; 264: Lilja H, Laurel! CB. The predominant protein in human seminal coagulate. Scand J Clin Lab Invest 1985;45: Wilson VB, Bunge RG. Infertility and semen non-liquefaction. J Urol1975;113: Vermeiden JPW, Bernardus RE, ten Brug CS, Statema Lohmeijer CH, Willemsen-Brugma AM, Schoemaker J. Pregnancy rate is significantly higher in in vitro fertilization procedure with spermatozoa isolated from nonliquefying semen in which liquefaction is induced by a-amylase. Fertil Steril1989;51: Cleland WW. Dithiothreitol, a new protective reagent for SH groups. Biochemistry 1964;3: Calvin HI, Bedford JM. Formation of disulphide bonds in the nucleus and accessory structures of mammalian spermatozoa during maturation in the epididymis. J Reprod Fertil1971;Suppl13: Hirsch SR, Zastrow JE, Kory RC. Sputum liquefying agents: a comparative in vitro evaluation. J Lab Clin Med 1969; 7 4: Upadhyaya M, HibbardBM, Walker SM. Use ofsputolysin for liquefaction of viscid human semen. Fertil Steril 1981;35: Wells RE, Denton R, Merrell EW. Measurement of viscosity of biological fluids by cone plate viscometer. J Lab Clin Med 1961;57: Evans EL, Kirkwood RB, Opsahl DG. The dynamic viscosity of some human blood. Biorheology 1971;8: Lorin Ml, Denning CR, Mandel ID. Viscosity of exocrine secretions in cystic fibrosis: sweat, duodenal fluid and submaxillary saliva. Biorheology 1972;9: World Health Organization. WHO Laboratory manual for the examination of human semen and semen-cervical mucus interaction. 2nd ed. Cambridge: The Press Syndicate of the University of Cambridge, 1987: SAS/STAT User's guide, release 6.03 edition Cary (NC): SAS Institute, Inc. 20. Ward WS, Coffee DS. DNA packaging and organization in mammalian spermatozoa: comparison with somatic cells. Bioi Reprod 1991;44: Vol. 62, No.6, December 1994 Gonzalez-Estrella et al. Dithiothreitol and human semen quality 1243