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The association between anogenital distance, reproductive and general health in adult females- a prospective cohort of 17 years
Reproductive Toxicology 90 (2019) 77–81
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The association between anogenital distance, reproductive and general health in adult females- a prospective cohort of 17 years
T
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Tamar Wainstocka, , Israel Yolesb,c, Ruslan Sergienkoa, Asnat Walfischb a
Department of Public Health, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel Department of Obstetrics and Gynecology, Soroka University Medical Center, Ben-Gurion University of the Negev, Beer-Sheva, Israel c Clalit Health Services, Central District, Israel b
A R T I C LE I N FO
A B S T R A C T
Keywords: Anogenital distance Early life exposures Endocrine disrupting agents Gynecological morbidities Prospective cohort
Background: Studies have suggested an association between prenatal hormonal environment and anogenital distance (AGD). We aimed to study the association between AGD and health characteristics in adult women. Methods: In this prospective cohort AGD was measured in 300 parturients during the years 2000-2001. In December 2017, study participants` hospital records were abstracted and medical diagnoses grouped into major categories, including gynecological, cardiovascular, and other. Associations between AGD and the morbidities categories were evaluated using multivariable survival models, which adjusted for confounding variables. Results: Participants were 44.3 ± 5.4 years old with mean AGD 40.3 mm ( ± 10.7 mm). Women with below vs. above mean AGD were more likely to develop gynecological morbidities (39.9% vs. 27.1%, adjusted Hazard ratio 1.82; 95%CI 1.08–3.06). Conclusions: Women with short AGD are at increased risk for gynecological morbidities. AGD should be studied further, and may possibly be used for screening of women at risk for these conditions.
1. Introduction During early human life, the endocrine system is involved in the formation and ensuring proper function of various systems, including metabolism, cardiovascular, neurological and mainly the reproductive systems [1]. Early life exposures to estrogen and testosterone have been associated with a variety of later morbidities including breast cancer [2]; cardiac, and possibly metabolic functions and morbidity throughout life [3–6]. The developing reproductive system is highly sensitive, particularly during early gestation, to hormonal balance and signaling [4,7], which affects its development and function [8]. In males, reproductive development depends on androgen exposure, and in females on lack of androgen [9,10]. Anogenital distance (AGD) measured between the anus and the genital fourchette, has been shown to depend on prenatal hormonal environment, and specifically androgen exposure, which differs between males and females [11,12]: Males have a longer AGD which is associated with higher early gestation exposure to androgen levels [13]. A shorter AGD is considered feminine, due to insufficient androgen exposure during this period [14]. Fetal hormonal environment and exposures are difficult to measure, as maternal levels of exposures may not adequately represent actual fetal exposures. Thus, AGD has been
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used as a marker of early hormonal environment. An association between AGD and reproductive characteristics and morbidity was demonstrated, among both males and females, including: testosterone levels [15,16], hypospadias severity [17,18], reproductive function [16,19,20], female fertility [21], polycystic ovary syndrome [22,23] and endometriosis [20]. Prenatal exposure to endocrine disrupting chemicals, defined as exogenous substance that alters functions of the endocrine system [25], has also been associated with offspring AGD [26–30]. As AGD is an established marker of an abnormal intrauterine endocrine environment on one hand, and is, on the other hand, associated with later reproductive and general morbidity, we sought to prospectively study its association with reproductive and cardiovascular morbidities in adult women, in which AGD was measured surrounding childbirth, 17 years ago [31]. 2. Methods A prospective cohort study was performed, in which 300 parturients were recruited during the years 2000–2001 at Soroka University Medical Center (SUMC). SUMC is the only tertiary hospital in Israel`s southern region. The initial study aim, was to assess the association
Corresponding author at: School of Public Health, Faculty of Health Sciences, Ben-Gurion University of the Negev, POB 653, Beer-Sheva, 84105, Israel. E-mail address: [email protected] (T. Wainstock).
https://doi.org/10.1016/j.reprotox.2019.08.007 Received 17 April 2019; Received in revised form 16 July 2019; Accepted 12 August 2019 Available online 14 August 2019 0890-6238/ © 2019 Elsevier Inc. All rights reserved.
