Journal of Assisted Reproduction and Genetics, Vol. 9, No. 3, 1992
MALE-FACTOR INFERTILITY
The Effects of Lignocaine on Human Sperm Motility STEPHEN J O H N BENNETT, 1'2 VIRGINIA BOLTON,1 and JOHN PARSONS 1
Submitted: January 22, 1992 Accepted: March 17, 1992
tected in the follicular fluid aspirated subsequently, with reported levels ranging from 0 to 156 txg/ml (2,3). Transient exposure of the oocyte to these levels of lignocaine within the follicle would seem unlikely to have any significant adverse effects on the outcome of in vitro fertilization (IVF), since units utilizing this form of analgesia can achieve results comparable to those of units employing alternative techniques. It has been suggested that follicular fluid might be used to supplement culture media used for IVF, since follicular fluid has been shown to enhance sperm capacitation and increase hyperactivated motility. However, if follicular fluid is to be used in the IVF culture system, contamination with lignocaine will result in prolonged exposure of both oocytes and sperm to the drug, with potentially adverse effects on sperm motility and fertilizing capacity. Therefore, we examined the effect of varying concentrations of lignocaine on sperm motility during 4 hr of incubation under capacitating conditions, using a Celltrack/S digital computerized sperm analyzer (Launch Diagnostics Ltd., Kent, England).
Objective: The purpose of this study was to assess the motility of human sperm incubated with various concentrations of lignocaine. Methods: Eleven semen samples with a sperm density >~20 × 106 ml and progressive motility >i40% were prepared using a swim-up technique. Aliquots from each sample were incubated for 4 hr under capacitating conditions with lignocaine concentrations of lO0, 10, 1, and 0.1 txg/ml and without additional lignocaine as a control. Digital computerized motion analysis was pelformed on all samples at 1, 2, and 4 hr after the addition of lignocaine. Results: After 2 hr of incubation a significant (P < 0.05) increase in the percentage of sperm with a curvilinear velocity >100 I~m/sec was observed in those samples incubated with 100 ~tg/ml lignocaine. This stimulatory effect was no longer apparent after a further 2 hr of incubation. No other significant changes were identified in any of the motility parameters examined. Conclusions: No adverse effects on human sperm motility were identified during incubation with low concentrations of lignocaine. A transient stimuIatory effect was observed at a lignocaine concentration of I00 p.g/ml. KEY WORDS: lignocaine; human; sperm; motility.
MATERIALS AND METHODS
INTRODUCTION
Semen samples (n = 1 l) were obtained from patients attending our laboratory for routine semen analysis. Only those samples with a sperm density t>20 million/ml and motility ~>40%, showing rapid linear progressive motility (WHO grade A motility), were included in this study. Isolated teratozoospermia was not considered a contraindication to inclusion since motility was the parameter under investigation. Semen samples were prepared using Earle's balanced salt solution (EBS; Life Technologies, Uxbridge, England) supplemented with 10% (v/v) al-
Lignocaine and other local anesthetic agents are known to inhibit sperm motility. Although the precise molecular basis for this action is not clear, previous work has shown the effect to be irreversible and dose dependent (1). When lignocaine is used to induce local anesthesia of the vaginal vault during transvaginal oocyte recovery, significant levels of the drug can be de1 Assisted Conception Unit, 7th Floor New Ward Block, King's College Hospital, Denmark Hill, London SE5 8RX, UK. 2 To whom correspondence should be addressed.
271
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BENNETT, BOLTON, AND PARSONS
buminar (Armour Pharmaceutical Company Ltd., Eastbourne, England). A standard centrifugation and swim-up technique (4) was used to produce final sperm preparations of >170% motility. Each prepared specimen was subdivided into five equal portions (0.2-0.5 ml), to each of which was added an equal volume of EBS + 10% albuminar containing lignocaine, to give final lignocaine concentrations of 100, 10, 1, 0.1, and 0 p~g/ml, respectively. The final motile sperm concentration in each sample was the same. The samples were incubated at 37°C in 5% CO2 in humidified air, and an aliquot from each sample was analyzed blind by a single operator (SB) 1, 2, and 4 hr following the addition of lignocaine. Samples were encoded with random numbers prior to analysis by an independent observer. Samples were analyzed in a 12-/xm Microcell counting chamber (Launch Diagnostics, Kent, England) using a heated microscope stage at 37°C. Computerized sperm motility analysis was performed using a frame speed of 60 frames/sec to calculate the percentage motility, mean curvilinear velocity (VCL), mean straight-line velocity (VSL), mean linearity (LIN), and mean amplitude of lateral head displacement (ALH). The percentage of cells exhibiting ALH ~>7 txm and CVL >~100 ~xm/sec was calculated, to give an indication of the degree of hyperactivation present. All results were analyzed using paired t tests.
