Reproductive Toxicology, Vol. 6, pp. 323-327, 1992
0890-6238/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.
Printed in the U.S.A. All rights reserved.
EFFECTS OF OOCYTE EXPOSURE TO LOCAL ANESTHETICS ON IN VITRO FERTILIZATION AND EMBRYO DEVELOPMENT IN THE MOUSE V. L. SCHNELL,* A . G . SACCO,t R. T. SAVOY-MOORE,t K. M. ATAYA,~ a n d K. S. MOGHISSI t *Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, University of Texas
Medical Branch, Galveston, Texas; tC.S. Mott Center, Wayne State University, Department of Obstetrics and Gynecology, Detroit, Michigan; ~Divisionof Reproductive Endocrinology, Department of Obstetrics and Gynecology, Metro Health Medical Center, Case Western Reserve University, Cleveland, Ohio Abstract - - The effect on fertilization and development of local anesthetics routinely used during ultrasoundguided oocyte retrieval in women undergoing in vitro fertilization was examined in a mouse in vitro fertilization system. Mouse oocytes were exposed in vitro to lidocaine, chloroprocaine, and bnpivacaine at concentrations of 0 (control), 0.01, 0.1, 1.0, 10.0, 100.0 ~tg/mL for 30 min, washed, and then inseminated. In vitro oocyte fertilization at 24 and 48 h and embryo development at 72 h were determined. Bupivacaine adversely affected mouse in vitro fertilization and embryo development only at the highest exposure concentration, 100/~g/mL, while lidocaine and chloroprocaine produced adverse effects at concentrations as low as 1.0 and 0.1 ~g/mL, respectively. Furthermore, an adverse dose-related effect on fertilization and embryo development was shown for lidocaine and chloroprocaine, but not for bupivacaine. These data demonstrate that the local anesthetics, iidocaine (L), chloroprocaine (C), and buprivacaine (B), adversely affect mouse in vitro fertilization and embryo development in the order of C > L > B . Key Words: anesthetics; in vitro fertilization; embryo; lidocaine; chloroprocaine; bupivacaine; toxicology.
INTRODUCTION
leagues (4) and Boyers and colleagues (5) have shown an adverse effect of general anesthetic duration (used for laparoscopic follicular aspiration) on oocyte fertilization and embryo development. Fishel and colleagues (6) also reported improved pregnancy rates if general anesthesia with enflurane rather than halothane was used for embryo transfer. Therefore, evaluation of general anesthetics and other agents used during follicular aspiration has improved IVF pregnancy rates. Ultrasound-guided ovarian follicle aspiration is rapidly replacing laparoscopy for oocyte retrieval in many patients (7). Decreased operating room time, general anesthetic dose, and recovery time, and improved access to ovarian follicles were benefits initially observed with ultrasound-guided oocyte retrieval. However, ultrasound-guided follicular aspiration requires that a local anesthetic, commonly lidocaine, be injected into the vaginal fornices (8). While the adverse effects of lidocaine on oocyte fertilization and embryo development have been well documented in nonmammalian animal models (913), little comparable data are available for mammals. The mouse in vitro fertilization and embryo development system is routinely used for quality con-
Initial technologic developments to improve pregnancy rates during in vitro fertilization (IVF) were aimed at the in vitro laoratory. More recently, attempts have been directed at the clinical aspects of in vitro fertilization and include l) ovulation induction protocols, 2) follicular aspiration for oocyte retrieval methods, 3) embryo transfer techniques, and 4) medical luteal phase support. Initially human oocytes for IVF were laparoscopically aspirated from human ovarian follicles under general anesthesia using 100% carbon dioxide to create a pneumoperitoneum. Subsequently decreasing the carbon dioxide concentrations used for pneumoperitoneum has been reported to improve pregnancy rates ( 1). Endler and colleagues (2) and Lefebuvre and colleagues (3) have reported cases of successful pregnancies when spinal and epidural anesthesia, respectively, were used for laparoscopic follicular aspiration for IVF. Hayes and colAddress correspondence to V.L. Schnell, M.D., Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, University of Texas Medical Branch, Galveston, TX 77550-2776. Received 3 September 1991; Revision received 2 December 1991; Accepted 4 December 1991. 323
324
Reproductive Toxicology
trol in clinical in vitro fertilization laboratories (14). All new media, serum, solutions, gloves, plastics, and surgical instruments are routinely tested in the mouse one- or two-cell embryo culture system, and many improvements in the IVF laboratory evolved from early mouse in vitro studies (15). The current study was undertaken to evaluate the effect of exposure of mouse oocytes to increasing concentrations of local anesthetics such as lidocaine (L), bupivacaine (B), and chloroprocaine (C) on subsequent in vitro fertilization rates, embryo cleavage, and embryo development. Our results indicate that in the dose range tested (0.1 to 100 ug/mL) mouse in vitro fertilization and development were adversely affected in the order C>L>B. MATERIALS AND M E T H O D S Outbred Swiss Webster female mice, 4 weeks of age and 8 week old proven breeder males (Charles River Inc., Wilmington, MA) were kept at 21 to 23 °C under controlled 12 h light/12 h dark. Mice were superovulated by a single intraperitoneal (IP) injection of 5 IU pregnant mares serum gonadotropin (PMSG) followed 48 hours later by 5 IU human chorionic gonadotropin (hCG, Sigma, St. Louis, MO). All subsequent procedures were done under a laminar flow hood at 37 °C in 5% CO2 in air. Eggs were recovered from excised oviducts I I h after hCG injection by cutting the ampullary tube to release cumulus-oocyte masses. Gentle pipetting of media containing these masses facilitated separation of individual oocytes with surrounding cumulus. Gamete preparation, IVF, and embryo culture were carried out in Ham's F-10 (GIBCO, Grand Island, NY) medium containing 0.075 g/L penicillin, 0.07 g/L streptomycin, 0.2452 g/L calcium lactate, and 2.10 g/L sodium bicarbonate and supplemented with 7.5% human serum. All media were standardized to 280 mOsm, pH 7.4, and filtered sterilized. Human sera for use in media preparation were obtained from women achieving pregnancy within the Wayne State University in vitro fertilization program and additionally tested for mouse embryo toxicity in a two-cell mouse embryo quality control protocol. Twenty-four hours prior to gamete retrieval, medium was dispensed into Falcon #3037 organ culture plates, covered with sterile paraffin oil, and equilibrated at 37 °C in 5% CO2 in air. In each of 3 separate experiments, oocyte-cumulus masses were randomized into dishes containing the following concentrations of bupivacaine ([B] Sigma], chloroprocaine ([C], Astra, Westford, MA], or lidocaine ([L], Sigma] in media: 0, 1.0, 10, and 100
Volume 6, Number 4, 1992
#g/mL. Concentrations tested were based on reported ranges of lidocaine levels detected in follicular fluid samples obtained by transvaginal oocyte retrieval (16; Wikland, personal communication, 1988). Osmolarity and pH were again readjusted, if necessary, following addition of anesthetics. In 3 separate experiments, 15 to 18 female mice were killed by cervical dislocation. Excised tubal segments were placed in media followed by oocyte-cumulus mass excision from bulging distal ampulla (range = 330 to 550 per experiment) and random division ofoocytes into control and anesthetic exposure groups. Following a 30-minute exposure, the oocytecumulus masses were removed from the test anesthetic and washed three times in culture medium prior to insemination. Each dish contained 15 to 30 oocytes. Male mice were killed by cervical dislocation and semen specimens expressed from the cauda epididymis into 2.0 mL of culture media. The semen specimen was transferred to a culture tube maintained at a 45 ° inclination and incubated 30 min at 37 *C in 5% CO2 in air. This incubation facilitated sedimentation of debris and of nonviable sperm while aiding capacitation (17). The highest quality sperm in 1.0 mL of culture media were gently pipetted from the surface of this inclined conical tube. A portion of the sample was used for counting viability with Eosin Y staining (0.5% in 0.15 M phosphate, pH 7.4) and motile sperm. 1 × 106 motile sperm/mL were added to culture dishes containing the oocytes. Oocytes were transferred into fresh media after 18 h coincubation with spermatozoa and fertilization was determined by the presence of two pronuclei. Embryo development was monitored every 24 h for 3 days and embryo development scores assigned as follows: atretic or fragmented = 0, 1-cell = 1, 2-cell = 2, 4-cell = 4, and >_ 8-cell = 8. Differences in fertilization by 48 h were determined using chi square analysis of independent samples. Embryo development scores attained by 72 h were analyzed using ANOVA followed by Scheff~'s procedure. The anesthetic dose-related effect on fertilization and embryo development was analyzed with Pearson's correlation. RESULTS Fertilization Pooled data from 3 separate experiments indicating fertilization rates (mean _+ standard deviation) for the control groups and for each anesthetic concentration group are shown in Figure 1. Control groups had fertilization rates of 66, 74, and 77% (mean =
Anesthetics and fertilization • V. L. SCHNELL ET AL.
325
control lidocaine • chloroprocaine [ ] bupivacaine []
b
80
b
[]
A
60 b a
o
N '~
40
20
0
0.01
0.1
I
10
100
a n e s t h e t i c concentration (ug/ml)
Fig. 1. Percent fertilization at 48 h (means + standard deviation) for each anesthetic concentration exposure group. The control groups had percent fertilization rates of 66%, 74%, and 77% (mean = 72%) in each of the 3 experiments. For statistical analyses the actual fertilization score (mean + standard deviation) and not percentages as show were compared using chisquared analysis of independent samples with Bonferroni correction for multiple comparisons (ap < 0.05 anesthetics compared with control, bp < 0.05 lidocaine and chloroprocaine compared with bupivacaine).
72%) in each of the 3 separate experiments. None of the 3 anesthetics adversely affected mouse in vitro fertilization at the lowest concentration (0.01 #g/mL) tested. C (0.1 to 100 ug/mL), L (1.0 to 100 ug/mL), and B (100 #g/mL) showed significantly lower fertilization rates as compared to the controls. A dose-related decrease in fertilization scores was observed for L (r = -0.833, P < 0.05) and C (r = -0.926, P < 0.01). Such a dose-related effect was not seen for B (r = -0.390) since the only adverse effect on fertilization occurred at 100 ug/mL. As indicated in Figure 1, B had significantly higher fertilization scores than L and C at dosages of 1.0 to 100 #g/mL (P < 0.05).