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variable, based on tertiles (defining three study groups). Demographic and background characteristics were available from the initial study or from the current hospital records, and included age, height, ethnicity and years of education. Univariable analysis compared hospital encounter rates between the study groups in all categories, as well as background and demographic characteristics, using chi square test, and presented as Odds Ratios (OR) and their Confidence Interval (CI) ; descriptive for continuous variables are described as mean ± standard deviation (SD) and compared using student t tests. All analyses were two sided, with p < 0.05 considered statistically significant. Kaplan Meier analysis was performed to compare cumulative risk for hospital encounters in each category, between the two study groups. Multivariable Cox proportional survival models were used to study the association between AGD (below vs. above mean) and time to hospital encounters in each category, while adjusting for confounding variables. Variables statistically different between the study groups (based on the univariable analysis), were tested for their association with the outcomes (based on their inclusion in a multivariable model). If they had changed the adjusted Hazard Ratio for the AGD by more than 10%- they would be defined as confounding variable, and included in the final model. None of the variables tested were confounding the studied association. Additionally, variables with clinical importance, were considered in the multivariable models. The study population consists of women from two ethnicities residing in Israel`s Southern region: Jewish and Bedouin. These populations differ not only in religion but in traditional values, education, income and occupation. Ethnicity therefore represents socio-economic differences between the study population, and this variable is clinically significant. Follow-up time was calculated from maternal date of birth to either end of study period (December 2017) or until the first encounter occurred independently in each of the studied categories.
between perineal length (AGD) prior to, and during delivery and perineal damage during the delivery (lacerations and episiotomies). AGD was measured three times surrounding delivery (latent phase, crowning, and 24 h post-partum). The anatomical landmarks used in the protocol are comparable to those developed by Salazar- Martinez et al. [32] for female AGD measurements. All measurements were performed by either one of two physicians. The agreement between the physicians was not evaluated. All pregnancies were singleton, at term and with cephalic presentation. Additional details and results of the initial study can be found in Walfisch et al. [31]. In December 2017 hospital records of all study participants were reviewed, including all hospital encounters at SUMC (emergency room visits, hospitalizations, and outpatient clinics visits), as well as demographic and background health characteristics. The SUMC database includes ICD-9 codes for all medical diagnoses. All diagnoses (n = 1441) were grouped by ICD-9 code categories, according to systems and organs, by an Obstetrician/ Gynecologist specialist (IY), into 19 different categories of medical conditions: cardiovascular (CVD), gynecological (including general, benign, and malignant), all other malignancies and benign tumors, infertility, infectious diseases, endo metabolic, lipid disorders, hematological, psychiatric, neurological, gall bladder related, suicide, and “other”. A List of the grouped diagnoses and ICD-9 codes is presented in Supplementary Table 1. In Israel all medical care expenses are fully covered by a national health law (1997), including pregnancy follow-up, birth, and all other hospitalization. This enables access to medical care to citizens of all backgrounds. The study has been carried out in accordance with the code of ethics of the World Medical Association, and its protocol approved by the local SUMC institutional review board committee. All study participants gave informed consent.
4. Results 3. Statistical analysis The study included 300 women. The mean age in 2017 was 44.3 ± 5.37 (range: 33–59) years; 151 (50.3%) were Jewish and 149 (49.7%) were Bedouins. AGD was normally distributed, with a mean of 40.3 mm ( ± 10.7 mm). The study groups (below and above mean AGD) were similar in background characteristics, including: most of women were married (96.8% and 95.2% among below and above mean AGD, missing n = 7), graduated from high-school or colleges (68.4% and 67.0% among below and above mean AGD, missing n = 7), and did not have any background health problems (93.8% and 91.6% among below and above mean AGD). Background health problems included Diabetes mellitus, Asthma, thyroid problems or thrombophilia. Additional background and demographic characteristics of the study population
The statistical analysis was done using SPSS software version 23.0 (IBM, Chicago, Illinois). The studied outcomes included hospital encounters (yes/no) and time to encounter, due to gynecological or any of the other morbidity categories, including CVD related. If there were encounters due to more than one category per participant, all were included and compared in both the univariable and the multivariable levels. The first AGD measured in the initial study (at admission for delivery) was defined as the independent variable and was studied as a categorical variable: above versus below mean AGD (defining the two study groups). AGD was additionally analyzed as a three categories Table 1 Selected morbidities categories by study group. Morbidities Categories
Below mean AGD n (%) 193 (64.3)
Above mean AGD n (%) 107 (35.7)
OR; 95%CI
p- value for Above vs. Below AGD mean
Cardio Vascular Total Gynecological Morbiditiesa Female malignancies Female benign Gynecological conditions Endo - metabolic Obesity Lipid disorders Hematological Psychiatric Neurological Gallbladder Malignancy (non-female related) Benign tumors (non-female related) Infections
