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 Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 13  |  Issue : 2  |  Page : 67-71

Effect of caffeine on foetal morphometric parameters of embryonic Wistar rat (Rattus norvegicus)


1 Department of Medicine, Neurology Unit, Aminu Kano Teaching Hospital, Bayero University, Kano, Nigeria
2 Department of Anatomy, University of Ibadan, Ibadan, Nigeria

Date of Web Publication1-Aug-2016

Correspondence Address:
Lukman Femi Owolabi
Department of Medicine, Neurology Unit, Aminu Kano Teaching Hospital, Bayero University, PMB 3452, Kano
Nigeria
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0331-8540.187362

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  Abstract 

Background: There is increasing body of evidence that maternal caffeine use during pregnancy predisposes the embryo and foetus to spontaneous abortion but data on its influence on foetal morphometry is scarce. Aim: This study aimed to evaluate experimentally the effects of caffeine on morphometric parameters of newborn of Wistar rats. Materials and Methods: Thirty, experimentally naive, adult female Wistar-albino rats weighing 100–150 g were mated. After confirming pregnancy by vaginal plug method, the pregnant rats were randomly assigned into four groups (Group A–D). The dams in Group A served a normal control. They had 0.2 ml/kg body weight of normal saline intraperitoneally from day 9 to 20. Experimental Group B rats had 25 mg/kg body weight of caffeine intraperitoneally. Group C had 50 mg/kg body weight of caffeine intraperitoneally and Group D dams had 100 mg/kg body weight of caffeine intraperitoneally. Dams in both experimental and control groups were sacrificed on day 20 after caffeine administration. The foetuses were weighed and their crown-rump lengths were measured. Their body mass index of the litters (foetuses) was calculated and their placentas were weighed. Results: The mean crown-rump length of the litters (Group A) was significantly more (5.61 ± 0.29) than the experimental which were 5.10 ± 0.22 cm, 4.72 ± 0.24 cm and 3.81 ± 0.16 cm for Group B, C and D litters, respectively (P < 0.0001). The mean foetal weight of Group A litters was significantly more (1.56 ± 0.38) than the experimental which were 1.25 ± 0.41 g, 1.15 ± 0.77 cm and 0.78 ± 0.34 cm for Group B, C and D litters, respectively (P < 0.0001). The mean placenta weight of the foetuses was significantly lower in the females from the experimental Group B, C and D compared to the control group (P < 0.0001). Conclusion: This study showed a significantly reduced foetal weight, crown-rump length and placenta weight in the offspring of experimental female rats treated with caffeine compared to the control.

Keywords: Caffeine, foetus, morphometry parameters, rat


How to cite this article:
Owolabi LF, Shokunbi MT. Effect of caffeine on foetal morphometric parameters of embryonic Wistar rat (Rattus norvegicus). Niger J Basic Clin Sci 2016;13:67-71

How to cite this URL:
Owolabi LF, Shokunbi MT. Effect of caffeine on foetal morphometric parameters of embryonic Wistar rat (Rattus norvegicus). Niger J Basic Clin Sci [serial online] 2016 [cited 2019 May 23];13:67-71. Available from: http://www.njbcs.net/text.asp?2016/13/2/67/187362


  Introduction Top


Coffee, which largely contains caffeine, is one of the most popular beverage with characteristic smell and taste that is consumed the world over.[1] Caffeine is one of the most frequently consumed stimulants among pregnant women and is present in many popular caffeinated beverages, foods and over-the-counter medications.[2] In the United State of America alone, over 75% of pregnant women reported intake of caffeine-containing products.[3] Caffeine crosses the umbilical cord and placenta easily and appears in the urine and plasma of neonates.[4] Therefore, foetal levels are believed to be in equilibrium with maternal concentrations [5] and a given in utero caffeine dosage may affect foetuses more than adults.[4],[6]

Many investigators have explored the potential harmful effects, such as birth defects and low birth weight, of caffeine on human pregnancy; however, the results are conflicting.[7],[8],[9] There is increasing body of evidence that maternal caffeine use during pregnancy predisposes the embryo and foetus to spontaneous abortion.[10],[11]

Even though it can be argued that advances in neonatal intensive care have been accompanied by a marked increase in the survival of very low birth weight infants caused by maternal caffeine consumption or consumption of any other agents that are capable of causing foetal toxicity,[5] this improved survival has been accompanied by increasing recognition of long-term neurological deficits.[5],[12]

Previous works have demonstrated that caffeine causes a number of physiological, pharmacological and cellular responses in a broad range of biological systems including central nervous system, gastrointestinal, renal, skeletal, smooth and cardiac muscle systems.[1],[12],[13]

Given the deleterious effect of caffeine on the various systems and considering the great number of individuals including the pregnant women consuming caffeine daily, this study aimed to evaluate experimentally the effects of coffee on morphometric parameters of the newborn of Wistar rats.


