protocol for a longitudinal twin birth cohort study to unravel the complex interplay between early-life environmental and genetic risk factors in health and disease: the chongqing longitudinal twin study (lotis)

Introduction non
Infectious Diseases (NCD)
In most countries, it is now the main burden of adverse health.
It is clear that the risk of this situation is largely from the early stages of life, possibly in the womb.
In view of its complex causes, it is necessary to understand the origin of NCD in
An in-depth analysis of the interaction between genetic variation and the environment, preferably over time.
Twin studies have played a key role in understanding these features for decades.
Their advantage is to be able to unlock the genetic and environmental factors that lead to the phenotype.
This is by comparing the genetically identical single eggs (MZ)
For twin twins with an average of 50% genetic variation, or by comparing MZ twins in a pair.
This study aims to identify genetic and environmental aspects of early
Intermediate phenotype associated with advanced adult disease (
Such as growth and nerve development)
And identify specific biomarkers and time points from which phenotype occurs during infancy, which is largely independent of potential genetic factors.
Research Method/design of vertical twins in Chongqing (LoTiS)
300 women with twins will be recruited to provide a wealth of nutrition for MZ pregnancy
Up to 3 years old.
Data will be collected at key points in pregnancy (×3)
After delivery and delivery (×9).
Biological samples of mothers and infants will be collected, including blood, urine, hair, nails and oral swabs, as well as measures such as fetal scanning and body measurements.
Other information in the questionnaire and medical records included pregnancy, diet, social population, maternal stress and infant growth and neurodevelopment.
This study was approved by the ethics committee of Chongqing Medical University (
Record Number: 201530)
And has been registered in the China clinical trial registry (
Registration number: ChiCTR-OOC-16008203).
Recruitment results and all follow-up analysis will be submitted to the peer for publication
Review journals.
Trial registration number ChiCTR-OOC-16008203; Results.
Introduction non
Infectious Diseases (NCD)
In most countries, it is now the main burden of adverse health.
It is clear that the risk of this situation is largely from the early stages of life, possibly in the womb.
In view of its complex causes, it is necessary to understand the origin of NCD in
An in-depth analysis of the interaction between genetic variation and the environment, preferably over time.
Twin studies have played a key role in understanding these features for decades.
Their advantage is to be able to unlock the genetic and environmental factors that lead to the phenotype.
This is by comparing the genetically identical single eggs (MZ)
For twin twins with an average of 50% genetic variation, or by comparing MZ twins in a pair.
This study aims to identify genetic and environmental aspects of early
Intermediate phenotype associated with advanced adult disease (
Such as growth and nerve development)
And identify specific biomarkers and time points from which phenotype occurs during infancy, which is largely independent of potential genetic factors.
Research Method/design of vertical twins in Chongqing (LoTiS)
300 women with twins will be recruited to provide a wealth of nutrition for MZ pregnancy
Up to 3 years old.
Data will be collected at key points in pregnancy (×3)
After delivery and delivery (×9).
Biological samples of mothers and infants will be collected, including blood, urine, hair, nails and oral swabs, as well as measures such as fetal scanning and body measurements.
Other information in the questionnaire and medical records included pregnancy, diet, social population, maternal stress and infant growth and neurodevelopment.
This study was approved by the ethics committee of Chongqing Medical University (
Record Number: 201530)
And has been registered in the China clinical trial registry (
Registration number: ChiCTR-OOC-16008203).
Recruitment results and all follow-up analysis will be submitted to the peer for publication
Review journals.
Trial registration number ChiCTR-OOC-16008203; Results.
The burden of not carrying
Non-communicable diseases
The best source of calcium (with choices);
The best source of vitamin C (with choices)
And what kind of food shortage in the early stage of pregnancy will increase the occurrence of test tube defects (with choices).
Examples of five questions to assess nutritional attitudes include whether women will look for nutritional information during pregnancy;
Whether their diet should be meal-based and supplemented, and whether to eat foods they don\'t like but are good for babies will be a problem.
The last part of this questionnaire asks about nutrition-related behavior.
There are five questions that ask about nutritional supplements taken during pregnancy and what foods to eat every day (with choices)
And the type and duration of exercise during pregnancy.
