Risk factors of bicycle traffic injury among middle school students in chaoshan rural areas of china

Zhen-Bin Lin
Yan-Hu Ji
†
Qing-Yu Xiao
Li-Bo Luo
†
Li-Ping Li
Bernard Choi
Risk factors of bicycle traffic injury among middle school students in chaoshan rural areas of china
10.1186/s12939-016-0512-8R E S E A R C H Open Access
Background: Bicycle injuries are a leading cause of accidental death among children in the world, and bicycle-related injuries are also very common in China, thus to find out bicycle injury risk factors is imperative. This study aims to identify the cyclist-, bicycle-and road-related risk factors of bicycle injury, to develop health education programs as an intervention and to provide a scientific basis for establishing policies against bicycle injury. Methods: We selected two middle schools randomly among seven schools in Chaoshan rural areas,where the main means of transportation for students from home to school was bicycle. The subjects were middle school students from 7th to 9th grades from Gucuo Middle School and Hefeng Middle School. Cyclists were surveyed through questionnaires about bicycle injury in the past 12 months. Results: Multivariable logistic analysis showed that compared with a combination-type road、 motor lane and a non-intact road were both risk factors of bicycle injuries. This was followed by riding with fatigue, non-motor lane and inattentive riding. Conclusion: Bicycle injuries are frequent in China. Three risk factors on bicycle traffic injury among middle school students in Chaoshan rural areas of China were identified. This study provides important data to develop intervention strategies for China and other developing countries.

Background

Bicycle riding is a popular means of transportation, recreation and exercise for children worldwide [1,2]. The use of bicycle is often the only way for young students to travel great distances at a fast speed [3]. However, compared to other road users, cyclists run a higher risk of being injured in a road crash [4]. As children have lower awareness of traffic rules and tend to undertake more risky behaviors than adults, bicycle riding is more hazardous for them. Children are much more likely to fall from a bicycle and suffer serious injuries [4]. In the United States, children with 19 years of age and under account for 57% of all bicycle injuries treated in emergency rooms and 15% of deaths. In the US, bicycle accidents are a leading cause of accidental death among children [5]. In developing countries such as China, bicycle is one of the most important means of transportation among teenagers, and consequently, bicycle-related injuries are very common. Li et al. have shown that bicycle-related accidents constitute 71.6% of all types of traffic injuries and fatalities among middle school students [6]. Therefore, effective measures should be taken for prevention of bicycle injury, mainly through interventions on risk factors.

In the review of previous epidemiological studies of cycling-related road crashes, we found that previous studies were usually designed just to investigate the most frequent patterns of accidents, accident circumstances and environment-related factors, or describing the characteristics of injured cyclists and the factors related to injury severity [6,7]. However, cyclists, vehicles, and roads all play important roles in a bicycle injury. Yet few studies have assessed the cyclist-, vehicle-and road-related factors associated with risk of causing crashes [7]. Moreover, research on bicycle injury were mostly carried out in urban areas and developed countries, and seldom in rural areas of developing countries. To fill this gap, we researched all three factors related to bicycle injuries (i.e. cyclists, bicycles and roads) and investigated major risk factors of bicycle injury in the Chaoshan rural areas of China. We want to provide data which are representative of many underdeveloped areas in developing countries where bicycles are very commonly used among middle school students. The study is expected to provide data for development of intervention strategies for effective prevention and control of bicycle injuries.

Methods

Subjects

This study involved a large group of junior high school students, through stratified cluster sampling. The subjects were Middlestudents from 7th to 9th grades from Gucuo Middle School and Hefeng Middle School. These two schools were selected randomly among seven schools in Chaoshan rural areas, where the main means of transportation for students from home to school was bicycle. Only students who stated 'go to school by bicycle' were included in this study. We hand out 2421 questionnaires and got 2075 back, they were all valid questionnaires. the effective rate was 85.7% (2075/2421). The prevalence of bicycle injury was 28.3% (588/2075). The injured students ranged from 11 to 19 years old, with an average age of 14.77 ± 1.07.

Data procedure

Informed written consent was given by the students, or the next of kin, caregivers or guardians on behalf of the minor/children participants involved in this study. Before the investigation, a questionnaire was designed based on the Haddon Matrix theory [8,9] and modified after a pilot study. Between November 2012 and March 2013, through stratified cluster sampling, students in two middle schools of Liangying Village in Chaoshan rural areas were recruited. Cyclists were surveyed through questionnaires about bicycle injury in the past 12 months. We collected data, including gender, age, basic knowledge of and attitude towards bicycle injury prevention (opinion on necessity of setting up traffic rules, recognition of traffic markings "for vehicles only", running a red light, opinion on whether bicycle injury is preventable, willingness to learn about prevention of bicycle injuries), lifestyle and bicycle riding behaviors (coffee or strong tea consumption, inattentive riding, riding speed, use of communication devices while riding, riding with fatigue, riding in bad mood), bicycle-related factors (periodic maintenance of bicycle, bicycle age), road-related factors (types of lanes, road situation, road surface, road materials). Validity and reliability were analyzed and the test-retest reliability and validity were both above 0.70.

During the investigation, trained interviewers explained the research purposes, the meaning of questionnaire items, and the filling methods to respondents. The questionnaires were collected and checked. Personal information of the respondents was completely confidential. The information collected was only used for summary analysis.

Statistical analysis

Respondents were classified into two groups: injured and uninjured. Injured refers to conditions meeting at least one of the following three criteria: 1.going to a medical institution for treatment, and being diagnosed as having an injury; 2. asking for one or more days off school because of an injury; 3. being given first-aid by family members, teachers or others because of an injury.

Using Epi Data 3.1 software, we entered data into a computer database, using double key entry to improve accuracy. Descriptive analysis was first used to study the variables. A chi-squared test was used to study significant differences between the two groups (injured and uninjured). Multivariable logistic analysis was used to assess the impact of variables on bicycle injury. A P value less than 0.05 was regarded as statistically significant. All statistical analyses were performed with SPSS 19.0.

Quality control

We trained interviewers to conduct the questionnaire survey, and applied methods of data collection and analysis in a consistent way. The quality control process of the study was carried out by assigned field coordinators during the course of investigation. The collected survey data was entered into the database after verification and a logical check was executed. The answers of 3% of respondents were verified through a phone call to ensure that the collected data was reliable.

Results

General conditions of the subjects between the respondents from the two junior high schools in Liangying Village, 85.7% of students went to school by bicycle. General conditions of the surveyed students from the two junior high schools in Liangying Village are given in Table 1. The prevalence of bicycle injury was 28.3%. Among the injured students, there were 357 males (60.7%) and 231 (39.3%) females. Chi-squared analysis comparing the distribution of gender and age in Table 1 suggested that male gender was related to the occurrence of bicycle injury (P <0.01, χ2 = 47.992). The relation between age and the occurrence of bicycle injury was non-significant (P = 0.961).

Cyclist-related factors Basic knowledge of and attitude towards bicycle injury prevention

Basic knowledge of and attitude towards bicycle injury prevention are presented in Table 2. Almost all the students (88.9%) thought it was highly necessary to set up traffic rules. Students who thought that setting up traffic rules was of little or no necessity had a higher prevalence of bicycle injuries (45.2%) than those who thought that setting up traffic rules was necessary (27.6%). 65.3% of students were willing to learn more about prevention of bicycle injuries. Although more than half (62.2%) of students believed that bicycle injury was preventable, some of the students (28.5%) were unsure whether or not bicycle injury was preventable.