32 (16.6) 77 (39.9) 3 (1.6) 22 (11.4) 70 (36.6) 50 (25.9) 8 (4.1) 3 (1.6) 141 (73.1) 2 (1.0) 29 (15.0) 16 (8.3) 7 (3.6) 6 (3.1) 74 (38.4)
18 (16.8) 29 (27.1) 1 (0.9) 7 (6.5) 25 (23.4) 31 (29.0) 5 (4.7) 3 (2.8) 75 (70.1) 2 (1.9) 13 (12.1) 5 (4.7) 2 (1.9) 8 (7.5) 33 (30.8)
1.02; 0.54-1.92 0.56; 0.33-0.94 0.6; 0.06-5.82 0.54; 0.22-1.32 0.54; 0.31-0.91 1.17; 0.69-1.98 1.13; 0.36-3.56 1.83; 0.36-9.21 0.86; 0.51-1.46 1.82; 0.25-13.10 0.78; 0.39-1.58 0.54; 0.19-1.52 0.51; 0.10-2.48 2.52; 0.85-7.46 0.72; 0.43-1.18
1.0 0.028 1.0 0.180 0.027 0.589 1.0 0.670 0.594 0.618 0.603 0.345 0.498 0.095 0.210
a
Including: female malignancies, benign and gynecological morbidities. 78
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are presented elsewhere [21,31]. Table 1 presents the incidence rates of selected hospital encounters with categories of morbidities, by the two study groups. Rate of encounters due to gynecological conditions was significantly higher among women with below mean AGD as compared to the above mean AGD group (36.6% vs. 23.4%, p = 0.03). Additionally, mean total number of gynecological related encounters was significantly higher among the below mean AGD group as compared to the above mean AGD group (mean number of encounters: 0.52 ± 1.23 vs. 1.17 ± 2.95; p = 0.009, in the above vs. below mean AGD groups, median = 0, p25-p75 = 0–1, in both groups). Rates of all other health categories encounters were not significantly different between the two study groups, including incidence rates of cardiovascular related encounters (16.6% vs. 16.8% among the below vs. above AGD groups, OR = 1.02; 0.54–1.92, p = 1.0), or mean number of cardiovascular related encounters (mean number of encounters: 0.24 ± 0.7 vs. 0.64 ± 3.9 ; p = 0.30, in above vs. below mean AGD, median = 0 and p25-p75 = 0-0 for both groups). When comparing the three categories of AGD (by tertiles), results remained similar: shorter AGD is associated with increased risk for gynecological morbidities but not with CVD morbidities (Figs. 1 and 2). Due to the small numbers in each of the three categories, the analysis focused on comparison across the two AGD levels. The Kaplan Meier survival analysis revealed a significant difference in the cumulative risk for gynecological morbidity between the two study groups (Fig. 3, log-rank, p = 0.01). No differences between the two study groups were found in any of the cumulative risks for other hospital encounters, including cardiovascular related encounters (Fig. 4, log-rank, p = 0.58). The crude HR for total Gynecological Morbidities and for Cardio Vascular Morbidities were: 1.67; 95%CI 1.02–2.72; and 1.03; 95%CI 0.55–1.84, respectively. Multivariable Cox proportional survival models (Table 2) adjusted for maternal ethnicity and age, and revealed that mothers with below mean AGD were more likely to experience gynecological morbidity related hospital encounter as compared to mothers with above mean AGD (adjusted HR = 1.71; 1.09–2.65). The association between AGD and cardiovascular related hospital encounters remained insignificant in the multivariable level.
Fig. 2. Incidence of Cardiovascular Morbidities by tertiles of AGD.
Fig. 3. Kaplan Meier Survival Analysis of Cumulative Risk for Gynecological Morbidities Among The Two Study Groups.
5. Discussion In this prospective cohort of 300 fertile women, short versus longer AGD was associated with an increased risk for gynecological morbidity in adult women (age range 33–59), but not with cardiovascular related or other morbidities. These results were persistent when studying AGD in two or three categories (based on above and below mean, or by tertiles). Gynecological morbidity included breast or ovarian cancers,
Fig. 4. Kaplan Meier Survival Analysis of Cumulative Risk for Cardiovascular Morbidities Among The Two Study Groups.
endometriosis, ovarian torsion, spontaneous abortions, vaginitis, and more. Early studies have suggested an association between prenatal exposures to endocrine disrupting chemicals and offspring reproductive
Fig. 1. Incidence of Gynecological Morbidities by tertiles of AGD. 79
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Table 2 Multivariable Cox Proportional Survival Models for the Association Between AGD, Cardio vascular and Total Gynecological Morbidities. Gynecological Morbidities
Cardio Vascular Morbidities
Variables
Adjusted HR (95%CI)
p-value
Adjusted HR (95%CI)
p-value
AGD, below vs. above mean Age Ethnicity, Bedouin vs. Jewish
1.71 (1.09, 2.65) 0.98 (0.94, 1.02) 0.99 (0.67, 1.46)
0.021 0.353 0.958
1.16 (0.64, 2.13) 0.97 (0.91,1.04) 0.96 (0.54, 1.69)
0.628 0.375 0.880
While an association between AGD and gynecological morbidity was expected, the direction of this association was inconsistent in previous publications. In our study, shorter AGD was associated with an increased risk for gynecological conditions, as also shown by Mendiola et al. [20] regarding endometriosis. However, longer AGD has been associated with polycystic ovary syndrome [22,23,40]. Due to the nature of this study and lack of power, these conditions were not studied independently. Since our study was based on fertile women, there was an under representation of severe morbidities and infertility. It is therefore possible that our results present an under estimation of the true association between AGD and gynecological morbidities. As in previous studies, our findings support the early life origin of disease in adulthood, and suggest AGD is determined during the critical period of these diseases origin. Further studies are recommended to clarify the etiology and direction of the association, with follow-up into the post-menopausal period, which will enable studying additional endocrine sensitive outcomes such as cardiovascular morbidity [45]. AGD measurements could be added to routine gynecological examination, or upon admission to the delivery room (as in our study), and provide critical data for defining women at risk for later gynecological morbidities, with the potential benefit of early diagnosis and treatment, and improved outcome.
