FACTORS AFFECTING SPERM MOTILITY. VII. SPERM VIABILITY AS AFFECTED BY CHANGE OF ph AND OSMOLARITY OF SEMEN AND URINE SPECIMENS

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1 FERTLTY AND STERLTY Copyright c 1981 The American Fertility Society Vol. 36, No.4, October 1981 Printed in U.SA. FACTORS AFFECTNG SPERM MOTLTY. V. SPERM VABLTY AS AFFECTED BY CHANGE OF ph AND OSMOLARTY OF SEMEN AND URNE SPECMENS AMNON MAH:.LER, M.D.* RAM DAVD, M.D.t ZEEV BLUMENFELD, M.D. OR S. BET'ER, M.D.* nfertility nstitute, Department of Obstetrics and Gynecology, and Department of Nephrology, Rambam Medical Center, Technion-Faculty of Medicine, Haifa, srael The effects of ph and osrrwlarity of semen and urine specimens on motility and velocity of human spermatozoa were studied objectively with the aid of the multiple exposure photography (MEP) method. The ph of fresh ejaculates ranged from 7.2 to 8.2 and specimens were slightly hyperosrrwtic ranging between 300 to 380 mosmlkg. Gradually changing the ph and osmolarity to either side of normal values led to progressive loss of sperm motility. However, sperm velocity was slightly increased by mild alkalinization and hyperosrrwlarity. Spermatozoa that became imrrwbilized by acidification regained their motility shortly after ph was restored to normal values. n the majority of instances spermatozoa lost their rrwtility when mixed with fresh urine specimens. Neutralization of urinary ph could not protect them from this effect unless urine osmolarity was also isotonically adjusted. t is suggested that patients with retrograde ejaculation should adequately increase their fluid intake before recovery of sperm from their bladder for artificial insemination. Fertil Steril 36:507, 1981 Ejaculated spermatozoa remain only for a short duration within seminal fluid. Soon they penetrate the cervical mucus, from which they migrate into the uterine cavity and the fallopian tubes. However, the majority of them become mixed with the contents of the vagina. n certain pathologic cases, like retrograde ejaculation, semen enters the bladder instead of being expelled from the body. While being mixed with one of these media, spermatozoa may be subjected to changes in the ph and osmolarity. t is generally accepted that spermatozoa cannot survive under substantial changes of both these variables. Received February 2, 1981; revised and accepted May 26, Reprint requests: Amnon Makler, M.D., nfertility nstitute, Department of Obstetrics and Gynecology, Rambam Medical Center, Technion-Faculty of Medicine, Haifa, srael. tpart of MD Thesis as required by the Technion-Faculty of Medicine. *Professor and Chief, Department of Nephrology. 507 So far, very few reports about the influence of these factors on sperm motility and viability have been published. 1-4 Since this subject was studied in the past mainly by subjective and inaccurate methods of motility evaluation, some important issues concerning physiologic and clinical aspects could not be provided. n the present study, the multiple exposure photography (MEP) method for objective and accurate sperm motility determination was utilized to investigate the effect of ph and osmolarity on human sperm motility. The aim of the study was to apply the provided information to research and clinical problems, especially that of retrograde ejaculation. MATERALS AND METHODS Fresh ejaculates from fertile and infertile patients attending our clinic were analyzed routinely within 1 hour, including ph and osmolarity

2 AY MAKLER ET AL X2 7.1 z.t 7.5 X6 X ad &1 &2 ph O OLARTY FG. 1. Distribution of values of osmolarity and ph among analyzed semen specimens. determinations. Specimens considered as normospermic according to standards established in our nstitute 5 were divided into several aliquots of 0.9 ml. The first one served as a control, and the others were incorporated in the following experiments. Experiment 1. Aliquots of 0.1 ml from stock solutions of either HC or NaC of concentrations from 0.1 to 0.5 N were added to the experimental test tubes. The same volume of normal saline was added to each control specimen. Final ph values and sperm motility were determined before and after 15, 60, and 240 minutes. Experiment 2. Aliquots of 0.1 ml from stock solutions of NaC of concentrations from 2% to 10% or distilled water from 0.1 to 1.0 ml were added to experimental test tubes. Equivalent volumes of isotonic NaC were added to control specimens. Final osmolarity and sperm motility were determined before and after 15, 60, and 240 minutes. Experiment 3. Specimens were mixed with 5 times their volume of fresh urine specimens. The latter were either original urine or urine specimens that had been neutralized to ph 7.4 or isotonically adjusted or had undergone both procedures. Control specimens had been diluted to the same extent with normal saline. Final values of ph and osmolarity as well as sperm motility were determined after 30 minutes. The ph was determined with a glass electrode ph meter (model October 1981 PBS720, E-Hama, srael). Osmolarity was assessed by the cryoscopic method using the Fiske Osmometer (model 130, Uxbridge, MA). Sperm motility was determined with the multiple exposure photography (MEP) method described in detail elsewhere. 