73.3% of the students could not recognize the traffic markings "for vehicles only". According to the chi-squared test, the relation between recognition of traffic markings "for vehicles only" and the occurrence of bicycle injury was non-significant (P = 0.509). Regarding the question of running a red light (If you come across an intersection with the red light on, but there is no other vehicle around, will you ignore the traffic rules and run the red light just to save time?), there was a higher prevalence among students who chose 'Yes' (47.3%) than those who chose 'No' (27.5%). Table 3 presents the behaviors and habits of bicycle riding elicited from the subjects. Most of the students (88.8%) rode at a speed between 10 km/h to 20 km/h. For students who rode either faster (≥20 km/h: 79.4%) or slower (≤10 km/h: 55.4%), the prevalence were much higher than those who rode at a moderate speed (>10 to <20 km/h: 23.6%). Students who rode a bicycle when they were sleepy or tired had a higher prevalence rate (40.3%) than those who did not (24.0%). Moreover, negative mood (anger, grief, etc.) was related to bicycle injury. Students who rode while having bad mood were  more likely to have bicycle injuries (37.1%) than those who did not (21.2%). Of the 1178 students who usually drank coffee or strong tea in their daily life, 31.7% had experienced bicycle injuries. In contrast, among 897 students who did not drink coffee or strong tea, only 24.0% had been injured in bicycle accidents.

Lifestyle and bicycle riding behaviors

Data in

Bicycle-related factors

Bicycle-related factors in bicycle injury among 2075 students in Chaoshan rural areas of China is showed in Table 4,which indicate that neither periodic maintenance of bicycle nor bicycle age was significantly related to bicycle injuries.

Road-related factors

We asked questions about the road conditions from the students' home to school, including types of lanes, road situation, road surface and road materials. It shows that 74.9% of students went to school on a combination-type road (roads that are not divided into specific lanes) in Table 5. The prevalence of injury on motor lanes and non-motor lanes were higher than that on combinationtype road (motor lane: 43.5%, non-motor lane: 47.8%, combination-type road: 22.1%). In addition, the injury prevalence of students who rode on non-intact roads was higher than those who rode on intact roads (43.1 and 23.3%, respectively). There was also a significant difference among injury prevalence on different road surfaces (dry: 18.8%, ponding: 46.5%, humid: 47.9%). Students riding on roads composed of sand and rocks or asphalt had higher rates of injury (sand and rocks: 50.6%, asphalt: 47.4%) than cement (21.3%).

Logistic regression analysis on bicycle traffic injury among middle school students

We performed univariate logistic regression analysis and set the occurrence of bicycle injury as the dependent variable, and cyclist-, bicycle-and road-related factors as  the covariates. Chi-squared results showed that male gender (P <0.01), opinion on necessity of setting up traffic rules (P = 0.004), running a red light (P <0.01), opinion on whether bicycle injury is preventable (P = 0.009), willingness to learn about prevention of bicycle injuries (P = 0.020), coffee or strong tea consumption (P <0.01), inattentive riding (P <0.01), riding speed (P <0.01), use of communication devices (like mobile phones) while riding (P <0.01), riding with fatigue (P <0.01), riding in bad mood (P <0.01), types of lanes (P <0.01), road situation (P <0.01), road surface (P <0.01), and road materials (P <0.01) were related to bicycle injury (Table 1, Table 2, Table 3, Table 4, Table 5). 5 statistically significant risk factors with P <0.01 were put into Multivariable logistic regression analysis. Odds ratios (OR) calculated for variables showed that the most important risk factors were: riding on motor lanes (OR = 2.414, 95% CI = 1.474~3.954), non-intact road situations (OR = 2.261, 95% CI = 1.632~3.133), riding with fatigue (OR = 1.714, 95% CI = 1.196~2.458), riding on non-motor lanes (OR = 1.648, 95% CI = 1.161~2.339) and inattentive riding (OR = 1.599, 95% CI = 1.122~2.278). (Table 6).

Discussion

Cyclist-related factors

A previous study [10] showed that the prevalence of bicycle injury among junior high school students in the Yangpu District of Shanghai, China is 8.71%. In the current research, the prevalence of bicycle injury in Chaoshan rural areas is 28.3%, which is much higher than that in the city. Therefore, bicycle injury prevention is a critical issue in the rural areas of China. Many studies show that younger age is a risk factor of bicycle injury. For instance, in America, one-fifth of bicycle-related injuries occurs in children 15 years and younger [11] with peak prevalence of bicyclerelated injuries and deaths occurring within the 9-15 yearsold age group [12]. Another study also found that risk of serious injury is increased by young age (<6 years) [13].   Table 5 However, in this study, the prevalence of bicycle injury in different age groups (11-13, 14-16, 17-19) are nearly the same (27.8%, 28.5%, 27.2%, P = 0.961, Table 1). Therefore, a bicycle education program should focus on junior high students of all age groups, and not only the younger students. Being male is associated with an increased risk for all types of injuries [14]. This study showed that the prevalence of injury in males (60.7%) is higher than that in females (39.3%).This supports previous research on bicycle injuries of children, where a male-to-female ratio of 3:1 has been reported [2]. This may be related to the nature of males. Firstly, males tend to have more impulsive, un-controlled behavioral styles that lead to a higher risk of unintentional injury [15]. Secondly, males are more likely to attribute injuries to bad luck, and females to their own behaviors and decisions, leading males to repeat injury risk behaviors more often than females [16]. So we should pay more attention to male students in order to reduce their high injury rate. Parental supervision is among the most effective behavioral techniques for reducing pediatric injury risk [17]. Adult supervisors can verbally intervene when male students begin to behave in a dangerous way. Supervision causes un-controlled children to reduce their tendency toward overestimation of ability and to judge their abilities more cautiously [18]. Moreover, bicycle injury education should be emphasized, especially for male students, in order to correct their wrong perspective (e.g. attributing injuries to bad luck) about injury. Also, the Bike Smart program (an eHealth product that utilizes video, animations, and still images to train children to acquire the key skills for bicycle safety) is a good way to improve males' cycling skills, because studies suggest that Bike Smart can be a low cost, effective component of safety training packages that include both skills-based and experiential training [12]. A walking school bus (a group of children walking to school with one or more adults) is a proven way to reduce cycling time, but few statistics are available to show whether it is really safer for children in China [19].

Basic knowledge of and attitude towards bicycle injury prevention

Attitudes towards bicycle safety play an important role in predicting child safety practices [20]. In this study, we investigated the attitudes of students on bicycle injuries (opinion on necessity of setting up traffic rules, opinion on whether bicycle injury is preventable, and willingness to learn about prevention of bicycle injuries). Results show that students who had a positive attitude towards bicycle injury prevention (thinking that setting up traffic rules was necessary, feeling that bicycle injury is preventable, and willing to learn about prevention of bicycle injuries) had a lower prevalence of injury than those who did not. Also, a previous study has suggested that better road safety knowledge is a protective factor for road traffic injuries among Chinese school children [21]. Specifically, a bicycling education program implemented in selected New Jersey schools and summer camps has shown that using road sign identification as an educational item in the program is effective in reducing bicycle crashes [22]. In our study, traffic marking recognition "for vehicle only" was also included. However, we found that it was only of borderline significance on bicycle injury (P = 0.509) according to chi-squared test. Why the result of our study was different from that of the New Jersey study could relate to the fact that there are few traffic signs on roads in Chaoshan rural areas, so even if students could recognize the markings in the questionnaire, it might not prevent them from bicycle injuries because they seldom encounter traffic markings on the road. Therefore, bicycle education programs focusing on recognition of traffic markings "for vehicles only" are not recommended before traffic signs are well established on the roads.