cancers, including testicular cancer and vaginal adenocarcinoma [33,34]. Since AGD has been identified by the US Environmental Protection Agency guidelines, as one of the endpoints for human reproductive toxicity studies, this measurement has been used as a marker of early life hormonal environment, including endocrine disrupting agents exposure. Indeed, studies have shown that early life endocrine disrupting chemicals exposures, such as Bisphenol A or nonsteroidal anti-inflammatory drugs, were associated with shorter AGD among newborns [19,28,35–37]. Hypospadias or undescended testes among infants have also been associated with shorter AGD [17,18,38], as well as prostate cancer diagnoses and poorer semen quality among adult males [39,40] and endometriosis among females [20]. Longer AGD, on the other hand, has been associated with polycystic ovaries [22]. The exact mechanism of by which prenatal hormonal environment is associated with gynecological morbidities in adulthood is unclear, and may involve epigenetic changes [41], or androgen and estrogen gene receptor sensitivity changes [12,42]. This study has several strengths. Most studies on AGD and female reproductive morbidities are based on a case-control design, with AGD comparison between cases and controls [18,20,38]. As far as the study team is aware, this is the first long term prospective study involving adult females, in which AGD was measured independent of background characteristics, years prior to the studied outcomes. In case exposure misclassification occurred, it would have been non-differential. This prospective approach, in which AGD was measured years prior to the gynecological morbidities diagnoses or menopause occurred, overcomes the possible effect of the diagnosis or treatment may have had on the AGD measured in case-control or cross-sectional studies [43]. Findings of the current prospective study present a higher level of epidemiological evidence of the studied association, and overcome this possible differential bias. One of the study`s limitation is the assumption that all women in the original study tended to go back to SUMC for any condition requiring medical attention, as this is the only tertiary medical center in the area. However, loss of follow-up may have occurred. Since loss of follow-up is unlikely to be associated with the exposure, it may have led to a possible non-differential participation bias, and an under-estimation of the true association is possible. Another study limitation is that the agreement between the physicians measuring the AGD was not evaluated. However, while there may have been differences in measurements between the physicians, it was non differential, and if it affected the studied association, it was biased toward the null. AGD can be measured in two approaches: 1) from the anterior clitoral surface to the center of the anus and 2) from the posterior fourchette to the center of the anus, which has been used in our study. The use of only the anus- fourchette measurement, which was available from a previous study, is another study limitation, which may affect the ability to compare findings from the current study to others. The expected association between AGD and CVD related morbidities was not observed in our study, and may be due to the protective effect of estrogen during the pre-menopausal period. Mean age of study participants was 44.3 ± 5.4, i.e. pre- menopausal, when the risk for cardiovascular disease is low due to the protective role of estrogen [44,45]. Longer follow-up is probably required to study the possible association between AGD and cardiovascular morbidity.
Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors. The authors declare they have no actual or potential competing financial interests. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.reprotox.2019.08.007. References [1] R.T. Zoeller, T.R. Brown, L.L. Doan, A.C. Gore, N.E. Skakkebaek, A.M. Soto, et al., Endocrine-disrupting chemicals and public health protection: a statement of principles from the Endocrine society, Endocrinology 153 (9) (2012) 4097–4110, https://doi.org/10.1210/en.2012-1422 PMID: 22733974. [2] J.R. Palmer, E.E. Hatch, C.L. Rosenberg, P. Hartge, R.H. Kaufman, L. Titus-Ernstoff, Risk of breast cancer in women exposed to diethylstilbestrol in utero: preliminary results (United States), Cancer Causes Control 13 (8) (2002) 753–758 PMID:12420954. [3] J.R. Bell, G.B. Bernasochi, U. Varma, A.J. Raaijmakers, L.M. Delbridge, Sex and sex hormones in cardiac stress–mechanistic insights, J. Steroid Biochem. Mol. Biol. (2013) 124–135, https://doi.org/10.1016/j.jsbmb.2013.05.015. [4] A.K. Vyas, V. Hoang, V. Padmanabhan, E. Gilbreath, K.A. Mietelka, Prenatal programming: adverse cardiac programming by gestational testosterone excess, Sci Rep. Jun 22 (6) (2016) 28335, https://doi.org/10.1038/srep28335 PubMed PMID: 27328820.
