Does exogenous female sex hormone administration affect the rate of tooth movement and root resorption? A systematic review of animal studies

Background The long-term use of contraceptive methods that contain estrogens, progestogens or combinations of the above among women aged 15 to 49 years is extensive. Both estrogens and progestogens affect bone metabolism. Objective To systematically investigate and appraise the quality of the available evidence from animal studies regarding the impact of exogenous administration of female sex hormones on the rate of orthodontic tooth movement and root resorption. Search methods Search without restriction in seven databases (including grey literature) and hand searching were performed until May 2021. Selection criteria We looked for controlled animal studies investigating the effect from exogenous administration of formulations containing female sex hormones on the rate of orthodontic tooth movement and root resorption. Data collection and analysis After study retrieval and selection, relevant data was extracted, and the risk of bias was assessed using the SYRCLE’s Risk of Bias Tool. The quality of available evidence was assessed with the Grades of Recommendation, Assessment, Development, and Evaluation. Results Three studies were identified, all being at unclear risk of bias. Overall, administration of progesterone and the combinations of estradiol with norgestrel and desogestrel were shown to significantly decrease the rate of orthodontic tooth movement when given for longer periods (>3 weeks). Inconsistent information was detected for shorter periods of consumption. Estradiol, with desogestrel use, resulted in less root resorption. The quality of the available evidence was considered to be low. Conclusions Exogenous administration of female sex hormones may decelerate in the long term the rate of tooth movement and decrease orthodontically induced root resorption in animals. Until more information becomes available, an orthodontist should be able to identify a patient consuming such substances and understand the potential clinical implications and adverse effects that may arise. Registration PROSPERO: CRD42017078208; https://clinicaltrials.gov/.


Objective
To systematically investigate and appraise the quality of the available evidence from animal studies regarding the impact of exogenous administration of female sex hormones on the rate of orthodontic tooth movement and root resorption.

Search methods
Search without restriction in seven databases (including grey literature) and hand searching were performed until May 2021.

Selection criteria
We looked for controlled animal studies investigating the effect from exogenous administration of formulations containing female sex hormones on the rate of orthodontic tooth movement and root resorption.

Introduction
In many parts of the world, a significant percentage of women have, during their lifetime, used contraception for long periods of time [1,2]. In the United States, more than 25% of women aged 15 to 49 years old are currently using oral contraceptive pills, long-acting reversible contraceptives, or other methods of contraception that contain various estrogens, progestogens or combinations [3]. Both estrogens and progestogens have been shown to affect bone turnover [4].
Exogenous estrogens and progestogens are not only used for hormonal birth control, but also as menopausal hormone therapy. Treatment with estrogens alone in hysterectomized women or in combination with a progestogen in non-hysterectomized women is indicated for the prevention of postmenopausal osteoporosis, as well as the treatment of menopausal symptoms [5]. Estrogens' administration is indicated in cases of vasomotor symptoms [6] and related genitourinary symptoms of menopause [7]. Estrogens and progestogens can also be used in the treatment of gynecological conditions, like endometrial hyperplasia [8], uterine bleeding [9] and premenstrual syndrome [10], to address infertility [11], as well as to support pregnancy by preventing miscarriage and preterm delivery [12,13].
In the field of orthodontics, thorough documentation of medical history, along with clinical examination and diagnostic records, are always imperative [14]. Specific information on any medication administered is not only significant in recording information about a patient's health status but might also provide further insights on clinical treatment as well. Medication administration may have varying effects on the rate of orthodontic tooth movement, during active treatment or the post-treatment period, as well as the associated root resorption development [15][16][17][18][19][20]. Thus, clinicians must be capable of identifying their patients' medication consumption patterns and understand the potential implications for their treatment.
Information relevant to the impact of exogenous administration of female sex hormones on the rate of orthodontic tooth movement and root resorption should ideally be retrieved from clinical trials. However, due to ethical reasons and difficulties in recruiting an adequate sample, there is a lack of human studies on this topic. As an alternative, animal experiments are expected to include pertinent information, while circumventing the ethical and practical considerations associated with experimentations on human subjects [21]. Murine reproductive research models have been used extensively for this reason [22]. This is because the estrus cycle of rats is analogous to the human menstrual cycle, not only in terms of the maintenance mechanism, but also regarding hormonal control [23].