Lifestyle and bicycle riding behaviors

Accidents involving child cyclists are often the result of the children playing, doing tricks, riding too fast or losing control [23]. These behaviors may sometimes help children gain experience and training in bicycle handling skills. However, practicing good habits is more important to enhance safety. Also, caution and rational behaviors should be emphasized more on the roads. A study in Taipei shows that speeding is a factor related to severe head injury (P = 0.031) [24]. In our study, while riding at a high speed (≥20 km/h) is correlated with a higher prevalence of injury (79.4%), riding at a low speed (≤10 km/h) is also related to a higher rate of injury (55.4%), compared to those riding at a moderate speed (>10 to <20 km/h, 23.6%). Slower speed may be associated with longer exposure time. Thus we advise students to ride at a moderate speed (>10 to <20 km/ h), rather than low (≤10 km/h) or high (≥20 km/h) speeds, for prevent bicycle injury prevention.

The data shows that coffee or strong tea consumption is related to bicycle injuries. it can make the riding students more excited, and cause the bicycle injury. Therefore, avoiding coffee and strong tea consumption is recommended for bicycle injury prevention. Injured students show bad habits, such as using communication devices (like mobile phones) while riding. This result supports prior research from Japan, which indicates that there is a significant relationship between phone usage while riding a bicycle and bicycle crash/near-crashes [25]. Also, mental status could play a role in bicycle injury. Being sleepy or being in bad mood (anger, grief, etc.) while riding could easily distract the riders and thus increases the risk of accidents. In order to reduce both the occurrence and severity of bicycle injury, all of these bad habits need to be avoided. In addition, law enforcers should do their job to prevent harmful behaviors in the first place, such as using mobile phones [26].

In this study, 65.3% of the students were willing to acquire more knowledge on bicycle injury prevention. Teachers, parents or other close relatives of children are recommended to be educated on bicycle injury prevention so as to pass on the knowledge to children to prevent potential injuries. More importantly, teachers and parents should set a good example by their own actions, such as traffic rule obedience, being cautious and attentive in using bicycles, etc.

Bicycle-related factors

Our research reveals that 8.1% of 2075 students perform regular bicycle maintenance (Table 4). Prior research shows that several defects result in single-bicycle crashes (i.e. a fall or obstacle collision): the chain broke off, the tire inflation was too low resulting in skidding while cornering, the fender or the front fork broke off, a wheel, saddle, or handlebars were loose or broke off [27]. However, in this study, there was no significant difference between the prevalence of bicycle injuries among students who maintained their bicycles periodically and those who did not (P = 0.593). Actually, the former group has slightly higher injury prevalence (30.2%) than the latter group (28.2%). The reason is probably that students who experience bicycle injuries care more about their bicycles, and thus are more likely to maintain their bicycles periodically after bicycle injury. Further research is required to identify whether there is a difference in habits of bicycle maintenance before and after injury and, also, whether bicycle injury prevalence changes before and after the formation of this habit. We do not recommend junior high school students to replace their old bicycle with a new one in order to prevent bicycle injury because we find that bicycle age is not related to bicycle injury prevalence (P = 0.398), and the prevalence of injury for different bicycle age groups are similar, around 30%.

Road-related factors

According to research done in Hungary, terrible road conditions are among the most important causes of bicycle traffic injury among rural children [2]. Our research finds that riding on a motor lane (OR = 2.414, 95% CI = 1.474~3.954) is a risk factor for bicycle injury. Riding on a motor lane violates traffic rules and increases the risk of a bicycle crash by motor vehicles. Previous research also indicates that in bicycle accidents, involvement in a collision with a motor vehicle increases the risk of severe injury by 3.6-fold [28].

The crash involvement rate per 1000 cyclists has been shown to be 11.8 on shared paths compared to 5.8 on cycle lanes [29]. It has been suggested that having a bicycle-friendly infrastructure (e.g. bike routes, painted bike lanes) is associated with a low injury risk [30]. Road infrastructure that separates motor vehicles from bicycles is common in some cycling countries [31,32]. Providing a one-way bicycle path for bicycle users can reduce the chance of severe injury [33]. However, in this study, riding on a non-motor lane (OR = 1.648, 95% CI = 1.161~2.339) is also a risk factor for bicycle injury compared with riding on a combination-type road. We attribute this finding to the fact that in rural areas of China, roads are seldom divided into specific lanes. All types of vehicles ride on the same lane (combination-type road). Therefore, cyclists are more cautious to avoid crashes with vehicles when they are on this type of road. On the contrary, cyclists may not be highly alert on non-motor lanes, thinking that these lanes are much safer. However, large numbers of drivers may not obey traffic rules and may take up the whole non-motor lane for convenience. Accidents happen under these circumstances. Therefore, separating motor vehicles from non-motor ones may help to improve cyclist safety in urban areas. But in Chaoshan rural areas of China specifically, more education is required to improve road users' awareness of traffic rule obedience and accident prevention before non-motor lanes can really play a role in bicycle injury prevention. Moreover, the traffic department should set up an effective reward and punishment system that would contribute to better administration of traffic rules and regulations.

In addition, insufficient infrastructure of rural roads and terrible road conditions are related to higher rates of bicycle injury in rural China. We show that non-intact road situations (OR = 2.261, 95%CI = 1.632~3.133) are important risk factors for bicycle injuries. The prevalence of students who ride on non-intact roads is much higher than those who ride on intact roads (43.1% and 30.4%). Moreover, compared to dry road surfaces, ponding or humid road surfaces are related to a higher prevalence of bicycle injuries (dry: 18.8%, ponding: 46.5%, humid: 47.9%). There is also a significant difference among prevalence on different road materials. Roads composed of sand and rocks, as well as asphalt, are associated with a higher bicycle injury rate compared with cement roads (sand and rocks: 50.6%, asphalt: 47.4% cement: 21.3%). the students are used with riding on the cement roads, which are most common in rural China. In summary, intactness, dryness and cement road play essential roles in bicycle injury prevention. All of these factors should be managed by the government. Government intervention can be effective in lowering the rates of injury [34]. In Australia, bicycle-related head injuries have steadily declined in the years after 2006, the year associated with an increase in spending on cycling infrastructure [35]. According to this research, the government must take measures to improve road conditions, which include repairing broken and uneven roads, separating road sections that are under construction with warning signs, equipping roads with effective drainage systems, and paving roads with cement instead of sand and rocks. Much work still needs to be done.