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Reproductive Toxicology journal homepage: www.elsevier.com/locate/reprotox
The association between anogenital distance, reproductive and general health in adult females- a prospective cohort of 17 years
T
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Tamar Wainstocka, , Israel Yolesb,c, Ruslan Sergienkoa, Asnat Walfischb a
Department of Public Health, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel Department of Obstetrics and Gynecology, Soroka University Medical Center, Ben-Gurion University of the Negev, Beer-Sheva, Israel c Clalit Health Services, Central District, Israel b
A R T I C LE I N FO
A B S T R A C T
Keywords: Anogenital distance Early life exposures Endocrine disrupting agents Gynecological morbidities Prospective cohort
Background: Studies have suggested an association between prenatal hormonal environment and anogenital distance (AGD). We aimed to study the association between AGD and health characteristics in adult women. Methods: In this prospective cohort AGD was measured in 300 parturients during the years 2000-2001. In December 2017, study participants` hospital records were abstracted and medical diagnoses grouped into major categories, including gynecological, cardiovascular, and other. Associations between AGD and the morbidities categories were evaluated using multivariable survival models, which adjusted for confounding variables. Results: Participants were 44.3 ± 5.4 years old with mean AGD 40.3 mm ( ± 10.7 mm). Women with below vs. above mean AGD were more likely to develop gynecological morbidities (39.9% vs. 27.1%, adjusted Hazard ratio 1.82; 95%CI 1.08–3.06). Conclusions: Women with short AGD are at increased risk for gynecological morbidities. AGD should be studied further, and may possibly be used for screening of women at risk for these conditions.
1. Introduction During early human life, the endocrine system is involved in the formation and ensuring proper function of various systems, including metabolism, cardiovascular, neurological and mainly the reproductive systems [1]. Early life exposures to estrogen and testosterone have been associated with a variety of later morbidities including breast cancer [2]; cardiac, and possibly metabolic functions and morbidity throughout life [3–6]. The developing reproductive system is highly sensitive, particularly during early gestation, to hormonal balance and signaling [4,7], which affects its development and function [8]. In males, reproductive development depends on androgen exposure, and in females on lack of androgen [9,10]. Anogenital distance (AGD) measured between the anus and the genital fourchette, has been shown to depend on prenatal hormonal environment, and specifically androgen exposure, which differs between males and females [11,12]: Males have a longer AGD which is associated with higher early gestation exposure to androgen levels [13]. A shorter AGD is considered feminine, due to insufficient androgen exposure during this period [14]. Fetal hormonal environment and exposures are difficult to measure, as maternal levels of exposures may not adequately represent actual fetal exposures. Thus, AGD has been
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used as a marker of early hormonal environment. An association between AGD and reproductive characteristics and morbidity was demonstrated, among both males and females, including: testosterone levels [15,16], hypospadias severity [17,18], reproductive function [16,19,20], female fertility [21], polycystic ovary syndrome [22,23] and endometriosis [20]. Prenatal exposure to endocrine disrupting chemicals, defined as exogenous substance that alters functions of the endocrine system [25], has also been associated with offspring AGD [26–30]. As AGD is an established marker of an abnormal intrauterine endocrine environment on one hand, and is, on the other hand, associated with later reproductive and general morbidity, we sought to prospectively study its association with reproductive and cardiovascular morbidities in adult women, in which AGD was measured surrounding childbirth, 17 years ago [31]. 2. Methods A prospective cohort study was performed, in which 300 parturients were recruited during the years 2000–2001 at Soroka University Medical Center (SUMC). SUMC is the only tertiary hospital in Israel`s southern region. The initial study aim, was to assess the association
Corresponding author at: School of Public Health, Faculty of Health Sciences, Ben-Gurion University of the Negev, POB 653, Beer-Sheva, 84105, Israel. E-mail address: [email protected] (T. Wainstock).
https://doi.org/10.1016/j.reprotox.2019.08.007 Received 17 April 2019; Received in revised form 16 July 2019; Accepted 12 August 2019 Available online 14 August 2019 0890-6238/ © 2019 Elsevier Inc. All rights reserved.
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variable, based on tertiles (defining three study groups). Demographic and background characteristics were available from the initial study or from the current hospital records, and included age, height, ethnicity and years of education. Univariable analysis compared hospital encounter rates between the study groups in all categories, as well as background and demographic characteristics, using chi square test, and presented as Odds Ratios (OR) and their Confidence Interval (CI) ; descriptive for continuous variables are described as mean ± standard deviation (SD) and compared using student t tests. All analyses were two sided, with p < 0.05 considered statistically significant. Kaplan Meier analysis was performed to compare cumulative risk for hospital encounters in each category, between the two study groups. Multivariable Cox proportional survival models were used to study the association between AGD (below vs. above mean) and time to hospital encounters in each category, while adjusting for confounding variables. Variables statistically different between the study groups (based on the univariable analysis), were tested for their association with the outcomes (based on their inclusion in a multivariable model). If they had changed the adjusted Hazard Ratio for the AGD by more than 10%- they would be defined as confounding variable, and included in the final model. None of the variables tested were confounding the studied association. Additionally, variables with clinical importance, were considered in the multivariable models. The study population consists of women from two ethnicities residing in Israel`s Southern region: Jewish and Bedouin. These populations differ not only in religion but in traditional values, education, income and occupation. Ethnicity therefore represents socio-economic differences between the study population, and this variable is clinically significant. Follow-up time was calculated from maternal date of birth to either end of study period (December 2017) or until the first encounter occurred independently in each of the studied categories.
