Objective
This study investigates fluoxetine (FLX) exposure as an etiology for altered gestational length and adverse pregnancy outcomes.
Study Design
Two experiments were performed exposing mice to drinking water (H 2 O) or H 2 O+FLX. Primary outcomes included gestational length, litter size, and live birth rate. In experiment 1, time-mated dams were monitored for spontaneous birth, and gestational length was calculated. In experiment 2, dams were dissected on day 14 to verify litter size and qualities of embryo implantation.
Results
There was no difference in gestational length between H 2 O dams (480.7 ± 13.2 hours) and H 2 O+FLX dams (483.5 ± 10.1 hours), P = .70. Mean litter size was decreased in H 2 O+FLX dams (4.1 ± 1.3/litter) compared to H 2 O dams (5.5 ± 1.9/litter), P = .04. H 2 O+FLX dams were less likely to have live births (25.4%) compared to H 2 O dams (49.3%), P = .01.
Conclusion
Antenatal FLX exposure did not statistically alter gestational length, but did affect litter size and spontaneous loss in mice. This warrants further investigation.
Clinical depression is a common condition among women of reproductive age. Between 14-23% of pregnant women will experience a depressive disorder during pregnancy. Although there is no standard screening protocol, prenatal depression is one of the most frequently encountered medical complications during pregnancy. The trend of antidepressant use during pregnancy has continued to rise over the last decade largely as a result of the increased use of selective serotonin reuptake inhibitors (SSRIs). In 2003, approximately 13% of women took an antidepressant at some point in pregnancy, a rate that has doubled since 1999.
While several research studies have focused on the potential maternal and fetal risks of SSRI use in pregnancy, including possible teratogenic effects, other studies have suggested that the increase in SSRI use may also affect prematurity rates. The rate of preterm birth in the United States is approximately 12.7% of live births, which is an increase of >15% in the last decade. This study will focus on the effect of SSRI exposure as an etiology for preterm birth in a mouse model.
Conflicting data exist linking SSRI use to an increased risk for preterm birth, shortened gestational length, or spontaneous fetal loss. While Chambers et al showed that third-trimester SSRI exposure was associated with higher rates of premature delivery, Malm et al published a conflicting population-based study of pregnant women exposed to SSRIs vs matched controls finding no increased risk of preterm birth. Two meta-analyses have demonstrated that women exposed to SSRIs in the first trimester had higher rates of spontaneous abortions when compared to a group of women who were not exposed.
Deficiencies within the literature exist demonstrating an association between antenatal fluoxetine (FLX) exposure and adverse pregnancy outcomes. Historically, studies have not accounted for obstetric factors relating to preterm birth, namely, history of a previous preterm birth or prior preterm premature rupture of membranes; nor have they differentiated among a spontaneous, indicted, or elective preterm birth. While these and many other factors are difficult to control in human studies, causal associations between FLX exposure and shortened gestational length can be approached using animal models. Accordingly, this study was initiated to study the impact of antenatal FLX exposure in a mouse model and to begin investigations of mechanistic pathways if an association was confirmed.
Materials and Methods
Two prospective, blinded observational experiments were performed. In experiment 1, 52 virgin female 129/SvEvTac mice (Taconic Farms, Germantown, New York) were time-mated (2 females and 1 male per cage) over 4 hours just prior to the end of the daily light cycle. Each female was examined for the presence of an ejaculatory plug in the vagina. The plug, composed of coagulated secretions from male accessory sex glands, indicated that coitus and ejaculation had occurred. Dams were randomly assigned to 1 of 2 cohorts. Group 1 (n = 29) had continuous access to drinking water (H 2 O) only, while group 2 (n = 23) had access to H 2 O+FLX. Dams were monitored every 12 hours for spontaneous birth and length of gestation, which were recorded and analyzed. The individual cages were monitored for the date and time of birth every 4 hours starting on day 18. The mouse dams and pups were utilized in subsequent experiments, independent of this study, and ultimately sacrificed.
