Introduction

Approximately 42% non-obstetrical surgeries are performed during the first trimester of pregnancy, 35% in the second and 23% in the third (Reitman & Flood, 2011; Mandim, Ruzi, Bernardes, & Teixeira, 2015). However, the number established for the first quarter may in fact be higher than in reality, since it is possible that early pregnancy may not be confirmed during this period. The incidence and types of surgeries performed in pregnant women was similar to that performed in young women who were not pregnant, and appendectomies were the most common (Van de Velde & De Buck, 2007).

The literature reports that anesthetics affect mitosis, DNA synthesis and induce neuroapoptosis response (Cattano, Young, Straiko, & Olney, 2008). Furthermore, in utero exposure to anesthetic of laboratory animals decreased acquiring spatial memory (Palanisamy et al., 2011). These data suggest that fetal brain development can be permanently affected by maternal anesthesia.

Propofol is anesthetic used for induction and maintenance of general anesthesia and sedation for obstetrical and pediatric procedures. Propofol interacts with GABA and glutamate receptors NMDA receptors. (Irifune et al., 2003). It is known that these receptors were modulated by anesthetics; however, information about the effects of propofol on central neurons is scarce.

In addition, propofol causes the death of brain cells in neonatal experimental animal (Vutskits, Gascon, Tassonyi, & Kiss, 2005; Cattano et al., 2008; Pesić et al., 2009; Bercker et al., 2009; Milanovic et al., 2010) and long-term behavioral deficits in the developing brain (Yu, Jiang, Gao, Liu, & Chen, 2013). Another example of propofol toxicity was verified by Cheng et al., (2010) who found that propofol prevented the proliferation of cardiac fibroblasts, by interfering with the generation of reactive oxygen species.

The safety of the propofol has not been established and the consult literature does not bring anything describing the effects of propofol in embryonic development and postnatal foetus. The objective of the present study was to investigate the toxicity of the drug in the different gestational periods and to evaluate the teratogenic effects on intrauterine and postnatal development of the offspring.

Material and methods

Animals

Swiss female mice (Mus musculus), with an approximate weight of 35 g, were maintained in a controlled lighting system (12-hours light / 12 hours dark cycle) at 22 ± 2°C, with water and food freely accessible. The animals were put to mate late in the afternoon in individual cages in a proportion of a female mice to a male mouse. In the following day, with a gap of 12 hours, the vagina of females were examined to verify the occurrence of the "vaginal plug", which would determine the day zero of pregnancy (DG 0). The Ethics Committee on Animal Experiments of the State University of Londrina (Protocol 13035/2008) approved all procedures in this study.

Experimental design

Evaluation of embryo-fetal development

Pregnant females were distributed in three treated groups and three control groups. Each group consisted of 15 animals. The animals of the treated groups received a 15 mg kg^-1 dose of propofol (PROPOVAN; Cristália, Itapira, Brazil) via the caudal vein on the fifth (group 1), tenth (group 2) and fifteenth day (group 3) of gestation to simulate the use of the anesthetic in a medical intervention during the first, second and third trimester corresponding to the stages of embryo implantation, embryonic period and fetal period respectively. The anesthetic dose used is the one recommended in the literature for mice anesthesia with propofol (Glen, 1980). Mice from the three control groups received a saline solution following the same scheme as the treated groups. Weight gain was monitored and on the eighteenth day, the females were euthanized by cervical dislocation. Procedures of laparotomy and hysterectomy were conducted to analyze embryo-fetal development.

Maternal weight gain (final weight – initial weight), number of resorptions (early and late resorptions were recorded), live and dead fetuses, fetal and placental weight were recorded. The number of implantation sites were determined using Salewski (1964) method. With these data, the rate of resorption (number of resorptions x 100 / number of implantations); rate of post-implantation loss (number of implantation – number of live fetuses x 100 / number of implantations); fetal viability (number of live fetuses x 100 / number of implantations) and the placental index (placental weight / fetal weight) were calculated.

The fetuses were measured by crown-rump length and weighed individually. Half of the fetuses of each litter was fixed in bodian for subsequent visceral analysis (Wilson, 1965). The other half of the litter was fixed in acetone for subsequent skeletal analysis utilizing the alizarin red technique (Staples & Schnell, 1964).

