Cotinine as an indicator of fetal exposure to active and passive smoking in pregnant women

In 2010, the so-called Anti-Tobacco Law was introduced in Poland, banning smoking in public places. Its idea was to limit the exposure of non-smokers to the impact of tobacco smoke. Before its introduction, smokers were allowed to smoke tobacco in public spaces without restrictions. This was a cause of exposure to various diseases, forcing people to inhale tobacco smoke in the workplace, restaurants, cafés, and many entertainment and culture-related facilities [1].

Nearly nine years after the passage of the Anti-Tobacco Law, the Public Opinion Research Centre (CBOS) assessed the effects of this law, and the obtained data were published in July 2019. The obtained data show that every fifth person in the country smokes cigarettes regularly, which constitutes 26% of adult Poles, and every twentieth person smokes tobacco occasionally. Smoking is becoming less and less popular. In 1997, 37% of Poles smoked, and since then there has been a decline in the number of smokers to 30% in 2010, and to 26% in 2019. The decrease in the number of smokers in the last period was mainly due to the reduction in smoking by men, 40% of whom were smokers in 2012, currently down to 23%. On the other hand, a slight increase can be observed in female smokers, from 23% in 2012 to 26% in 2019. People between 45 and 54 years of age smoke tobacco most frequently (35%), followed by people aged 55 to 64 (32%). Smoking is least popular among people aged 18 to 24. In the reproductive age of 25 to 44, approximately 33% of men and 19% of women smoke [2].

According to the “Health Behavior of Pregnant Women” report conducted throughout Poland, it was found that in 2017, 5.86% of pregnant women smoked tobacco, and 17.98% of pregnant women were exposed to the daily, passive inhalation of tobacco smoke [3].

The tolerance of people who do not smoke and are exposed to tobacco smoke has slightly decreased from 46% in 2012 to 42% today. As many as 50% of men and 34% of women are tolerant to smokers. People tolerant to smokers are younger: almost half of them (48%) are aged 18 to 24. Those most tolerant (84%) are smokers themselves. In contrast, 75% of those who never smoke and 65% of ex-smokers avoid being present in the company of smokers while they are smoking. The ban on smoking in public places is supported by 90% of all of those surveyed by CBOS.

Japanese researchers were the first to define second-hand smoke, and today this term is commonly used to refer to exposure to inhalation of cigarette smoke by non-smokers at home, at work or elsewhere for at least 2 hours a day [4]. The smoke exhaled by a smoker and the smoke from a burning cigarette in the atmosphere is a combination known as environmental tobacco smoke (ETS). Research on the impact of ETS has shown that regardless of the way smoke affects the body, the health consequences of smoking and being in the presence of smoke aerosol are the cause of many diseases, including cancer. The FDA (Food and Drug Administration) study assessing the knowledge of toxic substances contained in tobacco smoke showed high levels of ignorance about the type of chemical compounds and their impact on the health of smokers and people exposed to smoke in closed spaces [5].

Aerosol generated from electronic cigarettes, often used today, is inhaled by approximately 2% of young people aged 18–24. It contains a lower number of carcinogenic compounds in relation to the number found in tobacco smoke in traditional cigarettes and less nicotine. Nicotine is the most addictive substance found in cigarettes, which negatively affects the development of the brain, especially in young people before the age of 20. However, e-cigarette aerosol is an extremely harmful mixture of many chemicals, such as carcinogenic nitrosamines, acetaldehyde, acetone, acrolein, formaldehyde, nicotine, methylbenzaldehyde, and propanal. The use of e-cigarettes causes the emission of PM2.5, which consists of particulate matter (PM) with an aerodynamic diameter of 2.5 μm, and even smaller, ultrafine particles (UFPs) of 100 nm (PM0.1) or less. This type of particulate matter is the most dangerous for humans as it can get directly into the bloodstream. The concentration of PM2.5 and UFPs increases in the air surrounding an e-cigarette smoker. The fact that 30% of young people aged 15 to 19 are regular smokers of e-cigarettes is a cause for alarm. Like conventional cigarettes, e-cigarettes are included in the ban on smoking in public places and this ban was supported by 84% of respondents in the CBOS study [6, 7, 8].

