Menstrual Cycle Hormone Relaxin and ACL Injuries in Female Athletes: A Systematic Review

Abstract

Background

Female athletes are at increased risk for anterior cruciate ligament (ACL) injuries. The influence of hormonal variation on female ACL injury risk remains ill-defined. Recent data suggests that the collagen-degrading menstrual hormone relaxin may cyclically impact female ACL tissue quality. This review aims to identify any correlation between menstrual relaxin peaks and rates of female ACL injury.

Methods

A systematic review was performed, utilizing the MEDLINE, EMBASE, and CINAHL databases. Included studies had to directly address relaxin/female ACL interactions. The primary outcome variable was relaxin proteolysis of the ACL, at cellular, tissue, joint, and whole-organism levels. The secondary outcome variable was any discussed method of moderating relaxin levels, and the clinical results if available.

Results

AllThe numerous relaxin receptors on female ACLs upregulate local collagenolysis and suppress local collagen production. Peak serum relaxin concentrations (SRC) occur during menstrual cycle days 21-24; a time phase associated with greater risk of ACL injury. Oral contraceptives (OCPs) reduce SRC, with a potential ACLprotective effect.

Conclusion

A reasonable correlative and plausible causative relationship exists between peak relaxin levels and increased risk of ACL injury in females, and further investigation is warranted.

Level of Evidence: III

Introduction

Anterior cruciate ligament (ACL) injuries result from unpredictable, variable situations and individual patient risk factors.¹ Women additionally have sex-specific risk factors contributing to their ACL injury rate two- to tenfold higher than males.^2,3 Relative hamstring weakness, increased knee valgus (Q angle), and cyclic hormonal changes have been identified as fundamental female-specific risk factors predisposing women to this injury.^2,3

Neuromuscular training has been designed for female athlete ACL tear prophylaxis, to mitigate harmful hamstring and Q angle biomechanics.^2,3 Hormonal fluctuations, however, remain a vaguely-described risk factor with little investigative research.^2-4 Recent studies show concurrent fluctuations in ACL tissue quality and menstrual cycle hormone peaks; the peptide hormone relaxin is specifically implicated (Figure 1).^5-8 G protein-coupled relaxin receptors trigger collagen degradation of female ACLs—male ACLs lack receptors.^5-8 Despite these findings, no ACL-hormone protective interventions have been comprehensively investigated.^5-8 Meanwhile, one in five female collegiate athletes suffer an ACL injury.⁹

Figure 1.

Menstrual Cycle Hormone Peaks, Molecular Effects. Estrogen levels peak first, increasing expression of relaxin receptors in the body and increasing global synthesis of MMPs. The drop in estrogen triggers ovulation, and the remains of that ovarian follicle form the corpus luteum. As a temporary endocrine body, the corpus luteum secretes progesterone to prepare the endometrium for pregnancy and to sustain itself. It also synthesizes and releases relaxin, which binds receptors and activates MMPs recently upregulated by estrogen while suppressing de novo collagen synthesis. Relaxin is active during the luteal phase, chiefly CD21-24.

A handful of observational studies associate peak relaxin levels and increased ACL injury risk, an additive risk per menstrual cycle (Figure 2).^6,10,11 High relaxin positively correlates with injury rates, increasing almost logarithmically when relaxin is very high.⁶ As a corollary, artificially decreasing relaxin—by taking oral contraceptives (OCPS), for example— can decrease risk of ACL tear.^10,11 While these findings are helpful for building a causative hypothesis, advancing to interventional studies requires focused, high-quality clinical research. This review aims to summarize in a stepwise fashion the existing ACL/relaxin research to promote the development of future research questions.

Figure 2.

Estimated Lifetime Days of Relaxin-Induced Collagen Lysis, Female ACL Laxity. Relaxin, via G-protein coupled receptors, binds at the knee joint synovium to enact intra-articular changes. In stabilizing structures, the production of collagen-degrading enzymes is increased. Within the joint space, expression of inflammatory enzymes is also increased. This creates an environment not conducive to connective tissue stability, which recurs with each menstrual cycle and peak relaxin level.

Methods

Literature Search

Literature searches had broad parameters, including any English-language literature discussing both relaxin and musculoskeletal pathology. A thorough screening process led to the present systematic review focusing on ACL health. Initial search strategies were developed by the authors and an orthopedic health sciences librarian with expertise in literature reviews in June 2020, with a repeat search performed by the authors in March/ April 2022.

