Motherisk Int J 2020;1;23
Kamelia Mirdamadi1,2, Gideon Koren,3,4
- University of Toronto, Department of Pharmaceutical Sciences, Toronto, ON, Ca
- Hospital for Sick Children, Division of Clinical Pharmacology and Toxicology, Toronto, ON
- Ariel University, Department of Clinical Pharmacology, Israel
- Motherisk Israel Program
Leslie Dan Faculty of Pharmacy, Department of Pharmaceutical Sciences
144 College St. Rm. 1060
Kamelia Mirdamadi and Gideon Koren have no conflict of interest to disclose.
Financial disclosure: None
Non-financial disclosure: None
Background: Sleep disturbances during pregnancy are associated with higher risks of maternal depression, hypertension, gestational diabetes and preterm labor. Nocturnal plasma melatonin levels are known to increase as pregnancy proceeds, reaching their peak near term. While insomnia is frequently seen in pregnancy, the possible role of maternal melatonin levels in sleep pattern has not been investigated.
Objective: To compare nocturnal saliva melatonin levels in pregnant women with and without insomnia.
Subjects and Methods: A prospective observational cohort study with three groups: 1- Insomnia-treated pregnant women, 2- Pregnant women with insomnia abstained from pharmacotherapy, 3- Healthy pregnant women. All participants were enrolled in the first trimester and matched for gestational and maternal age. Saliva samples were collected by subjects one hour before bedtime (20-24hr) in 30 minute’ intervals at the end of each trimester. ELISA was used to quantify salivary melatonin levels.
Results: One-way ANOVA showed significantly lower melatonin levels in untreated pregnant women with insomnia compared to treated women, and healthy pregnant women in the third trimester, (p<0.04). The untreated group had significantly lower salivary melatonin levels than the treated group in the second and third trimesters, (p=0.03).
Conclusion: Significantly lower nocturnal salivary melatonin levels throughout pregnancy were observed in pregnant women with untreated insomnia. Future studies are warranted to evaluate the safety and efficacy of melatonin supplementation during pregnancy for the treatment of insomnia.
Keywords: pregnancy, sleep disorder, melatonin, saliva
Statement of Significance:
Little is known about the pathophysiology of gestational insomnia. To our knowledge salivary levels of melatonin during gestation have not been measured. Comparing salivary melatonin levels in insomniac women with healthy pregnancies may contribute to better management of the condition with melatonin, as opposed to psychotropic drugs with possible adverse prenatal effects Furthermore, melatonin has a good safety profile, is inexpensive and available over-the counter.
The results of this study may impact the quality of life and health many pregnant women worldwide who use pharmacologic sleep-aids.
Multiple awakenings during sleep and lower quality of sleep have been reported in nearly three quarters of pregnant women.(Baker, Wolfson, & Lee, 2009; Fernandez-Alonso, Trabalon-Pastor, Chedraui, & Perez-Lopez, 2012; Kizilirmak A,Timur S., Kartal B., 2012) Moreover, pregnant women diagnosed with depression are at significant increased risk for developing insomnia.(Kizilirmak A,Timur S., Kartal B., 2012) Lower quality of sleep occurs in depressed pregnant women as early as at 20 weeks gestation, suggesting an earlier onset of pregnancy-related sleep disturbances in those with depression.(Okun, Kiewra, Luther, Wisniewski, & Wisner, 2011) In normal pregnancies, maternal serum melatonin levels start to rise dramatically after 24 weeks and reaches their maximum at term.(Nakamura et al., 2001) Average serum melatonin levels during pregnancy have been measured as approximately 7.0 pg/ml in the first trimester, 9.0 pg/ml in the second trimester, and 18.0 pg/ml in the third trimester, suggesting an increasing physiological demand for the hormone as pregnancy progresses.(Kivela, 1991) However, there is a gap in knowledge on the synthesis and changes in the serum levels of melatonin in pregnant women with insomnia.