Study limitations

In this study, the accuracy of self-reports may be compromised because some health-risk behaviors are difficult to recall and some are so sensitive that respondents may not want to report them. Also, the use of self-report data on road traffic risk behaviors and injuries may be subject to social desirability biases [36]. Moreover, in recalling what happened, the injured cyclists usually examine their own behaviors more critically. Thus, they are more likely to admit to negative behaviors than the uninjured cyclists. Considering the reality of Chaoshan rural areas of China, where students hardly wear a helmet to go to school, we excluded the questions on helmet usage, although in foreign countries, it has been proven that a primary strategy to prevent bicycle-related injuries is the promotion of bicycle helmet use [37]. In addition, exposure time of bicycle injuries and the cluster effect are two factors which should be taken into consideration [38]. This current research does not cover these parts and we will take them for the further research.

Conclusion

Multivariable logistic analysis showed that compared with a combination-type road、 motor lane and a non-intact road were both risk factors of bicycle injuries. This was followed by riding with fatigue, non-motor lane and inattentive riding. the study investigate the epidemiological characteristics of bicycle injuries in the region and the related risk factors for the issue.

Abbreviation

Combination-type road: Road that is not divided into specific lanes; Motor lane: Lane that only allows motor vehicles; Non-intact road: Road that is uneven or under construction; Non-motor lane: Lane that only allows pedestrians and bicycles

Table 1
1General conditions of the middle school students in Chaoshan rural areas of ChinaSubject
The injured
n (%)

Sample
size n

Prevalence
of injury (%)

Gender  †

Male
357 (60.7)
1009
35.4

Female
231 (39.3)
1066
21.7

Total
588 (100)
2075
28.3

Age a

11-13
63 (10.7)
227
27.8

14-16
497 (84.5)
1745
28.5

17-19
28 (4.8)
103
27.2

Total
588 (100)
2075
28.3

 †

(P<0.01,χ2=47.992), a (P=0.961,χ2=0.079)

Table 2
2Basic knowledge of and attitude towards injury prevention among middle school students in Chaoshan rural areas of China If you come across an intersection with the red light on, but there is no other vehicle around, will you ignore the traffic rules and run red light just to save time?Whether bicycle injuries occurred

Yes (n)
No (n)
Prevalence of injury (%)
P

Table 3
3Lifestyle and bicycle riding behaviors of middle school students in Chaoshan rural areas of ChinaHave you ever had these behaviors and habits in the past 12 monthsWhether bicycle injuries occurred

Yes (n)
No (n)
Prevalence of injury (%)
P

Table 4
4Bicycle-related factors in bicycle injury among middle school students in Chaoshao rural areas of ChinaWhether bicycle injuries occurred

Yes (n) No (n) Prevalence
of injury (%)

P

Periodic maintenance of bicycle
0.593

Yes
51
118
30.2

No
537
1368
28.2

Bicycle age
0.398

<3 months
73
151
32.6

3~6 months
72
167
30.1

6~12 months
91
242
27.3

>12 months
352
927
27.5

Table 5
5Road-related factors in bicycle injury among middle school students in Chaoshan rural areas of Chinalane that only allows motor vehicles, θ lane that only allows pedestrians and bicycles, ** road that is not divided into specific lanes, † † road that is uneven or under constructionWhether bicycle injuries occurred

Yes (n)
No (n)
Prevalence of injury (%)
P

Types of lanes
<0.01

Motor lane β
50
65
43.5

Non-motor lane θ
194
212
47.8

Combination-type road **
334
1210
22.1

Road situation
<0.01

Intact
362
1189
23.3

Non-intact  † †
226
298
43.1

Road surface
<0.01

Dry
261
1131
18.8

Ponding
182
209
46.5

Humid
135
147
47.9

Road materials
<0.01

Cement
335
1235
21.3

Sand and rocks
217
212
50.6

Asphalt
36
40
47.4

β

Table 6
6Multivariable logistic regression analysis on bicycle traffic injury among middle school students in Chaoshan rural areas of ChinaVariable factor
P
OR
95%CI

Inattentive riding

No
-
1.0
Reference

Yes
0.009
1.599
1.122~2.278

Use of communication devices (like mobile phones) while riding

No
-
1.0
Reference

Yes
0.051
0.605
0.366~1.002

Riding with fatigue

Yes
-
1.0
Reference

No
0.003
1.714
1.196~2.458

Road situation

Non-intact
<0.01
2.261
1.632~2.339

Intact
-
1.0
Reference

Types of lanes

Non-motor lane
0.005
1.648
1.161~2.339

Motor lane
<0.01
2.414
1.474~3.954

Combination-type road
-
1.0
Reference

See footnotes to
AcknowledgmentsWe thank Prof Chen -bin for his help with analyzing the data.FundingThe authors have no support of funding to report.Availability of data and materialsData sharing not applicable to this article as no datasets were generated or analysed during the current study. Please contact author for data requests.Authors' contributions ZL and YJ designed the study and completed data collection and analyses. QX and LL provided input into the study design and data collection materials. LL and BC provided technical guidance. All authors have read and approved the final manuscript.Competing interestsThe authors declare that they have no competing interests.Consent for publication I have obtained consent to publish from the participant to report individual data.Ethics approval and consent to participateThe study protocol was approved by the Ethics Committee of the Shantou University Medical College. Submit your next manuscript to BioMed Central and we will help you at every step:
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Adipocyte alterations in endometriosis: reduced numbers of stem cells and microRNA induced alterations in adipocyte metabolic gene expression

Masoumeh Majidi Zolbin
Ramanaiah Mamillapalli
Sepide E Nematian
Laura Goetz
Hugh S Taylor
Adipocyte alterations in endometriosis: reduced numbers of stem cells and microRNA induced alterations in adipocyte metabolic gene expression
10.1186/s12958-019-0480-0R E S E A R C H Open Access
Background: Endometriosis is an estrogen dependent, inflammatory disorder occurring in 5-10% of reproductiveaged women. Women with endometriosis have a lower body mass index (BMI) and decreased body fat compared to those without the disease, yet few studies have focused on the metabolic abnormalities in adipose tissue in women with endometriosis. Previously, we identified microRNAs that are differentially expressed in endometriosis and altered in the serum of women with the disease. Here we explore the effect of endometriosis on fat tissue and identified a role for endometriosis-related microRNAs in fat metabolism and a reduction in adipocyte stem cell number.Methods: Primary adipocyte cells cultured from 20 patients with and without endometriosis were transfected with mimics and inhibitors of microRNAs 342-3p or Let 7b-5p to model the status of these microRNAs in endometriosis. RNA was extracted for gene expression analysis by qRT-PCR. PCNA expression was used as a marker of adipocyte proliferation. Endometriosis was induced experimentally in 9-week old female C57BL/6 mice and after 10 months fat tissue was harvested from both the subcutaneous (inguinal) and visceral (mesenteric) tissue. Adipose-derived mesenchymal stem cells in fat tissue were characterized in both endometriosis and non-endometriosis mice by FACS analysis.Results: Gene expression analysis showed that endometriosis altered the expression of Cebpa, Cebpb, Ppar-γ, leptin, adiponectin, IL-6, and HSL, which are involved in driving brown adipocyte differentiation, appetite, insulin sensitivity and fat metabolism. Each gene was regulated by an alteration in microRNA expression known to occur in endometriosis. Analysis of the stem cell content of adipose tissue in a mouse model of endometriosis demonstrated a reduced number of adipocyte stem cells.Conclusions: We demonstrate that microRNAs Let-7b and miR-342-3p affected metabolic gene expression significantly in adipocytes of women with endometriosis. Similarly, there is a reduction in the adipose stem cell population in a mouse model of endometriosis. Taken together these data suggest that endometriosis alters BMI in part through an effect on adipocytes and fat metabolism.