Evaluation of post-natal development

Females mice were distributed in six experimental groups: three treated groups and three control in which each group consisted of 10 animals. In the treated groups, the animals received propofol (15 mg kg^-1) through the caudal vein on the fifth, tenth and fifteenth day of gestation. The animals of the control group received a saline solution following the same scheme as the treated groups. The animals gave birth naturally and the litters were reduced to four females and four males to prevent competition of milk.

The day of birth was designed as post-natal day zero (DPN 0). On the first post-natal day (DPN 1), the external morphology of all the offspring was analyzed. The parameters related to physical and reflex development were evaluated according to Alder and Zbinden (1977). The mice were observed daily and weighed on the first, second, seventh, fourteenth and twenty-first post-natal day. The physical and reflex development parameters of 1 male and 1 female randomly selected of each litter was analyzed by the parameters: pinna detachment (starting from DPN 2), appearance of hair (starting from DPN 5), appearance of nipples (starting from DPN 6), upper and lower incisors eruption (starting from DPN 8), opening of the ear canal (starting from DPN 10) opening of the eyes (starting from DPN 12), testicular descent (starting from DPN 16) and opening of vaginal canal (starting from DPN 26). The anogenital distance, the distance from the anus to the base of the genitalia, was determined in pups of both sexes with the help of a caliper on DPN 1 and DPN 21. In addition, the presence of righting reflex posture; negative geotaxis and adult walking reflex were analyzed.

Statistical analysis

In the quantitative analyzes, Student's t-test was used. This test was used to compare the groups corresponding to the same day of treatment between the treated and control groups. The same procedure was appliedin the qualitative data, using Fisher's test. The statistical program used was GraphPad Prism. The level of significance was 5%.The ANOVA test was also used in the quantitative data, comparing the different days of treatment between the groups, verifying if there was divergence between the groups.

Results

Maternal toxicity and intrauterine development

The average weight of the maternal organs of the mice did not present significant alteration in any of the experimental groups (Table 1).

The rate of post-implantational embryonic loss and the rate of embryonic resorption had a statistically significant increase in females in the group anesthetized on the fifth gestational day, DG 5 (Table 2).

With respect to external and visceral morphology, the results showed fetuses with premature opening of the eyes and cleft palate, respectively (Table 3). However, these malformations were rare in occurrence and not considered statistically significant. Skeletal analysis showed lumbar rib and incomplete ossification of the cranial, sternum and supraoccipital bones (Table 3). However, such alterations were considered skeletal variations. The variations are usually reversible changes, characterized by not presenting risks to life and because they involve structural alterations of lesser functional significance. These results are in assent with those obtained by Mazze, Wilson, Rice and Baden (1985) who observed an increase in the frequencies of skeletal variation among the offspring of pregnant mice treated with small doses of the anesthetic isofluorane.

Post-natal development evaluation

The average weight gain per group of the mice whose mothers were exposed to propofol during the prenatal period can be observed (Table 4). The anesthetic significantly increased the weight gain of the pups whose mothers were treated on the fifteenth gestational day.

In relation to the physical and reflex development, the pups whose mothers received propofol on the fifteenth gestational day showed a significant delay in the pinna detachment (Table 5) and an increase in the anogenital distance of the male pups (Table 6).

Discussion

Maternal exposure to chemical agents during the gestational period can result in changes of the offspring’s development. These changes depend on inherent factors in the mother's body, placental function or direct action on the embryo and can cause embryonic and fetal death, malformations, congenital abnormalities and behaviour or physical disability of newborn. The literature related risk of impaired fertility, cleft palate, cardiac abnormalities, miscarriage, premature birth, delayed intrauterine growth and stillbirths in patients who had been anesthetized in the first month of pregnancy (Reitman & Flood, 2011).

There is an inconsistency in the literature regarding the adverse effects of the anesthetic propofol in relation to obstetric procedures. Although not recommended, propofol is considered safe for pregnant women and is widely used in non-obstetric surgeries and procedures during pregnancy (Ooi & Thomson, 2015). Recent studies in rats have concluded that exposure to propofol during development leads to retardation of physical and neurological reflex, suggesting nervous system injury (Li et al., 2014).