Since 2017, HnB (heat-not-burn) devices have been used by smokers, which, instead of liquid, heat the tobacco without burning it. The smoker experiences a feeling between smoking a regular cigarette and an e-cigarette. It is believed that this form of smoking delivers an aerosol to the lungs with a lower content of toxic substances than the smoke from conventional cigarettes. The use of HnB is comparable to conventional cigarettes, it quickly delivers nicotine and reduces the overall exposure to numerous toxic compounds contained in the smoke of conventional cigarettes. These devices are recommended for smokers undergoing nicotine addiction treatment [9].

People who are in the presence of smokers of conventional cigarettes are exposed to the ETS, especially sidestream smoke, and become so-called passive smokers or involuntary smokers. Using e-cigarettes eliminates the sidestream that contains carcinogenic nitrosamines, which are harmful to health. The remaining substances are present only in lower concentrations [10]. Currently used methods of combating tobacco dependence, in accordance with the recommendations of the World Health Organization (WHO), include monitoring attitudes towards smoking, protection against tobacco smoke, and offering help in the treatment of addiction. The results of the study by the Chemical Substances Office carried out in 2018 in Poland showed that 49.7% of smokers using HnB products did not return to smoking conventional cigarettes, as compared to 16.1% of e-cigarette smokers. This gives hope for an effective fight against this addiction [11].

However, all forms of smoking result in the release of smoke coming directly from cigarettes and e-cigarette aerosols exhaled by the smoker, which affect the health of those who inhale them. In 2012, the FDA published a list of 93 harmful and potentially harmful components of HPHC (Harmful and Potentially Harmful Constituents in Tobacco Products and Tobacco Smoke: Established List) present in tobacco products and tobacco smoke. All of them were found to be toxic to humans, both for smokers and for those inhaling smoke aerosol—that is, passive smokers [12].

The toxicological aspect of tobacco smoke metabolism includes the transformation of chemical compounds contained in the smoke: absorption, biotransformation in the body, and excretion of the products of transformation. The mutagenic effect, leading to numerous pathologies, including carcinogenesis, is the most dangerous. Cotinine, a metabolite of nicotine, causes a significant increase in the expression of VEGF (insulin-like growth factor-II) in endothelial cells, which suggests the influence of cotinine on the progression of cardiovascular diseases, as well as tumor progression and the formation of metastases [13]. The genotoxic effects of tobacco smoke mutagens inhibit DNA damage repair processes. The presence of cadmium in cigarette smoke may lead to fetal deformities [14]. And the presence of lead in inhaled cigarette smoke has a negative impact on the placental flow of micronutrients, which impairs the growth and development of the fetus and also of children [15].

Cigarette smoke contains over 4,000 ingredients and not all effects resulting from the contact of pregnant women with them have been discovered and described so far. In the light of current scientific discoveries, an expert committee appointed by the Society for Research on Nicotine and Tobacco concluded that cotinine is the best-known biomarker of tobacco smoke in people exposed to tobacco smoke. The sensitivity of serum cotinine determinations is 96–97%, and the specificity is 99–100%, which makes it highly diagnostic [16, 17, 18].

The randomized study selected a group of 127 pregnant women without the coexistence of chronic diseases, who entered a health facility for delivery after a full-term physiological pregnancy. The study was conducted from January to December 2016. Out of this group, 4 pregnant women withdrew from the study. A diagnostic survey was carried out among 123 pregnant women, including sociodemographic data, data illustrating eating habits and health behavior, attitudes towards smoking, as well as information on family, social, and professional contacts with tobacco smokers during pregnancy. Each participant in the study, after becoming acquainted with the essence of the study, gave written informed consent for participation. The study was positively assessed by the Bioethics Committee of the Medical University of Warsaw. In the surveyed group, 97 (79%) women declared that they had not smoked tobacco during pregnancy, and 26 (21%) pregnant women were smokers before pregnancy and continued to smoke while pregnant.

A different number of respondents than 18 (15%) admitted to being present in the environment of smokers. This fact was taken into account and the percentages were corrected when assessing the concentration of cotinine. Those pregnant women who declared that they had contact with smokers for at least 2 hours/day and those who had cotinine presence in their blood serum were considered as persons who are present in the company of smokers and are exposed to passive smoking.