Embryo development L (1.0 to 100.0), C (0.1 to 100.0 ug/mL), and B (100 ug/mL) significantly decreased embryo development at 72 h as compared to the control group (ANOVA, P < 0.01; Figure 2). Embryo development scores for B were significantly higher at concentrations as low as 0.1 ug/mL when compared to C and at 1.0 ug]mL when compared to both C and L (P < 0.01; Figure 2). L and C again had an inverse doserelated effect [L (r = -0.921, P < 0.01) and C (r = -0.917, P < 0.01)], not seen with B (r = -0.046). During the 3 separate experiments, the control group had 41%, 34%, and 40% blastocyst formation, respec-
tively, reflecting acceptable quality in the mouse IVF system (18). DISCUSSION Previously, Hayes and colleagues (4) and Boyers and colleagues (5) correlated decreased fertilization and subsequent embryo development in vitro with time from induction of general anesthesia during laparoscopic oocyte retrieval. Two agents used during general anesthesia, thiopental and thiamylal, have been detected in concurrent follicular fluid and serum samples in comparable concentrations (19). Ultrasound-guided follicular aspiration, under local anesthesia, has become more common recently. Lidocaine, frequently used for this procedure, was measured by Bailey-Pridham and colleagues in 16 follicular fluid aspirates following vaginal injection of l0 mL of 1% lidocaine hydrochloride (16). The lidocaine concentrations in the follicles aspirated between 4 to 15 min after induction ranges from 0 to 118 ~g/mL; in the last follicles, aspirated between 12 and 69 min after induction, lidocaine ranged from 0 to 12.2 ug/ mL. Two of the 4 oocytes exposed to lidocaine concentrations greater than 1.0 #g/mL in vivo failed to fertilize (16). While their numbers are small, they do suggest that oocytes in our in vitro mouse assay
326
Reproductive Toxicology
z uJ
Volume 6, Number 4, 1992
iiii!i-i- ~-2ii;-i!ii~-~-iiiii i ii~!(iqii~~ ~!~qiiiii;ii@~iiii~!iiiiiiii+~i~iii%~il;'i;Y i~i_
L
B 0
4.0-
C
--I tLI LtJ
r~
O >. a,¢o =Z UJ
a,b ,o 0.0
,~,b ........
. 0.01
.
.
.
0.1
ANESTHETIC
1.0
. 10.0
CONCENTRATION
100.0 (ug/ml)
Fig. 2. Embryo development scores (mean _+ standard deviation) at 72 h as a function of anesthetic and anesthetic concentration. Shaded area represents embryo development score 4.75 + 0.28 for the control mouse embryos (non-anesthetic-exposed). Significant differences in groups were determined with ANOVA followed by Scheff6's procedure for multiple comparisons (ap < 0.01 Lidocaine, Bupivacaine, Chloroprocaine compared with control, bp < 0.01 Bupivacaine compared with Lidocaine, Chloroprocaine).
mimic the human oocyte well, since the fertilization rate of mouse oocytes in the presence of 1.0 #g/mL lidocaine was 45% + 5%. Although fertilization is a species-specific event, many species share common basic mechanisms (20). For example, the oocyte membrane electrical potential changes and cortical granule dissolution occurs at the moment of sperm penetration in both the sea urchin and mammals (21). Local anesthetics depress facilitated diffusion of ions (Na +, K +, Ca + +) across the axonal cell membrane, preventing action potentials without altering resting membrane charge or intracellular metabolism (22). While such effects have not been studied in the mammalian oocyte, the role of altered cell membrane electrical potential is important in both nerve conduction and fertilization, and local anesthetics clearly can alter oocyte fertilization and embryo development (Figures 1 and 2; references 913). In the present study, mouse in vitro oocyte fertilization and embryo development were adversely affected by very low concentrations of lidocaine and chloroprocaine (1.0 and 0.1 #g/mL, respectively). While the exact mechanisms of action are unknown, there are several possibilities. Cortical granule attachment to the plasma membrane and subsequent exocytosis in sea urchin oocytes was inhibited by procaine and tetracaine (12). Intracellular effects of local anesthetics include increased pH resulting in polymerization of cortical actin (23) and detachment of cytoplasmic from membrane proteins (24) in sea urchins, and inhibition of membrane fusion in mouse splenic lymphocytes (25). As Lee and colleagues (9)
and O'Shea and colleagues (13) have shown, lidocaine exposure in vitro caused neural tube defects in chick embryos. Since we did not return in vitro exposed embryos to other female mice, we can only speculate that some embryos may have had later developmental problems. The activity of local anesthetics depends on several characteristics, including their chemical nature, hydrogen ion concentration, and intrinsic vasodilating capacity. Some characteristics for the anesthetics used in this study are shown in Table 1 (22). The high lipid solubility and protein binding of bupivacaine allow a lower dose to be used to block neuronal action potentials, and this effect will persist longer. In our study, bupivacaine was the least toxic agent, affecting fertilization and development only at 100 #g/mL. The damaging effects of 100 #g/mL bupivacaine were comparable to those produced by the 0. l #g/mL chloroprocaine and 1.0 #g/mL lidocaine. While it is tempting to suggest that bupivacaine, rather than lidocaine, should be employed in ultrasound-guided retrievals, experiments with longer exposure (up to 50
T a b l e I. C h a r a c t e r i s t i c s o f local a n e s t h e t i c s Lidocaine Potency Class Onset Duration pKa Protein binding
intermediate ester fast 90-200 min 7.7 none
Chloroprocaine
Bupivacaine
intermediate ester fast 30-60 min 8.7 64.3%
high amide slow 180-600 min 8. l 95.0%
Anesthetics and fertilization • V. L. SCHNELLET AL.