In the present study, it was found that weight gain was significantly reduced when mice were exposed to anesthesia on the fifth day of gestation however; there was no observable symptoms of maternal toxicity. The results suggested that this difference is related to the increase in post-implantation loss rate and the rate of embryonic resorption, which was statistically significant for females, which were exposed to propofol on the fifth day of gestation. The increased resorption rate and post implantation embryonic loss can be consequences of treatment with propofol on the fifth day of gestation, the time the implant is completed (Wang & Day, 2006). Based on this, it can be suggested that the presence of the anesthetic in the maternal circulation of the placenta associated with immaturity caused the occurrence of lethal events for the developing embryo.

Malformations and anomalies observed in fetuses during this research were rare in occurrence and not statistically significant. The fissure palate and open eyes are common malformations in mice and are included in the rate of malformations that are not related to environmental factors and probably have origins in genetic faults and unknown etiology (Niebyl & Simpson, 2008; Gritli-Linde, 2008; Dixon, Marazita, Beaty, & Murray, 2011; Yang et al., 2014).The reduced ossification of cranial bones observed in most experimental animals during the analysis of the skeleton, is considered transient and therefore can not interfere with the development of the offspring.

The manifestation of lumbar ribs is considered spontaneous and not related to treatment. This anomaly is of common occurrence in teratogenic studies of rodents and there is disagreement in the literature as to whether it represents a malformation or not (Damasceno, Volpato, Calderon, Aguilar, & Rudge, 2002). As a teratogenic event is by definition a permanent change (Wichramaratane, 1988), further study would be required to follow the development of additional ribs during adulthood.

The data from the postnatal analysis suggested that exposure to propofol during the prenatal period did not promote change in the weight gain of the offspring belonging to the groups that received anesthesia during the fifth and tenth gestational day. However, pups whose mothers were anesthetized on the fifteenth day of gestational showed significantly higher weight gain and growth in relation to the respective control group. A possible explanation for this finding would be the fact that 15^th GD presented the smallest number of pups. Thus, there was a greater supply of nutrients during gestation, favoring the development of the neonate. We did not find in the literature consulted data that relate to the increase of postnatal weight with anesthetics administered during pregnancy.

Besides the weight gain, the other physical parameters that have been altered were the pinna detachment and anogenital distance in male offspring. Gallavan, Holson, Stump, Knapp and Reynolds (1999) has shown the correlation between the anogenital distance and the body weight of the pups, demonstrated in their experiments with rats. The weight of pups, also can be influenced by litter size, maternal toxicity or neonatal toxicity. To minimize this interference, in this experiment, the size of each litter was reduced, for the proposed design (four females and four males).

The alteration of the anogenital distance also can be correlated with hormonal disturbances. As the consume of some substance which can cause endocrine disrupting (Birkett & Lester, 2003; Arnold et al., 2004), or exogenous factors such as the influence of uterine fetus positions during gestation, in which opposite sexes that are neighbours, are influenced by the secretion of different steroids (Richmond & Sachs, 1984). In the consulted literature, there is no report of the relationship of propofol with the endocrine system. Therefore, further studies need to be conducted to evaluate both situations.

Although other studies have reported neurotoxic potential exposure related to Propofol (Yu et al., 2013; Creeley et al., 2013; Yang et al., 2014; Li et al., 2014), according to the results of this study, the anesthetic does not seem to modify the reflection development of the puppies, because there were no differences between the response time for animal control or treated groups.

The main objective of the anesthesiologist is to ensure a safe anesthesia for pregnant women and simultaneously minimize the risk of premature birth or miscarriage (Mhuireachtaigh & O'Gorman, 2006). According to Reitman and Flood (2011), in cases of pregnancy, the anesthesiologist should avoid fetal exposure to unnecessary medication and regional anesthesia is usually preferred when it is practical for medical and surgical conditions.

Actually, the Guideline published by the UK National Collaborating Centre for Acute Care recommended pregnancy test for women in reproductive age.

It is difficult to measure whether the change observed in the newborn comes from the exposure to anesthesia or surgical procedure. In this present study, pregnant females received anesthesia and did not undergo any surgical procedure; therefore, all changes observed here are due to exposure to anesthetic.