The initial search strategy was developed for MEDLINE. The first search string looked generally for musculoskeletal pathology, with MeSH terms such as “musculoskeletal injury”, and text words and phrases including “ligament” and “cartilage”. The second search string focused on female variations in relaxin hormone levels and activity, with MeSH terms such as “relaxin receptor”, and text words and phrases including “cyclic hormonal variation” and “eumenorrheic”. The full search strategy for MEDLINE can be found in Appendix Item 1. The strategy was adapted for EMBASE and CINAHL, available upon request.

The reference list was screened for additional source materials via SCOPUS. All identified citations were uploaded to EndNote (EndNote X9.2, Clarivate Analytics, PA, USA) and duplicates removed by a combination of software screening and manual review. The review process, following PERSiST guidance, was compliant with the framework outlined in the 2020 PRISMA statement (see Appendix Item II for the PRISMA 2020 Checklist).¹² Titles and abstracts were screened by two authors independently against the inclusion criteria. A full-text assessment was then performed to identify final inclusions, with any residual discrepancies screened by the senior author. This review was not registered.

The initial expansive screen yielded 82 studies, notable for several high-quality studies addressing relaxin and risk of ACL injury in women. This information was condensed and presented in a scoping review as supporting evidence for musculoskeletal impacts of relaxin, and thus was not separately explored or reported in the scoping review. The authors rigorously screened the 82 initial studies and additional works from the repeat search, selecting papers which specifically addressed the relaxin-ACL relationship. Lower quality literature such as case studies, and studies of animal subjects only were excluded. Screening was once more performed independently by two junior authors, supervised by the senior authors, per PRISMA standards.

Statistical Analyses

Excel v.1808 (Microsoft Inc, Redmond, WA) was utilized to perform basic demographic calculations and all Student’s t-tests; which evaluated patient demographic data such as mean percent of female patients and mean patient age. Student’s t-tests were also utilized for study outcome variable assessment if data was sufficient for an appropriately powered analysis.

Outcome Variables

The literature facilitated examination of successive relaxin effects at increasingly complex levels of female ACL health. Thus, our primary outcome was comprehensively detailing sequential relaxin activity; from a cellular level, to a tissue level, to an isolated joint level, and finally at the population level. The timing of relaxin concentration measurements did not exclusively capture peak levels (Figure 1). Cell and tissue effects had to be demonstrated rather than just hypothesized, via biopsies, immunohistochemical testing, etc. Similarly, knee joint manifestations had to be confirmed with a validated physical exam or imaging test, or intra-operatively confirmed.

The secondary outcome of interest was possible methods of suppressing relaxin levels described in the literature, looking at both effectiveness from a basic science level and any translation to clinical effects. The population-level studies of women allowed us to build upon data from our investigation of the primary outcome— the immediate consequences of cyclic relaxin peaks at smaller cohort analysis levels—to visualize the larger-scale, longer-term impacts of potential relaxin-induced female knee injuries, on a more heterogenous group of women.

As a separate, literature-focused outcome we highlighted the current research support behind each “step” correlating increased relaxin and increases in ACL injuries. Currently, ACL/relaxin research is present in published literature, but not cohesive nor pursued beyond the level of clinical correlation, even for aspects as simple as a cohort analysis of peak relaxin measurement versus incidence of injury. With this review we aimed to concisely synthesize prior correlational research, emphasize prior research proposing a causative relationship, and turn the focus towards potential researchable tissue quality interventions to prevent injury.

Study Quality

Study quality was evaluated by two authors independently, prior to reaching consensus scores on the Modified Coleman Criteria Scale (MCMS). Oversight was provided by the senior authors. The average MCMS was 39±9.7 (range: 25 to 55) with a bimodal distribution; the lab studies and cross-sectional studies had scores between 25-30, while the prospective and retrospective cohort studies had scores between 45-55. There were no statistical MCMS score outliers within either of the two literature groups delineated by study type.

Results

From the original and repeat search results for studies discussing relaxin and musculoskeletal health on a general level, we identified 12 qualifying studies (Table 1) specifically addressing the interaction of relaxin and the ACL.^{5-8,10,11,13-19} While some modest overlap existed between studies, we were able to assess the literature according to the subcategories of basic science and clinical outcomes delineated within our outcome variables: the molecular level;^7,8,16 the level of the tissue and the joint (small study sizes);^{6,13-15,18,19} and the level of the joint from a population perspective, along with any population differences attributed to relaxin concentration, variability, and/or moderation (Table 2).^{10,11,14,17,18}

Table 1.