This information will be collected between 11 and 16 cycles of pregnancy.
The short baby sleep questionnaire was evaluated using a short baby sleep questionnaire, a reliable assessment tool for assessing and screening baby sleep problems.
The twins were evaluated at the age of 6 months, and the information collected included sleep measures such as night sleep time, daytime sleep time, number of times the baby wakes up at night, awake time at night, start time of night sleep, sleep position and preferred body position.
Bayley III baby development scale-
Revision of Bayley baby development scale in China
China\'s amendment is a standardized assessment of cars. fine and gross), language (
Acceptance and expression)
Cognitive development of infants and preschool children (
Age 2-30 months).
It is managed by trained professionals and consists of a series of game tasks.
The assessment was conducted at 18 months old.
Kaili preschool Temperament Questionnaire 12 was used to determine the sensitivity of children to external stimuli and the intensity of their response.
A range of questions were asked about food, animals, strangers, levels of attention, the ability to socialize and interact with others, mood swings, the ability to work with others and share toys.
The assessment was conducted at the age of 30 months.
The best source of food frequency Q calcium (with choices);
The best source of vitamin C (with choices)
And what kind of food shortage in the early stage of pregnancy will increase the occurrence of test tube defects (with choices).
Examples of five questions to assess nutritional attitudes include whether women will look for nutritional information during pregnancy;
Whether their diet should be meal-based and supplemented, and whether to eat foods they don\'t like but are good for babies will be a problem.
The last part of this questionnaire asks about nutrition-related behavior.
There are five questions that ask about nutritional supplements taken during pregnancy and what foods to eat every day (with choices)
And the type and duration of exercise during pregnancy.
This information will be collected between 11 and 16 cycles of pregnancy.
The short baby sleep questionnaire was evaluated using a short baby sleep questionnaire, a reliable assessment tool for assessing and screening baby sleep problems.
The twins were evaluated at the age of 6 months, and the information collected included sleep measures such as night sleep time, daytime sleep time, number of times the baby wakes up at night, awake time at night, start time of night sleep, sleep position and preferred body position.
Bayley III baby development scale-
Revision of Bayley baby development scale in China
China\'s amendment is a standardized assessment of cars. fine and gross), language (
Acceptance and expression)
Cognitive development of infants and preschool children (
Age 2-30 months).
It is managed by trained professionals and consists of a series of game tasks.
The assessment was conducted at 18 months old.
Kaili preschool Temperament Questionnaire 12 was used to determine the sensitivity of children to external stimuli and the intensity of their response.
A range of questions were asked about food, animals, strangers, levels of attention, the ability to socialize and interact with others, mood swings, the ability to work with others and share toys.
The assessment was conducted at the age of 30 months.
Food Frequency Questionnaire (FFQ)
FFQ is used to evaluate dietary intake, nutritional status, and dietary patterns associated with pregnancy outcomes.
Information about the frequency and quantity of food consumed in the first 3 months is recorded in face-to-
Interviews with trained researchers at two time points: 11-16 weeks of pregnancy and 30-34 weeks of pregnancy. Ninety-
Five questions related to consumption of bread, cereals, noodle meals, vegetables, tofu, fruits, poultry, meat dishes, biscuits, pastries, cakes, snacks, milk and dairy products.
15 questions related to eating behavior. Twenty-four-
24-hour food recall questionnaire
Hour food recall questionnaire 13 14 for recording detailed food intake within 24 hours
An hour until the last meal.
The questionnaire was designed to identify dietary patterns and related behaviors.
A trained researcher is equipped with standardized instruments and pictures to guide women to the weight and conduct a face-to-face questionnaire surveyto-face interview.
The women were asked to recall information about their food consumption.
The issue concerns the name and consumption of different foods, as well as the place where each food is consumed.
This assessment was carried out in conjunction with FFQ.
The child nutrition questionnaire consists of three parts.
The first is the feeding survey, with a total of 25 problems.
Examples include: What is your child\'s feeding pattern for the first 6 months?
What time is the solid introduced?
Is the food specially prepared for your child?
How many times does your child feed every day?