LIN, ALH) available for the remaining two sampies. Analysis of the percentage motility, VCL, VSL, LIN, ALH, and subgroup of sperm with ALH i>7 Ixm failed to demonstrate any statistically significant differences between any of the lignocaine concentrations tested and control samples at any stage of incubation. The mean percentage of sperm with VCL >~100 ~xm/sec was significantly increased after 2 hr of incubation at the highest concentration of lignocaine tested (100 ixg/ml; Table I), the percentage rising from 8.4% in control samples to 13.9% (P < 0.05). This difference was no longer apparent after 4 hr of incubation. Mean VCL also increased from 78.3 to 88.8 p,m/sec in conjunction with the increase in the percentage of sperm with VCL >/100 p,m/sec, but this difference was not significant at the P < 0.05 level.
DISCUSSION The range of lignocaine concentrations tested in this study was of the same order of magnitude as that found in follicular fluid when lignocaine was used for local anesthesia during oocyte collection (2,3). Even at the highest concentration tested (100 txg/ml), we were unable to demonstrate any adverse effects on a range of sperm motility parameters. This result was somewhat surprising since previous work had suggested that detectable adverse effects on sperm motility could be demonstrated at lignocaine concentrations as low as 0.1 txg/ml (1). However, an earlier paper showed that the concentration of lignocaine required to inhibit sperm motility by 50% was 4624 ixg/ml (5). This is consider-
RESULTS Complete data sets were obtained for nine sampies, with only partial data (percentage motility,
Table I. The Percentage of Sperm Showing a Curvilinear Velocity 1>100 ~m/sec After Incubation for Various Lengths of Time a 1-hr incubation
2-hr incubation
4-hr incubation
Sample no.
X
Y
X
Y
X
Y
1 2 3 4 5 6 7 8 9
1.57 13.16 3.92 5.88 8.82 8.87 5.94 33.00 6.62
2.21 11.76 8.00 7.84 14,00 11.00 7.55 8.77 12.50
1.63 12.93 1.96 7.41 10.68 10.91 5.26 18.42 6.67
10.66 23.65 7.84 0.00 12.62 15.84 20.00 27.35 7.50
4.00 6.48 0.00 0.00 4.72 12.00 8.26 16.66 5.60
3.39 3.05 0.00 0.00 9.90 17.92 7.96 12.61 2.61
Mean +-- SD Probability
9.75 ~ 9.31
9.29 - 3.51
8.43 - 5.40
13.94 -+ 8.66
6.41 +-- 5.38
0.88
0.03
6.38 -+ 6.15 0.98
" X = control samples; Y = samples incubated with 100 ixg/ml lignocaine.
Journal of Assisted Reproduction and Genetics, Vol. 9, No. 3, 1992
LIGNOCAINE AND SPERM MOTILITY
ably higher than tested here and the presence of an undetected minor effect cannot be excluded. The mean concentrations of lignocaine recorded from follicular fluid aspirates in previous series are several orders of magnitude lower than the maximum recorded level of 156 ~g/ml. If follicular fluid obtained from combining multiple aspirates is used in the culture system, the final lignocaine concentration to which sperm would be exposed would be considerably lower than the maximum level of I00 ~g/ml that we tested. Furthermore, unless this fluid is used undiluted, the addition of culture medium will reduce the final lignocaine concentration even further. Since we were unable to demonstrate any adverse effects on sperm motility even at lignocaine concentrations of 100 ~g/ml, it appears that the addition of follicular fluid contaminated with lignocaine to the culture system used for IVF would be unlikely to produce any adverse effects on sperm motility. The significance of the transient increase in VCL observed in samples incubated with 100 ~g/ml of lignocaine is uncertain. The statistically significant increase in the percentage of sperm with VCL/> 100 pxrdsec in the absence of any increase in the overall percentage motility suggests stimulation of hyperactivated motility in already motile sperm. However, the application of an alternative criterion for identifying hyperactivated cells, namely, ALH ~>7 (6), fails to support this suggestion. Alternatively, since local anesthetics at doses lower than those required to inhibit motility have been shown to stimulate sperm motility (and respiration) in several mammalian species (7), it is possible that this observation reflects a direct stimulatory action. If the
Journal of Assisted Reproduction and Genetics, Vol. 9, No. 3, 1992
273
latter explanation is correct, than the transient nature of the increase in motility remains unexplained. The fact that low levels of lignocaine appear to exert a stimulatory effect on human sperm motility is intriguing and merits further investigation.
