Evidence indicates a link between autism spectrum disorder (ASD) and gender diversity, yet this intersection remains insufficiently understood. Here, we investigated whether (1) ASD affects gender-related cognition , (2) autistic people have increased gender dysphoria and recall limited gender-typed behavior from childhood, and (3) transgender individuals have increased ASD-like traits and difficulties in mentalizing. A total of 106 non-autistic cisgender , 107 autistic cisgender , 78 non-autistic transgender , and 56 autistic transgender adults  participated in the study. The mean age of participants was 31.01 years (range = 18 to 70). Using an explicit as well as an implicit measure, for the first time, we found that ASD affected gender-related cognition only in autistic cisgender people. Sex differences were also observed in this group. Whereas autistic cisgender birth-assigned males showed a stronger implicit gender-group identification than non-autistic cisgender birth-assigned males, autistic cisgender birth-assigned females showed a weaker gender-group identification than non-autistic cisgender birth-assigned females. Furthermore, autistic cisgender people reported significantly more gender dysphoric feelings and recalled significantly less gender-typed behavior from childhood than non-autistic cisgender individuals. No difference was observed between non-autistic and autistic transgender people. We also found that relative to non-autistic cisgender individuals, both non-autistic transgender and autistic transgender people reported significantly more ASD-like traits. However, mentalizing difficulties were observed only in the latter group. This research enhances our understanding of the link between ASD and gender diversity.The online version contains supplementary material available at 10.1007/s10508-022-02386-5. In recent years, clinicians and researchers have provided increasing evidence for a link between autism spectrum disorder (ASD) and gender diversity , self-harm, and suicidality  in ASD. The sparse research conducted on this topic has shown that autistic adults show a reduced propensity to explicitly identify with personality traits and gender roles that are stereotypically attributed to males .Next, we attempted to extend the original findings further by examining gender-group identification in autistic people. Autistic people’s sense of belonging to one gender group over another was examined in cisgender and transgender people, separately, for the first time. This enabled us to understand how exactly ASD might affect gender-related cognition. Olson et al.  were theWithin each group (autistic cisgender/autistic transgender/non-autistic cisgender/non-autistic transgender) scores on the explicit and the implicit measures of gender self-concept would align with experienced gender, rather than birth-assigned sex .We expected autistic participants who identify either as cisgender or transgender to show a significantly weaker explicit and implicit gender self-concept than non-autistic people.The second aim of this study was to examine whether autistic people have increased gender dysphoric feelings and whether they recall limited gender-typed behavior from their childhood years. Recent evidence indicates a small association between ASD traits and gender variance in children from the general population .We expected autistic people who identify either as cisgender or transgender to recall significantly less gender-typed behavior from childhood than non-autistic cisgender people, autistic cisgender people to recall significantly more gender-typed behavior from childhood than non-autistic transgender individuals, and autistic transgender people to recall significantly less gender-typed behavior from childhood than non-autistic transgender individuals (autistic transgender < non-autistic transgender < autistic cisgender < non-autistic cisgender).The third aim of the current study was to examine whether transgender people (autistic and non-autistic) have increased ASD-like traits and difficulties in mentalizing. Previous studies have suggested there may be a selective difference in ASD-like traits between transgender and control birth-assigned females only , but potentially meaningful, difference between transgender and cisgender people.ASD is characterized by well-established difficulties with mentalizing .We expected non-autistic transgender individuals to perform significantly less well on the mentalizing task than non-autistic cisgender people, but significantly better than autistic cisgender people, and autistic transgender people to perform significantly less well on the task than autistic cisgender participants (autistic transgender < autistic cisgender < non-autistic transgender < non-autistic cisgender).n = 4), whereas participants who identified as trans nonbinary or masculine nonbinary (n = 4) were included in the transgender group. The number of birth-assigned females and males did not differ significantly between groups, χ2 = 0.81, p = 0.846, φ = 0.05, but differences in age were found, F = 26.99, p < 0.001, A total of 106 non-autistic cisgender adults , 107 autistic cisgender adults , 78 non-autistic transgender adults , and 56 autistic transgender adults  took part in the current study. The mean age of participants was 31.01 years (range = 18 to 70). Participants who identified as gender nonconforming or unknown were excluded from the study (https://osf.io/bke5j/?view_only=795bddddc1144ef295db601982f6a12f). We should note, however, that none of the hypotheses about the autistic transgender group have been included in the preregistration. Yet, they were all made before any statistical analyses were conducted, and if preregistered, they would be exactly the same as the ones presented in the current article .The current study was preregistered on Open Science Framework (preregistration can be viewed here: We assessed participants’ implicit gender self-concept using the Implicit Association Test (IAT) described by Greenwald et al. . ParticiIn the first block , the “self” category label was presented in the upper left corner of the screen and the “other” category label in the upper right corner. Participants were asked to sort words that belonged either to “self” or “other” category by pressing the “a” key of a keyboard for words related to “self” and the “l” key for words related to “other”. In the second block , categories referred to “female” and “male” categorization. The “female” category label was presented in the upper left corner and the “male” category in the upper right corner. Participants were instructed to press the “a” key for words belonging to “female” category and the “l” key for items belonging to “male” category. In the third block , the four categories were presented combined  and participants practiced the sorting task, by pressing the key that was assigned to each category in the preceding two blocks . In the fourth block , participants completed the first experimental condition of the combined sorting task. In the fifth block , the “female” category label was presented in the upper right corner and the “male” category label in the upper left corner, subsequently the assignment of the key for each category was reversed, compared to the first block. Participants were instructed to press “l” for items belonging to the “female” category and “a” for items belonging to the “male” category. In the sixth block , participants practiced the combined task using the switched key assignments. They were instructed to press the “a” key to categorize items that belonged either to the “self” or to the “male” category and the “l” key for words that belonged either to the “other” or “female” category. In the last block , they completed the second experimental condition of the combined sorting task.D) in response latencies between the two practical blocks  and the two experimental blocks . When participants respond faster in the “self-female and other-male” condition than in the “self-male and other-female” condition, they receive a positive D score indicating a female self-concept. When they respond faster in the “self-male and other-female” than in the “self-female and other-male” condition they receive a negative D score indicating a male self-concept. When error rate in the critical blocks  exceeded 20%, participants  were not included in the analyses , and it was administered using Inquisit Web Player 4.0.10. After the completion of the IAT, participants were automatically redirected to a Qualtrics survey to complete the rest of the tasks and questionnaires.The dependent measure for the IAT was the strength of the automatic associations, calculated with the scoring algorithm recommended by Greenwald et al. . That isWe assessed participants’ explicit gender self-concept using the measure designed by Greenwald et al. . ParticiParticipants were asked to indicate their birth-assigned sex, such as on an original birth certificate  and their gender identity . Participants whose birth-assigned sex was congruent to their gender identity were categorized as cisgender, and those whose birth-assigned sex was incongruent to their gender identity or identified as transgender were categorized as transgender. Participants completed the RMIE  = − 0.32, p < 0.001 (one-tailed). In keeping with the preregistered hypotheses, AQ score was also significantly associated with the strength of the explicit gender self-concept (note: scores from the explicit measure were untransformed) among both non-autistic cisgender birth-assigned males  and non-autistic cisgender birth-assigned females . The results of the analyses are included in the online supplementary material. Contrary to predictions, AQ did not correlate significantly with the strength of the implicit gender self-concept among non-autistic cisgender people (Hypothesis 1a), r(97) = − 0.06, p = 0.276 (one-tailed). Results remained nonsignificant when the analysis was performed for each birth-assigned sex separately .In keeping with the preregistration, a series of correlation analyses was conducted to investigate the relations between AQ and performance on the explicit and implicit measures of gender self-concept among non-autistic cisgender individuals . As predicted, AQ score was negatively and significantly related to the strength of the explicit gender self-concept (Hypothesis 1a), F = 2.18, p = 0.140, F = 0.05, p = 0.829, F = 0.14, p = 0.707, F = 2843.37, p < 0.001, F = 34.46, p < 0.001, F = 6.49, p = 0.011, F = 21.33, p < 0.001, A 2  higher than birth-assigned males . This indicates that birth-assigned females who identify as females showed an explicit female self-concept and birth-assigned males who identify as males showed an explicit male self-concept. The opposite pattern was observed among non-autistic and autistic transgender individuals, with birth-assigned females scoring significantly lower than birth-assigned males . This indicates that birth-assigned females who identify as males showed an explicit male self-concept