Details of All Included Studies

Author, Year	Study Type	N (%F)	Age (Years)	Variable(s) of Interest	Outcome Variable(s)	Study Results
Arnold et al., 200213	Prospective cohort	62 (92%)	M 20.2±2.2, F 19.3±1.5	Patient sex	SRC, knee anterior-posteri-or laxity	ANOVA showed a significant change to weekly SRC in women, but not in laxity of the knee.
DeFroda et al., 201910	Retrospec- tive cohort	165748 (100%)	15-19 (29.35% ACLR)	ACL reconstruction (ACLR)	Hormonal contraceptive (HCP) use	HCP use in 15-19-year-olds correlates with a 63% decrease in ACLRs; an OR of 0.37 (chi2 < 0.001, 95% CI 0.27-0.50) with NNT=6. In all age groups using HCPs, the ACLR OR was 0.82 (chi2 = 0.001, 95% CI 0.72-0.92).
Dragoo et al., 20116 (AJSM)	Prospective cohort	128 (100%)	Collegiate athletes	Serum relaxin level (SRC)	ACL tear	Cumulative ACL tear incidence was 21.9%, more likely with higher SRC, particularly >6pg/mL
Dragoo et al., 201114 (Int WJ)	Cross- sectional	169 (100%)	Collegiate athletes	Menstrual status	SRC	SRC was significantly lower in women using HCPs; the other groups were not significantly different
Dragoo et al., 20037	Lab study; cross- sectional	10 (50%)	F 15-36; M 16-35	ACL relaxin receptors	Positive stain; stain characteristics	Only ACL remnants from women expressed relaxin receptors; binding was specific, saturable, and uniform
Em at al., 201515	Case- control	BJHS 45; C 40 (100%)	BJHS 25.2±6.6; C 25.4±7.1	Benign Joint Hypermobility Syndrome (BJHS)	SRC, locomotor system exam findings	BJHS patients had non-significant elevation of SRC, with higher rates of arthralgia and myalgia (significant), and of pes planus and hyperkyphosis (strongly significant).
Faryniarz et al., 200616	Lab study; cross- sectional	15 (53.3%)	Female 26.6; male 32.3	ACL estrogen, relaxin receptors	Positive stain; positional relationship of receptors	Estrogen receptor levels were similar between the sexes, but relaxin receptor concentration was significantly greater in women; 4 of 5 female ACLs bound relaxin vs. 1 of 5 male ACLs
Galey et al., 20038	Lab Study	12 (66.7%)	46	ACL relaxin receptors	Positive stain; stain characteristics	ACL remnants from young female patients qualitatively demonstrated the most abundant hormone binding
Marshall-Gradisnik et al., 200417	Conference abstract-case series	10 (100%)	20.8±2.2	Active women on OCPs	SRC on CD 2, 16, and 26	Women on OCPs do not have significant changes in SRC during their cycles
Nose-Ogura et al., 201718	Prospective cohort	57 (100%)	24.4±4.6	Female athletes with luteal phase SRC>6.0pg/mL initiating HCPs	SRC during two subsequent menstrual cycle luteal phases	Of the 57 athletes, 36 (63.2%) had detectable luteal phase SRC. 21 (36.8%) had luteal phase SRC>6.0pg/ mL. Five of these 21 started OCPs, and by their second cycle, SRC decreased below the detection limit of 0.26pg/mL (p<0.01)
Pokorny et al., 200019	Case-control	55 (100%)	HCP 22.6±1.61; control 22.4±1.73	HCP use in women ages 20-25	Peripheral joint laxity; knee, 2nd digit DIP, 5th digit DIP	There was no significant difference in peripheral joint laxity with HCP use, including laxity of the knee. Laxity did not significantly differ by menstrual phase. Only 8 control and 11 test patients had an SRC drawn during the luteal phase.
Rahr-Wagner et al., 201411	Case- control (population registry)	13355 (100%); 4497 ACLR	23.8	ACL reconstruction and HCP use	Correlation of HCP use versus ACLR risk	Between “ever” and “never” HCP users, the adjusted RR of ACLR was 0.82 (95% CI, 0.75-0.90). Among long-term and recently-started HCP users, the adjusted RR of ACLR was 0.80 (95% CI, 0.740.91) and 0.81 (95% CI, 0.72-0.89). Results indicate HCPs are protective against ACL injury.
Average ± StDev	*67.3±52.2 patients; 88.5±19.7% female		25.2±7.5 years	Assessed: Athletic status (41.7%), OCP use (50%), ACL injury status (50%), generalized joint laxity (25%)		Conclusions regarding: SRC/joint laxity (25%), SRC/OCP use (25%), ACL/RLX binding (25%), and joint injury/ OCP use or joint injury/SRC (25%)

Table 2.

