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In addition to case-control studies, there are other observational studies: exposed-unexposed (cohort) prospective studies. If we should conduct two studies, one of a cohort and one case-control to assess the link between a prognostic factor and a disease, the study considered the best, closest to the truth, is the cohort study.

In exposed - unexposed prospective studies we can identify a group of subjects who are exposed to a prognostic factor (the exposed group (e.g. smoker students) and compares with persons with similar characteristics (sex, age, socio-economic status), but who are not exposed to the prognostic factor (the unexposed group - students non-smokers (e.g. university students from the same university)). None of the subjects should have the disease at study entry. These two groups are followed over time and the onset of the disease is recorded.

Exposed-unexposed studies are costly, time-consuming, but provide more accurate information than case-control studies, since bias linked to ambiguous patient records, patient subjectivity and differences in recalling exposure can be avoided.

Example of exposed-unexposed study

The study we will consider as an example was published in the Journal of Atherosclerosis and Thrombosis in December 2017 is indexed in PubMed and can be downloaded for free. The article has the title Exposure to Parental Smoking in Childhood is Associated with High C-Reactive Protein in Adulthood: The Cardiovascular Risk in Young Finns Stud, having as authors Di Wang, Markus Juonala, Jorma S.A. Viikari, Feitong Wu, Nina Hutri-Kähönen, Olli T. Raitakari and Costan G. Magnussen.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5742368/pdf/jat-24-1231.pdf

The scenario of the example study

This study aimed to determine whether children with smoker parents (passive smoking) have a long-term increased risk of having high levels of highly sensitive C-reactive protein (hsCRP) when they reach adulthood. It is known that an increased value for hsCRP (> 3 mg / L, according to the American Heart Association) is an increased risk for cardiovascular disease and arteriosclerosis.

To examine this, information from Cardiovascular Risk in the Young Finns Study was used - a prospective cohort that began in 1980 and which gathered data for 31 years.

The above article contains all the necessary information on how they include and monitor the participants in the study.

Next, starting from this study and generating some data that is consistent with the study's findings, we will step by step repeat the study protocol.

Starting from the Cardiovascular Risk database in the Young Finns Study, which contains information that has begun to be collected since 1980 on the factors that influence cardiovascular disease in five Finnish university centers, it is desirable to determine whether there is an association (and if any, quantify this link) between mother's smoking in a family where there are children and high level of hsCRP in their children (when reached the adulthood). In 2016, the children who had become adults in the meantime were recalled to the physician to investigate their hsCRP. Excluded from the study were participants with: hsCRP> 10 mg / L levels, chronic rheumatic disease, history of infection in the past two weeks, subjects with type 1 diabetes mellitus, pregnant women, women who are breastfeeding, women using oral contraceptives (some previous studies shown that they canaffect the level of hsCRP).

Study protocol

Aim of research: Assessment of the influence of mother smoking on the hsCRP level of the adult child

Objectives:

• Assessing the link between maternal smoking and the child's hsCRP (reached adulthood).
• Quantification of the link between mother's smoking and the hsCRP level of the child (reached adulthood).

Domain of research: Evaluation of prognostic (risk or protective) factors

Study type:

• A. Based on study objectives: Analytical
• B. Based on the researcher’s role: Observational

 Target population and study sample

Target population:

Demographic characteristics: parents with minors in Finland

Accessible population: parents with minor children from five university centers in Finland

Study sample

Inclusion criteria: parents with minor children from five university centers in Finland

Exclusion criteria:

• Biasing factors:
  • hsCRP> 10 mg/L
  • chronic rheumatic illness
  • history of infection in the past two weeks
  • type 1 diabetes
  • pregnant woman
  • women who are breastfeeding
  • women using oral contraceptives
• Factors that make data collection difficult or impossible:
• Ethic issues: - patients who did not sign the study participation agreement
• Sample size: The sample consists of 2511 participants. The size of the sample is large enough. Test power was 80%.