minutes) of mouse oocytes to this agent should be performed prior to such a recommendation. It would also be important to measure bupivacaine concentrations in follicular fluid aspirates when it is employed in vivo. In the field of assisted reproductive technology, there is currently a suggestion to return to natural cycle IVF, without ovulation induction medications or anesthetic (26,27). This procedure is performed at only a few IVF centers. The resulting number of pregnancies is limited, so it is too early to determine whether oocytes retrieved without any anesthetics will develop and implant better than those exposed to these potentially toxic agents. Ultimately, the test of our current findings will be such a comparison. REFERENCES 1. Edwards RG, Steptoe PC, Purdy JM. Establishing full-term human pregnancies using cleaving embryos grown in vitro. Br J Obstet Gynaecol. 1980;87:737-41. 2. Endler GC, Magyar DM, Hayes MF, Moghissi KS. Use of spinal anesthesia in laparoscopy for in vitro fertilization. Fertil Stedl. 1985;43:809-10. 3. Lefebvre G, Vauthier D, Seebacher J, Henry M, Thormann F, Darbois Y. In vitro fertilization: a comparative study of cleavage rates under epidural and general anesthesia--interest for gamete intrafallopian transfer [letter]. J In Vitro Fertil Embryo Transf. 1988;5:305-6. 4. Hayes MF, Sacco AG, Savoy-Moore RT, et al. Effect of general anesthesia on fertilization and cleavage of human oocytes in vitro. Fertil Steril. 1987;48:975-81, 5. Boyers SP, Lavy G, Russell JB, DeCherney AH. A paired analysis of in vitro fertilization and cleavage rates of first- versus last-recovered preovulatory human oocytes exposed to varying intervals of 100% CO2 pneumoperitoneum and general anesthesia. Fertil Steril. 1987;48:969-74. 6. Fishel S, Webster J, Faratian B, Jackson P. General anesthesia for intrauterine placement of human conceptuses after in vitro fertilization. J In Vitro Fertil Embryo Transf. 1987;4:260-4. 7. Lewin A, Laufer N, Robinowitz R, Margalioth EJ, Bar 1, Schenker JG. Ultrasonically guided oocyte collection under local anesthesia: the first choice method for in vitro fertilization--a comparative study with laparoscopy. Fertil Steril. 1986;46:257-6 I. 8. Dellenbach P, Nisand I, Moreau L, et al. Transvaginal sonographically controlled follicle puncture for oocyte retrieval. Fertil Steril. 1985;44:656-62.
327
9. Lee H, Nagele RG. Neural tube defects caused by local anesthetics in early chick embryos. Teratology. 1985;31:119-127. 10, Daley R, Schuel H. Multiple effects of procaine on fertilization in sea urchins. J Cell Biol. 1978;79:159. 11. Bozhkova VP, Sharova LV, Petriaevskaia VB, Kozlov DA, Khrust IR. Effect of local anesthetics and phorbol ester on intracellular pH and rate of development of sea urchin embryos. Ontogenez. 1988;19:73-81. 12. Hylander BL, Summers RG. The effect oflocal anesthetics and ammonia on cortical granule plasma membrane attachment in the sea urchin egg. Develop Biol. 1981;86:1-11. 13. O'Shea, Kaufman MH. Neural tube closure defects following in vitro exposure to xylocaine. J Exp Zool. 1980;214:235-8. 14. Ackerman S, Swanson RJ, Taylor S, Fenwick L. Toxicity testing for human IVF programs. J In Vitro Fertil. 1985;2:132-5. 15. Ackerman S, Swanson R J, Adams P, Wortham JWE. Comparison of strains and culture media used for mouse IVF. Gamete Res. 1983;37:103-7. 16. Bailey-Pridham DD, ReshefE, Drury K, et al. Follicular fluid lidocaine levels during transvaginal oocyte retrieval. Fertil Steril. 1990;53:171-3. 17. Fraser LR. Minimum and maximum intracellular Ca 2+ requirements during mouse sperm capacitation and fertilization in vitro. J Reprod Fertil. 1987;81:77-89. 18. Van de Sandt JJM, Schroder AC, Eppig JJ. Culture media for mouse oocyte maturation affect subsequent embryonic development. Mol Reprod Dev. 1990;25:164-7 I. 19. Endler GC, Stout M, Magyar DM, Hayes MF, Moghissi KS, Sacco AG. Follicular fluid concentrations of thiopental and thiamylal during laparoscopy for oocyte retrieval. Fertil Steril. 1987;48:828-33. 20. Longo FJ. Egg cortical architecture. In Schaten H, Schaten G, eds. The cell biology of fertilization. New York: Academic Press; 1989. 108-138. 21. Yanagimachi R. Mammalian fertilization. In Knobil E, Neill J, et al., eds. The physiology of reproduction. New York: Ravin Press; 1988: 135-185. 22. Seeman P. The membrane actions of anesthetics and tranquilizers. Pharmacol Rev. 1972;24:583-655. 23. Begg DA, Reghun LI. pH regulates the polymerization ofactin in the sea urchin egg cortex. J Cell Biol. 1979;83:241-8. 24. Poste G, Papahadjopoulos D, Nicolson GL. Local anesthetics affect transmembrane cytoskeletal control of mobility and distribution of cell surface receptors. Proc Natl Acad Sci USA. 1975;72:4430-4. 25. Poste G, Allison AG. Membrane fusion. Biochim Biophys Acta. 1973;300:421-65. 26. Paulson R J, Sauer MV, Lobo RA. In vitro fertilization in unstimulated cycles: a new application. Fertil Steril. 1989;51 : 1059-60. 27. Ramsewak SS,Anju K, Welsby R, Mowforth A, Lenton EA, Cooke ID. Is analgesia required for transvaginal single-follicle aspiration in in vitro fertilization? A double-blind study, J In Vitro Fertil. Embryo Transfer. 1990;7:103-6.