6, 7 Each of two or three drops from a well-mixed specimen were placed in each improved 10 jj.m chambers (manufactured by Sefi-Medical nstruments, Haifa, srael) and 8 to 12 fields containing 200 to 400 spermatozoa per specimen were photographed at a predetermined location. Each film was exposed for 1 second, during which time the sample was illuminated by six light pulses. mages of photographed sperm were projected onto sheets of paper, from which the percentage of motile sperm and their velocity were calculated with the specially designed microcomputer. 9 The results from all experiments were statistically evaluated, and significance was determined by Student's paired t-test. RESULTS The ph of fresh specimens ranged within narrow limits from 7.2 to 8.2, with an average of7.65. ~ '541 - U 6S =.,..'! U " AV -n' V"CTY _ looo 0-. FG. 2. Change in sperm velocity and percentage of motility 1 hour after being subjected to variations in ph and osmolarity of the seminal fluid. Each point indicates mean value of 32 specimens.

3 . Vol. 36, No.4 FACTORS AFFECTNG SPERM MOTLTY 509 " i o _ - -~ _ 240 FG. 3. Change of index of motility (product of sperm velocity and percentage of motility) with time after being subjected to variations in ph and osmolarity of the seminal fluid. Each point indicates the mean value of 32 specimens. Depression of sperm motility was not complete immediately but progressed gradually throughout the first 4 hours of incubation (Fig. 3). n no case was the harmful effect followed by spontaneous recovery of sperm motility or when osmolarity was readjusted. However, neutralization of specimens acidified below ph 5 was followed by substantial recovery of previously immobilized spermatozoa. This reversibility of immobilization occurred after specimens were acidified for 1 to 4 hours. n no case could neutralization restore immobilized spermatozoa in previously alkalinized specimens (Fig. 4). All randomly selected 38 urinary specimens had low ph and were hyperosmotic relative to arterial blood. n the majority of cases their mixture with spermatozoa induced complete inhibition of motility (Fig. 5). Motile spermatozoa were not found in urine specimens of ph below 5.5 or when osmolarity exceeded 810 mosm. On the contrary, motility was well preserved in urinary specimens of low osmolarity and high ph. Neutralizing the acidity of urine prior to its mixture with semen could not protect spermatozoa from the immobilization effect of intact urine. However, making urine isotonic decreased markedly its immobilization effect (Fig. 6). Adjustment of both osmolarity and ph of urine before its use for semen dilution further inhibited its harmful effect on sperm motility. All specimens exhibited slight hyperosmotic values that ranged from 300 to 380 with an average of 337 mosm/kg (Fig. 1). No correlation was found between these variables and seminal volume, sperm concentration, percentage of motility, sperm velocity, and abnormal forms. Neither was correlation found between osmolarity of semen and urinary specimens voided by the same patients at the same time. Sperm velocity and percentage of motility varied considerably as a result of change in these variables from ph of 5.25 to 9.75, or between 150 and 1000 mosmlkg. However, these parameters were not equally affected and not in a pure symmetrical form (Fig. 2). Velocity was more resistant than percentage of motility to the inhibitory effect induced by increments of ph in the alkaline zone, and in about 30% of the cases velocity exhibited some improvement by slight alkalinization. Hypoosmolarity inhibited both parameters more extensively than did hypertonic solutions. n most cases velocity was highest when osmolarity increased to the optimal value of 400 mosmlkg. DSCUSSON The narrow distribution of ph and osmotic values in all analyzed specimens and the lack of correlation with any of the routinely evaluated seminal parameters indicate that a routine determination of these variables in fresh semen specimens is of questionable value. We could not confirm reports by others in which ph values increased shortly after ejaculation, due to loss of CO 2 The ph remained stable for a long time and -,." , MOTU ~ V.LOC," 0 -- ~ wn 1.1,.". FG. 4. Sperm motility in specimens where ph was restored 15 minutes after being acidified or alkalinized (n = 12).