Included Studies per Subcategory of Relaxin Level of Effect

Subcategory	Author, Year	Findings	Subjective Synthesis
Molecular level effects of relaxin on the ACL	Dragoo et al., 20037	Only ACL remnants from women expressed relaxin recep- tors; binding was specific, saturable, and uniform	Compared to male ACL remnants, female ACL remnants are significantly more likely to bind greater amounts of relaxin, in a specific, saturable, and uniform distribution, among receptors in synovial tissue cells
	Faryniarz et al., 200616	Relaxin receptor concentration was significantly greater in women; 4 of 5 female ACLs bound relaxin
	Galey et al., 20038	ACL remnants from young female patients had abundant hormone binding
Effects of relaxin at the tissue level and knee joint level (small sample sizes)	Arnold et al., 200213	Women had a significant change to weekly SRC, but not anterior translation of the knee.	Women with higher SRC (at baseline, and/or during the luteal phase peak) were not more likely to show increased joint laxity such as increased anterior knee translation (2 studies) or increased knee hyperextension (1 study) Neither maneuver assesses torsional resistance Some measurements were not taken during peak relaxin levels However, higher peak levels of SRC were associated with increased ACL tear incidence This suggests that the collagenolysis trig- gered by relaxin in certain joints may not increase sub-failure tissue laxity, or laxity in all planes of mechanical stress, while still decreasing loads required for catastrophic tissue failure SRC is significantly lower among women using OCPs; OCPs appear to impact SRC within as little as two menstrual cycles
	Dragoo et al., 20116	Cumulative ACL tear incidence was 21.9%, more likely with higher SRC, particularly >6pg/mL
	Dragoo et al., 201114	SRC was significantly lower in women using OCPs
	Em et al., 201515	Relaxin was non-significantly higher in BJHS patients, who had significantly higher rates of pes planus, hyper- kyphosis
	Nose-Ogura et al., 201718	Roughly one-third of athletes had high luteal phase SRC (>6.0pg/mL). Five started OCPs and had SRCs <0.26pg/ mL by their second cycle (p>0.01)
	Pokorny et al., 200019	There was no significant difference in peripheral joint laxity between users and non-users of OCPs. However, only one-third had measurements taken during the luteal phase relaxin peak (CD 21-24).
Effects of relaxin at the knee joint level (large popu- lation studies), impact of relaxin concentration variability, and moderation on ACL injury	DeFroda et al., 201910	Among all age groups, the OR for ACL tear on OCPs was 0.82 (chi2= 0.001, 95% CI 0.72-0.92). This was driven by the 15-19 age group (odds ratio 0.37 (chi2< 0.001, 95% CI 0.27-0.50) who had a 63% reduction in tear risk. The NNT for OCP usage in 15-19yos= 6.	OCP use lowers baseline SRC in women and prevents luteal phase SRC peaks within a relatively short period of initiating treat- ment (2 menstrual cycles, ~2 months) OCPs show a protective effect against ACL tears in adolescents (63% reduction in rate of tear) and non-adolescents alike (18- 20% risk reduction) The NNT with OCPs to prevent ACL tear in the 15-19yo age group is 6 patients; pa- tients in all OCP studies did not report any serious adverse effects from OCP use
	Dragoo et al., 201114	SRC was significantly lower in women using OCPs
	Marshall- Gradisnik et al., 200410,11,14,17,18	Women on OCPs do not have significant changes in SRC during their cycles
	Nose-Ogura et al., 201718	Of athletes with high SRC (>6.0pg/mL), two OCP cycles decreased SRC below the detection limit of 0.26pg/mL (p<0.01)
	Rahr-Wagner et al., 201411	RR of OCP use vs. ACL injury was 0.82 (95% CI, 0.75- 0.90). RR of ACL injury was 0.80 (95% CI, 0.74-0.91) in long-term users and 0.81 (95% CI, 0.72-0.89) in recent users.

Average study size was calculated excluding data from DeFroda et al. and Rahr-Wagner et al.; both database-level analyses of population segments (165,748 and 13,335 patients, respectively) which skewed results.^10,11 The average study size of the remaining ten studies was 67.3±52.2 patients. Among all studies, average percent of female patients was 88.5%±19.7%. Average patient age was not available from DeFroda et al.,¹⁰ and was extrapolated to be 20 years old for both Dragoo et al. studies examining only “college female athletes”. With these adjustments, average patient age was 25.2±7.5.

In the twelve final studies, study design type was evenly dispersed: 25% of studies were case-control, 25% of studies were laboratory-based cross-sectional, 25% were prospective cohorts, and the remaining quarter were a retrospective cohort, a case series, and a non-laboratory cross-sectional study (Table 1). The properties of the population of interest were also divided in a fairly equal manner, with some study populations having more than one property specifically noted by researchers. In total, five studies (41.7%) focused on athletic participation, six studies (50%) focused on OCP use, six studies (50%) focused on current or past ACL injury status, and 3 studies (25%) focused on presence of generalized joint laxity (Table 1).