Objective
The aim of this systematic review was to systematically investigate and assess the quality of the existing available scientific information regarding the rate of tooth movement and orthodontically induced root resorption following the systemic administration of exogenous female sex hormones.

Protocol and registration
The present review was based on a protocol developed, registered, carried out and reported following relevant methodological guidelines (PROSPERO: CRD42017078208) (S1 Checklist) [24][25][26][27]. As the present study is a systematic review, ethical approval was not necessary.

Eligibility criteria
The Participant, Intervention, Comparator, Outcomes and Study design domains were used to describe the eligibility criteria (Table 1). We looked for prospective experimental controlled studies on healthy female animal subjects, assessing the rate of tooth movement and/or root resorption following the administration of exogenous female sex hormones. All types of orthodontic interventions to induce movement of teeth were considered, and the studies had to report on the amount of tooth movement either during the application of orthodontic forces or after their cessation. Tooth movement could be measured in various ways (with calipers, feeler gauges, etc. directly or from plaster models; from histological cuts directly on the optical microscope or from digital photos; radiographs of any kind i.e., lateral cephalometric radiographs, Cone Beam CT, micro-CT, etc.). The amount and extent of root resorption could be investigated histomorphometrically, by scanning electron or 3D surface microscopy or radiographically. Studies on male animals, as well as female animals under medication, with dietary deficiencies, co-morbidities or ovariectomy were excluded. Studies involving animals with additional clinical interventions such as tooth extraction, etc. were also eliminated, as well as studies presenting qualitative assessments. In terms of study design, we planned to select only experimental controlled animal studies and to exclude human, in vitro, ex-vivo or in silico studies; non-comparative studies, reviews, systematic reviews, meta-analyses and studies with fewer than 5 subjects per group analyzed, as per relevant methodological guidelines regarding the consideration of degrees of freedom for treatment comparisons [28,29]. (orthodont � OR "orthodontic force" OR "mechanical force") AND ("tooth movement" OR "orthodontic movement" OR "orthodontic anchorage" OR "root resorption") in Record Title OR (orthodont � OR "orthodontic force" OR "mechanical force") AND ("tooth movement" OR "orthodontic movement" OR "orthodontic anchorage" OR "root resorption") in Abstract-(Word variations have been searched)

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Scopus https://www.scopus.com TITLE-ABS ((orthodont � OR "orthodontic force" OR "mechanical force") AND ("tooth movement" OR "orthodontic movement" OR "orthodontic anchorage" OR "root resorption")) AND (LIMIT-TO (SUBJAREA,"DENT")) AND (LIMIT-TO (EXACTKEYWORD,"Tooth Movement")) made by reviewing the reference lists in reviews, included and excluded studies, as well as other related articles. Duplicates were removed using EndNote's duplicate identification strategy (EndNote X9™, Clarivate™, Philadelphia, PA, USA) and then manually by EGK. The corresponding authors of potentially included records were to be contacted, if needed, in order to provide additional data.

Selection process
The titles and abstracts of the retrieved records, followed by the full report of any record considered to meet the inclusion criteria, were assessed independently and in duplicate by EGK and MAM, who were both not blinded. If the abstract was unclear, the full paper was accessed to determine eligibility for inclusion.

Data collection and data items
Special data collection forms were used for data extraction, which was performed independently by the first two authors. The following information was recorded: bibliographic details; information on experimental design and verification of study eligibility; characteristics of the animals (number of animals in each group and sample size calculation; age and weight of animals) and tooth movement mechanics; data on medicinal administration; measurement of outcomes details (orthodontic tooth movement and/or root resorption) and reliability assessment; results. Results were to be extracted and categorized separately for each species or type of mechanics used, since differences are to be expected [30]. Results were also categorized depending on the time period of force application: less than 3 weeks, and 3 weeks or more. Orthodontic tooth movement has been shown to be divided into two phases, a non-linear phase (<3 weeks) and a linear phase thereafter, with the speed of movement peaking at 3 weeks following force application [30,31].