0890-6238/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.
Printed in the U.S.A. All rights reserved.
EFFECTS OF OOCYTE EXPOSURE TO LOCAL ANESTHETICS ON IN VITRO FERTILIZATION AND EMBRYO DEVELOPMENT IN THE MOUSE V. L. SCHNELL,* A . G . SACCO,t R. T. SAVOY-MOORE,t K. M. ATAYA,~ a n d K. S. MOGHISSI t *Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, University of Texas
Medical Branch, Galveston, Texas; tC.S. Mott Center, Wayne State University, Department of Obstetrics and Gynecology, Detroit, Michigan; ~Divisionof Reproductive Endocrinology, Department of Obstetrics and Gynecology, Metro Health Medical Center, Case Western Reserve University, Cleveland, Ohio Abstract - - The effect on fertilization and development of local anesthetics routinely used during ultrasoundguided oocyte retrieval in women undergoing in vitro fertilization was examined in a mouse in vitro fertilization system. Mouse oocytes were exposed in vitro to lidocaine, chloroprocaine, and bnpivacaine at concentrations of 0 (control), 0.01, 0.1, 1.0, 10.0, 100.0 ~tg/mL for 30 min, washed, and then inseminated. In vitro oocyte fertilization at 24 and 48 h and embryo development at 72 h were determined. Bupivacaine adversely affected mouse in vitro fertilization and embryo development only at the highest exposure concentration, 100/~g/mL, while lidocaine and chloroprocaine produced adverse effects at concentrations as low as 1.0 and 0.1 ~g/mL, respectively. Furthermore, an adverse dose-related effect on fertilization and embryo development was shown for lidocaine and chloroprocaine, but not for bupivacaine. These data demonstrate that the local anesthetics, iidocaine (L), chloroprocaine (C), and buprivacaine (B), adversely affect mouse in vitro fertilization and embryo development in the order of C > L > B . Key Words: anesthetics; in vitro fertilization; embryo; lidocaine; chloroprocaine; bupivacaine; toxicology.
INTRODUCTION
leagues (4) and Boyers and colleagues (5) have shown an adverse effect of general anesthetic duration (used for laparoscopic follicular aspiration) on oocyte fertilization and embryo development. Fishel and colleagues (6) also reported improved pregnancy rates if general anesthesia with enflurane rather than halothane was used for embryo transfer. Therefore, evaluation of general anesthetics and other agents used during follicular aspiration has improved IVF pregnancy rates. Ultrasound-guided ovarian follicle aspiration is rapidly replacing laparoscopy for oocyte retrieval in many patients (7). Decreased operating room time, general anesthetic dose, and recovery time, and improved access to ovarian follicles were benefits initially observed with ultrasound-guided oocyte retrieval. However, ultrasound-guided follicular aspiration requires that a local anesthetic, commonly lidocaine, be injected into the vaginal fornices (8). While the adverse effects of lidocaine on oocyte fertilization and embryo development have been well documented in nonmammalian animal models (913), little comparable data are available for mammals. The mouse in vitro fertilization and embryo development system is routinely used for quality con-
Initial technologic developments to improve pregnancy rates during in vitro fertilization (IVF) were aimed at the in vitro laoratory. More recently, attempts have been directed at the clinical aspects of in vitro fertilization and include l) ovulation induction protocols, 2) follicular aspiration for oocyte retrieval methods, 3) embryo transfer techniques, and 4) medical luteal phase support. Initially human oocytes for IVF were laparoscopically aspirated from human ovarian follicles under general anesthesia using 100% carbon dioxide to create a pneumoperitoneum. Subsequently decreasing the carbon dioxide concentrations used for pneumoperitoneum has been reported to improve pregnancy rates ( 1). Endler and colleagues (2) and Lefebuvre and colleagues (3) have reported cases of successful pregnancies when spinal and epidural anesthesia, respectively, were used for laparoscopic follicular aspiration for IVF. Hayes and colAddress correspondence to V.L. Schnell, M.D., Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, University of Texas Medical Branch, Galveston, TX 77550-2776. Received 3 September 1991; Revision received 2 December 1991; Accepted 4 December 1991. 323
324
Reproductive Toxicology
trol in clinical in vitro fertilization laboratories (14). All new media, serum, solutions, gloves, plastics, and surgical instruments are routinely tested in the mouse one- or two-cell embryo culture system, and many improvements in the IVF laboratory evolved from early mouse in vitro studies (15). The current study was undertaken to evaluate the effect of exposure of mouse oocytes to increasing concentrations of local anesthetics such as lidocaine (L), bupivacaine (B), and chloroprocaine (C) on subsequent in vitro fertilization rates, embryo cleavage, and embryo development. Our results indicate that in the dose range tested (0.1 to 100 ug/mL) mouse in vitro fertilization and development were adversely affected in the order C>L>B. MATERIALS AND M E T H O D S Outbred Swiss Webster female mice, 4 weeks of age and 8 week old proven breeder males (Charles River Inc., Wilmington, MA) were kept at 21 to 23 °C under controlled 12 h light/12 h dark. Mice were superovulated by a single intraperitoneal (IP) injection of 5 IU