The age of the respondents ranged from 18 to 38 years; the average age was 25.3 years. Neonatal records with the Apgar score and the assessment of the child's condition made immediately after birth were used to assess the condition of the newborn. All newborns who received not less than 7 Apgar points in the 5th minute after birth were considered to be in good condition at birth.

After admission to the labor ward, all subjects had blood taken for basic tests and for the assessment of cotinine levels. The umbilical cord blood was collected from the newborns of the mothers participating in the study immediately after birth for the assessment of cotinine levels. Cotinine concentration levels in smoking and non-smoking mothers and in their children are presented in Table 7. Generally, the norms are <10ng/ml for non-smokers and >10ng/ml for smokers.

Cotinine tests in blood serum were performed in the Drug and Metabolite Assay Laboratory, part of the Environmental Mass Spectrometry Laboratory of the Institute of Biochemistry and Biophysics of the National Academy of Sciences in Warsaw. Cotinine concentrations were determined by the UPLC/MS/MS analytical method. Ultra-performance liquid chromatography (UPLC) coupled with tandem mass spectrometry (MS/MS) was used.

In order to answer the research problems posed, statistical analyses were performed using IBM SPSS Statistics 23. An analysis of the basic descriptive statistics was performed, along with the Student's t test for independent samples, Mann-Whitney U tests, x² test, Fisher's exact test and Spearman's ρ rank correlation analysis. The classical threshold α=0.05 was considered the level of significance; however, the test statistical probability results of 0.05 p<0.1 were interpreted at the statistical tendency level.

The cotinine concentration in blood samples was determined using the UPLC/MS/MS analytical method, using ultra-efficient liquid chromatography (UPLC) coupled with tandem mass spectrometry (MS/MS).

The characteristics of the entire study group of 123 pregnant women are presented in Table 1.

Among 123 pregnant women surveyed, 97 (79%) of those surveyed stated that they did not smoke tobacco and were not in the company of smokers. Out of this group, 39% (16) stopped smoking at the beginning of pregnancy, from 4–10 hbd (hebdomas graviditatis, week of pregnancy) and 34% (42) stopped smoking 3 months before the planned pregnancy. In contrast, 26 (21%) of the respondents smoked before pregnancy and continued smoking during pregnancy. Women who smoked during pregnancy were significantly younger than non-smokers: 30.8% vs 8.3% (U = 709; Z = - 2.84; p = 0.005; r = 0.29). They also significantly more often were less educated than non-smokers: 65.5% vs 11.5% (U = 391; Z = - 5.43; p <0.001; r = 0.54).

After the biochemical test determining the level of cotinine concentration in the blood, the lack of cotinine was found in only 50 (41%) of the respondents, which means that 47 (38%) of the respondents had contact with and inhaled tobacco smoke and gave a false answer. Between 18 and 46 of respondents admitted to being present in a smoking environment in various parts of the survey. 13.4% of pregnant non-smokers came into contact with smoke at home, 7% at work, 4% at their parents’ home, and 2% at friends.

Out of all 123 respondents, only 33 (27%) of the respondents had no contact with smoking in their parents’ home. In the group of 26 pregnant smokers, 18 (69%) came from families where their parents smoked tobacco. In the group of 97 non-smokers, the parents of 72 (74%) were smokers (Table 2). As many as 31% of pregnant women rejected the habit of smoking when smoking had been present in their parent's home, whereas only 26% of pregnant women coming from non-smoking homes rejected this addiction. It seems that the choice of smoking in the group of smokers was influenced by the peer-environmental factor, and in the group of non-smokers by the health factor.

Smoking by the surveyed pregnant women turned out to be significantly (χ²(1) = 28.03; p < 0.001; V = 0.5) related to the presence of smokers in their environment. Smokers were almost always (96.2%) in the presence of smokers. The number of cigarettes smoked by them differed. 38.5% of the respondents smoked up to 5 cigarettes a day, the same percentage in the case of smoking up to 10 cigarettes a day, 15.4% smoked occasionally, and 3.8% smoked more than 20 cigarettes per day. Most of the respondents, 77%, declared smoking throughout their pregnancy.

For family members and friends, pregnancy was not a reason to avoid smoking in the company of pregnant women. When assessing who in the pregnant women's environment smoked tobacco in their company, it was found that most commonly the smokers were members of their immediate family, husbands, and parents; their friends smoked less often and only their grandparents did not smoke (Table 3).