Summary sentence

Reduced adipocyte stem cell numbers and microRNA driven changes in adipocyte gene expression contribute to the metabolic alterations and low BMI in women with endometriosis.

Background

Endometriosis is a gynecological disorder characterized by the deposition and proliferation of endometrial cells or tissue outside the uterine cavity [1,2] that occurs in 10% of reproductive-aged women [3]. While the most widely recognized symptoms of endometriosis are pelvic pain [4] and infertility [5], which are reported in more than 50% of patients, endometriosis is also associated with symptoms unrelated to the reproductive tract [6]. Women with endometriosis have a lower body mass index (BMI) than those without the disease [7]. In a mouse model of endometriosis we have previously shown that endometriosis decreases weight gain and body fat levels [8]. Similarly, lipid dysfunction and fat loss have been reported in humans with endometriosis [9].

Adipose dysfunction is characterized by morphological alterations including subcutaneous fat atrophy, fibrosis and metabolic alteration [10]. To maintain normal adipocyte numbers and function, adipocytes contain a population of mesenchymal stromal progenitor/stem cells (MSCs). MSCs are progenitor cells that are part of the stroma surrounding the mature adipocyte and preserve tissue homeostasis by regulating the number of mature adipose cells [11]. Endometriosis is known to interfere with the normal stem cell trafficking to the uterus that is necessary for endometrial growth and repair [12]. Altered stem cell mobility and engraftment characterize this disease [13][14][15][16]. Endometriosis is very effective at recruiting stem cells and lesions act as a sponge to attract these cells [17]. The reasons for diffuse symptoms and the precise pathophysiology of endometriosis are still not well understood [18]; however, inappropriate stem cell trafficking and stem cell defects are part of the pathophysiology of this disease and could affect adipose stem cell trafficking as well [19].

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression and bind to the 3′-untranslated region (UTR) of target mRNA [20], thus inducing translational inhibition and/or mRNA degradation of protein-coding genes, and regulating various cell activities, such as differentiation, proliferation, apoptosis, and motility [21,22]. MicroRNAs have been implicated in controlling MSC differentiation into adipocyte lineage and regulating cell proliferation [23]. Increased expression of a specific miRNA generally causes the repression of translation of the targeted mRNA, whereas decreases in miRNA expression exert the opposite effect [24]. MicroRNAs are known to be differentially expressed in the serum of women with endometriosis [25] and are considered to be potent regulators of gene expression [26]. In this way, they play a key role in the growth of endometriotic lesions. Previously, our laboratory has reported the upregulation of miR-342-3p [27] and downregulation of Let-7b [25,28] in the serum of women with endometriosis. Although these studies demonstrated that serum miRNAs are potential biomarkers for endometriosis, it is unclear if they contribute to the systemic manifestations of the disease. Specifically, the mechanisms involved in inducing the clinically observed low BMI phenotype in endometriosis are unclear.

Of the microRNAs known to be altered in endometriosis, Let-7b and miR-342 are thought to play a role in metabolism. MicroRNAs such as Let-7b are known to regulate glucose metabolism [29], and metabolic functions of obesity-derived adipocytes [30]. MiR-342-3p is involved in adipose differentiation in conjunction with brown fat-specific transcription factors [31], lipogenesis [32], and browning of adipose tissue [33]. In this study, we aim to determine the effects of both Let-7b and miR-342-3p on adipose tissue from patients with endometriosis. Here we report the effect of these two miRNAs on gene expression in adipose cells and the effect of endometriosis on adipose stem cell populations in a mouse model of endometriosis.

Materials and methods

Study population

Fat specimens were collected from 20 women with endometriosis (EMS) and without endometriosis (non-EMS, N = 10 per group) who were treated at Yale-New Haven Hospital (New Haven, CT, USA). The study population was selected from patients who underwent laparoscopy for multiple benign indications including adenomyosis, myoma, pelvic pain, infertility, or endometriosis and the participants gave written informed consent. Institutional Review Board (IRB) approval was obtained from Yale School of Medicine (New Haven, CT, USA).

Cell culture

Subcutaneous fat tissue in the abdominal region of the body was collected under sterile conditions from patients with and without endometriosis. The tissue was subsequently minced into fine pieces (1-2 mm), and then digested in buffer containing collagenase type 2 (0.83 mg/ml, #LS004174, Worthington Biochemical Corporation, NJ) in sterile Hank's balanced salt solution (HBSS, Life Technologies, CA, #14185-052) with 3% bovine serum albumin (BSA), calcium chloride (1.23 mM), magnesium chloride (1.03 mM) and zinc chloride (0.83 mM) for 75 min at 37°C. The cell pellet was collected by centrifugation at 300×g for 3 min, re-suspended in HBSS with 3% BSA, and filtered through a sterile 40 μm (BD Biosciences CA, #352340) nylon mesh filter. The cells were then re-suspended in the growth medium DMEM/ F12 medium (Life Technologies) containing 10% fetal bovine serum (FBS) and antibiotics (2% anti-anti), and plated and cultured in a T75 tissue culture flask that was maintained at 37°C and 5% CO2-atmosphere. After 48 h, the non-adherent cells were washed off with phosphate buffered saline (PBS) while the adherent adipocytes were grown to 75% confluence. After the third passage the cells were transfected with miRNAs.

Transfection of miRNAs

Adipocytes obtained and cultured from controls without endometriosis were transfected with mimics, inhibitors and respective controls of miRNAs 342-3p and Let -7b-5p. Mimics are the mature microRNA sequences that result in functional microRNAs while inhibitors are the small nucleotide sequences that bind to functional microRNAs and inhibit their function. Both mimics (#SMM-003) and inhibitors (#SMI-003) were obtained from Bioneer Inc. CA, USA. Negative control (NC) is the scrambled sequence that does not show an effect on the target genes of a mature microRNA. Briefly, cells were trypsinized, centrifuged and plated in a 6-well plate at 2 × 10 5 cells/well with 2 ml of growth medium without antibiotics. After 24 h the cells were transfected with the two miRNAs (50 nmol) mentioned above, using Lipofec-tamine™ 2000 and Opti-MEM® (Invitrogen, Carlsbad, CA) without serum or antibiotics according to manufacturer's protocol. After 24 h the transfection media was replaced by growth medium with 10% FBS and antibiotics. Cells were cultured for an additional 72 h before RNA was extracted for gene expression analysis. Transfection efficiency was assessed by directly measuring the transfected microRNA in the cell lysate by qPCR. All transfections were carried out in duplicate wells under sterile conditions.

Immunofluorescence studies

Immunofluorescence studies were carried out to determine PCNA expression as a marker of cell proliferation in both EMS and non-EMS groups. Cells were fixed at room temperature in 100% chilled methanol for 5 min, then washed three times with PBS and permeabilized with 0.25% Triton X-100 followed by blocking with 1% BSA in PBST (PBS + 0.1% Tween 20). Cells were incubated with anti-PCNA antibody (#ab18197, Abcam, 1:500 dilution) and anti-vimentin (#sc-373,717, Santacruz, 1:50 dilution) antibody in 5% BSA in PBST overnight at 4°C. The next day, the cells were washed with PBS and incubated with secondary antibody in 1% BSA and counterstained with DAPI (46-diamidino-2-phenylindole; #H-1200; Vector Laboratories, Burlingame, CA). The PCNA antibody stains the cell nucleus while the vimentin antibody stains the cytoplasm.