Melatonin, an indole-amine produced in the pineal gland, is a key player in the regulation of the sleep-wake cycle. There is a strong association between melatonin, sleep, energy, and mental ability.(Fuller PM,Gooley J.J., Saper C.B., 2006) Day-time activity, dark-light cycle, and sleep are all mediated by changes in melatonin and cortisol levels with a negative correlation between the two hormones.(Fuller PM,Gooley J.J., Saper C.B., 2006) With the onset of dark-time, the pineal gland starts the synthesis of melatonin and serum levels of melatonin rise significantly closer to bedtime allowing for sleep onset.(Cagnacci, 1996) Therefore, supplementation with melatonin at right time can adjust circadian rhythm after shift work, jetlag, blindness, or sleep disorders. While previous studies have shown a pattern of increased maternal serum melatonin levels as gestation proceeds reaching its peak near term, more fragmented sleep and lower melatonin levels have been observed in pregnant women with mood disorders and/or anxiety.(Wisner,Okun M.L.Kiewra K.Luther J.F.Wisniewski S.R., 2011) The objective of this study was to measure the endogenous salivary melatonin levels in pregnant women with treated and untreated insomnia.
This was a prospective observational cohort study. Twenty pregnant women with treated or untreated insomnia and eighteen healthy pregnant women with no insomnia were enrolled during their first trimester between April 2013 and June 2015 through The Motherisk Program (MR) telephone help- line at the Hospital for Sick Children in Toronto (HSC) (Fig. 1;). Groups were matched for maternal (+/- 2yr) and gestational age(trimester). Originally verbal and then written informed consents were obtained, and a detailed pregnancy questionnaire was filled, including the Brief Insomnia Questionnaire (BIQ).(Kessler RC,Coulouvrat C., Hajak G., Lakoma M.D., Roth T., Sampson N., Shahly V., Shillington A., Stephenson J.J., Walsh J.K., Zammit G.K., 2010) Women with previous or present exposure to known teratogenic drugs, to melatonin,those unable to communicate in English, and inability to provide clean saliva samples, were excluded. Shift workers and those with a history of drug abuse were also excluded. The study protocol was approved by the Research Ethics Board at HSC. Characteristics of the study population are presented in Table 1.
Saliva Sample Collection
Saliva samples were collected by the participants three times during pregnancy; at 12 -14 weeks, 24-26 weeks, and 34-36 weeks. At each time point participants were instructed to collect three saliva samples every 30 minutes before bedtime while in dim light and an instruction sheet was provided. In total, each participant collected 9 samples during the study period. Subjects were instructed to refrain from chocolate, banana, alcohol, caffeine, and drinks with artificial colorants on the day of sampling and no aspirin or other medicines containing NSAIDS were allowed. On the sampling night they were instructed not have sat closer than 6 feetfrom a TV, and if using a computer/laptop/tablet, the contrast should have been adjusted to low. No meals at least 30 minutes before sampling was allowed to avoid saliva contamination. Participants were to brush their teeth without toothpaste, and rinse with water 10 minutes before sampling.(Voultsios, Kennaway, & Dawson, 1997) For saliva sampling a cotton swab kit (Alpco,US) was supplied, which was chewed or held in the mouth for 1-2 minutes, and placed in the tube when sufficiently soaked. Each sampling time was documented on a log sheet provided to participants. Saliva samples were refrigerated or kept frozen within 30 minutes of sampling and were shipped to the laboratory at Sickkids hospital in a cold box within 1-3 days.