in line with their experienced (rather than birth-assigned) gender, whereas birth-assigned males who identify as females showed an explicit female self-concept in line with their experienced (rather than birth-assigned) gender. These results were entirely expected.Breaking down the three-way interaction, a simple effects analysis of birth-assigned sex within gender identity and diagnostic category indicated that in accordance with the preregistered Hypothesis 1b, the explicit measure of gender self-concept was sensitive to gender identity differences. Results of the analysis are illustrated in Fig. Next, we conducted a simple effects analysis of diagnostic category within birth-assigned sex and gender identity. The results of the analysis are illustrated in Fig. F = 6.41, p = 0.012, F = 7.64, p = 0.006, F = 13.83, p < 0.001, F = 302.40, p < 0.001, F = 4.98, p = 0.026, F = 0.15, p = 0.701, F = 2.80, p = 0.095, A 2  p < 0.001, one-tailed; autistic p = 0.003, one-tailed), and mean scores of birth-assigned males were significantly below zero . This indicates that birth-assigned females who identify as females showed an implicit female self-concept, whereas birth-assigned males who identify as males showed an implicit male self-concept. The opposite pattern was observed among non-autistic and autistic transgender individuals, with birth-assigned females scoring significantly lower than birth-assigned males . This indicates that birth-assigned females who identify as males showed an implicit male self-concept in line with their experienced (rather than birth-assigned) gender, and birth-assigned males who identify as females showed an implicit female self-concept in line with their experienced (rather than birth-assigned) gender. These results were entirely expected.Breaking down the three-way interaction, a simple effects analysis of birth-assigned sex within gender identity and diagnostic category indicated that in accordance with the preregistered Hypothesis 1b, the IAT was sensitive to gender identity differences. Results of the analysis are illustrated in Fig. D score on the IAT than non-autistic cisgender birth-assigned females, indicating a weaker implicit female self-concept. Contrary to predictions, we found that autistic cisgender birth-assigned males achieved a significantly lower D score on the IAT than non-autistic cisgender birth-assigned males, indicating a stronger implicit male self-concept. Also unexpectedly, a nonsignificant difference in the strength of implicit gender self-concept was found between autistic and non-autistic transgender individuals. Both autistic and non-autistic transgender adults displayed an implicit gender self-concept that was in keeping with their experienced gender, rather than birth-assigned gender, to the same degree.Next, we conducted a simple effects analysis of Diagnostic Category within Birth-assigned Sex and Gender Identity. The results of the analysis are illustrated in Fig. Table M = 3.25, SE = 0.04) reported significantly more gender dysphoric feelings than birth-assigned males , gender identity, such that transgender individuals  reported significantly more gender dysphoric feelings than cisgender people , and diagnostic category, such that autistic people  reported significantly more gender dysphoric feelings than non-autistic individuals . The analysis also revealed a significant Gender Identity A 2  t-tests was conducted. Results of the analyses are presented in Table To test Hypothesis 2a, the significant Gender Identity M = 2.89, SE = 0.04) recalled significantly less gender-typed behavior from childhood than birth-assigned males , gender identity, such that transgender participants  recalled significantly less gender-typed behavior from childhood than cisgender participants , and diagnostic category, such that autistic participants  recalled significantly less gender-typed behavior than non-autistic participants . The analysis also revealed a significant Gender Identity A 2  t-tests was conducted. Results of the analyses are presented in Table To test Hypothesis 2b, the significant Gender Identity M = 29.82, SE = 0.67) reported significantly more ASD-like traits than cisgender participants , and diagnostic category, such that autistic participants  reported significantly more ASD-like traits than non-autistic participants . The main effect of birth-assigned sex was nonsignificant, as were the Birth-Assigned Sex A 2  t-tests was conducted. Results of the analyses are presented in Table To examine Hypothesis 3a, a series of planned M = 25.11, SE = 0.44) performed significantly better on the task than birth-assigned females , gender identity, such that transgender participants  performed significantly better on the task than cisgender participants , and diagnostic category, such that non-autistic participants  performed significantly better on the task than autistic participants . The analysis also revealed a significant Birth-Assigned Sex A 2  t-tests was conducted. Results of the analyses are presented in Table To test Hypothesis 3b, the significant Gender Identity In the current study we examined three core aspects of the link between ASD and gender diversity as a whole, aiming to acquire a more holistic understanding of intersectional identities (autistic + gender diverse). We found that relative to non-autistic cisgender people, autistic cisgender people showed a less strong explicit identification with gender groups that corresponded to their experienced gender . This was accompanied by fewer memories of gender-typed behavior in childhood and increased  levels of current gender dysphoric feelings. In contrast, autistic transgender people showed a strong identification with gender groups that matched their experienced gender, and their levels of childhood gender-typed behavior and gender dysphoria were equivalent to levels reported by non-autistic transgender people. Lastly, the latter group presented elevated ASD-like traits. However, these traits were not accompanied by challenges with mentalizing. Below follows a more extensive discussion of the findings.The first aim of the current study was to examine whether ASD affects gender-related cognition. To do so, we first investigated the relation between ASD-like traits, on the one hand, and the strength of explicit and implicit gender self-concept, on the other hand, among non-autistic cisgender people. The approach followed was taken by Kallitsounaki and Williams  to enablContrary to predictions, the number of self-reported ASD-like traits was not significantly associated with the strength of the implicit gender self-concept. This finding is inconsistent with that of Kallitsounaki and Williams  who obseNext, we aimed to extend Kallitsounaki and Williams’  findingsTo our knowledge, this is the first study to investigate implicit gender self-concept in transgender adults and found that non-autistic and autistic transgender adults perceive themselves explicitly and implicitly in terms of their experienced gender. Olson et al.  rightly,As predicted, autistic cisgender individuals  showed a weaker explicit identification with the gender groups associated with their birth-assigned sex than non-autistic cisgender people. This was expected, given previous findings of lower explicit gender identification in autistic people  on the implicit measure of gender self-concept did not differ significantly from the performance of non-autistic transgender individuals. Both groups identified implicitly with their experienced gender, rather than their birth-assigned gender, to the same degree.In sum, autistic cisgender people were able to identify explicitly with the gender groups of their birth-assigned sex, yet they identified less strongly than did non-autistic cisgender people. They also identified implicitly with the gender groups associated with their birth-assigned sex, but the strength of the identification was weaker only among birth-assigned females. In contrast, autistic transgender and non-autistic transgender people identified with their experienced gender at least to the same degree, both explicitly and implicitly. On this basis, it could be argued that these results provide preliminary evidence of a selective influence of ASD in the consolidation of a collective gender self-concept that matches people’s birth-assigned sex.To put it simply, ASD seems to hinder the explicit  identification of people only with the gender groups associated with their birth-assigned sex. The explicit and implicit identification of autistic transgender people with the gender groups of their experienced gender seems to be unaffected by ASD. It is possible that the formation and consolidation of a gender self-concept that does not correspond to birth-assigned sex might be achieved through trajectories and mechanisms that are different from the ones followed when a gender self-concept corresponds to birth-assigned sex. To get a better understanding of the findings from the autistic adult population (cisgender and transgender), we believe it is essential to know how gender self-concepts develops in autistic children. Given that research on this topic is almost nonexistent, future research will be needed to elucidate whether the development of gender self-concept follows the same cognitive and developmental trajectories in autistic and non-autistic children . This is important because to date, only a very small number of studies  and one that taps recalled gender-typed behavior in childhood . Although both measures are commonly used in research, they do not capture non-binary gender identification that is common in ASD . Given that gender neutral pronouns are commonly used by gender diverse people, it could be argued that this methodological limitation might have attenuated the scores of transgender participants on the IAT. We should note, however, that in the current study we included mostly participants who identified with a binary gender, and thus they were more likely to use female or male pronouns than neutral pronouns. Also the IAT task is designed to be easier when two strongly associated concepts  share the same response option than when two unrelated or only weakly related concepts share the same response option  ASD appears to hinder the consolidation of a strong explicit  gender self-concept in autistic cisgender people only, (b) autistic cisgender people have increased current gender dysphoric feelings and recall limited gender-typed behavior from childhood, and (c) non-autistic transgender people show only the behavioral features of ASD, whereas autistic transgender people show features of ASD at the behavioral and cognitive level. The current study enhances our understanding of the link between ASD and gender diversity. We hope future research will further extend the evidence base required to better inform clinical judgement and improve best practice.In sum, the main findings of this study indicate that Supplementary file1 (DOCX 85 KB)Below is the link to the electronic supplementary material.


