Vol. 27, No.6, June 1976
FERTILITY AND STERILITY Copyright < 1976 The American Fertility Society
Printed in U.S.A.
A METHOD FOR COLLECTING MOTILE SPERMATOZOA FROM
HUMAN SEMEN ALEXANDER LOPATA, M.B., B.S., PH.D., MARGARET J. PATULLO, DIP. BroCHEM., ALLAN CHANG, M.B., B.S., M.R.C.O.G., F.A.G.O., AND BRIAN JAMES, M.Sc. Department of Obstetrics and Gynecology and Department of Pediatrics, Monash University, Queen Victoria Memorial Hospital, Melbourne, Victoria, Australia
Spermatozoa migrate rapidly into a diluent brought into contact with semen. When this process was used to collect sperm in a diluent contained in Pasteur pipettes that were dipped into semen for 1 hour, concentrations of spermatozoa ranging from 0.6 to 5.7 x 1(Ji cellslml were obtained. This method yielded sperm suspensions free of seminal plasma and populations of spermatozoa that had increased motility ratings compared with the original semen. In contrast, removal of the seminal plasma by a procedure involving three dilutions and two gentle centrifugations resulted in a decrease in the average motility ratings. Determinations were made of the electrolyte, energy substrate, and trace element content of preovulatory human tubal fluid. A sperm diluent having an electrolyte composition similar to that of tubal fluid was formulated and used for collecting spermatozoa. Populations of spermatozoa that migrated from semen into this diluent had significantly greater mean velocities than sperm in the semen. Moreover, the diluent proved to be a favorable medium for significantly delaying the deterioration of sperm progression over a 24-hour period.
At the ovulatory stage of the menstrual cycle the watery mucus in the cervical canal enables the invading spermatozoa to separate from the seminal plasma of the ejaculate. Motile spermatozoa are selected as they penetrate the mucus interface more readily than dead cells. 1, 2 Moreover, the cervical secretion prolongs survival of the sperm and favors their continued passage into the genital tract.2-4 Mechanical activity of the uterus and cervix has also been implicated in sperm transport. 3,4 Evidence often quoted in support of this concept is based on the findings that, in two anesthetized patients treated with oxytocin, carbon particles negotiated the cervical canal and Accepted January 28, 1976.
entered the fallopian tubes when a suspension of carbon particles in 30% dextran was applied to the cervix. 5 Further evidence implicating activity of the genital tract in sperm transport is based on the finding that spermatozoa enter the uterus and oviducts more rapidly than would be possible if their progress was entirely dependent on their intrinsic motility.4 Effective spermatozoal motility is essential, however, for penetration of the cumulus and cornona cell layers surrounding the oocyte, and for impregnating the ovum. It is now clear that in most mammals the passage of sperm through the egg investments is also facilitated by a physiologic change in the spermatozoa 6 -8 that predisposes to membrane breakdown and vesicle formation at the
677
678
June 1976
LOPATA ET AL.
fuging at 1000 rpm for 10 minutes to deposit the cells_ After the supernatant was removed the sperm pellet was resuspended with 4 ml of fresh medium and the gentle centrifugation was repeated. The sperm pellet was again resuspended and then diluted to obtain the required concentration of spermatozoa. This method of washing spermatozoa therefore required two centrifugations and at least three dilutions. Such mechanical procedures may be avoided, and motile sperm, free of seminal plasma and debris, can be collected with the apparatus shown in Figure 1. A similar system was suggested by Bedford. 16 It consists of five Pasteur pipettes, with 4-mm diameter tips, filled with approximately 1.5 ml of modified Tyrode's solution or other diluent that maintains FIG. 1. Apparatus used for collecting motile spermatozoa from semen. Pasteur pipettes with widened tips are filled with diluent, mounted vertically in a support, and lowered to establish contact with the semen. This enables progressive spermatozoa to migrate upward, into the columns of diluent.
acrosome 9 and that appears to induce increased sperm propulsion and penetrability.IO-12 Most of the present investigations were conducted to determine a suitable method of preparing spermatozoa for in vitro fertilization of human oocytes. Initially, sperm suspensions were prepared by the standard washing and dilution techniques in spite of their known disadvantages. 13. 14 Subsequently, a less traumatic method of collecting spermatozoa, using a diluent similar in composition to human tubal fluid, was assessed; the data are presented in this report. MATERIALS AND METHODS
At the outset, spermatozoa were separated from seminal plasma by diluting 1 ml of raw semen with 4 ml of modified Tyrode's solution l5 and centri-
TABLE 1. Composition of Preovulatory Human Tubal Fluid and of a Diluent Used for Collecting Spermatozoa Constituentfl
Tubal fluid'
Sperm diluent'}
Sodium (mM) Potassium (mM) Calcium (mM) Magnesium (mM) Chloride (mM) Lactate (mM) Pyruvate (mM) Protein (gm/100 ml) Iron (/LgIlOO ml) Copper (/Lg/100 ml) Zinc (/Lg/100 mIl Manganese (/Lg/100 ml)
149.2 4.5 1.38 0.19 119.7 2.52 0.18 0.11 58.9 27.8 34.9 2.1
148.2 5.1 2.04 0.20 100.5 21.44 0.33 0.18 9.3 7.1 18.3 2.2
a All electrolyte and trace element determinations were made with a Varian Techtron model 1200 atomic absorption/emission spectrophotometer. Sodium and potassium concentrations were determined by flame atomic emission spectroscopy; calcium, magnesium, and zinc by flame atomic absorption spectroscopy; iron, copper, and manganese by non-flame atomic absorption spectroscopy using a Varian Techtron model 63 carbon rod atomizer; chloride by precipitation of chloride ions with silver nitrate and measurement of unreacted silver by atomic absorption spectroscopy; lactate and pyruvate by ultraviolet determination according to the method in reference 18; protein according to the method of Lowry et aU 9 h Averages based on duplicate analyses of two specimens.