REFERENCES 1. Thorneycrofl IH, Wun W-SA, Ewell M, Wheeler C, McFarland C: The effects of local anaesthetics on sperm motility (ABSTR). Presented at the 44th annual meeting of American Fertility Society, Atlanta, GA, Oct 10-13, 1988. American Fertility Society Official Program, 1988, p 58 2. Wickland M, Enk L, Evers H, Hamberger L, Jakobsson A-H, Nilsson L, Sandquist U, Sjoblom P: Lidocaine does not influence the in vitro fertilization and embryo cleavage in an human IVF/ET program (ABSTR). Presented at the 5th annual meeting of European Society of Human Reproduction and Embryology, Malmo, Sweden, June 25-28, 1989. European Society of Human Reproduction and Embryology Official Program, 1989, p 85 3. Bailey-Pridham DD, Cook CL, Reshef E, Hurst HE, Drury K, Yussman MA: Follicular fluid Lidocaine levels during transvag~nal oocyte retrieval. Fertil Steril 1990;53:171 4. Lopata A, Patullo MJ, Chang A, James B: A method for collecting motile spermatozoa from human semen. Fertil Steril 1976;27:677 5. Yong CY, Wu P, Shieh CC, Chiang BN: Membrane stabilising activity and inhibition of human sperm motility. Lancet 1986;2:402 6. Robertson L, Wolf DP, Tash JS: Temporal changes in motility parameters related to acrosomal status: Identification and characterization of populations of hyperactivated human sperm. Biol Reprod 1988;39:797 7. Singh JP, Babcock DF, Lardy HA: Motility activation, respiratory stimulation and alteration of C a 2+ transport in bovine sperm treated with amine local anaesthetics and calcium transport antagonists. Arch Biochem Biophys 1983; 221:291
MALE-FACTOR INFERTILITY
The Effects of Lignocaine on Human Sperm Motility STEPHEN J O H N BENNETT, 1'2 VIRGINIA BOLTON,1 and JOHN PARSONS 1
Submitted: January 22, 1992 Accepted: March 17, 1992
tected in the follicular fluid aspirated subsequently, with reported levels ranging from 0 to 156 txg/ml (2,3). Transient exposure of the oocyte to these levels of lignocaine within the follicle would seem unlikely to have any significant adverse effects on the outcome of in vitro fertilization (IVF), since units utilizing this form of analgesia can achieve results comparable to those of units employing alternative techniques. It has been suggested that follicular fluid might be used to supplement culture media used for IVF, since follicular fluid has been shown to enhance sperm capacitation and increase hyperactivated motility. However, if follicular fluid is to be used in the IVF culture system, contamination with lignocaine will result in prolonged exposure of both oocytes and sperm to the drug, with potentially adverse effects on sperm motility and fertilizing capacity. Therefore, we examined the effect of varying concentrations of lignocaine on sperm motility during 4 hr of incubation under capacitating conditions, using a Celltrack/S digital computerized sperm analyzer (Launch Diagnostics Ltd., Kent, England).
Objective: The purpose of this study was to assess the motility of human sperm incubated with various concentrations of lignocaine. Methods: Eleven semen samples with a sperm density >~20 × 106 ml and progressive motility >i40% were prepared using a swim-up technique. Aliquots from each sample were incubated for 4 hr under capacitating conditions with lignocaine concentrations of lO0, 10, 1, and 0.1 txg/ml and without additional lignocaine as a control. Digital computerized motion analysis was pelformed on all samples at 1, 2, and 4 hr after the addition of lignocaine. Results: After 2 hr of incubation a significant (P < 0.05) increase in the percentage of sperm with a curvilinear velocity >100 I~m/sec was observed in those samples incubated with 100 ~tg/ml lignocaine. This stimulatory effect was no longer apparent after a further 2 hr of incubation. No other significant changes were identified in any of the motility parameters examined. Conclusions: No adverse effects on human sperm motility were identified during incubation with low concentrations of lignocaine. A transient stimuIatory effect was observed at a lignocaine concentration of I00 p.g/ml. KEY WORDS: lignocaine; human; sperm; motility.