  Materials and Methods Top


Forty experimentally naive, adult albino Wistar rats (thirty females and ten males) ranging from 10 to 12 weeks of age and weights between 100 g and 150 g were obtained from the breeding colony of the animals house of Physiology and Veterinary Department of the University of Ibadan.

The animals were acclimatised in the Anatomy Department Graduate Research Laboratory for 2 weeks and subsequently divided at random into experimental and control groups. The animals were kept in well-ventilated, metal cages at room temperature (29°–30°C) with a 12-h light/dark cycle, in the Department of Anatomy, and were fed with standard rat pellet and received tap water ad libitum. Feed and water consumption were monitored on a daily basis.

Subsequently, three female rats were then introduced into the cages containing 1 male rat (m: f = 1:3) for mating; female rats were paired with adult male rats over 2–4 days in a dark room for copulation. Mating was deemed to have taken place on the appearance of vaginal plug. Vaginal plug is one of the first signs that a female rat has mated successfully; it is the appearance in her vagina of a copulatory mucous plug, formed from mucus and secretions of the male rats' accessory sex glands. The plug holds the sperm in the female's vagina and also may prevent impregnation by other males. After confirming pregnancy by the vaginal plug method, the pregnant rats were then divided into a total of four groups with seven animals in each group.

  • Group A: Rats in this group served a normal control. They were given 0.2 ml/kg body weight of normal saline intraperitoneally from day 9 to 20
  • Group B: Rats in this group served as the first experimental group. These rats were given 25 mg/kg body weight of caffeine intraperitoneally from day 9 to 20.
  • Group C: Rats in this group served as the second experimental group. These rats were given 50 mg/kg body weight of caffeine intraperitoneally from day 9 to 20
  • Group D: Rats in this group served as the third experimental group. These rats were given 100 mg/kg body weight of caffeine intraperitoneally from day 9 to 20.


All the groups were subjected to the same feeding regimen and were also weighed daily.

On day 9 of gestation, a day critical for the development of teratologic effects in this species,[14] the dams were then treated with intraperitoneal (i.p) caffeine,[15] using 1 ml and 2 ml syringes, at doses of 25 mg/kg/day (Group B), 50 mg/kg/day (Group C) and 100 mg/kg/day (Group D) during pregnancy from 9 to 20 days of gestation. Group A dams were treated with normal saline at the rate of 50 mg/kg/day for the same period.

Other measures of embryo growth and development were also taken. The minimum dose of caffeine used in this study (25 mg/kg) is a dose which does not result in gross morphological deformities and is comparable to the upper limit of caffeine consumption in humans (equivalent to approximately 10–12 cups of brewed coffee).

The animals were generally observed, before and after caffeine administration. Their weight before and after drug administration were recorded. Dams in both experimental and control groups were sacrificed on day 20 after caffeine administration. The rats were anaesthetised using chloroform vapour before dissection. After cutting the covering tissues of the abdominal cavity, the uterus with the foetuses was incised. The numbers of live, dead and resorbed foetuses were counted. After incising the placentas, the foetal membranes and foetal vitality were assessed. The placentas were incised from the foetal membrane and the umbilical cord. They were evaluated macroscopically, and their weight was then determined. The foetuses were also weighed and their crown-rump lengths were measured. Their body mass index (BMI) was calculated as w/l 2 where: w = weight of the foetus; l = crown-rump length of the foetus. The number of litters per mother as well as gross abnormalities was also noted.

Statistical analysis

The data entry analysis was done using SPSS Version 20 (SPSS Inc. Chicago, IL, USA). Normality of data was assessed using D'Agostino and Pearson omnibus test. Continuous data were expressed as means (standard error of the mean). Multiple comparisons between treatment groups were performed using one-way analysis of variance and Tukey's post hoc test for a test of significance (two-tailed). The level of significance was fixed at <5% probability for chance.


  Results Top


No female rat died spontaneously during the study. There were no significant differences in the amount of feed and water consumed between the groups during the experimental period. From the gestational day 9 when the administration of caffeine was commenced, caffeine-treated pregnant rats were more active and they consumed more food than the control. However, at about gestational day 18, the group (Group D) that had daily caffeine of 100 mg/kg body weight became significantly less active, the reduced locomotor activity of these animals, was evident by sluggishness and reduced agility.