.
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Vol. 27, No.6
sperm viability. When the pipettes were mounted vertically in a support over the semen specimen, loss of diluent was prevented by tightly fitting rubber teats which were used to fill the pipettes. The pipette tips were lowered into a liquefied ejaculate to permit spermatozoa to swim into the columns of diluent. Sperm were usually collected for 30 to 60 minutes in the absence of light1 7 in an incubator at 37' C. The composition of an alternative diluent used for collecting spermatozoa is shown in Table 1. Its formulation (Table 2) is based on the electrolyte composition of human tubal fluid that was obtained at laparoscopy from patients treated with gonadotropins. 20 The concentrations of other components in the diluent are based on the composition of media used for mammalian oocyte culture. 2 1, 22 The osmolarity of the sperm diluent was 295 to 300 mOsmoles/kg, and its pH after gassing with 5% CO 2 was 7.6 ± 0.1. Care was taken to maintain trace elements at the low level present in human tubal fluid (Table 1). Semen specimens were obtained from the husbands of infertile women and from medical students. Ejaculates were TABLE 2. Compounds Used for Preparing the Sperm Diluent Compound
NaCI KCl KH2P0 4 NaHC03 CaCI 2 • 2H20 MgS04 ' 7H 2 0 Sodium pyruvate Sodium lactate Glucose SPPS" HCl (1 N) Penicillin G Streptomycin sulfate
679
COLLECTION OF MOTILE SPERMATOZOA
Concentration
5.3 gmlliter 0.35 gmlliter 0.05 gm/liter 3.0 gmlliter 0.3 gm/liter 0.05 gmlliter 0.036 gm/liter 2.4 gmlliter 1.0 gmlliter 2.0 mill iter 1.0 milliter 100,000 IU/liter 100,000 /-Lglliter
"SPPS, Stable plasma protein solution, made by Commonwealth Serum Laboratories, Melbourne. The solution contains human serum albumin (Cohn fraction V), 86 to 90%, and Cohn fraction IV-4, 10 to 14%. The solution was dialyzed against the diluent for 24 hours prior to use.
collected in sterile plastic jars and used within 1 hour of collection. Standard methods were used for determining sperm counts and motilities. To obtain a quantitive parameter of sperm movement, sperm velocities were determined. All measurements of spermatozoal velocities were made at room temperature, using 20-101,1 samples of sperm, spread uniformly between a slide and a 22 x 22 mm cover slip. Phasecontrast optics at a magnification of x 400 and an eyepiece graticule having a 5 x 5 square grid, calibrated with a stage micrometer, were used to make the measurements. Progressive spermatozoa moving across the ocular grid were timed individually with a stopwatch. The paths traversed and the corresponding times were recorded on a prototype of the grid prepared on data sheets; for ease of extrapolation 1 mm on the prototype square corresponded to 1 p,m of the grid. Measurements were made on random spermatozoa traversing the grid in duplicate 20-p,1 samples. In most samples, therefore, the mean sperm velocity was calculated from the velocity of at least 20 randomly progressive spermatozoa; in some experiments quadruplicate measurements were made. RESULTS
The average concentration of spermatozoa attained in the collection pipettes after 60 minutes ranged from 0.6 to 5.7 x 106 cells/ml (Table 3). There was close agreement in concentration of sperm cc~ lected from the same semen. The large range of sperm concentrations obtained from ejaculates of different individuals reflects variations in semen quality and spermatozoal motility. The average concentration of progressive spermatozoa in the column of diluent increased rapidly during the first 60 minutes of collection. Thereafter the cell concentration increased slowly for up
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LOPATA ET AL.
TABLE 3. Concentration and Motility of Spermatozoa That Entered the Modified Tyrode's Solution Contained in Pipettes Which Were Dipped in Semen for 60 Minutes Original semen
Collected spermatozoa
}6'S%
t
} 18'4%
t
1%
~721J
Donor
Count"
Motility
Count" 10 6
x 10 1i
%
T. S. B.D. E. V. B. A. F. R. C.O. C.A. S. T.
196 134 187 108 122 88 59 173
80 70 40 50 80 30 80 70
5.7 1.9 2.0 0.6 1.0 2.7 1.2 1.8
Mean
133
62.5
2.1
a
X
Motility %
95 95 75 100 95 50 80 95
to 120 minutes (Fig. 2). Figure 3 shows the pattern of sperm distribution in columns of diluent after 30 minutes and 60 minutes of collection. Prior to analysis the first two drops of diluent were discarded to remove contaminating seminal fluid from the tip of the pipette. The results for each time interval are mean values based on duplicate sperm counts at six levels of diluent in two pipettes. As may be seen in Figure 3, the lower half of the column of diluent contained approximately 85% of the spermatozoa at each time interval. Since sperm collection depended on movement of propulsive spermatozoa
Z
a:~E
Y2 HR COLLECTION
oX
1 HR COLLECTION
FIG. 3. Distribution of spermatozoa at different levels of diluent in Pasteur pipettes dipped into liquefied semen for 'h hour and 1 hour. Sperm counts were made after the first two drops of diluent were discarded to remove seminal fluid from the tips. For both collection times the values at each level of the columns of diluent represent the mean sperm concentration x 105 , based on duplicate counts in two pipettes. The percentage of spermatozoa present in the upper, middle, and lower one-third of diluent are shown to the right of each column.
against gravity, the method yielded populations of spermatozoa with up to 100% progressive motility (Table 3). A markedly improved percentage motility, compared with that in the original semen, was obtained for all specimens (Table 3). On the other hand, when the seminal TABLE 4. Motility of Spermatozoa after Their Washing in the Modified Tyrode's Solution to Remove the Seminal Plasma
1·0 08
Donor
Original semen Count
Motility
0·6
°0 ""- 04 UJ
20
75 .1
85.6
Average of duplicate counts.