Understanding women’s experience of birth planning is necessary for introducing and implementing this process in the Iranian maternity services. This study aims to explore perceptions of birth plan implementation in Iran from the perspective of women, their husbands, and clinicians.This qualitative study was conducted in Iran. Qualitative data were collected from November 2020 to March 2021 by conducting semi-structured in-depth interviews with ten mothers who prepared a birth plan, and 15 key informants  who were involved in the implementation process of birth plans. Data were analyzed using conventional qualitative content analysis.Data reduction process resulted in 380 codes that were categorized in 16 subcategories and five main categories. The main categories were “Guide and pattern of preparing for childbirth pathway”, “Maternal empowerment and sense of triumph”, “Facilitating and enhancing communication”, “Successful transition to parenthood and women’s satisfaction”, and “Challenges associated with implementation of the birth plan”. The overarching theme “Birth plan: The missing link in promotion of vaginal birth in Iran” was constructed from these categories.Findings of this study highlight the effectiveness of the implementation of birth plan along with childbirth preparation classes for increasing the likelihood of a successful vaginal birth and promoting empowerment and satisfaction in women during the childbirth process. The findings of this study could pave the way for developing, introducing, and implementing of birth plan in Iran.The online version contains supplementary material available at 10.1186/s12884-022-05305-7. The childbirth process is a sacred event which creates life-long memories . It affeEvery mother would like her childbirth process to be replete with beautiful and memorable moments. To achieve this goal, careful planning is required for and by women so as to experience the birth process safely and comfortably . ChildbiA birth plan is a written document in which pregnant women can describe their expectations and preferences regarding the care they would like to receive during labor and childbirth . Women uIn 1996, the World Health Organization (WHO) introduced the birth plan as one involving “good practices in the care provision during delivery and birth” in order to reorganize and humanize obstetric care . Based oResearch has shown that the prevalence of negative childbirth experiences among primiparous Iranian women is 37%, which is much higher than that in other countries . UnnecesThe use of a birth plan has become increasingly popular in western countries in defense of women’s autonomy during childbirth . AlthougGiven the conflicting results of previous studies with respect to the use of birth plans , 25, 26 The present study is part of mixed methods research conducted in fulfilment of a doctoral dissertation in midwifery. It aimed to explore women, their husbands and clinicians’ perspectives of birth plan implementation in Tehran, Iran. This research was situated within a feminist pragmatism theoretical framework as its research method because it was concerned with both the midwives, who have a female-dominated profession, and women’s birth choices. .Feminist pragmatism is rooted in feminist theories according to which construction of knowledge is inherently a political matter . HoweverTen women who had participated in the quantitative research phase and had a vaginal birth were selected. To further enrich the study, interviews were also conducted with 15 key informants  in line with the purpose of the study. Written informed consent was obtained from all participants. To ensure that participants were well informed about the content of the consent form, at first the lead investigator briefly explained the contents of the birth plan form as well as the goals and reasons for doing the research. A purposeful sampling method was used to select the participants for interview. The participants’ demographic data are shown in Table The study setting included four public health centers  affiliated to Tehran University of Medical Sciences, two private hospitals, and one hospital of armed forces  in Tehran, Iran. The main reason for choosing these hospitals was that the birth plan is not routinely implemented in Iran, and these hospitals facilitated pregnant women to give birth in maternity wards based on their birth plan. These hospitals manage 2000, 3000, 5000 births per year, respectively. The hospitals had to meet some conditions for the implementation of birth plan. These included the presence of the husband or a companion of the woman’s choice during the birth process, having a private midwife, having a private room, having a doula, and water birth in the maternity wards. Midwives in any Iranian hospital, including the mentioned hospitals, work under the supervision of obstetricians.Data were collected from November 2020 to March 2021 via in-depth semi-structured interviews with women ten days after childbirth  are summarized in Tables Participants identified birth plans as models and guides, which have significant positive effects on determining the path to the ultimate goal of vaginal birth. From the point of view of mothers as well as the birth attendants participating in this study, the birth plan is a guide that helps mothers to know their expectations and preferences, to predict the requirements of childbirth, and to make informed choices accordingly. This category involves three sub-categories: “Identifying the goal and making informed decisions”, “Enhancing the power and creativity of the mind”, and “Step-by-step guide to visualizing the vaginal birth process”.One of the participants commented: “It’s very important for the mother to know what’s going to happen to her; even for someone who wants to choose a cesarean section to go and investigate about it and know the process and to know what she wants to go through when having a birth plan, and how it’s going to be done; that’s very helpful and very important in making the path easier.” (Woman 4).According to the mothers in this study, a birth plan serves as a guide to remind them of things at the time of childbirth that they may forget.“The birth plan was somewhat a reminder of some of the things I had forgotten, such as which hospital I wanted to go to if my baby had any problem or whether the hospital has the facilities; for example, is there a cameraman available in the hospital at the time of the baby’s birth.” (Woman 1).The interviewees in this study believed that the birth plan was a model that could enhance their creativity and mentality to achieve a successful vaginal birth. To them, a birth plan was a written document that is effective in boosting and increasing the brain’s energy to achieve maximum positive results from childbirth. In fact, from participant’s point of view, since the birth plan is written, this writing process becomes a factor which helps the mother to adhere to the implementation of its provisions. They believed that the efficiency of thinking is enhanced by writing and having a plan.“You don’t know how writing does wonders. The brain releases many times more energy for doing the things that are written. When you write your plan, it is as if you’ve passed 80% of the path. Writing a birth plan for my wife brought the best results, and I would certainly say we could not have succeeded without this plan.” (Father 2).The participants believed that birth plan was a step-by-step guide to visualizing the birth process and specifying the direct path to reach the goal.“Nine months of hardship is summarized in four hours. When you have a plan, it is as if you have a guide and you reach the goal through a direct and main route, which is the vaginal birth.” (Woman 6).In their experience, the birth plan determines the steps to reach the goal and reveals a picture of the birth process to them in advance, and writing it removes the mental ambiguities about birth.“If we push someone into a pool full of water in a dark night, they may be worried, even if they know how to swim. Birth plan is an opportunity to prepare the woman for birth in a way that she can have a vivid picture of the process.” (Midwife 2).The women participating in this study reported that the birth plan helped them to focus only on the birth process at the time of birth and to make the right decision in admission to the hospital when their labor pain started. They also believed that the birth plan made them take responsibility for their choices.In this study, the mothers stated that they improved their self-efficacy and empowerment for childbirth through the birth plan. This category is divided into four subcategories: “Increasing the women’s skills in managing labor pain”, “Acquiring the required information from various sources”, “Increasing the mothers’ self-confidence, self-esteem, and self- efficacy and “Women’s positive attitude toward vaginal birth”.Women believed that the birth plan mentally prepared them for adapting to labor pain and consciously accepting it. In their experience, the birth plan helped them have the ability to endure the labor pain.“Basically, you need to plan for everything, especially when it comes to giving birth to a baby. Because I had no knowledge about childbirth and it was my first experience, the birth plan helped me a lot. When you have the knowledge and information, you are more determined and you make solid decisions, and this helps your mind to be ready and accept the situation, and the situation will become easier for you.” (Woman 4).“Labor pain is a strange pain, and everyone experiences it in a different way, and I can’t say that it was more than what I had imagined or less than that, but the important thing is that I was able to endure it. This endurance was achieved thanks to the grace of God and the birth plan. What really impressed me was the pre-planning and relaxation. Although I was not mentally prepared for that amount of pain, I was more conscious about it.” (Woman 6).From the point of view of the birth attendants, a birth plan increases the mother’s acceptance skills under unexpected circumstances during vaginal birth. They also believed that women’s minds are expanded by giving them the right to choose and increase their level of knowledge, and in this way, their inner commitment and responsibility to meet their needs in the birth plan is enhanced.