The number of cigarettes smoked among family members and friends varied. Tobacco was smoked significantly more often in the environment of pregnant women who were themselves smokers (Mann-Whitney U test, U = 448; Z = - 2.51; p = 0.012; r = 0.28).

Pregnant women were significantly more likely to be exposed to passive smoking at their parents’ home and at friends. The full data is presented in Table 4.

Moreover, it was shown that pregnant smokers were present for significantly longer periods of time (U = 133.5; Z = - 2.36; p = 0.019; r = 0.36) in the company of smokers, being exposed to tobacco smoke, than were non-smokers.

The effects of smoking in pregnant women as well as the effects of passive smoking on the mother and the newborn were investigated by assessing the concentration of cotinine, a metabolite of nicotine in the mother's blood and in the umbilical cord blood of the newborn immediately after birth. The cotinine concentration levels in mother and child turned out to be statistically significant higher for smokers (Table 5) as well as for women exposed to passive smoking and their children (Table 6).

It was checked whether the levels of cotinine in the blood of the mother and umbilical cord blood are different in the case of pregnant women who spent time with family members and friends who are smokers. In the Mann-Whitney U tests performed, significantly higher concentrations were recorded in pregnant women exposed to tobacco smoke (Table 6).

Among smokers, it was assessed whether there was a relationship between the duration of smoking during pregnancy and the level of cotinine concentration in the mother's blood and the umbilical cord blood. A series of analyses of the Spearman's ρ rank correlation was performed and all the tested compounds turned out to be statistically significant. Relationship of the duration of smoking during pregnancy with the level of cotinine in the mother's blood is shown in Spearman's rho - 0.695 ng/ml, p<0.001 and umbilical cord blood in Spearman's rho - 0.718ng/ml, p<0.001. These correlations were positive, indicating that the longer women smoked while pregnant, the higher was the level of cotinine in the mother's blood and in the umbilical cord blood. The strength of the observed correlations was high.

Comparing the child's birth weight with the level of cotinine concentration in the mother's blood and the umbilical cord blood in the performed analysis of the Spearman's ρ rank correlation, it was found that the child's weight was negatively correlated with the cotinine level. The higher the mother's cotinine level (maternal cotinine - Spearman's rho - 0.177g, significance 0.071, umbilical cord blood cotinine - Spearman's rho - 0.174, significance 0.077), the lower the child's birth weight. However, the strength of these relationships was low.

It was surprising that when comparing the levels of cotinine in the blood of mothers and in the umbilical cord blood of newborns, it was found that among 73 women with diagnosed cotinine in the blood, 23 (31.5%) newborns had levels of cotinine in the umbilical blood higher than their mothers! In 26 (35.6%) subjects, the concentrations in both groups were identical and in 24 (32.8%) subjects, the concentration in children was lower than in the mother's blood.

In this group, the mean concentration of cotinine (ng/ml) in the umbilical cord blood was M -15.15, SD - 36.28, and in the mothers’ blood - M - 13.97 SD - 34.87 (z-0.85 p = 0.395).

Searching for the differentiating features in the children with a higher cotinine concentration than in their mothers, it was found that these mothers were significantly more often smokers. Moreover, among women smokers, the highest percentage of women declaring smoking during pregnancy was recorded in the group of women whose cotinine level was lower than that in the umbilical cord blood of their children, while the lowest percentage of women declaring smoking was recorded in the group of women where the cotinine level was 75pg/ml and was identical in the mother and the child. The strength of the relationship found was moderately high (Table 7).

When examining the group of newborns with higher cotinine concentration than in their mothers, it was assessed whether there were smokers in the environment of persons with varying cotinine levels. It was found that passive smoke inhalation significantly correlates (χ²(2) = 8.86; p = 0.015; V = 0.34) with the concentration of cotinine in the blood of the subjects. The highest percentage of women who indicated spending time in the company of smokers was in those with a concentration of cotinine higher than in their child's blood. The strength of the observed effect was moderately high. The frequency of contacts between the examined women and smokers in the group of husbands, parents, grandparents, and friends was presented. There was one statistically significant result in the frequency of smoking by parents of pregnant women (χ²(2) = 7.35, p = 0.025, V = 0.32).