Lipolysis was evaluated with hormone-sensitive lipase (HSL) staining (#4107 s, Cell Signaling Technology, Danvers, MA, 1:100 dilution). HSL protein levels determined in subcutaneous fat tissue sections were obtained from patients with and without endometriosis. Fat tissue from both groups was fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5-μm serial sections. Tissue sections were deparaffinized in xylene, rehydrated through a series of ethanol washes, then placed in boiling sodium nitrate (pH 6.0) followed by staining with anti-HSL antibody. Nuclear staining on sections was carried out using vectashield fluorescent mounting media with DAPI (Vector Laboratories). Negative controls were determined with the same protocol using its respective host protein IgG as an isotype control. All stained sections on slides were visualized under LSM 710 confocal microscopy (Carl Zeiss, New York, NY) using ZEN software (Carl Zeiss, New York, NY).

Quantitative real time polymerase chain reaction (qRT-PCR)

Total RNA was extracted from post-transfected cells using TRIzol reagent (Invitrogen) and purified using RNeasy cleanup kit (Qiagen, Valencia, CA). For cDNA synthesis, purified RNA (1000 ng) was reverse-transcribed using iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA). Real-time quantitative PCR (real-time qPCR) was performed using SYBR Green (Bio-Rad) and optimized in the MyiQ single-color real-time PCR detection system (Bio-Rad). Reactions were performed in a 10 μl volume containing 1 μl cDNA, 1 μl of primer mix, 5 μl master mix (SYBR® Green I; Applied Biosystems, Inc., Foster City, CA) and 3 μl water. PCR cycling conditions were as follows: polymerase activation and the initial DNA denaturation step required a temperature of 95°C for 3 min followed by 40 cycles of 30 s denaturation at 95°C and 20 s of annealing and extension at 57°C. The specificity of the amplified transcript and absence of primer-dimers was confirmed by a melting curve analysis. Gene expression was normalized to beta actin (ACTB) as an internal control. Relative mRNA expression was calculated using the comparative cycle threshold (Ct) method (2 −ΔΔCT ). All experiments were carried out three times and each in triplicate. Primer sequences used for gene expression analysis are listed in Table 1 and were obtained from the W.M. Keck Oligonucleotide Synthesis Facility (Yale University, New Haven, CT).

Endometriosis induction in mice and fat tissue collection

Endometriosis was induced experimentally in 9-week old female C57BL/6 mice (n = 5) (Charles River Laboratories, Wilmington, MA) with intact ovaries as previously described [34,35]. Mice were housed in the Yale Animal Resources Center (YARC) at Yale School of Medicine and maintained (4-5/cage) in a room (21 ± 1C) with a 12-h light/dark cycle (7:00 a.m. to 7:00 p.m.) with ad libitum access to food (Purina Chow; Purina Mills, Richmond, IN, USA) and water. All animal experiments were conducted in accordance with an approved protocol from Institutional Animal Care and Use Committee (IACUC) of Yale University. IACUC guidelines were followed for animal care, surgery and for tissue collection. Briefly, donor uterine horns were split longitudinally to expose the lumen and endometriosis was induced by suturing two uterine fragments on the right and left peritoneal surface area using 5-0 polyglactin sutures (Ethicon, Somerville, New Jersey). Control female C57BL/6 mice (n = 5) underwent sham surgery placing sutures on the parietal pelvic peritoneum. Mice were sacrificed 10 months after endometriosis induction and fat tissue was harvested from both subcutaneous (inguinal) and visceral (mesenteric) areas. The tissue was immediately digested in digestion buffer at 37°C for 1 h and single cell suspension was prepared for cell analysis by flow cytometry.

Fluorescence-activated cell sorting (FACS) analysis

Adipose-derived mesenchymal stem cells in fat tissue were characterized in both endometriosis and non-endometriosis mice by FACS analysis. Single cell suspensions were prepared from fat tissue after digestion and stained with anti-CD140b APC/Cy 7 (#103027), anti-CD146 PE (#134703) and anti-LY-6A/E PE/CY5 (SCa-1, #108109) antibodies obtained from BioLegend, San Diego, CA. Antibodies were diluted according to the manufacturer's recommendations (1:500) in 3% BSA/HBSS. The stroma vascular fraction (SVF) was re-suspended in the antibody staining solution, incubated on ice for 30 min, and washed and centrifuged at 300×g for 3 min. The SVF pellet was re-suspended, filtered through a 40 μm nylon filter and subjected to FACS analysis. Isotype-matched negative controls were used to define background staining. Cell sorting was performed using BD FACS Aria II software (BD Biosciences, Franklin Lakes, NJ).

Statistical analysis

GraphPad Prism 7.0 (GraphPad Software for Science Inc., San Diego, CA) was used for all statistical analyses. Gene expression data are presented as mean ± SE (standard error). Statistical significance is determined based on two-tailed t-tests for gene expression and two-way ANOVA analysis for the stem cell comparisons. Student t-test was used for evaluating normally distributed variables. P < 0.05 was considered to be statistically significant.

Results

Altered adipocyte metabolic gene expression is regulated by microRNAs in endometriosis

Several microRNAs are expressed at altered abundance in women with endometriosis [28,36]. To test the effect of miRNAs Let-7b and 342-3p on adipocytes, we transfected these miRNA mimics as well as their inhibitors in primary adipocyte cells cultured from fat tissue of women without endometriosis and examined genes known to be key mediators of adipocyte differentiation that are predicted targets of these microRNAs. Expression levels (mRNA) of CCAAT/ enhancer binding protein alpha (Cebpa) and beta (Cebpb), peroxisome proliferator-activated receptor gamma (Ppar-γ), leptin, adiponectin (Adipoq), interleukin-6 (IL-6), and hormone-sensitive lipase (HSL) genes were analyzed by qRT-PCR.

The Let-7b inhibitor treatment models the endometriosis state of reduced Let-7b expression, while the Let-7b mimic serves to increase Let-7b activity. Similarly, miRNA 342-3p mimic models endometriosis with increased levels, while the corresponding inhibitor serves to create the opposite effect as is seen in endometriosis. The Let-7b mimic significantly reduced the mRNA levels of Cebpa, (4.0 fold) Cebpb (5.1 fold), and Ppar-γ (3.2 fold) as shown in Fig. 1a, b and c respectively while miR-342-3p mimic increased the mRNA levels of Ppar-γ (1.75 fold) as shown in Fig. 1c compared to controls. Let-7b inhibitor increased Ppar-γ expression significantly (2.1 fold) as shown in Fig. 1c, while miR-342-3p inhibitor significantly reduced the expression of Cebpa, (2.4 fold, Fig. 1a), Cebpb (1.7 fold, Fig. 1b), and Ppar-γ (2.1 fold) as shown in Fig. 1a, b and c compared to controls. As adipocytes are the main source of the leptin and adipoq secreted into blood in metabolic disorders, we tested the mRNA levels of leptin and adipoq. MiR Let-7b mimic reduced leptin mRNA levels (2.1 fold), while its inhibitor increased expression (6.37 fold, Fig. 2a). In contrast, leptin levels were increased after miRNA 342-3p mimic transfection (9.78 fold), and did not significantly change with its inhibitor (Fig. 2a). Adipoq expression was reduced by the Let-7b mimic (2.2 fold), and not significantly altered by any of the other transfections (Fig. 2b).