“This is the first time a birth plan is used. The woman is given the right to choose from among a lot of options. You have all these options for the birth process, which you need to choose from. When the mother sees these options and when she realizes that she knows nothing about them, she goes and collects information, so her level of knowledge is enhanced. When the level of knowledge rises, the mindset becomes wider, making the mother more successful in the selection process; and because the woman herself has made a decision, she will hold herself more responsible for this decision.” (Midwife 2).“Decision making entails responsibility. That’s why when the mother writes her birth plan, she is responsible for the choices she makes, so she is not passive in this process; this is why this way of intervention and involving the mother in her birth can be effective. (Midwife 5)The women in this study believed that the birth plan gave them an opportunity to think, and it motivated them to seek information from a variety of authoritative sources such as childbirth preparation classes, reputable websites and books, and other mothers’ experiences, which led to an increase in their knowledge and awareness.“We can’t write a plan without preparation; childbirth can be done by everyone, even animals. So, since we are humans, there must be a difference between us and the animals; it is wisdom and contemplation, so we must be prepared and have a precise plan for our birth. Now, everyone gets this wisdom from somewhere. I read a lot of books, and I highlighted the main points of the books, and used to tell my husband a summary of the important points.” (Woman 7).According to the women participating in this study, a birth plan increased their self-confidence and reduced their fear of birth. By the same token, the birth attendants believed that having a choice in the birth plan and the effort a woman makes for it would increase her self-confidence.“My body was fully prepared for the childbirth because I had increased my knowledge about vaginal birth. It was as if I knew how to relieve my pain by myself and how I could control myself. When you are aware of something, you are less afraid of it.” (Woman 6).“The birth plan empowers the woman because she has manipulated her environment; she has made a choice; she has made an effort. Behind this birth, the woman’s world grows; she gains self-confidence because she gets the answer to the choice and effort she has made, so she works with self-confidence.” (Midwife 3).The women participating in this study felt unique and believed that the birth plan empowered them and encouraged them to have a vaginal birth with a birth plan in subsequent births.“If I decide to get pregnant again, I will definitely go the same way and make up for the shortcomings of my previous pregnancy.” (Woman 1).The majority of the women in this study believed that a birth plan would provide an opportunity to deepen reciprocal relationships of trust and respect between women, their peers, and their caregivers. This relationship is characterized with respect to the mother’s values and in gaining her trust. According to the women in our study, the mother-midwife relationship is strengthened in light of midwife’s empathy and her enthusiasm in helping the mother, which is fulfilled by providing the right training and information for the mother and encouraging and informing her about her capabilities in the birth plan. This will in turn provide an opportunity for the mother to tell the obstetrician and the midwife what she wants or does not want with respect to her birth. Having people share the same objectives and by strengthening the teamwork of members of the birth team, the birth plan facilitates and enhances the tripartite mother-midwife-obstetrician relationship. The category of Facilitating and enhancing communication had the following sub-categories: “Building a relationship of woman-midwife mutual trust”, “Strengthening the intra-organizational relationship among the birth team”, “Facilitating and maintaining the tripartite mother-midwife- physician relationship”, and “Facilitating the women’s communication with peers”.Women stated that birth plan deepened their relationship with the midwives. They highlighted that midwives encouraged them about their capabilities in their birth plan.“After 32 weeks, I decided to refer to a midwife. I trusted her a lot. The positive energy she gave me which made me believe that I could do it, deepened our relationship.” (Woman 3).“This birth plan was an opportunity to talk to my midwife and doctor about anything I wanted or did not want.” (Woman 5).From the point of view of midwives, the spirit of birth plan is respect and honoring for mother.“The first thing that one feels in the spirit of this work is the respect for the mother and honoring her. When we ask a mother about something, we are actually respecting and honoring her, which is definitely pleasing to God, and this is a very important issue in my opinion, which we see in the birth plan.” (Midwife 5).From the point of view of the birth attendants, the birth plan is a tool to transfer the mother’s preferences from the realm of health to the realm of treatment. By establishing a purposeful relationship between the mother, the care team, and the treatment team, the intra-organizational communication is enhanced.“When a mother with a birth plan is referred to the hospital by a health center and that mother is welcomed by an already prepared team, she will definitely have peaceful mind and experience pleasant and desirable moments during her childbirth process. This will also remove stress from the treatment and health teams.” (Midwife 2).According to both the women and the birth attendants, the birth plan provides an opportunity to quickly transfer information between the mother, the midwife, and the obstetrician.“By reading the birth plan, the midwives exactly knew what I wanted and conveyed it to the obstetrician. And that was great because when you’re in labor you don’t feel like talking. In fact, they know what you want from the moment you enter the birth room.“ (Woman 2).Participants in our study believed that the birth plan facilitates the mother’s communication with other peers by providing the opportunity for communication and group discussion and exchange of information related to the birth plan.“During pregnancy, women are most motivated to get information. When mothers as a group discuss the birth plan, this creates more communication between pregnant mothers and provides an opportunity to learn through their experiences in similar situations.” (Midwife 3).The majority of the women in this study were of the opinion that having a birth plan was effective in creating satisfaction as well as positive and pleasant experiences from the birth and the postpartum period. In the postpartum period, they expressed a feeling of happiness, triumph and greater readiness for parental responsibility. For some women, giving birth according to their preferences was the reason for their happiness and experiencing no depression after childbirth. In addition, the majority of mothers attributed the positive experiences and success of using the birth plan to the participation of their husbands in the planning process and supporting them during childbirth, and the choice of the midwife as the birth attendant.This category included three subcategories: “Successful transition from pregnancy to parenthood”, “Husbands’ participation in planning and implementation of the birth plan”, “Choosing a midwife as the best birth attendant”.“I think having a birth plan is worth it. After giving birth, one seems to be more prepared to become a mother or a father, and I don’t know what’s going on. I don’t know if this is because of team work or because the father sees what the mother goes through and gets a better understanding of her. I mean the quality of the relationship between my husband and I has improved since my last birth.” (Woman 3).“Even though I was in so much pain and it was all annoying, I did not get postpartum depression. Although I had depression during pregnancy, I did not get postpartum depression and the main reason for this success is that I was able to give birth the way I wanted. I miss the day I gave birth.” (Woman 7).In the women’s experience, the birth plan enhanced their motivation to breastfeed the baby with love and affection.“Even though I knew that not all my plans might be fulfilled, it was worth it and the benefits were greater. For example, the moment you breastfeed, you milk your baby with love.” (Woman 6).Women stated that the birth plan helped them to know that their husbands could be on their side during childbirth. From the point of view of women, the participation of their husbands in the implementation of birth plan were very useful, and improved the progress of labor.“My husband and I both think it is important to have a birth plan. This gave me the power to choose and helped me to know that my husband could be by my side during childbirth. It was very important for me to have my husband by my side, because it was more encouraging than when I’m alone; it also affected the progress of my birth, and it also had a great impact on my mental condition.” (Woman 4).The women interviewed in this study described the pleasant experience of childbirth as a miracle, thanks to the presence of a capable midwife.“I was lucky enough to be able to carry out my birth plan with an experienced midwife, and I really think that the delivery itself is a miracle.” (Woman 3).According to the experiences of some of the participants, the birth plan was accompanied with challenges and problems during the implementation phase. Factors such as medical dominance in Iran, the existing regulations in the delivery rooms, and mothers’ lack of access to one-to-one midwifery care were among the problems and challenges of implementing the birth plan. This category includes two subcategories: “Inadequate attention to planning and policy making to promote vaginal birth”, and “Conflicts between midwives and gynecologists”.The participants’ statements indicate that the main problems against implementation of the birth plans roots in the higher levels of management and policy making in health system of Iran. Problems such as forced parturient to accept the conditions in the delivery room, the lack of midwives’ authority, leadership role and decision-making power in implementation of birth plans, neglecting to provide on- demand care for women in health policies.