Conclusion

The results of this study show that propofol administration causes an increase in resorption and post-implantation loss in anesthetized female in early pregnancy and promotes an increase in the anogenital distance of male offspring, delays pinna detachment and weight gain of pups whose mothers were anesthetized in late pregnancy. Results obtained in this study and in the literature on events observed in fetuses after maternal exposure to anesthetics, suggest that pregnancy test should be strongly recommended to women who are of childbearing age who would undergo a surgical procedure making use of anesthetics.

Acknowledgements

The Araucária Foundation supported this work.

References

Alder, S., & Zbinden, G. (1977). Methods for the evaluation of physical, neuromuscular, and behavioral development of rats in early postnatal life. In D. Neubert, H. J. Merker, T. E. Kwasigroch (Eds.), Methods in prenatal toxicology (p. 175-185). Stuttgart, DE: Georg Thiene.

Arnold, A. P., Xu, J., Grisham, W., Chen, X., Kim, H. K., & Itoh, Y. (2004). Minireview: sex chromosomes and brain sexual differentiation. Endocrinology, 145(3), 1057-1062.

Birkett, J. W., & Lester, J. N. (2003). Endocrine Disrupters in Wastewater and Sludge Treatment Process. London, UK: Lewis Publishers.

Bercker, S., Bert, B., Bittigau, P., Felderhoff-Muser, U., Buhrer, C., Ikonomidou, C., … Kerner, T. (2009). Neurodegeneration in newborn rats following propofol and sevofluraneanesthesia. Neurotoxicology Research, 16(2), 140-147.

Cattano, D., Young, C., Straiko, M. M., & Olney, J.W. (2008). Subanesthetic doses of propofol induce neuroapoptosis in the infant mouse brain. Anesthesia and Analgesia, 106(6), 1712-1714.

Cheng, T. H., Leung, Y. M., Cheung, C. W., Chen, C. H., Chen, Y. L., & Wong, K. L. (2010). Propofol depresses angiotensin II-induced cell proliferation in rat cardiac fibroblasts. Anesthesiology, 112(1), 108-118.

Creeley, C., Dikranian, K., Dissen, G., Martin, L., Olney, J., & Brambrink, A. (2013). Propofol-induced apoptosis of neurones and oligodendrocytes in fetal and neonatal rhesus macaque brain. British Journal of Anaesthesia, 110(1), 29-28.

Damasceno, D. C., Volpato, G. T., Calderon, I. M., Aguilar, R., & Rudge, M. V. (2002). Effect of acetylsalicylic acid on the reproductive performance and on offspring from Wistar rats. Revista da Associação Médica Brasileira, 48(4), 312-316.

Dixon, M. J., Marazita, M. L., Beaty, T. H., & Murray, J. C. (2011). Cleft lip and palate: understanding genetic and environmental influences. Nature Reviews Genetics, 12(13), 167-178.

Gallavan, R. H., Holson, J. F., Stump, D. G., Knapp, J. F., & Reynolds, V. L. (1999). Interpreting the toxicologic significance of alterations in anogenital distance: potential for confounding effects of progeny body weights. Reproductive Toxicology, 13(5), 383-390.

Glen, J. G. (1980). Animal studies of the anaesthetic activity of ICI 35 868. British Journal Anesthesiology, 52(8), 731-742.

Gritli-Linde, A. (2008). The etiopathogenesis of cleft lip and cleft palate: Usefulness and caveats of mouse models. Current Topics in Developmental Biology, 84(1), 37-38.

Irifune, M., Takarada, T., Shimizu, Y., Endo, C., Katayama S., Dohi, T., & Kawahara, M. (2003). Propofol-induced anesthesia in mice is mediated by GABAA and excitatory amino acid receptors. Anesthesiology and Analgesia, 97(2), 424-429.

Li, J., Xiong, M., Alhashem, H. M., Zhang, Y., Tilak, V., Patel, A., … Bekker, A. (2014). Effects of prenatal propofol exposure on postnatal development in rats. Neurotoxicology and Teratology, 43(1), 51-58.