We additionally investigated the potential for these circulating miRNAs associated with endometriosis to alter adipocyte function by investigating their effect on the expression of IL-6 and HSL, important mediators of fat metabolism. We found that IL-6 expression was decreased in cells transfected with the Let-7b mimic (69 fold) and the 342-3p mimic (45 fold), while IL-6 expression was increased by the Let-7b inhibitor (26.7 fold) and the 342-3p inhibitor (1.53 fold, Fig. 2c). HSL expression was also reduced by Let-7b mimic (92 fold), and increased by its inhibitor (1.57 fold), while both mimic and inhibitor of miRNA 342-3p decreased the levels of HSL by 6.5 fold and 1.62 fold, respectively (Fig. 2d). These findings indicate the potential for changes in Let-7b to drive increased fat metabolism in patients with endometriosis.

To confirm that the HSL mRNA levels determined by qRT-PCR from cultured adipocytes mimicked the changes seen in vivo, we performed immunofluorescence (IF) staining studies for HSL protein levels in fat tissue collected from patients with endometriosis and without endometriosis. Subcutaneous fat tissue sections were subjected to IF using anti-HSL antibody. HSL staining is stronger in tissue from women with endometriosis ( Fig. 3a) compared to those without endometriosis (Fig.  3b). There is no staining for HSL protein in isotype control (Fig. 3c).

To gain further insight into the adipocyte cell proliferation and metabolism in subcutaneous fat from patients with and without endometriosis, we assessed the cultured adipocytes for expression of PCNA, a marker of cell proliferation, and for vimentin and DAPI simultaneously. Nuclear PCNA regulates proliferation, while the nuclear-to-cytoplasmic relocalization of PCNA is a key factor in shaping the energy metabolism [37][38][39]. Immunostaining results showed weaker nuclear PCNA and increased cytoplasmic staining in fat tissue of endometriosis patients compared to nonendometriosis patients (Fig. 4a). Also, we found that the number of cells stained for nuclear PCNA was significantly reduced (p = 0.004) in endometriosis patients compared to non-endometriosis as shown in Fig. 4b. While the majority of staining was nuclear in adipocytes cultured from women without endometriosis, the majority of PCNA was cytoplasmic in cells from endometriosis patients.

Adipocyte stem cells (mASCs) are reduced in a mouse model of endometriosis

To determine whether endometriosis affects the stem cell properties of fat tissue, we performed fluorescence activated cell sorting (FACS) to quantify the number of adipocyte stem cells. A mouse model was used to obtain sufficient cells from multiple fat compartments that are not easily accessed in humans. Subcutaneous and visceral fat tissue from both groups (endometriosis and non-endometriosis) was separately harvested and digested. Isolated cells were stained for adipocyte stem cell markers including Sca1, CD146, and CD140b. FACS analysis showed that more stem cells were present in Fig. 1 Altered gene expression in brown adipocyte differentiation. Let-7b mimic and miR-342-3p inhibitor significantly reduced mRNA levels of Cebpa (a), Cebpb (b), and Ppar-γ (c). Ppar-γ mRNA levels were increased by Let-7b inhibitor and miR 342-3p mimic, alterations similar to those seen in endometriosis. Each bar represents the mean ± SE for data from three individual experiments and each experiment was performed in triplicate. ** denotes statistical significance (p < 0.01) compared to the negative controls while * denotes p < 0.05 subcutaneous and visceral fat in the non-endometriosis group than in the endometriosis group (Fig. 5A). The percentage of stem cells for each marker was calculated. Stem cells with Sca1 + and CD146 + markers were significantly higher (Sca1 + , p = 0.0093; CD146 + , p = 0.0013; Fig.  5B) in the subcutaneous fat of the non-endometriosis group than in the corresponding endometriosis groups. The same pattern was observed for visceral fat when using the Sca1 + marker. In general, the number of stem cells present in subcutaneous fat is higher than in visceral fat, and the number of stem cells with the Sca1 + marker is significantly reduced (p < 0.05) in all other groups compared to the sham subcutaneous group. Also, a significant difference was observed between the subcutaneous and visceral fat of the endometriosis group for the Sca1 + marker. No significant change was observed in stem cells with CD140b + , a marker for platelet-derived growth factor receptor (PDGFR), between endometriosis and sham groups, and between subcutaneous and visceral fat. There was also a change in the endothelial marker CD146 in either subcutaneous fat in the endometriosis group, indicating that the vascularity of the fat tissue may have also been impacted. Among all three stem cell markers analyzed, we observed the most significant change in the Sca1 marker between the sham and endometriosis groups in both subcutaneous and visceral fat of endometriosis.

Discussion

Endometriosis is an estrogen dependent disease in reproductive-aged women that causes pelvic pain and infertility. Recent evidence shows that it is a systemic disease [40]. The systemic manifestations of endometriosis are, at least in part, mediated by abnormal levels of circulating miRNAs in women with the disease [27]. A recent review has thoroughly discussed the role of various microRNAs in Fig. 2 Altered expression of genes affecting appetite and insulin sensitivity. a Leptin mRNA levels were reduced by Let-7b mimic and increased by Let-7b inhibitor and miR 342-3p mimic. b Adipoq mRNA levels were reduced by Let-7b mimic. c IL-6 mRNA levels were reduced by Let-7b and 342-3p mimics, and increased by Let-7b and 342-3p inhibitors. d HSL mRNA levels were reduced by Let-7b mimic and increased by Let-7b inhibitor, and decreased by both 342-3p mimic and inhibitor. Each bar represents the mean ± SE for data from three individual experiments and each experiment was performed in triplicate. * denotes statistical significance (p < 0.05) compared to the negative controls while ** denotes p < 0.01 the pathology of endometriosis [41]. Though several micro-RNAs are involved in endometriosis, including miR-125b-5p, miR-342-3p, miR-Let-7b, miR-3613-5p and miR-150-5p, this study focused on those thought to alter metabolism. However, we can not rule out the possible role of other microRNAs which were not tested in this study.

Here we demonstrate that miRNAs Let-7b and 342-3p, which we have previously shown to be differentially expressed in the serum of patients with endometriosis, influenced adipocyte gene expression. Inhibition of Let-7b and upregulation of 342-3p models the down-regulation and upregulation of these two miRNAs, respectively, as is seen in endometriosis patients. These miRNAs induced consistent changes in adipocyte metabolic gene expression that would be expected to dramatically change metabolism in these cells. The alterations would be expected to  promote the clinically observed low BMI phenotype seen in endometriosis patients.

MicroRNA alterations associated with endometriosis affect genes such as Cebpa, Cebpb, and Ppar-γ, which are involved in brown adipocyte differentiation, leptin and adipoq, which are involved in glucose metabolism, and IL-6 and HSL, which are involved in fat metabolism. MicroRNA 342-3p has been previously shown to have a role in brown adipocyte differentiation. Here we show that it regulates Ppar-γ, a transcription factor that has previously been implicated in the conversion of white adipose tissue to brown adipose tissue [42,43]. PPAR-γ has also been shown to regulate fatty acid and glucose metabolism. Its expression is decreased in obese individuals [44], but increased in mice with endometriosis compared to mice without endometriosis [8]. HSL has been shown to drive lipolysis [45]. Our data suggest that decreased miR-Let-7b significantly decreased HSL expression in human fat. Taken together, our data indicate that the circulating miRNA profile associated with endometriosis may promote browning of adipose tissue.