pregnant mares serum gonadotropin (PMSG) followed 48 hours later by 5 IU human chorionic gonadotropin (hCG, Sigma, St. Louis, MO). All subsequent procedures were done under a laminar flow hood at 37 °C in 5% CO2 in air. Eggs were recovered from excised oviducts I I h after hCG injection by cutting the ampullary tube to release cumulus-oocyte masses. Gentle pipetting of media containing these masses facilitated separation of individual oocytes with surrounding cumulus. Gamete preparation, IVF, and embryo culture were carried out in Ham's F-10 (GIBCO, Grand Island, NY) medium containing 0.075 g/L penicillin, 0.07 g/L streptomycin, 0.2452 g/L calcium lactate, and 2.10 g/L sodium bicarbonate and supplemented with 7.5% human serum. All media were standardized to 280 mOsm, pH 7.4, and filtered sterilized. Human sera for use in media preparation were obtained from women achieving pregnancy within the Wayne State University in vitro fertilization program and additionally tested for mouse embryo toxicity in a two-cell mouse embryo quality control protocol. Twenty-four hours prior to gamete retrieval, medium was dispensed into Falcon #3037 organ culture plates, covered with sterile paraffin oil, and equilibrated at 37 °C in 5% CO2 in air. In each of 3 separate experiments, oocyte-cumulus masses were randomized into dishes containing the following concentrations of bupivacaine ([B] Sigma], chloroprocaine ([C], Astra, Westford, MA], or lidocaine ([L], Sigma] in media: 0, 1.0, 10, and 100
Volume 6, Number 4, 1992
#g/mL. Concentrations tested were based on reported ranges of lidocaine levels detected in follicular fluid samples obtained by transvaginal oocyte retrieval (16; Wikland, personal communication, 1988). Osmolarity and pH were again readjusted, if necessary, following addition of anesthetics. In 3 separate experiments, 15 to 18 female mice were killed by cervical dislocation. Excised tubal segments were placed in media followed by oocyte-cumulus mass excision from bulging distal ampulla (range = 330 to 550 per experiment) and random division ofoocytes into control and anesthetic exposure groups. Following a 30-minute exposure, the oocytecumulus masses were removed from the test anesthetic and washed three times in culture medium prior to insemination. Each dish contained 15 to 30 oocytes. Male mice were killed by cervical dislocation and semen specimens expressed from the cauda epididymis into 2.0 mL of culture media. The semen specimen was transferred to a culture tube maintained at a 45 ° inclination and incubated 30 min at 37 *C in 5% CO2 in air. This incubation facilitated sedimentation of debris and of nonviable sperm while aiding capacitation (17). The highest quality sperm in 1.0 mL of culture media were gently pipetted from the surface of this inclined conical tube. A portion of the sample was used for counting viability with Eosin Y staining (0.5% in 0.15 M phosphate, pH 7.4) and motile sperm. 1 × 106 motile sperm/mL were added to culture dishes containing the oocytes. Oocytes were transferred into fresh media after 18 h coincubation with spermatozoa and fertilization was determined by the presence of two pronuclei. Embryo development was monitored every 24 h for 3 days and embryo development scores assigned as follows: atretic or fragmented = 0, 1-cell = 1, 2-cell = 2, 4-cell = 4, and >_ 8-cell = 8. Differences in fertilization by 48 h were determined using chi square analysis of independent samples. Embryo development scores attained by 72 h were analyzed using ANOVA followed by Scheff~'s procedure. The anesthetic dose-related effect on fertilization and embryo development was analyzed with Pearson's correlation. RESULTS Fertilization Pooled data from 3 separate experiments indicating fertilization rates (mean _+ standard deviation) for the control groups and for each anesthetic concentration group are shown in Figure 1. Control groups had fertilization rates of 66, 74, and 77% (mean =
Anesthetics and fertilization • V. L. SCHNELL ET AL.
325
control lidocaine • chloroprocaine [ ] bupivacaine []
b
80
b
[]
A
60 b a
o
N '~
40
20
0
0.01
0.1
I
10
100
a n e s t h e t i c concentration (ug/ml)
Fig. 1. Percent fertilization at 48 h (means + standard deviation) for each anesthetic concentration exposure group. The control groups had percent fertilization rates of 66%, 74%, and 77% (mean = 72%) in each of the 3 experiments. For statistical analyses the actual fertilization score (mean + standard deviation) and not percentages as show were compared using chisquared analysis of independent samples with Bonferroni correction for multiple comparisons (ap < 0.05 anesthetics compared with control, bp < 0.05 lidocaine and chloroprocaine compared with bupivacaine).
72%) in each of the 3 separate experiments. None of the 3 anesthetics adversely affected mouse in vitro fertilization at the lowest concentration (0.01 #g/mL) tested. C (0.1 to 100 ug/mL), L (1.0 to 100 ug/mL), and B (100 #g/mL) showed significantly lower fertilization rates as compared to the controls. A dose-related decrease in fertilization scores was observed for L (r = -0.833, P < 0.05) and C (r = -0.926, P < 0.01). Such a dose-related effect was not seen for B (r = -0.390) since the only adverse effect on fertilization occurred at 100 ug/mL. As indicated in Figure 1, B had significantly higher fertilization scores than L and C at dosages of 1.0 to 100 #g/mL (P < 0.05).