Study risk of bias assessment
The risk of bias in individual studies was assessed with the SYRCLE's risk of bias tool for animal studies [32] and the summary risk of bias within a study, according to Higgins and coworkers [26]. Assessments were subsequently entered into the Risk-of-bias VISualization (robvis) web application [33]. In all the aforementioned processes, disagreements were settled by discussion with AEA; following the relevant suggestions, kappa statistics were not calculated [26].

Effect measures, synthesis methods, certainty assessment and additional analyses
Although a synthesis of the results was planned, it was ultimately not carried out due to methodological diversity [26]. Due to inadequate information, analyses for "small-study effects" and publication bias, as well as subgroup analyses, were not performed [26]. Finally, despite the lack of extensive information, the quality of available evidence regarding the effect of exogenous female hormone administration on the rate of tooth movement and root resorption after 3 weeks of force application was assessed with the Grades of Recommendation, Assessment, Development, and Evaluation in order to adopt a structured and transparent approach in formulating an interpretation of the evidence [34].

Study selection
The flow of records through the reviewing process is shown in Fig 1. We initially identified 9980 references. We excluded 2856 as duplicates and another 7105 on the basis of their title and abstract. From the nineteen records that remained and were assessed for eligibility, sixteen records were excluded for the following reasons: papers investigating the effect of ovariectomy (5); papers investigating the phases of the menstrual/estrus cycle (5); studies where only the abstract was available (2); papers that constituted hypotheses only (2); a paper that was a review (1); a study that involved other substances (1) ( Table 3). Finally, 3 study records were assessed to be eligible for inclusion [35][36][37].

Study characteristics
The characteristics of the included studies are presented in Table 4. Two of the included studies experimented on rats [36,37], while the third study used rabbits [35]. The duration of force application varied from 2 to 60 days, and female sex hormones were administered for 9 weeks at maximum. In one study, the administration of hormones started one week before the initiation of tooth movement [36], and 6 weeks earlier in another [35]. Orthodontic movement was induced either by reciprocal stainless-steel springs on the incisors [35,36] or by closed coil NiTi spring between incisors and the first molar [37]. The forces exerted ranged between 30-50g.

PLOS ONE
The retrieved papers investigated the administration of progesterone [35] and the combinations of estradiol with the progestogens norgestrel [36] and desogestrel [37]. All studies reported the rate of tooth movement during the application of orthodontic forces, and only one assessed orthodontically induced root resorption [37]. The rate of tooth movement was measured clinically with calipers, and root resorption was assessed from histological cuts with light microscopy. No sample size calculations were performed, and only Yu et al. assessed the error of the method [37]. No study assessed tooth movement after the cessation of orthodontic forces.

Risk of bias within studies
All three studies were assessed to show an unclear risk of bias (Fig 2). All studies were considered to present low risk of bias regarding the items of group similarity and selective reporting,

PLOS ONE
as well as unclear risk for random sequence generation, allocation concealment, random housing of the animals and blinding of caregivers and investigators. Random selection of animals for outcome assessment was assessed at low risk only for Olyaee et al. [36]. Assessor blinding

PLOS ONE
Exogenous female sex hormone administration and orthodontic tooth movement/root resorption was appraised to be at low risk for Poosti et al. [35] and Yu et al. [37], while the handling of incomplete data was at low risk only for Olyaee et al. [36].