“I believe the success of the birth plan lies in the one-to-one midwifery care, which is pretty common in the world but still needs to be expanded and implemented in Iran.” (Midwife 6).“We don’t have full authority to decide on the birth plan, and this poses a very big challenge to the realization of this process, and the solution is to change the policies in such a way so that we (midwives) can be responsible for the birth, which is a long way to go.” (Midwife 4).“In Iran, we have not learned to be demanding people, and the parturient has to accept the conditions in the delivery room. For example, a pregnant mother can demand that her husband be with her during the birth. But in many good hospitals, this demand is ignored because the doctor and the maternity ward staff, who are all females, want comfortable clothing, and they consider the presence of a male stranger to be somewhat intruding, although we know that the presence of the husband is an important part of physiological childbirth.” (Midwife 1).Most midwives believe that achieving the goal and success in implementation of birth plan requires a strong teamwork. Some midwives believe that recognition of midwifery profession and midwives’ competencies by gynecologists, can lead to mutual respect and cooperation. Realization of the birth plan will benefit from such a constructive atmosphere.“Our gynecologists think that vaginal birth is exclusively their own duty. However, everywhere in the world, vaginal birth is the midwives’ responsibility, but in our country they do not accept this, and gynecologists consider themselves to be in charge of it, and as long as they interfere with our work, they affect vaginal birth, which also affects the birth plan.“(Midwife 4).This qualitative study was conducted with the aim of gaining a deeper understanding of women’s views and perceptions of using birth plan during pregnancy, childbirth, and the postpartum period in Iran. The findings demonstrated that the birth plan increased women’s positive childbirth experience and reduced their fear of vaginal birth. Moreover, it helped women to strengthen the reasoning power and better understanding about the birth process, helped women to engage actively in labour, be empowered to increase their skill for self-control and self- efficacy in labor and delivery, supported them to make informed decision, and facilitated communication with gynecologists, midwives, and other peers.In this study, using birth plan was a new experience for the participants, and they perceived it as a model and guide that affects their preparation for a vaginal birth. The birth plan helped women to better understand their expectations and preferences for childbirth and to make the best choice for controlling labor pain based on their preferences and the knowledge they had gained. In addition, the birth plan gave the women the capability to better manage their minds for birth and focus more on the birth process. The birth plan served as a model according to which the pregnant women were able to actively participate in decision-making for their childbirth through a step-by-step visualization of the childbirth process, from the onset of pain to the birth stage. Most importantly, childbirth in this study was cited to be a positive and enjoyable experience that was achieved when the demands and expectations in the birth program were met. In this study, 66% of the pregnant women were primiparous, and they often felt anxious about their pregnancy and childbirth. However, they reported that using a pre-arranged birth plan helped them clear up their mental ambiguities about birth. It was also believed to help the mothers identify their needs earlier and enter the birth process with a better mental preparation.The results of this study are supported by previous studies on the benefits of birth plans. In a study on 68 Chinese women, for example, women who used a birth plan stated that they perceived it as a guide or reminder to plan their care during birth . CapitulWomen in the present study reported that the birth plan helped them acquire the skills to accept unexpected circumstances in the birth process and to be responsible for their choices. In addition, they were of the opinion that the birth program enabled them to acquire the necessary knowledge and information from reputable sources. In order to make an informed choice, they would seek help from reputable websites, books, and the successful experiences of postpartum women. In this way, they would feel empowered and triumphant. Women’s enhanced knowledge and skills, along with their increased self-confidence and sense of overcoming the fear of vaginal birth granted them self-efficacy for vaginal birth. Pregnant women’s caregivers also believed that the birth plan increased these women’s self-confidence by giving them the right to choose and participate in the decision-making process. Anderson et al. found that postpartum self-esteem scores were higher in women who had a birth plan . A qualiParticipants in the present study stated that the birth plan facilitates and maintains a relationship of mutual respect and trust between women, their caregivers, and their peers. This relationship was maintained throughout labor and birth because the women believed that they were sufficiently prepared for unpredictable situations, and their caregivers were confident that these women would maintain the necessary flexibility and trust in their caregivers. Our findings are consistent with the results of previous studies reporting that all women and their caregivers considered the birth plan as a valuable communication and education tool , 37, 40.In our study, the majority of women were satisfied with having a birth plan. They experienced positive emotions such as happiness, success, greater readiness to accept the parenthood responsibility, and the mutual comfort between them and their husbands. A number of women in this study, including mothers with a history of previous cesarean section, were satisfied with preferring a midwife to a physician as the birth attendant. By choosing a midwife in their birth plan, they experienced reduced stress during pregnancy and enhanced childbirth. Some women even wanted to share the sweet and enjoyable experience of vaginal birth and recommended it to other expectant mothers. One of the mothers, for example, expressed her happiness about her success in the planned vaginal birth and wished that these conditions would be provided for all Iranian women. She described childbirth as a miracle in her life and asked the study researchers to let the voice of Iranian women be heard by policymakers; that they want their childbirth to be vaginal. Redshaw et al. (2019) suggested that a woman’s experiences of obstetric care may affect her future pregnancies as well as decisions about her subsequent deliveries. In thisOther studies have reported conflicting results on the effect of birth plan on women’s satisfaction. Farahat et al., for instance, reported that the mean score of childbirth satisfaction was significantly higher in women using a birth plan . In ShamDespite the women’s satisfaction, a number of women and midwives complained about the challenges and problems they faced in implementing the birth plan. Medical dominance in Iran, where the midwife is not in charge of vaginal births and there is no one-by-one midwifery care, accounts largely for the failure of planned vaginal births. In addition, according to the midwives participating in this study, childbirth preparation classes were not fully efficient. They argued that although childbirth preparation classes were successful in giving awareness to the mother, they failed to prepare the mother for a good childbirth. This is where the birth plan can be of great help by letting the mother contemplate and write down how her birth should be carried out. A number of midwives in this study also believed that professional challenges between midwives and obstetricians in Iran along with the midwives’ lack of decision-making power could also lead to the failure of the birth plan. However, one of the obstetricians interviewed in this study believed that the cumbersome rules related to blood money  force doctors to do something that they themselves might not be happy to do. Other studies reported different challenges of birth plan. For example, in Whitford et al.’s qualitative study, lack of a clear understanding of the goals of the birth plan, providing no support for the selected plans, and reluctance to do too much planning were the challenges of the birth plan . Aragon This study is worthwhile in that it is the first qualitative study to examine women’s perceptions of the impact of the birth plan on their childbirth experience while attending childbirth preparation classes in Iran. However, like any other study, it has a number of limitations. First, due to the COVID-19 pandemic and the lockdown during data collection, we had to conduct a number of interviews through telephone, which might have affected the results. Because the absence of face-to-face contact restricted the development of communication, and we couldn’t see body language and facial expressions of interviewees. Thus the resulting data may not be as rich as it would be from a face-to-face interview. Second,According to the results of this study, the birth plan, accompanied with childbirth preparation classes, increases the likelihood of a successful vaginal birth and the desire for having another child. The findings of this study highlight the effectiveness of the birth plan in terms of women’s empowerment for vaginal birth and their satisfaction during childbirth and in the postpartum period. Therefore, vaginal birth promotion policies can include using birth plans in prenatal care of low-risk pregnant women and in the educational content of childbirth preparation classes in Iran. This can be an effective intervention aimed at reducing the rate of cesarean section. Furthermore, it seems that the present study may greatly interest for managers, leaders, and policymakers and may support current population policies in Iran. However, further studies are required to identify barriers to assess the effectiveness of the birth plans.Additional file 1.