Q ~
7 ' 9%
Zc 0
0=
E a:0·2 UJ
:::!
Q.
rJ)
15
20
COLLECTION TIME(HR)
FIG. 2. Time course of accumulation of spermatozoa in columns of diluent contained in Pasteur pipettes that were dipped into liquefied semen. At each time interval the sperm concentration in the diluent was determined after the first two drops were discarded to avoid contamination with semen. Each point on the graph is the average value of duplicate sperm counts.
Motility of washed sperm
x 10 6
%
%
D.A. S. T. S.K. F.E. G.1. K.1. C.A. L. E. E. Y. G.L. B. U. B.N. S.1.
167 50 225 426 112 39 45 105 24 20 64 34 145
90 60 75 70 80 70 85 75 60 50 50 80
80 40 40 60 30 60 40 70 75 50 40 50 70
Mean
112
71.2
54.2
80
.
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Vol. 27, No.6
681
COLLECTION OF MOTILE SPERMATOZOA
TABLE 5. Comparison of the Mean Velocity of Spermatozoa in Semen and in a Diluent Resembling Tubal Fluid Factor
No. of observations
SD
Mean velocity /-Lmlsec
Specimen Au In semen In diluent Specimen Bb In semen In diluent
40 40
19.8 26.6
4.4 8.3
39 40
13.8 27.7
4.5 10.3
Analysis of variance
Source of variation df
SSQ
Due to semen Due to diluent Interaction Residual error
1 1 1 155
223.42 4,238.12 502.48 8,366.42
Total
158
13,330.44
Variance
F
223.42 4,238.12 502.48 53.98
4.14" 79.52d 9.31 d
aOriginal sperm count, 165 x 106/ml; sperm count in diluent, 0.37 x 106/ml; dilution, 1:446. bOriginal sperm count, 138 x 106/ml; sperm count in diluent, 0.41 x 106/ml; dilution, 1:336. cp < 0.05. dp < 0.005.
plasma was removed by the direct dilution and gentle centrifugation procedure described under "Materials and Methods," sperm motility generally deteriorated to varying extents (Table 4). Tables 5, 6A, and'6B show that there was a significant difference between the mean velocity of spermatozoa in the semen of different men. The tables also show that the diluent caused a highly significant increase in the mean sperm velocity of spermatozoa in all specimens studied; this effect occurred even when spermatozoa became diluted more than 400-fold. From comparison of the F ratios (Table 6B) of the main effects of diluent and semen (199.90 -;- 8.57 = 23.33) it may be concluded that the increases in mean spermatozoal velocity caused by the diluent were greater than the differences in the mean spermatozoal velocity between semen. Moreover, since there was no statistically significant interaction either between the effects of diluent and semen or between the effects of diluent and time, the changes in the mean velocity of spermatozoa appear to have been produced entirely by the action of the diluent.
The mean velocity of spermatozoa in the semen decreased significantly after 24 hours (Tables 6A and 6B). Furthermore, the significant interaction between the effects of semen and time indicates that the magnitude of the decrease in TABLE 6A. Comparison of the Effects of Time on the Mean Velocity of Spermatozoa in Semen and in a Diluent Resembling Tubal Fluid Factor
No. of
Mean
observations
velocity
SD
pm!sec
Specimen Ca In semen After 24 hr in semen In diluent After 24 hr in dil uen t Specimen D" In semen After 24 hr in semen In diluent After 24 hr in diluent Specimen Ec In semen After 24 hr in semen In diluent After 24 hr in dil uent
5 10 36 32
33.7 15.4 38.9 23.1
12.4 9.4 15.3 9.4
40 40 40 40
27.5 11.8 40.9 27.4
6.8 4.3 8.5 10.4
40 40 40 40
22.5 15.1 28.8 32.4
6.8 6.2 8.1 10.3
a Original sperm count, 130 x count in diluent, 0.37 x 106/m l; bOriginal sperm count, 170 x count in diluent, 1.4 x 106/m l; "Original sperm count, 145 x count in diluent, 1.6 x 106/m l;
106/ml; sperm dilution, 1:350. 106/m l; sperm dilution, 1:120. 106/m l; sperm dilution, 1:90.
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LOPATA ET AL.
TABLE 6B. Analysis of Variance for the Data of Table 6A Analysis of variance
Source of variation
dt
SSQ
Variance
F
684.48 15,969.85 10,175.26 Negligible 2,276.85 Negligible Negligible 79.89
8.57" 199.90" 126.72" NSb 28.50" NS NS
Due to semen Due to diluent Due to time Due to semen-diluent interaction Due to semen-time interaction Due to diluent-time interaction Due to semen-diluent-time interaction Residual error
396
1,368.96 15,969.85 10,175.26 Negligible 4,553.85 Negligible Negligible 31,637.55
Total
402
63,705.47
2 1 1 2
"p < 0.005. bNS, Not significant.