Data collection method

• A. Based on the studied population: sampling
• B. Based on the duration of data collection: longitudinal; prospective
• C. Based on the grouping method: exposed-unexposed groups;

The target disease: increased hsCRP (leading to arteriosclerosis and heart disease) in the adult child;

The prognostic factor: mother's smoking

 Defining variables

Dihotomial: Smoker Mother (Yes / No), Increased hsCRP (Yes / No)

 Data description and analysis plan

Will be use frequency tables and pie charts to describe dichotomous qualitative data.

To describe the association between mother's smoking and the high level of hsCRP of the adult child data contingency tables and column type graphs will be used.

Statistical analysis

• Objectives:
  • testing the existence of the link between mother's smoking and the high level of hsCRP of the adult child - the Chi-square test
  • the quantification of the importance of this link – calculating the relative risk (RR) and attributable risk (RA)

Expected results. Data analysis and data presentation

Table 1. Contingency table: the association between mother's smoking and the high level of hsCRP of the adult child

• Column chart

Figure 1. Contingency table: the association between mother's smoking and the high level of hsCRP of the adult child

• Risk Ratio (RR) and its 95% confidence interval:

Considering the general contingency table:

Formula: RR=[a/(a+b)] / [c/(c+d)]

After making the calculation RR=2.43.

RR and 95% confidence interval: RR=2.43 (95% CI 1.93 – 3.07).

• Risk Difference (RD/RA) and its 95% confidence interval

Formula: RA= [a/(a+b)] - [c/(c+d)]

After making the calculation: RA=10.43%

RA and 95% confidence interval: RA=10.43 (95% CI 7.58 – 13.29).

p-value and name of the applied test: p0.001 (Chi-square test)

Interpretation of data. Discussions

Interpreting the results from statistical point of view

The objective of the study was to assess the relationship between mother's smoking and the child's hsCRP (when reached adulthood).

From a statistical point of view, test of the association was made by formulating two hypotheses (the null hypothesis and the alternative hypothesis), the null hypothesis being the one tested. In the case of its rejection, we affirm that we are in favor of the alternative hypothesis.

Null hypothesis: There is no significant association between maternal smoking and the child's hsCRP (above the threshold).

Alternative hypothesis: There is a significant association between mother's smoking and the child's hsCRP (above the threshold)

Because p 0.05, we reject the null hypothesis, therefore there is a significant association between mother's smoking and the child's hsCRP level (above the threshold value).

From a statistical point of view, quantification of the association was achieved by the punctual estimators of RR and RA (RD) and 95% confidence intervals.

• RR: The risk that a child whose mother was a smoker has elevated hsCRP values at maturity was 2.43 times higher than children whose mothers were not smokers.
• RA (RD): The risk that a child whose mother was a smoker has elevated hsCRP values at adulthood was with 10% higher than children whose mothers were not smokers.

Observation. RR, respectively RD refers to this (study) sample.

95% confidence interval interpretations

RR: We are 95% sure that population RR is in the range [1.93 – 3.07]. If we repeat the study with all the possible samples with the same numbers of subjects as in our study in 95% of them the RR will be in the range [1.93 – 3.07].

RA (RD): We are 95% sure that population RR is in the range [7.58 – 13.29]. If we repeat the study with all the possible samples with the same numbers of subjects as in our study in 95% of them the RA will be in the range [7.58 – 13.29].

Interpret the results from a clinical point of view

• RR: moderate importance
• RA: moderate importance

Observation. The confidence interval for RR and RA (RD) refers to what is happening in the population. Because the confidence intervals for RR and RA (RD) are narrow, the results are accurate.

The relationship between maternal smoking and the high level of hsCRP of the mature child is a relatively clinically important one, because both ends of the confidence interval have clinically important value.

Study conclusion

Children who are exposed to passive smoking (smoking mother) have double risk of increased general inflammation (high hsCRP) in adulthood. This can lead to arteriosclerosis and cardiovascular disease.

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