between perineal length (AGD) prior to, and during delivery and perineal damage during the delivery (lacerations and episiotomies). AGD was measured three times surrounding delivery (latent phase, crowning, and 24 h post-partum). The anatomical landmarks used in the protocol are comparable to those developed by Salazar- Martinez et al. [32] for female AGD measurements. All measurements were performed by either one of two physicians. The agreement between the physicians was not evaluated. All pregnancies were singleton, at term and with cephalic presentation. Additional details and results of the initial study can be found in Walfisch et al. [31]. In December 2017 hospital records of all study participants were reviewed, including all hospital encounters at SUMC (emergency room visits, hospitalizations, and outpatient clinics visits), as well as demographic and background health characteristics. The SUMC database includes ICD-9 codes for all medical diagnoses. All diagnoses (n = 1441) were grouped by ICD-9 code categories, according to systems and organs, by an Obstetrician/ Gynecologist specialist (IY), into 19 different categories of medical conditions: cardiovascular (CVD), gynecological (including general, benign, and malignant), all other malignancies and benign tumors, infertility, infectious diseases, endo metabolic, lipid disorders, hematological, psychiatric, neurological, gall bladder related, suicide, and “other”. A List of the grouped diagnoses and ICD-9 codes is presented in Supplementary Table 1. In Israel all medical care expenses are fully covered by a national health law (1997), including pregnancy follow-up, birth, and all other hospitalization. This enables access to medical care to citizens of all backgrounds. The study has been carried out in accordance with the code of ethics of the World Medical Association, and its protocol approved by the local SUMC institutional review board committee. All study participants gave informed consent.
4. Results 3. Statistical analysis The study included 300 women. The mean age in 2017 was 44.3 ± 5.37 (range: 33–59) years; 151 (50.3%) were Jewish and 149 (49.7%) were Bedouins. AGD was normally distributed, with a mean of 40.3 mm ( ± 10.7 mm). The study groups (below and above mean AGD) were similar in background characteristics, including: most of women were married (96.8% and 95.2% among below and above mean AGD, missing n = 7), graduated from high-school or colleges (68.4% and 67.0% among below and above mean AGD, missing n = 7), and did not have any background health problems (93.8% and 91.6% among below and above mean AGD). Background health problems included Diabetes mellitus, Asthma, thyroid problems or thrombophilia. Additional background and demographic characteristics of the study population
The statistical analysis was done using SPSS software version 23.0 (IBM, Chicago, Illinois). The studied outcomes included hospital encounters (yes/no) and time to encounter, due to gynecological or any of the other morbidity categories, including CVD related. If there were encounters due to more than one category per participant, all were included and compared in both the univariable and the multivariable levels. The first AGD measured in the initial study (at admission for delivery) was defined as the independent variable and was studied as a categorical variable: above versus below mean AGD (defining the two study groups). AGD was additionally analyzed as a three categories Table 1 Selected morbidities categories by study group. Morbidities Categories
Below mean AGD n (%) 193 (64.3)
Above mean AGD n (%) 107 (35.7)
OR; 95%CI
p- value for Above vs. Below AGD mean
Cardio Vascular Total Gynecological Morbiditiesa Female malignancies Female benign Gynecological conditions Endo - metabolic Obesity Lipid disorders Hematological Psychiatric Neurological Gallbladder Malignancy (non-female related) Benign tumors (non-female related) Infections
32 (16.6) 77 (39.9) 3 (1.6) 22 (11.4) 70 (36.6) 50 (25.9) 8 (4.1) 3 (1.6) 141 (73.1) 2 (1.0) 29 (15.0) 16 (8.3) 7 (3.6) 6 (3.1) 74 (38.4)
18 (16.8) 29 (27.1) 1 (0.9) 7 (6.5) 25 (23.4) 31 (29.0) 5 (4.7) 3 (2.8) 75 (70.1) 2 (1.9) 13 (12.1) 5 (4.7) 2 (1.9) 8 (7.5) 33 (30.8)
1.02; 0.54-1.92 0.56; 0.33-0.94 0.6; 0.06-5.82 0.54; 0.22-1.32 0.54; 0.31-0.91 1.17; 0.69-1.98 1.13; 0.36-3.56 1.83; 0.36-9.21 0.86; 0.51-1.46 1.82; 0.25-13.10 0.78; 0.39-1.58 0.54; 0.19-1.52 0.51; 0.10-2.48 2.52; 0.85-7.46 0.72; 0.43-1.18
1.0 0.028 1.0 0.180 0.027 0.589 1.0 0.670 0.594 0.618 0.603 0.345 0.498 0.095 0.210
a
Including: female malignancies, benign and gynecological morbidities. 78