Periodontal disease (PerioD) is a chronic, complex inflammatory condition resulting from the interaction between subgingival dysbiotic bacteria and the host immune response leading to local inflammation. Since periodontal inflammation is characterized by multiple cytokines effects we investigated whether Periodontal Inflamed Surface Area (PISA), a continuous measure of clinical periodontal inflammation is a predictor of composite indexes of salivary cytokines.In a cross-sectional study of 67 healthy, well-educated individuals, we evaluated PISA and several cytokines expressed in whole stimulated saliva. Two salivary cytokine indexes were constructed using weighted and unweighted approaches based on a Principal Component Analysis [named Cytokine Component Index (CCI)] or averaging the (standardized) level of all cytokines [named Composite Inflammatory Index (CII)]. In regression analysis we found that PISA scores were significantly associated with both salivary cytokine constructs,  independent of age, gender and BMI showing that single scores summarizing salivary cytokines correlated with severity of clinical periodontal inflammation.Clinical periodontal inflammation may be reflected by a single score encompassing several salivary cytokines. These results are consistent with the complexity of interactions characterizing periodontal disease. In addition, Type I error is likely to be avoided. Periodontal disease (PerioD) is a chronic, inflammatory condition present in more than 50% of the population . It resuThe significance of local periodontal inflammation resides not only in its disease maintenance and contribution to its progression ,9 but alMany studies investigated salivary cytokines in relation to a variety of continuous and discrete measures of periodontal disease ,16. HoweInclusion Criteria: All included subjects were required to be ≥ 45 years, with ≥12 years of education and be cognitively unimpaired. Exclusion Criteria: Individuals were excluded if they had significant comorbidities, including stroke, diabetes, uncontrolled hypertension, head trauma, any neurodegenerative disease, and chronic depression. Subjects taking anti-inflammatory medications for chronic conditions , or antibiotics or having periodontal treatment within 3 months of the periodontal evaluation were also excluded.This is a cross-sectional study in which the subjects were recruited from an existing cohort participating in studies relating periodontal disease to Alzheimer’s disease ,19 whoseSalivary cytokines were sampled from whole saliva.Stimulated whole-saliva samples were collected on ice in a sterile 50 ml plastic conical centrifuge tube. After rinsing with drinking water to clear food debris, the subject chewed on a piece of unflavored gum and spit the saliva in the sterile tube until 5mls were collected. Then, the saliva was centrifuged for 25 minutes at 2,500rpm at 4C. The aliquoted supernatant was stored at -80°C until cytokine analysis.Salivary cytokines  levels were assessed in stimulated saliva using electrochemiluminescence method (MSD platform). Human V-Plax proinflammatory panel-Cat# K15049D measuring the above cytokines was performed according to the manufacture protocol. In the final step, after the Read Buffer was added, the signal from the MSD plates was read on the Meso Scale Discovery (MSD) Sector Imager 2400 at 620 nm. Cytokines expressed in pg/ml were quantified using a standard curve of appropriate standards provided by the kit. Each sample was assayed in duplicate and the average was used for analysis. Our analyses excluded cytokines with >10% of samples scoring outside the dynamic range of the standard curve: INF-gamma, IL-2 and IL-4: INF-gamma with 11 subjects (16%), IL-2: 16 subjects (24%), and IL-4: 48 subjects (72%) testing bellow the detection limit.Subjects received an oral-periodontal examination, as previously described . Briefly2 [http://www.parsprototo.info/). Since PD measures the past and present disease and BOP is the most reliable measure for assessing gingival inflammation [2 as threshold [Results of the periodontal exam were summarized as the Periodontal Inflamed Surface Area (PISA) scores expressed in mm2 . PISA isammation , therefohreshold .Demographic information , systemic factors (comorbidities), oral  and smoking behaviors were obtained by a standardized examiner-conducted interview at the time of the oral examination .All our analyses were performed using IBM SPSS Statistics software, version 27 . Continuous data are presented as means and standard deviation (SD) and categorical data as percentages. Normality was tested by Kolmogorov-Smirnov and log10 transformation was used to normalize the distributions for each salivary cytokine. Group differences for cytokines were assessed using Students t-test.To reduce the dimensionality of our data (numbers of cytokines) we used weighted and unweighted approaches based on a Principal Component Analysis (PCA) or averaging the (standardized) level of all cytokines. Thus, we created two salivary cytokine indexes: 1. salivary cytokine component index (CCI) and 2. salivary cytokine composite inflammatory index (CII).l-known analysis that is based on reducing the dimensionality of data such as multiple-correlated variables into a reduced number of uncorrelated components called Principal Components. PCA has been previously used in cytokine analyses [1) Salivary CCI was derived from Principal Component Analysis (PCA) that included 6 salivary cytokines . Principal Component Analysis (PCA) is a welanalyses ,24.a priori decision was to retain all the components with the eigenvalue greater than unity. An eigenvalue is a measure of the variance accounted for by one component. Component loadings are the correlations between the cytokines and the component.Our data supports the use of PCA: cytokines are presented as continuous data and there was a linear relationship between them. Linearity between all variables was evaluated using a correlation matrix while sampling adequacy was evaluated using the Kaiser-Meyer-Olkin (KMO). The correlation matrix showed that all variables have at least one correlation with another variable greater than the 0.3 cut-off. The overall KMO measure of 0.723 is considered fair. Similarly, for each individual variable the KMO measure ranged between 0.907 and 0.646 considered adequate. Bartlett’s test of sphericity was statistically significant (p<0.0005) indicating that the components of the analysis were correlated. The 2) Salivary CII was derived using statistical means. We constructed a salivary CII defined as the statistical mean of the 6 salivary cytokines: IL-1β, IL-6, IL-8, IL-10, IL-13 and TNF-α.Correlations and linear regression models were used to assess PISA relationships with salivary cytokines . In initial models, we evaluated the association of potential confounders with the cytokines: age, gender, education, BMI, behaviors , systemic conditions . Gender and BMI were significant and were included in the multivariate models. Although age was not significant, due to its reported association with both cytokines and PISA, it was also included in the final model.To determine whether PISA was uniquely associated with salivary cytokines, regression analyses were performed in which either the individual salivary cytokines, the salivary CCI or the salivary CII were predicted from the PISA score and the relevant covariates. The part correlations (part R), unstandardized regression coefficients (β) and the 95% confidence interval (95%CI) of the β were obtained. Statistical significance was set at p<0.05.The characteristics of our population are shown in The levels of salivary cytokines are shown in Regression analysis showed that after age, gender and BMI adjustment, PISA maintained significant associations with IL-1β , IL-6 , IL-8 , IL-13  and TNFα .Thus, the addition of PISA to age, gender and BMI, led to a statistically significant increase in R2 for each cytokine ranging from 10 to 18%.PCA was applied with the goal of reducing the number of salivary cytokines into fewer components. PCA showed only one component with an eigenvalue greater than unity and it explained 52% of the total variance. In regression analyses, the CCI score was predicted by PISA score , gender  and BMI , yielding a total R2 of 0.37, p<0.001). We re-run the analyses using age, education and BMI categories and the results were similar. In regression analyses, the salivary CII score was predicted by PISA score , gender  and BMI , yielding a total R2 of 0.29, (p = 0.001). Re-running the analyses using age, education and BMI categories, resulted in the same outcome. In this study we found that PISA scores were significantly associated with both salivary cytokine constructs, salivary CCI and salivary CII and these associations were independent of age, gender and BMI. Smoking was not significant in the model suggesting that smoking was not a confounder of the relationship between PISA and salivary cytokine indexes. These results support our hypothesis that a single score can be used to summarize levels of salivary cytokines and that this score is highly correlated with the severity of periodontal inflammation. PISA association with salivary CCI was stronger (R2 = 0.37) than its association with salivary CII (R2 = 0.29) because the former index accurately weighted the component scores. However, salivary CII is easy to construct and use and its numerical value is easy to interpret. Periodontal disease results from a complex interaction between periodontal bacteria and immune response supporting multiple cytokines as predictors of PISA. Our population