Embryo development L (1.0 to 100.0), C (0.1 to 100.0 ug/mL), and B (100 ug/mL) significantly decreased embryo development at 72 h as compared to the control group (ANOVA, P < 0.01; Figure 2). Embryo development scores for B were significantly higher at concentrations as low as 0.1 ug/mL when compared to C and at 1.0 ug]mL when compared to both C and L (P < 0.01; Figure 2). L and C again had an inverse doserelated effect [L (r = -0.921, P < 0.01) and C (r = -0.917, P < 0.01)], not seen with B (r = -0.046). During the 3 separate experiments, the control group had 41%, 34%, and 40% blastocyst formation, respec-
tively, reflecting acceptable quality in the mouse IVF system (18). DISCUSSION Previously, Hayes and colleagues (4) and Boyers and colleagues (5) correlated decreased fertilization and subsequent embryo development in vitro with time from induction of general anesthesia during laparoscopic oocyte retrieval. Two agents used during general anesthesia, thiopental and thiamylal, have been detected in concurrent follicular fluid and serum samples in comparable concentrations (19). Ultrasound-guided follicular aspiration, under local anesthesia, has become more common recently. Lidocaine, frequently used for this procedure, was measured by Bailey-Pridham and colleagues in 16 follicular fluid aspirates following vaginal injection of l0 mL of 1% lidocaine hydrochloride (16). The lidocaine concentrations in the follicles aspirated between 4 to 15 min after induction ranges from 0 to 118 ~g/mL; in the last follicles, aspirated between 12 and 69 min after induction, lidocaine ranged from 0 to 12.2 ug/ mL. Two of the 4 oocytes exposed to lidocaine concentrations greater than 1.0 #g/mL in vivo failed to fertilize (16). While their numbers are small, they do suggest that oocytes in our in vitro mouse assay
326
Reproductive Toxicology
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Volume 6, Number 4, 1992
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Fig. 2. Embryo development scores (mean _+ standard deviation) at 72 h as a function of anesthetic and anesthetic concentration. Shaded area represents embryo development score 4.75 + 0.28 for the control mouse embryos (non-anesthetic-exposed). Significant differences in groups were determined with ANOVA followed by Scheff6's procedure for multiple comparisons (ap < 0.01 Lidocaine, Bupivacaine, Chloroprocaine compared with control, bp < 0.01 Bupivacaine compared with Lidocaine, Chloroprocaine).
mimic the human oocyte well, since the fertilization rate of mouse oocytes in the presence of 1.0 #g/mL lidocaine was 45% + 5%. Although fertilization is a species-specific event, many species share common basic mechanisms (20). For example, the oocyte membrane electrical potential changes and cortical granule dissolution occurs at the moment of sperm penetration in both the sea urchin and mammals (21). Local anesthetics depress facilitated diffusion of ions (Na +, K +, Ca + +) across the axonal cell membrane, preventing action potentials without altering resting membrane charge or intracellular metabolism (22). While such effects have not been studied in the mammalian oocyte, the role of altered cell membrane electrical potential is important in both nerve conduction and fertilization, and local anesthetics clearly can alter oocyte fertilization and embryo development (Figures 1 and 2; references 913). In the present study, mouse in vitro oocyte fertilization and embryo development were adversely affected by very low concentrations of lidocaine and chloroprocaine (1.0 and 0.1 #g/mL, respectively). While the exact mechanisms of action are unknown, there are several possibilities. Cortical granule attachment to the plasma membrane and subsequent exocytosis in sea urchin oocytes was inhibited by procaine and tetracaine (12). Intracellular effects of local anesthetics include increased pH resulting in polymerization of cortical actin (23) and detachment of cytoplasmic from membrane proteins (24) in sea urchins, and inhibition of membrane fusion in mouse splenic lymphocytes (25). As Lee and colleagues (9)
and O'Shea and colleagues (13) have shown, lidocaine exposure in vitro caused neural tube defects in chick embryos. Since we did not return in vitro exposed embryos to other female mice, we can only speculate that some embryos may have had later developmental problems. The activity of local anesthetics depends on several characteristics, including their chemical nature, hydrogen ion concentration, and intrinsic vasodilating capacity. Some characteristics for the anesthetics used in this study are shown in Table 1 (22). The high lipid solubility and protein binding of bupivacaine allow a lower dose to be used to block neuronal action potentials, and this effect will persist longer. In our study, bupivacaine was the least toxic agent, affecting fertilization and development only at 100 #g/mL. The damaging effects of 100 #g/mL bupivacaine were comparable to those produced by the 0. l #g/mL chloroprocaine and 1.0 #g/mL lidocaine. While it is tempting to suggest that bupivacaine, rather than lidocaine, should be employed in ultrasound-guided retrievals, experiments with longer exposure (up to 50
T a b l e I. C h a r a c t e r i s t i c s o f local a n e s t h e t i c s Lidocaine Potency Class Onset Duration pKa Protein binding
intermediate ester fast 90-200 min 7.7 none
Chloroprocaine
Bupivacaine
intermediate ester fast 30-60 min 8.7 64.3%
high amide slow 180-600 min 8. l 95.0%
Anesthetics and fertilization • V. L. SCHNELLET AL.