mean sperm velocity after 24 hours was different in different semen specimens. Since there was no statistically significant interaction between semen specimens and diluent (Table 6B) it may be concluded that the change in mean spermatozoal velocity produced by the diluent was of the same order of magnitude in different specimens. Similarly, the statistically insignificant interaction between the effects of time and diluent indicates that the changes in mean velocity of spermatozoa of all men studied were of similar magnitude after 24 hours in the diluent. It should be noted, however, that in two patients the mean velocity of spermatozoa in the diluent decreased after 24 hours, whereas in the third patient the mean velocity increased after the same period of time. DISCUSSION
Progressive spermatozoa rapidly entered a diluent contained in pipettes that were dipped into a semen specimen. This is a gentle method for collecting a suspension of spermatozoa largely free of seminal plasma. Such sperm suspensions may be used directly for insemination of oocytes in vitro or for intrauterine instillation of motile spermatozoa during artificial insemination. Centrifugation of the dilute sperm suspensions (1000 rpm for 10 minutes) yields
approximately a 10-fold increase in concentration of progres~ive spermatozoa. The concentrated samples may then be used for metabolic studies or for lowtemperature storage after protective substances such as glycerol or egg yolk are added. It has been reported that dilution of spermatozoa with various physiologic solutions immediately reduces motility and survival ratings and decreases the average speed of sperm progression. 14, 23 In contrast to this, passage of spermatozoa into columns of diluent having a composition similar to tubal fluid yielded sperm with greatly improved motility and survival ratings, which in all cases had an increased average speed of progression as compared with sperm in the original semen. Therefore, this method of sperm collection should be of value in the preparation of spermatozoa for storage at low temperatures, since sperm with better forward progression have improved survival and forward progression ratings after freezing and storage. 23 Dilute suspensions of spermatozoa (0.5 to 1.0 x 106 cells/ml) obtained directly from columns of diluent have been used for insemination of preovulatory oocytes in vitro.24 The viscous cumulus surrounding the oocytes was dispersed within 6 hours of incubation. At 12 to 18 hours after insemination numerous spermatozoa had penetrated the corona radiata
Vol. 27, No.6
COLLECTION OF MOTILE SPERMATOZOA
683
FIG. 4. Spennatozoa attached to the zona pellucida of an oocyte placed in a diluent resembling tubal fluid and containing 1.5 x 106 sperm/ml. After 12 hours the cumulus that invested the egg was completely dispersed, and some of the corona cells were removed as the egg was transferred onto a slide. The egg was photographed in a droplet of diluent, using phase-contrast optics at a magnification of x 400.
cell layer and become attached to, or embedded in, the zona pellucida (Fig. 4). The passage of spermatozoa through the egg investments and their interaction with the zona pellucida indicate that the diluent permitted development of the acrosome reaction. Acknowledgments. We are grateful to Professor Carl Wood for his helpful advice and encouragement. Mrs. Margaret Dooley provided technical assistance with a part of this work.
REFERENCES 1. Moghissi KS: Spenn migration through cervical mucus. In Pathways to Conception, Edited by AI Sherman. Springfield Ill, Charles C Thomas, 1971, p 214
2. Moghissi KS: Sperm migration through the human cervix. In Cervical Mucus in Human Reproduction: World Health Organization Colloquium, Edited by M Elstein, KS Moghissi, R Borth. Copenhagen, Scriptor, 1973, p 128 3. Hafez ESE: Transport of spennatozoa in the female reproductive tract. Am J Obstet Gynecol 115:703, 1973 4. Hafez ESE, Thibault CG: International symposium on the biology of spermatozoa: Transport, survival, and fertilizing ability. Fertil Steril 25 :825, 1974 5. Egli GE, Newton M: The transport of carbon particles in the human female reproductive tract. Fertil Steril 12:151,1961 6. Austin CR: Capacitation and release of hyaluronidase. J Reprod Fertil1:310, 1960 7. Bedford JM: The importance of capacitation for establishing contact between eggs and sperm in the rabbit. J Reprod Fertil 13:365, 1967 8. Bedford JM: Spenn capacitation and fertilization in mammals. BioI Reprod (Suppl 2) 2:128, 1970
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LOPATA ET AL.
9. Barros C, Bedford JM, Franklin LE, Austin CR: Membrane vesiculation as a feature of the mammalian acrosome reaction. J Cell BioI 34:C1,1967 10. Yanagimachi R: The movement of golden hamster spermatozoa before and after capacitation. J Reprod Fertil 23:193, 1970 11. Bedford JM: Morphologic aspects of sperm capacitation in mammals. In Advances in the Biosciences: Schering Symposium on Mechanisms Involved in Conception, Berlin, March 13 to 15, 1969, Vol 4, Edited by G Raspe. New York, Pergamon Press, 1970, p 35 12. Bedford JM: Sperm transport, capacitation and fertilization. In Reproductive Biology, Edited by H Balin, S Glasser. Amsterdam, Excerpta Medica Foundation, 1972, p 338 13. Chang MC: Effect of dilution on fertilizing capacity of rabbit spermatozoa. Science 104:361, 1946 14. Harvey C: The speed of human spermatozoa and the effect on it of various diluents, with some preliminary observations on clinical material. J Reprod Fertil1:84, 1960 15. Edwards RG, Bavister BD, Steptoe PC: Early stages of fertilization in vitro of human oocytes matured in vitro. Nature 221:632, 1969 16. Bedford JM: Personal communication, 1973
June 1976
17. Hamner CE, Williams WL: The effect of light on the respiration of spermatozoa. Biochem Biophy.s Res Commun 5:316, 1961 18. Sigma Technical Bulletin 726/826-UV. StLouis Mo, Sigma Chemical Co, October 1968 19. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J BioI Chem 193:265, 1951 20. Talbot JM, Dooley M, Leeton J, Lopata A, McMlllfter R, Wood C, Brown JB, Evans JH: Gonadotrophin stimulation for oocyte recovery and in vitro fertilization in infertile women. Aust NZ J Obstet Gynaecol. In press 21. Biggers JD, Whitten WR, Whittingham DG: The culture of mouse embryos in vitro. In Methods in Mammalian Embryology, Edited by JC Daniel Jr. San Francisco, WH Freeman & Co, 1971, p 86 22. Gwatkin RBL: Defmed media and development of manpnalian eggs in vitro. Ann NY Acad Sci 139:79, 1966 23. Freund M, Wiederman J: Factors affecting the dilution, freezing and storage of human semen. J Reprod Fertil11:1, 1966 24. Lopata A, Brown JB, Leeton J, McMaster R, Talbot .rM, Wood C: The maturation of human ovarial\ eggs and their fertilization and cleavage in vitro. Unpublished data
FERTILITY AND STERILITY Copyright < 1976 The American Fertility Society
Printed in U.S.A.