The second part is 24-
The third part is a food frequency questionnaire designed specifically for LoTiS babies.
The assessment will be conducted separately for all 12-month-old twins.
Drug use we are collecting all information about drug use before and after pregnancy, as well as a history of illness at the time of recruitment.
Drug prescriptions during pregnancy are also recorded.
During the three prenatal examinations, samples of maternal blood peripheral blood will be collected by intravenous puncture to assess the complex lipid, trace nutrient and endocrine status of the maternal, to collect live lymphocytes and to extract DNA/RNA.
A total of 15 ml of blood will be collected at each visit: 6 ml in a 10 ml coagulant tube (for serum)
And anti-coagulation tube in 10 mL 6 mL (
Plasma, white blood cells, red blood cells)
, The remaining 3 mL in the 5 mL Heparin anti-coagulation tube (for plasma).
Samples will be transported to the laboratory within 2 hours of collection for treatment and storage as serum, plasma, red blood cells and white blood cells.
Cord blood and placenta tissue cord blood collected from the umbilical vein were made into 10 mL eb anti-coagulation tube, 5 mL Heparin anti-coagulation tube and 10 ml coagulation tube.
20 pieces will be randomly selected using 4mm skin biopsy punch (
2mm x 2mm x 4mm per piece)
Placenta tissue from the surface of the fetus and mother.
The sample will be placed immediately in a pre-cooling state (4°C)
Physiological saline solution for transport to the laboratory for cold storage within 30 minutes of collection at a temperature of-80 °c.
The oral glucose tolerance test will be performed at the second prenatal examination when a woman is 22 to 25 weeks pregnant to detect gestational diabetes.
The measures recorded included fasting blood glucose levels, 60 min blood glucose levels after meals, and 120 min blood glucose levels after meals.
Urine samples are collected for each mother\'s prenatal examination to provide information on environmental exposure (
Such as double phenol).
Clean the vulva using a clean wipes before collection.
10 ml of urine will be collected using a 15 ml sterile centrifugal tube.
After being transported to the laboratory, the sample will be centrifuge for 10 minutes in 30 minutes/min, then wait to be assigned to a 250l QR code tube and stored at-80 °c within 30 minutes of processing
At the first prenatal check-up, hair will be collected for metabolic OMICS and toxin measurements to assess the condition of acute and chronic exposure to a range of minerals and toxins.
The sample included about 30 hairs close to the scalp.
After the birth of the twins, about three clusters of baby hair will also be cut close to the scalp and stored for future analysis.
Tin paper will be used to wrap the hair with a label at the time of collection to identify the position of the near end of the scalp.
Samples will be transported to the laboratory by cold storage within 2 hours of collection
-Long-term storage at 20 °c
Samples of nail mother\'s nails were collected during the first and third prenatal examinations to measure chronic environmental exposure (
(Such as heavy metals).
After collection, the nails will be wrapped in foil immediately and then shipped to the laboratory within 2 hours of collection for immediate storage at 20 °c.
In all three maternal prenatal examinations, cheek cells will be collected using oral swabs.
DNA will be extracted from the swab for subsequent genetic and/or epigenetic testing.
At 6 weeks old, cheek cells will be collected from each twin in the same way.
Participants will be required not to eat food and liquid within 30 minutes prior to collection.
To collect the cells, the oral mucosa of each cheek will be rubbed about 10 times with a sterile oral swab.
The swab will then be stored immediately in a sterile test tube and stored in a refrigerator at 80 °c.
DNA will be extracted from the swab within one month of the sample collection and stored again at-80 °c.
During the three prenatal examinations, maternal blood peripheral blood will be collected by intravenous puncture to assess the complex lipid, trace nutrient and endocrine status of the mother, to collect live lymphocytes and to extract DNA/RNA.
A total of 15 ml of blood will be collected at each visit: 6 ml in a 10 ml coagulant tube (for serum)
And anti-coagulation tube in 10 mL 6 mL (
Plasma, white blood cells, red blood cells)
, The remaining 3 mL in the 5 mL Heparin anti-coagulation tube (for plasma).