The most common properties examined in concert were athletic participation and OCP use, representing three of eight studies with more than one population property of interest (37.5%) (Table 1). It should be noted that both overlap studies which assessed OCP use plus current or former ACL injury were the population registry-based studies, providing a robust amount of data for this combination.^10,11 In reviewing all twelve studies, conclusions were drawn in the following categories: the SRC/joint laxity relationship (25% of studies), the SRC/ OCP relationship (25% of studies), the ACL/RLX binding relationship (25% of studies), and the SRC/ACL injury or OCP use/ACL injury relationship (25% of studies) (Table 1).

Methodological errors related to orthopaedic and gynecologic concepts were identified in two of the twelve studies. Both studies detailed errors related to knee/ACL laxity testing; Arnold et al. and Pokorny et al. assessed ACL laxity by measuring anterior-posterior translation of the tibia relative to the femur.^13,19 As the majority of ACL tears occur secondary to torsional/rotational force, the shear force of anterior-posterior testing is not a valid substitute. Pokorny et al.¹⁹ also describes sub-optimal timing for obtaining SRCs; only 8/25 control patients and 11/30 test group patients had SRC measured on a predicted peak day (cycle day 21-24) (Table 1).

Discussion

This review aimed to efficiently present existing information regarding the effects of relaxin on ACL injury in women. Based on preliminary literature analysis, our review outcome variables were structured to assess molecular level, tissue level, joint level, and population level impact of relaxin—including variable concentrations and moderation—on incidence of ACL injury (Table 3). This focused synopsis was designed to direct future research on this topic towards promising options for mitigation of relaxin-induced ACL damage.

Table 3.

Resources on Relaxin Properties, Effects Beyond the Reproductive Tract

Subcategory	Author, Year	Relevant Findings
Properties of Relaxin and Relaxin Receptors	Bryant-Greenwood et al., 19825 Goldsmith et al., 199520 Grossman et al., 201021 Kapila et al., 199822 Kleine et al., 201723 Lubahn et al., 200624 MacLennan et al., 198325 Powell et al., 201526 Wolf et al., 201227 Wolf et al., 201328	Relaxin (RLX) is a peptide hormone in men and women, largely H2 relaxin which binds the prevalent relaxin family peptide receptor-1 (RXFP-1) with high affinity The corpus luteum in women is the largest production site for relaxin in women Relaxin preferentially exerts paracrine effects, meaning that location of receptors rather than serum levels reflect activity Relaxin is biologically and immunologically active in pregnant and non-pregnant females, peaking during the luteal phase of the menstrual cycle RXFP-1 expression is primed by estrogen to respond at SRC 10-100x lower than normal Estrogen, progesterone, and relaxin receptors modulate MMP production and activity in chondroblasts, fibrochondroblasts, myofibroblasts, and ligaments
Functional and Physiologic Roles of Relaxin	Ando et al., 196029 Dragoo et al., 20037 Galey et al., 20038 Goldsmith et al., 199520 Grossman et al., 201021 Nose-Ogura et al., 201718 Powell et al., 201526	Relaxin controls extracellular matrix (ECM) turnover by stimulating collagenase (MMP-1/-13) and gelatinase (MMP-2/-9) expression, and suppressing collagen synthesis A major function of relaxin is increasing MMP-1 to degrade type 1 collagen while suppressing type 1 collagen de novo synthesis MMP signaling through MAPK and NF-kB pathways triggers acetylation/methylation to impact transcription MMP-1/-13 and -2/-9 synergistically digest collagen ° Fibrillar bundles dissociate, water uptake increases, and viscosity/density decreases ° Local total collagen decreases Degradation at nanoscale level means macro-level effects are not always ap preciable Relaxin has dose-dependent and differential functioning; dependent on location and other hormones Binding of estrogen, progesterone, and relaxin synovial joint receptors increases expression of inflammatory MMPs ° Relaxin also upregulates MMP-1/-13 and MMP-2/-9 In ligaments and fibrocar tilage
Physiology and Pathophysiology of Matrix Metal- loproteinases	Klein et al., 201130	MMPs modulate and turnover ECM protein components, and in states of dysregulation, are linked to disease development MMP-1: Collagenase-1; Interstitial collagenase One of few MMPs able to degrade fibrillar collagen, unwinding triple helix Key role in ECM turnover, excessive or insufficient action can cause fibrosis issues Local sampling (i.e. synovial fluid in RA patients) more accurate than serum levels MMP-2: Gelatinase A; 72-kDa type IV collagenase Proteolyticallly degrades gelatine (denatured collagen), constitutive expression, not induced by most inflammatory stimuli MMP-9: Gelatinase B; 92-kDa type IV collagenase Proteolytically degrades gelatine, activated by other MMPs/oxidative stress, attracts immune cells Crucial for multiple stages of female reproductive cycle, i.e. remodeling endometrial tissue during menstrual cycle MMP-13: Collagenase-3 Preferential cleavage of type II collagen Involved in bone development and remodeling, has been linked to cartilage destruc tion in OA, RA
Mechanics of Collagen-Based Structures, Catastrophic ACL Failure	Fidler et al., 201831 Meyer et al., 200832 Szcesny et al., 201433 Shoulders et al., 200934	Major ECM component collagen is characterized by fundamentally important triple helix, with physical (steric) and chemical (hydrogen bonds) stability Tendon/ligament composition: 20% fibroblast + 80% ECM The ECM is 60-70% fibres The ECM is 60-70% fibres Ligament fibres are 70-80% T1 collagen Tendon fibres are 95-99% T1 collagen Microscale and macroscale behavior of ex vivo ligaments shows collagen fibrils are loaded via interfibrillar shear, from sliding of fibrils, along with fibril elongation Interfibrillar shear load transfer between discontinuous fibrils is underlying mechanism of tendon fascicle mechanics, producing post-yield drop in tissue stiffness Non-linear elastic response of collagenous tissues under uniaxial tension is due to fibrils Roughly 90% of ACL injuries occur due to non-contact internal torsion of the tibia relative to the femur