had stage III and IV periodontitis. Therefore, it is unknown if this relationship would be similar within or between other stages of periodontitis and health.PISA as assessed by PD and BOP represents the overall clinical periodontal inflammation. On the other hand, salivary cytokines are a correlate of the molecular make-up of the oral environment. Periodontal inflammation is best represented by the presence of inflammatory cytokines in gingival crevicular fluid (GCF) the proximal periodontal fluid. However, sampling of GCF poses challenges: it is difficult and time consuming; it reflects gingival inflammation at a specific site and therefore numerous sites must be sampled ; samplinSeveral studies have shown that PISA is associated with several systemic diseases including CVD , dementiWe used two salivary cytokine indexes and both included the cytokines IL-1β, IL-6, IL-8 and TNF-α. These are well known pro-inflammatory cytokines and their association with periodontal inflammation is not surprising. In fact, numerous studies investigating the levels of these cytokines in association with periodontal disease found positive results  althoughIL-10 is an anti-inflammatory cytokine with regulatory immune functions. In periodontitis it has protective functions and inhibits pro-inflammatory cytokine production. In our study, IL-10 loaded on the PCA inflammatory component in tandem with the other pro-inflammatory molecules. Its contribution to the inflammatory component was not anticipated. However, it can be explained by its up-regulation in response to an increased inflammatory milieu. Increased inflammation may trigger a feedback mechanism to counteract further inflammation. In addition, IL-10 could have a proinflammatory role .Interestingly, IL-13 loading on CCI was the strongest. IL-13 is a pleomorphic cytokine with both anti-inflammatory and proinflammatory functions. In its role as anti-inflammatory cytokine, IL-13 inhibits the secretion of pro-inflammatory cytokines including TNF-α, IL-1β, IL−6, and IL−8 ,43. HoweSeveral strengths characterized our study. Our sample was quite homogeneous consisting of elderly, well-educated and relatively healthy individuals. Periodontal measures and saliva collection were standardized and salivary cytokine assays were determined blindly to the periodontal assessment.There are several limitations related to our study that include the design, population characteristics, and sample size. As a cross-sectional study, it shows only a correlate of periodontal inflammation to salivary cytokines. The number of subjects was moderate. An additional weakness was the periodontal status of our population: all had severe periodontitis with various degree of clinical periodontal inflammation. The ranges of PISA in our study: mild-moderate range with few severe forms. Due to their homogeneity, and limitations described these results may not apply to general population. It is desirable to test our hypothesis in a diverse population composed of periodontically healthy subjects, gingivitis and various stages and grades of periodontitis.In conclusion, we showed that two salivary cytokine indices could be used to better characterize the inflammatory consequences of periodontal inflammation. However, subsequent larger studies should be done to validate these indices in a heterogeneous population. Studies that manipulate periodontal inflammation are also needed to support a causal link with cytokine responses.S1 Table(XLSX)Click here for additional data file.


Multivariate logistic regression models were computed to explore independent associations and expressed as (adjusted) odds ratios (aOR) with 95% confidence intervals (CI). The findings of this study indicate no statistically significant relationship between blood mercury, manganese, or lead levels and stillbirth, preterm birth, low birth weight, and low Apgar score. However, the covariate diabetes mellitus ) was independently associated with preterm birth and the covariate hypertension ) with low birth weight. Nevertheless, the observed high proportions of pregnant women with blood levels of mercury, manganese, and lead above the reference levels values of public health concern warrants environmental health research on risk factors for adverse birth outcomes to develop public health policy interventions to protect pregnant Surinamese women and their newborns from potential long-term effects.Globally, adverse birth outcomes are increasingly linked to prenatal exposure to environmental contaminants, such as mercury, manganese, and lead. This study aims to assess an association between prenatal exposure to mercury, manganese, and lead and the occurrence of adverse birth outcomes in 380 pregnant women in Suriname. The numbers of stillbirths, preterm births, low birth weights, and low Apgar scores were determined, as well as blood levels of mercury, manganese, lead, and relevant covariates. Descriptive statistics were calculated using frequency distributions. The associations between mercury, manganese, and lead blood levels, on the one hand, and adverse birth outcomes, on the other hand, were explored using contingency tables, tested with the χ Globally, stillbirth, preterm birth, low birth weight, and low Apgar score are among the most common adverse birth outcomes. Each year, roughly 15 million babies are born prematurely , approxiSeveral factors have been associated with adverse birth outcomes. Some of these include socio-demographic characteristics of the mother, such as age at the birth of the child, marital status, education, occupation, household income, and nutritional status ,10,11, aThe Republic of Suriname is a South American country that borders the Atlantic Ocean to the north and is located between French Guiana and Guyana. Suriname’s major means of subsistence are gold mining and agriculture, in addition to crude oil drilling, fisheries, forestry, and ecotourism . TogetheHoplias aimara [Regrettably, these developments have been achieved at the expense of severe environmental pollution, massive land degradation, and extensive deforestation . The uses aimara ,28. The s aimara  that is s aimara ,31. Leads aimara ,33. Thiss aimara .When these heavy metals enter the food chain and are ingested by pregnant women, they increase the risk of adverse birth outcomes. Prenatal exposure to mercury has been associated with reduced placental and fetal growth ,36, whilThe hospital-based maternal mortality rate in Suriname over the past thirty years was 127 per 100,000 , the stiThe influence of several factors that were not the focus of the current study but may have an impact on adverse birth outcomes have also been assessed. These covariates included medical conditions, such as diabetes mellitus ,47, hypeIn this study, the possible association of prenatal exposure to mercury, manganese, and lead, on the one hand, and adverse birth outcomes in pregnant women in Suriname, on the other hand, was analyzed. As mentioned before, for the current study, the data analyzed were from the Caribbean Consortium for Research in Environmental and Occupational Health (CCREOH)’s cohort of 1200 pregnant women in 2017. The number of 1200 participants was calculated based on a multiple linear regression model using a coefficient of determination of 0.10 and an R2 differential of 0.02 to have 80% power at a 0.05 level of significance, taking into account the potential loss of participants . In thisThe current study is based on a subset of 400 participants whose blood metal levels were available and have been analyzed. The CCREOH study has been approved by the Institutional Review Board of the Ministry of Health of Suriname (protocol number VG 023-14) and the Institutional Review Board of Tulane University, New Orleans, LA, USA (protocol number 83 093). Each participant entered the study voluntarily, gave informed consent after the purpose of the study was thoroughly explained, and agreed to donate venous blood.Enrollment of the participants and collection of maternal socio-demographic and dietary data was conducted by trained recruiters through face-to-face-interviews using questionnaires on encrypted iPads, as described in the CCREOH’s study protocol .The follData on birth outcomes of the study participants were retrieved from their birth and medical records at the hospitals and primary healthcare clinics where the delivery took place. Stillbirth was defined as a fetus born with no signs of life from 22+ completed weeks of gestation or a birthweight of 500+ g ,63,64,65Whole blood samples from the 400 study participants were collected during the first and second trimesters of their pregnancy using trace element vacutainers with potassium EDTA. The blood samples were frozen and shipped to the Wisconsin State Laboratory of Hygiene (WSLH) at the University of Wisconsin–Madison . The Trace Element Research Group of that institution determined the concentrations of mercury, manganese, and lead in the samples using sector field inductively coupled plasma mass spectrometry.The detection limits for mercury, manganese, and lead were 0.05 µg/L, 0.07 µg/L, and 0.06 µg/L, respectively. Blood levels of mercury were categorized as <3.5 µg/L and ≥3.5 µg/L, using 3.5 µg/L as the reference level ,69,70. TThe covariates considered relevant to the current study included elevated levels of blood glucose  , raised 2-test, and expressed with a p value. Fisher’s exact test was performed to adjust for sensitivity. Logistic regression models were computed to explore crude and independent associations of the categorized blood levels of the heavy metals and the covariates with the