Effects of exogenous administration of female sex hormones
For the first two weeks after the application of orthodontic forces, progesterone administration in rabbits did not result in any statistically significant difference between the experimental and control group, even for the animals that started the treatment 6 weeks before the placement of the orthodontic appliance. In studies involving rats, the combination of estradiol with desogestrel produced a reduction in tooth movement after 2 weeks, whereas the combination with norgestrel resulted in significant differences in the measurements conducted in both the first and second weeks (Table 4).
Regarding the measurements after the third week of force application, Poosti et al. [35] demonstrated that progesterone resulted in significant decreases in the amount of tooth movement in rabbits, but only in the group that had been exposed to the exogenous hormone for 6 weeks before the initiation of movement. The combination of estradiol with desogestrel also led to a reduction in tooth movement measured in the rats 3 and 4 weeks after the commencement of the experiment [37] (Table 4).
The only study that included assessments of root resorption experimented on rats, and observed reductions in all measurements from the first until the fourth week of force application (Table 4).
Regarding the effect of exogenous female hormone administration on the rate of orthodontic tooth movement and orthodontically induced root resorption, the quality of available evidence was considered as low (Table 5).

Discussion
The long-term consumption of contraceptives that contain estrogens, progestogens or combinations of them among women aged 15 to 49 years is extensive [1][2][3]. Both estrogens and progestogens affect bone metabolism [4]. Overall, the hormonal formulations investigated resulted in reductions in the amount of tooth movement when administered for longer periods (>3 weeks). The same was noted for orthodontically induced root resorption. Although the quality of evidence assessment provides insight on the strength of the relevant recommendations, and the information provided is not fully translatable to human clinical scenarios, until more research information becomes available, it could be good practice to identify orthodontic Oral contraceptive pills and long-acting reversible contraceptives include two main categories-progestin-only pills, and combinations of estrogens and progestogens [38]. Progestinonly pills function primarily through thickening the cervical mucus, and, secondarily, through inhibiting ovulation to variable degrees, reducing cilia activity in the fallopian tubes, and altering the endometrium [39]. Contraceptives containing both estrogens and progestogens function primarily though inhibition of ovulation via feedback mechanisms in the hypothalamicpituitary-ovarian axis and thickening of the cervical mucus [38]. The papers used in the present review investigated the effect of progesterone [35] as well as combinations of estrogen with progestogens [36,37], and their results suggested reductions in the speed of orthodontically induced movement and root resorption when administered for longer periods.
Estrogen serum levels are of ovarian origin, and not only regulate female reproductive and sexual function [40], but are also indispensable in maintaining adequate bone mass and mineralization [41,42] through receptors detected in human cells [43]. Especially during pregnancy, estrogens have been found to exert a significant role regarding bone mass preservation [42]. Prevention of bone remodeling by estrogens is a result of osteoclastogenesis prevention from marrow precursors, as well as of induction of the Fas/FasL system that enhances osteoclast apoptosis [44,45]. Estrogens lead to a further inhibition in bone resorption through effects on the receptor activator of nuclear factor-Kappa B (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) system and the production of some pro-resorptive cytokines (e.g., IL-1, IL-6, IL-7, TNF) [46][47][48][49][50][51][52]. Moreover, estrogens directly affect osteoblastic cells contributing to bone preservation [53,54].
Progesterone is a steroid sex hormone produced in the ovaries and involved in the menstrual cycle, pregnancy and embryogenesis. It has been shown to possess bone protective qualities [55], directly regulated by progesterone receptors in osteoblasts [56]. These are found in both osteoblasts and osteoclasts [57][58][59] and are upregulated by estrogen levels, possibly alluding to the fact that the bone effects of estrogens are being partially regulated via progestogens [43,57,58]. In addition, progesterone may participate in the control of bone matrix, through its inhibitory action on metalloproteinases [60,61]. Serum osteocalcin is significantly correlated with progesterone levels, indicating a further interdependence with bone-forming activities [62].
The combinations of estrogens with progestogens for hormonal contraception are extensively researched medications, as they were originally designed for long-term use by young, healthy women, unlike other drugs that are intended for therapeutic effect [63]. Regarding bone mineral metabolism, the effects of these formulations seem to differ between adolescent and adult women. In younger individuals, although this claim is contested, a small overall negative effect seems probable if their use commences very early after menarche. On the contrary, combined oral contraceptives seem to exert a positive effect on bone turnover in adult females [63]. Overall, there seems to be no association between the risk of fractures in the period before menopause and oral contraceptives use [64].
Results from studies on ovariectomized animals provide data that corroborate further the information located in the included studies. After surgical removal of the ovaries, there is a lack of endogenous progesterone and estrogens, resulting in bone loss that initiates in two weeks post-surgery at the earliest, reaches 50% approximately after 4 to 8 weeks, and becomes stable within 12 weeks [65]. Ovariectomy results in osteopenia and osteoporosis and, subsequently, a higher rate of tooth movement [66], while estrogen administration reduces the speed of movement in osteoporotic rats [67,68], as it is closely related to the activity of osteoclasts [69]. The absence of estrogens has been associated with greater root resorption following orthodontic tooth movement as well [68].
Although the information located from these animal studies was not extensive, some points might be relevant to the treatment of adolescent and adult females using contraceptive methods that involve exogenous hormone administration. An eventual protective effect against root resorption could be important. Given the potentially decelerating effect on tooth movement, an orthodontist should always consider the impact on space closure, frequency of the appointments, and estimation of the duration of orthodontic treatment. Both active and anchor segments' resistance to movement might be increased, making space closure more challenging, and requiring altered biomechanical systems because of the altered bone turnover [70]. Furthermore, in some stages of treatment, there could be no issue in seeing patients at longer time intervals. Finally, it could be possible that the overall treatment duration might be increased in such circumstances. Although no direct information came out of the material studied, it could be possible that the retention period could be more uneventful in individuals receiving female sex hormones exogenously. Following the removal of orthodontic appliances, the mechanical equilibrium changes immediately, and leads to events analogous to those seen during active treatment, acting in opposite directions [71]. Possible implications could be magnified given the frequently long periods of consumption of such formulations in real life scenarios.