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are presented elsewhere [21,31]. Table 1 presents the incidence rates of selected hospital encounters with categories of morbidities, by the two study groups. Rate of encounters due to gynecological conditions was significantly higher among women with below mean AGD as compared to the above mean AGD group (36.6% vs. 23.4%, p = 0.03). Additionally, mean total number of gynecological related encounters was significantly higher among the below mean AGD group as compared to the above mean AGD group (mean number of encounters: 0.52 ± 1.23 vs. 1.17 ± 2.95; p = 0.009, in the above vs. below mean AGD groups, median = 0, p25-p75 = 0–1, in both groups). Rates of all other health categories encounters were not significantly different between the two study groups, including incidence rates of cardiovascular related encounters (16.6% vs. 16.8% among the below vs. above AGD groups, OR = 1.02; 0.54–1.92, p = 1.0), or mean number of cardiovascular related encounters (mean number of encounters: 0.24 ± 0.7 vs. 0.64 ± 3.9 ; p = 0.30, in above vs. below mean AGD, median = 0 and p25-p75 = 0-0 for both groups). When comparing the three categories of AGD (by tertiles), results remained similar: shorter AGD is associated with increased risk for gynecological morbidities but not with CVD morbidities (Figs. 1 and 2). Due to the small numbers in each of the three categories, the analysis focused on comparison across the two AGD levels. The Kaplan Meier survival analysis revealed a significant difference in the cumulative risk for gynecological morbidity between the two study groups (Fig. 3, log-rank, p = 0.01). No differences between the two study groups were found in any of the cumulative risks for other hospital encounters, including cardiovascular related encounters (Fig. 4, log-rank, p = 0.58). The crude HR for total Gynecological Morbidities and for Cardio Vascular Morbidities were: 1.67; 95%CI 1.02–2.72; and 1.03; 95%CI 0.55–1.84, respectively. Multivariable Cox proportional survival models (Table 2) adjusted for maternal ethnicity and age, and revealed that mothers with below mean AGD were more likely to experience gynecological morbidity related hospital encounter as compared to mothers with above mean AGD (adjusted HR = 1.71; 1.09–2.65). The association between AGD and cardiovascular related hospital encounters remained insignificant in the multivariable level.
Fig. 2. Incidence of Cardiovascular Morbidities by tertiles of AGD.
Fig. 3. Kaplan Meier Survival Analysis of Cumulative Risk for Gynecological Morbidities Among The Two Study Groups.
5. Discussion In this prospective cohort of 300 fertile women, short versus longer AGD was associated with an increased risk for gynecological morbidity in adult women (age range 33–59), but not with cardiovascular related or other morbidities. These results were persistent when studying AGD in two or three categories (based on above and below mean, or by tertiles). Gynecological morbidity included breast or ovarian cancers,
Fig. 4. Kaplan Meier Survival Analysis of Cumulative Risk for Cardiovascular Morbidities Among The Two Study Groups.
endometriosis, ovarian torsion, spontaneous abortions, vaginitis, and more. Early studies have suggested an association between prenatal exposures to endocrine disrupting chemicals and offspring reproductive
Fig. 1. Incidence of Gynecological Morbidities by tertiles of AGD. 79
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Table 2 Multivariable Cox Proportional Survival Models for the Association Between AGD, Cardio vascular and Total Gynecological Morbidities. Gynecological Morbidities
Cardio Vascular Morbidities
Variables
Adjusted HR (95%CI)
p-value
Adjusted HR (95%CI)
p-value
AGD, below vs. above mean Age Ethnicity, Bedouin vs. Jewish
1.71 (1.09, 2.65) 0.98 (0.94, 1.02) 0.99 (0.67, 1.46)
0.021 0.353 0.958
1.16 (0.64, 2.13) 0.97 (0.91,1.04) 0.96 (0.54, 1.69)
0.628 0.375 0.880
While an association between AGD and gynecological morbidity was expected, the direction of this association was inconsistent in previous publications. In our study, shorter AGD was associated with an increased risk for gynecological conditions, as also shown by Mendiola et al. [20] regarding endometriosis. However, longer AGD has been associated with polycystic ovary syndrome [22,23,40]. Due to the nature of this study and lack of power, these conditions were not studied independently. Since our study was based on fertile women, there was an under representation of severe morbidities and infertility. It is therefore possible that our results present an under estimation of the true association between AGD and gynecological morbidities. As in previous studies, our findings support the early life origin of disease in adulthood, and suggest AGD is determined during the critical period of these diseases origin. Further studies are recommended to clarify the etiology and direction of the association, with follow-up into the post-menopausal period, which will enable studying additional endocrine sensitive outcomes such as cardiovascular morbidity [45]. AGD measurements could be added to routine gynecological examination, or upon admission to the delivery room (as in our study), and provide critical data for defining women at risk for later gynecological morbidities, with the potential benefit of early diagnosis and treatment, and improved outcome.