Before mating, the mean weight of the control group and the experimental was (141.14 ± 1.57) for the control group (A) and 140.86 ± 1.07 g, 140.57 ± 1.9 g and 140.29 ± 1.38 g for Groups B, C and D, respectively, the difference was, however, not statistically significant (P = 0.743) [Table 1] and [Figure 1]. On a gestational day 18, which was the 10th day of caffeine administration, the average body weight of the control group (239.14 ± 1.95 g) was greater than that of experimental groups; Group B (218.57 ± 3.95 g), Group C (210.29 ± 8.67 g) and Group D (178.29 ± 6.05 g) and the difference was statistically significant (P < 0.0001). The difference was pronounced between the control group and each of the experimental groups [Table 1] and [Figure 1].
Table 1: Distribution of the mean weight of the female rats before mating and gestational day 18

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Figure 1: Distribution of mean weight before and at gestation day 18

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The mean crown-rump length of the offspring (litters) of the control group (Group A) was also more (5.61 ± 0.29) than that of the experimental which were 5.10 ± 0.22 cm, 4.72 ± 0.24 cm and 3.81 ± 0.16 cm for Group B, C and D litters, respectively, and the difference was statistically significant (P < 0.0001). The difference was most pronounced between the control group and Group C and D [Table 2].
Table 2: Distribution of the morphometric measurements of the litters/foetuses by group

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The mean foetal weight litters from the control group (Group A) was also more (1.56 ± 0.38) than the cases which were 1.25 ± 0.41 g, 1.15 ± 0.77 cm and 0.78 ± 0.34 cm for Group B, C and D litters, respectively (P < 0.0001). The difference was most pronounced between the control group and Group C and D [Table 2]. The mean BMI of the respective groups was shown in [Table 2]. The mean weight of the placenta was significantly lower in the females from the experimental group receiving caffeine compared to control group (P < 0.0001) [Table 3].
Table 3: Distribution of the mean weight of placentas by group

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  Discussion Top


This study has largely shown that caffeine influences foetal morphometric parameters suggesting that caffeine interferes with intrauterine growth in the rat. Our observation corroborates the earlier reports that showed that caffeine consumption during pregnancy has a deleterious effect on the development of foetuses.[15],[16],[17],[18]

The weight of the litters was significantly lower in experimental groups on the one hand, and on the other hand, it was lower in the group receiving the higher dose of caffeine. This finding is in agreement with reports from previous work.[15],[16],[17],[19],[20] In separate studies, Wilkinson and Pollard [19] using caffeine administered at a dose of 25 mg/kg on the 8th and 9th day of gestation as well as Nakamoto et al.,[18] using caffeine administered at dosses between 0.5 and 2 mg/kg between the 8th and 22nd day of gestation showed that caffeine in these doses administered to pregnant females rat results in foetuses being of low weight.

In a study on humans, Fortier et al. showed a rise of 5.2% in the risk of giving birth to an underweight baby if the daily intake of caffeine during pregnancy was >300 mg/day compared to a 1.3% risk in mothers who did not consume caffeine. The difference in weight between the two groups of offspring was about 105 g. They concluded that caffeine intake during pregnancy was a risk factor for intrauterine growth retardation.[21]

The findings in the current study have clinical implication in humans as in utero caffeine exposure has been associated with increased risk of abnormal foetal growth including small-for-gestational-age.[21],[22],[23] Small-for-gestational-age has been associated with higher risk of obesity and metabolic syndrome after birth and is considered to be an early indicator of adverse foetal programming for childhood and adult metabolic diseases.[24],[25],[26],[27] Thus, emerging evidence from both human and experimental studies provides support to examine an association between in utero caffeine exposure and the risk of childhood obesity

A pharmacologically active agent, caffeine, (1, 3, 7-trimethylxanthine), as psycho-stimulant, is one of the most widely consumed substances in the world.[28] Caffeine can have access into foetal circulation through penetration of the placental barrier.[29] Its clearance from the body is prolonged in pregnant women; moreover, due to low levels of metabolic enzymes, its rate of metabolism is low in the foetus.[5] Consequently, there is a prolonged in utero caffeine exposure of the foetus when compared with adults. It has also been hypothesised that caffeine may influence cell development culminating in gross structural and physiological subnormality through increasing cellular cyclic adenosine monophosphate concentrations,[30] and decrease intervillous placental blood flow via increasing circulating catecholamines.[31]

In light of this hypothesised mechanism of causation and in conformity with other studies,[16],[28],[30],[32],[33] the current study also showed a significant reduction in placental weight. The reduction in placental blood flow could partly explain the relatively small size of the placenta following maternal caffeine consumption.[31] This reduction in placenta weight also explains the high risk of miscarriage, foetal death and impaired neurological development in offspring of mothers who consume caffeine during pregnancy.[3],[34]

In view of the findings in our study and other studies, both experimental and human, it will be a sensible advice for women contemplating conception to reduce their caffeine intake before conception and that once pregnancy is confirmed, they should make every effort to stop caffeine consumption. However, Like in many other previous studies, the absence of serum assay of caffeine was not conducted in this study, this appears to be a limitation of this study. Thus, interpretation and generalisation of our findings should be done with caution.


  Conclusion Top


This study showed a significantly reduced foetal weight, crown-rump length and placenta weight in offspring of experimental female rats treated with caffeine compared with their control counterparts.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

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