4 is 'C'$ 510 MAKLER ET AL. October 1981 pm 1.D o o 0 00 : " o i " 01 _onu lo.ot.l1 _Nl_ -----~- --!~.~--+~~----!.-: "" FG. 5. Viability of spermatozoa 30 minutes after being mixed with various urine specimens. Each one of the 37 points indicates ph and osmolarity of the tested urine specimens. dropped only after extensive bacterial growth, as was shown by us elsewhere.12 Osmolarity values of intact specimens were surprisingly higher than those reported by Keitel.13 t appears that the prostate or seminal vesicle can modify the concentration of body fluids and render them hypertonic. The physiologic implication of this finding is still obscure, and further investigation is needed for determination of whether it depends on state of hydration, geographic, climatic, or ethnic variations, though no correlation was found between semen and urine specimens of the same patients. While determination of semen ph and osmolarity of intact semen per se is not of much value, very useful information has been collected from the study of sperm motility when subjected to variations of ph and osmolarity. Generally speaking, sperm could resist an alkaline and hypertonic environment better than one of acidity or hypotonicity. n a substantial number of cases sperm velocity improved by a small shift into the alkaline or hypertonic zones. Nevertheless, we could not dis- - - pm UR'.' _on -on,. OTL 0 _VlLOCtTY ~. - u... FG. 6. Motility of spermatozoa diluted in urine before and after their ph and osmolarity have been adjusted, as compared with specimens diluted in normal saline (n = 12). cover even a single case where asthenospermic or nonmotile sperm showed any "revitalization" or improvement of motility when ph and osmolarity were varied in either direction from the original values. From a biologic standpoint, optimum levels of ph and osmolarity for both sperm velocity and viability coincided with those naturally found along the female reproductive tract, starting with cervical mucus,14, 15 followed by the uterine cavity16 and the fallopian tubes.17 Only the vaginal contents are highly acidic,18, 19 inducing an immobilization effect. However, as was shown by others in animals20 and by us in the human being, this effect is almost completely reversible when ph is restored to original levels. t is tempting to believe that the vaginal acidity is, after all, not spermicidic, as it is commonly assumed; temporarily immobilized sperm may subsequently migrate from the depth of the vagina into the cervical canal long after ejaculation. The finding that sperm immobilization does not necessarily reflect a permanent stage and that spermatozoa could be triggered by changing their environmental conditions is an encouraging fact for those who seek certain ways to improve or to induce revitalization of asthenospermic patients. Treatment of retrograde ejaculation is based mainly on the recovery of active spermatozoa from the bladder t has been suggested that urinary ph be neutralized as much as possible by proper diet or medicaments.24 However, the importance of reducing urine hyperosmolarity by encouraging the patients to increase adequately their fluid intake has been ignored.25 Chang and Thorsteinsson26 discussed the harmful effect of hypertonic urine on sperm motility in the rabbit with only minimal reduction of its fertilizing capacity. As it was shown by us, optimal adjustment of both urinary ph and osmolarity could prevent the synergistic harmful effect of these variables on sperm motility. t is hoped that this may increase the chance for recovery of motile spermatozoa and the rate of subsequent conceptions in properly treated patients. REFERENCES 1. Blackshaw AW, Emmens CW: The interaction of ph, osmotic pressure and electrocyte concentration on the motility of ram, bull, and human spermatozoa. J Physiol 114:6, Chang MC, Thorsteinsson T: Effect of osmotic pressure and hydrogen-ion concentration on the motility and fertilizing capacity of rabbit spermatozoa. Fertil Steril 9:510, 1958