Mandim, B. L. S, Ruzi, R. A., Bernardes, C. P., & Teixeira, R. R. (2015). Anesthesia for non - obstetrical surgery during pregnancy. Genetic Medicine, 3(1), 1-4.

Mazze, R. I., Wilson, A. I., Rice, S. A., & Baden, J. M. (1985). Fetal development in mice exposed to isoflurane. Teratology, 32(3), 339-345.

Mhuireachtaigh, R., & O’Gorman D. A. (2006). Anesthesia in pregnant patients for non-obstetric surgery. Journal of Clinical Anesthesia, 18(1), 60-66.

Milanovic, D., Popic, J., Pesic, V., Loncarevic-Vasiljkovic, N., Kanazir, S., Jevtovic-Todorovic, V., & Ruzdijic, S. (2010). Regional and temporal profiles of calpain and caspase-3 activities in postnatal rat brain following repeated propofol administration. Developmental Neuroscience, 32(4), 288-301.

Niebyl, J., & Simpson, J. (2008). Teratology and drugs in pregnancy. Iowa, IA: The Global Library of Women is Medicine.

Ooi, M., & Thomson, A. (2015). Morbidity and mortality of endoscopist-direct nurse-administered propofol sedation (EDNAPS) in a tertiary referral center. Endoscopy International Open, 3(5), 393-397.

Palanisamy, A., Baxter, M. G., Keel, P. K., Xie, Z., Crosby, G., & Culley, D. J. (2011). Rats exposed to isoflurane in utero during early gestation are behaviorally abnormal as adults. Anesthesiology, 114(13), 521-528.

Pesić, V., Milanović, D., Tanić, N., Popić, J., Kanazir, S., Jevtović-Todorović, V., & Ruzdijić, S. (2009). Potential mechanism of cell death in the developing rat brain induced by Propofol anesthesia. International Journal of Developmental and Neuroscience, 27(3), 279-287.

Reitman, E., & Flood, P. (2011). Anaesthetic considerations for non-obstetric surgery during pregnancy. British Journal Anesthesiology, 107(1), 72-78.

Richmond, G., & Sachs, B. (1984). Further evidence for masculinization of female rats by males located caudally in utero. Hormones and Behavior, 18(4), 484-490.

Salewski, E. (1964). Farbemethode zum makroskopischen Nachweis von Implantationsstellen am Uterus der Ratte. Naunym-Schmiedeberg Archives of Pharmakology, 247(1), 367-367.

Staples, R. E., & Schnell, V. L. (1964). Refinements in rapid clearing technic in the KOH-alizarin red S method for fetal bone. Stain Technology, 39(1), 61-63.

Van de Velde, M., & De Buck, F. (2007). Anesthesia for non-obstetric surgery in the pregnant patient. Minerva Anestesiologica, 73(4), 235-240.

Vutskits, L., Gascon, E., Tassonyi, E., & Kiss, J. Z. (2005). Clinically relevant concentrations of propofol but not midazolam alter in vitro dendritic development of isolated gamma-aminobutyric acid- positive interneurons. Anesthesiology, 102(5), 970-976.

Wang, H., & Day, S. K. (2006). Roadmap to embryo implantation: clues from mouse models. Nature Reviews Genetics, 7(3), 185-199.

Wichramaratane, G. A. (1988). The post-natal fate of supernumerary ribs in rat teratogenic studies. Journal of Applied Toxicology, 8(2), 91-94.

Wilson, J. G. (1965). Methods for administering agents and detecting malformations in experimental animals. In J. G. Wilson, J. Warkany. (Ed.), Teratology principles and techniques (p. 262-277). Chicago, IL: University of Chicago Press.

Yang, T., Jia, Z., Bryant-Pike, W., Chandrasekhar, A., Murray, J. C., Fritzch, B., & Bassuk, A. G. (2014). Analysis of PRICKLE1 in human cleft palate and mouse development demonstrates rare and common variants involved in human malformations. Molecular Genetic Genomic Medicine, 2(2), 138-151.

Yu, D., Jiang, Y., Gao, J., Liu, B., & Chen, P. (2013). Repeated exposure to Propofol potentiates neuroapoptosis and long-term behavioral deficits in neonatal rats. Neuroscience Letters, 534(1), 41-46.