A METHOD FOR COLLECTING MOTILE SPERMATOZOA FROM
HUMAN SEMEN ALEXANDER LOPATA, M.B., B.S., PH.D., MARGARET J. PATULLO, DIP. BroCHEM., ALLAN CHANG, M.B., B.S., M.R.C.O.G., F.A.G.O., AND BRIAN JAMES, M.Sc. Department of Obstetrics and Gynecology and Department of Pediatrics, Monash University, Queen Victoria Memorial Hospital, Melbourne, Victoria, Australia
Spermatozoa migrate rapidly into a diluent brought into contact with semen. When this process was used to collect sperm in a diluent contained in Pasteur pipettes that were dipped into semen for 1 hour, concentrations of spermatozoa ranging from 0.6 to 5.7 x 1(Ji cellslml were obtained. This method yielded sperm suspensions free of seminal plasma and populations of spermatozoa that had increased motility ratings compared with the original semen. In contrast, removal of the seminal plasma by a procedure involving three dilutions and two gentle centrifugations resulted in a decrease in the average motility ratings. Determinations were made of the electrolyte, energy substrate, and trace element content of preovulatory human tubal fluid. A sperm diluent having an electrolyte composition similar to that of tubal fluid was formulated and used for collecting spermatozoa. Populations of spermatozoa that migrated from semen into this diluent had significantly greater mean velocities than sperm in the semen. Moreover, the diluent proved to be a favorable medium for significantly delaying the deterioration of sperm progression over a 24-hour period.
At the ovulatory stage of the menstrual cycle the watery mucus in the cervical canal enables the invading spermatozoa to separate from the seminal plasma of the ejaculate. Motile spermatozoa are selected as they penetrate the mucus interface more readily than dead cells. 1, 2 Moreover, the cervical secretion prolongs survival of the sperm and favors their continued passage into the genital tract.2-4 Mechanical activity of the uterus and cervix has also been implicated in sperm transport. 3,4 Evidence often quoted in support of this concept is based on the findings that, in two anesthetized patients treated with oxytocin, carbon particles negotiated the cervical canal and Accepted January 28, 1976.
entered the fallopian tubes when a suspension of carbon particles in 30% dextran was applied to the cervix. 5 Further evidence implicating activity of the genital tract in sperm transport is based on the finding that spermatozoa enter the uterus and oviducts more rapidly than would be possible if their progress was entirely dependent on their intrinsic motility.4 Effective spermatozoal motility is essential, however, for penetration of the cumulus and cornona cell layers surrounding the oocyte, and for impregnating the ovum. It is now clear that in most mammals the passage of sperm through the egg investments is also facilitated by a physiologic change in the spermatozoa 6 -8 that predisposes to membrane breakdown and vesicle formation at the
677
678
June 1976
LOPATA ET AL.
fuging at 1000 rpm for 10 minutes to deposit the cells_ After the supernatant was removed the sperm pellet was resuspended with 4 ml of fresh medium and the gentle centrifugation was repeated. The sperm pellet was again resuspended and then diluted to obtain the required concentration of spermatozoa. This method of washing spermatozoa therefore required two centrifugations and at least three dilutions. Such mechanical procedures may be avoided, and motile sperm, free of seminal plasma and debris, can be collected with the apparatus shown in Figure 1. A similar system was suggested by Bedford. 16 It consists of five Pasteur pipettes, with 4-mm diameter tips, filled with approximately 1.5 ml of modified Tyrode's solution or other diluent that maintains FIG. 1. Apparatus used for collecting motile spermatozoa from semen. Pasteur pipettes with widened tips are filled with diluent, mounted vertically in a support, and lowered to establish contact with the semen. This enables progressive spermatozoa to migrate upward, into the columns of diluent.
acrosome 9 and that appears to induce increased sperm propulsion and penetrability.IO-12 Most of the present investigations were conducted to determine a suitable method of preparing spermatozoa for in vitro fertilization of human oocytes. Initially, sperm suspensions were prepared by the standard washing and dilution techniques in spite of their known disadvantages. 13. 14 Subsequently, a less traumatic method of collecting spermatozoa, using a diluent similar in composition to human tubal fluid, was assessed; the data are presented in this report. MATERIALS AND METHODS
At the outset, spermatozoa were separated from seminal plasma by diluting 1 ml of raw semen with 4 ml of modified Tyrode's solution l5 and centri-
TABLE 1. Composition of Preovulatory Human Tubal Fluid and of a Diluent Used for Collecting Spermatozoa Constituentfl
Tubal fluid'
Sperm diluent'}
Sodium (mM) Potassium (mM) Calcium (mM) Magnesium (mM) Chloride (mM) Lactate (mM) Pyruvate (mM) Protein (gm/100 ml) Iron (/LgIlOO ml) Copper (/Lg/100 ml) Zinc (/Lg/100 mIl Manganese (/Lg/100 ml)
149.2 4.5 1.38 0.19 119.7 2.52 0.18 0.11 58.9 27.8 34.9 2.1
148.2 5.1 2.04 0.20 100.5 21.44 0.33 0.18 9.3 7.1 18.3 2.2
a All electrolyte and trace element determinations were made with a Varian Techtron model 1200 atomic absorption/emission spectrophotometer. Sodium and potassium concentrations were determined by flame atomic emission spectroscopy; calcium, magnesium, and zinc by flame atomic absorption spectroscopy; iron, copper, and manganese by non-flame atomic absorption spectroscopy using a Varian Techtron model 63 carbon rod atomizer; chloride by precipitation of chloride ions with silver nitrate and measurement of unreacted silver by atomic absorption spectroscopy; lactate and pyruvate by ultraviolet determination according to the method in reference 18; protein according to the method of Lowry et aU 9 h Averages based on duplicate analyses of two specimens.