Samples will be transported to the laboratory within 2 hours of collection for treatment and storage as serum, plasma, red blood cells and white blood cells.
Cord blood and placenta tissue cord blood collected from the umbilical vein were made into 10 mL eb anti-coagulation tube, 5 mL Heparin anti-coagulation tube and 10 ml coagulation tube.
20 pieces will be randomly selected using 4mm skin biopsy punch (
2mm x 2mm x 4mm per piece)
Placenta tissue from the surface of the fetus and mother.
The sample will be placed immediately in a pre-cooling state (4°C)
Physiological saline solution for transport to the laboratory for cold storage within 30 minutes of collection at a temperature of-80 °c.
The oral glucose tolerance test will be performed at the second prenatal examination when a woman is 22 to 25 weeks pregnant to detect gestational diabetes.
The measures recorded included fasting blood glucose levels, 60 min blood glucose levels after meals, and 120 min blood glucose levels after meals.
Urine samples are collected for each mother\'s prenatal examination to provide information on environmental exposure (
Such as double phenol).
Clean the vulva using a clean wipes before collection.
10 ml of urine will be collected using a 15 ml sterile centrifugal tube.
After being transported to the laboratory, the sample will be centrifuge for 10 minutes in 30 minutes/min, then wait to be assigned to a 250l QR code tube and stored at-80 °c within 30 minutes of processing
At the first prenatal check-up, hair will be collected for metabolic OMICS and toxin measurements to assess the condition of acute and chronic exposure to a range of minerals and toxins.
The sample included about 30 hairs close to the scalp.
After the birth of the twins, about three clusters of baby hair will also be cut close to the scalp and stored for future analysis.
Tin paper will be used to wrap the hair with a label at the time of collection to identify the position of the near end of the scalp.
Samples will be transported to the laboratory by cold storage within 2 hours of collection
-Long-term storage at 20 °c
Samples of nail mother\'s nails were collected during the first and third prenatal examinations to measure chronic environmental exposure (
(Such as heavy metals).
After collection, the nails will be wrapped in foil immediately and then shipped to the laboratory within 2 hours of collection for immediate storage at 20 °c.
In all three maternal prenatal examinations, cheek cells will be collected using oral swabs.
DNA will be extracted from the swab for subsequent genetic and/or epigenetic testing.
At 6 weeks old, cheek cells will be collected from each twin in the same way.
Participants will be required not to eat food and liquid within 30 minutes prior to collection.
To collect the cells, the oral mucosa of each cheek will be rubbed about 10 times with a sterile oral swab.
The swab will then be stored immediately in a sterile test tube and stored in a refrigerator at 80 °c.
DNA will be extracted from the swab within one month of the sample collection and stored again at-80 °c.
Assessment body measures the mother\'s body measurements will be performed in all three prenatal examinations to assess weight gain and fetal development during pregnancy.
Height, weight and blood pressure will be recorded at the first visit.
For subsequent visits, weight, blood pressure, height of the stomach, abdominal circumference, and fetal heart will be recorded.
Baby body measurements will be performed at birth and repeated in each of the eight follow-up items
Visited by trained pediatric nurses to assess growth and development. A stadiometer (
Shanghai, China)
Will be used to evaluate maternal height and weight and to record the corresponding baby information on the growth curve.
Standard tape measure will be used to determine head circumference, chest circumference, abdominal circumference, and digital body fat calipers will be used to determine the thickness of skin folds.
Body composition information about the body composition of twins will be collected at birth, with babies aged 6 weeks, 6 months and 1, 2 and 3 years.
This information will be used in conjunction with body measurements to gain insight into body composition.
The body composition analyzer collects parameters of whole body water, fat mass, no fat mass, out-of-cell fluid, in-cell fluid and body mass index (
Imp sfb1, Australia).
Delivery record information from delivery records will be collected.
This will include details of the delivery method (
C-section or vagina)
Any preoperative and postoperative diagnosis, as well as the size and weight of the placenta, the length of the umbilical cord, the volume and features of the amniotic fluid.
The baby\'s gestational age, gender, weight, length and Apgar score at the time of delivery will also be recorded.
Using tidal breath analysis, breathing function will be measured by trained professionals when the baby is 2 years old.