In experiment 2, 76 virgin female 129/SvEvTac mice (Taconic Farms) were mated (2 females and 1 male per cage) just prior to the end of the daily light cycle. After plug confirmation, dams were randomly assigned to 1 of 2 cohorts. Group 1 (n = 40) had continuous access to H 2 O, while group 2 (n = 36) had access to H 2 O+FLX. Plugged dams were sacrificed, dissected, and analyzed on day 14 following mating to verify pregnancy and assess for litter sizes and qualities of embryo implantation. H 2 O or H 2 O+FLX was initiated after plug confirmation and continued until the end of pregnancy. FLX was dissolved in H 2 O at the dose of 4 mg/50 mL to achieve an approximate 10 mg/kg/d dosing. FLX was freely available in the animal’s H 2 O in opaque bottles to protect from light and changed weekly. FLX was chosen because it is the most commonly prescribed antidepressant during pregnancy and because of its extended half-life. The dosing regimen utilized by our laboratory and others has repetitively produced therapeutically relevant blood levels in mice. FLX and the metabolite levels have been measured in serum and whole brain and serum, and it has been previously shown that steady state for both FLX is achieved by 5 days of receiving FLX in the H 2 O (10 mg/kg/d), as serum levels after 5 or 90 days of treatment are equivalent. FLX levels were determined by liquid chromatography with fluorescence detection. Detection of a copulatory plug designated gestational day and time 0. The mated mice were housed individually and were weighed weekly and at the time of delivery. The average length of gestation of a 129/SvEvTac mouse is 480 hours, and the average 129/SvEvTac dam produces a litter size of 6.4 ± 0.88 pups (Taconic Farms).
Animals were maintained in a 12-hour light and dark cycle in a temperature- and humidity-controlled environment. Unlike the animal care technicians, the individuals observing the pup deliveries were blinded and unaware of the composition of the subjects’ H 2 O. The animals were fed with standard food pellets. Animal testing was conducted in accordance with the National Institutes of Health laboratory animal care guidelines and with Columbia University Institutional Animal Care and Use Committee approval.
Based on pilot data, a power analysis was performed, with an alpha of 0.05, and a beta of 0.80, and it was determined that a minimum of 21 dams would be needed in each group for each experiment to demonstrate a decrease in gestational age by 1.1 days or 5%, for a total sample size of 42 dams. The live birth rate was calculated as the ratio of pregnant dams to the total number of plugged dams. The data are expressed as mean ± SD. The significance of differences among the groups was assessed using 1-way analysis of variance test and Fisher’s exact test. The criterion for significance for all analyses was P ≤ .05. The criterion for a trend was .05 < P < .1.
Results
The Table presents the findings from experiment 1 (length of gestation), including differences in mean weight gain at gestational day 14 and mean litter size. In all, 52 females showed evidence of vaginal plugs following the brief exposure to males. Of these, 17 dams ultimately had live births. There was no significant difference in gestational length between H 2 O dams (n = 13; 480.7 ± 13.2 hours) and H 2 O+FLX dams (n = 4; 483.5 ± 10.1 hours), P = .70. However, dams exposed to H 2 O+FLX were less likely to have live births, 17.4% (4/23), than dams exposed to H 2 O alone, 44.8% (13/29), P = .04.
| Outcome | Experiment 1 (n = 52) | Experiment 2 (n = 76) | Total (n = 128) | ||||||
|---|---|---|---|---|---|---|---|---|---|
| Drinking water only (n = 29) | Drinking water plus fluoxetine (n = 23) | P | Drinking water only (n = 40) | Drinking water plus fluoxetine (n = 36) | P | Drinking water only (n = 69) | Drinking water plus fluoxetine (n = 59) | P | |
| Length of gestation, h | 480.7 ± 13.2 | 483.5 ± 10.1 | .70 | NA | NA | NA | NA | NA | NA |
| Mean weight gain at gestational day 14, g | 6.1 ± 2.7 | 4.9 ± 1.3 | .44 | 6.1 ± 2.4 | 4.9 ± 1.0 | .12 | 6.0 ± 2.5 | 5.2 ± 1.2 | .27 |
| Live birth rate (confirmed pregnancy/plugged) | 44.8% (13/29) | 17.4% (4/23) | .04 | 52.5% (21/40) | 30.6% (11/36) | .05 | 49.3% (34/69) | 25.4% (15/59) | .01 |
| Mean litter size (pups/litter) | 5.8 ± 1.7 | 4.5 ± 1.7 | .22 | 5.5 ± 1.9 | 4.1 ± 1.3 | .02 | 5.5 ± 1.9 | 4.1 ± 1.3 | .04 |
| Missed abortion rate | NA | NA | NA | 0.0% (0/21) | 18.2% (2/11) | .20 | NA | NA | NA |
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