.
pz-------------------~------------------
Vol. 27, No.6
sperm viability. When the pipettes were mounted vertically in a support over the semen specimen, loss of diluent was prevented by tightly fitting rubber teats which were used to fill the pipettes. The pipette tips were lowered into a liquefied ejaculate to permit spermatozoa to swim into the columns of diluent. Sperm were usually collected for 30 to 60 minutes in the absence of light1 7 in an incubator at 37' C. The composition of an alternative diluent used for collecting spermatozoa is shown in Table 1. Its formulation (Table 2) is based on the electrolyte composition of human tubal fluid that was obtained at laparoscopy from patients treated with gonadotropins. 20 The concentrations of other components in the diluent are based on the composition of media used for mammalian oocyte culture. 2 1, 22 The osmolarity of the sperm diluent was 295 to 300 mOsmoles/kg, and its pH after gassing with 5% CO 2 was 7.6 ± 0.1. Care was taken to maintain trace elements at the low level present in human tubal fluid (Table 1). Semen specimens were obtained from the husbands of infertile women and from medical students. Ejaculates were TABLE 2. Compounds Used for Preparing the Sperm Diluent Compound
NaCI KCl KH2P0 4 NaHC03 CaCI 2 • 2H20 MgS04 ' 7H 2 0 Sodium pyruvate Sodium lactate Glucose SPPS" HCl (1 N) Penicillin G Streptomycin sulfate
679
COLLECTION OF MOTILE SPERMATOZOA
Concentration
5.3 gmlliter 0.35 gmlliter 0.05 gm/liter 3.0 gmlliter 0.3 gm/liter 0.05 gmlliter 0.036 gm/liter 2.4 gmlliter 1.0 gmlliter 2.0 mill iter 1.0 milliter 100,000 IU/liter 100,000 /-Lglliter
"SPPS, Stable plasma protein solution, made by Commonwealth Serum Laboratories, Melbourne. The solution contains human serum albumin (Cohn fraction V), 86 to 90%, and Cohn fraction IV-4, 10 to 14%. The solution was dialyzed against the diluent for 24 hours prior to use.
collected in sterile plastic jars and used within 1 hour of collection. Standard methods were used for determining sperm counts and motilities. To obtain a quantitive parameter of sperm movement, sperm velocities were determined. All measurements of spermatozoal velocities were made at room temperature, using 20-101,1 samples of sperm, spread uniformly between a slide and a 22 x 22 mm cover slip. Phasecontrast optics at a magnification of x 400 and an eyepiece graticule having a 5 x 5 square grid, calibrated with a stage micrometer, were used to make the measurements. Progressive spermatozoa moving across the ocular grid were timed individually with a stopwatch. The paths traversed and the corresponding times were recorded on a prototype of the grid prepared on data sheets; for ease of extrapolation 1 mm on the prototype square corresponded to 1 p,m of the grid. Measurements were made on random spermatozoa traversing the grid in duplicate 20-p,1 samples. In most samples, therefore, the mean sperm velocity was calculated from the velocity of at least 20 randomly progressive spermatozoa; in some experiments quadruplicate measurements were made. RESULTS
The average concentration of spermatozoa attained in the collection pipettes after 60 minutes ranged from 0.6 to 5.7 x 106 cells/ml (Table 3). There was close agreement in concentration of sperm cc~ lected from the same semen. The large range of sperm concentrations obtained from ejaculates of different individuals reflects variations in semen quality and spermatozoal motility. The average concentration of progressive spermatozoa in the column of diluent increased rapidly during the first 60 minutes of collection. Thereafter the cell concentration increased slowly for up
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TABLE 3. Concentration and Motility of Spermatozoa That Entered the Modified Tyrode's Solution Contained in Pipettes Which Were Dipped in Semen for 60 Minutes Original semen
Collected spermatozoa
}6'S%
t
} 18'4%
t
1%
~721J
Donor
Count"
Motility
Count" 10 6
x 10 1i
%
T. S. B.D. E. V. B. A. F. R. C.O. C.A. S. T.
196 134 187 108 122 88 59 173
80 70 40 50 80 30 80 70
5.7 1.9 2.0 0.6 1.0 2.7 1.2 1.8
Mean
133
62.5
2.1
a
X
Motility %
95 95 75 100 95 50 80 95
to 120 minutes (Fig. 2). Figure 3 shows the pattern of sperm distribution in columns of diluent after 30 minutes and 60 minutes of collection. Prior to analysis the first two drops of diluent were discarded to remove contaminating seminal fluid from the tip of the pipette. The results for each time interval are mean values based on duplicate sperm counts at six levels of diluent in two pipettes. As may be seen in Figure 3, the lower half of the column of diluent contained approximately 85% of the spermatozoa at each time interval. Since sperm collection depended on movement of propulsive spermatozoa
Z
a:~E
Y2 HR COLLECTION
oX
1 HR COLLECTION
FIG. 3. Distribution of spermatozoa at different levels of diluent in Pasteur pipettes dipped into liquefied semen for 'h hour and 1 hour. Sperm counts were made after the first two drops of diluent were discarded to remove seminal fluid from the tips. For both collection times the values at each level of the columns of diluent represent the mean sperm concentration x 105 , based on duplicate counts in two pipettes. The percentage of spermatozoa present in the upper, middle, and lower one-third of diluent are shown to the right of each column.
against gravity, the method yielded populations of spermatozoa with up to 100% progressive motility (Table 3). A markedly improved percentage motility, compared with that in the original semen, was obtained for all specimens (Table 3). On the other hand, when the seminal TABLE 4. Motility of Spermatozoa after Their Washing in the Modified Tyrode's Solution to Remove the Seminal Plasma
1·0 08
Donor
Original semen Count
Motility
0·6
°0 ""- 04 UJ
20
75 .1
85.6
Average of duplicate counts.