Lung function tester (
CareFusion, Germany)
Lung function parameters that will be used to determine the respiratory frequency, tidal volume, inhalation time, Expiration Time, time to reach the peak tidal flow, expiration volume of the peak tidal flow, the ratio of inspiration time to expiration time and the ratio of time to total expiration to reach peak expiration flow.
When the twins are 6 weeks old, an experienced ultrasound doctor will measure their cardiovascular function through abdominal ultrasound.
Using the commercial system, the middle thickness of the aortic endometrium and the diameter of the aorta will be obtained (
Voluson E8, General Electric Healthcare, Austria)
It\'s equipped with a C4-8 transducer (
Frequency 4-8 MHz).
Early assessment of the liver and kidney function of the mother (11–16 weeks)and late (30–34 weeks)pregnancy.
Serum Collection from women under 8 years of age
Fast for an hour.
These serum were examined in the clinical laboratory of the First Affiliated Hospital of CQMU or Chongqing maternal and child health center.
These measures will include total protein, clearance protein, immunoglobulin, total bile acid, gluten ammonia transfer enzyme, Valley propylene ammonia transfer enzyme, urea and creatinine urine.
According to the standard guidelines, an ultrasound scan of the fetus will be obtained for each prenatal examination.
Trained medical ultrasound inspectors using commercial systems (
General Electric Healthcare company Voluson E8)
Equipped with a sensor (
Frequency 5-10 MHz)
This assessment will be conducted.
Measures taken include the estimation of fetal weight, double-top diameter, head circumference, abdominal circumference and insertion point of umbilical cord.
Bedside ultrasound will be performed before delivery to confirm the final intrauterine location of twin1 and twin 2.
Body measures the mother\'s body measures will be performed in all three prenatal examinations to assess weight gain and fetal development during pregnancy.
Height, weight and blood pressure will be recorded at the first visit.
For subsequent visits, weight, blood pressure, height of the stomach, abdominal circumference, and fetal heart will be recorded.
Baby body measurements will be performed at birth and repeated in each of the eight follow-up items
Visited by trained pediatric nurses to assess growth and development. A stadiometer (
Shanghai, China)
Will be used to evaluate maternal height and weight and to record the corresponding baby information on the growth curve.
Standard tape measure will be used to determine head circumference, chest circumference, abdominal circumference, and digital body fat calipers will be used to determine the thickness of skin folds.
Body composition information about the body composition of twins will be collected at birth, with babies aged 6 weeks, 6 months and 1, 2 and 3 years.
This information will be used in conjunction with body measurements to gain insight into body composition.
The body composition analyzer collects parameters of whole body water, fat mass, no fat mass, out-of-cell fluid, in-cell fluid and body mass index (
Imp sfb1, Australia).
Delivery record information from delivery records will be collected.
This will include details of the delivery method (
C-section or vagina)
Any preoperative and postoperative diagnosis, as well as the size and weight of the placenta, the length of the umbilical cord, the volume and features of the amniotic fluid.
The baby\'s gestational age, gender, weight, length and Apgar score at the time of delivery will also be recorded.
Using tidal breath analysis, breathing function will be measured by trained professionals when the baby is 2 years old.
Lung function tester (
CareFusion, Germany)
Lung function parameters that will be used to determine the respiratory frequency, tidal volume, inhalation time, Expiration Time, time to reach the peak tidal flow, expiration volume of the peak tidal flow, the ratio of inspiration time to expiration time and the ratio of time to total expiration to reach peak expiration flow.
When the twins are 6 weeks old, an experienced ultrasound doctor will measure their cardiovascular function through abdominal ultrasound.
Using the commercial system, the middle thickness of the aortic endometrium and the diameter of the aorta will be obtained (
Voluson E8, General Electric Healthcare, Austria)
It\'s equipped with a C4-8 transducer (
Frequency 4-8 MHz).
Early assessment of the liver and kidney function of the mother (11–16 weeks)and late (30–34 weeks)pregnancy.
Serum Collection from women under 8 years of age
Fast for an hour.