Overview of Relaxin, Matrix Metalloproteinases, and Collagen

An appropriate discussion of relaxin impacts in one focal area necessitates a working knowledge of the endocrine properties of the hormone outside of a reproductive context and location, the physiologic and pathophysiologic functions of downstream factors signaled by relaxin, and the normal structural properties of the tissues on which relaxin activity is focused. Select resources are cited for this purpose (Table 3).

Relaxin is a peptide hormone only active beyond the reproductive system in women, who produce large amounts from the corpus luteum immediately following menstrual cycle ovulation. Its paracrine profile means that widespread presence of receptors is more indicative of activity than circulating serum levels. Relaxin has endocrine and immunologic functions, and it works synergistically with estrogen to increase target tissue RLX binding and levels of relaxin-activated effectors such as matrix metalloproteinases (MMPs). Specifically relevant to joint health, female ligaments, chondroblasts, and fibrochondroblasts bind relaxin (Figure 1) (Table 3).

Relaxin is known as the modulator of extracellular matrix (ECM) turnover due to its activation of MMPs, which collectively digest all protein components of the ECM. Specifically, relaxin upregulates active levels of collagenases (MMP-1/-13) which uniquely unwind the collagen triple helix, leaving it vulnerable to gelatinases (MMP-2/-9), also upregulated by relaxin. Collagen content decreases when relaxin is active, particularly type 1 collagen; targeted by MMP-1 for digestion while relaxin also suppresses de novo collagen synthesis (Table 3).

The type 1 collagen targeted by relaxin-activated MMP-1 is a major structural component of tendons and ligaments. When under stress, at the micro and macro level collagen transfers stress via the sliding fibril model to neighboring fibrils as shear force (interfibrillar shear). Interfibrillar shear causes the non-linear behavior of collagenous tissues under stress; the persistent decrease in tissue stiffness even when the load is removed. The collagen of the ACL predominantly fails from excess torsional forces (Figure 3), and joint motion patterns alter significantly following injury (Table 3).

Figure 3.

Hypothesized "Triple Hit" Scenario Resulting in Increased Female ACL Risk. The trio of factors which impact high rates of female ACL tear are biomechanical, anatomical, and hormonal. The biomechanical factor can be ascribed to relative weakness of hamstrings, and the anatomical factor is the result of an increased Q-angle. The hormonal factor has previously been ill-defined, but relaxin-induced degradation of collagen could be the major contributing component.

Molecular Effects of Relaxin on the Female ACL

Among all three studies of ACL remnants, harvested during ACL reconstruction procedures on males and females and immunostained for relaxin receptors, researchers found either exclusive or very heavily favored binding of relaxin to samples from female ACLs only. This finding laid the groundwork for considering relaxin’s role in this injury with such a predominant sex-preference. The relaxin was noted to bind to synovial cells and fibroblasts contained in the samples, reinforcing hypotheses that relaxin receptors are located synovially, and that fibroblasts (along with chondrofibroblasts and ligaments) are a type of articular cell to which relaxin will bind (Tables 2,3).