5 Vol. 36, No.4 FACTORS AFFECTNG SPERM MOTLTY Emmens CW: The effect of variations in osmotic pressure and electrocyte concentration on the motility of rabbit spermatozoa at different hydrogen-ion concentration. J Physiol 107:129, Mitchell JA, Nelson L, Hafez ESE: Motility of spermatozoa. n Human Semen and Fertility Regulation in Men, Edited by ESE Hafez. St. Louis, C. V. Mosby Co, 1976, p Makler A, tskovitz J, Brandes JM, Apldi E: Sperm velocity and percent of motility in 100 normospermic specimens analyzed by the MEP method. Fertil Steril 31:155, Makler A: A new multiple exposure photography method for objective human spermatozoal motility determination. Fertil Steril 30:192, Makler A: Use of the elaborated multiple exposure photography method in routine semen analysis and for research purposes. Fertil Steril 33:160, Makler A: The improved 10 J.Lm chamber for rapid sperm count and motility evaluation. Fertil Steril33:337, Makler A: Use of a microcomputer in combination with the MEP technique for human sperm motility determination. J Urol 124:372, Amelar RD, Dubin L, Walsh PC: Male nfertility. Philadelphia, W. B. Saunders Co, 1977, p Schirren S: Practical Andrology. Berlin, Verlag Bruder Hartmann, 1972, p Makler A, Urbach Y, Lefler E, Mertzbach D: Factors affecting sperm motility. V. Sperm viability under influence of bacterial growth in human ejaculation. n press 13. Keitel HG, Jones HS: The mineral and water composition of normal human sperm. J Lab Clin Med 47:917, Moghissi KS: Cyclic changes of cervical mucus in normal and progestin-treated women. Fertil Steril 17:663, MacDonald RR, Lumbley ill: Endocervical ph measured in vivo through the normal menstrual cycle. Obstet Gynecol 35:202, Feo LG: The ph of the human uterine cavity in situ. Am J Obstet Gynecol 70:60, Blandau R, Jensen L, Rumery R: Determination of the ph values of the reproductive tract fluids of the rat during heat. Fertil Steril 9:207, Rakof AE, Feo LG, Goldstein L: The biologic characteristics of the normal vagina. Am J Obstet Gynecol 47:467, Horne HW, Thibault JP: Sperm migration through the human female reproductive tract. Fertil Steril 13:135, Mann T: The biochemistry of semen and of the male reproductive tract. London, Methuen & Co, Ltd, 1964, p Hotchkiss PR, Pinto AB, Kleegman SJ: Artificial insemination with semen recovered from the bladder. Fertil Steril 6:37, Bourne RB, Kretzschmar WA, Esser JH: Successful artificial insemination in a diabetic with retrograde ejaculation. Fertil Steril 22:275, Glezerman M, Lunenfeld B, Potashnik G, Oelsner G, Beer R: Retrograde ejaculation: pathophysiologic aspects and report of two successfully treated cases. Fertil Steril 27: 796, Schram JD: Retrograde ejaculation: a new approach to therapy. Fertil Steril 27:1216, Crich JP, Jequier AM: nfertility in men with retrograde ejaculation: the action of urine on sperm motility and a simple method for achieving antegrade ejaculation. Fertil Steril 30:572, Chang MC, Thorsteinsson T: Effect of urine on motility and fertilizing capacity of rabbit spermatozoa. Fertil Steril 9:231, 1958