ELISA was used to measure salivary melatonin levels according to manufacturer, (BÜHLMANN Direct Saliva Melatonin ELISA kit (EK-DSM) Salem, NH, USA).(de Almeida et al., 2011) In brief, previously frozen saliva was thawed and centrifuged for 5 minutes at 3,000 rpm. 200 µl of saliva was added to polypropylene tubes with the pre-treatment solution (sodium hydroxide) and vortexed for 5 seconds. After a 10-minute wait, the neutralizing solution (hydrochloric acid) was added and vortexed for 5 seconds. Samples were then centrifuged for 5 minutes at 10,000 rpm. Pretreated samples and controls were placed in the ELISA plate and incubated for 16-20 hours in 2-8°C. Biotin conjugate was added to the wells and the plate was incubated for another three hours in 2-8 °C. Saliva melatonin competes with biotinylated melatonin for the binding sites of this highly specific antibody. Samples were then washed and incubated on a plate rotator in room temperature for one hour. The enzyme label, streptavidin, and horseradish peroxidase (HRP) were then added to the wells. This binds to the melatonin-biotin antibody complex captured on the coated wells. The unbound fraction of the enzyme label was then washed, and tetramethylbenzidine (TMB) substrate was added to the wells and incubated for thirty minutes in room temperature on a plate rotator. Stop solution was added to the wells and the plate was read at 450 nm on a SpectraMax® Plus 384 microtiter plate reader. Analytical Sensitivity (Limit of Detection – LOD) of the assay was 0.5 pg/ml.
SPSS version 23 was used for all statistical analyses. Based on data distribution, parametric and non -parametric tests were used to compare mean melatonin concentrations between pregnant women with insomnia and healthy pregnant women in each trimester and among sub-groups (treated insomnia, untreated insomnia, healthy) in each trimester. To further investigate the pattern of change of salivary melatonin levels during pregnancy, repeated measure analysis of variance was used for all three groups. All data are presented in mean± SEM.
Among the 38 participants, 30 (79%) were Caucasian. The mean age of participants was 34 years old. In the treated group (3/7) had only insomnia and (4/7) had other psychiatric comorbidities ( Table 2) Most patients continued treatment throughout pregnancy. One patient started aripiprazole for the treatment of bipolar disorder in the second trimester. One patient stopped zopiclone very early in pregnancy.
In one-way ANOVA (Fig. 2); pregnant women with untreated insomnia had significantly lower melatonin levels (p=0.04) in the third trimester compared with the treated group and healthy pregnant women. The untreated group had also significantly lower salivary melatonin levels than the treated group in the second and third trimester (p=0.03).
To further investigate the pattern of change of salivary melatonin levels during pregnancy, repeated measure analysis was used for all three groups separately (Fig. 3A trend towards elevated levels of melatonin was observed in the treated women in the second and third trimester, (p=0.07) and in healthy pregnant women in the third trimester (p<0.05). Salivary melatonin levels remained significantly lower and unchanged in the untreated group with insomnia throughout pregnancy. The data did not show a significant difference between the healthy and insomniac groups in the first and third trimesters (Fig. 4) ; No significant difference was found in melatonin levels among the groups in the first trimester.
To the best of our knowledge this is the first study examining the levels of endogenous salivary melatonin in pregnant women with insomnia. Insomnia was associated with comorbid mental disorders in both treated and untreated pregnant women with insomnia. Our results demonstrate significantly lower salivary melatonin levels throughout pregnancy in women with untreated insomnia. Limited data demonstrated that sleep deprivation during pregnancy is a major risk factor to developing longer and painful labor, increased risk of preterm birth and cesarean section.(Chang, J. J., Pien, Duntley, & Macones, 2010) It has been further suggested that acute inflammation and a rise in pro-inflammatory cytokines as a result of dysregulated sleep pattern are potential contributing factors to this mechanism.(Miller, Maletic, & Raison, 2009) Melatonin is a strong anti-inflammatory and anti-oxidant hormone produced in the maternal pineal gland and in significantly larger amounts by the placenta.(Chang, Jen Jen, Pien, Duntley, & Macones, 2010) Therefore, significantly lower melatonin levels throughout pregnancy and persistent insomnia in untreated pregnant women may indicate a potential role of melatonin in regulating sleep-wake cycle during pregnancy and the impact of insomnia and sleep disorders on modulating melatonin levels and subsequent adverse maternal and neonatal outcomes. The rise in salivary melatonin levels reaching its peak in the third trimester observed in the healthy group is consistent with previous studies where serum melatonin levels were measured.(Kivela, 1991)