On the other hand, when assessing the number of cigarettes smoked by people from the environment of the pregnant respondents, depending on the level of cotinine concentration in their blood and that of their children, it was found that the exposure of the studied women to cigarette smoke was significantly the highest (χ²(2) = 9.08; p = 0.011; V = 0.35) in the group with a higher concentration of cotinine in the umbilical cord blood of children than in the mother's blood. Mothers whose children had higher cotinine levels than themselves were surrounded by smokers who smoked 20 or more cigarettes during their meetings.

When assessing the child's condition according to the Apgar scale as well as birth weight in the three examined subgroups of cotinine concentration, no significant differences were found, the results were similar.

In a WHO survey, smoking during pregnancy is confirmed by 1% of women in Algeria, up to 20% of pregnant women in Belgium and Germany, and >30% in Bosnia and Herzegovina [19]. There is a steady decline in the number of pregnant women smoking in the US. In 2014, 10.1% of American women smoked, while the smoking prevalence index in 2020, extrapolated from the then trend, was 6.1% [20]. As many as 15–30% of pregnant women in many countries, also in Poland, admit to smoking in surveys, but conceal the actual frequency of smoking. The biochemical assessment of nicotine markers increases the number of identified smokers by an average of about 10%. In Polańska's study, 14% of pregnant women were smoking according to the survey; this percentage increased to 20% after the biochemical assessment of cotinine in blood serum. An even greater increase, from 44% to 83%, occurred among women interviewed by these authors about passive smoking vs biochemical assessment of the cotinine biomarker. In our study, 38% of respondents provided false information about exposure to secondhand smoke. Assessment of exposure to passive smoking is difficult to establish. Polańska et al., assessing the survey information on smoking during pregnancy provided by the respondents, stated that the specificity of the response is 97.9% and the sensitivity is 60%. In contrast, data on exposure to ETS has a sensitivity of 50% and specificity of 40% [21]. Krzyścian et al. estimated that about 60% of pregnant women are exposed to inhaling tobacco smoke, and Polańska et al. estimated this percentage at 74% of the pregnant women. A similarly high percentage (60%) of people exposed to passive smoking was indicated by Kharazzi et al. [20, 21, 22, 23].

Pregnant women conceal the fact that their household members smoke. Perhaps this is due to the fact, as was shown by this study, that they are exposed to inhaling tobacco smoke significantly often at home and the most frequent smokers are the child's father and parents of the surveyed women. Therefore, in order to make a reliable assessment of a child's exposure to tobacco smoke, the interview is not a sufficient method. It should be verified with the biochemical assessment of the nicotine biomarker—serum cotinine or other biological material [21, 22, 23, 24, 25].

Most studies assessing the harmful effects of tobacco smoke concern the health condition of children and adolescents or the course of chronic diseases in adults [26]. Studies on the harmfulness of smoking by pregnant women based on the assessment of nicotine metabolites in the blood and of the mother and child are less frequently conducted, although this assessment provides reliable and undeniable data. As early as 2000, Pichini et al. showed that the determination of cotinine in cord blood is the best method for illustrating the exposure of pregnant women to tobacco smoke from active and passive smoking [27].

In our study, we found that the cotinine levels in the blood of 123 newborns studied shortly after birth were closely related to the length of the mother's smoking period. The longer a woman smoked while pregnant, the higher was the level of cotinine in both her blood and the baby's umbilical cord blood. When assessing the effect of cotinine on the birth weight of newborns, it was found that the correlations were negative. The higher the level of cotinine in the mother's blood, the lower was the birth weight of the child, but the strength of these relationships was low. Wierzyńska et al., when assessing the birth weight of newborns of mothers smoking up to 20 cigarettes a day, also did not find such a relationship [28]. On the other hand, Garn et al., analyzing over 40,000 births, found that in pregnant women who smoke 41 to 60 cigarettes a day, the risk of low birth weight increases and the score of the newborn child's general condition on the Apgar scale, in the 1st and 5th minute after birth, is low. Moreover, these authors found that the negative effects of smoking increase proportionally with the number of cigarettes smoked [29].