MATERIALS AND METHODS
INTRODUCTION
Semen samples (n = 1 l) were obtained from patients attending our laboratory for routine semen analysis. Only those samples with a sperm density t>20 million/ml and motility ~>40%, showing rapid linear progressive motility (WHO grade A motility), were included in this study. Isolated teratozoospermia was not considered a contraindication to inclusion since motility was the parameter under investigation. Semen samples were prepared using Earle's balanced salt solution (EBS; Life Technologies, Uxbridge, England) supplemented with 10% (v/v) al-
Lignocaine and other local anesthetic agents are known to inhibit sperm motility. Although the precise molecular basis for this action is not clear, previous work has shown the effect to be irreversible and dose dependent (1). When lignocaine is used to induce local anesthesia of the vaginal vault during transvaginal oocyte recovery, significant levels of the drug can be de1 Assisted Conception Unit, 7th Floor New Ward Block, King's College Hospital, Denmark Hill, London SE5 8RX, UK. 2 To whom correspondence should be addressed.
271
1058-046802/0600-0271506.50/0 © 1992PlenumPublishingCorporation
272
BENNETT, BOLTON, AND PARSONS
buminar (Armour Pharmaceutical Company Ltd., Eastbourne, England). A standard centrifugation and swim-up technique (4) was used to produce final sperm preparations of >170% motility. Each prepared specimen was subdivided into five equal portions (0.2-0.5 ml), to each of which was added an equal volume of EBS + 10% albuminar containing lignocaine, to give final lignocaine concentrations of 100, 10, 1, 0.1, and 0 p~g/ml, respectively. The final motile sperm concentration in each sample was the same. The samples were incubated at 37°C in 5% CO2 in humidified air, and an aliquot from each sample was analyzed blind by a single operator (SB) 1, 2, and 4 hr following the addition of lignocaine. Samples were encoded with random numbers prior to analysis by an independent observer. Samples were analyzed in a 12-/xm Microcell counting chamber (Launch Diagnostics, Kent, England) using a heated microscope stage at 37°C. Computerized sperm motility analysis was performed using a frame speed of 60 frames/sec to calculate the percentage motility, mean curvilinear velocity (VCL), mean straight-line velocity (VSL), mean linearity (LIN), and mean amplitude of lateral head displacement (ALH). The percentage of cells exhibiting ALH ~>7 txm and CVL >~100 ~xm/sec was calculated, to give an indication of the degree of hyperactivation present. All results were analyzed using paired t tests.
LIN, ALH) available for the remaining two sampies. Analysis of the percentage motility, VCL, VSL, LIN, ALH, and subgroup of sperm with ALH i>7 Ixm failed to demonstrate any statistically significant differences between any of the lignocaine concentrations tested and control samples at any stage of incubation. The mean percentage of sperm with VCL >~100 ~xm/sec was significantly increased after 2 hr of incubation at the highest concentration of lignocaine tested (100 ixg/ml; Table I), the percentage rising from 8.4% in control samples to 13.9% (P < 0.05). This difference was no longer apparent after 4 hr of incubation. Mean VCL also increased from 78.3 to 88.8 p,m/sec in conjunction with the increase in the percentage of sperm with VCL >/100 p,m/sec, but this difference was not significant at the P < 0.05 level.
DISCUSSION The range of lignocaine concentrations tested in this study was of the same order of magnitude as that found in follicular fluid when lignocaine was used for local anesthesia during oocyte collection (2,3). Even at the highest concentration tested (100 txg/ml), we were unable to demonstrate any adverse effects on a range of sperm motility parameters. This result was somewhat surprising since previous work had suggested that detectable adverse effects on sperm motility could be demonstrated at lignocaine concentrations as low as 0.1 txg/ml (1). However, an earlier paper showed that the concentration of lignocaine required to inhibit sperm motility by 50% was 4624 ixg/ml (5). This is consider-
RESULTS Complete data sets were obtained for nine sampies, with only partial data (percentage motility,
Table I. The Percentage of Sperm Showing a Curvilinear Velocity 1>100 ~m/sec After Incubation for Various Lengths of Time a 1-hr incubation
2-hr incubation
4-hr incubation
Sample no.