Our results suggest similar salivary melatonin levels between insomnia-treated pregnant women with psychotropic drugs and healthy controls, consistent with previous research that demonstrated an increased in serum melatonin levels with some psychotropic drugs. Bupropion, mirtazapine, quetiapine, and trazodone are associated with elevated serum melatonin levels.(Antonioli, Rybka, & Carvalho, 2012; Cohrs et al., 2004; Morera, Abreu-Gonzalez, Henry, & Garcia-Hernandez, 2009) However, hypnotics such as zopiclone and zolpidem do not affect melatonin levels(Mann et al., 1996), and benzodiazepines have sleep-promoting effects through peripheral GABAergic system.(Monteleone, Forziati, Orazzo, & Maj, 1989) Selective serotonin reuptake inhibitors (SSRI) modulate melatonin levels as well.(Antonioli et al., 2012) Carvalho et. al. (2009) demonstrated increased melatonin levels after treatment of depression with fluoxetine and duloxetine.(Carvalho, Gorenstein, Moreno, Pariante, & Markus, 2009) Nevertheless, previous observational studies demonstrated that many of the commonly used psychotropic interventions have been classified as Class C (Risk cannot be ruled out), D (Positive Evidence of Risk), and X (Contraindicated in Pregnancy) during pregnancy by The Food and Drug Administration (FDA).(Hashmi, Bhatia, Bhatia, & Khawaja, 2016) Efficacy and safety of melatonin supplementation for the treatment of insomnia have been documented in non-pregnant individuals. Therefore, more investigation into the safety of exogenous melatonin in pregnancy is required. Melatonin is inexpensive, is available over-the counter and it has a low side effect profile.
Saliva melatonin was selected for the purpose of this study as it is a non-invasive method, and there is a significant positive correlation between saliva and serum melatonin levels.(Voultsios et al., 1997) However, some limitations to this study have to be acknowledged. As pregnancy progressed and patients start to experience more pain and discomfort, there was an increased rate of participant drop-outs from the study. Our sample size is relatively small, owing to the complexity of recruiting these pregnant patients. We were lucky in being part of a large pregnancy health line that counseled pregnant women on pregnancy risks of medications, or untreated condition in pregnancy. The fact that despite a limited sample size we did document statistical differences suggests that the effect size is clinically important and robust.
This study documented lower salivary melatonin levels in pregnant women with untreated insomnia. In light of the findings of this study, assessment of the efficacy and reproductive safety of melatonin supplementation as an alternative medicine for the treatment of insomnia during pregnancy is warranted.
The authors would like to thank The Motherisk staff at The Hospital for Sick Children in Toronto, Canada in helping with subject recruitment. Authors also thank Dr. Shinya Ito with helping the end stage of the study.
ANOVA: analysis of variance
BIQ: brief insomnia questionnaire
Ect: ectopic pregnancy
ELISA: enzyme-linked immunosorbent assay
FDA: food and drug administration
HRP: horseradish peroxidase
HSC: hospital for sick children
LOD: limit of detection
NSAIDS: non-steroidal anti-inflammatory drugs
Pg/ml: picogram per millilitre
SA: spontaneous abortion
SEM: standard error of the mean
SSRI: selective serotonin reuptake inhibitor
TA: therapeutic abortion
TMB: tetramethyl benzidine
Table and Figure Legends:
Table 1: Demographics of the study population.
Table 2: Disease composition of exposed and disease-match group. All patients in the treated and untreated groups met the BIQ criterion for insomnia.
Figure 1: Total number of study population in each trimester. T: trimester
Figure 2:Changes of maternal salivary melatonin levels at nighttime in the normal singleton Pregnancy (solid black), pregnancy with untreated insomnia (Red bordered), and pregnancy with treated insomnia (blue bordered).
Figure3: Repeated measure analysis shows a trend towards significance increase in the levels of salivary melatonin levels in the treated group in the third trimester.
Figure 4: Plot of salivary melatonin levels in the first (A) and third (B) trimesters in the healthy and insomniac groups. There is no significance difference in salivary melatonin levels between the groups in the first and third trimester.
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