This fact was confirmed by Sochaczewska et al., who showed that the level of cotinine in the blood of the newborn increases with the increase in the number of cigarettes smoked by the pregnant woman. This fact was also confirmed by our analysis. Sochaczewska stated that after smoking 5 cigarettes, the concentration of cotinine in the umbilical blood was about 2.35 ng/ml, and after smoking 15 cigarettes was 59.1 ng/ml [30]. Moreover, in terms of the analyzed amount of 15 cigarettes smoked, these researchers showed no correlation between the concentration of cotinine, erythropoietin, pCO₂ and the condition of the newborn according to the Apgar scale in the assessment of fetal hypoxia [30].

Since the beginning of the 1950s, many health effects of active and passive smoking by pregnant women have been known, described, and documented [31]. Exposure to tobacco smoke during pregnancy is the cause of many complications, such as placenta previa and premature separation of placenta, preeclampsia, and preterm labor. Smoking also contributes to the activation of oncogenic HPV and an increase in the incidence of cervical cancer [32, 33, 34]. Prenatal exposure to cotinine leads to health risks to the unborn child, manifested by low birth weight (LBW), birth of a small fetus in relation to gestational age (SGA), IUGR, PPARD, stillbirth risk, and infertility [28, 35, 36, 37, 38].

Smoking while pregnant affects the child's health in many ways and has potential lifelong consequences. The mechanisms are largely unknown, but most likely epigenetics plays a role. It has been shown that as a result of smoking during pregnancy, the fetal DNA strands are chemically modified and many diseases develop and manifest themselves in different ways and throughout the child's life. These studies provided evidence that DNA changes not only contribute to the incidence of birth defects such as cleft lip and palate in children, but also may contribute to lung and nervous system diseases [39].

Studies comparing the concentration of cotinine in the blood of the mother and the umbilical cord blood of the newborn mostly show higher values of the marker in the blood of the mother than in the newborn. In our study, every third child (31.5%) of a mother-smoker had a higher level of cotinine in the umbilical cord blood than in the mother's blood.

Detailed analysis of this group showed that this phenomenon concerns newborns of mothers who are smokers and who live in an environment heavily polluted with tobacco smoke, among smokers who smoke 20 or more cigarettes/day.

It is believed that the measurements of the concentration of cotinine in the umbilical cord blood are lower than the measurements of this marker in the mother's blood. This fact is explained by the significant role of placental enzymes in the metabolism of nicotine and its metabolites [22]. This belief is based on studies and an analysis of cotinine concentration levels in women who usually smoke up to 15 cigarettes a day. It seems that the reason for the significant increase in cotinine concentration in newborns is the summation of the effects of cotinine resulting from active smoking by the mother and the exposure of pregnant women to ETS, as shown in the results of our study.

There are no studies to explain other reasons for high cotinine levels in a certain group of newborns. The studies by Dempsey et al. on the effects of cotinine on the bodies of newborns showed that the half-life of nicotine differed significantly in this age group compared to adults. These authors showed significantly higher values in newborns, which in their opinion may be the result of different enzymatic specificity and liver enzyme deficiency at this age [40].

High levels of cotinine concentrations in newborns can cause many diseases both in infancy and later in life, not excluding myocardial infarctions and strokes as a result of prenatal exposure to nicotine leading to increased oxidative stress and vascular hypertension [41].

Pregnant women should be compulsorily educated and persuaded to stop smoking during the first 20 weeks of pregnancy. In such a case, quitting smoking has a positive effect on reducing the likelihood of negative effects caused by the toxic effects of tobacco smoke [42].

Reducing exposure to tobacco smoke in public spaces is regulated by laws prohibiting the consumption of tobacco in public places. The effects of these bans have been assessed in many European countries as well as in Australia and the USA. Researchers describing this phenomenon agreed that the introduction of legal regulations that restrict smoking significantly contributed to the reduction of the impact of the ETS and thus limited the exposure of pregnant women and their children to negative health effects. Educational programs illustrating the harmful effects of smoking involving pregnant women themselves and those around them have so far failed to bring the expected effect [32, 43].

In the face of new data on the harmful effects of nicotine and cotinine on children, preventive healthcare should be intensified and targeted at family members of pregnant women. Almost every third fetus is exposed to the products of tobacco smoke, the effects of which may be visible at various times in a child's life. The inclusion of cotinine level assessment in pregnant women in the program of the standard of perinatal examinations should be considered in order to protect children from diseases resulting from the effects of nicotine in the embryonic period.