Q ~
7 ' 9%
Zc 0
0=
E a:0·2 UJ
:::!
Q.
rJ)
15
20
COLLECTION TIME(HR)
FIG. 2. Time course of accumulation of spermatozoa in columns of diluent contained in Pasteur pipettes that were dipped into liquefied semen. At each time interval the sperm concentration in the diluent was determined after the first two drops were discarded to avoid contamination with semen. Each point on the graph is the average value of duplicate sperm counts.
Motility of washed sperm
x 10 6
%
%
D.A. S. T. S.K. F.E. G.1. K.1. C.A. L. E. E. Y. G.L. B. U. B.N. S.1.
167 50 225 426 112 39 45 105 24 20 64 34 145
90 60 75 70 80 70 85 75 60 50 50 80
80 40 40 60 30 60 40 70 75 50 40 50 70
Mean
112
71.2
54.2
80
.
p-------------------~~~--~-~--~-~~----
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681
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TABLE 5. Comparison of the Mean Velocity of Spermatozoa in Semen and in a Diluent Resembling Tubal Fluid Factor
No. of observations
SD
Mean velocity /-Lmlsec
Specimen Au In semen In diluent Specimen Bb In semen In diluent
40 40
19.8 26.6
4.4 8.3
39 40
13.8 27.7
4.5 10.3
Analysis of variance
Source of variation df
SSQ
Due to semen Due to diluent Interaction Residual error
1 1 1 155
223.42 4,238.12 502.48 8,366.42
Total
158
13,330.44
Variance
F
223.42 4,238.12 502.48 53.98
4.14" 79.52d 9.31 d
aOriginal sperm count, 165 x 106/ml; sperm count in diluent, 0.37 x 106/ml; dilution, 1:446. bOriginal sperm count, 138 x 106/ml; sperm count in diluent, 0.41 x 106/ml; dilution, 1:336. cp < 0.05. dp < 0.005.
plasma was removed by the direct dilution and gentle centrifugation procedure described under "Materials and Methods," sperm motility generally deteriorated to varying extents (Table 4). Tables 5, 6A, and'6B show that there was a significant difference between the mean velocity of spermatozoa in the semen of different men. The tables also show that the diluent caused a highly significant increase in the mean sperm velocity of spermatozoa in all specimens studied; this effect occurred even when spermatozoa became diluted more than 400-fold. From comparison of the F ratios (Table 6B) of the main effects of diluent and semen (199.90 -;- 8.57 = 23.33) it may be concluded that the increases in mean spermatozoal velocity caused by the diluent were greater than the differences in the mean spermatozoal velocity between semen. Moreover, since there was no statistically significant interaction either between the effects of diluent and semen or between the effects of diluent and time, the changes in the mean velocity of spermatozoa appear to have been produced entirely by the action of the diluent.
The mean velocity of spermatozoa in the semen decreased significantly after 24 hours (Tables 6A and 6B). Furthermore, the significant interaction between the effects of semen and time indicates that the magnitude of the decrease in TABLE 6A. Comparison of the Effects of Time on the Mean Velocity of Spermatozoa in Semen and in a Diluent Resembling Tubal Fluid Factor
No. of
Mean
observations
velocity
SD
pm!sec
Specimen Ca In semen After 24 hr in semen In diluent After 24 hr in dil uen t Specimen D" In semen After 24 hr in semen In diluent After 24 hr in diluent Specimen Ec In semen After 24 hr in semen In diluent After 24 hr in dil uent
5 10 36 32
33.7 15.4 38.9 23.1
12.4 9.4 15.3 9.4
40 40 40 40
27.5 11.8 40.9 27.4
6.8 4.3 8.5 10.4
40 40 40 40
22.5 15.1 28.8 32.4
6.8 6.2 8.1 10.3
a Original sperm count, 130 x count in diluent, 0.37 x 106/m l; bOriginal sperm count, 170 x count in diluent, 1.4 x 106/m l; "Original sperm count, 145 x count in diluent, 1.6 x 106/m l;
106/ml; sperm dilution, 1:350. 106/m l; sperm dilution, 1:120. 106/m l; sperm dilution, 1:90.
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TABLE 6B. Analysis of Variance for the Data of Table 6A Analysis of variance
Source of variation
dt
SSQ
Variance
F
684.48 15,969.85 10,175.26 Negligible 2,276.85 Negligible Negligible 79.89
8.57" 199.90" 126.72" NSb 28.50" NS NS
Due to semen Due to diluent Due to time Due to semen-diluent interaction Due to semen-time interaction Due to diluent-time interaction Due to semen-diluent-time interaction Residual error
396
1,368.96 15,969.85 10,175.26 Negligible 4,553.85 Negligible Negligible 31,637.55
Total
402
63,705.47
2 1 1 2
"p < 0.005. bNS, Not significant.