adverse birth outcomes and expressed as crude and adjusted odds ratios (OR) with 95% confidence intervals (CI). Only variables that were statistically significantly associated at the bivariate level at p < 0.10 were included in the multivariate models. In the crude models, each variable was tested individually against the relevant birth outcome. In the adjusted models, the variables were evaluated together against the relevant birth outcome to establish associations between the variables. All analyses were conducted using IBM “Statistical Package for the Social Sciences” (SPSS) version 25.Descriptive statistics were calculated using frequency distributions. The potential associations between prenatal exposure to mercury, manganese, and lead, on the one hand, and adverse birth outcomes, on the other hand, were explored using contingency tables, tested with the χDue to missing data on adverse birth outcomes, 20 participants were excluded from the analyses. The socioeconomic characteristics of the remaining 380 women are presented in A total of 74 of the 380 evaluable women had experienced an adverse birth outcome . The mosAs shown in Assessment of the covariates considered relevant to the current study , showed p values > 0.10). Maternal age 35 years and over, underlying anemia, multiparity, and smoking were also not statistically significantly associated with any of the adverse birth outcomes .The results from bivariate associations between the blood levels of the heavy metals or the covariates, on the one hand, and the occurrence of adverse effects, on the other hand, are shown in p value of 0.065), though not with that of stillbirth, low birth weight, and low Apgar score . The presences of diabetes mellitus, alcohol consumption, and the use of prescription medicines were also statistically significantly associated with preterm birth . The same held true for alcohol consumption, as well as underlying hypertension and the occurrence of low birth weight . Together, these observations suggest that high lead blood concentrations, as well as the presence of diabetes mellitus, alcohol consumption until at least 3 months before pregnancy, and the use of prescription medicines may represent independent variables for the occurrence of preterm birth, and that underlying hypertension and, the use of alcohol until at least 3 months before pregnancy may represent independent variables for the occurrence of low birth weight.On the other hand, blood concentrations of lead ≥3.5 µg/dL were statistically significantly associated with the occurrence of preterm birth  times greater odds of giving birth to a baby with a low birth weight than women with a normal tension, while pre-pregnancy alcohol use was associated with a decreased risk of 0.43 (95% CI 0.22–0.85) when compared to the abstinence of alcohol . Thus, tNext, the influence of underlying diabetes mellitus, hypertension, prescribed medicine, or alcohol intake on the occurrence of preterm birth or low birth weight was explored in the adjusted models. In these analyses, the aOR for an association between blood lead levels ≥ 3.5 µg/dL and preterm birth decreased from 1.88 (95% CI 0.98–3.60) to 1.38 (95% CI 0.40–4.79), and the association between prescription medicine use, as well as pre-pregnancy alcohol use, and preterm birth changed from 2.53 (95% CI 1.28–5.00) to 2.1 (95% CI 0.43–10.16) and 0.50 (95% CI 0.27–0.93) to 0.36 (95% CI 0.11–1.145), respectively . The OR Together, these findings suggest that there was no association between prenatal exposure to mercury, manganese, or lead and adverse birth outcomes, but that the covariates of high blood glucose levels and preterm birth, and high blood pressures and low birth weight, showed to be independently related to the adverse birth outcomes.In this study, the association of blood levels of mercury, manganese, and lead, on the one hand, and the occurrence of adverse birth outcomes, including still birth, preterm birth, low birth weight, and low Apgar score, on the other hand, was assessed in 380 pregnant Surinamese women. Covariates, such as underlying hypertension, diabetes mellitus, and anemia, as well as multiparity, age over 35 years, alcohol use until at least 3 months before pregnancy, smoking until at least 3 months before pregnancy, and the use of prescription medicines, such as anti-anemic drugs, were also included in the analyses.The women enrolled in the study were part of a larger group of 1200 women who had given birth and in whom the effects of environmental contaminants on birth outcomes and neurological development had been studied . Their aThe frequency of adverse birth outcomes in the current sub-cohort (19.5%) and the frequency distribution of the birth outcomes  were also roughly within the same range as those previously mentioned for Suriname ,80 and fWhile investigating this group of pregnant Surinamese women, no statistically significant associations were found between blood levels of mercury, manganese, or lead and the occurrence of stillbirth, preterm birth, low birth weight, or low Apgar score. This was unexpected since blood concentrations of the heavy metals were above public health action levels in 40.5%, 63.9%, and 21.3%of the women, respectively. As mentioned before, these compounds are present in various (banned) agricultural pesticides that are, nevertheless, abundantly used in Suriname . ResidueNevertheless, that the high number of blood levels above the reference levels of these compounds was apparently not associated with adverse birth outcomes in the current study may be tentatively explained by the relatively small number of participants included in the study. The study was initially designed and powered for 1200 participants based on the effect size for the longitudinal study with this number of women. However, as mentioned before, at the time of the current study, blood specimens’ data were only available for 400 women. Consequently, the effect size was smaller than the effect size for which the study was originally powered. Obviously, the resulting lower power is a limitation of the current study. The remaining blood specimen data are anticipated to be available in the near future.In addition, the high frequency of women with blood metal levels above public health action reference levels may not lead to immediate adverse health effects in pregnant women and newborns, but rather manifest as neurodevelopmental disorders in children many years after birth ,90,91, cThe association of each of the studied covariates in the current study with adverse birth outcomes has been well documented ,101,102.There is also no explanation at hand for the apparent risk reduction in preterm birth in women who had used alcohol before their pregnancy and who were at the same time suffering from diabetes mellitus or had used prescription medicines, such as anti-anemic drugs. However, some studies have shown an independent relationship of diabetes mellitus and preterm birth in pregnant women ,105, whiThe apparent attenuation of the risk of preterm birth in women who had been exposed to higher lead concentrations and had also used prescription medicines may tentatively be attributed to interactions of both substances and lead to changes in their pharmacological fate and modification of the risk for this adverse birth outcome. This may hold true for, for instance, iron supplements, which are often prescribed for anemia in pregnant Surinamese women . This caIn summary, the results of the current study suggest that there were no associations between prenatal exposure to mercury, manganese, or lead and adverse birth outcomes. However, the small sample size of the study decreases its statistical power, warranting caution when trying to extrapolate these findings to the general population. For the same reason, the absence of an association between the high mercury, manganese, or lead blood levels and adverse birth outcomes must be considered with care. The same holds true for the confounding effects of underlying diabetes mellitus and/or hypertension, as well as the use of prescription medicines and alcohol use.Nevertheless, our data warrant more careful assessments of pregnant women for risk factors for adverse birth outcomes, such as prenatal exposure to hazardous environmental compounds; underlying (chronic) diseases, such as diabetes mellitus; the use of certain prescription medicines; and pre-pregnancy alcohol use, not only separately but also when these risks are present in various combinations. As shown by the current data, these practices will likely help improve environmental and health policies to ensure the safety of (pregnant) women and their newborns in Suriname. Future studies should include an increased number of pregnant women and specifically enroll women with underlying anemia, diabetes mellitus, and/or hypertension, as well women using prescription medicines, such as anti-anemic drugs in order to eliminate type II errors.The findings of this study suggest that there were no statistically significant relationships between mercury, manganese, and lead blood levels above reference (high) values and adverse birth outcomes, including stillbirths, preterm birth, low birth weight, and low Apgar score. These findings and the alarming frequency of blood levels of mercury, manganese, and lead above the reference levels, indicate an urgent need for environmental health research and public health policy interventions to evaluate and protect pregnant Surinamese women more meticulously for potential risk factors for adverse birth outcomes and the effects in the long term for mothers and their newborns.