Strengths and limitations
The employed search strategy was comprehensive, all-inclusive, up to May 2021 and followed established guidelines to reduce bias. Most importantly, as similar investigations might encounter significant practical obstacles in human subjects, the current review summarized the available information from animal models that have been used extensively in female reproduction research [22].
There also some limitations that ought to be further elaborated. The level of available evidence was assessed to be low, because of the peculiarities of the eligible studies, regarding nature, characteristics, and the retrieved data. In addition, we were not able to proceed with meta-analysis or other analyses due to the scarcity of information.
Furthermore, it should be highlighted that the presented information is applicable to animals and can be extrapolated to the clinic environment only indirectly. The administered dosages and durations of the tested formulations differ from those used in humans [72]. Also, the route of administration differs, which might lead to different pharmacokinetics and bioavailability [73]. Since different animal species and strains were used in the reviewed experiments, it is important to expect variability in the content and the properties of the bony tissue [74]. The specific biomechanics used to produece the movement of the teeth in the animal experiments are not comparable directly to those used in humans. In addition, in the clinic a patient's medical status may affect the homeostasis of the periodontal ligament and the movement of the teeth [75,76]. Finally, the eligible studies did not include power sample calculations, possibly leading to problems regarding the precision of the presented results. Thus, the extent of the effect from exogenous female sex hormones administration on orthodontic tooth movement and root resorption is still unclear in human beings. However, executing such studies in humans could prove to be challenging, both ethically and practically.

Recommendations for future research
Since female sex hormones are used widely nowadays among female adolescents and adults, further research is warranted regarding their effect on the phenomena associated with tooth movement and orthodontically induced root resorption. Future studies should follow relevant guidelines to improve standardization and control the various sources of bias. Finally, to increase generalizability parameters like dosages, duration, and routes of administration, together with the mechanics of movement should be close, as possible, to daily clinical practice.

Conclusions
Based on the compiled information from animal studies, the exogenous female sex hormone administration might result in reductions in orthodontically induced tooth movement and root resorption when administered for longer periods. Although results that derive from animal studies cannot be directly extrapolated to human clinical scenarios, the clinician needs to be aware of the relevant implications in orthodontic treatment.