Relaxin binding to the female ACL is pathologically significant because relaxin triggers collagen digestion and suppresses collagen synthesis in a paracrine manner. Thus, during the menstrual cycle, estrogen would peak and increase expression of relaxin receptors along with levels of relaxin-activated MMPs. When relaxin subsequently peaks, it binds to the upregulated receptors surrounding the knee and works by two mechanisms to degrade local collagen: activation of MMPs which will digest even triple-helical collagen, and transcription suppression to prevent synthesis of de novo collagen. Given that ligaments are between 42% to 56% type 1 collagen in composition, there is high availability of target tissue for MMPs to digest. Therefore, the micro-scale effects of relaxin on the female ACL are degradation of type 1 collagen, a major structural component of the ligament. The collagen loses density and viscosity, and the total amount decreases; all of which would be expected to decrease collagen strength (Tables 2,3).

Effects of Relaxin at the ACL Tissue Level, and Knee Joint Level of Small Studies

Two included studies assessed ACL laxity in patients at sub-failure stress levels, comparing it to SRC.^13,15 However, both studies utilized the anterior-posterior (AP) displacement of the tibia relative to the femur as a proxy for ACL laxity, when in fact this scenario would better demonstrate the “dashboard sign” of a PCL laceration. The most important component of ACL strength for resisting common injury mechanisms is torsional resistance/rotational strength, which AP testing does not assess. It may also be considered that the molecular changes to the ACL collagen do not cause corresponding linear changes in elasticity of the ligament. It is entirely possible that relaxin activity lowers the torsional force required for catastrophic tissue failure; thus obvious signs may not be present at sub-failure forces (Tables 2,3).

From a clinical correlation perspective, a longitudinal study of 128 collegiate female athletes in various sports by Dragoo et al.⁶ showed a significant, positive correlation between peak relaxin levels and incidence of ACL tear. Collectively, one in five women completing a college athletics career suffered an ACL tear, which is a vast number when scale upwards to female athletes at numerous colleges across the country. When dividing this group by injury status, those with ACL tears had significantly greater peak SRC levels, even when athletes with undetectable SRC levels were not included in the statistical modeling. Furthermore, a subgroup analysis was performed on all athletes with detectable peak SRC (>0.26 pg/mL), showing that athletes with very high peak levels (>6.0 pg/mL) were, significantly, even more likely to suffer a tear than their peers with detectable SRC peaks below 6.0 pg/mL (Table 3).¹⁸

In fact, when an SRC > 6.0 pg/mL was utilized in post-hoc analysis as an ACL injury risk “cutoff value”, researchers found that female athletes with a peak SRC > 6.0 had 4.4 times increased risk of an ACL tear (Table 3).⁶ The positive correlation calculated in this study supports the hypothesis that molecular degeneration of the ACL from relaxin increases the risk of catastrophic tissue failure; the finding is bolstered by a second calculated positive correlation in which both variables have trended in the same direction; the increased SRC > 6.6 pg/mL significantly correlated with an even greater injury risk.⁶ A cohort study on OCP use of female collegiate athletes by Nose-Ogura et al.¹⁸ reinforces that female athletes with a 4.4 times increased risk of ACL tears are not uncommon; as 21.7% of their patients had an SRC of 6.0 pg/mL or higher (Table 3).¹⁸

Effects of Relaxin at Knee Joint Level of a Population, Variability and Moderation Effects

The inclusion in the review literature of two population registry-based studies helped greatly to assess the broader impact of relaxin on women’s musculoskeletal health, and also provided data from large sample pools for evaluation of potential impacts of SRC moderation.^10,11 A number of smaller cohort studies were able to determine that SRC could be intentionally lowered with OCPs; Dragoo et al.,¹⁴ Marshall-Gradisnik et al.,¹⁷ Nose-Ogura et al.¹⁸ So it was then a critical finding when both DeFroda et al.¹⁰ and Rahr-Wagner et al.¹¹ found that lowering SRC via OCPs had a significant protective effect against ACL tears at a population level (Table 3).

Rahr-Wagner et al., using the statistical model of risk reduction (RR), found an adjusted RR of 0.82 (95% CI, 0.75-0.90) between women who had started an OCP at any point in the past and women who never used OCPs. Between these two groups, OCP use decreased the relative risk of an ACL tear by approximately 80%.¹¹ DeFroda et al. utilized an odds ratio (OR) model, comparing OCP use vs. non-use among patients undergoing ACL reconstruction. Among all age groups in the study, the odds ratio of requiring ACL reconstruction while taking an OCP was was 0.82 (chi² = 0.001, 95% CI 0.72-0.92); OCP users were approximately 20% less likely to require surgery compared to their counterparts who did not take OCPs (Table 3).¹⁰

Researchers attributed much of this effect to the strong statistical findings among the 15-19 year-old age group where the OR for requiring ACL reconstruction while on OCPs was 0.37 (chi² < 0.001, 95% CI 0.27-0.50), or nearly 60% less likely. Given these findings, they calculated that the number of patients needed to treat (NNT) in the 15-19yo age group to prevent one ACL tear was six. So if OCPs were utilized to prevent ACL tears in girls between ages 15 and 19, six young women would need to be treated to prevent one ACL tear. (Table 3).¹⁰

How Can the Associated Risk Between Relaxin Levels and Female ACL Tears be Reduced?