These findings are consistent with the results reported by Goetz et al. showing an increase in leptin and ppar-γ expression in a murine model of endometriosis [8]. Leptin is a protein secreted by adipocytes and has been widely shown to improve insulin sensitivity and suppress appetite [8,[46][47][48]. Adipoq is a fat-derived hormone that has been demonstrated to improve insulin sensitivity, and is involved visceral fat from sham and EMS mice. Mesenchymal stem cell markers: Sca-1 + , CD140b + , CD146 + . (B) Quantification of mesenchymal stem cell markers by flow cytometry. The number of Sca-1 + and CD146 + expressing stem cells are significantly lower in sham-visc, EMS-s.c and EMS-visc groups compared to sham-visc. EMS-visc has low Sca-1 + population compared to EMS-s.c group. Each bar represents the mean ± SE for data from three individual experiments and each experiment was performed in duplicate. * denotes statistical significance (p < 0.05) between sham-s.c vs other groups, EMS-s.c vs EMS-visc (Sca-1 + ) in brown adipose differentiation pathways as well [49][50][51][52]. Circulating miRNAs associated with endometriosis may drive appetite reductions and improvements in insulin sensitivity. Similarly, miR-342-3p up-regulation has been demonstrated with metformin treatment [53]. IL-6 is a cytokine that is secreted by adipose tissue. Since it has been shown to specifically stimulate fat metabolism, it is considered to be a lipolytic factor [54,55].

We identified a lower proliferative ability of fat cells from endometriosis patients. Adipose tissue harvested from endometriosis patients showed alterations in cell proliferation corresponding to decreased nuclear PCNA expression. Interestingly, nuclear-to-cytoplasmic relocalization of PCNA has been demonstrated in non-proliferating cells; cytosolic PCNA is a key factor in shaping the energy metabolism, enhancing glycolysis [37,38,56]. Direct interaction between PCNA and NAMPT in the cytoplasm modulates the intracellular NAD+ level and glycolysis [39]. We identified a shift to cytosolic PCNA in endometriosis that likely also increases glycolysis, thus altering availability of lipid precursors. Longer doubling times and decreased nuclear expression of proliferation marker PCNA have also been associated with miR-342-3p in other conditions [57,58]. Similarly miR-Let-7b acts as a key regulator of cell proliferation of stem cells [59].

Finally, we identified a reduction in adipocyte stem cells in the subcutaneous fat in a mouse model of endometriosis. We identify a dysfunctional phenotype of endometriosis adipose stem cells (ASCs) which can alter their differentiation and proliferation in the process of tissue regeneration. A reduction in Sca1 positive stem cells may interfere with fat cell expansion in endometriosis.

In conclusion, we have demonstrated for the first time significant alterations in the adipocytes of women with endometriosis. Fat cells have altered lipid and glucose metabolism as that predict a propensity toward lower fat mass and explain the lower BMI seen in women with endometriosis. The altered circulating microRNAs found in women with the disease affect adipocyte gene expression, and may be a mechanism by which endometriosis lesions communicate with fat leading to altered fat metabolism in these women. Further the decreased adipocyte stem cell numbers also suggest a second mechanism driving decreased adiposity. Endometriosis has widespread manifestations affecting multiple tissues. These include a lower BMI and less body fat; understanding the molecular and cellular etiology of this disorder may provide new targets for treatment and the ability to limit the systemic effects of the disease.

Fig. 3
3Analysis of hormone sensitive lipase (HSL) enzyme by immunofluorescence. HSL protein staining in tissue sections from subcutaneous fat: a EMS, b non-EMS and c Negative isotype control

Fig. 4
4Analysis of PCNA expression by immunofluorescence. a Showing PCNA, and vimentin protein staining by their respective antibodies, with nuclear staining by DAPI in adipocytes from subcutaneous fat tissue of women with and without endometriosis. There is more vimentin protein staining in endometriosis patients than in non-endometriosis patients. b Reduction in nuclear PCNA protein expression in EMS compared to non-EMS. Each bar represents the mean ± SE. * denotes statistical significance (p = 0.0040) between EMS and non-EMS, (N = 10)

Fig. 5
5Analysis of mesenchymal stem cell markers by flow cytometry. (A) Flow cytometry histograms. (a) and (b): subcutaneous fat; (c) and (d):

Abbreviations ACT: Beta actin; Adipoq: Adiponectin; BMI: Body mass index; BSA: Bovine serum albumin; CD146: Cluster of differentiation 146; Cebpa & Cebpb: CCAA T/enhancer binding protein alpha & beta; EMS: Endometriosis; non-EMS: Non-endometriosis; FACS: Fluorescence-activated cell sorting; HSL: Hormone-sensitive lipase; IL-6: Interleukin-6; miRNAs: MicroRNAs; MSCs: Mesenchymal stromal progenitor/stem cells; PBS: Phosphate buffered saline; PCNA: Proliferating cell nuclear antigen; PCR: Polymerase chain reaction; Ppar-γ: peroxisome proliferator-activated receptor gamma; qRT-PCR: Quantitative real-time polymerase chain reaction; RNA: Ribonucleic acid; UTR: 3′-untranslated region

Table 1
1Sequences of primers usedHSL
TGCTAGGCACATAGCCTCCT
GCTGGGCTATGGGTGTCTTT
NM_005357.3

CEBPA
GCAAACTCACCGCTCCAATG
CTTCTCTCATGGGGGTCTGC
NM_004364.4

CEBPB
GCCGGTTTCGAAGTTGATGC
TTACACGTGGGTTGCGTCAG
NM_005194.3

Leptin
TGCGGATTCTTGTGGCTTTG
CTGACTGCGTGTGTGAAATGT
NM_000230.2

ADIPOQ
TTCCATACCAGAGGGGCTCA
CCCTTGAGTCGTGGTTTCCT
NM_004797.3

PPARG
CCGTGGCCGCAGAAATGAC
CACGGAGCTGATCCCAAAGT
NM_005037.5

ADIPONECTIN
TTCCATACCAGAGGGGCTCA
GAGTCGTGGTTTCCTGGTCA
NM_001177800

IL6
ACCCCCAGGAGAAGATTCCA
GTCTTCCCCCACACCAAGTT
NM_000600

β-actin
GAAGATCAAGATCATTGCTC
AACGCAACTAAGTCATAGT
NM_001101

AcknowledgementsWe thank Joshua Huttler for proof reading manuscript.FundingThis work was supported by NIH U54 HD052668 and R01 HD076422.Availability of data and materialsThe data and materials are available from the corresponding author on reasonable requests.LG performed statistical analysis. and wrote the manuscript. HST designed the study, interpreted results, data analysis and revised the manuscript. All authors read and approved the final manuscript.Ethics approval and consent to participateThis study was approved by the Institutional Review Board (IRB) of the Yale School of Medicine (New Haven, CT, USA). All participants signed an informed consent.Consent for publication Not applicableCompeting interestsThe authors declare that they have no competing interest.
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