X
Y
X
Y
X
Y
1 2 3 4 5 6 7 8 9
1.57 13.16 3.92 5.88 8.82 8.87 5.94 33.00 6.62
2.21 11.76 8.00 7.84 14,00 11.00 7.55 8.77 12.50
1.63 12.93 1.96 7.41 10.68 10.91 5.26 18.42 6.67
10.66 23.65 7.84 0.00 12.62 15.84 20.00 27.35 7.50
4.00 6.48 0.00 0.00 4.72 12.00 8.26 16.66 5.60
3.39 3.05 0.00 0.00 9.90 17.92 7.96 12.61 2.61
Mean +-- SD Probability
9.75 ~ 9.31
9.29 - 3.51
8.43 - 5.40
13.94 -+ 8.66
6.41 +-- 5.38
0.88
0.03
6.38 -+ 6.15 0.98
" X = control samples; Y = samples incubated with 100 ixg/ml lignocaine.
Journal of Assisted Reproduction and Genetics, Vol. 9, No. 3, 1992
LIGNOCAINE AND SPERM MOTILITY
ably higher than tested here and the presence of an undetected minor effect cannot be excluded. The mean concentrations of lignocaine recorded from follicular fluid aspirates in previous series are several orders of magnitude lower than the maximum recorded level of 156 ~g/ml. If follicular fluid obtained from combining multiple aspirates is used in the culture system, the final lignocaine concentration to which sperm would be exposed would be considerably lower than the maximum level of I00 ~g/ml that we tested. Furthermore, unless this fluid is used undiluted, the addition of culture medium will reduce the final lignocaine concentration even further. Since we were unable to demonstrate any adverse effects on sperm motility even at lignocaine concentrations of 100 ~g/ml, it appears that the addition of follicular fluid contaminated with lignocaine to the culture system used for IVF would be unlikely to produce any adverse effects on sperm motility. The significance of the transient increase in VCL observed in samples incubated with 100 ~g/ml of lignocaine is uncertain. The statistically significant increase in the percentage of sperm with VCL/> 100 pxrdsec in the absence of any increase in the overall percentage motility suggests stimulation of hyperactivated motility in already motile sperm. However, the application of an alternative criterion for identifying hyperactivated cells, namely, ALH ~>7 (6), fails to support this suggestion. Alternatively, since local anesthetics at doses lower than those required to inhibit motility have been shown to stimulate sperm motility (and respiration) in several mammalian species (7), it is possible that this observation reflects a direct stimulatory action. If the
Journal of Assisted Reproduction and Genetics, Vol. 9, No. 3, 1992
273
latter explanation is correct, than the transient nature of the increase in motility remains unexplained. The fact that low levels of lignocaine appear to exert a stimulatory effect on human sperm motility is intriguing and merits further investigation.
REFERENCES 1. Thorneycrofl IH, Wun W-SA, Ewell M, Wheeler C, McFarland C: The effects of local anaesthetics on sperm motility (ABSTR). Presented at the 44th annual meeting of American Fertility Society, Atlanta, GA, Oct 10-13, 1988. American Fertility Society Official Program, 1988, p 58 2. Wickland M, Enk L, Evers H, Hamberger L, Jakobsson A-H, Nilsson L, Sandquist U, Sjoblom P: Lidocaine does not influence the in vitro fertilization and embryo cleavage in an human IVF/ET program (ABSTR). Presented at the 5th annual meeting of European Society of Human Reproduction and Embryology, Malmo, Sweden, June 25-28, 1989. European Society of Human Reproduction and Embryology Official Program, 1989, p 85 3. Bailey-Pridham DD, Cook CL, Reshef E, Hurst HE, Drury K, Yussman MA: Follicular fluid Lidocaine levels during transvag~nal oocyte retrieval. Fertil Steril 1990;53:171 4. Lopata A, Patullo MJ, Chang A, James B: A method for collecting motile spermatozoa from human semen. Fertil Steril 1976;27:677 5. Yong CY, Wu P, Shieh CC, Chiang BN: Membrane stabilising activity and inhibition of human sperm motility. Lancet 1986;2:402 6. Robertson L, Wolf DP, Tash JS: Temporal changes in motility parameters related to acrosomal status: Identification and characterization of populations of hyperactivated human sperm. Biol Reprod 1988;39:797 7. Singh JP, Babcock DF, Lardy HA: Motility activation, respiratory stimulation and alteration of C a 2+ transport in bovine sperm treated with amine local anaesthetics and calcium transport antagonists. Arch Biochem Biophys 1983; 221:291