Our secondary study outcome focused on potential approaches for moderating relaxin levels and thus ACL injury risk due to relaxin, and what the clinical effectiveness of these interventions might look like. In multiple studies in the present review, oral/hormonal contraceptives were selected as an intuitive intervention for an issue related to menstrual cycle hormones, and preliminary results show that their use can indeed reduce the risk of ACL injury.

However, it is recognized that OCPs are not a risk-free intervention, and that medical, social, and financial contraindications exist as likely barriers to widespread adoption. Still, the research performed using OCPs was very valuable for efforts to alleviate the sex disparity of this injury, for two main reasons: first, they demonstrate that SRC is a modifiable factor and that lowering it does not have adverse reproductive effects; and second, they demonstrate that lowering of the SRC is an approach which successfully reduces some of the risk of ACL injury (Table 3). Having these two important standards established is encouraging for future research on decreasing female ACL tear risk.

All included studies support and verify that peak relaxin levels occur during predictable days of the menstrual cycle, days 21-24. One way to build upon that is to follow the cohort studies which evaluated the use of a high SRC result as a “cutoff value”. The significant results lend credence to the idea of a “risk stratification” approach.⁶ For example, a female athlete with musculoskeletal pains during the luteal phase of her menstrual cycle may wish to obtain a peak SRC measurement. If the measurement is above the cutoff value, signifying increased risk, she then has more information when considering activity modification or OCP use.⁹ Conversely, a teammate with occasional mild symptoms and a low peak SRC may feel more comfortable making the informed decision to not pursue a preventive route. Appendix Item III depicts an example risk stratification approach in further detail.

Pharmaceutical advances, the synovial location of relaxin receptors, and the innate properties of compounds such as testosterone to be “collagen-protective” in direct opposition to effects of relaxin raise the possibility of eventually developing a targeted/local intervention. However, there is no reason for not concurrently pursuing easy-access, low-cost interventions, such as menstrual cycle tracking, which requires only patient education. This could prove simple and effective for populations such as recreational athletes and women who exercise regularly without competing in a sport. Having access to data on which days their joint health may be more at risk would allow them to modify their activities accordingly.

Limitations

Limitations of this literature review mirror limitations on this research topic as a whole. Only a small number of lower quality studies were available for review, with no large and well-designed randomized control trials. There are multiple studies to confirm relaxin preferentially binding female ACLs (three studies; Dragoo et al., Faryniarz et al., Galey et al.^7,8,16); relaxin levels cycling with other menstrual hormones and being suppressed by OCPs (four; Arnold et al., Dragoo et al., Marshall-Gradisnik et al., Nose-Ogura et al.^13,14,17,18); and OCPs reducing the risk of ACL tear (two; DeFroda et al. and Rahr-Wagner et al.^10,11

However, for the important conceptual linkage point of women with naturally elevated relaxin levels having an increased risk of ACL injury, there is only one study by Dragoo et al.⁶ This study has not, since the time of its publication, been replicated. This pool of literature also supports high relaxin levels not only having a correlational relationship with high ACL injury incidence rates, but possibly a causal relationship as well. Yet in the three years since the most recently published study, by DeFroda et al.,¹⁰ which showed a 63% risk reduction in ACL tear for teen girls using OCPs, not one interventional cohort study has gone to publication.

Additionally, because this topic crosses medical disciplines, it can be difficult to ensure that both major variables are measured in reliable and valid ways. For example, regarding ACL tension necessary to prevent ligament rupture, torsional strength is much more important than shear strength. However, studies in the present review assessed knee/ACL laxity by largely historical measures focused on anterior-posterior translation of the tibia relative to the femur, a shear force test, when the pivot-shift test of torsional force would have been more appropriate. Similarly, some studies in the present review measure SRC in women as though it were a static variable, rather than measuring the critically important peak SRC value by completing the test on CD21-24.

Conclusion

A reasonable correlative and plausible causative relationship exists between peak relaxin levels and increased risk of ACL injury in females, and further investigation is warranted.