cancers, including testicular cancer and vaginal adenocarcinoma [33,34]. Since AGD has been identified by the US Environmental Protection Agency guidelines, as one of the endpoints for human reproductive toxicity studies, this measurement has been used as a marker of early life hormonal environment, including endocrine disrupting agents exposure. Indeed, studies have shown that early life endocrine disrupting chemicals exposures, such as Bisphenol A or nonsteroidal anti-inflammatory drugs, were associated with shorter AGD among newborns [19,28,35–37]. Hypospadias or undescended testes among infants have also been associated with shorter AGD [17,18,38], as well as prostate cancer diagnoses and poorer semen quality among adult males [39,40] and endometriosis among females [20]. Longer AGD, on the other hand, has been associated with polycystic ovaries [22]. The exact mechanism of by which prenatal hormonal environment is associated with gynecological morbidities in adulthood is unclear, and may involve epigenetic changes [41], or androgen and estrogen gene receptor sensitivity changes [12,42]. This study has several strengths. Most studies on AGD and female reproductive morbidities are based on a case-control design, with AGD comparison between cases and controls [18,20,38]. As far as the study team is aware, this is the first long term prospective study involving adult females, in which AGD was measured independent of background characteristics, years prior to the studied outcomes. In case exposure misclassification occurred, it would have been non-differential. This prospective approach, in which AGD was measured years prior to the gynecological morbidities diagnoses or menopause occurred, overcomes the possible effect of the diagnosis or treatment may have had on the AGD measured in case-control or cross-sectional studies [43]. Findings of the current prospective study present a higher level of epidemiological evidence of the studied association, and overcome this possible differential bias. One of the study`s limitation is the assumption that all women in the original study tended to go back to SUMC for any condition requiring medical attention, as this is the only tertiary medical center in the area. However, loss of follow-up may have occurred. Since loss of follow-up is unlikely to be associated with the exposure, it may have led to a possible non-differential participation bias, and an under-estimation of the true association is possible. Another study limitation is that the agreement between the physicians measuring the AGD was not evaluated. However, while there may have been differences in measurements between the physicians, it was non differential, and if it affected the studied association, it was biased toward the null. AGD can be measured in two approaches: 1) from the anterior clitoral surface to the center of the anus and 2) from the posterior fourchette to the center of the anus, which has been used in our study. The use of only the anus- fourchette measurement, which was available from a previous study, is another study limitation, which may affect the ability to compare findings from the current study to others. The expected association between AGD and CVD related morbidities was not observed in our study, and may be due to the protective effect of estrogen during the pre-menopausal period. Mean age of study participants was 44.3 ± 5.4, i.e. pre- menopausal, when the risk for cardiovascular disease is low due to the protective role of estrogen [44,45]. Longer follow-up is probably required to study the possible association between AGD and cardiovascular morbidity.
Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors. The authors declare they have no actual or potential competing financial interests. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.reprotox.2019.08.007. References [1] R.T. Zoeller, T.R. Brown, L.L. Doan, A.C. Gore, N.E. Skakkebaek, A.M. Soto, et al., Endocrine-disrupting chemicals and public health protection: a statement of principles from the Endocrine society, Endocrinology 153 (9) (2012) 4097–4110, https://doi.org/10.1210/en.2012-1422 PMID: 22733974. [2] J.R. Palmer, E.E. Hatch, C.L. Rosenberg, P. Hartge, R.H. Kaufman, L. Titus-Ernstoff, Risk of breast cancer in women exposed to diethylstilbestrol in utero: preliminary results (United States), Cancer Causes Control 13 (8) (2002) 753–758 PMID:12420954. [3] J.R. Bell, G.B. Bernasochi, U. Varma, A.J. Raaijmakers, L.M. Delbridge, Sex and sex hormones in cardiac stress–mechanistic insights, J. Steroid Biochem. Mol. Biol. (2013) 124–135, https://doi.org/10.1016/j.jsbmb.2013.05.015. [4] A.K. Vyas, V. Hoang, V. Padmanabhan, E. Gilbreath, K.A. Mietelka, Prenatal programming: adverse cardiac programming by gestational testosterone excess, Sci Rep. Jun 22 (6) (2016) 28335, https://doi.org/10.1038/srep28335 PubMed PMID: 27328820.
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