minutes) of mouse oocytes to this agent should be performed prior to such a recommendation. It would also be important to measure bupivacaine concentrations in follicular fluid aspirates when it is employed in vivo. In the field of assisted reproductive technology, there is currently a suggestion to return to natural cycle IVF, without ovulation induction medications or anesthetic (26,27). This procedure is performed at only a few IVF centers. The resulting number of pregnancies is limited, so it is too early to determine whether oocytes retrieved without any anesthetics will develop and implant better than those exposed to these potentially toxic agents. Ultimately, the test of our current findings will be such a comparison. REFERENCES 1. Edwards RG, Steptoe PC, Purdy JM. Establishing full-term human pregnancies using cleaving embryos grown in vitro. Br J Obstet Gynaecol. 1980;87:737-41. 2. Endler GC, Magyar DM, Hayes MF, Moghissi KS. Use of spinal anesthesia in laparoscopy for in vitro fertilization. Fertil Stedl. 1985;43:809-10. 3. Lefebvre G, Vauthier D, Seebacher J, Henry M, Thormann F, Darbois Y. In vitro fertilization: a comparative study of cleavage rates under epidural and general anesthesia--interest for gamete intrafallopian transfer [letter]. J In Vitro Fertil Embryo Transf. 1988;5:305-6. 4. Hayes MF, Sacco AG, Savoy-Moore RT, et al. Effect of general anesthesia on fertilization and cleavage of human oocytes in vitro. Fertil Steril. 1987;48:975-81, 5. Boyers SP, Lavy G, Russell JB, DeCherney AH. A paired analysis of in vitro fertilization and cleavage rates of first- versus last-recovered preovulatory human oocytes exposed to varying intervals of 100% CO2 pneumoperitoneum and general anesthesia. Fertil Steril. 1987;48:969-74. 6. Fishel S, Webster J, Faratian B, Jackson P. General anesthesia for intrauterine placement of human conceptuses after in vitro fertilization. J In Vitro Fertil Embryo Transf. 1987;4:260-4. 7. Lewin A, Laufer N, Robinowitz R, Margalioth EJ, Bar 1, Schenker JG. Ultrasonically guided oocyte collection under local anesthesia: the first choice method for in vitro fertilization--a comparative study with laparoscopy. Fertil Steril. 1986;46:257-6 I. 8. Dellenbach P, Nisand I, Moreau L, et al. Transvaginal sonographically controlled follicle puncture for oocyte retrieval. Fertil Steril. 1985;44:656-62.
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9. Lee H, Nagele RG. Neural tube defects caused by local anesthetics in early chick embryos. Teratology. 1985;31:119-127. 10, Daley R, Schuel H. Multiple effects of procaine on fertilization in sea urchins. J Cell Biol. 1978;79:159. 11. Bozhkova VP, Sharova LV, Petriaevskaia VB, Kozlov DA, Khrust IR. Effect of local anesthetics and phorbol ester on intracellular pH and rate of development of sea urchin embryos. Ontogenez. 1988;19:73-81. 12. Hylander BL, Summers RG. The effect oflocal anesthetics and ammonia on cortical granule plasma membrane attachment in the sea urchin egg. Develop Biol. 1981;86:1-11. 13. O'Shea, Kaufman MH. Neural tube closure defects following in vitro exposure to xylocaine. J Exp Zool. 1980;214:235-8. 14. Ackerman S, Swanson RJ, Taylor S, Fenwick L. Toxicity testing for human IVF programs. J In Vitro Fertil. 1985;2:132-5. 15. Ackerman S, Swanson R J, Adams P, Wortham JWE. Comparison of strains and culture media used for mouse IVF. Gamete Res. 1983;37:103-7. 16. Bailey-Pridham DD, ReshefE, Drury K, et al. Follicular fluid lidocaine levels during transvaginal oocyte retrieval. Fertil Steril. 1990;53:171-3. 17. Fraser LR. Minimum and maximum intracellular Ca 2+ requirements during mouse sperm capacitation and fertilization in vitro. J Reprod Fertil. 1987;81:77-89. 18. Van de Sandt JJM, Schroder AC, Eppig JJ. Culture media for mouse oocyte maturation affect subsequent embryonic development. Mol Reprod Dev. 1990;25:164-7 I. 19. Endler GC, Stout M, Magyar DM, Hayes MF, Moghissi KS, Sacco AG. Follicular fluid concentrations of thiopental and thiamylal during laparoscopy for oocyte retrieval. Fertil Steril. 1987;48:828-33. 20. Longo FJ. Egg cortical architecture. In Schaten H, Schaten G, eds. The cell biology of fertilization. New York: Academic Press; 1989. 108-138. 21. Yanagimachi R. Mammalian fertilization. In Knobil E, Neill J, et al., eds. The physiology of reproduction. New York: Ravin Press; 1988: 135-185. 22. Seeman P. The membrane actions of anesthetics and tranquilizers. Pharmacol Rev. 1972;24:583-655. 23. Begg DA, Reghun LI. pH regulates the polymerization ofactin in the sea urchin egg cortex. J Cell Biol. 1979;83:241-8. 24. Poste G, Papahadjopoulos D, Nicolson GL. Local anesthetics affect transmembrane cytoskeletal control of mobility and distribution of cell surface receptors. Proc Natl Acad Sci USA. 1975;72:4430-4. 25. Poste G, Allison AG. Membrane fusion. Biochim Biophys Acta. 1973;300:421-65. 26. Paulson R J, Sauer MV, Lobo RA. In vitro fertilization in unstimulated cycles: a new application. Fertil Steril. 1989;51 : 1059-60. 27. Ramsewak SS,Anju K, Welsby R, Mowforth A, Lenton EA, Cooke ID. Is analgesia required for transvaginal single-follicle aspiration in in vitro fertilization? A double-blind study, J In Vitro Fertil. Embryo Transfer. 1990;7:103-6.