These serum were examined in the clinical laboratory of the First Affiliated Hospital of CQMU or Chongqing maternal and child health center.
These measures will include total protein, clearance protein, immunoglobulin, total bile acid, gluten ammonia transfer enzyme, Valley propylene ammonia transfer enzyme, urea and creatinine urine.
According to the standard guidelines, an ultrasound scan of the fetus will be obtained for each prenatal examination.
Trained medical ultrasound inspectors using commercial systems (
General Electric Healthcare company Voluson E8)
Equipped with a sensor (
Frequency 5-10 MHz)
This assessment will be conducted.
Measures taken include the estimation of fetal weight, double-top diameter, head circumference, abdominal circumference and insertion point of umbilical cord.
Bedside ultrasound will be performed before delivery to confirm the final intrauterine location of twin1 and twin 2.
Sample storage and biological samples other than hair and nails all samples will be kept in pre-printed QR code 500l cyrovials (
Micro-, TheNetherlands).
Hair and Nail samples will be wrapped in foil at the time of collection and kept in a re-sealed bar-coded bags.
Each sample will be scanned by the Tracxer code reader (Micronics)
Enable the sample and its location to be tracked in the database (
Medscientific AB, Sweden).
Hair and Nail samples will be stored in the refrigerator at 20 °c (
Komeing, China).
Preserve the placenta tissue in a liquid nitrogen tank (Locator 4 Plus).
All other samples (
Urine, serum, DNA)
Will be stored in the-80 ° c freezer (
907, semmerfisher Science, USA).
-80 ° c freezer (
907, semmerfisher Science, USA)
Temperature Sensor (PT100)
And monitoring system (Smart-
Vue, heat FisherScientific, France)
And the standby liquid nitrogen cooling system (
DPL452, Tyler Wharton Business School, USA).
The database used to store all information is designed and implemented by Medscinet AB.
The company has been used to set up a similar data collection and storage base for other people. based studies.
The data will be stored in both Mandarin and English for extraction or annotation in both languages.
Data analysis the extensive longitudinal data and biological sample sampling of Chongqing LoTiS program provides considerable space to solve a series of research problems, from observation and mapping of growth trajectories to an epidemiology survey of a series of maternal factorsbetween pairs), or non-
Common environmental factors (within pairs)
Results for future generations.
A key part of future research will be a series \'-
Type data for Omics, especially genomics, epigenomics, transcription Omics, and metabolic Omics.
The generation and analysis of this will initially be done using a standard method applicable to each data type to generate a \"clean\" data set for everyone.
Subsequent standard twin modeling methods can then be used to divide the variation of specific phenotype traits
The output of Omics is component variance component of additive genetic, non-additive genetic
Additive inheritance of the common environment (
(For example, mother factor)and non-
Common environmental impact (
Features of placenta).
Paired comparison of similarity measures (
Intra-class correlation coefficient)
Is an easy way to evaluate the possibility of genetic effects on any specific phenotype.
Additional logistic regression methods will also be adopted as needed to incorporate double correlations into each model.
The ethical and communication data for the study will be published in the Journal of Medical Research, and any molecular data will be published in the appropriate public repository.
Many studies have been discussed suggesting that genetically identical mice raised under the same environmental conditions show variation in normal distribution in most phenotypes, which is most likely due to non-
Genetic process.
Longitudinal measurements of this mechanism in twins can provide a unique opportunity to identify genes that are sensitive to environmental factors associated with complex diseases.
LoTiS is specifically designed to combine the power of twin research designs to conduct longitudinal sampling of the phenotype results of exposure, biological samples and later NCD, starting before birth.
Headquartered in China, whether it is providingEuropean-
Based on genetic profiles, but also to address the unique nature of exposure/outcomes inherent in rapid Westernisation in developing economies in exposure and incidence of a range of non-communicable diseases.
And the comparison between the DZ and the MZ twins will reveal the genetic and environmental variation on the early-
The ability of life-table use of MZ twins to compare to control genetic variation will prove particularly valuable in helping to unravel the complexity of gene-environment-epigenetic interactions and their contribution to human health and disease.
In addition to the funding entity, the study was supported by the Ministry of Education\'s plan 111R.