mean sperm velocity after 24 hours was different in different semen specimens. Since there was no statistically significant interaction between semen specimens and diluent (Table 6B) it may be concluded that the change in mean spermatozoal velocity produced by the diluent was of the same order of magnitude in different specimens. Similarly, the statistically insignificant interaction between the effects of time and diluent indicates that the changes in mean velocity of spermatozoa of all men studied were of similar magnitude after 24 hours in the diluent. It should be noted, however, that in two patients the mean velocity of spermatozoa in the diluent decreased after 24 hours, whereas in the third patient the mean velocity increased after the same period of time. DISCUSSION
Progressive spermatozoa rapidly entered a diluent contained in pipettes that were dipped into a semen specimen. This is a gentle method for collecting a suspension of spermatozoa largely free of seminal plasma. Such sperm suspensions may be used directly for insemination of oocytes in vitro or for intrauterine instillation of motile spermatozoa during artificial insemination. Centrifugation of the dilute sperm suspensions (1000 rpm for 10 minutes) yields
approximately a 10-fold increase in concentration of progres~ive spermatozoa. The concentrated samples may then be used for metabolic studies or for lowtemperature storage after protective substances such as glycerol or egg yolk are added. It has been reported that dilution of spermatozoa with various physiologic solutions immediately reduces motility and survival ratings and decreases the average speed of sperm progression. 14, 23 In contrast to this, passage of spermatozoa into columns of diluent having a composition similar to tubal fluid yielded sperm with greatly improved motility and survival ratings, which in all cases had an increased average speed of progression as compared with sperm in the original semen. Therefore, this method of sperm collection should be of value in the preparation of spermatozoa for storage at low temperatures, since sperm with better forward progression have improved survival and forward progression ratings after freezing and storage. 23 Dilute suspensions of spermatozoa (0.5 to 1.0 x 106 cells/ml) obtained directly from columns of diluent have been used for insemination of preovulatory oocytes in vitro.24 The viscous cumulus surrounding the oocytes was dispersed within 6 hours of incubation. At 12 to 18 hours after insemination numerous spermatozoa had penetrated the corona radiata
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COLLECTION OF MOTILE SPERMATOZOA
683
FIG. 4. Spennatozoa attached to the zona pellucida of an oocyte placed in a diluent resembling tubal fluid and containing 1.5 x 106 sperm/ml. After 12 hours the cumulus that invested the egg was completely dispersed, and some of the corona cells were removed as the egg was transferred onto a slide. The egg was photographed in a droplet of diluent, using phase-contrast optics at a magnification of x 400.
cell layer and become attached to, or embedded in, the zona pellucida (Fig. 4). The passage of spermatozoa through the egg investments and their interaction with the zona pellucida indicate that the diluent permitted development of the acrosome reaction. Acknowledgments. We are grateful to Professor Carl Wood for his helpful advice and encouragement. Mrs. Margaret Dooley provided technical assistance with a part of this work.
REFERENCES 1. Moghissi KS: Spenn migration through cervical mucus. In Pathways to Conception, Edited by AI Sherman. Springfield Ill, Charles C Thomas, 1971, p 214
2. Moghissi KS: Sperm migration through the human cervix. In Cervical Mucus in Human Reproduction: World Health Organization Colloquium, Edited by M Elstein, KS Moghissi, R Borth. Copenhagen, Scriptor, 1973, p 128 3. Hafez ESE: Transport of spennatozoa in the female reproductive tract. Am J Obstet Gynecol 115:703, 1973 4. Hafez ESE, Thibault CG: International symposium on the biology of spermatozoa: Transport, survival, and fertilizing ability. Fertil Steril 25 :825, 1974 5. Egli GE, Newton M: The transport of carbon particles in the human female reproductive tract. Fertil Steril 12:151,1961 6. Austin CR: Capacitation and release of hyaluronidase. J Reprod Fertil1:310, 1960 7. Bedford JM: The importance of capacitation for establishing contact between eggs and sperm in the rabbit. J Reprod Fertil 13:365, 1967 8. Bedford JM: Spenn capacitation and fertilization in mammals. BioI Reprod (Suppl 2) 2:128, 1970
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9. Barros C, Bedford JM, Franklin LE, Austin CR: Membrane vesiculation as a feature of the mammalian acrosome reaction. J Cell BioI 34:C1,1967 10. Yanagimachi R: The movement of golden hamster spermatozoa before and after capacitation. J Reprod Fertil 23:193, 1970 11. Bedford JM: Morphologic aspects of sperm capacitation in mammals. In Advances in the Biosciences: Schering Symposium on Mechanisms Involved in Conception, Berlin, March 13 to 15, 1969, Vol 4, Edited by G Raspe. New York, Pergamon Press, 1970, p 35 12. Bedford JM: Sperm transport, capacitation and fertilization. In Reproductive Biology, Edited by H Balin, S Glasser. Amsterdam, Excerpta Medica Foundation, 1972, p 338 13. Chang MC: Effect of dilution on fertilizing capacity of rabbit spermatozoa. Science 104:361, 1946 14. Harvey C: The speed of human spermatozoa and the effect on it of various diluents, with some preliminary observations on clinical material. J Reprod Fertil1:84, 1960 15. Edwards RG, Bavister BD, Steptoe PC: Early stages of fertilization in vitro of human oocytes matured in vitro. Nature 221:632, 1969 16. Bedford JM: Personal communication, 1973
June 1976
17. Hamner CE, Williams WL: The effect of light on the respiration of spermatozoa. Biochem Biophy.s Res Commun 5:316, 1961 18. Sigma Technical Bulletin 726/826-UV. StLouis Mo, Sigma Chemical Co, October 1968 19. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J BioI Chem 193:265, 1951 20. Talbot JM, Dooley M, Leeton J, Lopata A, McMlllfter R, Wood C, Brown JB, Evans JH: Gonadotrophin stimulation for oocyte recovery and in vitro fertilization in infertile women. Aust NZ J Obstet Gynaecol. In press 21. Biggers JD, Whitten WR, Whittingham DG: The culture of mouse embryos in vitro. In Methods in Mammalian Embryology, Edited by JC Daniel Jr. San Francisco, WH Freeman & Co, 1971, p 86 22. Gwatkin RBL: Defmed media and development of manpnalian eggs in vitro. Ann NY Acad Sci 139:79, 1966 23. Freund M, Wiederman J: Factors affecting the dilution, freezing and storage of human semen. J Reprod Fertil11:1, 1966 24. Lopata A, Brown JB, Leeton J, McMaster R, Talbot .rM, Wood C: The maturation of human ovarial\ eggs and their fertilization and cleavage in vitro. Unpublished data
